Electrospun Plla Nanofiber Coating of Scaffolds For

ELECTROSPUN PLLA NANOFIBER COATING OF SCAFFOLDS FOR

APPLICATIONS IN BONE TISSUE ENGINEERING

A Dissertation

Presented to

The Graduate Faculty of The University of Akron

In Partial Fulfillment

Of the requirements for the Degree

Doctor of Philosophy

Phillip E. McClellan

December, 2015

ELECTROSPUN PLLA NANOFIBER COATING OF SCAFFOLDS FOR

APPLICATIONS IN BONE TISSUE ENGINEERING

Phillip E. McClellan

Dissertation

Approved: Accepted:

______Advisor Department Chair Dr. William J. Landis Dr. Coleen Pugh

______Committee Member Dean of the College Dr. Darrell H. Reneker Dr. Eric J. Amis

______Committee Member Interim Dean of the Graduate School Dr. Nita Sahai Dr. Chand Midha

______Committee Member Date Dr. Nic Leipzig

______Committee Member Dr. Edward Evans

ABSTRACT

In the field of tissue engineering, electrospun nanofibers gained notoriety for their capability to mimic the extracellular matrix of native tissues and organs.

However, few reports have been published that detail methods of producing electrospun nanofibrous materials with macroscopic three-dimensional complexity. There is a potential method of incorporating the benefits of electrospun nanofibers into prefabricated tissue engineering scaffolds in the form of a thin coating.

Nanofibers of poly-L-lactic acid (PLLA) were applied successfully to tissue engineering scaffolds composed of polycaprolactone/poly-L-lactic acid

(PCL/PLLA, 75/25) or sterile, human allograft bone by a modified electrospinning method. The electrospun PLLA nanofibers conform to the shape of the scaffolds, resulting in a thin layer of nanofibers over all the surfaces of the material. These scaffolds were then wrapped with human periosteal tissue and implanted in athymic (nude) mice. The mice, then, acted as bioreactors for growing and developing over various time periods the engineered electrospun PLLA nanofiber-coated constructs. Specimens containing PCL/PLLA as the underlying scaffold material were implanted for 10 weeks in vivo and specimens containing

iii allograft bone as scaffolds were implanted for 20 and 40 weeks. Harvested specimens were analyzed using histochemical and immunohistochemical methods to examine proliferation of cells, growth of new tissue, presence of mineral within the tissue, and presence of osterix, a bone-specific transcription factor necessary for osteoblast differentiation.

Mineralized tissue was present in the electrospun PLLA nanofiber-coated

PCL/PLLA constructs wrapped with periosteum after 10 weeks of implantation in vivo. Hematoxylin and eosin stains showed presumably new layers of tissue present between the layers of electrospun nanofibers and the underlying allograft bone in the allograft bone scaffolds coated with PLLA and then wrapped with human periosteal tissue. Osterix was identified by immunohistochemical staining and thereby verified presence of osteoblasts and preosteoblasts within the periosteal tissue and the electrospun PLLA nanofiber layers after 10, 20, and 40 weeks of implantation in vivo. The summary of these novel results suggests that electrospinning nanofibers such as PLLA on polymeric scaffolds or allograft bone can enhance tissue ingrowth from a periosteal wrap over such scaffolds or allografts for wider applications in bone tissue engineering.

DEDICATION

To my mother, father, and sister who have encouraged and supported me.

ACKNOWLEDGMENTS

First, I want to thank Dr. William J. Landis, my advisor, for his time and dedication in helping me to achieve my goals in graduate research. His dedication to students is astounding. I also thank Robin Jacquet for her assistance in developing the immunohistochemistry protocol for osterix as well as her help with the implantation of the constructs into nude mice. She devotes a considerable portion of her time and effort to ensuring the students in Dr. Landis’ laboratory have the tools and knowledge they need in order to succeed. Beth

Lowder was instrumental in providing knowledge of histological and cell culture techniques essential throughout the course of this research. I thank Dr. Bojie

Wang for providing access to and teaching me proper use of the scanning electron microscope in his laboratory. I appreciate the help I have received from all of my lab group members as well. Dr. Hitomi Nakao assisted with the nude mouse implantation surgeries. Qing Yu and Dr. Nakao helped with the harvesting of the constructs. I am especially grateful for the aid Dr. Darrell

Reneker provided through the course of the electrospinning portions of this project. I also thank the other members of my thesis committee, Dr. Nita Sahai,

Dr. Nic Leipzig, and Dr. Edward Evans, for providing insights which directed the

vi course of my research. Finally, I thank Dr. Susan Chubinskaya (Rush University,

Chicago, IL), the Gift of Hope Organ and Tissue Donor Network (Itasca, IL), and donor families for tissue access.

vii

TABLE OF CONTENTS

Page

LIST OF TABLES ...... xi

LIST OF FIGURES ...... xii

CHAPTER

I. BACKGROUND LITERATURE REVIEW...... 1

1.1 Bone fracture repair and regeneration ...... 1

1.2 Segmental bone defects ...... 3

1.3 Emergence of tissue engineering ...... 6

1.4 Periosteal tissue as a source of osteoprogenitor cells ...... 8

1.5 Brief history of electrospinning ...... 10

1.6 Basic principles of the electrospinning process ...... 12

1.7 Electrospun nanofibers used in biological systems ...... 13

1.8 Biodegradable nanofibers for tissue engineering ...... 15

1.9 Emulsion and coaxial electrospinning ...... 18

1.10 Fabricating three-dimensional nanofiber scaffolds ...... 19

1.10.1 Mechanically expanded nanofiber mats ...... 20

1.10.2 Nanofiber collector alteration ...... 20

1.10.3 Polymer solution manipulation ...... 22

viii

II. APPLICATION OF PLLA NANOFIBERS TO SCAFFOLDS OF PCL/PLLA ... 23

2.1 Materials and methods ...... 25

2.1.1 Preparation of PLLA nanofiber-coated PCL/PLLA scaffolds ...... 25

2.1.2 Scaffold drying and immersion in liquid ...... 26

2.1.3 Nanofiber morphology ...... 27

2.2 Results ...... 27

2.3 Figures ...... 30

2.4 Discussion ...... 37

III. TESTING OF PLLA NANOFIBER-COATED PCL/PLLA SCAFFOLDS IN VIVO ...... 45

3.1 Materials and methods ...... 46

3.1.1 Material and chemical sources ...... 46

3.1.2 Human periosteal tissue collection ...... 47

3.1.3 Scaffold sterilization and construct preparation ...... 47

3.1.4 Contruct harvest, fixation, processing, and embedding ...... 48

3.1.5 Construct sectioning and staining ...... 49

3.2 Results ...... 49

3.3 Figures ...... 51

3.4 Discussion ...... 59

IV. EXAMINATION OF PLLA NANOFIBER-COATED HUMAN ALLOGRAFT BONE SCAFFOLDS IN VIVO ...... 67

4.1 Materials and methods ...... 69

4.1.1 Human allograft bone scaffold preparation ...... 69

4.1.2 Preparation of periosteum-wrapped allograft constructs ...... 70 ix

4.1.3 Construct implantation, harvest, and analysis...... 70

4.1.4 Slide preparation and histochemical staining ...... 71

4.2 Results ...... 72

4.3 Figures ...... 74

4.4 Discussion ...... 87

V. IMMUNOHISTOCHEMICAL LABELING OF OSTERIX ...... 92

5.1 Materials and methods ...... 96

5.1.1 Sectioning and immunohistochemical staining of osterix ...... 96

5.1.2 Microscopy ...... 98

5.2 Results ...... 99

5.3 Figures ...... 101

5.4 Discussion ...... 106

IV. CONCLUSIONS AND FUTURE WORK ...... 112

BIBLIOGRAPHY ...... 119

APPENDIX...... 132

LIST OF TABLES

Table Page

5.1. Epitope retrieval solution, temperature, and time parameters………...... 98

LIST OF FIGURES

Figure Page

2.1. Schematic of modified electrospinning apparatus ...... 30

2.2. The proposed electric field diagram of the modified electrospinning design of this study ...... 31

2.3. Photograph of a PCL/PLLA scaffold (1) placed onto a fine-point stainless steel needle (2). The tip of the needle (3) protrudes slightly from the surface of the scaffold...... 32

2.4. Illustration showing comparisons between a typical PCL/PLLA scaffold lacking PLLA nanofiber coating (a), covered with PLLA nanofibers (b), and coated with PLLA nanofibers post-immersion for 24 hours in 100% ethanol...... 32

2.5. Scanning electron micrograph illustrating morphological characteristics of an uncoated, porous PCL/PLLA scaffold mounted on a stub (ST) using copper tape...... 33

2.6. Scanning electron micrograph highlighting uniform coverage of PLLA nanofibers electrospun over three surfaces (A, B, and C) of underlying PCL/PLLA scaffold material ...... 33

2.7. Scanning electron micrograph illustrating PLLA nanofiber coating on surfaces D-F of the PCL/PLLA scaffold ...... 34

2.8. Scanning electron micrograph showing collection of PLLA nanofibers on side A of the PCL/PLLA scaffold and located where the stainless steel needle protruded from the surface of the scaffold and was exposed to the ejected jet of PLLA nanofibers ...... 35

2.9. Scanning electron micrograph illustrating significant collection of nanofibers on side F of this PCL/PLLA scaffold and at the site where the needle was inserted into the scaffold during the electrospinning process. 35

xii

2.10. Scanning electron micrograph showing incomplete coverage of PLLA nanofibers over surface F (facing away from ejected jet of nanofibers) of the PCL/PLLA scaffold ...... 36

2.11. Scanning electron micrograph highlighting nanofiber coating over side E of the PCL/PLLA scaffold and porous structure of scaffold beneath ...... 36

2.12. Histogram of nanofiber diameter size distribution ...... 37

3.1. Intact human knee from a 51-year-old female donor ...... 51

3.2. Aseptic dissection and removal of overlying tissue from an intact human knee in a laminar flow hood to access and harvest periosteal tissue ...... 51

3.3. The basic design for the three experimental groups used in this study .... 52

3.4. Scaffolds implanted subcutaneously on the left and right sides of the back of an athymic mouse (left image) ...... 53

3.5. Diagram of results for staining two different sections in the same region of a specimen using alizarin red (left) and von Kossa (right) ...... 53

3.6. Results from toluidine blue, alizarin red, and von Kossa staining for a representative Group 1 scaffold harvested after 10 weeks in vivo ...... 55

3.7. Histological results from a representative Group 2 sample...... 56

3.8. Histological results of toluidine blue staining for a representative sample from Group 3 ...... 57

3.9. Histology results for alizarin red staining of a representative specimen from Group 3 ...... 58

3.10. Alizarin red (left) and von Kossa (right) staining of a representative specimen from Group 3 ...... 59

4.1. Diagram of experimental groups used in this study ...... 74

4.2. Schematic illustrating various regions of engineered constructs (1 and 2) assessed for thickness measurements of new layers of tissue formed between the electrospun PLLA nanofiber layers and underlying allograft bone scaffolds ...... 75

4.3. Samples from Groups 1 (left) and 2 (right) harvested after 20 weeks of implantation and stained with H & E ...... 76

xiii

4.4. Constructs from Groups 3 (left) and 4 (right) harvested after 20 weeks in vivo and stained with H & E ...... 77

4.5. Samples from Groups 1 (left) and 2 (right) harvested after 40 weeks of implantation in vivo and stained with H & E ...... 78

4.6. Murine fibrous tissue (F) stained with H & E is visible and grows into and beneath the layer of electrospun PLLA nanofibers (NF) coating the surfaces of the allograft bone (AB) in the Group 2 specimen shown in regions A and B in Figure 4.5 ...... 79

4.7. Specimens collected after 40 weeks of implantation in vivo for Groups 3 (left) and 4 (right) and stained with H & E ...... 80

4.8. Enlargement of region A of Figure 4.7 shows vascular elements (highlighted with arrows), including red blood cells, present within the open structure of allograft bone (AB) in this specimen from Group 3 constructs...... 81

4.9. Enlargement of region B of Figure 4.7 shows vascularity within the periosteum (P) from this specimen from Group 3 constructs ...... 81

4.10. Enlargement of region C of Figure 4.7 showing a transverse section of a blood vessel with red blood cells visible (highlighted with arrows) between periosteum (P) and allograft bone (AB) within this specimen from Group 3 constructs ...... 82

4.11. Enlargement of region D from Figure 4.7 shows a presumably new layer of periosteal tissue, separate from the original periosteum (P), formed between the electrospun PLLA nanofiber layer (NF) and the allograft bone scaffold (AB) in this specimen ...... 82

4.12. Similar to Figure 4.11, a presumed new layer of tissue (highlighted by arrows) is present between the electrospun PLLA nanofibers (NF) and the allograft bone (AB), separated from the original periosteum (P) ...... 83

4.13. Enlargement of region F in Figure 4.7 of this representative specimen from Group 4 shows supposed new layers of tissue (arrows) between the electrospun PLLA nanofiber layer (NF) and the allograft bone (AB) ...... 83

4.14. A histogram for the measured thickness of the new layers of tissue formed between the electrospun PLLA nanofibers and the allograft bone following 20 weeks of implantation in vivo (Group 4) ...... 84

xiv

4.15. A histogram for the measured thickness of the new tissue layers formed between the electrospun PLLA nanofibers and the allograft bone after 40 weeks of implantation in vivo (Group 4) ...... 85

4.16. A histogram of tissue layer thickness measurements for the new layers of tissue formed after 20 weeks of implantation in vivo (Group 4) and plotted in an effort to account for possible discrepancies in layer thickness measurements as a result of open cavities between allograft bone and the electrospun PLLA nanofibers ...... 86

4.17. A histogram of measurements for thickness of the new layers of tissue formed after 40 weeks of implantation in vivo (Group 4) ...... 87

5.1. Light micrograph of a PCL/PLLA (75/25) scaffold wrapped with periosteum, implanted for 10 weeks in an athymic mouse (Group 3, Chapter 3), and stained with anti-osterix (SP7) ...... 101

5.2. Light micrograph of a negative control IHC specimen from Group 3, Chapter 3 constructs ...... 102

5.3. Light micrograph illustrating a PCL/PLLA scaffold that shows osterix- positive osteoblasts (arrows) which appear to have migrated from the periosteum (P) to the electrospun PLLA nanofiber layer (NF) and underlying scaffold (PCL/PLLA) over the course of 10 weeks of implantation in an athymic mouse ...... 102

5.4. Osterix-positive cells (highlighted by arrows) are present within the electrospun PLLA nanofiber layer (NF) of a different area of the representative specimen shown in Figure 5.3 ...... 103

5.5. IHC staining results for a representative allograft bone scaffold (Group 1, Chapter 4) harvested after 40 weeks of implantation in vivo ...... 104

5.6. Results for electrospun PLLA nanofiber-coated allograft bone specimens (Group 2, Chapter 4) harvested after 20 (A) and 40 (B) weeks of implantation in vivo and stained with osterix antibody ...... 104

5.7. Osterix immunostaining results for representative periosteum-wrapped allograft bone constructs (Group 3, Chapter 4) harvested after 20 (A) and 40 (B) weeks of implantation in vivo show the presence of osterix-positive cells clustered within the periosteal tissue (P) around the allograft bone (AB) in these specimens ...... 105

5.8. IHC immunostaining results for representative specimens of electrospun PLLA nanofiber-coated allograft bone constructs wrapped with periosteum (Group 4, Chapter 4) ...... 106 xv

CHAPTER I

BACKGROUND LITERATURE REVIEW

Bone tissue engineering techniques have evolved throughout many years of medical research. The goal of this project was to advance the field by introducing nanofiber coatings into existing methods to accelerate regeneration and repair of segmental bone defects.

1.1 Bone fracture repair and regeneration

Bone is a mineralized tissue with a highly organized structure and is a critical component in vertebrate organisms.1,2 Although it was once believed to be static, only providing a support structure, it is now known that bone is a dynamic tissue, altered constantly in response to forces, mechanical and chemical, within and outside the body.3-5 As a structural component, it provides a solid base to which muscles and tendons can attach,6-8 and it is essential to protection of impact- sensitive organs, such as, lungs, heart, and brain. Bone exhibits the capacity for repair of small structural defects or fractures. The process by which bone tissue repairs itself is studied extensively.9-16 Immediately following fracture, the body restricts the size of blood vessels surrounding the break to prevent excess blood loss. Blood from the surrounding tissue collects around the injury site and forms

1 a clot and localized inflammation occurs at the site of the break as well.

Inflammation accomplishes two important functions. First, it leads to pain in the damaged location which prompts subconscious protection of the injury. Second, the excess liquid assists in stabilization of the break, limiting motion around the fracture site and acting as a cushion. The inflammation phase stimulates the release of multiple cytokines (such as tumor necrosis factor α and various interleukins (IL-1α, IL-1β, IL-6, IL-18)) responsible for the recruitment of a number of specialized cells (monocytes, lymphocytes, macrophages, and others) that begin initial repair of the fracture. During this stage macrophages and osteoclasts break down, and recycle materials from the damaged bone and surrounding tissue.

The next aspect of fracture repair is referred to as, appropriately enough, the reparative phase. This phase overlaps with the inflammation stage and is associated with the formation of a significant amount of new tissue. Whereas the first portion of regeneration is concerned primarily with stabilization of the fracture site and clearing of the damaged cells and tissue, the reparative stage focuses on establishing the bare framework to rebuild the tissue. Size, location, and mechanical stability of the fracture site are determining factors for whether a soft or hard callus is formed. A hard callus composed of bone will ultimately form if the mechanical stability of the tissue is high and the break is small enough, but these conditions are not the case for many fractures. Instead, a soft callus of fibrocartilage is generated to serve as a mechanically flexible template for new bone formation. In either case, extracellular matrix is laid down in thick layers

2 around the bone to generate the callus. In situations where a soft callus is created by chondrocytes and fibroblasts, it is slowly transformed from fibrocartilage to bone through the process of endochondral ossification to generate a hard callus. The hard callus is slowly altered over time to fit the original structure, size, and contour of the bone during the final phase, referred to as remodeling.

1.2 Segmental bone defects

While small fractures or defects in the structure of a bone heal with almost no scarring in most cases, destruction or removal of sizable portions of bone usually result in non-healing wounds.17-21 Methods devised to treat these large, segmental bone defects have improved significantly over the decades. The

Ilizarov method is used in many attempts to heal large defects in a bone. The technique was developed by a Soviet physician, Gavrii A. Ilizarov, in the 1950s as a means to address problems associated with bone defects, namely those of non-unions.22-24 A non-union refers to incomplete repair at the wound site, a significant issue that results in structural weakness within the bone structure and requires more time and surgeries for proper repair. The Ilizarov method of repair relies on distraction osteogenesis to rebuild/repair damaged bone tissue.25 In this approach, a large external fixator is attached to the damaged bone in multiple places. The fixator typically consists of several metal rings connected to pins that are inserted in the damaged bone at numerous sites. Separate rings are linked together using threaded rods, and the rods are adjusted to maintain

3 tension on healthy parts of the bone as well as to keep the defect area immobilized. The complete fixator also acts as a structural support, relieving pressure normally applied across the damaged portion of the bone.26,27

Application of tension to the intact portions of the bone around the defect induces growth of normal tissue and slow closure of the gap between the healthy bone pieces. The process has been very effective at healing segmental bone defects, but it is painful, time-consuming, and prone to allowing infections to enter the body through pin insertion sites in the skin. A possible method of mitigating these problems requires internalization of the entire fixation device instead of just inserting the pins through the skin.

Metals, such as titanium and stainless steel, can be implanted in the body and joined with existing bone structure to provide permanent, internal support structure.19 Other methods of accelerating bone regeneration were developed as well. Urist and Van de Putte published work in the 1960s highlighting the potential of using demineralized bone matrix (DBM) as an osteoinductive material to regrow damaged or missing bone.28,29 Removing the mineral from the bone tissue frees proteins (type I collagen and fibronectin, for example) and growth factors (primarily bone morphogenetic proteins (BMPs)) associated with regeneration of bone. In fact, DBM is still utilized in commercial products such as

DBX DBM (Synthes, West Chester, PA and the Musculoskeletal Transplant

Foundation, Jessup, PA), DynaBlast (Keystone Dental, Burlington, MA), Puros

DBM (Zimmer Dental, Carlsbad, CA), and StimuBlast (Arthrex, Naples, FL). The primary issue in using DBM stems from the limited supply of donor material.

DBM can be mixed with a filler material, for example hyaluronan, to help with the limited supply problem.30 Even with addition of a filler, there is still a shortage of

DBM, a result that has led researchers to develop procedures using one or two specific growth factors. Clinical treatments involving addition of single growth factors (usually BMPs) have met with some success in replacing DBM in grafting procedures.31

Other laboratories have focused on developing synthetic, biodegradable, biocompatible materials to employ in place of costly growth factors and natural tissue.32,33 Advantages of using these materials are cost, availability, and versatility. In addition, biodegradable materials act exactly as their name implies, that is, they degrade over time in a biological environment. Biocompatability is tangentially related to biodegradability and is important for any material placed into the body, biodegradable or not. Controlling the rate of degradation of a compound opens up many new avenues to repairing bone in the body. In fact, many different types of screws, pins, and patches were developed and are still in use today for skull defect repair (Synthes, West Chester, PA), ligament reconstruction (Depuy Synthes, Raynham, MA), and bone fixation (Biomet,

Warsaw, IN). No matter the specific application, the purpose of a biocompatible, biodegradable material is to serve as a support structure that is slowly broken down and replaced with natural tissue while any byproducts of degradation are disposed of by natural metabolic processes.

Even with new materials, better techniques, and more effective treatment options, non-unions remain a prevalent issue. Current methods are not suitable

5 for every long-term repair. For instance, inserting metal components as support might be insufficient to fix the bone of an individual who is still growing. Multiple surgeries may be required to remove or replace the metal during the lifetime of individuals. In other cases, the material used as the support is not integrated appropriately with the native bone tissue, resulting in a non-union.

Circumventing these issues is difficult in many cases and highly improbable in instances where large parts of the original bone are damaged beyond natural repair. New techniques or adaptations of current methods must seriously consider difficulties in repairing bone and preventing non-union situations to be effective.

1.3 Emergence of tissue engineering

The need to treat and repair severely damaged tissues has led to the development of a field known as tissue engineering. As its name indicates, tissue engineering refers to the growing, building, or engineering of replacement biological tissues. Once a concept relegated to fictional stories, the possibility of generating new body parts and organs was realized as researchers began collaborating with others outside their primary discipline. The combination of elements in biology, chemistry, physics, and engineering led to the formulation and development of materials that would provide basic structure and the possibility of directing living cells to generate an entire tissue or organ. Early work in tissue engineering was crude, utilizing off-the-shelf materials and simple structures to ensure the concept was fundamentally sound.34 Bioresorbable

6 sutures (typically made from polyglycolic acid, PGA) were in use already, and the natural progression was to utilize the same materials to support growth of cells and tissue. Freed et al. produced promising initial results when culturing chondrocytes for 8 weeks on PGA fibers (13 µm in diameter).35 Bovine chondrocytes attached to the fibers and retained their distinct spherical morphological characteristics in addition to being located within lacunae near the

PGA fibers and secreting glycosaminoglycans (GAG), a marker of chondrocyte activity. After demonstrating cells could survive and grow on simple biodegradable polymers, the next step was to focus on directing the cells to develop in a particular fashion and potentially rebuild an entire organ or tissue.

Significant results of the initial work using established biodegradable synthetic polymers35 led to the design and use of new biodegradable polymers. Complex, protein- and carbohydrate-based materials were synthesized to be degraded in a specific situation or with a unique enzyme.36 Other polymers were created with specific amino acid sequences for cell recognition and response.37-40 Structure of the scaffolds formed is variable as well and dependent on the tissue being repaired. Different scaffold structures include hydrogels, sponges, thin films, fibers, and numerous other materials. Advancements were not limited to formulation and fabrication of tissue engineering materials and scaffolds. New techniques for isolating and expanding the quantity of cells harvested from relatively small pieces of tissue have also increased the potential of producing viable tissue-engineered organs.41-43 Manipulation of the harvested cells is a possibility as well. Cell phenotypes are not as static as they were once believed

7 to be. Addition of specific growth factors or modification of the cellular environment can affect the type of tissue capable of being formed.43-46

Mesenchymal stem cells are targeted as having significant potential in the field of tissue regeneration.47-51 However, significant difficulty may be encountered with these cells as a consequence of their multipotent capability. They often require administration of growth factors or the presence of specific conditions in order to differentiate appropriately in vitro.49,51 Also, these cells secrete cytokines and other immune system suppression proteins which can lead to deleterious effects in vivo.52

1.4 Periosteal tissue as a source of osteoprogenitor cells

In the field of bone regeneration a valuable resource of progenitor cells is the periosteum. Periosteum is a very thin membrane-like layer of highly vascularized tissue present on the surface of bones throughout the body. Periosteum contains a fibrous extracellular matrix interspersed with progenitor cells capable of forming both cartilage and bone.53-60 Two layers of tissue comprise the periosteum. The first layer is a fibrous layer with a significant amount of collagenous matrix and elongated fibroblasts spread throughout its structure.

The second layer is known as the cambium layer. It faces the underlying bone and contains a significant number of cells responsible for growth and regeneration of cartilage and bone. During the reparative phase of bone fracture repair, progenitor cells from the periosteum proliferate and migrate to the fracture site and secrete extracellular matrix to build cartilage that makes up the soft

8 callus. As noted earlier, the soft callus is slowly replaced with mineralized bone tissue through the process of endochondral ossification.

As periosteum is critical to natural bone repair and growth processes, it is logical to utilize it for tissue engineering applications of bone and cartilage.

There are multiple methods employed to isolate osteoprogenitor cells from the periosteum and expand their number using cell culture techniques. Tissue can either be digested enzymatically to release the periosteal cells, or tissue pieces can be placed into a culture dish where the cells from the periosteum migrate from the tissue to the bottom of the plate where they proliferate.59 Of particular interest is the discovery that these periosteal-derived cells (PDCs) display regenerative properties similar to MSCs even when harvested from older patients.60 De Bari et al. detailed expansion of PDCs in monolayer cultures with addition of transforming growth factor β1 (TGFβ1). After 15 passages, PDCs from patients up to 95 years of age stained positive with alcian blue, an observation indicating chondrogenic potential of these cells.60 This result is important because it highlights the capability of utilizing autologous tissue to repair a tissue or organ defect, decreasing the chance of rejection or graft vs host disease occurring in cases where the cell source is not that from the individual who needs the repair.

Isolation and culture procedures can be time consuming and costly, requiring the addition of growth factors or specific nutrients to maintain osteogenic potential of the cells in vitro.54,60 Simpler procedures of harvesting and utilizing the entire periosteal tissue to regenerate bone tissue were developed to counter

9 the time and cost issues.61-63 Harvesting and utilization of the entire tissue has implications for clinical approaches where a decrease in the number of surgeries and time of hospitalization is essential to improving overall patient health and recovery. Intact periosteum also provides a barrier to protect the osteoprogenitor cells as they acclimate to their new environment. Much like DBM, the matrix of the periosteum contains structural components (blood vessels, Sharpey’s fibers, fibrous matrix) and proteins (collagen, growth factors) that could accelerate growth and development of new tissue.

1.5 Brief history of electrospinning

Coinciding with the emergence of tissue-engineered constructs is the re- emergence of a technique mentioned somewhat fleetingly over the last 100 or so years, electrospinning. Electrospinning is an ingenious method of producing nanoscale fibers in a rather simple fashion. While the technique has existed for approximately a century as noted, it did not rise to prevalence as a viable method of producing usable materials until the 1990s. The earliest published work with electrospinning was reported in 1887 when Charles V. Boys discussed a method of generating thin fibers using an electric current. He was focused primarily on producing single fibers for a specific use as evidenced by his remarks in the paper: “Fibres spun by the electrical method are so brittle that they do not seem to be of any practical use.”64 Just two years following the turn of the 20th century,

Cooley and Morton were each issued patents describing the dispersion of fluid

10 droplets utilizing electric potential.65,66 John Zeleny is also recognized for his work using the technique which was eventually coined “electrospraying”.67,68

Electrospinning was not investigated further until the 1930s and 1940s when

Anton Formhals applied for and was issued about 11 patents over the course of a decade. These detailed different methods and contraptions for generating and collecting electrospun fibers.69-79 Unfortunately, the devices and ideas of

Formhals did not generate significant interest in any major fields of research or production at the time. Even when Geoffrey Taylor examined the physics underlying the generation of electrospun fibers and electrosprayed droplets in the

1960s, there was not much interest in the process beyond the academic world.

The work of Taylor provided a detailed explanation and images of charged droplets as they formed conical structures on exiting a small orifice under electrically charged conditions.80,81 The cone formed just prior to ejection of droplets or fibers is referred to as a Taylor cone as a result of his descriptions.

While working at DuPont, Peter K. Baumgarten published work in 1971 detailing electrospinning of acrylic microfibers, but nothing significant was put into production by the company using his research at the time.82 The technique languished until the early 1990s when interest was renewed largely as a result of the need for a process which would enable the fabrication of fibers on the nanometer scale. In this regard, Reneker, Yarin, and other researchers began to examine more closely the fibers which were formed by this process and investigate the physics governing their formation.83-94

1.6 Basic principles of the electrospinning process

Electrospinning is made possible by an imbalance between two forces, surface tension and electric field strength. A solution or melt with sufficiently high viscosity passes through an orifice, usually at the end of a needle or metal cone with a small hole at its tip, to generate a small droplet. When surface tension dominates intermolecular interactions, the droplet of solution at the tip of the needle or cone is stable and remains in place. As the electric field strength increases, excess electrical charges, either positive or negative, accumulate on the surface of the suspended droplet and begin to overcome the surface tension.

A Taylor cone80,81 is formed as the drop distorts and a small strand of solution, or jet, is ejected from its tip, generating a flow-modified Taylor cone. The excess electrical charges within the jet repel one another, the result of which leads to rapid elongation of the expelled strand of solution. The jet of solution travels to a collector, which is usually in the form of a flat, electrically conductive plate. As the strand of solution travels to the collector, the solvent evaporates to leave solid fibers.

Many parameters may be manipulated to affect the electrospinning process.

A few factors often altered include applied voltage, distance between the electrospinning tip and collector, solvent volatility, electrical conductivity of the solution, and solution viscosity. Altering one or more of these parameters has a significant effect on the morphological characteristics of the formed nanofibers.

For example, increasing only solution viscosity increases the diameter of the fibers formed. Decreasing solution conductivity produces a similar effect.

Environmental factors such as temperature and humidity may also have a marked effect on the morphology of the fibers formed,83,84 and they have led to the creation of electrospinning designs that have tips and collectors either submerged in liquid95 or sealed in chambers with tightly controlled atmospheric conditions.96

1.7 Electrospun nanofibers used in biological systems

A very early attempt at utilizing electrospun materials in a biological setting occurred briefly during the 1970s when a polyurethane nanofiber vascular graft was fabricated and implanted into the thoracic aorta of minipigs for up to a full year.97 Early results were promising. The polyurethane nanofiber graft remained intact, showed no aneurism formation, and allowed for infiltration of cells and extracellular matrix from the surrounding vascular tissue. It also displayed similar structural and mechanical characteristics to the native blood vessel. However, these results did not appear to generate significant interest in the field. As mentioned previously, it was not until the 1990s that electrospun nanofibers worked their way back into research laboratories.89 Even then, nanofibrous mats of polymer were slow to merge with the field of tissue engineering. Instead, the focus was on generating nanofibrous materials for filtration devices,98 sensors,99 protective clothing,100 and other applications that would benefit from the high surface area-to-volume ratio of the nanofibrous mats. Attempts to incorporate electrospun nanofibers into biological environments truly began in the field of wound healing.

Biological applications of electrospun fibers concentrated on the field of surface wound healing for a few reasons. First, the materials used can be non- biodegradable and consist of polymers which are already used to form nanofiber mats.101,102 Second, the electrospun mats do not need to be subjected to the same rigorous testing protocols as a product used for implantation into the human body. Third, the morphological characteristics of the nanofibrous mats can be adjusted to allow air to pass through the mats while controlling or preventing the flow of moisture or dirt.100 Perhaps the most interesting feature of the nanofiber mats is the ability to couple them with antibacterial or regenerative agents to increase the rate of wound healing.

Small molecules or particles may become trapped within nanofibers as the solutions are electrospun. For instance, titanium dioxide nanoparticles have been added to nanofibers of polyurethane, which demonstrated increased antibacterial activity and significant water vapor permeability but exhibited relatively low cytotoxicity to cultured L929 (mouse fibroblast) cells. Water vapor permeability was examined because the desired application of these nanofibers was the treatment of burn wounds, which require moisture to prevent dehydration and promote tissue regeneration.101

Skin ulcers associated with leishmaniasis were treated effectively through the use of electrospun mats which were designed to release nitric oxide upon the addition of a small amount of water.102 The primary difficulty encountered in utilizing non-biodegradable materials is that they remain in place if they are implanted into the body. In order to circumvent this issue, biodegradable

14 polyesters such as PGA, PLLA, and PCL were examined with the intent of creating biodegradable nanofibers that may be implanted into a human body and dissolved slowly over time with few, if any, deleterious effects.

1.8 Biodegradable nanofibers for tissue engineering

A significant advance in the application of biodegradable polymers for tissue engineering was made in utilizing them to generate nanofibrous materials which could, in theory, mimic the native extracellular matrix of certain tissues. In fact, comparison of scanning electron micrographs demonstrates a marked similarity between normal extracellular matrix isolated from bovine vertebrae103 and electrospun fibers of PLLA. In 2001, Boland et al.104 detailed the production and mechanical testing of electrospun fibers composed of PGA, a biocompatible, bioresorbable polyester, which is used often in products such as dissolving sutures. One of the major benefits of the electrospinning process derives from the simplicity and tenability of the process. Simple alterations to the polymer solution or the electrical or environmental parameters can result in pronounced effects on fiber diameter, morphology, and chemical composition.105 Nanofiber sizes and composition could be tailored in specific cases for maximum benefits.

Other biodegradable synthetic polymers such as PLA,106,107 PCL,107-109 and polyethylene oxide (PEO)106,109 were electrospun with similar results.

Electrospun nanofibrous mats provide clear advantages in wound healing applications, particularly with regard to vascular and skin grafts. Typically, nanofiber mats are of a simple, two-dimensional nature, ideal for a thin, flat organ

15 such as skin.110 When rolled into a simple tube, nanofibrous sheets have the potential to make excellent vascular grafts.97,111-114 Vaz et al. detailed the fabrication of a multi-layered tube having nanofibers of PCL and PLA for the inner and outer layers, respectively.114 Their bilayer tubes showed sufficient strength and elasticity for potential vascular utilization. Histological examination of the potential vascular graft seeded with mouse fibroblasts (3T3 cell line) highlighted attachment and proliferation on and into the nanofibrous matrix over the course of 4 weeks in vitro. Gaudio et al. reported fabrication of a more complex structure when they generated a simple trileaflet heart valve using a slightly modified nanofiber collector design. Even lacking complexity of multiple layers (ventricularis, spongiosa, and fibrosa) of specialized tissue found in normal heart valves, functional testing of the PCL nanofiber heart valve demonstrated pressure and flow characteristics similar to those of normal valves.115

Natural polymers can be integrated into the nanofiber structure as well. Lee et al. fabricated a bilayer nanofiber scaffold composed of PCL and collagen for vascular scaffolds.111,112 Inclusion of collagen nanofibers in the constructs led to differences in the mechanical properties of the scaffolds under dry and wet conditions that more closely mimicked those of a native porcine coronary artery.111 Collagen has the added benefit of being a component of the ECM of blood vessels and holds the potential to increase rates of cell attachment and proliferation. Human aortic endothelial cell adhesion was examined on the nanofibers at multiple timepoints (1, 12, 24, and 72 hours) using immunofluorescent staining and confocal microscopy.112 Cells grown on smaller

16 nanofibers (average diameter = 0.27 µm) exhibited better organization of their cytoskeleton as well as more focal adhesion. Skin and blood vessel regeneration are not the only fields which benefit from inclusion of nanofibrous materials. Flat, electrospun nanofibrous mats have also been tested for their capability of supporting and promoting the growth and development of bone cells and tissue.116-118

Wutticharoenmongkol and colleagues demonstrated addition of small particle components to individual nanofibers by generating nanofibrous mats of PCL which contained calcium carbonate or hydroxyapatite nanoparticles for bone tissue regeneration.118 The nanofibrous mats produced were referred to as a

“guided bone regeneration membrane,” and the composite nanoparticles of the mats were postulated to facilitate the proliferation and differentiation of osteoblasts. Early results indicated the scaffolds did not exhibit any cytotoxic effects on human osteoblasts or rat fibroblasts.118 In addition, the osteoblasts grown on nanofibers containing calcium carbonate were examined under scanning electron microscopy and demonstrated early signs of mineralization with the appearance of small crystalline granules.118 A notable potential consequence of inducing and maintaining osteoblast differentiation was mentioned in the publication.118 The study found the osteoblasts which had differentiated slowed considerably with respect to proliferation. A similar situation was mentioned by Owen et al. in a study of rat osteoblasts grown in vitro.119

1.9 Emulsion and coaxial electrospinning

Modifications to the electrospinning process led to generation of nanofibers having more complex, multi-layered architectures with the capability of adding therapeutic agents to make drug delivery devices capable of accelerating or supporting regeneration of cells and tissue. The two prevalent techniques in this area are emulsion and coaxial electrospinning. In the case of both approaches, the nanofibers produced consist of an outer sheath and inner core of differing composition. Emulsion electrospinning relies on a chemical means of separation through the creation of an emulsion within a single solution. There is subsequent organization of the emulsified droplets into two distinct phases as the solvent evaporates from the electrospun fibers.120 This technique has proven successful in encapsulating a variety of molecules, including nerve growth factor,121 proteinase K,122 lysozyme,123 cytochrome C,124 and bovine serum albumin.125

Emulsion electrospinning is also being investigated because it accommodates the use of water as the solvent instead of more hazardous chemicals.126

Compared to emulsion electrospinning, coaxial electrospinning generates core- sheath fibers by physical separation through the utilization of two electrospinning tips and two solutions.120 Certain processing parameters and solution properties such as inner and outer solution flow rate, viscosity, and electrical conductivity are often taken into account when attempting to utilize the coaxial technique.

1.10 Fabricating three-dimensional nanofiber scaffolds

The examples mentioned in prior sections of this paper illustrate significant interest in developing methods and devices which utilize electrospun nanofibers to facilitate tissue repair and regeneration in the human body. A current, major field of focus in tissue engineering is the inclusion of nanofibers within more complex three-dimensional structures that could replace an entire organ.

Facilitating cellular growth in three dimensions is desirable because the native environment of a cell rarely consists of a two-dimensional matrix. Even “flat” tissues such as skin, blood vessels, and heart valves contain multiple layers of extracellular matrix and exhibit three-dimensionality in a limited form. Tissues and organs can hold intricacies well beyond that of a simple, two-dimensional sheet of nanofibers. Greater benefits are realized if nanofibers are produced or added to structures having increased three-dimensional complexity.

Researchers are still working on ways of transitioning from two to three dimensions using electrospun fibers. In this context, difficulty arises as a result of the manner in which the nanofibers are collected typically, for example, on a charged, flat plate conductor of opposing charge to the source of the fibers.

There are three distinct aspects of the electrospinning process that can be modified to create nanofibers having greater macroscopic three-dimensional complexity. First, the nanofibrous mats may be modified post-production.127

Essentially, nothing in the electrospinning process is changed. It is a simple and rather crude process, but it is effective. The second area of the process subject to alteration is the collection process. Alteration of the nanofiber collector allows

19 for the creation of simple three-dimensional structures.128,129 The third portion of the electrospinning process that is open to modification is the polymer solution components. Adding molecules to accelerate charge transfer from the electrospun nanofibers to the collector is an elegant means of achieving a three- dimensional nanofiber sponge scaffold.130

1.10.1 Mechanically expanded nanofiber mats

The first and simplest approach to overcome the two-dimensionality limitation of electrospinning noted above is to take the completed mats and physically pull them apart.127 Shim et al. generated microfibrous mats composed of PLLA which they separated post-production using a metal comb. The main advantage to this method is the lack of a need to alter anything already in place for the electrospinning apparatus. A significant disadvantage to the technique is a lack of control over the porosity of the scaffold as well as partial destruction of the material in the form of broken fibers. The broken fibers could lead to structural weakness and cause difficulties when attempting to replicate load-bearing tissues. Testing of mechanically expanded nanofiber mats with MC3T3-E1 cells

(murine osteoblasts) in vitro showed significant cell migration into the open structure, a feature difficult to achieve in sheets of tightly packed nanofibers.127

1.10.2 Nanofiber collector alteration

As an advance to the electrospinning process, modification of the nanofiber collector provides for generation of PCL nanofibers resembling a cotton ball.128

Instead of collecting the fibers on a flat plate, Blakeney et al. utilized a spherical foam dish backed by a stainless steel lining.128 Multiple stainless steel probes were inserted into the foam dish and connected to the stainless steel lining.

Blakeney et al. coined the product of this technique Focused Low density,

Uncompressed nanoFibrous (FLUF) scaffolds.128 Their method was unique, innovative, and patented. A significant advantage of this method over the

“mechanically separated” fibers is the absence of structural damage to the individual fibers and better control over the final scaffold porosity. Seeded INS-1

(pancreatic beta) cells infiltrated the FLUF scaffolds over the course of 7 days, in contrast to the nanofiber sheets of PCL, where the cells only grew on the surface of the material. The minor disadvantage of this technique is the requirement of a modified nanofiber collector.128

Another modification of the nanofiber collection method referred to as “two- pole air gap” collection was detailed by Jha and colleagues.128 Instead of collecting the nanofibers on the surface of a physical conductor, they set up two electrodes separated by an air gap, and nanofibers of PCL were collected in the air gap between the electrodes. Nanofibers displayed a degree of alignment in the conduits which were tested in vivo using a rat model where the sciatic nerve was damaged (10 mm section removed) and the nerve conduit was inserted into the section of missing neural tissue.129 Observations of the rats over seven weeks showed improvement in their gait at six weeks, especially when compared to two weeks post-surgery where the rats were walking on three limbs and motion of the leg closest to the nerve injury was not present.129 On recovery of

21 the conduits after seven weeks in vivo, histological examination of the scaffolds showed axon regeneration within the nerve guide.129

1.10.3 Polymer solution manipulation

A third process by which three-dimensional nanofibers can be generated involves manipulation of the polymer-solvent solution prior to its addition to the electrospinning device. Cai et al. added sodium dodecyl sulfate (SDS) to the zein/ethanol solution before placing it into the syringe of the electrospinning apparatus.130 The addition of SDS increased surface electrical conductivity and led to more rapid dispersal of the charge from the zein nanofibers to the collector.

Quick dispersal of charge was then countered by a rapid collection of the opposing electrical charge from the collector. The positively charged nanofibers were repelled slightly from the flat collector surface before they had the chance to lie flat. The advantage of this method is that few modifications are required to the electrospinning apparatus. The major disadvantage is the need to modify the polymer-solvent system with a surfactant molecule (SDS) which could impact the final properties of the nanofibers. Confocal microscopy of MC3T3 cells (murine fibroblasts) cultured in vitro on the zein nanofiber scaffolds showed increased cell proliferation over seven days and cell shapes that more closely resembled those found in native tissue.130 Both of these benefits were a direct result of the three- dimensional structure of the scaffolds. The open network of fibers provided more volume where the cells could proliferate.130

CHAPTER II

APPLICATION OF PLLA NANOFIBERS TO SCAFFOLDS OF PCL/PLLA

The three methods for generating three-dimensional scaffolds described in

Chapter 1.10 of this thesis are limited for a common and simple reason: each technique produces structures without the mechanical integrity and strength sufficient to replace load-bearing elements within the body. Bone tissue engineering necessitates a delicate balance between cellular infiltration and growth and the structural properties of resulting tissue and organs. Solid materials provide structural support but have no open network to allow cells to infiltrate them. Porous materials offer rapid cellular infiltration but can exhibit compromised mechanical strength. Devising a method of fabricating scaffolds to include advantages of both solid and porous configurations, while limiting disadvantages of both, is a challenge. Of considerable interest in this context is the capability of incorporating nanofiber technology in pre-existing scaffolds as a measure to accelerate cellular growth and infiltration during initial seeding and growth phases of engineered tissues.

As an example of this approach, previous work highlighted growth of human phalanges by wrapping human periosteum around scaffolds composed of

PCL/PLLA and implanting the constructs in athymic mice for up to 60 weeks.62,63

Tissue-engineered constructs demonstrated the presence of mineralized bone tissue after 60 weeks in vivo. In this work, the time necessary to produce a small tissue is extensive and an aspect of this issue originates with initial attachment and infiltration of cells from periosteum. It is essential that progenitor cells within the cambium layer of the periosteal tissue migrate from their native environment and begin secreting extracellular matrix components within the scaffold. Initial matrix deposition occurs slowly in many cases, but adding a thin layer of nanofibers to the surface of a pre-existing material has the potential to increase and improve initial cellular migration and matrix production processes in the resulting constructs.

The essential nature of electrospinning relies on charge conduction over the surface of a material to be coated, and preliminary attempts to produce nanofiber

“coatings” over the surface of the scaffolds proved difficult with scaffold materials, such as PCL/PLLA, that are poorly electrically conductive.34-36 Multiple methods exist, however, to facilitate the transfer of electrons over the surface of poorly conducting materials. For example, the underlying chemical structure of the material can be modified to expose charge carrying molecules to the surface.

Additionally, solutions can be added to the surfaces that increase the rate of charge transfer, or the material can be physically connected or linked to another, larger material that itself conducts surface charge from the initial material.

In applying the latter concept, and to facilitate and enhance efficient collection of electrospun nanofibers on the surfaces of pre-formed, poorly conductive

PCL/PLLA polymer scaffolds, a simple modification was applied: a very fine

24 stainless steel needle was inserted through the scaffold. The presence of the needle resulted in a much more effective electrical connection between the scaffold and flat plate collector. In this electrospinning system, the needle provides an attractive electrical potential near the surface of the scaffold and also an electrical connection to ions migrating on the surface of the polymer scaffold and collected nanofibers. This situation thereby maintains a potential that results in a collection of more fibers. Further, addition of the needle allows the scaffold to be supported above the collector so as to promote nanofiber deposition over the top, sides, and bottom of the PCL/PLLA scaffold. This design offers a new means of producing an electrospun three-dimensional nanofiber coating closely covering all surfaces of a particular scaffold. Deposition of electrospun nanofibers in this manner expands electrospinning technology to a wide array of applications in which three-dimensional coatings are advantageous. Detailed in this and the following chapters are methods of producing nanofiber-coated scaffolds composed of PCL/PLLA and human allograft bone, preparation of human periosteum-wrapped constructs, testing of periosteum-wrapped constructs in vivo, and histological and immunohistochemical analysis of said constructs.

2.1 Materials and methods

2.1.1 Preparation of PLLA nanofiber-coated PCL/PLLA scaffolds

The basic electrospinning apparatus developed and utilized by Reneker and

Yarin83 was experimentally adapted with the addition of a single, fine-point

25 needle to the flat plate collector. A 1.0% (w/v) solution of PLLA (700 kDa,

Polysciences, Warrington, PA) in chloroform (ACS reagent grade ≥99.8%,

Sigma-Aldrich, St. Louis, MO) was prepared and stirred continuously over a 12 hour period. The solution was then loaded into a 5 mL syringe (Luer-Lok tip, BD,

Franklin Lakes, NJ) having a blunt-tip needle (25 gauge, ½ inch in length, BD) attached to it. The syringe was placed into the syringe pump (NE-300, New Era

Pump Systems, Farmingdale, NY), and the positive electrode from a high-voltage power supply (ES60-10W, Gamma High Voltage Research, Ormond Beach, FL) was connected. A fine (0.5 mm diameter, 35 mm length) stainless steel needle was attached to a flat, grounded collector, and the end of the needle was inserted by hand through each of a number of poorly conductive, pre-formed rectangular-shaped tissue scaffolds (0.6 cm x 0.5 cm x 0.5 cm in dimensions) composed of PCL/PLLA (75/25 ratio of PCL/PLLA; Gunze Co., Japan).

Nanofibers of PLLA were generated and deposited onto the surface of the scaffolds using an applied voltage of 13.1 kV and working distance of 6 cm between the electrospinning tip and the tip of the grounded needle. Figure 2.1 is an illustration of the basic arrangement for the electrospinning design utilized in this process.

2.1.2 Scaffold drying and immersion in liquid

Scaffolds were coated in a one-by-one fashion. The time required to coat each scaffold sufficiently ranged from 55 to 70 minutes. Immediately after a scaffold was coated, it was removed carefully from the stainless steel needle

26 without greatly disturbing the nanofibers on its surfaces. The scaffold was then transferred to a small, covered petri dish and left to dry in a fume hood at ambient temperature (~22°C) overnight (~14 hours). Dried nanofiber-coated scaffolds were then placed into separate wells of a 12-well culture plate and stored at 4°C. Nanofiber-coated scaffolds were removed from storage and immersed in 100% ethanol for a period of 24 hours.

2.1.3 Nanofiber morphology

Uncoated and electrospun-coated scaffolds were mounted on copper studs following immersion in ethanol and subsequent drying. They were then sputter- coated with silver for examination by scanning electron microscopy (JEOL-7401,

Japanese Electron Optics Laboratory, Peabody, MA; SEM). The SEM was operated using multiple accelerating voltages (1.0-2.0 kV) and specimen images were recorded and collected digitally. Nanofiber diameter size distribution was determined using measurements collected in ImageJ. Histograms were constructed using Analysis Toolpak in Microsoft Excel 2010.

2.2 Results

Figure 2.2 shows a representative specimen mounted onto the end of the fine-point, stainless steel needle used in these studies, and Figure 2.3 illustrates the general electric field surrounding the needle-supported scaffold. The basic electric field diagram was generated in part from the program, Electric Field

(version 2.01, www.physics-software.com). Figure 2.4 shows a macroscopic

27 comparison between representative examples of uncoated, coated, and coated/post-immersion PCL/PLLA tissue scaffolds. There are differences in size and shape between uncoated (Figure 2.4a) and the two nanofiber-coated scaffolds (Figure 2.4b,c), these two latter samples being covered and increased in dimensions as visualized in part by PLLA nanofibers electrospun over the sample surfaces and collected around the supporting needle. Electrospinning was accomplished with use of a small stainless steel needle inserted left-to-right through the two relevant scaffolds shown in the figure. The small, irregular surface features visible on coated scaffolds (Figure 2.4b,c) are the direct result of initial PLLA nanofiber deposition at the tip of the needle during the electrospinning process. Additionally, the coated scaffold (Figure 2.4c) demonstrates that electrospun PLLA nanofibers remain attached to the underlying scaffold after 24 hours of exposure to 100% ethanol.

Figure 2.5 illustrates the structure of the PCL/PLLA scaffold having no nanofibers covering its surface. The scaffold is porous, and its surface features are readily distinguishable on SEM from the features of a scaffold coated with electrospun PLLA nanofibers.

For ease of explanation, the six surfaces or sides of the rectangular scaffolds coated with nanofibers are labeled A - F in SEM images presented (Figures 2.6 –

2.11). The needle was inserted through the scaffold surfaces A and F. Letters designate the following: A – the “top” or surface of a scaffold that faced the electrospinning tip, B through E – the four scaffold surfaces parallel to the

28 inserted needle, and F – the “bottom” or surface of a scaffold facing away and physically hidden from the electrospinning tip.

Figures 2.6 and 2.7 show nanofiber coverage over all six surfaces of the electrospun scaffolds. The image in Figure 2.6 demonstrates complete coverage on the top (A) and two separate surfaces (B and C) of the scaffold. Figure 2.7 illustrates coverage over the other two surfaces (D and E) as well as the scaffold bottom (F). Figure 2.8 presents an image of the “peak” of PLLA nanofibers formed over scaffold surface A as a result of the stainless steel needle protruding from this scaffold side facing the electrospinning tip. The F scaffold surface, opposing the A scaffold surface of the specimen, is visible in Figure 2.9. The grouping of nanofibers around the central portion of this image demonstrates the collection of PLLA around the point of protrusion of the metal needle at this surface.

Figure 2.10 shows incomplete coverage of nanofibers on the F surface of a specimen as well as illustrating the edge between surfaces F and C where the nanofibers are in close contact with the scaffold. Nanofibers of PLLA are visible in the foreground of Figure 2.11. The underlying PCL/PLLA scaffold may be seen through the gaps between individual fibers on a surface E when utilizing higher magnification in the SEM.

Figure 2.12 is a histogram illustrating unimodal diameter size distribution of nanofibers on the surface of the PCL/PLLA scaffolds. Mean fiber diameter was

1.15 µm with a standard deviation of 0.32 µm.

2.3 Figures

Figure 2.1. Schematic of modified electrospinning apparatus. PCL/PLLA scaffold (1) is mounted onto a fine point needle (2) with the tip of the stainless steel needle protruding approximately 2 mm from the surface of the scaffold facing the ejected jet of nanofibers. The needle is attached to a flat, conductive surface (3) which is connected to the negative terminal of a high voltage power supply (V). A solution of PLLA in chloroform is added to a syringe (4) having a blunt tip, 18 gauge needle connected to the positive terminal of the high voltage power supply.

Figure 2.2. The proposed electric field diagram of the modified electrospinning design of this study. A small amount of surface charge collects on the PCL/PLLA material (2) as a result of the connection between the needle (3) and a flat collector (4) attached to the negative side of a high voltage power supply (V).

The positive electrode from the high voltage power supply is connected to the end of a syringe containing PLLA/chloroform solution (1).

Figure 2.3. Photograph of a PCL/PLLA scaffold (1) placed onto a fine-point stainless steel needle (2). The tip of the needle (3) protrudes slightly from the surface of the scaffold.

Figure 2.4. Illustration showing comparisons between a typical PCL/PLLA scaffold lacking PLLA nanofiber coating (a), covered with PLLA nanofibers (b), and coated with PLLA nanofibers post-immersion for 24 hours in 100% ethanol.

Figure 2.5. Scanning electron micrograph illustrating morphological characteristics of an uncoated, porous PCL/PLLA scaffold mounted on a stub

(ST) using copper tape.

Figure 2.6. Scanning electron micrograph highlighting uniform coverage of PLLA nanofibers electrospun over three surfaces (A, B, and C) of underlying PCL/PLLA scaffold material. During the electrospinning process, side A was oriented on the needle so that it was directly facing the ejected jet of PLLA nanofibers. Sides B

33 and C of the scaffold were parallel to the electrospinning tip during the electrospinning procedure.

Figure 2.7. Scanning electron micrograph illustrating PLLA nanofiber coating on surfaces D-F of the PCL/PLLA scaffold. Surfaces D and E are analogous to sides denoted as B and C in Figure 2.6. Orientation of side F was such that it faced away from the ejected jet of nanofibers during the electrospinning procedure.

Figure 2.8. Scanning electron micrograph showing collection of PLLA nanofibers on side A of the PCL/PLLA scaffold and located where the stainless steel needle protruded from the surface of the scaffold and was exposed to the ejected jet of

PLLA nanofibers.

Figure 2.9. Scanning electron micrograph illustrating significant collection of nanofibers on side F of this PCL/PLLA scaffold and at the site where the needle was inserted into the scaffold during the electrospinning process.

Figure 2.10. Scanning electron micrograph showing incomplete coverage of

PLLA nanofibers over surface F (facing away from ejected jet of nanofibers) of the PCL/PLLA scaffold. Nanofiber coverage over surface C is more uniform but difficult to see from the angle of the image. The mounting stub is visible in the upper right of the image, labeled ST.

Figure 2.11. Scanning electron micrograph highlighting nanofiber coating over side E of the PCL/PLLA scaffold and porous structure of scaffold beneath.

Nanofibers display random orientation and relatively uniform distribution of fiber diameter.

25 Nanofiber Diameter Size Distribution

5 Percentage of nanofibers measured of nanofibersPercentage 0

Nanofiber Diameter (µm)

Figure 2.12. Histogram of nanofiber diameter size distribution. Fiber diameters ranged from 0.30 microns to 2.55 microns in diameter. Mean diameter of fibers was 1.15 microns with a standard deviation of 0.32 microns.

2.4 Discussion

Tissue engineering research is replete with reports describing methods to fabricate nanofibers utilizing various electrospinning techniques. Most of these techniques, however, produce nanofibers that are absent of the critical feature of macroscopic three-dimensional complexity. While the blood vessel tube models,110,111,113,114 mechanically expanded fibers,127 and FLUF scaffolds128 noted in Chapter 1 of this dissertation are valuable in their own right, they also do

37 not possess sufficient mechanical strength for effectiveness in the engineering of bone tissue. The technique demonstrated here is meant to combine the desirable attributes of electrospun nanofibers (capability to increase cell attachment and proliferation) with those of current tissue scaffolds (appropriate mechanical strength and three-dimensional complexity).

The fine stainless steel needle in the electrospinning design (Figure 2.1) collects, by surface diffusion of ions, nanofibers over the full, three-dimensional surface of a poorly conductive scaffold such as PCL/PLLA. As with insulating specimens typically utilized for electrospinning, the absence of a needle and ionic conductor, or another electrically conductive implement, in this regard results in electrospun nanofibers deposited inefficiently, or not at all, over the complete surfaces of a three-dimensional scaffold. In such circumstances, nanofibers collect on the conductive, grounded surfaces around the scaffold. The particular fibers collecting on surfaces of an otherwise poorly conductive PCL/PLLA are principally deposited only onto the surface facing the electrospinning unit.

Without the needle, the surfaces of this type of scaffold remain largely devoid of any nanofibers and directly coating the bottom surface of the scaffold is not possible because that surface has no exposure to potential nanofiber deposition.

A further benefit to the arrangement of the scaffold and needle is readily apparent: The scaffold is suspended above the flat collector such that the nanofibers are attracted to the side and bottom surfaces of the polymer scaffold in addition to the top (upper surface) of the scaffold with its exposed needle tip.

Separation or dissociation of the scaffold and fibers appears to be prevented

38 when the coated scaffolds are placed in a liquid environment (for example, 70-

100% ethanol for sterilization or cell growth medium for cell culture).

Diameter size distribution of the fibers was unimodal with limited variability in nanofiber diameters seen over the six surfaces of the PCL/PLLA scaffolds. The average fiber diameter of 1.15 µm is sufficient for a bone tissue engineering application. Smaller nanofiber diameters would more closely mimic native extracellular matrix fiber sizes, but reduction of the electrospun fiber diameter results in a significant decrease in the size of pores within the nanofiber matrix generated. This reduction in pore size could hinder migration of cells through the coating of electrospun nanofibers and slow regeneration of tissue.

The modified electrospinning technique represents a new method for applying nanofibers to three-dimensional surfaces. This process is not limited to a single scaffold size, shape, or composition. Larger scaffolds of greater three- dimensional complexity could be coated with nanofibers in a fashion similar to that exemplified by the PCL/PLLA scaffolds. Also, more than one needle could be utilized to attract charged nanofibers, in the case of very large or exceptionally complex 3D scaffold structures. Other modifications of the basic electrospinning process can be incorporated into this technique as well. For example, coaxial or emulsion techniques which have been utilized to sequester and deliver therapeutic agents120-126 could be used to produce a bioactive nanofiber coating on the surface of a scaffold in the shape of a specific organ (for example, a human ear or femur).

The present electrospinning design is subject to a number of limitations. First, while examples of rectangular-shaped scaffolds are coated on all their six surfaces, the thickness of the coating is not uniform on each surface. The “peak” or maximal number of nanofibers that collect on the end of the needle, for example, contributes to a thicker coating on the surface of the scaffold closest to the electrospinning tip. Conversely, the nanofiber coating on the opposite surface of the rectangular scaffold and opposite the electrospinning tip can be sparse with portions of that surface remaining relatively uncovered by fibers.

While electrospinning times were typically about one hour in this study, additional time of coating can increase fiber coverage on this and other scaffold surfaces.

In another context, a second limitation to the design is the significant amount of time required to produce a single, nanofiber-coated scaffold of relatively small size. Conditions given above were found to be optimal for deposition of the fibers directly to the scaffold surfaces used in the present work. To shorten deposition times, applied voltage may be increased in the electrospinning system.

Slight applied voltage increases of up to 2 kV beyond the stated parameter of

13.1 kV reduce deposition time and enhance fiber coating to only a small degree.

If greater voltages (>15 kV) are used, the fibers begin to collect on the flat collector, often bypassing the scaffold and needle entirely.131

Relatively lengthy electrospun fiber deposition times (approximately an hour per sample) are necessary because scaffolds must be coated in a singular fashion to prevent the possibility of “webs” of nanofibers forming between multiple needles and scaffolds. Both issues of longer deposition and web

40 formation may be addressed by altering the ground needle arrangement and system design. For instance, the needle may be reoriented such that it is perpendicular, rather than parallel, to the electrospinning tip and combined with continuous rotation of the scaffolds attached to the needles. Rotation of the scaffolds would provide the benefit of evenly coating each of their surfaces. In addition, aligned fibers could be generated, an approach that may or may not be desired for a specific application. As mentioned previously, increasing the number of needles connecting the object to the ground electrode could allow coating of larger, irregular-shaped objects. A needle may also be altered with respect to its composition, density and size according to specific design requirements. For example, a longer needle would facilitate mounting of longer objects or a needle of larger diameter would support heavier specimens.

The shape of the needle is another variable which may be altered to allow more efficient coverage of electrospun nanofibers over the entirety of the object.

The electric field describing the electrospinning device will change with needle shape. As an example, for a needle having a large base in contact with the conductive plate and gradually tapered to a fine point as it protrudes from the object and faces the electrospinning tip, there would be a conceptual increase in nanofiber deposition on the bottom surface of the object (away from the tip) as a result of alterations in the electric field. In practice, a needle could be designed having in mind the consequent shape of the electric field as well as that of the object to be coated in order to optimize nanofiber coverage over all the object surfaces. With respect to chemical composition of the needle, highly insulated

41 sample objects may benefit from the use of a more conductive element, such as copper or titanium, rather than stainless steel. Also, other possibilities exist to increase the number of poorly conductive specimens which may be coated at a time. Utilizing any method which increases the quantity of ions at the surface of the object could decrease the amount of time required to coat the object while preventing “webs” from forming between multiple specimens.

To the knowledge of the authors, any comparable technique for applying an electrospun coating to a poorly conductive object in three dimensions would require generation of a corona discharge around it to attract charged fibers.132,133

Such a corona discharge design would increase the complexity of coating a three-dimensional object with nanofibers, and it would not account for coating all surfaces of the object unless the object were suspended or supported to allow nanofibers to collect on all surfaces. It is thought that the fundamental basis of faithfully and reproducibly coating such specimens in three dimensions rests with needle insertion through the object and its conduction of sufficient electric charge to attract charged nanofibers over the full surface complement of the object. The resulting electrospun nanofibers are in contact with the object such that they do not separate, delaminate or otherwise dissociate from the object surfaces when the coated structure is tested by immersion in 100% ethanol for 24 hours.131

With the design of this new approach to electrospinning, there are numerous studies underway in this laboratory to optimize its effects with respect to biological, mechanical, and other aspects of the device and nanofiber coating. In this regard, cell adhesion, cell proliferation, and matrix secretion of bone,

42 cartilage, skin and additional connective tissues are now being examined with scaffolds coated with electrospun PLLA, collagen and other fibers. In addition, the relationships between applied voltage, humidity, temperature, scaffold conductivity, coating time and fiber deposition and distribution are being investigated as well as the effectiveness and efficiency of electrospinning objects varying in their size, shape, topology and composition. Results from these studies will hopefully produce greater insights into properties and features of the electrospinning method that refine coating parameters and use of poorly conductive three-dimensional specimens.

The technique development here has been employed to coat poorly conductive polymer scaffolds with electrospun nanofibers, and the method can be applied in a wide variety of tissue-engineering applications. The approach is straightforward and effective and it may readily overcome the common problems of low efficiency and partial coverage of electrospun fibers over complicated sample surfaces. Further, the method is not limited to poorly conductive scaffolds or tissue-engineering applications. For instance, small, complex electronic components may benefit from application of a hydrophobic nanofiber coating which would allow air to pass through for cooling purposes while maintaining a barrier to prevent direct contact with water. In fact, such a design could produce electrical components capable of being completely submerged in water to facilitate more efficient heat transfer, or “water-cooling,” in a fashion similar to that of modern internal combustion engines. Scaffolds used for filtration may be improved with accurate and complete three-dimensional

43 electrospun coatings, and there may be many other applications of this novel methodology anywhere current electrospinning technology in two dimensions is utilized.

The PCL/PLLA scaffolds coated with electrospun PLLA nanofiber that were generated using the protocol detailed in the preceding pages of this Chapter were tested for bone tissue regeneration in vivo with the experiments reported in the next Chapter of this dissertation.

CHAPTER III

TESTING OF PLLA NANOFIBER-COATED PCL/PLLA SCAFFOLDS IN VIVO

Initial application and analysis of PLLA nanofiber coatings on PCL/PLLA constituted a small piece of a larger experimental design. PCL/PLLA scaffolds and PLLA nanofibers were selected, in part, for their biocompatibility. Both materials are utilized to great effect in many areas of tissue engineering.106-109

Their properties such as biodegradability are well-known and used to produce materials which do not become permanent additions to the body. As mentioned briefly in Chapters 1 and 2 of this dissertation, the same PCL/PLLA scaffolds were used in efforts to grow human phalanges in athymic mice.62,63 These spongy scaffolds were appropriate to determine a baseline reference point for whether nanofibers help or hinder growth and infiltration of cells over time in vivo.

A critical factor to success of these scaffolds previously was their porosity, which allowed cells to infiltrate and begin secreting their appropriate extracellular matrix.

PLLA nanofibers deposited onto the surface of these scaffolds should serve to enhance initial cell attachment and proliferation characteristics on the materials.

Migration of the cells into and through the nanofiber coating is a critical factor to the success of these constructs.

The following experiment was designed to examine potential augmentation of initial cell infiltration and proliferation effects from adding the PLLA nanofibers to the surface of the PCL/PLLA materials and the study utilized human periosteum as the source of the cells. The structure of the periosteum is of particular interest for bone tissue regeneration. On the side of periosteal tissue normally facing the bone (cambium layer) in vivo, osteoprogenitor cells are present and primarily responsible for deposition and growth of extracellular matrix which mineralizes to form new bone tissue. Harvesting periosteum and wrapping it around the scaffolds with the layer of osteoprogenitor cells facing the material mimic the location and structure of this tissue as it exists within the body. It must be noted that the periosteum is wrapped and sutured to the scaffolds such that it remains in contact with the material at all times. In this case, progenitor cells utilize the scaffold as a “base” from which to migrate, infiltrate, and begin regeneration of new bone tissue.

3.1 Materials and methods

3.1.1 Material and chemical sources

Cell culture medium (M199) and penicillin/streptomycin were purchased from

Mediatech, Inc. (Corning, Manassas, VA). Primocin came from Invitrogen (Life

Technologies, Grand Island, NY). Defined fetal bovine serum (FBS) was from

Hyclone (GE Healthcare Life Sciences, Logan, UT).

3.1.2 Human periosteal tissue collection

A human knee from a 51-year-old female was immersed in sterile PBS in a sterile container. The sealed container was placed on ice and transported overnight from Rush University Medical Center, Chicago, IL, to the laboratory in the Goodyear Polymer Building at the University of Akron, Akron, OH. The knee was removed from its container and placed on sterile surgical towels in a laminar flow hood (Figure 3.1). Muscle, adipose tissue, and fascia were excised surgically from the knee in the hood to expose the thin layer of periosteum covering the underlying bone (Figure 3.2). The periosteum was sectioned into thin strips, (0.6 cm X 2.5 cm), removed from the bone, and placed into small petri dishes having the periosteal cambium layer face down in complete cell culture medium (M199, 10% FBS, penicillin/streptomycin, primocin). Explant cultures of periosteum were maintained within an incubator (5% CO2, 37°C) for up to one week with complete cell culture media changes every 48 hours.

3.1.3 Scaffold sterilization and construct preparation

PLLA nanofiber-coated PCL/PLLA scaffolds were sterilized by immersion in a series of solutions as follows: 100% ethanol for 15 minutes, 50% ethanol for 15 minutes, and sterile, 1X PBS for 20 minutes. Sterile scaffolds were then rinsed through three changes of complete cell culture medium and transferred to the

CO2 incubator for a period of 5 hours. Following this prescribed incubation time, culture plates containing sterile, media-soaked scaffolds and petri dishes containing periosteal strips were removed and placed in a laminar flow hood.

Strips of periosteum were wrapped tightly, cambium layer facing the scaffold, around PCL/PLLA scaffolds shaped as rectangular solids (0.6 cm x 0.5 cm x 0.5 cm in dimensions) and either having or lacking the PLLA nanofiber coating.

Periosteal tissue was wrapped in a manner so the cambium layer maintained contact with four contiguous sides of each scaffold prior to being sutured to the scaffolds. Formed constructs were maintained in culture for up to one week further prior to subcutaneous implantation in athymic (nude) mice. A third group of scaffolds having the PLLA nanofibers but lacking periosteum was placed in complete cell culture medium for 24 hours prior to implantation. Experimental groups are diagrammed in Figure 3.3.

3.1.4 Contruct harvest, fixation, processing, and embedding

Mice were sacrificed after a period of 10 weeks, and constructs were removed

(Figure 3.4) and fixed in 10% neutral-buffered formalin (NBF, Electron

Microscopy Sciences, Hatfield, PA) for one week. Following fixation, samples were rinsed through three changes of deionized water and three changes of 70% ethanol (Pharmaco-aaper, Shelbyville, KY), 15 minutes per rinse. After the third

70% ethanol rinse, samples were placed into plastic cassettes and processed

(Model ASP300S tissue processor, Leica, Buffalo Grove, IL) through a series of ethanol and xylene washes prior to infiltration with paraffin wax (Type 9, Thermo

Scientific, Waltham, MA). Paraffin-infiltrated samples were embedded in paraffin wax blocks.

3.1.5 Construct sectioning and staining

Formalin-fixed, paraffin-embedded samples were sectioned (5-7 µm thickness,

Model RM2255 microtome, Leica, Buffalo Grove, IL) with a c-profile, stainless steel knife. Sections were floated on a 42°C water bath, mounted onto glass slides (Superfrost Excell, Thermo Scientific, Waltham, MA), and placed on a warming platform at 42°C for a minimum of 3 hours. Slides were then stored in slide boxes prior to staining. Separate slides from each of the three groups of specimens were stained with toluidine blue to examine basic tissue and cellular structure, alizarin red to detect the presence and location of calcium ions, and von Kossa (with safranin O counterstaining) to examine the presence and location of phosphate ions. Figure 3.5 is a diagram depicting positive results for alizarin red and von Kossa stains.

3.2 Results

Figure 3.6 presents a series of light micrographs showing results from chemical staining (toluidine blue, alizarin red, and von Kossa) of a PLLA nanofiber-coated PCL/PLLA scaffold following 10 weeks implantation in and harvest from an athymic mouse. Toluidine blue highlights infiltration of murine fibrous tissue into the PCL/PLLA material. Alizarin red and von Kossa staining showed no significant calcium or phosphate deposits associated with the scaffold.

Three composite light micrograph images of histology of PCL/PLLA scaffolds wrapped with human periosteum and harvested following 10 weeks of implantation in a nude mouse appear in Figure 3.7. Periosteum and overlying

49 fascia were highlighted by the dark blue color of the toluidine blue stain. Light blue staining showing murine fibrous tissue encapsulation was observed around the outer perimeter of the sample. Calcium and phosphate ions were detected within periosteal tissue about the PCL/PLLA scaffold by alizarin red and von

Kossa staining, respectively.

The light micrograph in Figure 3.8 demonstrates the results of toluidine blue staining of a PLLA nanofiber-coated PCL/PLLA scaffold wrapped with human periosteum harvested after 10 weeks of implantation in an athymic mouse.

Figures 3.8A and 3.8B showed infiltration of periosteal cells and deposition of extracellular matrix within the nanofibers covering the surface of the PCL/PLLA scaffold.

Figure 3.9 shows a composite light micrograph image of alizarin red staining of a representative PLLA nanofiber-coated PCL/PLLA scaffold wrapped with human periosteum and harvested following 10 weeks of implantation in an athymic mouse. Figures 3.9A and 3.9B illustrate the presence of calcium deposits within the PLLA nanofibers over the surface of the PCL/PLLA scaffold.

The two composite light micrograph images in Figure 3.10 show results for alizarin red and von Kossa staining of a PLLA nanofiber-coated PCL/PLLA scaffold wrapped with human periosteum and retrieved after 10 weeks of implantation in an athymic mouse. Areas of significant overlap between the two stains are marked by asterisks around the von Kossa sample. These overlap regions are indicative of newly forming mineral (calcium phosphate) in the engineered constructs.

3.3 Figures

Figure 3.1. Intact human knee from a 51-year-old female donor. Scale bar = 1 cm.

Figure 3.2. Aseptic dissection and removal of overlying tissue from an intact human knee in a laminar flow hood to access and harvest periosteal tissue.

Figure 3.3. The basic design for the three experimental groups used in this study.

Group 1 scaffolds are composed of PCL/PLLA and an electrospun PLLA nanofiber coating. Group 2 consisted of PCL/PLLA scaffolds wrapped with human periosteum only. Group 3 was fabricated by coating PCL/PLLA materials with electrospun PLLA nanofibers prior to wrapping with human periosteal tissue.

Scaffolds and constructs were subsequently implanted in athymic (nude) mice.

N represents the number of individual specimens examined in each group.

Figure 3.4. Scaffolds implanted subcutaneously on the left and right sides of the back of an athymic mouse (left image). Panels to the right show harvested constructs immediately following removal from the mouse and prior to fixation in

10% NBF.

P P * * NF NF

PCL/PLLA PCL/PLLA

Figure 3.5. Diagram depicting conceptual staining features of two different sections in the same region of a specimen using alizarin red (left) and von Kossa

(right). Alizarin red stains regions dark red in specimens positive for calcium while the remaining tissue stains light orange (left image). The von Kossa stain highlights phosphate deposits in specimens as black with Safranin-O red

53 counterstaining the remaining tissue light red or pink (right image). Only regions where the positive results of these two stains overlap (asterisk in each image) are likely mineralized bone tissue. Regions where positive results do not overlap between the two stains (x, left image) are only highlighting the presence of calcium, not mineralized bone tissue. P = periosteum. NF = electrospun PLLA nanofibers. PCL/PLLA = synthetic PCL/PLLA scaffold.

Toluidine Blue Nanofiber Layer

PCL/PLLA

Alizarin Red von Kossa (Ca2+) 3- (PO4 )

PCL/PLLA PCL/PLLA

Figure 3.6. Results from toluidine blue, alizarin red, and von Kossa staining for a representative Group 1 scaffold harvested after 10 weeks in vivo. Toludine blue staining demonstrates infiltration of murine tissue into the porous structure of the

PCL/PLLA scaffold coated with electrospun PLLA fibers. Alizarin red and von

Kossa staining illustrate the absence of significant calcium and phosphate deposits, respectively. Positive calcium staining would otherwise be dark red and phosphate staining would be black. Alizarin red stains tissue lacking calcium 55 light orange (lower left image). The von Kossa counterstain, safranin O, stains background tissue dark pink/red (lower right image). Scale bar = 1 mm.

Murine fibrous Toluidine Blue Tissue

Periosteum with overlying fascia PCL/PLLA

Alizarin Red von Kossa 2+ (Ca ) 3- (PO4 )

PCL/PLLA PCL/PLLA

Figure 3.7. Histological results from a representative Group 2 sample.

Periosteal tissue and overlying fascia are marked in toluidine blue staining by dark blue coloring while murine fibrous tissue is stained lighter blue. Calcium

(dark red) and phosphate (black) deposits are visible in the periosteal tissue for the alizarin red and von Kossa stains, respectively. Scale bar = 1 mm.

P PCL/PLLA

B PCL/PLLA A

B NF

P Nanofiber Layer

Figure 3.8. Histological results of toluidine blue staining for a representative sample from Group 3. Periosteal tissue (P) is visible on the perimeter of the

PCL/PLLA scaffold. An electrospun PLLA nanofiber layer (NF) over all surfaces of the PCL/PLLA scaffold is denoted with arrows in the composite image (left).

Panels A and B highlight infiltration of cells from the periosteum into the nanofiber layer (NF) as well as the PCL/PLLA scaffold beneath the layer. In these images, the thickness of the electrospun PLLA nanofiber layer is ~50-100

µm, a typical result for the operation of the electrospinning system utilized in this study. Scale bar = 1 mm (composite image) and 100 µm (panels A and B).

P PCL/PLLA

PCL/PLLA NF P

PCL/PLLA

B A NF P

Nanofiber Layer

Figure 3.9. Histology results for alizarin red staining of a representative specimen from Group 3. Significant calcium deposits (dark red) are visible in the periosteal tissue (P) surrounding the PCL/PLLA scaffold. Enlarged images

(panels A and B) show the presence of calcium deposits within the nanofiber layer (NF) of the construct (arrows). Tissue negative for calcium presence is stained light orange the images. Scale bar = 1 mm (composite image) and 50

µm (panels A and B).

Alizarin Red von Kossa 2+ 3- (Ca ) * (PO4 )

P P PCL/PLLA PCL/PLLA *

* *

Nanofiber Layer *

Figure 3.10. Alizarin red (left) and von Kossa (right) staining of a representative specimen from Group 3. Asterisks on the perimeter of the von Kossa image mark areas of significant overlap between calcium (deep red on left image) and phosphate (black on right image) deposits within the periosteum (P) and the nanofiber layer (NF) coating the scaffold. Scale bar = 1 mm.

3.4 Discussion

The decision to utilize human periosteal tissue for the experiments described here was critical in regard to clinical applicability of nanofiber-coated scaffolds.

Many laboratories spend a significant portion of time using immortalized cell lines to determine the safety of new molecules and materials they develop in numerous and varied studies.114,118,127-130 For new, untested materials, this process is an effective and efficient means to gauge biocompatibility of a new compound. However, for purposes of tissue engineering, a material requires

59 testing utilizing the parameters and conditions it will encounter in a clinical setting.

With respect to bone tissue engineering, periosteal tissue is known to be a suitable source of osteogenic cells.53-63 As stated in Chapter 1 of this dissertation, periosteal cells can be isolated and expanded in culture in a manner similar to other cell types (MSCs, chondrocytes, or fibroblasts, for example).

However, harvesting small pieces of intact periosteum from a primary source (a knee from a 51-year-old female in the case of this series of experiments) and wrapping them around a scaffold is a more efficient manner of utilizing periosteal tissue as this approach eliminates the requirements of cell isolation and expansion ex vivo, decreasing the time necessary for a surgical repair.

This section of the dissertation involved a study of three distinct sets of experiments, each of which resulted in PCL/PLLA scaffolds that were processed in separate ways and then implanted in athymic mice for 10 weeks. In the first of the studies, these implanted scaffolds, coated with an electrospun layer of PLLA nanofibers on all its surfaces, were retrieved from the mice and found by toluidine blue staining to have infiltrates of murine tissue in the porous structures of both the nanofiber layer and the PCL/PLLA scaffolds, themselves. In this case, the mouse appears to have invaded the PLLA-coated PCL/PLLA material with its vasculature and cells and may be attempting to degrade, erode, or incorporate it into its own living tissues. The PLLA nanofiber coating over the PCL/PLLA scaffold apparently provides porosity suitable for vascular invasion and cell attachment without hindering migration of the cells into the underlying PCL/PLLA scaffold. As detected by alizarin red and von Kossa staining, there is no

60 evidence for calcium or phosphate deposition, respectively, in these PLLA- coated scaffolds.

In the second set of studies, the same PCL/PLLA scaffolds as described in the first experiments above were utilized, but these were absent of an electrospun PLLA nanofiber layer. Instead, the scaffolds were wrapped with a living, thin periosteal membrane from a human donor. In this instance, constructs retrieved after 10 weeks of implantation in additional mice demonstrated by toluidine blue staining successful infiltration and proliferation of periosteal cells associated with the PCL/PLLA scaffolds. Thus, these experiments establish the viability of the cells present within the donor periosteal tissue over the implantation time in this mouse model.

The third set of studies involved the PCL/PLLA scaffolds coated with electrospun PLLA nanofibers and also wrapped with human donor periosteum.

After 10 weeks of implantation in mice, these specimens, stained once again with toluidine blue, showed the presence of periosteal cells associated with the

PCL/PLLA scaffolds as had the specimens in the second set of experiments.

Both the second and third set of investigations indicated that the donor periosteal cells had little difficulty mobilizing and moving into the PCL/PLLA scaffolds and their infiltration led to extracellular matrix deposition (See the following paragraphs). As in the first set of studies, the third set demonstrated that cells, whether from the host mouse or the donor periosteum, remained viable and capable of migrating through a 50-100 μm thick electrospun layer of PLLA

61 coating the PCL/PLLA scaffolds. Further, these specimens contained deposits of calcium and phosphate, the basic elements required to build new bone.

While toluidine blue staining is effective at showing cells and tissue present within the scaffolds and nanofibers, this stain alone does not provide information necessary to ascertain if PLLA nanofibers are beneficial to cellular growth and infiltration of cells from the periosteum. Two other stains were then used to examine early bone tissue formation in the second and third sets of studies as a measure of the effect of PLLA fiber coatings on the PCL/PLLA scaffolds. As noted previously, alizarin red stains tissues dark red in locations where calcium ions are present, and von Kossa stains tissues black in the presence of phosphate ions in specimen sections. Often, either alizarin red or von Kossa is used alone to confirm the presence of mineral within a tissue or cell culture or in a section of tissue. Either stain alone can only suggest mineral formation, and both should be used to strengthen evidence that constituents of hydroxyapatite

(calcium and phosphate) are present in the same sections of a culture or a tissue.

Overlapping dark red and black regions in images of cultures or tissue sections are strong indicators for the formation of hydroxyapatite mineral. In the studies here, no staining for calcium and phosphate was found in the first set of experiments (Group 1) but staining was clearly observed for the second (Group

2) and third (Group 3) sets. Overlapping regions for calcium and phosphate staining appeared qualitatively to be greater or more extensive in Group 3 specimens compared to Group 2 specimens. These regions were readily identified and noted in Group 3 specimens but were not marked for Group 2 as

62 they were not as prevalent and well-defined. This result suggested that the presence of electrospun PLLA nanofiber layers in specimens compared to samples without the layers induced an increased number of cells from the periosteal tissue and/or an increased activity of the cells, either condition leading to the observed increased production of mineralized matrix in Group 3 compared to Group 2 constructs. Multiple specimens from each group were assessed for mineral presence in the case of these experiments although comparative measures were a challenge as histological examination provides primarily qualitative data as noted above. Quantitative data regarding the extent of mineralization may be attainable utilizing a technique such as microCT.

Chemical assays designed to quantitate the concentration of hydroxyapatite in specimens would also prove useful in confirming the histological assessment of the constructs presented here.

Scaffolds from Group 3 demonstrate the potential of adding nanofibers as a coating to scaffold materials. Cells and extracellular matrix have infiltrated the

PLLA nanofibers. Migration of these cells to the intermediate, nanofiber layer suggests they mobilize rapidly and begin depositing matrix to act as an additional layer under the cambium layer of the original periosteum. The PLLA nanofibers, then, could provide a means by which cells migrate both into and along the surfaces of a scaffold or other material. Movement and relocation of cells from the original periosteum to nanofibers on the surfaces of a scaffold present new avenues for engineering hard tissues. For instance, the difficulty in harvesting periosteum from another site within the body to “seed” an engineered scaffold

63 may be circumvented if the cells migrate from periosteal tissue along the edges of a wound into nanofiber coatings so that they cover the underlying material in its entirety. For large sections of bone, small pieces of periosteum could be transferred to areas of the nanofiber-coated material so cells might migrate from multiple areas over the scaffold. Of course, the size of a defect or the nature of the tissue being repaired requires consideration for each case.

The experiments here were limited from a few of standpoints. First, tissue was harvested from a single, human donor. Second, a single time-point of 10 weeks in vivo was sampled for comparisons of tissue growth among the three experimental groups described earlier. Third, materials selected, PCL/PLLA for the scaffolds and PLLA for the nanofibers, represent a small fraction of compounds available and being developed for bone tissue regeneration. With respect to the first limitation, donor human tissue is always a limited resource and has a significant impact leading to the second and third restrictions. A single time-point was selected to maintain a sample size sufficient to detect differences between the three groups. PLLA and PCL are used extensively in tissue engineering and represent a suitable and acceptable base to which other materials can be compared in future studies.

The experimental results presented above demonstrate a potential for nanofiber-coated materials in tissue-engineering applications. Cellular infiltration and growth on nanofiber-coated PCL/PLLA scaffolds is indicative of results one may desire to achieve with a material not conducive to initial cellular attachment.

The challenge of constructing materials that permit cellular infiltration while

64 maintaining certain mechanical properties is vast. An approach to incorporate nanofibers into existing techniques and scaffolds helps to bridge the gap between current technology and future endeavors in bone tissue regeneration.

For example, hard, solid materials which lack the porous surface of scaffolds presented here stand to benefit greatly from this technology. By providing a surface where cells will attach and proliferate, nanofiber coatings offer a simpler method to increase rates of bone regeneration and the potential to harvest smaller pieces of periosteal tissue from a patient. Migration of osteoprogenitor cells is not limited to movement through a nanofiber coating to the underlying scaffold. Cells can also advance peripherally along the nanofibers, expanding overall area of the tissue layer beyond the original size of the harvested periosteal tissue. Utilization of the entire tissue, instead of culturing cells for an extended period, will decrease the time and number of surgeries required to repair a patient defect.

Histological results obtained in these experiments in vivo led directly to the design of the subsequent experiments for this dissertation. A hard, solid material

(human allograft bone) was selected for the next set of studies in order to examine the growth and infiltration of periosteal cells on a scaffold exhibiting mechanical properties closer to those of native bone tissue. It was also important to examine nanofiber-coated allograft bone from a clinical standpoint.

Sterile human allograft bone is used currently in procedures attempting repair of segmental bone defects. Improving the effectiveness of an established method

65 is essential to integration of a new technique or material into current medical practice.

CHAPTER IV

EXAMINATION OF PLLA NANOFIBER-COATED HUMAN ALLOGRAFT BONE

SCAFFOLDS IN VIVO

Chapters 2 and 3 of this dissertation detailed methods of applying electrospun nanofibers of PLLA to scaffolds composed of PCL/PLLA (75/25) and histological analysis of such coated scaffolds subsequently wrapped with human periosteum and implanted for 10 weeks in nude mice. Results indicated the presence of cells and tissue within the PLLA nanofibrous coating as well as the underlying

PCL/PLLA scaffolds. Compared to PCL/PLLA scaffolds wrapped with donor periosteum but without a PLLA coating, the increasing extent of mineralization within both the periosteal tissue and a PLLA coating validated the potential of adding an electrospun nanofiber layer to porous, synthetic scaffolds, such as

PCL/PLLA, for bone tissue engineering.

It must be noted that bone is composed of matrix and mineral components having mechanical properties which differ from PCL/PLLA sponge scaffolds used in the experiments from Chapters 2 and 3. In this regard, it was necessary to investigate a nanofibrous coating applied to constructs exhibiting mechanical strength of native bone tissue. Current approaches in research for repairing critical-size bone defects are usually compared to the gold standard for clinical

67 treatment, autologous bone grafting. Allograft bone is utilized in situations in which autografting is not feasible (for example, a large portion of bone is required or bone from the patient is not suitable). Previous work in this laboratory demonstrated regenerative effects of wrapping periosteal tissue around sterile allograft bone over the course of 10 and 20 weeks in vivo.134 Histological examination of the harvested samples in these experiments provided some evidence of vascularization and new bone tissue formation within the structure of the bone after 20 weeks.134 However, the extent of new tissue formation was difficult to determine in these studies although gene expression analyses showed an increase in markers associated with bone matrix production (type I collagen, bone sialoprotein, osteocalcin, alkaline phosphatase, and decorin) from 10 to 20 weeks in vivo.

As a next step to utilizing electrospun polymeric materials coating scaffolds for bone tissue engineering, PLLA nanofibers were applied to sterile allograft bone prior to wrapping the bone with human donor periosteal tissue. As with its predecessor of PCL/PLLA scaffolds, the purpose of these studies with allograft bone was to investigate whether electrospun PLLA nanofibers increased the rate of new bone formation by providing a layer of synthetic polymer matrix to support migration and proliferation of osteoprogenitor cells from periosteal tissue wrapped over the nanofibers and allograft. Nanofibers of PLLA were applied to allograft bone samples using the methodology detailed in Chapter 2. Cadaveric human periosteum was harvested from three different donors, wrapped around the nanofiber-coated allograft bone samples, and implanted for periods of 20 and

40 weeks in athymic mice. Harvested constructs were analyzed by histology and any new tissue formation was documented.

4.1 Materials and methods

4.1.1 Human allograft bone scaffold preparation

Sterile human allograft bones (Musculoskeletal Transplant Foundation,

Jessup, PA) were cut into small rectangular shaped pieces (~0.5 x 0.5 x 0.5 cm) using a diamond cutting wheel attached to a Dremel rotary tool. The allograft bone scaffolds were placed into separate wells of a sterile cell culture plate and stored at room temperature prior to application of the nanofiber coating.

Immediately prior to mounting each bone scaffold onto the stainless steel needle of an electrospinning apparatus, a hole was drilled through the scaffold using a

0.8 mm diameter drill bit attached to the Dremel rotary tool.

As with PCL/PLLA (75/25) scaffolds described previously, sterile human allograft bone scaffolds were coated with PLLA nanofibers by mounting them onto the fine-point stainless steel needle. Nanofibers of PLLA were electrospun onto the allograft scaffolds utilizing parameters defined in Chapter 2 of this dissertation. After electrospinning, nanofiber-coated allograft bone scaffolds were dried and stored in the same manner as nanofiber-coated PCL/PLLA scaffolds.

PLLA nanofiber-coated allograft bone scaffolds were sterilized by immersion in 70% ethanol for 1 hour. The sterilized scaffolds were placed into separate wells of a 12-well culture plate and left in a laminar flow hood to dry for 1 hour. 69

Once dry, the scaffolds were immersed in complete cell culture medium (M199,

10% FBS, pen/strep, primocin) and placed into a CO2 incubator at 37°C overnight.

4.1.2 Preparation of periosteum-wrapped allograft constructs

Periosteal tissue was harvested from knees of three different human donors

(56-year-old F, 59-year-old F, 68-year-old M) and cut into thin strips before being placed into petri dishes containing complete cell culture medium. Cultures were maintained for up to one week with medium changes every 48 hours. Allograft scaffolds soaked in complete cell culture medium were wrapped with strips of periosteal tissue with its cambium layer facing toward the surface of each scaffold. Periosteum was sutured into place with nylon thread and constructs were placed into separate wells of a 12-well culture plate with complete cell culture medium. Constructs were maintained for up to one week in culture with medium changes every 48 hours. Experimental groups are shown in Figure 4.1.

4.1.3 Construct implantation, harvest, and analysis

Constructs were implanted subcutaneously into athymic mice (two constructs per mouse, one on each side of its back). Mice were sacrificed at 20 and 40 weeks of implantation, and constructs were removed, photographed, and fixed in

10% NBF for one week. Following fixation, harvested constructs were decalcified by daily changes of Immunocal (Decal Corp, Tallman, NY) over a period of 14 days. Decalcified samples were rinsed through water and ethanol

70 before being processed through an ethanol and xylene series of dehydration steps and infiltrated with paraffin wax. Paraffin-infiltrated constructs were embedded in paraffin blocks and stored at 4°C.

4.1.4 Slide preparation and histochemical staining

Paraffin-embedded samples were sectioned using a microtome (5-7 µm thickness, Model RM2255, Leica, Buffalo Grove, IL) with a tungsten carbide knife.

Sections were floated on a 42°C water bath, mounted on glass slides (Superfrost

Excell, Thermo Scientific, Waltham, MA), and placed on a warming tray at 42°C for up to 3 hours. Slides were stored in boxes prior to staining with hematoxylin and eosin (H & E). Light micrograph images were recorded using an inverted microscope (Model IX70, Olympus, Melville, NY) and camera (Model CC12,

Olympus, Melville, NY) under varying levels of magnification.

For sections of Group 4 constructs after 20 and 40 weeks of implantation in vivo, thickness measurements of presumed new tissue layers formed between the electrospun PLLA nanofibers and underlying allograft bone scaffolds were made perpendicular to the bottom layer of the electrospun PLLA nanofibers at intervals of approximately 30 µm along the layers using ImageJ (National

Institutes of Health, imagej.nih.gov/ij/). These data were plotted in histograms

(Figures 4.14 and 4.15) using Analysis ToolPak in Excel (Microsoft Corporation,

Redmond, WA) with the x-axis consisting of “bins” for tissue layer thickness measurements having 2 µm intervals from 2 to 72 µm and the y-axis

71 representing the percentage of total tissue layer thickness measurements collected.

Additional histograms were plotted (Figures 4.16 and 4.17) in an attempt to account for possible discrepancies in the structure of the bone adjacent to the electrospun PLLA nanofibers and periosteal tissue. Layers of new tissue in open areas or voids were typically characterized by material exhibiting the extracellular matrix features of fibrous tissue (random collagen fiber orientation, primarily) and by electrospun PLLA nanofibers which failed to follow the surface contours of the underlying allograft bone scaffold (for example, Figure 4.13). These regions were excluded from each data set. The remaining measurements obtained from regions of the specimens where the layers of tissue maintained morphologies similar to the original periosteal tissue (elongated cells within an extracellular matrix composed of fibers running parallel to the underlying allograft bone) were plotted in the same manner as the histograms in Figures 4.14 and 4.15. Figure

4.2 is a schematic that illustrates how the measurements were gathered and correlated.

4.2 Results

Figures 4.3 and 4.4 are composite light micrographs illustrating H & E staining results for specimens from Groups 1-4 after implantation for 20 weeks in vivo. In

Groups 1 and 2 (Figure 4.3), murine fibrous tissue is visible around the periphery of the specimens. Compared to Group 1 specimens, addition of electrospun

PLLA nanofibers for Group 2 specimens appears to result qualitatively in a slight

72 increase in the presence or amount of fibrous tissue encapsulating the allograft bone. Periosteal tissue is present for Group 3 and 4 (Figure 4.4) constructs surrounding the allograft bone scaffold with cells infiltrating PLLA nanofibers in

Group 4 constructs.

Specimens from Groups 1-4 are compared microscopically after 40 weeks of implantation and H & E staining (Figures 4.5 and 4.7). Compared to specimens from Group 1, those from Group 2 show a very slight qualitative increase in the presence or amount of the fibrous tissue encapsulation from the mouse (Figure

4.5). In Group 3 and 4 specimens (Figure 4.7), the periosteum is intact and present around the allograft bone as it was for 20 week constructs.

Micrographs shown in Figure 4.6 provide more detail of the electrospun PLLA nanofibers, fibrous mouse tissue, and allograft bone in a representative Group 2 construct. Cells from the mouse appear to have infiltrated and produced extracellular matrix encapsulating the PLLA nanofibers to a considerable extent.

Figures 4.8-4.10 show enlargements of a 40 week sample from Group 3 constructs. Vascular elements are present within both periosteal tissue and cavities that comprise the allograft bone structure.

Group 4 specimens are marked by the appearance of apparently new layers of tissue that are observed adjacent to the electrospun PLLA nanofiber coating the allograft scaffolds. The new tissue layers are shown in Figures 4.11-4.13 for one such Group 4 specimen. Measurements of the thickness of the new tissue layers were obtained from multiple images such as these in Figures 4.11-4.13 to generate two histograms comparing new tissue thicknesses from 20 to 40 week

73 implants (Figures 4.14 and 4.15). Average tissue thickness after 20 weeks of construct implantation was 11.46 ± 8.28 µm and after 40 weeks was 22.38 ±

13.28 µm. Subtracting measurements of tissue thickness collected in open areas or cavities in the bone (Figure 4.13) resulted in thickness measurements that yielded slightly different histograms (Figures 4.16 and 4.17) and averages of 9.61

± 5.37 µm and 16.38 ± 8.44 µm after 20 and 40 weeks of implantation, respectively.

4.3 Figures

Figure 4.1. Diagram of experimental groups used in this study. Group 1 consisted of only sterile allograft bone. Group 2 consisted of allograft bone coated with electrospun PLLA nanofibers. Group 3 was characterized by allograft bone wrapped with human periosteum. Group 4 comprised allograft

74 bone coated with electrospun PLLA nanofibers prior to wrapping the scaffold with periosteal tissue. N represents the number of individual specimens examined in each group.

Periosteum

Nanofiber Layer

Layers of New 1 Tissue 2

Allograft Bone

Figure 4.2. Schematic illustrating various regions of engineered constructs (1 and 2) assessed for thickness measurements of new layers of tissue formed between the electrospun PLLA nanofiber layers and underlying allograft bone scaffolds. The example region marked “2” in the image represents an artifactual open area or void associated with the perimeter of the allograft bone where the presumed new tissue exhibits morphological characteristics of fibrous tissue

(random arrangement of collagen fibers in the extracellular matrix) and the electrospun PLLA nanofiber layers failed to conform to the surface contours of the allograft bone. In this and other such similar regions it was not possible to distinguish between layers of new tissue (horizontal parallel lines), presumably derived from the human periosteum wrapped about allograft bone, and fibrous tissue (X’s), potentially of murine origin. The uncertainty in the precise identification of new tissue layers of periosteal derivation led to the exclusion of 75 the regions from each of the respective data sets used to plot the histograms in

Figures 4.16 and 4.17.

Nanofiber Layer

Fibrous Tissue

Figure 4.3. Samples from Groups 1 (left) and 2 (right) harvested after 20 weeks of implantation and stained with H & E. Murine fibrous tissue encapsulates the allograft bone (AB) scaffolds from both Groups and is visible infiltrating PLLA nanofibers around Group 2 constructs. In general, specimens from Group 2 compared to those from Group 1 yield slight qualitative increases in the presence or amount of the fibrous tissue surrounding the allograft bone. Scale bar = 1 mm.

Periosteum Nanofiber Layer

AB AB

Figure 4.4. Constructs from Groups 3 (left) and 4 (right) harvested after 20 weeks in vivo and stained with H & E. Periosteal tissue is present on two sides of the Group 3 sample shown (left) as a result of sectioning in a transverse fashion. Periosteal tissue is found around most of the Group 4 scaffold documented here as the section was obtained close to the outer face of this specimen. A hole generated from using the rotary tool and 0.8 mm diameter drill bit is present in the Group 4 scaffold (arrow) at the center of the allograft bone

(AB), and it was created to facilitate mounting of this and other allograft bone scaffolds onto the fine-point, stainless steel needle for application of the electrospun PLLA nanofibers. Scale bar = 1 mm.

Fibrous Tissue

B AB AB A

Nanofiber Layer

Figure 4.5. Samples from Groups 1 (left) and 2 (right) harvested after 40 weeks of implantation in vivo and stained with H & E. As with 20 week implanted constructs, murine fibrous tissue is visible around the periphery of allograft bone

(AB) with infiltration of cells and tissue into the electrospun PLLA nanofibers about Group 2 scaffolds (right). In general, there appears quantitatively to be slightly greater amounts of fibrous tissue in specimens from Group 2 compared to Group 1 after 40 weeks of implantation in nude mice. Enlargements of regions

A and B from this Group 2 specimen are shown in Figure 4.6. As with the specimen from Group 4 in Figure 4.4, a hole created to insert the fine-point, stainless steel needle for the electrospinning process is visible in the allograft bone of Group 2. Scale bar = 1 mm.

A B AB NF NF

F AB

Figure 4.6. Murine fibrous tissue (F) stained with H & E is visible and grows into and beneath the layer of electrospun PLLA nanofibers (NF) coating the surfaces of the allograft bone (AB) in the Group 2 specimen shown in regions A and B in

Figure 4.5. Cells are visible within cavities inside the allograft bone tissue (region

B). The space between AB and the nearby layer of fibrous tissue within NF is an artifact attributable to sectioning the construct. Scale bar = 100 µm (region A) and 200 µm (region B).

Periosteum

AB F AB C B E A D

Nanofiber Layer

Figure 4.7. Specimens collected after 40 weeks of implantation in vivo for

Groups 3 (left) and 4 (right) and stained with H & E. Periosteal tissue is present around the periphery of the allograft bone (AB) in both Groups. Cells and new tissue are found infiltrating the electrospun PLLA nanofiber layer in Group 4 specimens. Regions A-E of these two representative specimens are enlarged and shown in Figures 4.8 - 4.12. Scale bar = 1 mm.

AB AB

Figure 4.8. Enlargement of region A of Figure 4.7 shows vascular elements

(highlighted with arrows), including red blood cells, present within the open structure of allograft bone (AB) in this specimen from Group 3 constructs. The specimen has been stained with H & E. Scale bar = 50 µm.

B AB

Figure 4.9. Enlargement of region B of Figure 4.7 shows vascularity within the periosteum (P) from this specimen from Group 3 constructs. Red blood cells are visible within the cross sections of two blood vessels (highlighted with arrows).

The specimen was stained with H & E. AB = Allograft bone. Scale bar = 50 µm.

C AB

Figure 4.10. Enlargement of region C of Figure 4.7 showing a transverse section of a blood vessel with red blood cells visible (highlighted with arrows) between periosteum (P) and allograft bone (AB) within this specimen from Group 3 constructs. Scale bar = 50 µm.

NF P

Figure 4.11. Enlargement of region D from Figure 4.7 shows a presumably new layer of periosteal tissue, separate from the original periosteum (P), formed between the electrospun PLLA nanofiber layer (NF) and the allograft bone scaffold (AB) in this specimen. Scale bar = 200 µm.

NF P

Figure 4.12. Similar to Figure 4.11, a presumed new layer of tissue (highlighted by arrows) is present between the electrospun PLLA nanofibers (NF) and the allograft bone (AB), separated from the original periosteum (P). There may be greater infiltration of cells and tissue growth in this construct compared to that shown in Figure 4.11, perhaps as a result of a slight increase in the porosity of the electrospun PLLA nanofiber layer. Scale bar = 200 µm.

* Figure 4.13. Enlargement of region F in Figure 4.7 of this representative specimen from Group 4 shows supposed new layers of tissue (arrows) between

83 the electrospun PLLA nanofiber layer (NF) and the allograft bone (AB). Tissue that is present in the open structure around AB in the bottom portion of the image

(asterisk) appears to be less dense and more fibrous than the layer of tissue in close contact with AB. Scale bar = 50 µm.

Thickness of new periosteal tissue layers (20 weeks) 20

18 16 14 12 10 8 6

4 Percentage of total measurements total of Percentage 2 0

Tissue Layer Thickness (µm)

Figure 4.14. A histogram for the measured thickness of the new layers of tissue formed between the electrospun PLLA nanofibers and the allograft bone following 20 weeks of implantation in vivo (Group 4). From 1275 total measurements, the average thickness of the new tissue layers was 11.46 ± 8.28

µm.

Thickness of new periosteal tissue layers (40 weeks) 20

18 16 14 12 10 8 6

4 Percentage of total measurements total of Percentage 2 0

Tissue Layer Thickness (µm)

Figure 4.15. A histogram for the measured thickness of the new tissue layers formed between the electrospun PLLA nanofibers and the allograft bone after 40 weeks of implantation in vivo (Group 4). From 1267 total measurements, the average thickness of the new tissue layers was 22.38 ± 13.28 µm.

Thickness of new periosteal tissue layers (20 weeks) 20

18 16 14 12 10 8 6 4 Percentage of total measurements total of Percentage 2 0

Tissue Layer Thickness (µm)

Figure 4.16. A histogram of tissue layer thickness measurements for the new layers of tissue formed after 20 weeks of implantation in vivo (Group 4) and plotted in an effort to account for possible discrepancies in layer thickness measurements as a result of open cavities between allograft bone and the electrospun PLLA nanofibers. The calculated average tissue thickness from

1160 total measurements under these constraints was 9.61 ± 5.37 µm.

Thickness of new periosteal tissue layers (40 weeks) 20

16 14 12

10 8 6

4 Percentage of total measurements total of Percentage 2 0

Tissue Layer Thickness (µm)

Figure 4.17. A histogram of measurements for thickness of the new layers of tissue formed after 40 weeks of implantation in vivo (Group 4). Measurements were made to account for possible discrepancies as a result of open cavities between allograft bone and the electrospun PLLA nanofibers. The calculated average tissue thickness from 928 total measurements using these constraints was 16.38 ± 8.44 µm.

4.4 Discussion

As stated previously with respect to the potential clinical applications of the constructs described here, it is important to examine nanofiber-coated human allograft bone samples wrapped with human periosteum. Procedures utilizing sterilized allograft bone for the repair of segmental bone defects have met with

87 varying degrees of success, and they represent a standard for which it is possible to compare experimental results of techniques designed to modify or replace them. The periosteal tissue in this set of experiments was harvested from three different donors, two females and one male (ages 56, 59, and 68, respectively). Once more, it should be noted that these donors were somewhat elderly and the osteogenic potential of their periosteal tissue was very likely diminished compared to tissue obtained from younger donors. On the other hand, constructs of bone allografts wrapped with periosteal tissue from these donors were viable after 20 and 40 weeks of implantation in vivo. Such a result may be considered somewhat surprising, but it is in part attributable to the persistence of the periosteum and its component cells as well as the care, skill, and precision of the laboratory group members conducting the experiments presented here.

Nanofibers deposited onto the surface of the allograft bone samples were composed of the same PLLA as in the experiments with PCL/PLLA scaffolds described in Chapters 2 and 3 of this dissertation, and allograft bone segments were coated with nanofibers utilizing the same electrospinning conditions.

Preparation of the allograft bone to facilitate deposition of the nanofibers was not the same as that followed for the synthetic (PCL/PLLA) material. In this regard, first, the allograft bone was delivered in large sections (5-10 cm in length) that were subsequently cut into smaller pieces using a rotary tool with a diamond cutoff wheel attachment. The bones were not only cut into small fragments, but also drilled to produce a small hole (0.8 mm) for mounting them on the fine-point

88 stainless steel needle in the electrospinning process. The cutting and drilling did not affect the use of the allografts in these studies.

Implantation times in athymic mice were 20 and 40 weeks for specimens.

These time points were selected in order to examine potential long-term formation of tissue. Previous experiments highlighted a relatively short implantation time of 10 weeks and were sufficient to determine early responses of the periosteal tissue and cells to the electrospun PLLA nanofibers, and these investigations served as the basis for the work over longer periods of time.

As with the experiments using synthetic scaffolds of PCL/PLLA, the cells from the donor human periosteal tissue are seen migrating from the periosteum through the PLLA nanofibers and laying down an apparently entirely new extracellular matrix between the nanofibers and the allograft bone. Compared to specimens from Group 3, the Group 4 specimens exhibited pronounced migration of cells from the periosteal tissue. New layers of tissue were not evident within Group 3 specimens after 20 and 40 weeks of implantation in vivo.

The apparent absence of new layers of tissue in Group 3 constructs could be attributed to the difficulty in differentiating new tissue from the original periosteal tissue. It is also likely that the periosteum was wrapped around allograft bone in a manner that maintained contact between the two but restricted cellular migration and deposition of new tissue. PLLA nanofiber layers present between the periosteal tissue and underlying allograft bone in Group 4 provided an environment suitable for migration of osteoprogenitor cells from the periosteum.

The migration of these cells presumably resulted in the new layers of tissue

89 observed between the electrospun PLLA nanofibers and the underlying allograft bone scaffolds. Thus, it may be concluded, as with constructs with PCL/PLLA scaffolds described in Chapter 3 previously, the presence of electrospun PLLA nanofiber layers coating the allograft scaffolds here induced increasing numbers of cells from the overlying periosteum and/or increased migration of the periosteal cells, and led in this case to new layers of tissue. These new layers of tissue would appear to be critical to the formation of new bone tissue in possible clinical applications with such engineered constructs. Analysis of histogram measurements over the course of 20 weeks in vivo shows the average thickness of the new layers of tissue formed was approximately 11.5 ± 8.3 µm. By 40 weeks, layer thickness increased to an average of 22.4 ± 13.3 µm. Accounting for areas of the allograft bone scaffolds having more open structures on the periphery of the specimens, there was a shift in tissue thicknesses at 20 and 40 weeks to 9.61 ± 5.37 µm and 16.38 ± 8.44 µm, respectively. While the shift in thickness of the apparently new layers of tissue from 20 to 40 weeks of implantation does not appear to be significant in statistical terms, there appears to be a distinct visual (qualitative) difference in tissue structure that occupies the open cavities of the scaffolds (where space exists between the electrospun PLLA nanofibers and the allograft bone): Tissue in these regions seems to exhibit characteristics of fibrous tissue with a more random and less dense arrangement of cells and extracellular components.

The source of this new layer or layers of tissue along and within the electrospun PLLA nanofibers is intriguing. Given the apparent variability in its

90 structural characteristics, this layer may possibly derive from the implantation host murine cells, but it may also originate from the human periosteal cells. The cells may be, in addition, of a bone lineage. In order to gain insight into these various possibilities, an immunohistochemical (IHC) study of the specimens was conducted to identify osterix, an osteoblast-specific transcription factor.

CHAPTER V

IMMUNOHISTOCHEMICAL LABELING OF OSTERIX

IHC is utilized in many fields of biological research. It is a method that provides a means of identifying and locating a protein within a tissue through microscopic visualization. Protein identification and localization occur using nanoscale-sized markers such as functionalized gold particles or fluorescent labels in conjunction with light or electron microscopy techniques.135-139 IHC has versatility in its application in that it has wide variability of approaches to recover, tag, and visualize any number of epitopes. For commonly examined proteins, there are many standardized protocols already developed. However, difficulty arises when one attempts to use an antibody or tissue not investigated routinely.

In such cases, often a great deal of time and effort is expended refining and developing new methods to ensure that appropriate results may be achieved in a reproducible fashion.

A critical factor in tagging or labeling a protein successfully in formalin-fixed, paraffin-embedded tissues is the epitope retrieval process. During fixation and embedding processes of tissues for microscopy, many proteins have their three- dimensional structure altered to the point where an antibody fails to recognize or cannot locate the epitope (amino acid sequence or sequences) to which it will

92 bind.140,141 Numerous methods have been designed to recover protein structure from a fixed and embedded tissue in this case, and these methods are generally separated into two classes: heat-induced epitope retrieval (HIER)142-144 or protease-induced epitope retrieval (PIER).145-147 Both methods function as their titles suggest. HIER relies on elevated temperature to unfold the protein and

“open up” its epitope for antibody recognition of the target motif. PIER uses a class of enzymes, referred to as proteases, which cleave proteins and accomplish the same goal as HIER. The challenge of either method stems from the enormously wide array of potential conditions for both retrieval techniques.

HIER conditions include buffer composition and pH in addition to temperature variability. Achieving various temperature targets is accomplished in multiple ways. Vegetable steamers, water baths, microwaves, autoclaves, and pressure cookers are all employed with varying degrees of success.142-144 Microwaving and pressure-cooking methods comprise the majority of HIER protocols for two reasons, control and time. Both can be regulated tightly for temperature and only require minutes to unmask an epitope. However, high temperature invariably causes some tissue damage and creates issues hindering the identification, localization, and quantitation of an intended target. Should time be less a factor, methods using a water bath produce similar results under “gentler” conditions.

Tissue structure is preserved, and the location and quantity of the target protein is easier to examine.

PIER is different from HIER in that it involves lower temperatures and more tightly controlled solution parameters. Problems arise when a tissue is digested

93 extensively. Tissue structure is altered, and the target protein is cleaved to the point where antibody recognition fails in a worst-case scenario. However, there are numerous enzymes available as potential candidates for use with this method. Pronase, hyaluronidase, multiple types of collagenases, and pepsin, for example, may be employed to retrieve epitopes.145-147 PIER effectiveness depends on the chosen enzyme being able to remove proteins that block an epitope or to cleave parts of the protein to expose the epitope to the primary antibody. Some proteins require this method when physical heating methods are insufficient or impractical.

The antibody of interest for the experiments in this dissertation tags osterix

(SP7, Osx), an osteoblast-specific zinc-finger transcription factor that is critical to the production and development of bone tissue.148-151 Nakashima et al. showed that inactivation of the Osx gene in mice resulted in little to no bone formation.148

In locations where bones were supposed to grow and mineralize in Osx-null mice, there were instead groups of undifferentiated mesenchymal cells and tissue. The only locations showing mineral deposition and potential bone growth were the result of chondrocytes that provided a template upon which osteoblasts could build.148 Other researchers have shown that osterix helps with regulation of other proteins crucial for osteoblast differentiation. These include bone morphogenetic protein 6 (BMP-6)149 and calmodulin-dependent kinase II (CaMKII).150 Also affected by osterix are Wnt, β-catenin, and RunX2.152-154 All of these molecules are involved in the development of osteoblasts and maintenance of mature bone tissue. RunX2 is also associated with the development of cells having

94 chondrogenic potential.155,156 In fact, preosteoblasts display a degree of bipotentiality, requiring osterix to direct osteoblast instead of chondroblast development and maturation.157

Osterix is a bone-specific intracellular protein detected within the cytoplasm of only osteoblasts and preosteoblasts, and it does not appear in any measurable amount in extracellular spaces of the tissue. In these experiments, the purpose of determining the presence and location of osterix is to ascertain whether or not human donor periosteal tissue retains its osteogenic potential following implantation in athymic mice. The periosteal tissue was removed from its native bone and wrapped around either synthetic polymer scaffolds or sterile human allograft bone. The goal was to ensure that the human tissue was not altered significantly during the process.

A small number of retrieval methods was chosen to attempt unmasking of the osterix epitope. HIER using varying buffers and temperatures and PIER with different proteases were examined for their efficacy in revealing osterix protein for labeling while at the same time causing as little damage as possible to tissue samples. Sample tissues examined provided a degree of difficulty as they were not tissue alone. They also contained synthetic polymers (polycaprolactone,

PCL, and poly-L-lactic acid, PLLA) which are more sensitive to heating methods and solution parameters in the IHC process. As PCL and PLLA can undergo acid- or base-catalyzed degradation, the time and heating parameters of HIER required minor modification. The HIER technique was found to be most suitable under these conditions to obtain desired results. As otherwise part of the normal

95 procedure with polymers, reducing background staining of the polymers with 3,3’- diaminobenzidine (DAB) was unnecessary. It aided, however, in confirming and identifying the presence and location of osterix within the cells present on and within the polymer structures. The primary goal for these experiments was the development of a protocol to localize human osterix within tissue-engineered constructs.

5.1 Materials and methods

5.1.1 Sectioning and immunohistochemical staining of osterix

Formalin-fixed paraffin-embedded specimens (Group 3, Chapter 3 and

Groups 1-4, Chapter 4) were sectioned (5-7 µm thickness) with a microtome

(Model RM2255, Leica, Buffalo Grove, IL), floated on a 42°C water bath, mounted onto glass slides (Superfrost Excell, Thermo Scientific, Waltham, MA), and left on a warming platform to dry at 42°C for a minimum of 3 hours. Slides were then stored in slide boxes prior to immunohistochemical staining.

In preparation for IHC, sections on slides were placed flat and face up on an aluminum slide tray, and the tray was removed to a 60°C oven for a minimum of

5 hours. The slides were then inserted into a glass slide holder for one additional hour. Sections were deparaffinized through three fresh changes of xylene (10 minutes each, E K Industries, King of Prussia, PA). They were rehydrated using

100% ethanol (1 x 10 minutes, 1 x 5 minutes) and 95% ethanol (2 x 5 minutes each). Individual slides were removed from the slide holder after the second

95% ethanol and placed face up on a benchtop to dry. Drying of sections utilized

96 a heat gun (5 amp, 93°C/149°C, Master Appliance Corp, Racine, WI) set in such a manner that cool air passed over the slides for a period of 10 minutes. This procedure was followed by a small period of heating where the underside of each slide was exposed to hot air from the heat gun for approximately 3 seconds at least twice as each slide was cycled through the process. The cool air/heating cycle was repeated once more before the slides were rehydrated through two changes of deionized water (5 minutes each). Rehydrated slides were then moved to a Coplin jar and fixed in 10% neutral-buffered formalin (NBF, VWR,

Radnor, PA) overnight at 4°C.

The following morning, NBF was exchanged for fresh Immunocal (two changes, 1 hour each, Decal, Tallman, NY). Slides were rinsed with deionized water (2 x 5 minutes each) following the second Immunocal rinse and before addition of a peroxide blocking solution (0.3% H2O2/30% CH3OH, 30 minutes).

Antigen retrieval methods varied slightly and are listed in the table below.

Following retrieval, slides were transferred, face up, to a humidified chamber, and Background Buster (INNOVEX, Richmond, CA) was added as the protein block (30 minutes). Protein blocking was trailed immediately with addition of primary antibody (OSX (Y-21): sc-133871, dilution 1:50, Santa Cruz

Biotechnologies, Dallas, TX), and negative control sections (specimens receiving no primary antibody) were covered with TTBS in place of the primary antibody.

The chamber containing the slides was placed in a 4°C refrigerator overnight.

The next morning, slides were washed (Tween 20/tris-buffered saline (TTBS),

3 x 5 minutes each) before addition of secondary antibody (ImmPRESS, 30

97 minutes, Vector Laboratories, Burlingame, CA). Excess secondary was washed from the slides (TTBS, 3 x 5 minutes each) before another peroxide block (0.3%

H2O2/30% CH3OH, 10 minutes). The peroxide block was rinsed from the slides

(TTBS, 3 x 5 minutes each) before addition of 3,3’-diaminobenzidine (DAB, 1 tablet + 7 mL PBS + 3 drops 3% H2O2, MaxTag DAB tablets, Rockland, Limerick,

PA) solution. Slides were counterstained with Mayer’s hematoxylin (Electron

Microscopy Sciences, Hatfield, PA), cleared, mounted with DPX (Electron

Microscopy Sciences, Hatfield, PA), and coverslipped for viewing under the microscope.

Table 1. Epitope retrieval solution, temperature, and time parameters.

Antigen Retrieval Solution Temperature (°C) Time 10 minutes – Sodium citrate/EDTA/Tween 20 (pH 6.0) 60-120 72 hours Sodium citrate/SDS buffer (pH 6.0) 65 1-24 hours 10% ammonium hydroxide 25 1-3 hours Tris-HCl (pH 9.0) 65 1 hour Boric acid (pH 7.0) 65 44 hours Pronase/type 1 collagenase 37 30 minutes Type 1 collagenase 37 30 minutes Pronase 37 30 minutes

5.1.2 Microscopy

Micrograph images were obtained using an inverted light microscope (IX70,

Olympus, Melville, NY) under 40X magnification with a camera (CC12, Olympus,

Melville, NY). Multiple images were collected from the same section area at differing focal points and then “focus-stacked” using Adobe Photoshop auto- blend function to produce a clear picture. Osterix-positive cells appear as brown

98 (DAB staining). Other cells within the structure stain blue (Mayer’s hematoxylin counterstain).

5.2 Results

Optimal immunostaining results in this series of experiments were achieved utilizing sodium citrate/EDTA/Tween 20 buffer as the epitope retrieval solution.

Figure 5.1 presents a composite of 16 focus-stacked light micrograph images and illustrates positive staining of osterix present within osteoblasts in a representative construct (Group 3, Chapter 3) following high-temperature, elevated-pressure epitope retrieval (120°C, 15 psi, 10 minutes). Periosteal tissue, electrospun PLLA nanofibers, and portions of the PCL/PLLA scaffold delaminated from the slides during these epitope retrieval conditions and were lost in the buffer solution in this instance.

Figure 5.2 is a light micrograph of a representative negative control slide from the same construct as above (Group 3, Chapter 3) processed under different thermal parameters (60°C, 72 hours). The structure of the specimen section was preserved using these conditions and Mayer’s hematoxylin counterstain shows cell nuclei within periosteal tissue, the electrospun PLLA nanofibers, and the

PCL/PLLA scaffold.

Positive IHC immunostaining results for osterix in a different section from the representative construct from Figure 5.2 are shown in Figure 5.3. Osterix- positive cells (brown) are present within the periosteum, the electrospun PLLA

99 nanofibers, and the PCL/PLLA scaffold following identical epitope retrieval conditions as noted previously (60°C, 72 hours).

Figure 5.4 is a light micrograph illustrating the presence of osterix-positive cells. Such cells were located within the electrospun PLLA nanofibrous layer of a different area of the tissue section shown in Figure 5.3.

Figures 5.5-5.8 highlight results of representative specimens that were each processed under the same epitope retrieval conditions (60°C, 48 hours). Figure

5.5 shows staining of a sample harvested after 40 weeks of implantation in a nude mouse (Group 1, Chapter 4). The specimen demonstrated a negative result for the presence of osterix. Limited edge-effect staining from DAB was found in the allograft bone in an enlarged image (Figure 5.5B).

IHC staining for allograft bone samples coated with electrospun PLLA nanofibers and harvested from athymic mice at 20 and 40 weeks (Group 2,

Chapter 4) is shown in Figure 5.6 (A and B, respectively). Background DAB staining was present in the extracellular spaces of the murine fibrous tissue within the specimen (Figure 5.6B).

Figure 5.7 presents allograft bone constructs wrapped with periosteum (Group

3, Chapter 4) and harvested after 20 and 40 weeks implantation in vivo.

Prominent clusters of osterix-positive cells were observed within the periosteal tissue of these representative specimens.

Results similar to those shown in Figure 5.7 were found for allograft bone samples coated with electrospun PLLA nanofibers and wrapped with periosteum

(Group 4, Chapter 4) following 20 and 40 weeks of implantation in vivo (Figures

100

5.8A and B, respectively). Osterix-positive cells infiltrated the electrospun PLLA nanofibers on the surface of the allograft bone scaffold in the representative specimens.

5.3 Figures

PCL/PLLA

Figure 5.1. Light micrograph of a PCL/PLLA (75/25) scaffold wrapped with periosteum, implanted for 10 weeks in an athymic mouse (Group 3, Chapter 3), and stained with anti-osterix (SP7). Diaminobenzidine (DAB) as an indicator molecule shows osterix as brown within several cells in the image field (arrows).

The counterstain is Mayer’s hematoxylin to indicate the cell nuclei as blue. The periosteal tissue and electrospun PLLA nanofibers should be present in the upper right portion of the image, but they separated from the slide and were lost in the retrieval buffer as a result of the high temperature and elevated pressure

(120°C, 15 psi, 10 minutes) used during the epitope retrieval procedure. Scale bar = 50 µm.

101

PCL/PLLA

NF P

Figure 5.2. Light micrograph of a negative control IHC specimen from Group 3,

Chapter 3 constructs. Cell nuclei are stained with Mayer’s hematoxylin (blue).

Structural damage to the periosteal tissue (P), electrospun PLLA nanofiber layer

(NF), and underlying scaffold (PCL/PLLA) was limited, compared to the results shown in Figure 5.1, using different thermal conditions (60°C, 72 hours) for epitope retrieval. Scale bar = 50 µm.

P NF

PCL/PLLA

Figure 5.3. Light micrograph illustrating a PCL/PLLA scaffold that shows osterix- positive osteoblasts (arrows) which appear to have migrated from the periosteum

102

(P) to the electrospun PLLA nanofiber layer (NF) and underlying scaffold

(PCL/PLLA) over the course of 10 weeks of implantation in an athymic mouse.

The tissue section was counterstained using Mayer’s hematoxylin. Scale bar =

50 µm.

Figure 5.4. Osterix-positive cells (highlighted by arrows) are present within the electrospun PLLA nanofiber layer (NF) of a different area of the representative specimen shown in Figure 5.3. Periosteal tissue is not shown in this image because the section was collected close to an end of the specimen where the periosteum was absent. The nanofiber layer is characterized in this image by fine, brown-staining, randomly dispersed, thread-like PLLA fibers. Many cells that are osterix-negative but whose nuclei are stained blue with Mayer’s hematoxylin are also found within the NF layer. Scale bar = 50 µm.

103

A B

F AB AB

Figure 5.5. IHC staining results for a representative allograft bone scaffold

(Group 1, Chapter 4) harvested after 40 weeks of implantation in vivo. Mayer’s hematoxylin stain highlights nuclei of cells, presumably fibroblasts, from murine fibrous tissue (F) encapsulating the allograft bone (AB). The boxed area in panel

A is enlarged in panel B. There is minor edge-effect staining of DAB (arrow) present on the allograft bone in the enlarged image (B). Scale bar = 200 µm (A) and 50 µm (B).

A B NF

F F

Figure 5.6. Results for electrospun PLLA nanofiber-coated allograft bone specimens (Group 2, Chapter 4) harvested after 20 (A) and 40 (B) weeks of

104 implantation in vivo and stained with osterix antibody. Significant background staining from DAB is present within the extracellular matrix of the representative

40 week specimen (B). The lack of apparent intracellular DAB staining suggests either the activity of endogenous peroxidases was not blocked completely during the peroxidase blocking step or the protein blocking step was insufficient to prevent the secondary antibody from binding to proteins within the endogenous murine tissue (F). NF = Electrospun PLLA nanofibers. Scale bar = 50 µm.

A B P P

Figure 5.7. Osterix immunostaining results for representative periosteum- wrapped allograft bone constructs (Group 3, Chapter 4) harvested after 20 (A) and 40 (B) weeks of implantation in vivo show the presence of osterix-positive cells clustered within the periosteal tissue (P) around the allograft bone (AB) in these specimens. Scale bar = 50 µm.

105

A P B NF P

AB NF

Figure 5.8. IHC immunostaining results for representative specimens of electrospun PLLA nanofiber-coated allograft bone constructs wrapped with periosteum (Group 4, Chapter 4). These specimens were harvested after 20 (A) and 40 (B) weeks of implantation in vivo. Arrows in both images denote osterix- positive cells migrating from the periosteum (P) through the electrospun PLLA nanofiber layer (NF). AB = Allograft bone. Scale bar = 50 µm.

5.4 Discussion

This dissertation chapter describes the successful labeling of the osterix protein in cells from human donor periosteum used to wrap various scaffolds of interest, that is, PCL/PLLA scaffolds (Chapter 3) and human allograft bone

(Chapter 4), each coated with an electrospun PLLA nanofiber layer. Such suitable labeling was accomplished using the protocol detailed in the materials and methods of this chapter. The procedure was modified from IHC techniques designed for labeling proteins within soft tissues rather than tissue such as periosteum, giving rise to mineralized matrices. A few steps included in this modified IHC protocol are not required for soft, unmineralized specimens and it is 106 useful to point out differences. For instance, the slides holding specimens wrapped with periosteum were immersed in 10% NBF overnight prior to the epitope retrieval process in order to limit potential structural damage of the tissue incurred during decalcification. The decalcification process, using the formic- acid-based decalcifier, Immunocal, was critical for removing residual calcium and phosphate potentially present in the tissue sections that could interfere with binding of the osterix antibody.

Following the decalcification steps, a series of routine epitope retrieval protocols was employed in an effort to determine a suitable approach to labeling osterix within the tissue specimens. In the cases where sodium citrate/SDS was utilized as the epitope retrieval buffer, the sections delaminated from the slides entirely, likely a result of SDS being a strong, anionic surfactant.158 For slides treated with 10% ammonium hydroxide and Tris-HCl, only half retained sections through the rest of each protocol and all of these were absent of any visible labeling of osterix. Enzyme-treated slides did not exhibit section delamination, but all immunostaining results using the enzyme-based protocols were likewise negative for the presence of osterix. For the sodium citrate/EDTA/Tween 20 buffer, conditions for successful epitope retrieval were achieved initially with the slides in the buffer solution in an autoclave at 120°C and 15 psi for 10 minutes.

The high temperature and pressure, however, caused complications as the electrospun PLLA nanofibers and PCL/PLLA scaffold were removed partially and the periosteum was removed entirely from slides. In these instances, determining the precise location of osterix-positive cells in the overall structure of

107 the tissue was not possible. On the other hand, osterix labeling seen in autoclaved specimens did suggest that heating was an appropriate parameter for retrieving the osterix epitope.

Obtaining clear images of autoclaved slides using light microscopy was frequently challenging as portions of the tissue no longer lay flat on the surface of slides as a result of tissue section delamination from the high heat and pressure of the retrieval process. Consequently, multiple images, at varying focal points, were collected and “focus-stacked” to generate a single, clear image of the tissue.

There are obvious limits to this technique as it uses a three-dimensional set of images projected onto a single, two-dimensional plane, and the potential exists for overlapping areas of tissue to distort the final image and alter interpretation of results. Specific concerns regarding tissue integrity and clear imaging subsequently led to epitope retrieval being achieved at decreased temperature for increased time in an effort to counteract the damaging effects of high temperature and pressure on the sections and generate conclusive results with regard to the presence of osterix-positive cells in tissue specimens.

Epitope retrieval using sodium citrate/EDTA/Tween 20 buffer for 72 hours at

60°C yielded successful labeling of osterix protein and maintained structural integrity of the tissue within the sections. Osterix-positive cells were visible within the periosteum, the electrospun PLLA nanofibers, and the interior of underlying

PCL/PLLA scaffolds (Group 3, Chapter 3), a result suggesting osteoblasts and preosteoblasts migrated into the scaffolds from the periosteal tissue after 10 weeks of implantation in vivo. Osterix labeling in specimens containing allograft

108 bone (Chapter 4) also showed positive results for osterix staining. These data were obtained using decreased epitope retrieval time for these specimens compared to specimens with PCL/PLLA scaffolds (Chapter 3). However, epitope retrieval times of less than 48 hours produced no visible labeling of the osterix protein. Allograft bone samples (Group 1, Chapter 4) and allograft bone specimens coated with electrospun PLLA nanofibers (Group 2, Chapter 4) demonstrated negative results for the presence of osterix within cells of the murine fibrous tissue encapsulating the bone. Background staining of DAB was present in tissue of allograft bone coated with electrospun PLLA nanofibers at 40 weeks likely as a result of endogenous peroxidase activity or secondary antibody binding to murine tissue. In these specimens with DAB staining visible only within extracellular matrix of the fibrous tissue, it is probable that osterix is absent from within the cells of the specimens.

Allograft bone scaffolds wrapped with human periosteum (Group 3, Chapter

4) showed osterix labeling within periosteal tissue after 20 and 40 weeks of implantation in vivo, a result that appears to confirm that periosteum was an appropriate source of osteoprogenitor cells. Osterix-positive cells were also present within the periosteal tissue and the electrospun PLLA nanofiber layers covering the allograft bone scaffolds (Group 4, Chapter 4) after 20 and 40 weeks of implantation in vivo. Of particular interest were the cells additionally found in the layers of electrospun PLLA nanofibers. Many of these cells exhibited the presence of osterix after 40 weeks of implantation, an observation suggesting that the periosteal cells migrate, differentiate into osteoblasts, and form new

109 tissue over extended periods of time within the PLLA nanofiber-coated allograft bone scaffolds. The apparent migration of preosteoblasts and osteoblasts from the periosteum into and through the electrospun PLLA nanofiber layers would indicate that the human donor periosteal tissue, not the vasculature of the host mice, was the source of the presumed new layers of tissue about the allograft bone coated with electrospun PLLA nanofibers. This result illustrates the critical role of the periosteum during the bone regeneration process for tissue engineering applications. There was no presence of osteoprogenitor cells detected in the specimens lacking the periosteal tissue, indicating the periosteum was the major contributor of new bone tissue formation in these experiments.

These preceding results highlight the significant benefit of applying electrospun PLLA nanofibers as coatings for bone tissue-engineering scaffolds in order to promote cellular migration and subsequent regeneration of bone tissue in vivo. Future studies for tissue engineering to augment or repair bone defects, including large segmental gaps as noted earlier in this dissertation, should focus efforts on improving cell migration and tissue regeneration qualities of electrospun nanofiber-coated materials. Electrospinning nanofibers of a composition closely compatible with bone tissue, for example, collagen or elastin nanofibers, might be very useful in this context.

It is a challenge to improve IHC procedures for proteins that are found in specimens also containing synthetic polymer materials as well as mineral. With the field of tissue engineering expanding to new avenues in biology, medicine and other areas, developing histochemical methods to study living tissue,

110 synthetic materials, and mineral is important for furthering the landscape of regenerative medicine. Epitope retrieval is a critical step in immunohistochemical techniques applied to fixed and embedded samples or those that may be completely untreated. A balance between unmasking of epitopes of a protein of interest and preservation of the structure of the tissue specimen must be established in order to produce quality IHC staining results. Heat was shown to be a critical component in retrieval of the osterix epitope for the tissue samples presented in this Chapter, but extreme autoclave heat and pressure during the retrieval process altered the structure of the original tissue to the degree that it was not possible to identify osterix or determine the exact location of osterix- positive cells in tissue sections. Heat applied at a lower temperature during epitope retrieval and for an extended time preserved the original tissue structure of specimens while retrieving the desired osterix epitope.

An increased epitope retrieval time necessary to complete the osterix staining protocol outlined in the preceding pages of this Chapter is significant. It points to the need for researchers to examine all aspects of the IHC process and make decisions in developing protocols to tag a protein without significantly impacting original tissue structure. Apparently simple modifications to established protocols, such as increasing the amount of time during epitope retrieval, could prove to be a key factor in whether an IHC technique succeeds in labeling a target protein.

IHC continues to be a versatile tool for identifying and localizing proteins critical for identification of cells and tissues within many research and clinical fields.

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CHAPTER VI

CONCLUSIONS AND FUTURE WORK

The goal of the work detailed in the preceding Chapters of this dissertation was to design and develop a simple method of incorporating electrospun nanofibers into existing tissue engineering scaffolds for treatment of segmental bone defects. Chapter 1 highlighted and summarized approaches to augment or repair segmental bone defects, methods of tissue engineering, various techniques in electrospinning, and attempts to utilize electrospun nanofibers in regenerative medicine applications. The nanofiber-coated materials presented in this thesis were fabricated as an advancement of current practices and as a means of illustrating the merits of continued research in the field of electrospinning. Electrospun nanofibers hold an amazing capability to mimic native extracellular matrix, but little has been accomplished in integrating them into three-dimensional structures necessary to rebuild entire tissues and organs.

The method of applying PLLA nanofibers to prefabricated scaffolds of PCL/PLLA detailed in Chapter 2 offers a middle ground between preformed three- dimensional materials and flat sheets of nanofibers commonly available in the field. PLLA nanofiber application also may be extended to more intricate and

112 robust constructs which have not been generated solely through the electrospinning process.127-130

Chapter 2 presented a novel methodology for applying electrospun nanofiber coatings of PLLA to all surfaces of a solid of any shape and, in particular, to prefabricated, three-dimensional, spongy scaffolds composed of PCL/PLLA.

Producing nanofibers from a simple, biodegradable polyester (PLLA) as a coating for an existing material demonstrated a small, but significant, advance to practices employed currently for treating segmental bone defects. Minimal alteration was required with regard to storage and preparation methods prior to surgery and techniques utilized by surgeons, promoting rapid adoption of these electrospun PLLA nanofiber-coated PCL/PLLA scaffolds as potential bone engineering materials. Addition of growth factors and other specialized molecules to the individual nanofibers or nanofibrous mats which could accelerate tissue regeneration is possible using the modified electrospinning approach explained in Chapter 2. The addition of these components, however, could be affected by storage and preparation conditions and are not necessarily required to achieve a desired result. Such complex methods with the potential for effective regeneration may require extensive time and effort to be integrated into conventional medical practices. Off-the-shelf scaffolds are likely candidates for general reconstruction procedures where specialized techniques are too intricate or expensive to be applied effectively.

In Chapter 3, the electrospun PLLA nanofiber-coated PCL/PLLA scaffolds were wrapped with human periosteal tissue and implanted into athymic (nude)

113 mice for a period of 10 weeks. Histochemical stains were selected to investigate cellular infiltration and extracellular matrix production in the PLLA nanofibers as well as the underlying PCL/PLLA materials. The stains showed the presence of new tissue and mineral formation following the 10 weeks of implantation in vivo.

Cellular infiltration and extracellular matrix secretion was shown within the electrospun PLLA nanofiber layer and the underlying PCL/PLLA scaffolds. A slight increase in the amount of mineral formed within the PLLA nanofiber layers of the constructs at 10 weeks was an important discovery that showed the potential advantage of using electrospun nanofiber coatings of PLLA to accelerate new bone formation.

In Chapter 4, sterile, human allograft bone specimens were coated with PLLA nanofibers, wrapped with human periosteum, and implanted for 20 and 40 weeks in nude mice. Histological analyses of the harvested tissues were based on hematoxylin and eosin (H & E) stained specimens. Alizarin red and von Kossa stains were not selected to investigate mineral formation in these allograft bone specimens as they were in the case of the PCL/PLLA scaffolds because the allograft bone was demineralized prior to paraffin wax embedding in order to preserve morphology of the tissue and ensure collection of consistent sections.

H & E staining highlighted the presence of supposed new layers of tissue following 20 and 40 weeks of implantation in vivo. It was presumed that the new layers of tissue were the result of cellular activity of the osteoprogenitor cells derived from the human periosteal tissue used for these constructs. This assumption was examined with additional experimental methods to confirm that

114 the presence of new tissue within constructs described in Chapters 3 and 4 was the direct result of the actions of osteoprogenitor cells from the human periosteal tissue and not the result of murine fibrous tissue infiltration in vivo.

Chapter 5 detailed work investigating the source of osteoprogenitor cells as just noted. Chapter 5 also expanded current immunohistochemical (IHC) techniques to account for factors specific to constructs containing mineralized tissue (bone) and synthetic polymer materials (PCL and PLLA) for the purpose of labeling osterix, an osteoblast-specific transcription factor. The simple modification of existing IHC procedures for unmineralized tissues was made by decreasing the temperature and extending the time for epitope retrieval in construct IHC processing. As a result, there was clear labeling of the osterix protein within cells of the periosteum, the electrospun PLLA nanofibers, and underlying scaffolds of constructs. The reduced temperature and longer times in the newly designed IHC protocol also prevented significant structural damage or alteration to the structure of the tissue. The immunostaining results for osterix in the nanofiber-coated constructs demonstrated cellular infiltration and migration into and through the PLLA nanofibers of constructs. IHC further appeared to confirm that the apparently new layers of tissue present in specimens detailed in

Chapters 3 and 4 could be attributed to the cells derived from the human periosteum and not from the vascularity or other potential sources from the athymic mice hosting the construct implants. Importance of the periosteum in the bone regeneration process should not be overlooked in this context. Periosteal tissue is rich in osteoprogenitor cells responsible for endogenous repair and

115 regeneration of bone tissue.53-61 Utilizing this tissue as a resource for bone tissue-engineering applications is the logical extension of techniques employed by orthopaedic surgeons utilizing the Ilizarov method for bone repair.22-25

The results presented in Chapters 2 through 5 indicate the merit of utilizing electrospun nanofiber coatings for approaches in bone tissue engineering and suggest it is possible to extrapolate the use of electrospun nanofiber-coated materials for other purposes in tissue engineering. As explained in Chapter 1, electrospinning was discovered over a century ago and did not generate significant interest until research in the 1990s returned it to the forefront as a method for producing nanoscale fibers appropriate to many fields. Interest in the electrospinning technique continues to expand as additional applications are discovered for nanofibrous materials.

In the same span of time, tissue engineering has become an expanding discipline, perched on the edge of breakthroughs in regenerative medicine that were labeled science fiction as recently as thirty years ago. Growth of entire organs is no longer out of reach for the medical field. Replacing lost, damaged, or diseased portions of the human body continues to represent a significant milestone for efforts in the field of tissue-engineering research. It requires the efforts of many individuals from varying research backgrounds to realize such a goal. Combining engineering, chemistry, and biology to generate viable ideas and methods for accomplishing such a difficult task is advancing the field of regenerative medicine at a remarkable pace. It is a matter of time before organ

116 transplants from donor patients and simple prosthetics are no longer the only option for patients in need of them.

The experiments presented in this dissertation constitute a small fraction of possibilities in the field of tissue engineering. A narrow focus on improving current techniques and procedures for repairing segmental bone defects was important to advancing research in the area. Decisions to utilize human tissue and a limited set of simple biodegradable polyesters in these series of studies were part of a strategy to determine the merit of incorporating nanofiber-coated constructs in situations applicable in the medical field while working within confines of limited tissue availability. Advancements in cell isolation, expansion, and seeding offer promise for future applications in tissue engineering but have been relatively slow to be incorporated into actual practice. Until newer methods are adopted in more wide-spread fashion, increasing the effectiveness of current techniques for limited tissue availability is necessary.

As research in tissue engineering continues to progress, it is important to consider each new development a stepping stone to future insights. Answers in one part of the field lead to new questions and the potential for greater improvement in approaches, methodology, and applications. Nanofiber-coated scaffolds provide direction toward improving cellular infiltration and growth in bone tissue-engineered constructs. Further expansion of the modified electrospinning technique presented in this dissertation could include nanofibers and underlying scaffolds with more complex and specialized chemical structures designed for specific purposes. It is possible to electrospin natural materials like

117 collagen and elastin. A scaffold covered in nanofibers composed of these materials would conceivably exhibit properties influencing more rapid growth of tissue in comparison to the PLLA nanofibers described in the preceding experiments. Other avenues for expanding this field of research could concentrate on repairing segmental defects in appropriate animal models. The athymic mice used for the experiments detailed in this dissertation served only as simple bioreactors to validate the merits of electrospun nanofiber coatings for bone tissue engineering. Designing a suitable animal bone defect model for testing would further highlight the potential of nanofiber-coated scaffolds and be a logical next step toward utilizing this technology in a clinical application. There remains room for enhancing the effectiveness of nanofiber-coated materials, and it is of value that efforts continue in order to advance this and related fields of research.

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