The objectives of this study were to characterize fresh porcine menisci and develop a decellularization protocol with a view to the generation of a biocompatible and biomechanically functional scaffold for use in tissue engineering/regeneration of the meniscus. Menisci were decellularized by exposing the tissue to freeze-thaw cycles, incubation in hypotonic tris buffer, 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer plus protease inhibitors, nucleases, hypertonic buffer followed by disinfection using 0.1% (v/v) peracetic acid and final washing in phosphate-buffered saline. Histological, immunohistochemical, and biochemical analyses of the decellularized tissue confirmed the retention of the major structural proteins. There was, however, a 59.4% loss of glycosaminoglycans. The histoarchitecture was unchanged, and there was no evidence of the expression of the major xenogeneic epitope, galactose-alpha-1,3-galactose. Biocompatibility of the acellular scaffold was determined by using contact cytotoxicity and extract cytotoxicity tests. Decellularized tissue and extracts were not cytotoxic to cells. Biomechanical properties were determined by indentation and tensile tests, which confirmed the retention of biomechanical properties following decellularization. In conclusion, this study has generated data on the production of a biocompatible, biomechanically functional scaffold for use in meniscal repair.

The aim of this study was to develop a technique to decellularize a porcine cartilage bone construct with view to using this as a biological scaffold for cartilage substitution. The decellularization protocol applied freeze/thaw cycles; this was followed by cyclic incubation in hypotonic tris buffer and 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer plus protease inhibitors. Nucleases (RNase and DNase) were used to digest nucleic acids followed by disinfection using 0.1% (v/v) peracetic acid. Histological analysis confirmed the absence of visible cells within the decellularized tissue. DNA analysis revealed the near-complete removal of genomic DNA from the decellularized tissues. The decellularization process had minimal effect on the collagen content of the cartilage. However, there was a significant reduction in the glycosaminoglycan content in the decellularized tissues. There was no evidence of the expression of the major xenogeneic epitope, galactose-α-1,3-galactose. Biomechanical indentation testing of decellularized tissues showed a significant change in comparison to the fresh cartilage. This was presumed to be caused by the reduction in the glycosaminoglycan content. Biocompatibility of the acellular scaffold was determined using contact cytotoxicity assays and a galactosyltransferase knockout mouse model. Decellularized porcine cartilage tissue was found to exhibit favorable compatibility in both in vitro and in vivo tests. 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.

Previously, we have described the development of an acellular porcine meniscal scaffold. The aims of this study were to determine the immunocompatibility of the scaffold and capacity for cellular attachment and infiltration to gain insight into its potential for meniscal repair and replacement. Porcine menisci were decellularized by exposing the tissue to freeze-thaw cycles, incubation in hypotonic tris buffer, 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer plus protease inhibitors, nucleases, hypertonic buffer followed by disinfection using 0.1% (v/v) peracetic, and final washing in phosphate-buffered saline. In vivo immunocompatibility was assessed after implantation of the acellular meniscal scaffold subcutaneously into galactosyltransferase knockout mice for 3 months in comparison to fresh and acellular tissue treated with a-galactosidase (negative control). The cellular infiltrates in the explants were assessed by histology and characterized using monoclonal antibodies against: CD3, CD4, CD34, F4/80, and C3c. Static culture was used to assess the potential of acellular porcine meniscal scaffold to support the attachment and infiltration of primary human dermal fibroblasts and primary porcine meniscal cells in vitro. The explants were surrounded by capsules that were more pronounced for the fresh meniscal tissue compared to the acellular tissues. Cellular infiltrates compromised mononuclear phagocytes, CD34-positive cells, and nonlabeled fibroblastic cells. T-lymphocytes were sparse in all explanted tissue types and there was no evidence of C3c deposition. The analysis revealed an absence of a specific immune response to all of the implanted tissues. Acellular porcine meniscus was shown to be capable of supporting the attachment and infiltration of primary human fibroblasts and primary porcine meniscal cells. In conclusion, acellular porcine meniscal tissue exhibits excellent immunocompatibility and potential for cellular regeneration in the longer term.

Glycosaminoglycans (GAGs) have been shown to be responsible for the interstitial fluid pressurization of articular cartilage and hence its compressive stiffness and load-bearing properties. Contradictory evidence has been presented in the literature on the effect of depleting GAGs on the friction properties of articular cartilage. The aim of this study was to investigate the effect of depleting GAGs on the friction and deformation characteristics of articular cartilage under different tribological conditions. A pin-on-plate machine was utilized to measure the coefficient of friction of native and chondroitinase ABC (CaseABC)-treated articular cartilage under two different models: static (4 mm/s start-up velocity) and dynamic (4 mm/s sliding velocity; 4 mm stroke length) under a load of 25 N (0.4 MPa contact stress) and with phosphate-buffered saline as the lubricant. Indentation tests were carried out at 1 N and 2 N loads (0.14 MPa and 0.28 MPa contact stress levels) to study the deformation characteristics of both native and GAG-depleted cartilage samples. CaseABC treatment rendered the cartilage tissue soft owing to the loss of compressive stiffness and a sulphated-sugar assay confirmed the loss of GAGs from the cartilage samples. CaseABC treatment significantly increased (by more than 50 per cent) the friction levels in the dynamic model (p < 0.05) at higher loading times owing to the loss of biphasic lubrication. CaseABC treatment had no effect on friction in the static model in which the cartilage surfaces did not have an opportunity to recover fluid because of static loading unlike the cartilage tissue in the dynamic model, in which translation of the cartilage surfaces was involved, ensuring effective biphasic lubrication. Therefore the depletion of GAGs had a smaller effect on the coefficient of friction for the static model. Indentation tests showed that GAG-depleted cartilage samples had a lower elastic modulus and higher permeability than native tissue. These results corroborate the role of GAGs in the compressive and friction properties of articular cartilage and emphasize the need for developing strategies to control GAG loss from diseased articular cartilage tissue.

SummaryObjectiveTo investigate the ability of high-field (9.4 T) magnetic resonance (MR) imaging to delineate porcine knee meniscal tissue structure and meniscal tears.Materials and methodsPorcine knees were obtained from a local abattoir, and eight medial menisci with no visible defects were dissected. Lesions simulating longitudinal tears were created on two of the menisci. MR images of the menisci were obtained at 9.4 T using a three-dimensional (3D)-FLASH sequence. A detailed 3D internal architecture of the intact and injured menisci was demonstrated on high-resolution MR images.ResultsHigh-resolution 3D MR imaging allowed visualisation of internal architecture of the meniscus and disruption to the internal structural network in damage models. The architecture of the porcine knee meniscus revealed by the MR scans appeared similar to the structures visualised by histology in previously reported studies.ConclusionHigh-field MRI is a non-destructive technique to examine the internal structural components and damage/wear of meniscal tissue. It has tremendous potential in the field of functional cartilage/meniscus biomechanics and biotribology.