Abstract

Decellularised porcine superflexor tendon (pSFT) has been demonstrated to be a suitable scaffold for anterior cruciate ligament reconstruction[1]. While the role of collagen in tendons is well known, the mechanical role of glycosaminoglycans (GAGs) is less clear and may be altered by the decellularisation process.

To determine the effects of decellularisation on pSFT GAG content and mechanical function and to investigate the consequences of GAG loss in tensile and compressive loading.

pSFTs were decellularised following previous techniques [2]. For GAG removal, native pSFTs were treated with chondroitinase ABC (ChABC; 0.1U/mL, 72h). Cell and GAG removal was validated using histology and quantitative assays. Native, decellularised and ChABC treated groups (n=6) were biomechanically characterised. In tension, specimens underwent stress relaxation and strength testing using previous protocols [1]. Stress relaxation data was fitted to a modified Maxwell-Weichert model to determine time-dependent (E1 & E2) and time-independent moduli (E0). The toe and linear region moduli (Etoe, Elinear), in addition to tensile strength (UTS) and failure strain were determined from strength testing. In compression, specimens underwent confined loading conditions (ramp at 10 s-1 to 10% strain and hold). The aggregate modulus (HA) and zero-strain permeability (k0) were determined using previous techniques [3]. Data was analysed by one-way ANOVA with Tukey post-hoc test to determine significant differences between test groups (p<0.05).

Quantitative assays showed no GAG reduction post-decellularisation, but a significant reduction after ChABC treatment. HA was only significantly reduced in the ChABC group. k0 was significantly higher for the ChABC group compared to decellularised. E0 was significantly reduced in the decellularised group compared to native and ChABC groups, while E1 and E2 were not different between groups. Etoe, Elinear, UTS and failure strain were not different between groups.

Decellularisation does not affect GAG content or impair mechanical function in pSFT. GAG loss adversely affects pSFT compressive properties, revealing major mechanical contribution under compression, but no significant role under tension.

This study aims to assess the changes in mechanical behaviour over time in ‘haemarthritic’ articular cartilage compared to ‘healthy’ articular cartilage.

Pin-on-plate and indentation tests were used to determine the coefficient of friction (COF) and deformation of ‘healthy’ and ‘haemarthritic articular cartilage. Osteochondral pins (8 mm) were extracted from porcine tali and immersed in exposure fluid for two hours prior to test. Pins were articulated against a larger bovine femoral plate for 3600 seconds under a load of 50 N. Osteochondral pins (8 mm) were loaded during indentation testing for 3600 seconds under a load of 0.25 N. To mimic the effect of a joint bleed in vitro; serum, whole blood and 50% v/v were used as exposure and lubricant fluids. COF and deformation were expressed as mean (n=3) and statistically analysed using a one-way ANOVA and post-hoc Tukey test (p>0.05).

The serum condition yielded a COF of 0.0428 ± 0.02 with 0.08mm ± 0.04 deformation. The 50% v/v condition produced a higher COF of 0.0485 ± 0.02 and 0.21mm ± 0.04 deformation. The lowest COF and deformation were produced by the whole blood condition (0.0292 ± 0.02 and 0.06mm ± 0.006 respectively). Statistical analysis indicated no significant difference across the friction test conditions but a significant difference across all indentation test conditions (ANOVA, p>0.05). Combination of creep deformation and wear was observed on the articular surface up to 24 hours post-test in 50% v/v and whole blood conditions.

The average haemophilia patient can experience multiple joint bleeds per year of which this study demonstrates the effect of just one joint bleed. This study has provided evidence of potential reversible and irreversible mechanical changes to articular cartilage surface during a joint bleed.

Abstract

Decellularised porcine superflexor tendon (pSFT) provides an off-the-shelf, cost-efficient option for ACL reconstruction (ACLR). During decellularisation, phosphate buffered saline (PBS) is used for washing out cytotoxic solutes and reagents, maintaining tissue hydration. It has been shown to increase water content in tendon, swelling the tissue reducing mechanical properties. End stage PBS washes in the standard protocol were substituted with alternative solutions to study tissue swelling and its impact on the mechanical behaviour and matrix composition of pSFTs. 25%, 100% Ringers and physiological saline test groups were used (n=6 for all groups). pSFTs were subject to tensile and confined compression testing. Relative hydroxyproline (HYP), glycosaminoglycan (GAG) and denatured collagen content (DNC) were quantified. Modified decellularised tendon groups were compared to tendons decellularised using the standard protocol and native tendons. Specimen dimensions reduced (p=0.004) post-decellularisation only in 25% Ringers group. In all other modified groups, less swelling was apparent but not statistically different from standard group. Only 25% Ringers group had higher linear modulus (p=0.0035) and UTS (p=0.013) compared to standard group. All decellularised groups properties were reduced compared to native pSFTs. Stress relaxation properties showed a significant reduction in decellularised groups compared to native. Compression testing showed no significant differences in peak stress for modified decellularised groups compared to native. A reduction (p=0.036) was observed in standard group. Quantification of GAGs and DNC showed no significant differences between groups. HYP content was higher (p<0.0001) for saline group. A significant reduction in tissue swelling could be related to improved mechanical properties of decellularised pSFTs. Alternative solutions in end stage washes had no significant effect on quantities of matrix components, but altered structure/function could explain the differences in tensile and compressive behaviour, and should be further studied. In all decellularised groups, pSFTs retained suitable mechanical properties for ACLR.

Abstract

Abstract

Decellularised extracellular matrix scaffolds show great promise for the regeneration of damaged musculoskeletal tissues (cartilage, ligament, meniscus), however, adequate fixation into the joint remains a challenge. Here, we assess the osseo-integration of decellularised porcine bone in a sheep model. This proof-of-concept study supports the overall objective to create composite decellularised tissue scaffolds with bony attachment sites to enable superior fixation and regeneration.

Porcine trabecular bone plugs (6mm diameter, 10mm long) were decellularised using a novel bioprocess incorporating low-concentration sodium dodecyl sulphate with protease inhibitors. Decellularised bone scaffolds (n=6) and ovine allograft controls (n=6) were implanted into the condyle of skeletally mature sheep for 4 and 12 weeks. New bone growth was visualised by oxytetracycline fluorescence and standard resin semi-quantitative histopathology.

Scaffolds were found to be fully decellularised and maintained the native microarchitecture. Following 4-week implantation in sheep, both scaffold and allograft appeared well integrated. The trabecular spaces of the scaffold were filled with a fibro-mesenchymal infiltrate, but some areas showed a marked focal lymphocytic response, associated with reduced bone deposition. A lesser lymphocytic response was observed in the allograft control. After 12-weeks the lymphocytic reaction was minimised in the scaffold and absent in allografts. The scaffold showed a higher density of new mineralized bone deposition compared to allograft. New marrow had formed in both the scaffold and allografts.

Following the demonstration of osteointegration this bioprocess can now be transferred to develop decellularised composite musculoskeletal tissue scaffolds and decellularised bone scaffolds for clinical regeneration of musculoskeletal tissues.

Cartilage lesions occur as a result of joint trauma, and progressively degenerate over time leading to osteoarthritis (OA). Early intervention therapies to repair the initial tissue damage have the potential to delay or prevent the onset of OA. We have developed two acellular treatments; 1) an injectable proteoglycan-like self-assembling hydrogel for the repair of ICRS grade 1 lesions, and 2) a decellularised xenogeneic osteochondral scaffold for surgical grafting in grade 2–4 lesions. We produced an in vitro glycosaminoglycan depleted grade 1 lesion model using porcine cartilage. Peptide-chondroitin sulphate mixture was injected and spontaneously gelled in situ. Cartilage resistance to deformation was increased by 50 %. Decellularised porcine osteochondral scaffolds which maintain the native tissue composition and architecture whilst being immunocompatible were successfully developed and are currently undergoing in vivo assessment in an ovine critical size condylar defect model. Incorporation of the subchondral bone in osteochondral scaffolds is intended to improve osseointegration; implanted decellularised bone-only scaffolds in sheep exhibited superb osteoinductive and osteoconductive properties in a proof-of-concept study. We envisage that our early intervention therapies will be employed clinically to maintain or restore functional hyaline-like cartilage across the whole range of early chondral pathologies and prevent the onset of OA.