This study aimed to determine the optimal starting material for the development of an acellular osteochondral graft. Osteochondral tissues from three different species were characterised; pig (6 months), cow (18 months) and two ages of sheep (8 -12 months and > 4 year old). Tissues from the acetabulum and femoral head of the hip, and the groove, medial and lateral condyles and tibial plateau of the knee were assessed. Histological analysis of each tissue allowed for qualification of cartilage histoarchitecture, glycosaminoglycan (GAG) distribution, assessment of cellularity and cartilage thickness. Collagen and GAG content were quantified and cartilage water content was defined. Following biomechanical testing, the percentage deformation, permeability and equilibrium elastic modulus was determined.Results showed that porcine cartilage had the highest concentration of sulphated proteoglycans and that the condyles and groove of the knee showed higher GAG content than other joint areas. Cartilage from younger tissues (porcine and young ovine) had higher cell content and was thicker, reflecting the effects of age on cartilage structure. Cartilage from
It is proposed that an acellular natural osteochondral scaffold will provide a successful repair material for the early intervention treatment of cartilage lesions, to prevent or slow the progression of cartilage deterioration to osteoarthritis. Here, we investigated the efficacy of methods for the decellularisation of bovine osteochondral plugs. The plugs were subject to four freeze/thaw cycles followed by two cycles of washes in hypotonic solution and low concentration (0.1 % w/v) sodium dodecyl sulphate with protease inhibitors. Plugs were treated with nuclease (DNase and RNase) treatment followed by sterilization in peracetic acid. Full tissue decellularisation was achieved as confirmed by histological analysis and DNA quantification, however the resultant acellular matrix had reduced glycosaminoglycan content which led to an increased percent deformation of cartilage. Furthermore, the acellular scaffold was not reproducibly biocompatible. Additional terminal washes were included in the process to improve biocompatibility, however, this led to visible structural damage to the cartilage. This damage was found to be minimised by reducing the cut edge to cartilage area ratio through decellularisation of larger cuts of osteochondral tissue.
Peptide-based hydrogels are of interest for their potential use in regenerative medicine. Combining these hydrogels with materials that may enhance their physical and biological properties, such as glycosaminoglycans, has the potential to extend their range of biomedical applications, for example in the repair of early cartilage degeneration. The aim of this study was to combine three self-assembling peptides (P -4, P -8, and P -12) with chondroitin sulphate at two molar ratios of 1:16 and 1:64 in 130 and 230 mM Na salt concentrations. The study investigates the effects of mixing self-assembling peptide and glycosaminoglycan on the physical and mechanical properties at 37°C. Peptide alone, chondroitin sulphate alone, and peptide in combination with chondroitin sulphate were analysed using Fourier transform infrared spectroscopy to determine the β-sheet percentage, transmission electron microscopy to determine the fibril morphology, and rheology to determine the elastic and viscous modulus of the materials. All of the variables (peptide, salt concentration, and chondroitin sulphate molar ratio) had an effect on the mechanical properties, β-sheet formation, and fibril morphology of the hydrogels. P -4 and P -8-chondroitin sulphate mixtures, at both molar ratios, were shown to have a high β-sheet percentage, dense entangled fibrillar networks, as well as high mechanical stiffness in both (130 and 230 mM) Na salt solutions when compared with the P -12/chondroitin sulphate mixtures. These peptide/chondroitin sulphate hydrogels show promise for biomedical applications in glycosaminoglycan depleted tissues.
Intervertebral disc (IVD) degeneration is a major cause of back pain. Current surgical interventions have limitations. An alternative approach is to replace degenerated IVDs with a natural biological scaffold. The removal of cellular components from human IVDs should render them nonimmunogenic upon implantation. The aim of this initial proof of technical feasibility study was to develop a decellularization protocol on bovine IVDs with endplates (EPs) and assess protocol performance before application of the protocol to human IVDs with attached EP and vertebral bone (VB). A decellularization protocol based on hypotonic low concentration sodium dodecyl sulfate (0.1% w/v) with proteinase inhibitors, freeze/thaw cycles, and nuclease and sonication treatments was applied to IVDs. Histological, biochemical, and biomechanical comparisons were made between cellular and decellularized tissue. Cell removal from bovine IVDs was demonstrated and total DNA levels of the decellularized inner annulus fibrosus (iAF), outer annulus fibrosus (oAF), and EP were 40.7 (±11.4), 25.9 (±3.8), and 29.3 (±3.1) ng.mg −1 dry tissue weight, respectively ( n = 6, ±95% confidence level [CL]). These values were significantly lower than in cellular tissue. No significant difference in DNA levels between bovine cellular and decellularized nucleus pulposus (NP) was found. Glycosaminoglycans (GAGs) were largely retained in the NP, iAF, and oAF. Cyclic compression testing showed sufficient sensitivity to detect an increase in stiffness of bovine IVD postdecellularization (2957.2 ± 340.8 N.mm −1 ) (predecellularization: 2685.4 ± 263.1 N.mm −1 ; n = 5, 95% CL), but the difference was within natural tissue variation. Total DNA levels for all decellularized tissue regions of human IVDs (NP, iAF, oAF, EP, and VB) were below 50 ng.mg −1 dry tissue weight (range: 2 ng.mg −1 , iAF to 29 ng.mg −1 , VB) and the tissue retained high levels of GAGs. Further studies to assess the biocompatibility and regenerative potential of decellularized human IVDs in vitro and in vivo are now required; however, proof of technical feasibility has been demonstrated and the retention of bone in the IVD samples would allow incorporation of the tissue into the recipient spine. Impact statement Intervertebral disc (IVD) degeneration is a major cause of back pain. Current surgical treatments have limitations and relatively poor outcomes. An implantable cell-free biological scaffold, which will not invoke adverse immune responses, has the potential to preserve the natural mobility of the patient's spine and be regenerated with endogenous cells, preventing further degeneration and improving surgical outcomes. This study demonstrates, for the first time, that it is possible to create a cell-free human IVD biological scaffold wi...
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