Development of a Z-Stack Projection Imaging Protocol for a Nerve Allograft

DEVELOPMENT OF A Z-STACK

PROJECTION IMAGING

PROTOCOL FOR A NERVE

ALLOGRAFT

SELVAANISH SELVAM

Submitted in partial fulfillment of the requirements for the degree of Master of Science

Dissertation Advisor: Dr. George F. Muschler

Department of Biomedical Engineering

CASE WESTERN RESERVE UNIVERSITY

August 2018 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis of

Selvaanish Selvam

candidate for the degree of (Master of Science) *.

Committee Chair

Dr. George F. Muschler

Committee Member

Dr. Eben Alsberg

Committee Member

Dr. Robert Kirsch

Committee Member

Cynthia Boehm

Date of Defense

May 4th 2018

*we also certify that written approval has been obtained

For any proprietary material contained therein

Table of Contents

List of Tables……………………………………………………………………..4

Figures List……………………………………………………………………….5

Acknowledgments………………………………………………………………..6

List of Abbreviations………………………………………………………….....7

Abstract…………………………………………………………………………...8

Introduction……………………………………………………………………..10

Methods………………………………………………………………………….21

Data and Analysis………………………………………………………………31

Conclusions and Future Directions……………………………………………51

References……………………………………………………………………….53

List of Tables

Table 1: Overall summary of ratios and retention rates……………………..31

Table 2: Selection Ratio for grafts (3X – 10-minute rinse) ………………….35

Table 3: Selection Ratio for grafts (15-minute soak) ………………………...35

List of Figures:

Figure 1: 2D 10X DAPI stained image using current techniques…….……….9

Figure 2: Peripheral Nerve Anatomy………………………………………….10

Figure 3: Nerve grade injury…………………………………………………..11

Figure 4: Different types of peripheral nerve repair…………….…………...13

Figure 5: Z-stack of images creating a Z-stack projection image……...……18

Figure 6: Jablonski diagram for Fluorescence molecules……………………19

Figure 7: Nerve allograft…..…………………………………………………...22

Figure 8: Nerve graft after rinse……………………………………………….22

Figure 9: Nerve graft with media at Day 0 of cell culture……………………23

Figure 10: Cell Culture at day 0………………..…………………………...…25

Figure 11: Cell Culture at day 3………………………………..……..……….25

Figure 12: C2 Graft……………………………………………..……...………39

Figure 13: E1 Graft………………………………………………………….….40

Figure 14: E2 Graft…………………………………………….………….……41

Figure 15: F2 Graft………………………………….…………….……………43

Figure 16: F3 Graft……………………………………………………………..44

Figure 17: Week 3 (G1, G2, and G3 graft)…………………………………....45

Figure 18: Z-stack projection image of D1 Graft…………………………….47

Figure 19: Z-stack projection image of D2 Graft…………………………….49

Acknowledgement

I would like to thank Dr. George Muschler, Cynthia Boehm, Dr. Nicolas Piuzzi, Wes

Bova, and Viviane Luangphakdy and Ratnam Mantripragada from the Cleveland clinic in helping me formulate, solve, and present this project.

I also would like to thank Dr. Robert Kirsch, Dr. Eben Alsberg, Amrish Selvam, and the rest of Case Western Reserve University in helping me complete my project.

List of Abbreviations

2D: Two Dimensional

BMA: Bone Marrow Aspirate

BMC: Bone Marrow Aspirate Concentrate

CTP: Connective Tissue Progenitor

CTPs: Connective Tissue Progenitor cells

DAPI: 4',6-diamidino-2-phenylindole stain

FDA: Food and Drug Administration

HBSS: Hank’s Balanced Salt Solution

NGF: Nerve Growth Factor

PBS: Phosphate-Buffered saline

Development of a Z-Stack Projection

Imaging Protocol for a Nerve Allograft

Abstract

SELVAANISH SELVAM

Peripheral nerve injuries have traditionally been treated with a variety of different surgical procedures, but the use of allografts for these injuries remain to be a largely unexplored concept. As a result, there has yet to be a successful protocol created for imaging cells on the surface of nerve allografts. In this thesis, I developed a z-stack projection imaging protocol for nerve allografts and demonstrated the ability to take clear and focused images of cell retention and proliferation on the surface of the allograft. I also optimized the preparation of the nerve graft to improve cell and connective progenitor cell (CTP) retention rates.

Problem Statement and Purpose Bone Marrow Aspirate has been used in many graft applications and has shown significant clinical improvement. 1 However, when using a fluorescence light microscope to image the graft, limited focusing capabilities and the uneven topography of the graft make it difficult to obtain a clear image of the cells on the surface of the graft. Figure 1 shows an example of a graft using current 2D image techniques. The figure indicates cell attachment, but the image acquired by the present technique produces a blurry and inconclusive image of the graft. With such an unfocused image little to no analysis can be done to understand the number of cells retained and proliferating on the nerve graft.

Figure 1. A 2D 10X image of a DAPI stained nerve graft. The few small white spots in the middle appear to be cells but cannot be confirmed due to the quality of the image. Large portions of the image remain distorted and increases the need for a better image acquisition protocol for grafts.

The purpose of this experiment will be to resolve this imaging issue by developing and introducing a workable z-stack imaging protocol to have a clear view of cells on the top surface of the nerve graft. To test this imaging protocol, nerve allografts will be imaged.

The preparation of this graft will also be optimized to improve overall cell and connective tissue progenitor (CTP) retention rates.

Introduction Nerves are a bundle of fibers that relay information from the brain and spinal cord to muscles and skin. They use electrical and chemical signals to send information quickly to different parts of the body to help establish motor and sensory functions. 2,3

Various injuries to the structure of nerves can cause detrimental damage to the patient and their quality of life. Before looking into the different classifications of these nerve injuries it is paramount to understand nerve anatomy.

Each nerve fiber is composed of connecting axons and are protected by a layer of connective tissue known as the endoneurium. When multiple nerve fibers are combined together the bundle is known as a fascicle. The fascicle is encircled by a sheath of connective tissue called a perineurium and is known as the smallest structure capable of accepting sutures.4 Multiple fascicles and blood vessels combined together make up the peripheral nerve architecture. The peripheral nerve is surrounded by a loose outer sheath

of blood vessels known as the

epineurium. Figure 2 is a

descriptive image that shows

the previously described

structures under one diagram

Figure 2. Schematic illustration of peripheral nerve anatomy and vital structures.5

Categorization of Nerve Injury When specific structures of the nerve anatomy are injured, various levels of nerve pathophysiology are affected. The most minimal form of peripheral nerve injury is

neurapraxia which is

defined as focal

demyelination with no

damage to the connective

tissues or the axons. This

can lead to muscle

weakness and can be

resolved within a few

days after injury. The

Figure 3. Image representing functioning nerve with no second level of nerve damage, Grade IV damaged nerve, and Grade V damaged nerve.6 injury is known as axonotmesis and can be classified under Grade II, Grade III, and Grade IV of the

Sunderland nerve injury scale. 6 Axonotmesis is categorized as damage to axons along with focal demyelination. Grade II axonotmesis is classified with no damage to surrounding connective tissue and the endoneurium is kept intact. Grade III is observed when the endoneurium is damaged, but the perineurium is still kept intact. Grade IV classification extends damage to the perineurium, but the epineurium is unbroken. The most severe peripheral nerve injury category is Grade V or neurotmesis and involved complete transection of the nerve. In this level of injury, the perineurium is damaged and axons are fully disconnected.6–8 Grade IV and Grade V injuries require surgical techniques for recovery.4 Figure 3 shows a diagram of a proper nerve along with Grade

IV and Grade V nerve injuries. Large portions of the nerve are seen transected and muscle function is compromised.

Natural Nerve Reinnervation Process

Following a peripheral nerve injury several natural mechanisms try to reinnervate the proximal end of the injured nerve to the distal end. If injuries affect greater than 90% of the axons then axonal regeneration is the primary method for reinnervation.9 The three primary steps to achieve full recovery are: Wallerian degeneration, axonal regeneration, and end-organ reinnervation. 7 A phenomenon known as Wallerian degeneration occurs within the first week of injury and causes the breakdown of axonal cytoskeleton. In the distal stump, granular disintegration occurs after an influx of extracellular ions and results in a process resembling apoptosis. On the other hand, the proximal stump is also broken down but much more limited and breakdown typically only progresses to the first node of Ranvier on the proximal end. Once Wallerian degeneration is fully complete the environment is now conducive to nerve regeneration since myelin debris and existing damaged nerves have mostly been removed. Regeneration begins when a growth cone is formed on the distal tip of the proximal stump. Neurotropic factors and guidance molecules like Collapsin-1 help guide the growth cone towards the distal end of the injured nerve. Schwann cells are also critical in promoting suitable axonal regeneration as they display nerve growth factors (NGFs) in the distal section of the axon. Once the growth cone reaches the endoneurial tube, maturation begins. This last step remyelinates axons and also increases overall axon diameter. When these changes occur, ATP is released causing Schwann cells to change their phenotype to become myelinating agents and conclude the reinnervation process.8,10 Nerve innervation is key to restoring muscle

12 and skin sensations and can be achieved by connecting the newly grown motor nerve to muscle or a sensory nerve to skin.3,4,11 Once this connection is established sensory-motor functions begin to rejuvenate. Electromyography tests can show if nerve innervation has occurred and if no progress in nerve regeneration has occurred then nerve surgery is suggested.

Surgical Techniques Utilized for Nerve Reinnervation

However, sometimes due to the size of the nerve gap, scar tissue, or neuroma formation surgical nerve repair becomes necessary for proper nerve reinnervation.

12 This becomes abundantly clear in Grade IV and Grade

V injuries. Many different surgical techniques are available including direct Nerve Repair, nerve transfers, autografts, and allografts. These different methods are shown on figure 2. 12The type of technique used depends Figure 4. Different types of nerve repair shown on of a variety of factors including time elapsed, patient age, a human hand. mechanism of damage, and proximity of lesion to distal targets. 12,13

Direct nerve repair is used to connect the proximal and distal nerve ends without any tension. It is commonly used in patients having injury in the proximal portion of the nerve and has many different types including: end-to-end repair, epineural sleeve repair, and end-side repair. This type of nerve repair is done when the gap is small and can be connected with minimal tension and allows for a quick recovery time. However if the gap is larger than 3cm then other repair methods have to be implemented. 12,14

Nerve Transfers is another surgical technique commonly used by surgeons to repair peripheral nerve injuries. Nerve Transfers transfer nearby less important or redundant functioning nerves to the injured site’s nerve motor endplate. When using this technique, the proximity of donor nerves to the target site allows for faster reinnervation and this technique can be implemented when the proximal stump is unavailable.

However, like direct nerve repair, this technique can only be used for small gaps and has donor nerve site morbidity which may cause a loss of function for the donor muscle. This is a common surgery used when the ulnar nerve is injured. 12,15,16

Nerve Autografts are immunogenically inert systems that are able to bridge the gap between axons. The graft used for nerve autografts is harvested from the patients and undergo Wallerian degeneration to provide a supportive architecture for axon regeneration. Schwann cells can be grafted onto the autograft to provide a basement membrane so extracellular matrix proteins like laminin and fibronectin can survive.12,17

Although nerve grafts are nonimmunogenic and can bridge gaps larger than 3 cm long, sensory information is usually lost when this technique is implemented and donor site morbidity raises a huge concern for the health of the patient.

Nerve Allograft A promising alternative are nerve allografts. Nerve allografting is a technique used to transplant a nerve from one individual to another from the same species.

Allografts require an immunosuppressive treatment to help prevent rejection of the graft inside the body. Allografts can also be combined with bone marrow aspiration (BMA) to help catalyze the axon regeneration process. There are numerous advantages to nerve allografts compared to other nerve repair techniques including: lack of donor site, unlimited length of nerve tissue, patient specific nerve type, used for gaps larger than 3

14 cm. 12,18 The only limitation to this technique is the potential side effects that could result from a host immune response if the allograft is not properly immunosuppressed.

Due to the overwhelming advantages present for allografts the nerve graft used for imaging in this project was an FDA approved nerve allograft from a tissue engineering company. This graft is a commercially available decellularized, pre- degenerated and sterilized extracellular matrix processed from donated human cadaver peripheral nerve tissue. Many cellular and non-cellular factors such as blood and axons have been removed to inhibit immunogenicity. The three-dimensional scaffold along with the basal lamina structure are all preserved allowing vital proteins including collagen, laminin, fibronectin, and proteoglycans to be retained. This nerve graft is special in that it is completely decellularized meaning there are no residual cells or cellular debris before implantation. Decellularized grafts have shown reduction in panel reactive antibodies (PRA) which decrease antigenicity and also provide the capacity to recellularize thus increasing the durability of the graft.19,20

Bone Marrow Aspirate Concentrate The nerve graft will be supplied with autologous Bone Marrow Aspirate

Concentrate (BMC). BMC is the concentrated form of bone marrow aspirate (BMA) and is prepared by centrifugation of the BMA. The centrifugation step is critical in that it forces the heavier red blood cell (RBC) layer to the peripheral and the lighter cell fractions to be concentrated in the center. The lighter cell fractions containing serum, lighter platelets, and connective tissue progenitor cells (CTPs) are then aspirated off of the top of the red cell mass and the BMC sample is created.21 In this study the concentration process reduces the total BMA volume from 60 mL to 7 mL. A more detailed process of how the BMC was prepared for this study is described under the

15 protocol section. BMC is advantageous compared to BMA due to the increase in concentration of nucleated cells (including connective tissue progenitor cells) and the decreased number of red blood cells (RBCs). This allows a greater number of cells and

CTPs to be implanted in a given volume.22 BMC allows cells of value to be collected while eliminating cells that may not contribute to the regenerative process. 23

BMC is part of the bone marrow that has many growth factors and anti- inflammatory proteins.24,25 BMC also has an increased concentration of one essential ingredient, connective tissue progenitor cells. (CTPs) CTPs are defined as a heterogenous population of proliferative cells that can differentiate into connective tissue phenotypes.

26,27 This term describes the cells as not a uniform population, but rather a combination of stem and progenitor cells from native tissues. This diversity allows the collection of multipotent stem cells that are capable of self-renewal as well as semi-mature cells that have already committed to various lineages and cannot exhibit self-renewal qualities. 27 It has also been suggested that CTPs contain cells that are localized in the quiescent state

(G0) and thus have similar self-renewal and regeneration capabilities found in stem cells.28 Previous research using bone marrow aspirate concentrate prior to allografting has shown significant improvements in the quality and speed of regeneration. 29–31 This can be attributed to the increased concentration of CTPs in the wound. The increased number of CTPs catalyzes the formation of colonies and eventual regeneration of cells.

The ability to concentrate and characterize CTPs easily in image applications is also one of the key advantages of using BMC. 31

Z-Stack Projection Image Technology The concept behind creating the new imaging protocol is the Z-Stack projection.

Z-stack projection is an image acquisition method in which multiple images are taken at

16 different locations on the z-axis and merged together to produce a more focused image with a great depth of field. The depth of field (DOF) is the thickness of the plane of focus.32 In simpler terms, the depth of field is the distance between the closest and farthest objects that are in focus. When using a high magnification microscope, the lens has a high numerical aperture value (NA) and allows more light but decreases overall

DOF. This relationship can be described by the Shilaber equation.

Equation 1. Shilaber equation. NA represents numerical aperture value. n represents reflective index of medium, λ represents the wavelength of light.

So, in order to increase the overall DOF, a stack of focused images is taken along the z-axis and termed as a z-stack. Each focused image still has out-of-focus information and so a z-stack projection is done using a mathematical software that eliminates out-of- focus regions and combines all the images to one 2D z-stack projection image.32 This final image will contain all the focused parts of each z-stack and illuminate the cells using cell segmentation features. This process is explained in Figure 3. 33

In this experiment, the z-stacking

technique is administered using a Leica

DM6000B microscope along with Fiji

and ImageJ software. Since DAPI

staining will be used at a magnification

of 10x, 460 nm will be the wavelength

and the numerical aperture value will be

.25.32 When these values are plugged Figure 5. Individual Z-stack images with out- of-focus regions eliminated and combined to into the Shilaber equation a DOF of form projection image. 7.126 micrometers is achieved. This means that when using standard imaging techniques at 10X the DOF is only 7.126 microns thick. Any information above or below this region will be out of focus. This complication can be solved by creating a z-stack projection image which will increase the

DOF greatly and allow normally out of focus regions to come into focus.

Camera used for Image Acquisition

The camera used for this experiment was the Retiga 2000R charged coupled device (CCD) Camera. This camera allows for a precise quantitative analysis in low light fluorescence imaging of biological molecules. Since this camera will be used for low light applications it is imperative to limit image noise which is caused by electrical interference. The Retiga camera used has a large sensor (1/1.8 inches) to collect more photons and produce a signal with much less noise compared with a camera fitted with a small sensor. 34 The Retiga camera also takes advantage of a large aperture size to maximize the number of photons hitting the sensor. A unique feature of this camera is the

18 ability to expose from 10 microseconds to 17.9 minutes, which is adequate for fluorescent imaging. Combining these various traits, the Retiga 200R CCD camera is a viable camera choice for fluorescent imaging of cells on nerve allografts.

Stain used for fluorescent imaging

The type of stain used in this experimentation is a fluorescence stain.

Fluorescence is a three-stage process and works by first absorbing high energy light. This causes electrons in the fluorescence molecule to raise from the ground state to an excited state. These electrons quickly lose a little bit of energy due to the vibration of molecules and relax to a lower energy level. The electrons eventually drop from the first singlet level to ground level and emit light by releasing all of the stored energy of the photon.

Since some of the energy has been lost as heat under the vibration of molecules the energy of the light emitted is always lower than that of the energy absorbed. 35,36 Figure 7 describes this process under a jablonski diagram.

Figure 6. Jablonski diagram for fluorescence molecules.36

In this experiment, a blue-fluorescent 4',6-diamidino-2-phenylindole (DAPI) nucleic acid stain is used. DAPI stains double-stranded deoxyribonucleic acid (dsDNA) and associates with the adenine-thymine (AT) bonds in the minor groove of DNA. The displacement of water molecules from the attachment of DAPI and the minor groove causes a ~20-fold increase in fluorescence enhancement. 37 DAPI was excited with a 405 nm wavelength light and later absorbed at a wavelength of 460 nm.

Variables and Goals of Experiment There are several variables this project will be manipulating to allow testing for the newly developed z-stack projection image protocol. The first variable that will be manipulated is the type of rinse that is used when preparing the nerve graft. Either a 15- minute soak with 1X HBSS with Calcium and Magnesium will be used or three separate

(3X) 10-minute soaks with 1X HBSS with Calcium and Magnesium will be used.

The other variable of the experiment is the length of time the grafts interact with the bone marrow aspirate concentrate. These times are either 10 minutes, 30 minutes, or

60 minutes. The purpose of manipulating these variables is to better prepare the nerve graft for optimal CTP retention and proliferation.

Pictures will be taken of the graft at various time points of incubation and qualitatively analyzed for cell proliferation. Lastly, a final 2D z-stack projection image will be produced to visualize cell adherence and proliferation on the growth of the allograft. This image will be taken by following the newly developed z-stack projection image protocol.

Materials and Methods BMC protocol With Institutional Review Board approval, bone marrow aspirate (BMA) was obtained from 17 healthy volunteer donors (mean age 32 years old, range 22 to 40 years old; 13 male and 4 female). Bone marrow aspirates (BMA) were obtained from the iliac crest of these 17 patients. Bone marrow aspirate concentrate (BMC) was manually prepared with 8 samples. In these, the heparinized aspirate was placed into a 50 ml tube,

(Falcon 2098) containing 20 ml of α-MEM (Gibco, #11900-073) with 2U/ml heparin and centrifuged at 400 x g for ten minutes. After centrifugation, the buffy coat was manually isolated and suspended in 20 ml of α-Minimum Essential Medium (α-MEM). The BMC then undergoes a cell counting protocol via a hemacytometer. 2 million cells are taken from the BMC to undergo fixation and would eventually follow the same protocol as the effluent sample. The rest of the BMC is stored for use in the nerve graft. Before the BMC can be placed onto the nerve graft, the graft must first be prepared.

The nerve graft is kept in a negative 80-degree Celsius freezer until it is ready for experimentation. Each graft is 30 mm in length and 5 mm in diameter. (Figure 7) Each nerve graft was cut into 12 pieces each with a length of 2.5 mm and a diameter of 5 mm.

The unused nerve graft pieces were placed back into the freezer for future use. The rest of the other nerve grafts were thawed using a Lactated Ringers + Dextrose Ringers 5% solution for 10 minutes in a petri dish at room temperature (23°C). The graft was saturated with either 3 separate 10-minute rinses or one 15-minute soak with 1X Hank’s

Balanced Salt Solution (HBSS) with Calcium and Magnesium. (Figure 8)

Figure 7. Nerve allograft. 30mm x 5 mm diameter provided in sterile packaging and stored at -80oC.

Hank’s balanced salt solution is an isotonic solution that helps maintain physiological pH and osmotic pressure. Calcium and Magnesium are divalent cations that are vital for cell protein activity. Using the balanced salt solution with calcium and magnesium helps the cells proliferate. HBSS is a versatile solution and has been used for washing cells before experiments, transporting cells, and diluting cells for counting. 39

Figure 8. Nerve Graft E1 and E2 after rinse. The left petri dish is nerve graft E1 and the right petri dish represents nerve graft E2. Both images were recorded after the HBSS rinsing.

After the rinse phase, the graft is incubated with Bone marrow aspiration concentrate for 10, 30, or 60 minutes. The graft plus cells were incubated at 37°C and placed on an orbital shaker set to 70 rpm, to provide gentle mixing of cells and graft.

After incubation, the graft is removed from the incubation dish and placed into a new 12- well plate containing growth media consisting of alpha-minimum essential medium Eagle

(a-MEM) with 10% fetal bovine serum, 1 U/mL penicillin, 0.1 mg/mL streptomycin, 10-8

M dexamethasone, and 50ug/mL ascorbate and is moved to the cell culture protocol. 40

(Figure 9)

Figure 9. Nerve Graft E1 and E2 at Day 0 of cell Culture. Nerve graft E1 (left) and E2 (right). Nonadherent effluent sample removed media, (2ml) placed into each chamber.

The nonadherent BMC is collected and placed into 15 mL Falcon tubes and labeled as the effluent sample. The volume of the effluent sample is recorded and a cell counting protocol is done via a hemacytometer. This cell count along with the initial cell count of the BMC sample allows the ability to calculate the number of cells loaded onto the graft. From this a cell loading efficiency percentage can be calculated and reviewed for analysis. After the cell counting protocol, the effluent sample was plated at a density of 500,000 cells per chamber and goes onto the cell culture protocol for effluent sample.

Cell Culture Protocol for Effluent Sample Effluent samples are incubated for 6 days with media changes every 2 days. The incubator is set at 37°C to mimic physiological temperature. The media used for the incubation media changes is similar to the growth media used before, but ascorbic acid

(vitamin C) is also added. Vitamin C is an important water soluble anti-oxidant that is vital for the growth of cells.41 This incubation time period was selected from previous studies conducted by the Muschler laboratory. A longer incubation time causes too much growth and CTP counts become inaccurate due to colonies merging together. An incubation time under 6 minutes does not represent enough growth to obtain accurate representation of the sample. During every media change the samples were viewed under a brightfield light microscope to qualitatively confirm cell growth. At day 6, cultures are fixed and harvested. (Figure 10 and 11)

Fixation of the effluent sample and BMC sample starts with a 2mL rinse of PBS per chamber and is rinsed 3 separate times to completely remove all media. 2mL of a 1:1

Acetone: Ethanol mixture is used per chamber for 10 minutes. The slides are left to air dry for several hours and are then imaged for CTP counts via a brightfield microscopy.

With the CTP counts of the original BMC sample and effluent sample, the total number of CTPs loaded on the graft can be calculated along with CTP loading efficiency. Now by having both the cell loading efficiency and CTP loading efficiency the selection ratio can be calculated.

Figure 10. Cell Culture at day 0 of BMC, effluent samples E1 and E2, and nerve graft E1 and E2. The first 2 slides are the original BMC sample, the next two slides represent effluent sample E1, the two slides after that show effluent sample E2. The nerve graft E1 is represented in the 7th slide and nerve graft E2 is shown in the 8th slide. All cells except nerve slides are plated at a density of 500,000 per chamber.

Figure 11. Cell Culture at day 3 of BMC sample, effluent samples E1 and E2, and nerve graft. The first 2 slides were the BMC sample, the next two slides represent effluent sample E1, the two slides after that show effluent sample E2. The nerve graft E1 is represented in the 7th slide and nerve graft E2 is shown in the 8th slide.

Cell Culture Protocol for Nerve Allograft The nerve graft is incubated in the same incubator as the effluent sample, but for longer time lengths (several weeks to 3 months). This was specifically done to imitate physiological conditions of the body and allow for a more complete cell proliferation on the graft. The cell growth and retention rates were imaged at various time periods. This was done by slicing a small portion of the graft and staining the graft with a DAPI stain

25 protocol (described further in report). After staining of the graft, a fluorescence light imaging microscope (Leica DM 6000B) was used to take 2D images of the top surface of the graft. The orientation of the graft was consistently kept the same from start to finish of the incubation protocol.

After the end of the incubation cycle, the nerve grafts are first rinsed using 1X

HBSS with Calcium and Magnesium to remove any excess media. Then 10% formalin is added to the graft and submerged for one day to acquire complete preservation. Formalin is used as a reagent is helps preserve and disinfect the graft. The following day, PBS is used again to rinse the graft.

DAPI Staining After fixation of the nerve graft, it is rinsed again thoroughly with phosphate buffered saline (PBS) to rehydrate the graft and maintain physiological pH. DAPI (4’, d- diamidino-2-phenylinodole) was prepared according to manufacturer’s instructions and a droplet of DAPI was placed onto each nerve graft. DAPI has the ability to pass through an intact cell membrane and stain A-T portion of DNA, providing a bright fluorescent of the nuclei when analyzed under a microscope using UV. The nuclei are emitted around

460 nm. The graft was left in the dark for 30 minutes to allow DAPI ample time to stain the entire graft. It was also crucial to keep everything in the dark as DAPI is highly photosensitive and photobleaching is a common problem associated with using fluorescence imaging.42 Once stained with DAPI, samples are covered with foil and minimal exposure to light is maintained for the rest of the experiment. After 30 minutes, the DAPI stain is removed and PBS is used again to rinse the cells and maintain the osmolarity of cells. The grafts are kept covered and protected from light and allowed to dry slightly before proceeding to z-stack imaging.

PBS is a buffer solution that maintains a particular level of pH. Along with being non-toxic to cells, PBS has been used for dilutions and rinse cycles. PBS and HBSS are both isotonic solutions and helps regulate the pH and osmolarity of the sample. One major difference between the two is that HBSS contains glucose and other vital inorganic molecules which would be beneficial for live cells. 43 Since staining revolves around cells that have already been fixed and are not alive, PBS can be used.

Z-stack Image Protocol The prepared DAPI stained graft is imaged using the Leica DM6000B fluorescence microscope. The first step before loading the graft onto the microscope is to create a force on top of the graft. This slightly compresses the graft, creating a more even surface topography. This force was created by placing another slide on top of the nerve graft and weighing the slide down with 6 pennies. The total downward force achieved was 0.2 Newtons.

Next, the graft is loaded onto the microscope stage and the Surveyor program is opened. A suitable pattern formation is created and loaded. Several other parameters are turned on including turbo scan cruise channel scanning, z-stack method, and multichannel scanning. The next step involves gathering the correct gain and exposure settings for optimal cell imaging. Every graft has different values for these two parameters, but on average the grafts have an exposure time of 50 ms and a gain level of

5 dB. This allows adequate, but not too much light to enter the lens off the microscope.

The step size is set at 2 microns and this value is used by the microscope to move 2 microns between each captured image.

The range of the z-stack is identified by calculating the minimum point of the z- plane where the graft goes out of focus and the maximum point of the z-plane when the

27 graft again goes back to out of focus. The maximum and minimum points are subtracted and the range is calculated. The midpoint where the microscope actually starts the z-stack image scan is calculated by dividing the range by 2 and adding it to the minimum z-axis position value.

Once all variables have been thoroughly calculated and checked, the graft undergoes the z-stack scan at 10x magnification and a stack of images are created. This stack is unprocessed still needs to be consolidated into one picture. This is done by using

ImageJ. By using the Stack image option on ImageJ all the pictures from the z-stack scan are combined together to form a z-stack image where photos taken at every z-plane can be analyzed.

The next step is to image process this newly created z-stack for cell identification and processing in ImageJ. By using a protocol established by Wes Bova in Dr.

Muschler’s laboratory the z-stack can be converted to one focused z-stack projection image.

After establishing the scale for the image, the z-stack can be processed. A thorough review of the z-stack is done to make sure there is no image tearing. The z-stack can then be converted to a single 2-dimensional (2D) image using the maximum intensity feature in imageJ. This projection method analyzes the neighborhood around each pixel and from the culminative list of the neighbors determines a maximum value. The image is then projected in a 2D format and each pixel is replaced with the maximum value for each neighborhood. The maximum intensity projection method allows for a clear view of the external section of the nerve allograft.44,45 The image obtained after this plugin is known as the max intensity image.

With the z-stack selected, a stack focuser plugin is run to create a focused 2D image from a stack of images at different focal planes. Several features of this plugin include finding edges of each image slice by running a median 3x3 filter to reduce noise, creating a map of how far the influence of a focused edge extends, and copies specific pixel values from the original image to the new image based on the maximum value threshold. 46 The final focused stack image will later be merged with the final nuclei segmented image.

Now that a focused 2D image has been established, nuclei segmentation can begin. To identify the nuclei of cells the max intensity image is selected and a normalized local contrast filter is run. This filter processes each pixel based on block-statistics, specifically the mean and standard deviation. 47 The filter ultimately produces an image with a more universal contrast to aid in nuclei identification. 48 A block radius of 40 x 40 and a standard deviation of 3 provided an image with the highest quality.

With the normalized local contrast image chosen, an adaptive threshold plugin is selected to segment all cells regardless of their respective intensities. The adaptive threshold plugin changes the threshold dynamically across the image, a crucial difference from traditional threshold plugins which use just one global threshold for all pixels. The adaptive threshold uses local pixel intensity and allows a clear segmentation of cells among a non-homogenous background.49 The plugin works by dividing the image into a matrix of overlapping sub images and finds the most optimal threshold within the specific sub image by analyzing its histogram. The chosen size of the pixel block should be large enough to cover enough foreground and background pixels, but also not large enough to impede the rule of uniform illumination.49,50 The chosen block size for nerve allografts

29 was 19 pixels. A global threshold plugin is run after the adaptive threshold plugin to further eliminate debris and parts of the graft that should not be segmented. By this step most of the cells on the nerve allograft have appropriately been segmented and analysis can be accomplished. 51

Using the newly thresholded image, analysis can be performed on the cells retained on the graft. The plugin works by scanning and finding edges of objects. ImageJ then outlines the object using a wand tool and measures the area of each object as well as the overall number of objects in the image. There is also an option to fill each object as a mask and produces a final image of just objects within the image with no background noise. Several parameters can also be manipulated to analyze the particles more accurately. The size field allows only objects within the size range to be analyzed and for this experiment a parameter range of 20 – infinity pixels^2 is used as the images were taken at 10x. The circularity field uses a formula of 4pi(area/perimeter^2) and the range used that produced the best image quality was 0.4-1.00. Anything outside of this range was ignored. 52 Since the default mask is in black and white and can be sometimes difficult to locate the image is inverted using invertLUT and colored green. Now that all the objects(cells) have been accurately identified and analyzed the focused stacked image can be merged with the green analyzed image using the function merge channels. This produces the final 2D z-stack projection image showing DAPI stained cells on the top surface layer of the nerve allograft with completed cell segmentation.

Some of the variables that we tried, but found to be inferior were having a adaptive threshold being less than 15 and above 20, having the gain above 5, using the low intensity threshold, and having a larger range of circularity.

Results and Discussion

After completing all the methods for the experiment, the cell retention, CTP retention, and selection ratios were compared between all graphs. Table 1 summarizes all the data.

Table 1. Overview table showing multiple variables and results of the study.

The identifier (ID) represents a unique date of experimentation and a number that represents a sequence of graft that was used. For each unique date of experimentation, a different BMA sample was used. The second column, type of sample, represents either unprocessed BMA or the concentrated form of BMA, BMC. The third column represents the sample and graft incubation time period. Samples either received 10 minutes, 30, minutes, or 60 minutes of contact with BMC. These values were chosen because 10 minutes to one hour provided adequate time for cells to adhere onto the graft. Previous testing showed that a BMC incubation time period of over 60 minutes has little to no advantage. The third column described the type of rinse used whether it was three

31 separate 10-minute rinses, or one 15-minute soak. The Cell Loading Efficiency

(calculated variable) was found by dividing the number of cells retained on the allograft after the rinse (measured variable) over the number of cells loaded onto the graft

(measured variable). A similar calculation was used for the CTP loading efficiency

(calculated variable) as well, except CTP counts via brightfield microscopy was used.

The selection ratio (calculated variable) is the CTP loading efficiency over the cell loading efficiency. In other words, it accesses the quality of the graft in how many CTPs it is able to retain. This means that the higher the selection ratio is, the higher the CTP content for the number of cells loaded. It is interesting to note that a graft can have low cell loading efficiency but still display a high selection ratio if the cells retained were mostly CTP. This can also be reversed and a selection ratio can be low even if there is a high number of cells retained due to a low CTP loading efficiency. So, a low selection ratio would indicate that most cells retaining on the graft are not CTP cells. A selection ratio greater than one indicates CTPs were slightly more likely to be retained than non- progenitor nucleated cells.40 A selection ratio of less than one indicates non-progenitor nucleated cells were slightly more likely to be retained than CTPs.

The data for the E1 graft appears to be an anomaly as it shows a negative CTP loading efficiency value. For this specific graft the number of CTPs for the effluent sample after the 6-day culture was higher than the CTP count for the initial BMC sample after the 6-day culture. This abnormality caused the cell loading efficiency to be negative.

Possible reasons for this happening cane be related to either an incorrect CTP count or a complication in the cell culture phase of the experiment.

From this data table three graphs comparing various variables were created. The creation of graphs helped visualize specific patterns among the graft.

Selection Ratio for BMC + nerve graft + 3X Rinse 4.00 N = 7 BMC samples 3.50 3.00 2.50 2.00 1.50

SELECTION RATIO SELECTION 1.00 0.50 0.00 10 30 60 BMC + GRAFT INCUATION TIME (MINUTES)

A2 B2 C2 D2 E2 F1 F2 F3 H1 H2 H3

Graph 1. Selection ratio of BMC infused grafts that have undergone three separate 1X HBSS rinses. Graph 1 shows the selection ratio for BMC incubated grafts that have undergone three separate 10-minute rinses. The selection ratios are compared among 10, 30, or 60- minute BMC + graft incubation times. From this graph it clear that as the BMC incubation time increases from 10 to 30 to 60, the overall selection ratio on average decreases. The average selection ratio for a 10-minute BMC incubation time is 2.09, a

30-minute BMC incubation time is 1.39, and 60-minute BMC incubation time is 1.44.

Selection Ratio for BMC + nerve graft + 15 minute Rinse 8.00 N = 2 BMC samples 7.00 6.00 5.00 4.00 3.00

SELECTION RATIO SELECTION 2.00 1.00 0.00 10 30 60 BMC + GRAFT INCUBATION TIME (MINUTES)

G1 G2 G3 H4 H5 H6

Graph 2. Selection ratio of BMC infused grafts that have undergone a 15-minute soak with 1X HBSS. Graph 2 shows the selection ratio for BMC incubated grafts that have undergone one 15-minute soak. The selection ratios were also compared among 10, 30, or 60-minute

BMC + graft incubation times. Graph 2 showed a similar trend as graph 1 with average selection ratios decreasing with increasing BMC incubation time. The average selection ratio for a 10-mintue BMC incubation time is 4.07, a 30-minute BMC incubation time is

3.16, and 60-minute BMC incubation time is 1.94.

Selection Selection Selection Ratio for 10 Ratio for 30 Ratio for 60 ID minutes minutes minutes A2 1.33 B2 1.31 C2 2.94 D2 3.34 E2 1.07 F1 3.17 F2 1.46 F3 1.31 H1 1.45 H2 1.33 H3 1.58 Average: 2.09 1.39 1.44 Variance 1.01 0.01 0.04 Table 2. Selection Ratio for grafts that went through 3X - 10-minute rinse

Selection Ratio Selection Ratio Selection Ratio ID for 10 minutes for 30 minutes for 60 minutes G1 7.18 G2 5.43 G3 2.55 H4 0.95 H5 0.89 H6 1.33 Average: 4.07 3.16 1.94 Variance 19.38 10.32 0.75 Table 3. Selection Ratio for grafts that went through 15-minute soak.

Table 2 and 3 show the data tables that were used to create the graph along with statistical analysis of the average and variance. From this data it is clear that the variances of selection ratios for the grafts that underwent the 3X – 10 min rinse were on average smaller than those grafts that endured the 15-minute rinse. This can partly be attributed to the small sample size of grafts in the 15-minute rinse. Low variances indicate that the

35 selection ratio data was similar to the mean and to other ratios, whereas larger variances indicate the selection ratios as far from the mean and each other.

From both these graphs it is clear that for both rinse types the overall selection ratio decreases with increasing incubation time. This indicates that incubating the nerve allografts for a longer period of time with BMC has no advantage in retaining CTPs over non-progenitor nucleated cells. A possible explanation for this phenomenon is that since

BMC is used there is a high number of concentrated cells so when the concentrated cells spend more time with the graft, oxygen and nutrition depletion start to occur after extended period of times. This may have caused cells that have originally attached to the graft to detach and look for other sources of nutrition and may have adhered to other cells or the plastic petri dish. Any of these variables could have caused the drop in selection ratio as the incubation time increases. However, it is important to note that when running a T-test: two-sample assuming unequal variances among the various samples all the p- values were over 0.05. This indicates that the results were not statistically significant and are rather trends.

A separate graph was used to compare the type of rinse and see if the cell loading efficiency, CTP loading efficiency, or selection ratio changed with rinse type.

Graft H vs different efficiency ratios

H6 H5 H4 H3

NERVE GRAFTS NERVE H2 H1

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 EFFICIENCY AND RATIO PECENTATAGES (%)

Selection Ratio CTP Loading Efficiency (%) Cell Loading Efficiency (%)

Graph 3. This graph shows Graft H only and its representative selection ratios, CTP loading efficiencies, and cell loading efficiencies Graph 3 represents only Graft H and its representative efficiency and ratio percentages. H1-H3 were grafts rinsed with three separate 10-minute rinses of 1X HBSS with Calcium and Magnesium. H4-H6 showed grafts rinsed with one 15-minute soak of

1X HBSS with Calcium and Magnesium. H1 and H4 had a BMC incubation time of 10 minutes. H2 and H5 had a BMC incubation time of 30 minutes. H3 and H6 had a BMC incubation time of 60 minutes. Graph 3 allows the ability to compare between different rinse cycles. When looking at graft H1 and H4, both of which had a BMC incubation time of 10 minutes, the selection ratio, cell loading efficiency, and CTP loading efficiency were all higher for H1. When comparing grafts with a BMC incubation time of

30 minutes, H2 and H5, a similar trend was observed with all three ratios and efficiencies being higher for the H2 graft instead of the H5 graph. For the 60-minute BMC incubation period, H3 and H5 were compared. Although the CTP loading efficiency and cell loading efficiency was higher for the H6 graft, the overall selection ratio still remained higher for

H3. This demonstrated again that by employing multiple rinses the likelihood of CTPs

37 retaining over non-progenitor cells is increased. Since, H1, H2, and H3 all were rinsed three separate times for 10 minutes, the data indicates rinsing opposed to just a 15-minute soak might prove more beneficial for retaining CTPs over non-progenitor cells.

A similar T-test: two-sample assuming unequal variances was run among the various samples and all the p-values were again over 0.05. This indicates that the results were not statistically significant and are rather trends.

Nerve Graft Analysis During this time, nerve grafts were kept in incubation for several weeks and imaged via 10X fluorescence microscopy at various time points. A total of 8 different grafts were imaged using the standard imaging procedure and 2 unique grafts were imaged using the newly developed z-stack projection image protocol. In the next section images of these grafts will be displayed and compared.

C2 Graft The C2 graft was administered using the BMC sample with a sample + matrix incubation time period of 10 minutes. The type of rinse used for this particular graft was three-separate 10-minute rinses.

Figure 12. (A) represents the graft at 3 days of incubation. (B) represents the graft at 6 days of incubation. (C) represents the graft at 14 days of incubation. (D) and (E) are brightfield images acquired of the graft after 3 weeks of incubation via a light microscope. Images without nuclear segmentation, illustrating cell retention onto the allograft nerve surface. Retained CTPs have proliferated to generate progeny (daughter cells) that populate the entire surface of the graft. Figure 12. (A), (B), and (C) were images taken after 3, 6, and 14 days of incubation respectively via the current fluorescence imaging technique after proper DAPI staining. (D) and (E) are brightfield images of the nerve graft after 14 days. Image (A),

(B), and (C) all show very little cell retention, but the common theme among these images is the poor resolution of the graft. Image (A) shows a properly focused cell attachment in the middle of the image, but other parts of the graft are not in focus because they are out of the DOF range for a 10x microscope. A similar problem is visible for images (B) and (C) as well. In image (C) the bottom part of the image is clearly out of focused and the problem can be related back to the graft being at a different z-plane than

39 the DOF. That is why the z-stack projection image is necessary as it increases the DOF greatly allowing portions of the graft normally bleary to become focused.

Since present imaging techniques do not provide an adequate image of cell retention, brightfield images of the graft were taken to see if cells were really attaching to the graft. As shown on Figure 12, 3 weeks of incubation provided sufficient time for cell growth. (D) demonstrates a “web-like” structure forming on the side of the graft demonstrating cell proliferation. (E) further confirms cell proliferation as the cells are seen growing on the outer edges of the graft. Although brightfield images revel cell proliferation on the outer edges of the graft, the actual graft itself appears black due to its thick architecture. This inhibits clear imaging of cell attachment within the graft. A z- stack projection image would be able to resolve these issues and provide a much higher resolution of the graft with cell attachment.

E1 Graft The E1 graft was administered using the unprocessed sample with a sample + matrix incubation time period of 10 minutes. The type of rinse used for this particular graft was three-separate 10-minute rinses.

Figure 13. (A) E1 graft at 6 weeks of incubation. (B) E1 graft at 11 weeks of incubation. Images without nuclear segmentation, illustrating cell retention onto the allograft nerve

40 surface. Retained CTPs have proliferated to generate progeny that populate the entire surface of the graft Figure 13. (A) and (B) were images taken after 6 weeks and 11 weeks of incubation respectively via the current fluorescence imaging technique after proper DAPI staining. (A) shows more cell retention then images acquired form the C2 graft, but number of cells retaining on the graft still cannot be accurately determined due to the right and left side of the image being out of focus. Again, the uneven topography produced by the valleys and dips of the graph make it difficult for cells in those regions to appear in the DOF. Image (B) shows much more cell retention confirming the growth of cells in the graft, but the image becomes unfocused towards the bottom. The unfocused regions prevent accurate quantitative analysis of the graft and other than confirming cell proliferation from 6 weeks to 11 weeks no other conclusions can logically be shown due to the low resolution of the images. It makes sense for the graft to display higher cell retention for image (B) compared to image (A) because cells would have more time to incubate and grow providing a higher number of cells to be imaged.

E2 Graft The E2 graft was administered using the BMC sample with a sample + matrix incubation time period of 10 minutes. The type of rinse used for this particular graft was three-separate 10-minute rinses.

Figure 14. (A) E2 graft at 6 weeks of incubation. (B, C) E2 graft at 11 weeks of incubation. Images without nuclear segmentation, illustrating cell retention onto the

41 allograft nerve surface. Retained CTPs have proliferated to generate progeny that populate the entire surface of the graft. Figure 14. (A), (B), and (C) were images taken after 6 weeks, 11 weeks, and 11 weeks of incubation respectively via the current fluorescence imaging technique after proper DAPI staining. (A) shows a large number of cells retaining on the graft in the central portion of the photo. Cells also appear to be retained on the right side of the image as well, but with a lowered focus. The portion of graft located on the left side of image

(A) appears to be so out of focus that cell adherence is not visible. A z-stack projection image would eliminate this issue and provide a much more comprehensive view of the allograft. Images (B) and (C) both show tremendous amount of cell proliferation and cell attachment is much more clear than previous images. In all these images, there is still room for improvement as the right side of image (B) and bottom part of image (C) starts to become unfocused as the graft dips back into a region outside of the DOF. By using a z-stack projection image and increasing the DOF these cells would be clearly focused and a quantitative analysis could be performed.

When comparing the E2 graft (Figure 14) with the E1 graft (Figure 13) the only distinction is the type of sample, but even with current imaging techniques it is possible to qualitatively tell the difference. At the same time point, 6 weeks, image (A) from E2 expresses a greater amount of cell proliferation then image (A) from E1. This same pattern is followed for the 11-week sample. The contrast in the number of cells proliferating between the two grafts is justified because BMC is a concentrate of BMA with usually higher CTP prevalence. This means that it is expected for grafts infused with

BMC to have higher cell proliferation rates than grafts infused with unprocessed BMA.

F2 Graft The F2 graft was administered using the BMC sample with a sample + matrix incubation time period of 30 minutes. The type of rinse used for this particular graft was three-separate 10-minute rinses.

Figure 15. A) F2 graft at 5 weeks of incubation. (B) F2 graft at 10 weeks of incubation. Images without nuclear segmentation, illustrating cell retention onto the allograft nerve surface. Retained CTPs have proliferated to generate progeny that populate the entire surface of the graft. Figure 15. (A) and (B) were images taken after 5 weeks and 10 weeks of incubation respectively via the current fluorescence imaging technique after proper DAPI staining. Image (A) shows poor resolution when imaging with current imaging techniques. Most of the graft appears out of focus, with only a few cells appearing in the center portion of the image indicating some cell attachment. An image like (A) is commonly found when grafting allografts using this technique and provides little valuable information to the research community. Image (B) shows the vast improvement in cell proliferation as the incubation time increases from 5 to 10 weeks. Much of the graft is in focus and cells are clearly visible, but in the bottom right of the image the graft descends into a valley just a few microns deep and this is enough to kick that part of the graft out of the DOF and producing an out of focus section in the image. An out of focus

43 section will not appear on a z-stack projection image because the mathematical software used to construct the projection image will eliminate all out-of-focus regions.

F3 Graft The C2 graft was administered using the BMC sample with a sample + matrix incubation time period of 60 minutes. The type of rinse used for this particular graft was three-separate 10-minute rinses.

Figure 16. (A) F3 graft at 5 weeks of incubation. (B) F3 graft at 10 weeks of incubation. Images without nuclear segmentation, illustrating cell retention onto the allograft nerve surface. Retained CTPs have proliferated to generate progeny that populate the entire surface of the graft. Figure 16. (A) and (B) were images taken after 5 weeks and 10 weeks of incubation respectively via the current fluorescence imaging technique after proper DAPI staining. Image (A) shows a quality focused portion of cells, but the rest of the graft quickly goes out of focus since it is again not in the DOF. The increase in incubation time continues to express greater cell proliferation as shown in image (B). However, like in previous images, image (B) has cells that go out of focus and large sections of the graft are not clearly visible.

Comparing F2 (Figure 15) and F3 (Figure 16), the only change is the sample + matrix incubation time period changing from 30 minutes (F2) to 60 minutes (F3). From

44 these images alone, it appears that 60 minutes allows for more cell retention and proliferation than 30 minutes as a greater number of cells are visible among both time points for F3 than in F2. When taking a look at the data table, however, the selection ratio is higher for F2 even with less cell loading efficiency. This can be contributed to the higher CTP loading efficiency for F2. This further proves the importance of calculating the CTP loading efficiency. Even though cell retention appeared high for F3, it does not mean that a high number of CTPs will be present on the graft and it’s important to compare not only cell retention values, but CTP loading efficiencies as well. Comparing both these values using the selection ratio provides a more holistic and thorough analysis of what type of cells are adhering to the graft.

Week 3 (G1, G2, G3 graft) All three G1, G2, and G3 grafts were administered using the BMC sample. The type of rinse used for all three grafts was the 15-minute soak. G1 had a sample + matrix incubation time of 10 minutes, G2 had a sample + matrix incubation time of 30 minutes, and G3 had a sample + matrix incubation time of 60 minutes.

Figure 17. (A) G1 graft with 10-minute incubation time. (B) G2 graft at 30-minute incubation time. (C) G3 graft at 60-minute incubation time. Images without nuclear segmentation, illustrating cell retention onto the allograft nerve surface. Figure 17. (A), (B), and (C) were images of G1, G2, and G3 respectively taken after 3 weeks of incubation via the current fluorescence imaging technique after proper

DAPI staining. Image (A) and Image (B) show very little cell attachment as most of the

45 image remains out of focus. Large sections of the graft are not clear and even the small focused regions around the center of the image do not indicate high cell adherence as they are not focused enough for proper image analysis. Image (C) shows increased cell attachment when compared with image (A) and image (B). Although the attachment of cells seems to be higher for 60 minutes, it is still important to evaluate the CTP prevalence as mentioned in the previous section.

The lower visibility of cell retention among these three grafts may also be contributed to the use of a 15-minute rinse instead of three separate 10-minute rinses.

These images further prove that preparing the graft with separate rinses is more beneficial in retaining cells than using a one-time 15-minute soak.

Z-stack projection image of D1 The D1 graft was administered using the unprocessed BMA sample with a sample

+ matrix incubation time period of 10 minutes. The type of rinse used for this particular graft was three-separate 10-minute rinses.

Figure 18. D1 graft imaged using the z-stack projection image acquisition protocol. (B) D1 graft viewed at a different angle using the new z-stack protocol. Two Z-Stack image projections from a single nerve allograft. This graft was loaded with unprocessed BMA cells using a 10-min incubation time and 3 separate washing steps, and then cultured for six weeks. The nerve was stained using DAPI. Nuclei are detected using fluorescence at 405 nm excitation and 460 nm emission. Only cells on the top surface of the graft with a direct light path for excitation and emission are visualized. Segmented nuclei are artificially colored green in this image. Cells on the contralateral surface and any cells that may be internal within the graft are not visualized due to optical opacity of the nerve tissue. Figure 18. (A) and (B) were images taken after 6 weeks of incubation respectively via the newly created z-stack projection image protocol after proper DAPI staining. The biggest difference witnessed from using this new protocol is the high level of acuity achieved at a greater depth of the graft. A total of 83 and 71 separate slices were used for image (A) and image (B) respectively. This means that The DOF has increased to 166 microns for image A and 142 microns for image B since a step size of 2 was used. Since

47 the nerve graft has a diameter of 5 mm, these images show cells on the top 3.2% of the nerve graft surface in image A and 2.8% of the nerve graft surface in image B. The increased DOF provides a more integrated view of the surface of the graft and the use of cell segmentation software to highlight the DAPI nucleated cells allows for the potential of cell counting software to be used. It is quite clear from these images how proliferative the cells were at just 6 weeks on the surface.

Cells do not appear plentiful in the top right section of the graft and this may be contributed to the use of unprocessed BMA instead of BMC. The lowered concentration of CTPs make it more difficult for the cells to proliferate the graft as quickly as a graft injected with BMC. This difference will be analyzed and compared further in the next section.

Z-stack projection image of D2 The D2 graft was administered using the BMC sample with a sample + matrix incubation time period of 10 minutes. The type of rinse used for this particular graft was three-separate 10-minute rinses.

Figure 19. (A) D2 graft imaged using the z-stack projection image acquisition protocol. (B) D2 graft viewed at a different angle using the new z-stack protocol. Two Z-Stack image projections from a single nerve allograft. This graft was loaded with unprocessed BMA cells using a 10-min incubation time and 3 separate washing steps, and then cultured for six weeks. The nerve was stained using DAPI. Nuclei are detected using fluorescence at 405 nm excitation and 460 nm emission. Only cells on the top surface of the graft with a direct light path for excitation and emission are visualized. Segmented nuclei are artificially colored green in this image. Cells on the contralateral surface and any cells that may be internal within the graft are not visualized due to optical opacity of the nerve tissue. Figure 19. (A) and (B) were images taken after 6 weeks of incubation respectively via the newly created z-stack projection image protocol after proper DAPI staining. A total of 117 and 171 separate slices were used for image (A) and image (B) respectively.

This means that The DOF has increased to 234 microns for image A and 342 microns for image B since a step size of 2 was used. Since the nerve graft has a diameter of 5 mm, these images show cells on the top 4.6% of the nerve graft surface in image A and 6.8%

49 of the nerve graft surface in image B. Both these images show high cellular and graft focus. The z-stack projection image not only correctly identifies the cells, but also disregards highlighting debris and graft material. The long strand of black debris in the middle of the graft is not identified as a cell in either image which further proves the accuracy of this technique. In image (B) graft material can be seen close to the central region of the image and this also is not identified by the new technique.

Both images also display cellular retention throughout the entire graft providing more evidence for the use of BMC injection instead of unprocessed BMA. From the z- stack projection image, cells seem to be more in focus and retained on the outside peripheral surface of the graft compared to the inside surface of the graft. This may indicate that the graft may need further modifications in the center to allow a more distributed flow of cell attachment.

Conclusions and Further Directions: The overall objective of creating a z-stack projection image protocol for nerve allografts has been successfully accomplished through this project. Not only was a z- stack created, but a clear projection image of the z-stack with cell segmentation using

ImageJ was utilized and produced. This new protocol can now be used for imaging nerve allografts for future clinical and research studies.

From administrating cell culture and implementing cell and CTP counting techniques the overall selection ratios for all 22 nerve grafts were calculated. From this data, the overall selection ratio on average decreased from 10 minutes to 30 minutes to 60 minutes and proved that 10 minutes of BMC interaction time with the nerve graft is adequate for proper cell and CTP proliferation. While the power of these observations could define 10 minutes as “optimal”, there was no evidence to suggest that longer periods of incubation were necessary to achieve cell and CTP attachment and retention on the nerve tissue. Shorter periods of incubation were not tested.

The study also proved that by soaking the nerve graft in 1X HBSS for 10 minutes

3 separate times it resulted in a trend towards higher selection ratios regardless of the

BMC incubation time. Therefore, a nerve graft prepared with three separate 10-minute

1X HBSS rinses along with a BMC incubation time of 10 minutes provides the highest likelihood of retaining CTPs over non-progenitor nucleated cells. Again, this data represents a trend and is not statistically significant.

So far in this study none of the grafts were cut open so only the surface of the graft has been imaged. A further step in this project would be to utilize histological assessment to determine the extent to which cells and CTPs that are initially retained on the surface

51 of a nerve allograft will invade radially into the substance of the allograft. This can lead to a better insight on how cells react with the nerve and if there are any particular defects associated with the nerve.

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