DEVELOPMENT AND CHARACTERIZATION OF AN ACELLULAR PORCINE CARTILAGE BONE MATRIX FOR USE IN TISSUE ENGINEERING

Abstract

Introduction: The aim of this study was to develop a technique to decellularise a porcine cartilagebone construct with a view to using this as a biological scaffold for transplantation into human osteochondral defect as a cartilage substitute.

Methods: Decellularisation was based on a modification of the technique of Booth et al (2002). Cartilage bone matrix (n=9) were decellularised by exposing the tissue to 2 cycles of dry freeze-thaw followed 2 more cycles with the addition of hypotonic (10mM tris-HCl, pH8.0) buffer. Samples were then cycled through hypotonic buffer, followed by ionic detergent (0.1% [w/v] sodium dodecyl sulphate [SDS]) in the presence of protease inhibitors (aprotinin 10 KIU/ml) and 0.1% (w/v) ethylene diamine tetraacetic acid (EDTA). This was followed by washes in PBS with aprotinin and incubation in nuclease solution containing DNase (50U/ml) and RNase (1U/ml). Decontamination using 0.1% (v/v) peracetic acid in PBS was then incorporated to achieve disinfection of the tissue samples. Finally, samples were washed in PBS. Three decellularisation protocols were used depending on the number of hypotonic/SDS cycles: this was either done once, three or six times referred to as DC1, DC3 and DC6 respectively. Fresh & decellularised cartilage were compared histologically using haematoxylin and eosin staining, to visualize cellular content, sirius red, to visualise collagen fibres & alcian blue, to visualise glycosaminoglycans (GAG). Immunohistochemistry staining for galactose-α-1,3-galactose (α-gal), collagen I, II & VI was performed for fresh and decellularised samples. DNA assay: Genomic DNA was extracted using a DNA isolation kit for tissues (Roche Applied Sciences). Collagen and DMB sulphated sugar assay, as described by Stapleton et al. (2008), were performed to measure collagen and GAG content. The biphasic property of fresh and decellularised cartilage was determined using a pin on plate indentation test.

Results: H& E staining revealed the absence of visible whole cells. Sirius red stain gave evidence of the retention of collagen following decellularisation. In contrast, the acellular matrix showed evidence of loss of GAGs. There was no evidence of the expression of α-gal in the acellular scaffold. DNA analysis revealed the absence of genomic DNA in comparison to fresh tissues (ANOVA, p< 0.05). The decellularisation process had minimal effect on the collagen content of the cartilage. Nevertheless there was a significant difference in the sulphated sugar content of the fresh tissue when compared to the decellularised tissue (ANOVA, p< 0.05), indicating loss of 92% GAG. Biomechanical testing of decellularised tissues showed a significant change (ANOVA, p< 0.05) in comparison to the fresh cartilage.

Discussion: In conclusion this study has generated data on the production of an acellular cartilage bone matrix scaffold for use in osteochondral defect repair. To our knowledge, this is the first study that has successfully removed whole cells and α-gal from xenogeneic cartilage and bone tissue. Future studies are required to investigate methods to recellularise the acellular matrix using an appropriate cell type and mechanical conditioning and to investigate replenishing GAG loss following decellularisation.