Decellularization to Produce Biological Synovial Extracellular Matrix Scaffolds THESIS Presented in Partial Fulfillment of the R

Decellularization to Produce Biological Synovial Extracellular Matrix Scaffolds

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Nathalie Ann Reisbig

Graduate Program in Comparative and Veterinary Medicine

The Ohio State University

2016

Master's Examination Committee:

Alicia Bertone, DVM, PhD, DACVS, DACVSMR (advisor)

Maxey Wellman, DVM, MS, PhD, Diplomate ACVP

Matthew Brokken, DVM, MSc, DACVS, DACVSMR

Abstract

The objective of this study was to evaluate four methods to generate a decellularized synovium scaffold (SynECM) for use as a biologic transplant. Villous synovium was harvested and frozen (-80oC) from the femoropatellar and medial femorotibial joints of four adult normal horses <7 years of age. Fresh-thawed equine stifle synovial tissue was decellularized by four methods: 1) 0.1% peracetic acid (PAA) solution (1XPAA), 2) PAA treatment repeated (2XPAA), 3) 1% Triton X followed by DNAse (Triton), and 4) 2M

NaCl followed by DNAse (NaCl). Tissue from each method was evaluated for morphology (histologic, scanning electron microscopy), viability (culture and exclusion staining) and decellularization efficiency (presence of residual cells, DNA content, and

DNA fragmentation). All four methods resulted in non-viable synovial extracellular matrix scaffolds. Single PAA treatment retained synovium villous matrix integrity but with excess cell residue containing high cellular DNA content and DNA fragments >

25,000 base pairs (bp). 2XPAA treatment also had retained matrix integrity, but low

DNA content with short DNA fragments (< 300 bp). The Triton and the NaCl preparations damaged villous structure leaving little to no discernible synovial villi, no identifiable residual cells and short (<300 bp) DNA fragments. The results indicated that two serial treatments of 0.1% PAA was the best method evaluated for obtaining SynECM ii with desirable morphology and low DNA content. An additional advantage of 2XPAA preparation is that PAA sterilizes ECMs during the decellularization process.

iii

Acknowledgments

The author wishes to thank Dr Becky Lovasz for technical assistance.

Vita

2002...... Diploma, Ringstabekk Skole, Oslo, Norway

2004...... International Baccalaureate, Oslo, Norway

2012...... Vet. Med. Leipzig, Germany

2013 to present ...... Resident, School of Veterinary Medicine,

The Ohio State University

Publications

Prospective Randomized Blinded Clinical Trial Evaluating Dental Pulp Treatment for Equine Lameness. Alicia L. Bertone, DVM, PhD, DACVS, DACVSMR; Nathalie Reisbig, MedVet; Allison H. Kilborne, DVM; Navid Salmanzadeh, DVM; Rebecca Lovasz DVM; Mari Kaido, BVSc; Lisa J. Zekas, DVM, DACVR; Matthew Brokken, DVM, MSc, DACVS, DACVSMR; Joy Sizemore BSc; Logan Scheuermann BSc. NAVRMA Abstracts; 164, 2015

Objective Gait Analysis in Naturally Lame Horses. Mari Kaido, BVSc; Allison H. Kilborne, DVM; Joy Sizemore, BSc; Nathalie Reisbig, MedVet; Turi Aarnes, DVM, ACVA; Alicia L. Bertone, DVM, PhD, DACVS, DACVSMR. ACVS Abstracts; 2015.

Prospective Randomized Blinded Controlled Clinical Trial for the Injection of Living Dental Pulp Cell Particles for the Treatment of Equine Lameness Conditions Alicia L. Bertone, DVM, PhD, DACVS, DACVSMR; Nathalie Reisbig, MedVet; Allison H. Kilborne, DVM; Navid Salmanzadeh, DVM; Rebecca Lovasz DVM; Mari Kaido, BVSc; Joy Sizemore BSc; Logan Scheuermann BSc; Lisa J. Zekas, DVM, DACVR; Matthew Brokken, DVM, MSc, DACVS, DACVSMR. ACVS Abstracts; 2015.

Characterization of Living Synovial Extracellular Matrix Scaffolds for Gene Delivery Reisbig N, Hussein H, Pinnell E, Bertone AL. ACVS Abstracts; 2015.

Decellularization to Produce Biological Synovial Extracellular Matrix Scaffolds. NA Reisbig, H Hussein, E Pinnell, AL Bertone. European College of Veterinary Surgeons Annual Abstracts, Berlin, 2015. v

Fields of Study

Major Field: Comparative and Veterinary Medicine

Table of Contents

Abstract ...... ii

Acknowledgments...... iv

Vita ...... v

Publications ...... v

Fields of Study ...... vi

List of Tables ...... ix

List of Figures ...... x

Chapter 1 Introduction ...... 1

Chapter 2 Materials and Methods ...... 5

Decellularization and Creation of Scaffolds (SynECM) ...... 6

SynECM Characterization...... 8

Quantification of Residual Living Cells ...... 8

Histological Evaluation ...... 8

Scanning Electron Microscopy (SEM) ...... 10

DNA Isolation and Quantification ...... 10

vii

Gel Electrophoresis ...... 10

Data Analysis ...... 11

Chapter 3 Results ...... 12

Chapter 4 Discussion ...... 22

Footnotes ...... 27

References ...... 28

viii

List of Tables

Table 1. The methods used for decellularizing synovium...... 7

Table 2. Histological criteria scale (0-4) for the decellularized synovium and control ...... 9

Table 3. Overview of results ...... 14

List of Figures

Figure 1. Hematoxylin and eosin staining ...... 15

Figure 2. Microscopy of hematoxylin-eosin and picrosirius red stained SynECMs...... 16

Figure 3. Scanning electron microscopy (280x) of the SynECMs...... 18

Figure 4. Histomorphological scoring of control synovium and SynECMs ...... 19

Figure 5. DNA content of SynECMs ...... 20

Figure 6. DNA gel electrophoresis of SynECMs...... 21

Chapter 1 Introduction

Decellularized extracellular matrices (ECMs) have been of intense interest in regenerative research for use as tissue or organ replacement1 and in reconstructive surgical procedures.2 The field of decellularizing ECM has grown and developed to include tissues spanning from decellularized sheaths (skin, esophageal mucosa3, small intestinal submucosa4, human umbilical vein) to full organ decellularization (liver, heart5, lung6). ECM is composed of the molecules secreted by the resident cells in the tissue thereby providing a biologic matrix with the potential to retain the ECM 3-dimensional structure and composition that supports the cell of original phenotype. The ECM functions as a medium for signal transfer to and between cells, influencing the proliferation and migration of resident cells to express the tissue phenotype.7, 8 The ECM is regarded as being in a state of dynamic equilibrium8 that is central to normal tissue and organ development.9 Decellularized ECM with homologous transfer should readily integrate into the natural biologic turnover of ECM for that tissue10.

Generating decellularized tissue requires a process that removes cells and antigenic components, yet retains the constituents of 3-D structure and composition, without residual toxicity, such that the ECM promotes the residence and growth of homologous cells that could be transplanted without adverse reaction.11 Numerous studies have shown

1 that the method of decellularization affected the properties of an ECM.12 Fortunately, most ECM components are relatively conserved across mammalian species reducing the risk of a host immune response to allogenic or xenogeneic ECM,13,14 however it has been shown that insufficient removal of cell components can be a source of inflammation and contribute to poor results after implantation.15 To avoid adverse reactions16 from ECMs, guidelines have been recommended for the maximum amount of residual cells (lack of visible nuclear material), DNA (<50 ng dsDNA per mg ECM dry weight) and base pair

(bp) fragment length (<200bp DNA ). The focus on nucleic material when evaluating decellularized tissue is paramount as it is directly correlated to adverse host reactions.16

Many decellularization agents and protocols or combinations of agents have been published.16 Methods range from mechanical, agitation and pressure, to a multitude of chemical agents including acids and bases, hypo- and hypertonic solutions and detergents.1 Biological agents, like enzymes, have been found to incompletely remove cell fragments, but are a good adjunctive treatment. There is concern that enzyme residues may cause adverse reactions in vivo, although this is a concern for most of the decellularizing agents.

Cartilage ECM has not worked satisfactorily as a structural scaffold for the repair of cartilage in vivo.17 Synovium, an integral part of diarthrodial joints, could be an alternative to cartilage ECM. The synovium, has 2 distinct layers, an intima and a subintima. The subintima consists of capillaries, with a deeper plexus of small arterioles and venules. The connective tissue matrix in normal synovium includes a fine fibrillar matrix with a few type I collagen fibers in the intima, beneath which is a layer relatively

2 rich in type I collagen which forms the physical membrane. The deepest area of the normal synovium consists of a loose connective tissue layer.18

A known important function of the synovium is the interaction with cartilage, including chondrocytes, for both regenerative and degenerative processes 19,20. Synovium contains highly metabolic, relatively hardy cells that readily proliferate and secrete joint restorative substances such as hyaluronan, growth factors and Interleukin-1 receptor antagonist (Il-1 ra).19,21 The loose synovium ECM could be used either as a direct transfer of chondrocytes (not studied to date) or as a living transplant with synovial cells, particularly engineered synovial cells, in the vicinity of cartilage damage. The latter, especially if engineered synovium cells were used could promote differentiation and maturation of local cells in the cartilage defect to form articular cartilage. In this case, the synovial living scaffold is not a structural scaffold, but is a cell receptacle, a vehicle, that would permit a biologic transplant to release gene products juxta-position to cartilage injury. However, to our knowledge, there have been no viable purified synovium ECMs

(SynECM) or living ECMs with synovium cells produced to date.

The objective of this study was to develop a method of producing SynECM to serve as a biologic scaffold. Four methods, treatment with 0.1% peracetic acid (PAA) solutiona once (1XPAA) or twice (2XPAA), 1% Triton X-100 (t-octylphenoxypolyethoxyethanol)b followed by DNAsec (Triton), or 2M NaCld followed by DNase (NaCl), were compared among each other and to a sham-treated control specimen. The resulting SynECMs were evaluated morphologically using histologic sections and scanning electron microscopy, viability using culture and trypan blue exclusion staining, and the efficiency of

3 decellularization using DNA content and fragment size. We hypothesized that all methods would decellularize the synovium and that the PAA methods would be superior in retaining the synovial structure.

Chapter 2 Materials and Methods

Four horses (<7 years), euthanized for reasons unrelated to orthopedic problems, with no current history of apparent lameness, had villous synovium harvested aseptically from the femoropatellar and medial femorotibial joints, dissected from the underlying fat and fibrous layer of the joint capsule, and placed in culture mediae for transport. The joints were macroscopically inspected for any signs of pathology including but not restricted to abnormal synovial fluid, thickened inflamed synovium, hemorrhage in or around the joint or visible cartilage damage. Any joints exhibiting pathological changes were excluded as a source of synovium for study. All the villus synovium that could be obtained from stifles bilaterally was harvested, pooled by horse and divided under a dissecting microscope into 1 x 1 cm synovial sheets. These sheets were then cut with an 8mm biopsy punch to standardize the size of the specimens for study. The thickness was consistent at 6 mm ±1mm. All the specimens for each horse were then randomly assigned to the control (no processing) or each of the four decellularizing methods such that at least fifteen specimens were allotted from each horse to each method. This resulted in a minimum of seventy-five synovial specimens (8 mm diameter) per horse such that each method (control and four decellularized) would have three final specimens fixed for histology, cultured for viability, collagenase digested to release cells, and DNAse

5 digested for DNA content and fragment size. At least one specimen was allocated to electron microscopy (SEM). This was repeated for each of four horses, resulting in ~ three hundred total specimens (five methods x five analyses x 12 test items obtained as three specimens from each of four horses). Specimens were frozen at -80oC in an ethanol container with no media and used within 1 month of freezing. Extra specimens were maintained at -80oC and if needed as replacements were used within 2 months. Data were analyzed for differences among horses and methods for each outcome.

Decellularization and Creation of Scaffolds (SynECM)

Fresh-thawed synovial specimens were randomly assigned to control (no processing) or one of four decellularization methods (Table 1) each performed in batch of 12 specimens for each method for each horse. The 1XPAA method used 0.1% PAA solution and mechanical agitation for 6 hours22 before a washing process. The 2XPAA method used a modification of 1XPAA in which the decellularization process using 0.1% PAA was repeated after washing. The Triton method is described in detail in a previous article 23 but, in brief, involved exposure to 1% Triton X-100 and 24 hours mechanical agitation, washing, then followed by overnight incubation with DNAse and sterilizing with 0.2%

PAA for 2 hours. The NaCl method was performed in a similar manner to Triton, except

2M sodium chloride solution replaced the 1% Triton X-100. 23

Table 1. The methods used for decellularizing synovium.

Step Method 1(5) Method 2 Method 3(6) Method 4(6)

1% Triton X-100 2M NaCl 0.1% PAA /4%ethanol 24h mechanical 24h mechanical 1 6 h mechanical agitation, agitation, 100 rpm agitation, 100 rpm 100 rpm (37oC) (37oC) (37oC)

2 Wash* Wash* Wash** Wash**

Step 1 3 DNase** DNase** Repeated

4 Wash* Wash* Wash*

*15 min 2xPBS, 2xdeionized water

** Incubate (37°C) overnight in PBS + 70 U/ml DNAse

SynECM Characterization

Quantification of Residual Living Cells

Control and decellularized tissue from each method were placed in Corning Costar

TranswellTM 6 mm insertsf designed to fit into standard 12-well culture plates. Each method was incubated for 3 days at 37oC using alpha-MEM containing 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin. On day 3, wells were evaluated (NR) under a microscope for identification and counting of cells adhered to the plate or growing out from the scaffold. It was anticipated no cells would survive the freeze thaw, but culture plates were verified without growth by a blinded investigator (AB). The scaffolds were then digested in a 0.02% collagenase solution mixed with Hank’s Balanced Salt Solution (HBSS) at 37oC. After 5 hours, the digested cells were filtered through a 70μm cell strainer, stained with trypan blue, counted and evaluated (NR) for viability (% unstained cells).

Histological Evaluation

Scaffolds were fixed in 10% neutral buffered formalin, thin sectioned to 8 μm, stained with hematoxylin and eosin (H&E) and picrosirius red and scored as per Table 2 (NR and

ALB) in a blinded fashion. Score sheets for H&E stained sections were subsequently decoded for method and horse for the presence of villous integrity and cell content (Table

2). Collagen integrity, linearity, and alignment was evaluated in picrosirius red stained sections (NB and AB) and scored under polarized light microscopy as per Table 2.

Table 2. Histological criteria scale (0-4) for the decellularized synovium and control

Staining Structure 4 3 2 1 0 Villi clearly Villi clearly Villi clearly Villi clearly Villi not clearly Integrity of identifiable identifiable identifiable identifiable identifiable on Villi along whole along > 75% of along 75-50% along 50-25% H&E surface area. surface area. surface area. of surface area. of surface area. Cell nucleous Cell nucleous Cell nucleous Cell nucleous No cell found on outer found on outer found on outer found on outer nucleous found Cell loss layer of Villi layer of Villi layer of Villi layer of Villi on outer layer < 25% of Villi. 25-50% of 50-75% of >75% of Villi. of Villi. Villi. Villi. Collagen Collagen Collagen Collagen Collagen Collagen clearly intact clearly intact clearly intact clearly intact clearly intact Picrosirious structure and and aligned and aligned > and aligned 75- and aligned 25- and aligned < integrity (pink fibrils). 75% (pink 50% (pink 50% (pink 25% fibrils). fibrils). fibrils). (pink fibrils).

Scanning Electron Microscopy (SEM)

SynECM sheets were processed for SEM. One representative sample per method was imaged. Briefly; the pre-cut SynECM was thawed and fixed in 2.5% glutaraldehyde,

0.1M phosphate buffer saline (PBS) and 0.1M sucrose for 24 hours at 4oC. Samples were post fixed with 100% osmium tetroxide for 1 hour at 22oC, washed twice in PBS prior to imaging. The samples were then treated by ethanol gradient (50%, 70%, 80%, 95%, and

100%) for dehydration. Following the dehydration process, the samples were dried using hexamethyldisilazane gradient and sputter coated with a gold/palladium micro-layer and observed by SEMk equipped with a field-emission gun electron source. Surface topography was evaluated subjectively and presence of villi noted as present or absent

(NR and AB).

DNA Isolation and Quantification

DNA was extracted from control and decellularized scaffolds (5mg tissue wet weight) in triplicate using QIAamp DNA Mini extraction Kit.g The scaffolds were fully pulverized in lysis buffer and Proteinase K and all subsequent steps were performed according to the manufacturer’s instruction. DNA content was then quantified using NanoDrop 1000

Spectrophotometerh and values were expressed as ng DNA/mg of the scaffold.

Gel Electrophoresis

To determine DNA fragment size, equal concentrations and volumes (100 ng/10 μl) of purified DNA from each sample were separated on a 3% agarose gel with 0.5% ethidium bromide and visualized with ultraviolet trans-illumination using a reference 1Kbp and

25Kbp ladder (Figure 5).i

Data Analysis

Numerical data (viability, cellular content, and DNA content) were analyzed using commercial softwarel for normality and a two-factor ANOVA was performed with horse and method as factors. Horse was found not to be a significant factor and horse data was analyzed as a one-way ANOVA for method. Differences between methods, including control, were determined with Tukey’s post-hoc test. Data were expressed as mean ±

SEM. For all analyses, values of p< 0.05 were considered significant.

Chapter 3 Results

Frozen then thawed untreated synovium (control) had residual nonviable cells (no cell growth after 3-days of incubation) that stained positive with trypan blue. For all four methods of decellularized scaffolds there were no residual intact cells that stained positive with trypan blue or cell growth after a 3-day incubation (Table 3).

Histological examination of the control SynECMs showed a 1-2 cell layer lining of synoviocytes, a central arteriole and venule in the loose connective tissue consisting of fat cells and a fine fibrillar matrix in the interstitium (Figure 1A). Histologic morphology and SEM of the SynECMs differed among decellularization methods. 1XPAA and

2XPAA retained the synovium villous matrix integrity that was lost with the Triton and

NaCl preparations (Figures 1, 2 and 4). SEM confirmed that the fine villous synovium structure was maintained in the 1XPAA and 2XPAA methods (Figure 3).

Despite the removal of intact cells from the synovial sheets by all decellularization methods, there was significant DNA residue (Figure 5) and very large fragments exceeding 25,000 bps in SynECM from 1XPAA (Figure 6). This agrees with the histological examination that showed cellular debris on the surface of the 1XPAA. The other preparation methods all had very low residual DNA content (p <0.001) and small size of the residual DNA, base pair lengths of <300 bps (Table 3). Cellular debris was not

12 noted in the histological examination, confirming the lack of cells on the surface and lack of DNA fragments above 300 bps in these groups.

Table 3. Overview of results

SynECM Characterization Control Method 1 Method 2 Method 3 Method 4

Residual cell content No viable cells No viable cells No viable cells No viable cells No viable cells

No villous No villous Histological analysis H&E Villous integrity Villous integrity Villous integrity integrity integrity

Histological analysis Collagen integrity Collagen integrity Collagen integrity No villous No villous

Picrosirius and alignment and alignment and alignment integrity integrity

No villous No villous Histological analysis SEM Villous integrity Villous integrity Villous integrity integrity integrity

DNA content 1245 ± 330 ng/mg 1209 ± 63 ng/ml 101 ± 12 ng/mg 143 ± 15ng/mg 140 ± 24 ng/ml

DNA length > 25,000 bp > 300 bp < 300 bp < 300 bp < 300 bp

Figure 1. Hematoxylin and eosin staining

Hematoxylin and eosin staining in the control (A) () points to the 2-3 layer intima.

1XPAA (B), 0.1% peracetic acid incubated for 6h retained the villous structure (arrow) but also retained a significant amount of cells, 2XPAA (C), 0.1% peracetic acid incubated for 12h retained the villous structure (arrow) in similar fashion to 1XPAA but did not retain cells. Triton (D), and NaCl (E) showed no cells and a loss of synovial architecture.

All images are oriented so that the surfaces are in the upper left.

Figure 2. Microscopy of hematoxylin-eosin and picrosirius red stained SynECMs.

A) control (untreated synovium villus), B) 1XPAA, villus structure retained, some cellular debris on surface (arrow), cellular debris was observed as nuclear fragmentation, pyknotic cells, pyknotic and fragmented nuclei, C) 2XPAA, villus structure retained, no cells or cellular debris visible (arrow), D) Triton and E) NaCl, no villi were found so imaged tissue is surface of flat synovium (no cells are visible). The control, 1XPAA and

2XPAA are the central portion of synovium villi, where the structures of synovium were clearly distinguishable; first the outer cell layers, with no cells in C), then the denser collagen with lymphatic tissues, and in the center a loose structure normally containing an artery and vein. In all the samples, the 2XPAA (C) had a slightly denser structure than the control (A) and 1XPAA (B). In the Triton and NaCl samples there were no distinguishable synovium villi. Images shown (D and E) are of bundles of collagen that were found in the samples.

Hematoxylin-eosin stain Picrosirius stain

Figure 3. Scanning electron microscopy (280x) of the SynECMs.

A) Control (synovium that had not been treated), B) 1XPAA, C) 2XPAA, D) Triton and

E) NaCl. The control (A) shows the surface of a synovium villus, a relatively smooth structure of spherical cells (black arrow) in a collagen architecture. The SEMs of 1XPAA and 2XPAA preparations (B and C) both showed a structure of numerous distinct crisscrossing collagen fibers with fewer (B) or no (C) cells on the surface. In the Triton and NaCl preparations (D and E) the collagen fibrous architecture was lost leaving indiscriminate masses of material.

Figure 4. Histomorphological scoring of control synovium and SynECMs

Histomorphological scoring of control synovium and synovial scaffolds decellularized with 1XPAA, 2XPAA, Triton and NaCl (Mean± SEM). All decellularization methods significantly decreased cell count compared to the control synovium as evaluated by

H&E staining (p<0.0001). Decellularization using Triton and NaCl significantly decreased villous and collagen integrity scoring compared to the control synovium

(p<0.01 and p<0.001 repectively). Decellularization using 1XPAA or 2XPAA showed an increased overall histological score with significant cell loss and maintained villous and collagen integrity compared to the control synovium, with the highest overall score seen in 2XPAA decellularization.

Figure 5. DNA content of SynECMs

DNA content (Mean± SEM) of the control, 1XPAA, 2XPAA, Triton and NaCl. There was no significant difference between the control and 1XPAA (a) or between the methods 2XPAA, Triton or NaCl (b). There was a significant difference between the control and 1XPAA (a) method versus the methods 2XPAA (b), Triton (b) or NaCl (b)

(p<0.001).

Figure 6. DNA gel electrophoresis of SynECMs

Gel electrophoresis of SynECM from the decellularization preparations;

1 kb + standard, showing a ladder starting at 300 going up to 25 kb, control,> 25 kb,

1XPAA > 25 kb, 2XPAA, Triton and NaCl having no presence of DNA >300 bp

Chapter 4 Discussion

In this study, four procedures of decellularizing synovium were investigated for the effectiveness of removal of cells, the retention of DNA, and ECM ultrastructure. Only one of the four procedures, 2XPAA (0.1% PAA treatment performed twice), had low residual cellularity, low DNA content as well as retained the synovium villous architecture, and was considered the best method evaluated for obtaining SynECM.

The other three procedures, 1XPAA, Triton, and NaCl, were chosen because they have previously been used to obtain ECM from tissues that are structurally similar to synovium. 0.1% PAA was used in small intestinal submucosa1 ECM preparation, 1%

TritonX/DNase for urinary bladder matrix1, and 2M NaCl/DNAse for human umbilical vein23. These tissues, like synovium, have an architecture that are relatively loose and are only 2-3 cell layers thick. In our study, none of the three published methods gave the retained villous structure, low residual cells and low DNA content required for the generation of an ECM scaffold that could support living cells. Our findings are in line with previous studies that have shown the importance of tailoring the decellularization method to the specific tissue of interest.24

It is widely accepted that the goal of preparing ECM scaffolds is to maintain a good structural matrix as well as to keep as much of the biological factors (proteins, growth

22 mediators, anti-inflammatory cytokines) that promote cell adhesion and differentiation, yet remove underlying molecules that may promote host reactivity11. Since 1XPAA (a single wash with 0.1%PAA) retained the villous synovium structure, but had residual

DNA content, a 2XPAA (a double wash of 0.1%PAA) appeared to be a natural step in achieving a good scaffold for cell seeding and transplantation. Ongoing current work in this laboratory has shown that 2XPAA prepared SynECM seeded with synoviocytes, produce substantial proliferation and translocation of cells into the SynECM.m The synovium villous structure appears to be a good marker of the structural integrity of a

SynECM.

The disintegration of the synovium villous architecture by the Triton and NaCl methods was unanticipated. These methods are used in many ECM preparations that have tissue architectures similar to synovium.23 Triton X-100 is one of the most widely used nonionic surfactants that permeabilize living cell membranes, lysing cells allowing for the extraction of proteins and other cellular organelles.25, 26 In our study, the synovium villous structure was lost during the Triton X-100 treatments, indicating the synovium architecture could be dependent on lipid interactions between cells and the synovium

ECM. A similar interpretation could be made for the loss of synovium villi in the 2 M

NaCl preparations. 2 M NaCl is known to disrupt membranes through high osmotic pressure and cause denaturation of proteins. The 2XPAA procedure in contrast to the

Triton and NaCl methods, retained the synovium villi. PAA is thought to disrupt the chemi-osmotic function of the lipoprotein cytoplasmic membrane, rupture cell walls27,28 and oxidize sensitive sulfhydryl bonds as well as double bonds in enzymes, proteins and

23 other biological compounds. It appears that a relatively less disruptive removal of cell components by the 0.1% PAA washings removed the necessary cell components but retained the structure of the SynECM.

The disruptive agent combined with the treatment time may also be a contributing factor to the loss of synovium villi in the Triton and NaCl preparations. Both methods had a 36 hour processing time whereas the 2XPAA method was complete within 12 hr.

This study was not designed to investigate the individual factors and determine their contributions to preparing the optimum synECM. Even the extra wash cycle undergone in the 2XPAA method may not be necessary; the increase in time from 6h in the 1XPAA to

12h for the 2XPAA may be sufficient to prepare viable synovium ECM. Further work could be done to identify the optimal synovium decellularization process. However, this study shows that the 2XPAA method is an easy and useful synovium decellularization with promising results for synovial cell seeding.

Similar or higher concentrations of PAA (0.1-0.2%) have been used in many ECM decellularization protocols as a final wash to sterilize the ECM. In those studies, PAA sterilized the ECM but left the important structural29 and biological components intact for successful growth of cells and as an in-vivo scaffold.30 Thus, an additional advantage of the 2XPAA method is the elimination of this extra wash cycle for sterility since PAA is considered to be less toxic during cell seeding and is used to sterilize ECMs in many preparations.31,32 The loss of biologically active proteins after different decellularization protocols has been studied for several tissue types.33,34 The effect of PAA as a

24 sterilization step on the biological protein content has been studied in some tissues 35, but to our knowledge never as a single step procedure as for synovium.

Although the methods used in this article have been shown to remove a sufficient amount of chemicals, residual presence of chemicals and the potential toxicity were not addressed in this work. The chemicals used were chosen due to their abilities to damage cells, hence any residue may cause cell damage or death, decreasing the cells ability to migrate and thrive24,36. Further work could include assays to quantify the presence of residual chemicals in the decellularized tissue.

Further work may address some of the limitations of our study. The study of the double

0.1% PAA in a bioreactor with constant bathing of all cell surfaces in a controlled environment and/or studying the biocompatibility of this method in vivo with cellular survival and health may give valuable information on the potential of the SynECM. Our initial work with synovial cell engraftment in the 2XPAA has been promising without toxicity noted.m Also, the scaffolds were not compared based on biochemical content

(such as glycosaminoglycans and growth hormones) or biomechanical factors, both of these components being important bioactivities for the re-seeding and transplantation of scaffolds.

In summary, the 0.1% PAA, performed twice (2XPAA), was considered to have the best scaffold potential due to the low cellularity, low DNA content and retained villous architecture. PAA, performed once, retained excessive DNA and cells. The loss of synovial morphology and ECM integrity with Triton and NaCl preparations was less optimal. Other advantages of the PAA are elimination of an extra cycle for sterility (PAA

25 is used to sterilize ECM) and potentially less toxicity for cell seeding.

Footnotes

aSigma Aldrich, Steinheim Germany bSigma Aldrich, Steinheim, Germany cCollagenase Type II, Gibco, USA dSigma Aldrich, Steinheim, Germany eDMEM, Gibco, USA f Corning Costar Transwell, Sigma Aldrich, Steinheim, Germany g Qiagen, Hilden, Germany hNanoDrop 1000 Spectrophotometer, Thermo Scientific, USA

I GelDoc, Kodak Inc., j MediaCybernetics, Silver Spring, MD k FEI™ NOVA NanoSEM 400, Hillsboro, OR, USA l SPSS, IBM, New York m Reisbig et al. 2015: Characterization of Living Synovial Extracellular Matrix Scaffolds for Gene Delivery. Abstract presented at ACVS, Nashville, TN

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