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A comparison of decellularization methods applied to porcine osteochondral xenografts for articular cartilage repair.

INTRODUCTION

Many young physically active individuals frequently suffer from joint injuries, which result in osteochondral and cartilage legions. These legions lead to pain, swelling, and loss of functionality of the joint. One of the most popular treatments is osteochondral allograft transplantation (OAT). This treatment has the advantage of immediately restoring a resistance to mechanical stress. The big disadvantage to OAT is that there is a low supply of allografts available. This why using a xenograft is a very appealing alternative because they are in high supply and have a low cost. Decellularization of the xenograft can reduce the immunogenicity of the graph, and it can also help integration into the host tissue. Several studies have already shown that osteochondral xenografts (OCXG) have potential to repair articular cartilage, but they still have room for improvement [1, 2]. The aim of this study is to evaluate the ability of previously published decellularization protocols on the removal of antigenic cellular material, structural biochemistry, and biomechanics of porcine OCXGs, and to study the in vivo response of the OCXGs in a rabbit articular cartilage defect model.

MATERIAL AND METHODS

Tissue Samples:

[empty set]5.0 mm osteochondral dowels were harvested using a trephine from adult porcine stifle joints obtained from a local meat processor. All samples were rinsed with PBS and frozen at -20 [degrees]C , and when samples were ready for processing or testing they were thawed.

Method 1 Decellularization was based on the method described by Khier et al[3]. First, OCXGs were exposed to two freeze/thaw cycles. The plugs were then exposed to two more cycles of freeze/thaw in hypotonic buffer. OCXGs were then incubated with agitation in detergent. This was then followed by two washes in PBS with aprotinin. After, the wash samples were then incubated in nuclease. Once completed OCXGs were then washed in PBS with protease inhibitors. Decontamination was achieved by incubating samples in 0.1% (v/v) peracetic acid in PBS for 3h at room temperature.

Method 2

This method was designed for the decellularization of myocardium and was developed by Wang et al [4]. Osteochondral plugs were decullarized in a solution of 0.25% SDS, 1mM PMSF, 0.01%, 20 ug/ml RNase, and 0.2 mg/ml DNase under agitation for 11 days under room temperature. During the 11 days solution was changed every 3 days and samples received 10min ultrasonic treatment (50Hz). After 11 days samples were washed in PBS twice for 1h. Following that they were sterilized in two washes

of 70% EtOH for 2h each and then washed with sterile PBS.

Method 3

This method was adapted from Xie et al [5] and is a partial demineralization and deproteinization. First, OCXGs were immersed in 10% hydrogen peroxide for 24h at 38[degrees]C. Next the plugs were partially demineralized in 0.6 N hydrochloric acid for 3-4 h at room temperature. Then they were incubated at room temperature for 1h in chloroform/ methanol (1:1) and then in 0.25% trypsin for 12h at 4[degrees]C. Lastly, they went through multiple extensive washes with distilled water and were lyophilized. They were gas sterilized for the in vivo experiment.

Animal Testing

All experiments involving New Zealand White rabbits were approved by the Mississippi State Animal Care and Use Committee. This experiment involved 6 healthy male rabbits weighing approximately 4kg. Rabbits were anesthetized by intubation using 1% isoflurane and oxygen. Full-thickness cylindrical defects, 5mm diameter by 7mm depth, were created bilaterally in the central part of the patellofemoral groove of both stifle joints. The defects were then filled with one of the decellularized OCXGs matching the defects size so the grafts and the host cartilage was flush. The xenografts were selected so no rabbit received the same type in both knees and each method was represented 4 times. Three rabbits containing 2 constructs from each method were euthanized at 2 weeks and 8 weeks after surgery to assess the inflammatory response and cellular infiltration of the xenograft. Following this the joints were radiographed to assess bone healing.

Histology, Biomechanical Testing, and Biochemistry

Samples were fixed in formalin and decalcified in formic acid. Sections were then embedded in paraffin and stained with H&E, Toluidine Blue, and Picrosirius Red. Biomechanical testing of the non-implanted tissue required the separation of the cartilage from the bone with a scalpel. The cartilage pieces (n=5) were then loaded to 10% strain under confined compression, and the aggregate modulus was found by fitting the stress relation curve to the biphasic model. The bone cylinders (n=5) were then loaded to failure in unconfined compression at a rate of 0.01[mms.sup.-1]. Five samples from each group were digested in papain buffer (pH 6.5) for 24h at 60[degrees]C. DNA was then quantified using Hoechst 33258. Glycosaminoglycan (GAG) was quantified using the Biocolor Blyscan Assay based on dimethyl- methylene blue dye-binding. Bone (n=5) cylinders were tested for their calcium content using the Stanbio Calcium (CPC) LiquiColor kit after extraction with HCL.

H&E staining showed that cell nuclei were completely removed by method 1, but many nuclei remained after processing by methods 2 and 3. The Toluidine blue staining showed that method 1 almost completely stripped the tissue of proteoglycans, but methods 2 and 3 only partially removed the proteoglycans from the tissue (Fig. 1). Picrosirius red stating showed that method 1 removed a lot of the collagen in the tissue (Fig. 1). The results from the biochemistry and biomechanical testing shown in Table 1

Animal Experiment

After 2 weeks of implantation the grafts were strongly demarcated from the surrounding bone and had evoked a sclerotic reaction. By 8 weeks the grafts had begun to integrate with the native bone as shown in Figure 2. Qualitatively, method 1's grafts were the best integrated followed by method 3 and then method 2, but the variation was minor. Synovitis was present in all of the synovial membrane/ joint capsules to some degree, but it was generally mild.

DISCUSSION

The goal of decellularizing the OCXGs was to remove as much antigenic cellular material as possible while still leaving an extra cellular matrix still intact and having the xerograph still retain its mechanical properties. Method 1 was able to remove completely remove all cells for the cartilage phase, while method 2 and 3 only removed some of the nuclei. Method 1 removed almost all the DNA in the OCXG while, method 3 removed 75% of the DNA, and method 2 only removed 50% of it. The histology and biochemistry showed that method 1 removed almost all of the GAG molecules from the tissue and methods 2 and 3 only removed about half of the GAG in their OCXGs. Method 3 stripped about 75%-50% of the calcium from the bone of the OCXGs, but methods 1 and 2 showed little to no calcium loss. Method 3 reduced the aggregate modulus of the cartilage by about 50%, but method 2 was not far behind reducing it by 60%. The biggest reduction was seen in method 1, which reduced the modulus by 85%. The decellularization of the bone did not affect the Young's modulus in method 2. The other methods reduced the Young's modulus by about 50%. The animal experiment showed that none of methods had any sever adverse immune responses. Also, all the OCXGs had started to integrate into native bone and cartilage with an acceptable level of joint resurfacing. Considering all the factors method 1 is not acceptable because it compromised too much of the mechanical strength by stripping out most of the collagen and the GAG. Methods 2 and 3 look very promising they have a good balance of DNA removal and retention of the collagen and GAG. Also, they preserve the mechanical strength of tissues. Method 2 does have a little more priority over method 3 because it did not harm the bone matrix as much. It is believed that methods 2 and 3 could be refined to create better OCXGs because they only have just been adapted to decellularization of cartilage and bone.

CONCLUSIONS

OAT is a viable option when repairing osteochondral and cartilage lesions. The drawback is that allografts are hard to come by. Xenografts are widely available and come at cheap cost. Decellularization of xenografts promotes cell infiltration and integration into the tissue, while not causing a severe immunogenic response from the host. The evaluation of these three decellularization techniques shows that methods 2 and 3 were the most advantageous and promising to use. They stripped DNA and cells from the tissue, while still preserving some of the GAG and the mechanical properties of the xenograft. Method 2 was just slightly favored because it had less of an impact on the bone phase. With further research these two methods could be improved upon since there were adapted to this task.

ACKNOWLEDGMENTS

Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number 1R15AR057934. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

REFERENCES

[1] Toolan B, Frenkel S, Pereira D, Alexander H. Development of a novel osteochondral graft for cartilage repair. J Biomed Mater Res. Vol. 41, no. 2, pp. 244-250.Aug, 1998

[2] von Rechenberg B, Akens M, Nadler D, et al. Mosaicplasty with photooxidized, mushroomshaped, bovine, oteochondral xenografts in experimental sheep. Vet Comp Orthop Traumatol. Vol. 19, no. 3, pp. 147-56. 2006

[3] Kheir E, Stapleton T, Shaw D, Jin Z, Fisher J, Ingham E. Development and characterization of an acellular porcine cartilage bone matrix for use in tissue engineering. J Biomed Mater Res A. vol. 99, no. 2, pp 283-94. Nov, 2011.

[4] Wang B, Borazjani A, Tahai M, Curry AL, Simionescu DT, Guan J, To F, Elder SH, Liao J. Fabrication of cardiac patch with decellularized porcine myocardial scaffold and bone marrow mononuclear cells. J Biomed Mater Res A. vol. 94, no.4, pp. 1100-10. Sep, 2010.

[5] Xie H, Yang F, Deng L, Luo J, Qin T, Li X, Zhou GQ, Yang Z. The performance of a bone-derived scaffold material in the repair of critical bone defects in a rhesus monkey model. Biomaterials. vol. 28, no.22, pp.3314-24. Aug, 2007.

Mark Mosher (1), Ryan Butler (3), Steven Elder (1), Andrew Claude (3), Jim Cooley (2), Eric Gilbert (1), Anuhya Gottipati (1), Jun Laio (1), Robert Meyer (3)

(1) Agricultural & Biological Engineering, Mississippi State University, Mississippi State, Mississippi

(2) Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi

(3) Department of Clinical Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi

Table 1. Biochemical and Biomechanical data of OCXG. (* designates a
statistical difference wrt controls)

                  Method 1                   Method 2

DNA normalized    0.1982 [+ or -] 0.0364 *   0.8498 [+ or -] 0.2017 *
to wet

GAG normalized    3.742 [+ or -] 3.25 *      54.736 [+ or -] 9.96 *
to wet

Calcium           137.0 [+ or -] 11.45       93.21 [+ or -] 33.31
normalized
to dry

Aggregate         0.0213 [+ or -] 0.0026 *   0.0552 [+ or -]0.0039 *
Modulus of
Cartilage
(MPa)

Young's           79.32 [+ or -] 39.69       167.93 [+ or -] 44.56
Modulus of Bone

                  Method 3                   Frozen/Thawed Controls

DNA normalized    0.4186 [+ or -] 0.2067 *   1.5076 [+ or -] 0.3310
to wet

GAG normalized    44.765 [+ or -] 16.81 *    99.018 [+ or -] 28.26
to wet

Calcium           30.79 [+ or -] 23.62 *     115.93 [+ or -] 21.34
normalized
to dry

Aggregate         0.077 [+ or -]0.0084 *     0.1397 [+ or -] 0.04
Modulus of
Cartilage
(MPa)

Young's           97.58 [+ or -] 49.25 *     167.88 [+ or -] 82.58
Modulus of Bone
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Article Details
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Author:Mosher, Mark; Butler, Ryan; Elder, Steven; Claude, Andrew; Cooley, Jim; Gilbert, Eric; Gottipati, An
Publication:Journal of the Mississippi Academy of Sciences
Article Type:Report
Geographic Code:1USA
Date:Apr 1, 2014
Words:1959
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