Repopulation of decellularised porcine pulmonary valves in the right ventricular outflow tract of sheep: Role of macrophages.

The primary objective was to evaluate performance of low concentration SDS decellularised porcine pulmonary roots in the right ventricular outflow tract of juvenile sheep. Secondary objectives were to explore the cellular population of the roots over time. Animals were monitored by echocardiography and roots explanted at 1, 3, 6 (n = 4) and 12 months (n = 8) for gross analysis. Explanted roots were subject to histological, immunohistochemical and quantitative calcium analysis (n = 4 at 1, 3 and 12 months) and determination of material properties (n = 4; 12 months). Cryopreserved ovine pulmonary root allografts (n = 4) implanted for 12 months, and non-implanted cellular ovine roots were analysed for comparative purposes. Decellularised porcine pulmonary roots functioned well and were in very good condition with soft, thin and pliable leaflets. Morphometric analysis showed cellular population by 1 month. However, by 12 months the total number of cells was less than 50% of the total cells in non-implanted native ovine roots. Repopulation of the decellularised porcine tissues with stromal (α-SMA+; vimentin+) and progenitor cells (CD34+; CD271+) appeared to be orchestrated by macrophages (MAC 387+/ CD163low and CD163+/MAC 387-). The calcium content of the decellularised porcine pulmonary root tissues increased over the 12-month period but remained low (except suture points) at 401 ppm (wet weight) or below. The material properties of the decellularised porcine pulmonary root wall were unchanged compared to pre-implantation. There were some changes in the leaflets but importantly, the porcine tissues did not become stiffer. The decellularised porcine pulmonary roots showed good functional performance in vivo and were repopulated with ovine cells of the appropriate phenotype in a process orchestrated by M2 macrophages, highlighting the importance of these cells in the constructive tissue remodelling of cardiac root tissues.

The effect of decellularisation on the real time mechanical fatigue of porcine aortic heart valve roots.

Decellularised heart valve roots offer a promising option for heart valve replacement in young patients, having the potential to remodel and repair. Replacement heart valves have to undergo billions of opening and closing cycles throughout the patient's lifetime. Therefore, understanding the effect of cyclic loading on decellularised heart valve roots is important prior to human implantation. The aim of this preliminary study was to investigate the influence of low concentration sodium dodecyl sulphate (SDS) decellularisation treatment on the in vitro real time mechanical fatigue of porcine aortic heart valve roots under physiological real time cyclic loading conditions. This required a specific real time in vitro method to be developed, since previous methods relied on accelerated testing, which is non-physiological, and not appropriate for valve replacement materials that exhibit time dependent characteristics. The effects of the real time fatigue on hydrodynamic function and mechanical properties of the heart valve roots were assessed. The mechanical fatigue of decellularised porcine aortic heart valve roots (n = 6) was assessed and compared to cellular porcine aortic heart valve roots (n = 6) in a modified Real time Wear Tester (RWT) at a physiological frequency and under cyclic pressure conditions for a maximum of 1.2 million cycles. Periodically, the heart valve roots were removed from the RWT to assess the influence of cyclic loading on valve competency (static leaflet closure). At the end of testing further hydrodynamic performance parameters were ascertained, along with determination of leaflet material properties. A real time mechanical fatigue assessment method was developed and applied; with two cellular and two decellularised porcine aortic leaflets in different heart valve roots showing tears in the belly region. The decellularised aortic heart valve roots exhibited comparative functionality to the cellular heart valve roots under in vitro static and pulsatile hydrodynamic conditions. However, the material properties of the decellularised aortic leaflets were significantly altered following cyclic fatigue assessment and showed increases in elastin and collagen phase slopes and ultimate tensile strength compared to the cellular porcine aortic leaflets in the circumferential direction. This preliminary study demonstrated that low concentration SDS decellularised porcine aortic heart valve roots can withstand physiological cyclic deformations up to 1.2 million cycles in a RWT whilst maintaining their overall hydrodynamic function and leaflet mechanical properties. This is the first full report of preclinical mechanical fatigue assessment of decellularised porcine aortic heart valve roots under physiological real time conditions.

Decellularised human bone allograft from different anatomical sites as a basis for functionally stratified repair material for bone defects.

Tissue engineered bone solutions aim to overcome the limitations of autologous and allogeneic grafts. Decellularised tissues are produced by washing cellular components from human or animal tissue to produce an immunologically safe and biocompatible scaffold, capable of integration following implantation. A decellularisation procedure utilising low concentration sodium dodecyl sulphate (0.1% w/v) was applied to trabecular bone from human femoral heads (FH) and tibial plateaus (TP). Biological (histology, DNA quantification), biomechanical (compression testing) and structural (µCT) comparisons were made between decellularised and unprocessed cellular tissue. Total DNA levels of decellularised FH and TP bone were below 50 ng mg-1 dry tissue weight and nuclear material was removed. No differences were found between cellular and decellularised bone, from each anatomical region, for all the biomechanical and structural parameters investigated. Differences were found between cellular FH and TP and between decellularised FH and TP. Decellularised FH had a higher ultimate compressive stress, Young's modulus and 0.2% proof stress than decellularised TP (p = 0.001, 0.002, 0.001, Mann Whitney U test, MWU). The mineral density of cellular and decellularised TP bone was significantly greater than cellular and decellularised FH bone respectively (cellular: p = 0.001, decellularised: p < 0.001, MWU). The bone volume fraction and trabecular thickness of cellular and decellularised FH bone were significantly greater than cellular and decellularised TP bone respectively (cellular: p = 0.001, 0.005; decellularised: p < 0.001, <0.001, MWU). Characterisation of decellularised trabecular bone from different anatomical regions offers the possibility of product stratification, allowing selection of biomechanical properties to match particular anatomical regions undergoing bone graft procedures.

Integration and functional performance of a decellularised porcine superflexor tendon graft in an ovine model of anterior cruciate ligament reconstruction.

The objective was to evaluate the performance of decellularised porcine superflexor tendon (pSFT) as an anterior cruciate ligament (ACL) reconstruction device. The ACL of adult sheep was reconstructed with decellularised pSFT or ovine allograft SFT and animals sacrificed at 4, 12 and 26 weeks (n = 4 per group) for biological evaluation and 26 weeks (n = 6) for biomechanical evaluation of the grafts. Both grafts showed good in vivo performance with no major differences at macroscopic evaluation post euthanasia. Histopathology revealed an inflammatory reaction to both grafts at 4 weeks, which reduced by 26 weeks. There was advanced cellular ingrowth from 12 weeks, ligamentisation of intra-articular grafts, ossification and formation of Sharpey's fibers at the graft/bone junctions. Immunohistochemistry showed that at 4 and 12 weeks, the host response was dominated by CD163+ M2 macrophages and a cell infiltrate comprising α-SMA + myofibroblasts, CD34+ and CD271+ progenitor cells. At 26 weeks the biomechanical properties of decellularised pSFT and oSFT grafts were comparable, with all grafts failing in the intra-articular region. This study provides new insight into constructive remodelling of tendons used for ACL replacement and evidence of integration and functional performance of a decellularised xenogeneic tendon with potential as an alternative for ACL reconstruction.

Translation of mechanical strain to a scalable biomanufacturing process for acellular matrix production from full thickness porcine bladders.

Bladder acellular matrix has promising applications in urological and other reconstructive surgery as it represents a naturally compliant, non-immunogenic and highly tissue-integrative material. As the bladder fills and distends, the loosely-coiled bundles of collagen fibres in the wall become extended and orientate parallel to the lumen, resulting in a physical thinning of the muscular wall. This accommodating property can be exploited to achieve complete decellularisation of the full-thickness bladder wall by immersing the distended bladder through a series of hypotonic buffers, detergents and nucleases, but the process is empirical, idiosyncratic and does not lend itself to manufacturing scale up. In this study we have taken a mechanical engineering approach to determine the relationship between porcine bladder size and capacity, to define the biaxial deformation state of the tissue during decellularisation and to apply these principles to the design and testing of a scalable novel laser-printed flat-bed apparatus in order to achieve reproducible and full-thickness bladder tissue decellularisation. We demonstrate how the procedure can be applied reproducibly to fresh, frozen or twice-frozen bladders to render8×8 cm2patches of DNA-free acellular matrix suitable for surgical applications.

Augmentation of the insufficient tissue bed for surgical repair of hypospadias using acellular matrix grafts: A proof of concept study.

Acellular matrices produced by tissue decellularisation are reported to have tissue integrative properties. We examined the potential for incorporating acellular matrix grafts during procedures where there is an inadequate natural tissue bed to support an enduring surgical repair. Hypospadias is a common congenital defect requiring surgery, but associated with long-term complications due to deficiencies in the quality and quantity of the host tissue bed at the repair site. Biomaterials were implanted as single on-lay grafts in a peri-urethral position in male pigs. Two acellular tissue matrices were compared: full-thickness porcine acellular bladder matrix (PABM) and commercially-sourced cross-linked acellular matrix from porcine dermis (Permacol™). Anatomical and immunohistological outcomes were assessed 3 months post-surgery. There were no complications and surgical sites underwent full cosmetic repair. PABM grafts were fully incorporated, whilst Permacol™ grafts remained palpable. Immunohistochemical analysis indicated a non-inflammatory, remodelling-type response to both biomaterials. PABM implants showed extensive stromal cell infiltration and neovascularisation, with a significantly higher density of cells (p < 0.001) than Permacol™, which showed poor cellularisation and partial encapsulation. This study supports the anti-inflammatory and tissue-integrative nature of non-crosslinked acellular matrices and provides proof-of-principle for incorporating acellular matrices during surgical procedures, such as in primary complex hypospadias repair.

An experimental simulation model to assess wear of the porcine patellofemoral joint.

A range of surgical techniques and osteochondral interventions have been developed for early stage chondral/osteochondral repair interventions in the knee however, methods for functional, pre-clinical assessment of these therapies are limited. In this study, a method for simulating physiological loading and motion in the porcine patellofemoral joint was developed using a 6-axis simulator. As an example of how the method can be used, the influence of surgical positioning of osteochondral allografts in the patella on cartilage wear, deformation and damage and graft stability was investigated in this porcine patellofemoral joint model. The functional performance of allografts implanted either optimally (flush with the cartilage surface) or 1 mm proud of the cartilage surface was compared to a positive control (stainless steel pin implanted 1 mm proud of the cartilage surface), a negative control (no intervention) and a defect model. Allografts implanted flush with the surrounding cartilage could restore the articulating surface of the patella resulting in low wear, damage and deformation of the opposing cartilage surface, similar to that of the negative control group. Implanting the graft proud of the patella surface resulted in cartilage lesions on the femoral trochlea (ICRS grade 2) and a cartilage volume difference of 2.0 ± 3.9 mm3; the positive controls resulted in more severe lesions, a higher volume difference (14.2 ± 7.4 mm3) which in some cases exposed subchondral bone (ICRS grade 4). Defects in the patella caused deformation of the opposing cartilage surface. All grafts implanted in the patella subsided over the duration of the study. This study demonstrated a method that can be used to evaluate osteochondral repair strategies in the patellofemoral joint applying physiological loading and motions.

Development of a specimen-specific in vitro pre-clinical simulation model of the human cadaveric knee with appropriate soft tissue constraints.

A human cadaveric specimen-specific knee model with appropriate soft tissue constraints was developed to appropriately simulate the biomechanical environment in the human knee, in order to pre-clinically evaluate the biomechanical and tribological performance of soft tissue interventions. Four human cadaveric knees were studied in a natural knee simulator under force control conditions in the anterior posterior (AP) and tibial rotation (TR) axes, using virtual springs to replicate the function of soft tissues. The most appropriate spring constraints for each knee were determined by comparing the kinematic outputs in terms of AP displacement and TR angle of the human knee with all the soft tissues intact, to the same knee with all the soft tissues resected and replaced with virtual spring constraints (spring rate and free length/degree). The virtual spring conditions that showed the least difference in the AP displacement and TR angle outputs compared to the intact knee were considered to be the most appropriate spring conditions for each knee. The resulting AP displacement and TR angle profiles under the appropriate virtual spring conditions all showed similar shapes to the individual intact knee for each donor. This indicated that the application of the combination of virtual AP and TR springs with appropriate free lengths/degrees was successful in simulating the natural human knee soft tissue function. Each human knee joint had different kinematics as a result of variations in anatomy and soft tissue laxity. The most appropriate AP spring rate for the four human knees varied from 20 to 55 N/mm and the TR spring rate varied from 0.3 to 1.0 Nm/°. Consequently, the most appropriate spring condition for each knee was unique and required specific combinations of spring rate and free length/degree in each of the two axes.

Decellularized Intervertebral Discs: A Potential Replacement for Degenerate Human Discs.

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 with attached bone using decellularization technology, the first step toward the development of an implantable regenerative device for IVD replacement.

Developing a Tooth in situ Organ Culture Model for Dental and Periodontal Regeneration Research.

In this study we have realized the need for an organ culture tooth in situ model to simulate the tooth structure especially the tooth attachment apparatus. The importance of such a model is to open avenues for investigating regeneration of the complex tooth and tooth attachment tissues and to reduce the need for experimental animals in investigating dental materials and treatments in the future. The aim of this study was to develop a porcine tooth in situ organ culture model and a novel bioreactor suitable for future studies of periodontal regeneration, including application of appropriate physiological loading. The Objectives of this study was to establish tissue viability, maintenance of tissue structure, and model sterility after 1 and 4 days of culture. To model diffusion characteristics within the organ culture system and design and develop a bioreactor that allows tooth loading and simulation of the chewing cycle. Methods: Twenty-one porcine first molars were dissected aseptically in situ within their bony sockets. Twelve were used to optimize sterility and determine tissue viability. The remainder were used in a 4-day organ culture study in basal medium. Sterility was determined for medium samples and swabs taken from all tissue components, using standard aerobic and anaerobic microbiological cultures. Tissue viability was determined at days 1 and 4 using an XTT assay and Glucose consumption assays. Maintenance of structure was confirmed using histology and histomorphometric analysis. Diffusion characteristics were investigated using micro-CT combined with finite element modeling. A suitable bioreactor was designed to permit longer term culture with application of mechanical loading to the tooth in situ. Result: XTT and Glucose consumption assays confirmed viability throughout the culture period for all tissues investigated. Histological and histomorphometric analysis confirmed maintenance of tissue structure. Clear microbiological cultures indicated maintenance of sterility within the organ culture system. The novel bioreactor showed no evidence of medium contamination after 4 days of culture. Finite element modeling indicated nutrient availability to the periodontium. Conclusion: A whole tooth in situ organ culture system was successfully maintained over 4 days in vitro.

Biomechanical assessment of the stability of osteochondral grafts implanted in porcine and bovine femoral condyles.

Osteochondral grafts are used clinically to repair cartilage and bone defects and to restore the congruent articulating surfaces of the knee joint following cartilage damage or injury. The clinical success of such osteochondral grafts is heavily reliant on the biomechanical and tribological properties of the surgical repair; however, a limited number of studies have investigated these factors. The aim of this study was to evaluate the influence of graft harvesting and implantation technique as well as bone properties on the primary stability of press-fit implanted osteochondral grafts using a series of uniaxial experimental push-in and push-out tests. Animal (porcine and bovine) knees were used to deliver models of different bone properties (elastic modulus and yield stress). The study showed the graft harvesting method using either a chisel or drill-aided trephine to have no influence on primary graft stability; however, the preparation technique for the graft recipient site was shown to influence the force required to push the graft into the host tissue. For example, when the length of the graft was equal to the recipient site (bottomed), the graft was more stable and dilation of the recipient site was shown to reduce short-term graft stability especially in immature or less dense bone tissue. The push-out tests which compared tissue of different skeletal maturities demonstrated that the maturity of both the graft and host bone tissue to influence the stability of the graft. A higher force was required to push out more skeletally mature grafts from mature bone tissue. The study demonstrates the importance of surgical technique and bone quality/properties on the primary stability and ultimately, the success of osteochondral grafts in the knee.

Stratifying the mechanical performance of a decellularized xenogeneic tendon graft for anterior cruciate ligament reconstruction as a function of graft diameter: An animal study.

OBJECTIVES: This study investigated the biomechanical performance of decellularized porcine superflexor tendon (pSFT) grafts of varying diameters when utilized in conjunction with contemporary ACL graft fixation systems. This aimed to produce a range of 'off-the-shelf' products with predictable mechanical performance, depending on the individual requirements of the patient. METHODS: Decellularized pSFTs were prepared to create double-bundle grafts of 7 mm, 8 mm, and 9 mm diameter. Femoral and tibial fixation systems were simulated utilizing Arthrex suspension devices and interference screws in bovine bone, respectively. Dynamic stiffness and creep were measured, followed by ramp to failure from which linear stiffness and load at failure were measured. The mechanisms of failure were also recorded. RESULTS: Dynamic stiffness was found to increase with greater graft diameter, with significant differences between all groups. Conversely, dynamic creep reduced with increasing graft diameter with significant differences between the 7 mm and 9 mm groups and the 8 mm and 9 mm groups. Significant differences were also found between the 7 mm, 8 mm, and 9 mm groups for linear stiffness, but no significant differences were found between groups for load at failure. The distribution of failure mechanisms was found to change with graft diameter. CONCLUSION: This study showed that decellularized pSFTs demonstrate comparable biomechanical properties to other ACL graft options and are a potentially viable option for ACL reconstruction. Although grafts can be stratified by their diameter to provide varying biomechanical properties, it may be more appropriate to alter the fixation technique to stratify for a greater diversity of biomechanical requirements.Cite this article: Bone Joint Res 2019;8:518-525.

Development of a pre-clinical experimental simulation model of the natural porcine knee with appropriate ligamentous constraints.

A robust and stratified pre-clinical natural knee model, which has the capability to more appropriately simulate the biomechanical environment in vivo, will deliver more efficient and reliable assessment of soft tissue interventions before clinical studies. In order to simulate the biomechanical function of the natural knee without the natural ligaments in place, there is a requirement to develop appropriate spring constraints for the natural knee model. Therefore, this study was to investigate the effect of spring constraints on the function and output of the natural porcine knee model, and determine the spring constraint which most closely replicated the function of the natural ligaments. Two linear compression springs with stiffnesses of 9 N/mm (spring-9) and 20 N/mm (spring-20) were set at different free lengths in the anterior-posterior (A/P) axis in a natural knee simulator. The kinematic (A/P displacement) and tribological properties (shear force) output of the simulator were compared at different spring settings. The most appropriate spring setting was determined by comparing the A/P displacement and shear force output at different spring settings with those of the all ligaments model. Spring-9 with a free length of 4 mm showed the minimal difference (-0.03±0.68 mm) in A/P displacement output and spring-20 with a free length of 5 mm showed the minimal difference (-0.10±0.73 mm) in A/P displacement output compared to the all ligament control. There was no statistical difference between the two minimal differences either in A/P displacement or in shear force (paired t-test, p = 0.58, and p = 0.68 respectively) when both spring settings matched most closely to the A/P kinematics of the intact knee. This indicated that both conditions were appropriate spring constraints settings in the A/P direction for the natural porcine knee model.

Decellularisation affects the strain rate dependent and dynamic mechanical properties of a xenogeneic tendon intended for anterior cruciate ligament replacement.

Development of new replacement grafts for anterior cruciate ligament (ACL) repair requires mechanical testing to ensure they can provide joint stability following implantation. A decellularised porcine superflexor tendon (pSFT) has been developed previously as an alternative to current reconstruction methods and subjected to biomechanical analysis. The application of varied strain rates to biological tissues is known to alter their biomechanical properties, however the effects of decellularisation on strain rate dependent and dynamic mechanical behaviour of tissues have not been explored. This study utilised tensile testing to investigate the material properties of native and decellularised pSFTs at three different strain rates (1%.s-1, 10%.s-1 and 100%.s-1). In addition, dynamic mechanical analysis (DMA) was used to ascertain the relative contributions of the solid and fluid phase components of the tissues. Ultimate tensile strength was significantly reduced in decellularised compared with native untreated pSFTs but was unaffected by strain rate. In contrast, toe region moduli increased with increasing strain rate for native tissues, but this effect was not observed in decellularised pSFTs. Linear region moduli were unaffected by strain rate, but were significantly reduced in decellularised pSFT compared with native tissue. Following DMA, significant reductions in dynamic modulus, storage modulus and loss modulus were seen in decellularised compared with native pSFT. Interestingly, the damping ability of the tendons was unaffected by decellularisation, suggesting that solid and fluid phases of the tissue were affected equally. These results, alongside previous studies, suggest that decellularisation affects collagen crimp, tissue swelling and collagen fibre sliding. However, despite these findings, the biomechanical properties of decellularised pSFT remain sufficient to act as an off-the-shelf solution for ACL reconstruction.

Simple geometry tribological study of osteochondral graft implantation in the knee.

Robust preclinical test methods involving tribological simulations are required to investigate and understand the tribological function of osteochondral repair interventions in natural knee tissues. The aim of this study was to investigate the effects of osteochondral allograft implantation on the local tribology (friction, surface damage, wear and deformation) of the tissues in the natural knee joint using a simple geometry, reciprocating pin-on-plate friction simulator. In addition, the study aimed to assess the ability of osteochondral grafts to restore a low surface damage, deformation and wear articulation when compared to the native state. A method was developed to characterise and quantify surface damage wear and deformation of the opposing cartilage-bone pin surface using a non-contacting optical profiler (Alicona Infinite Focus). Porcine 12 mm diameter cartilage-bone pins were reciprocated against bovine cartilage-bone plates that had 6 mm diameter osteochondral allografts, cartilage defects or stainless steel pins (positive controls) inserted centrally. Increased levels of surface damage with changes in geometry were not associated with significant increases in the coefficient of dynamic friction. Significant damage to the opposing cartilage surface was observed in the positive control groups. Cartilage damage, deformation and wear (as measured by change in geometry) in the xenograft (2.4 mm3) and cartilage defect (0.99 mm3) groups were low and not significantly different (p > 0.05) compared to the negative control in either group. The study demonstrated the potential of osteochondral grafts to restore the congruent articular surface and biphasic tribology of the natural joint. An optical method has been developed to characterise cartilage wear, damage and deformation that can be applied to the tribological assessment of osteochondral grafts in a whole natural knee joint simulation model.

Decellularization of human donor aortic and pulmonary valved conduits using low concentration sodium dodecyl sulfate.

The clinical use of decellularized cardiac valve allografts is increasing. Long-term data will be required to determine whether they outperform conventional cryopreserved allografts. Valves decellularized using different processes may show varied long-term outcomes. It is therefore important to understand the effects of specific decellularization technologies on the characteristics of donor heart valves. Human cryopreserved aortic and pulmonary valved conduits were decellularized using hypotonic buffer, 0.1% (w/v) sodium dodecyl sulfate and nuclease digestion. The decellularized tissues were compared to cellular cryopreserved valve tissues using histology, immunohistochemistry, quantitation of total deoxyribose nucleic acid, collagen and glycosaminoglycan content, in vitro cytotoxicity assays, uniaxial tensile testing and subcutaneous implantation in mice. The decellularized tissues showed no histological evidence of cells or cell remnants and >97% deoxyribose nucleic acid removal in all regions (arterial wall, muscle, leaflet and junction). The decellularized tissues retained collagen IV and von Willebrand factor staining with some loss of fibronectin, laminin and chondroitin sulfate staining. There was an absence of major histocompatibility complex Class I staining in decellularized pulmonary valve tissues, with only residual staining in isolated areas of decellularized aortic valve tissues. The collagen content of the tissues was not decreased following decellularization however the glycosaminoglycan content was reduced. Only moderate changes in the maximum load to failure of the tissues were recorded postdecellularization. The decellularized tissues were noncytotoxic in vitro, and were biocompatible in vivo in a mouse subcutaneous implant model. The decellularization process will now be translated into a good manufacturing practices-compatible process for donor cryopreserved valves with a view to future clinical use. Copyright © 2016 The Authors Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.

Development and characterisation of a low-concentration sodium dodecyl sulphate decellularised porcine dermis.

The aim of this study was to adapt a proprietary decellularisation process for human dermis for use with porcine skin. Porcine skin was subject to: sodium chloride (1 M) to detach the epidermis, trypsin paste to remove hair follicles, peracetic acid (0.1% v/v) disinfection, washed in hypotonic buffer and 0.1% (w/v) sodium dodecyl sulphate in the presence of proteinase inhibitors followed by nuclease treatment. Cellular porcine skin, decellularised porcine and human dermis were compared using histology, immunohistochemistry, GSL-1 lectin (alpha-gal epitope) staining, biochemical assays, uniaxial tensile and in vitro cytotoxicity tests. There was no microscopic evidence of cells in decellularised porcine dermis. DNA content was reduced by 98.2% compared to cellular porcine skin. There were no significant differences in the biomechanical parameters studied or evidence of cytotoxicity. The decellularised porcine dermis retained residual alpha-gal epitope. Basement membrane collagen IV immunostaining was lost following decellularisation; however, laminin staining was retained.

The effects of irradiation dose and storage time following treatment on the viscoelastic properties of decellularised porcine super flexor tendon.

Decellularised porcine super flexor tendon (pSFT) offers a promising solution to the replacement of damaged anterior cruciate ligament. It is desirable to package and terminally sterilise the acellular grafts to eliminate any possible harmful pathogens. However, irradiation techniques can damage the collagen structure and consequently reduce the mechanical properties. The aims of this study were to investigate the effects of irradiation sterilisation of varying dosages on the viscoelastic properties of the decellularised pSFT. Decellularised pSFT tendons were subjected to irradiation sterilisation using either 30kGygamma, 55kGygamma, 34kGy E-beam, 15kGygamma, 15kGy E-beam and (15+15) kGy E-beam (fractionated dose). Specimens then underwent stress relaxation testing at 0 and 12months post sterilisation to determine whether any effect on the viscoelastic properties was progressive. Significant differences were found which demonstrated that all irradiation treatments had an effect on the time-independent and time-dependent viscoelastic properties of irradiated tendons compared to peracetic acid only treated controls. No significant differences were found between the irradiated groups and no significant differences were found between groups at 0 and 12months. These results indicate the decellularised pSFT graft has a stable shelf-life.

Decellularization and Characterization of Porcine Superflexor Tendon: A Potential Anterior Cruciate Ligament Replacement.

The porcine superflexor tendon (SFT) was identified as having appropriate structure and properties for development of a decellularized device for use in anterior cruciate ligament reconstruction. SFTs were decellularized using a combination of freeze-thaw and washes in hypotonic buffer and 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer plus proteinase inhibitors, followed by nuclease treatment and sterilization using peracetic acid. The decellularized biological scaffold was devoid of cells and cell remnants and contained only 13 ng/mg (dry weight) residual total DNA. Immunohistochemistry showed retention of collagen type I and III and tenascin-C. Quantitative analysis of sulfated sugar and hydroxyproline content revealed a loss of glycosaminoglycans compared with native tissue, but no loss of collagen. The decellularized SFT was biocompatible in vitro and in vivo following implantation in a mouse subcutaneous model for 12 weeks. Uniaxial tensile testing to failure indicated that the gross material properties of decellularized SFTs were not significantly different to native tissue. Decellularized SFTs had an ultimate tensile strength of 61.8 ± 10.3 MPa (±95% confidence limits), a failure strain of 0.29 ± 0.04, and a Young's modulus of the collagen phase of 294.1 ± 61.9 MPa. Analysis of the presence of the α-Gal (galactose-α-1,3-galactose) epitope by immunohistochemistry, lectin binding, and antibody absorption assay indicated that the epitope was reduced, but still present post decellularization. This is discussed in light of the potential role of noncellular α-Gal in the acceleration of wound healing and tissue regeneration in the presence of antibodies to α-Gal.

The effects of irradiation on the biological and biomechanical properties of an acellular porcine superflexor tendon graft for cruciate ligament repair.

Acellular xenogeneic tissues have the potential to provide 'off-the-shelf' grafts for anterior cruciate ligament (ACL) repair. To ensure that such grafts are sterile following packaging, it is desirable to use terminal sterilization methods. Here, the effects of gamma and electron beam irradiation on the biological and biomechanical properties of a previously developed acellular porcine superflexor tendon (pSFT) were investigated. Irradiation following treatment with peracetic acid was compared to peracetic acid treatment alone and the stability of grafts following long-term storage assessed. Irradiation did not affect total collagen content or biocompatibility (determined using a contact cytotoxicity assay) of the grafts, but slightly increased the amount of denatured collagen in and decreased the thermal denaturation temperature of the tissue in a dose dependant fashion. Biomechanical properties of the grafts were altered by irradiation (reduced ultimate tensile strength and Young's modulus, increased failure strain), but remained superior to reported properties of the native human ACL. Long term storage at 4°C had no negative effects on the grafts. Of all the conditions tested, a dose of minimum 25 kGy of gamma irradiation had least effect on the grafts, suggesting that this dose produces a biocompatible pSFT graft with adequate mechanical properties for ACL repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2477-2486, 2017.