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Osteochondral xenograft development for articular cartilage repair.

INTRODUCTION

Multiple studies have been conducted with the objective of repairing articular cartilage lesions in symptomatic patients. Some of this research indicates that osteochondral xenografts may be able to restore the same biomechanical properties that these individuals once possessed [1]. Fresh allogenic transplants are very effective, but their supply and shelf life are limited. They also carry risks of immune rejection and disease transmission. Xenografts have the potential advantages of abundant supply, low cost, longer shelf life, product uniformity, and low risk of disease transmission. Biological tissue transplantation provides a huge advantage in that it could match normal mechanical properties compared to traditional arthroscopic surgeries such as microfracture. However, before an osteochondral xenograft can be used to treat patients it has to overcome biological hurdles such as preventing host rejection from immunologic responses. Immunologic responses can range from biomechanical failure of the xenograft to host death due to antigens present on the graft [2]. By removing antigens through decellularization, the resulting xenograft articular cartilage becomes much less immunogenic scaffold which is more compatible for host acceptance and may even provide the opportunity for host cell migration [3,4,5,6]. Decellularization techniques include, but are not limited to, enzymatic digestion like DNase and detergents like Sodium dodecyl sulfate (SDS) [1].

The problem with decellularization of osteochondral xenografts is that it typically disrupts the structure of the extracellular matrix and degrades its biomechanical properties [3]. This study specifically focuses on the use of genipin to restore the tissue's biomechanical properties after decellularization. Glycosaminoglycan (GAG), which makes an important contribution to the biomechanical strength and structural integrity of the xenografts, is extracted when tissue is decellularized with high detergent concentration and with lengthy intervals of time [5,7]. However, we have previously demonstrated that crosslinking the collagen of a xenograft with genipin, from gardenia jasminoides Ellis fruit, increases compressive stiffness and inhibits enzymatic degradation. It also protects against immunogenic responses by masking antigens [1,8]. Moreover, genipin is a chemical crosslinker with low cytotoxicity; in comparison to glutaraldehyde it is reportedly 5,000 to 10,000 times less toxic [9]. The purposes of this study were to characterize the effect of decellularization on the biochemistry of porcine articular cartilage, determine the effect of genipin concentration on the degree of crosslinking, quantify the effect of genipin crosslinking on the stiffness of the cartilage-bone interface, and evaluate the effect of genipin-fixed cartilage on the viability of primary autologous chondrocytes. Overall we hypothesized that the degree of crosslinking would be proportional to genipin concentration, that genipin fixation would reinforce the cartilage-bone interface, and that genipin-fixed cartilage would support attachment and survival of autologous chondrocytes.

METHODS

Decellularization and degree of crosslinking. The decellularization solution was 10 mM Tris-HCl (pH 8), 2% SDS, 0.5 mg/ml DNase , 1 mM EDTA, 5 mM Mg[Cl.sub.2], 0.5 mM Ca[Cl.sub.2], 0.1 mM PMSF, and 1% antibiotic-antimycotic mixture. Full-thickness discs of porcine articular cartilage were decellularized for 48 hours at 37[degrees]C with gentle agitation. Following decellularization, some samples were incubated in various concentrations of genipin in PBS ranging from 0.0008% to 0.1%. Incubation was for 24 hours at 37[degrees]C with gentle agitation. The degree of crosslinking was determined using the ninhydrin assay as previously described [10].

DNA, GAG, and collagen contents. Native and decellularized porcine articular cartilage was freeze dried and digested in 100 mM sodium phosphate buffer/10 mM Na2EDTA/10 mM L-cysteine/0.125 mg/mL papain overnight at 60[degrees]C. The Hoechst assay for DNA was performed by adding 50 pl of digestate to 2 ml of 10 mM Tris-HCl/1 mM EDTA/100 mM NaCl/0.2 [micro]g/ml Hoechst 33258 and reading the raw fluorescence. Calf thymus DNA was used as a standard. The Blyscan Assay Kit for GAG was used according the manufacturer's instructions. The chloramine-T assay for hydroxyproline was used to determine collagen content by assuming 12.5% of the collagen is hydroxyproline [11].

Cartilage-bone shear test. Cylindrical osteochondral plugs where extracted from porcine stifle joints (5 mm diameter x ~ 9 mm) were immediately washed with phosphate buffered saline. Some of the osteochondral samples were then decellularized or decellularized and crosslinked as above. The cartilage was cut sharply along the diameter and half the cartilage removed by cutting as close to the bone as possible. This exposed a rectangular shelf of cartilage, 5 mm x cartilage thickness (Fig. 1). Samples were then placed into polyurethane molds keeping the osteochondral interface exposed. Once the osteochondral plugs were firmly set, all the samples, including native osteochondral plugs, were subjected to a shear test in an Instron 1011 Universal Testing machine. The embedded bone and the cartilage shelf were aligned perpendicular to the axis of the Instron. A broad, flat ram attached to the actuator was positioned approximately 1 mm above the shelf of cartilage so that it just touched the exposed bone/calcified cartilage. The ram was then advanced at 5 mm/min until failure. Shear stiffness was calculated as the slope of the linear region of the force vs. displacement curve.

Biocompatibility. Porcine articular cartilage disks were decellularized as above. Residual decellularization solution was removed by several washes with PBS. Disks were sterilized by incubating for 3 hours 1% peracetic acid at room temperature followed by extensive washing in PB S until the pH of the rinsate had neutralized. Two control discs were further incubated in PBS. Two experimental discs were fixed in 0.1% sterile aqueous genipin as above. Residual genipin was removed by extensive washing with PBS. Decellularized controls and genipin-fixed discs were placed into separate wells of a 24-well plate and equilibrated in complete culture medium (DMEM, 10% fetal bovine serum, 1% antibiotic-antimycotic mixture) by overnight incubation at 37[degrees]C. They were then air dried in a biosafety cabinet for 90 min. Fresh porcine cartilage from the same joint was digested in collagase to liberate the cells, which were seeded into a T-175 flask with complete culture medium. Twenty-five microliters of a 5 x [10.sup.6] cells/ml suspension was pipetted onto the surface of each disc. Additional culture medium was added after allowing 2 hours for cell attachment. Five days after seeding, cell viability was assessed using a fluorometric Live/Dead Cell Staining Kit (Promokine). Images were captured using a Leica DFC 420C camera attached to a Leica DM2500 microscope.

RESULTS

Table 1 displays the biochemical components of the articular cartilage disk and effects of decellularization and genipin fixation on biomechanics. Figure 2 exhibits degree of articular cartilage crosslinking with the concentration of aqueous genipin. As the figure demonstrates, the degree of articular cartilage cross linking is directly proportional to the concentration of aqueous genipin present in the solution.

Figure 3 illustrates a sample of the mechanical shearing of cartilage from bone. Data collected for shear strength only includes samples that had articular cartilage completely shear off the cartilage/bone interface. The shear stiffness was determined and recorded by analyzing their corresponding curves like the example in figure 3. There was no statistical difference among the groups with respect to shear stiffness and shear strength by one-way analysis of variance ([alpha] =.05). After decellularization of native articular cartilage (figure 4D), the tissue was then subjected to chemical crosslinking to genipin (figure 4C). It is important to note that in figure 4C the red is brighter from the genipin itself. Moreover, it is observed from figure 4 cells were successful in being biocompatible with genipin fixation. By comparing the images between figures 4A and figure 4B it can be interpreted that there were no dead cells present on the genipin fixed samples.

DISCUSSION

The data collected from processing ECM content exemplifies the effect of decellularization of osteochondral porcine tissue. In the process of removing antigens, approximately 80% of the DNA was removed from native porcine cartilage. However the high detergent effects of using SDS with decellularization degrades glycosaminoglycan content (GAG). GAG reduction after decellularization was roughly 50%. Conversely, there was no significant effects on collagen content after decellularization. These findings indicate that immunogenic responses may be lowered and could prevent host rejection [2]. Furthermore, crosslinking with genipin, depending on the degree of crosslinking, may mask further antigens present on the articular cartilage and provide structural integrity. In regards to scaffold integrity and masking of antigens, higher degree of genipin fixation would be the most viable option.

DISCUSSION

The data collected from processing ECM content exemplifies the effect of decellularization of osteochondral porcine tissue. In the process of removing antigens, approximately 80% of the DNA was removed from native porcine cartilage. However the high detergent effects of using SDS with decellularization degrades glycosaminoglycan content (GAG). GAG reduction after decellularization was roughly 50%. Conversely, there was no significant effects on collagen content after decellularization. These findings indicate that immunogenic responses may be lowered and could prevent host rejection [2]. Furthermore, crosslinking with genipin, depending on the degree of crosslinking, may mask further antigens present on the articular cartilage and provide structural integrity. In regards to scaffold integrity and masking of antigens, higher degree of genipin fixation would be the most viable option.

Neither decellularization nor genipin crosslinking significantly affected shear stiffness and strength. However, there is a trend toward decreasing mechanical properties with decellularization and a modest recovery after genipin crosslinking. The detergent minimally affected collagen content of the articular cartilage disk which contributes to the overall shear stiffness and strength. Due to the small sample size our data concerning the interfacial strength are not conclusive. However, they demonstrate that any differences are likely to be minor and clinically insignificant.

Due to genipin's properties of being a natural cross linker, it allows cells to adhere and grow to the osteochondral samples (Figure 4). Compared to other crosslinkers, like glutaraldehyde, this growth is possible because of genipins low toxicity levels to cells. Furthermore, after thorough washing of any residual detergents, genipin's crosslinking makes the ECM stronger and it makes cell growth easier of the decellularized cartilage for chondrocytes. Our study confirmed genipin's low toxicity and specifically shows that it supports chondrocyte attachment and survival.

CONCULSIONS

In general, chemically crosslinking decellularized samples with .1% aqueous genipin may allow the development of osteochondral xenografts from porcine tissue. It is possible that immunogenic responses from the host organism will be significantly reduced or non-existent, however this cannot be determined until in vivo studies are conducted [1,8]. Additionally if host cells are able to migrate to the osteochondral xenograft, like in this study, it may be possible to regenerate additional mechanical properties. These findings also indicated that the mechanical data for decellularized and genipin-fixed cartilage did not significantly change in regards to native cartilage. Therefore, genipin-fixed xenografts may be able to withstand physiological pressures.

ACKNOWLEDGEMENTS

This research was supported through the College of Agriculture and Life Sciences Undergraduate Research Scholars Program (URSP) at Mississippi State University and Mississippi State University's Bagley College of Engineering.

REFERENCES

[1] Revell, Christopher M., and Kyriacos A. Athanasiou, "Success Rates and Immunologic Responses of Autogenic, Allogenic, and Xenogenic Treatments to Repair Articular Cartilage Defects," Tissue Engineering. Part B. Reviews, vol. 15, no. 1, pp. 1-15, 2009.

[2] Cissell DD, Hu JC, Griffiths LG, Athanasiou KA, "Antigen removal for the production of biomechanically functional, xenogeneic tissue grafts," Journal Biomechanics, vol. 47, no. 9, pp. 1987-1996, 2014.

[3] Holger Koch, Cora Graneist, Frank Emmrich, et al., "Xenogenic Esophagus Scaffolds Fixed with Several Agents: Comparative In Vivo Study of Rejection and Inflammation," Journal of Biomedicine and Biotechnology, vol. 2012, pp.1-11, 2012.

[4] Tavassoli, A., Mahdavi-Shahri, N., Matin, M., Fereidoni, M., & Shahabipour, F, "Bovine articular cartilage decellularized matrix as a scaffold for use in cartilage tissue engineering," The Iranian Journal of Veterinary Science and Technology, vol 4, no. 1, pp. 1-8, 2012.

[5] Elder, Benjamin D., Sriram V. Eleswarapu, and Kyriacos A. Athanasiou, "Extraction Techniques for the Decellularization of Tissue Engineered Articular Cartilage Constructs," Biomaterials, vol. 30, no.12, pp. 3749-3756, 2009.

[6] Schwarz, S., Elsaesser, A. F., Koerber, L., Goldberg-Bockhorn, E., Seitz, A. M., Bermueller, C., Durselen, L., Ignatius, A., Breiter, R. and Rotter, "Processed xenogenic cartilage as innovative biomatrix for cartilage tissue engineering: effects on chondrocyte differentiation and function," Journal of Tissue Engineering and Regenerative Medicine, vol. Published online in Wiley Online Library, no. DOI: 10.1002/term. 1650, 2012.

[7] Zhang X, Chen X, Yang T, et al, "The effects of different crossing-linking conditions of genipin on type I collagen scaffolds: an in vitro evaluation," Cell & Tissue Bank, vol. 15, no. 4, pp. 531-541, 2014.

[8] Hiraishi N, Sono R, Sofiqul I, et al, "In vitro evaluation of plant-derived agents to preserve dentin collagen," Dental Materials, vol. 29, no. 10, pp. 1048-54, 2013.

[9] Cheng N-C, Estes BT, Young T -H, Guilak F, "Genipin-Crosslinked Cartilage-Derived Matrix as a Scaffold for Human Adipose-Derived Stem Cell Chondrogenesis," Tissue Engineering, Part A, vol. 19. No. 3-4, pp. 484-496, 2013.

[10] Cui L, Jia J, Guo Y, Liu Y, Zhu P, "Preparation and characterization of IPN hydrogels composed of chitosan and gelatin cross-linked by genipin," Carbohydrate Polymers, Vol. 99, pp. 31-38, 2014.

[11] Reddy GK, Enwemeka CS, "A simplified method for the analysis of hydroxyproline in biological tissues," Clinical Biochemistry, vol. 29, no. 3, pp. 225-229, 1996.

A. Garza (1), C. Young (2), S. Moore (2), S. Elder (2)

(1) Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, Mississippi State University

(2) Department of Agricultural and Biological Engineering, Mississippi State University

Table 1 Biochemical and biomechanical properties of decellularized
and genipin crosslinked cartilage.

                                Native             Decellularized

DNA (ng DNA/mg dry wt)   335.5 [+ or -] 25.0    63.3 [+ or -] 16.2 *

GAG ([micro]g GAG/mg     242.6 [+ or -] 21.8    150.5 [+ or -] 17.3 *
dry wt)

Collagen ([micro]g       151.7 [+ or -] 37.9     191.9 [+ or -] 37.1
collagen/mg dry wt)

Shear stiffness          13.1 [+ or -] 2.8      11.3 [+ or -] 2.4
(N/mm)                          (n=4)                   (n=6)

Shear strength (N)        15.6 [+ or -] 4.1      13.8 [+ or -] 3.0
                                (n=4)                   (n=4)

                         Decellularized + Genipin

DNA (ng DNA/mg dry wt)              N/A

GAG ([micro]g GAG/mg                N/A
dry wt)

Collagen ([micro]g                  N/A
collagen/mg dry wt)

Shear stiffness)          12.3 [+ or -] 1.7 (n=7)
(N/mm

Shear strength (N)        13.6 [+ or -] 3.1 (n=4)

* signifies (p<.05) independent t-test
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Author:Garza, A.; Young, C.; Moore, S.; Elder, S.
Publication:Journal of the Mississippi Academy of Sciences
Article Type:Report
Date:Apr 1, 2015
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