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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

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.

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 render $8 \times 8$ cm2 patches of DNA-free acellular matrix suitable for surgical applications.

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.

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.

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

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.

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.

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.

In order to pre-clinically evaluate the performance and efficacy of novel osteochondral interventions, physiological and clinically relevant whole joint simulation models, capable of reproducing the complex loading and motions experienced in the natural knee environment are required. The aim of this study was to develop a method for the assessment of tribological performance of osteochondral grafts within an in vitro whole natural joint simulation model. The study assessed the effects of osteochondral allograft implantation (existing surgical intervention for the repair of osteochondral defects) on the wear, deformation and damage of the opposing articular surfaces. Tribological performance of osteochondral grafts was compared to the natural joint (negative control), an injury model (focal cartilage defects) and stainless steel pins (positive controls). A recently developed method using an optical profiler (Alicona Infinite Focus G5, Alicona Imaging GmbH, Austria) was used to quantify and characterise the wear, deformation and damage occurring on the opposing articular surfaces. Allografts inserted flush with the cartilage surface had the lowest levels of wear, deformation and damage following the 2 h test; increased levels of wear, deformation and damage were observed when allografts and stainless steel pins were inserted proud of the articular surface. The method developed will be applied in future studies to assess the tribological performance of novel early stage osteochondral interventions prior to in vivo studies, investigate variation in surgical precision and aid in the development of stratified interventions for the patient population.

The aims of this study were to develop a biological large diameter vascular graft by decellularisation of native human aorta to remove the immunogenic cells whilst retaining the essential biomechanical, and biochemical properties for the ultimate benefit of patients with infected synthetic grafts. Donor aortas (n = 6) were subjected to an adaptation of a propriety decellularisation process to remove the cells and acellularity assessed by histological analysis and extraction and quantification of total DNA. The biocompatibility of the acellular aortas was determined using standard contact cytotoxicity tests. Collagen and denatured collagen content of aortas was determined and immunohistochemistry was used to determine the presence of specific extracellular matrix proteins. Donor aortas (n = 6) were divided into two, with one half subject to decellularisation and the other half retained as native tissue. The native and decellularised aorta sections were then subject to uniaxial tensile testing to failure [axial and circumferential directions] and suture retention testing. The data was compared using a paired t-test. Histological evaluation showed an absence of cells in the treated aortas and retention of histoarchitecture including elastin content. The decellularised aortas had less than 15 ng mg¯¹ total DNA per dry weight (mean 94% reduction) and were biocompatible as determined by in vitro contact cytotoxicity tests. There were no gross changes in the histoarchitecture [elastin and collagen matrix] of the acellular aortas compared to native controls. The decellularisation process also reduced calcium deposits within the tissue. The uniaxial tensile and suture retention testing revealed no significant differences in the material properties (p > 0.05) of decellularised aorta. The decellularisation procedure resulted in minimal changes to the biological and biomechanical properties of the donor aortas. Acellular donor aorta has excellent potential for use as a large diameter vascular graft.

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 30 kGy gamma, 55 kGy gamma, 34 kGy E-beam, 15 kGy gamma, 15 kGy E-beam and (15 + 15) kGy E-beam (fractionated dose). Specimens then underwent stress relaxation testing at 0 and 12 months 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 12 months. These results indicate the decellularised pSFT graft has a stable shelf-life.

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.

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, 2016.

Anterior cruciate ligament rupture is rising in its prevalence amongst the young and those with physically active lifestyles. Acellular human patellar tendon (PT) grafts offer a promising restoration solution, returning knee joint stability and overcoming some of the current disadvantages of autologous or allogeneic grafts. However, it is necessary to ensure that the decellularisation bio-processes involved do not cause structural changes in the microstructure of the tendon tissue that may adversely affect the mechanical properties, particularly with respect to the physiological range of loading.Sixteen cadaveric human PT grafts were sourced and processed from eight donors, with full ethical approval and consent for use in research. Eight specimens were allocated for decellularisation, while the remaining eight contralateral specimens were used as native controls. Testing consisted of 12 preconditioning cycles followed by uniaxial extension until failure occurred. Stress–strain data was then fitted to a bi-linear model using least squares regression by a custom-written Matlab script. The elastic moduli for the toe region and linear region of each specimen were determined, in addition to the transition point co-ordinates and strain energy density for increasing strain. No significant differences were found between groups for all of the parameters investigated. Hence, the shape and magnitude of the stress–strain profile was found to be the same for both groups throughout loading.The results of this study indicated that decellularisation appeared to have no effect on the material properties of human PT grafts under quasistatic conditions. Therefore, acellular human PT grafts can offer a viable additional solution for ACL replacement compared to current autologous and allogeneic treatment options.

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.

Supplemental Digital Content is available in the text, Study Design. An in vitro study of the intervertebral disc (IVD) structure using 9.4T magnetic resonance imaging (MRI). Objective. Investigate the potential of ultrahigh-field strength MRI for higher quality 3-dimensional (3D) volumetric MRI datasets of the IVD to better distinguish structural details. Summary of Background Data. MRI has the advantages of being nondestructive and 3D in comparison to most techniques used to obtain the structural details of biological tissues, however, its poor image quality at higher resolution is a limiting factor. Ultrahigh-field MRI could improve the imaging of biological tissues but the current understanding of its application for spinal tissue is limited. Methods. 2 ovine spinal segments (C7–T1, T2–T3) containing the IVD were separately imaged using 2 sequences; 3D spin echo (multislice-multiecho) pulse sequence for the C7–T1 sample and 3D gradient echo (fast-low-angle-shot) pulse sequence for the T2–T3 sample. The C7–T1 sample was subsequently decalcified and imaged again using the same scanning parameters. Histological sections obtained from the decalcified sample were stained followed by digital scanning. Observations from corresponding MRI slices and histological sections were compared as a method of confirmation of morphology captured under MRI. The signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR) and relative-contrast values were calculated for quantitative evaluation of image quality. Results. Measurements from histology sections and corresponding MRI slices matched well. Both sequences revealed finer details of the IVD structure. Under the spin echo sequence, the annulus lamellae architecture was distinguishable and the SNR and CNR values were higher. The relative contrast was considerably higher between high (nucleus) and low (bone) signal constituents, but between the nucleus and the annulus the relative contrast was low. Under the gradient echo sequence, although the relative contrasts between constituents were poor, the fiber orientation was clearly manifested. Conclusion. The obtained positive results demonstrate the potential of ultrahigh-field strength MRI to nondestructively capture the IVD structure. Level of Evidence: N/A

A review of research undertaken to evaluate the biomechanical stability and biotribological behaviour of osteochondral grafts in the knee joint and a brief discussion of areas requiring further improvement in future studies are presented. The review takes into consideration osteochondral autografts, allografts, tissue engineered constructs and synthetic and biological scaffolds.

The successful development of early-stage cartilage and meniscus repair interventions in the knee requires biomechanical and biotribological understanding of the design of the therapeutic interventions and their tribological function in the natural joint. The aim of this study was to develop and validate a porcine knee model using a whole joint knee simulator for investigation of the tribological function and biomechanical properties of the natural knee, which could then be used to pre-clinically assess the tribological performance of cartilage and meniscal repair interventions prior to in vivo studies. The tribological performance of standard artificial bearings in terms of anterior–posterior (A/P) shear force was determined in a newly developed six degrees of freedom tribological joint simulator. The porcine knee model was then developed and the tribological properties in terms of shear force measurements were determined for the first time for three levels of biomechanical constraints including A/P constrained, spring force semi-constrained and A/P unconstrained conditions. The shear force measurements showed higher values under the A/P constrained condition (predominantly sliding motion) compared to the A/P unconstrained condition (predominantly rolling motion). This indicated that the shear force simulation model was able to differentiate between tribological behaviours when the femoral and tibial bearing was constrained to slide or/and roll. Therefore, this porcine knee model showed the potential capability to investigate the effect of knee structural, biomechanical and kinematic changes, as well as different cartilage substitution therapies on the tribological function of natural knee joints.

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 older sheep had a much higher elastic modulus and was less permeable than other species.

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 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.

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.

Shortage of donor organs combined with ever increasing liver disease has directed attention towards growing implantable liver tissue in the laboratory setting. Recent developments in decellularisation and recellularisation techniques have opened exciting facets for liver whole organ reengineering. The aim of this study was to develop a decellularisation protocol for human liver, assess the structural and biochemical integrity and assess the biocompatibility of the acellular matrix. Discarded human livers were split and the left lateral and right posterior lobes with a mean weight of 399 ± 162 g were used (n=8 lobes). Perfusion decellularisation was carried out through the hepatic artery and portal vein. Hypotonic buffer, 0.1 % (w/v) sodium dodecyl sulphate, hypertonic buffer and nuclease solutions were used to remove cells plus nuclear material. Following several iterations of a previously patented Leeds University decellularisation protocol, greater than 91.0 % (w/w) DNA was removed from all areas of liver tissue. Fluoroscopy confirmed integrity of vascular tree. Histological analysis demonstrated lack of cells and maintenance of the portal triad histioarchitecture. Immunohistochemistry demonstrated positive staining for key extracellular proteins such as collagen type I and III, fibronectin and laminin. Scanning electron microscopy showed an intact extracellular matrix. Biochemical assessment confirmed significant reduction in glycosaminoglycan content, but there was increase in collagen content, the latter likely due to extraction by dry weight of other soluble components in the matrix. The acellular tissues and extracts were not cytotoxic to either murine 3T3 or baby hamster kidney cells. Preliminary recellularisation (n=3 lobes) showed the matrix to be biocompatible to primary cryopreserved human hepatocytes seeded at 30,000 cells. The study has developed a suitable protocol for decellularisation of human liver lobes, without adversely affecting the extracellular matrix. The biocompatible acellular scaffold has the potential to be recellularised with hepatocytes, with a view to engineering a transplantable auxiliary liver graft.

Medical interventions for the treatment of spinal disc degeneration include total disc replacement and fusion devices. There are, however, concerns regarding the generation of wear particles by these devices, the majority of which are in the nanometre sized range with the potential to cause adverse biological effects in the surrounding tissues. The aims of this study were to develop an organ culture model of the porcine dura mater and to investigate the biological effects of CoCr nanoparticles in this model. A range of histological techniques were used to analyse the structure of the tissue in the organ culture. The biological effects of the CoCr wear particles and the subsequent structural changes were assessed using tissue viability assays, cytokine assays, histology, immunohistochemistry, and TEM imaging. The physiological structure of the dura mater remained unchanged during the seven days of in vitro culture. There was no significant loss of cell viability. After exposure of the organ culture to CoCr nanoparticles, there was significant loosening of the epithelial layer, as well as the underlying collagen matrix. TEM imaging confirmed these structural alterations. These structural alterations were attributed to the production of MMP-1, -3, -9, -13, and TIMP-1. ELISA analysis revealed that there was significant release of cytokines including IL-8, IL-6, TNF-α, ECP and also the matrix protein, tenascin-C. This study suggested that CoCr nanoparticles did not cause cytotoxicity in the dura mater but they caused significant alterations to its structural integrity that could lead to significant secondary effects due to nanoparticle penetration, such as inflammation to the local neural tissue.

The meniscus plays an important role in the biomechanics and tribology of the knee joint. Damage to or disease of the meniscus is now recognized to predispose to the development of osteoarthritis. Treatment of meniscal injury through arthroscopic surgery has become one of the most common orthopedic surgical procedures, and in the United States this can represent 10 to 20% of procedures related to the knee. The meniscus has a limited healing capacity constrained to the vascularized periphery and therefore, surgical repair of the avascular regions is not always feasible. Replacement and repair of the meniscus to treat injuries is being investigated using tissue engineering strategies. Promising as these approaches may be, there are, however, major barriers to overcome before translation to the clinic.

Tissue engineered therapies are emerging as solutions to several of the medical challenges facing aging societies. To this end, a fundamental research goal is the development of novel biocompatible materials and scaffolds. Self-assembling peptides are materials that have undergone rapid development in the last two decades and they hold promise in meeting some of these challenges. Using amino acids as building blocks enables a great versatility to be incorporated into the structures that peptides form, their physical properties and their interactions with biological systems. This review discusses several classes of short self-assembling sequences, explaining the principles that drive their self-assembly into structures with nanoscale ordering, and highlighting in vitro and in vivo studies that demonstrate the potential of these materials as novel soft tissue engineering scaffolds.

The introduction of metal-on-metal total disc replacements motivated studies to evaluate the effects of cobalt-chromium (CoCr) nanoparticles on cells of the dura mater. Porcine fibroblasts and epithelial cells isolated from the dura mater were cultured with clinically-relevant CoCr nanoparticles and the ions, generated by the particles over 24 h, at doses up to 121 μm(3)per cell. Cell viability and production of proinflammatory cytokines was assessed over 4 days. The capacity of the particles to induce oxidative stress in the cells was evaluated at 24 h. The CoCr particles and their ions significantly reduced the viability of the dural epithelial cells in a dose-dependent manner but not the fibroblasts. Both cell types secreted IL-8 in response to particle exposure at doses of 60.5 μm(3) (epithelial cells) and 121 μm(3) (fibroblasts, epithelial cells) per cell. No significant release of IL-6 was observed in both cell types at any dose. Reactive oxygen species were induced in both cell types at 50 μm(3) per cell after 24 h exposure. The data suggested novel differences in the resistance of the dural epithelial cells and fibroblasts to CoCr nanoparticle/ion toxicity and demonstrated the inflammatory potential of the particles. The data contributes to a greater understanding of the potential biological consequences of the use of metal-on-metal total disc prostheses.

Patients with cobalt chrome (CoCr) metal-on-metal (MOM) implants may be exposed to a wide size range of metallic nanoparticles as a result of wear. In this study we have characterised the biological responses of human fibroblasts to two types of synthetically derived CoCr particles [(a) from a tribometer (30 nm) and (b) thermal plasma technology (20, 35, and 80 nm)] in vitro, testing their dependence on nanoparticle size or the generation of oxygen free radicals, or both. Metal ions were released from the surface of nanoparticles, particularly from larger (80 nm) particles generated by thermal plasma technology. Exposure of fibroblasts to these nanoparticles triggered rapid (2 h) generation of reactive oxygen species (ROS) that could be eliminated by inhibition of NADPH oxidase, suggesting that it was mediated by phagocytosis of the particles. The exposure also caused a more prolonged, MitoQ sensitive production of ROS (24 h), suggesting involvement of mitochondria. Consequently, we recorded elevated levels of aneuploidy, chromosome clumping, fragmentation of mitochondria and damage to the cytoskeleton particularly to the microtubule network. Exposure to the nanoparticles resulted in misshapen nuclei, disruption of mature lamin B1 and increased nucleoplasmic bridges, which could be prevented by MitoQ. In addition, increased numbers of micronuclei were observed and these were only partly prevented by MitoQ, and the incidence of micronuclei and ion release from the nanoparticles were positively correlated with nanoparticle size, although the cytogenetic changes, modifications in nuclear shape and the amount of ROS were not. These results suggest that cells exhibit diverse mitochondrial ROS-dependent and independent responses to CoCr particles, and that nanoparticle size and the amount of metal ion released are influential.

Vitamin E (VE) has been added to ultrahigh-molecular-weight polyethylene (UHMWPE) acetabular cups and tibial trays primarily to reduce oxidative damage to the polymer. The aim of this study was to investigate the relative wear rates of UHMWPE-containing VE compared with virgin UHMWPE. The ability of VE to reduce the amount of inflammatory cytokines produced from stimulated peripheral blood mononuclear cells (PBMNCs) was also investigated. Stimulation was achieved by exposure of PBMNCs to either lipoplysaccharide (LPS) or VE-containing UHMWPE (VE-UHMWPE). In the present study, results showed that the wear rates of UHMWPE with or without VE were not significantly different. Particles generated by UHMWPE with and without VE were not significantly different in size distribution. The production of osteolytic mediators, tumor necrosis factor-alpha, interleukin 1β (IL-β), IL-6, and IL-8 were significantly reduced in (PBMNCs) stimulated with either LPS + VE compared with LPS or VE-UHMWPE particles compared to virgin UHMWPE particles. This trend was also observed when VE was added as a liquid to UHMWPE wear particle-stimulated PBMNCs. The exact mechanism of how VE affects the release of inflammatory mediators from particle-stimulated macrophages is not yet understood. It is likely to involve the anti-inflammatory and/or antioxidant effects of VE.

Background aimsMesenchymal stromal cells (MSCs) are regenerative and immuno-privileged cells that are used for both tissue regeneration and treatment of severe inflammation-related disease. For quality control of manufactured MSC batches in regard to mature fat cell contamination, a quantitative method for measuring adipogenesis is needed.MethodsFour previously proposed methods were validated with the use of bone marrow (BM) MSCs during a 21-day in vitro assay. Oil red staining was scored semiquantitatively; peroxisome proliferator activated receptor-γ and fatty acid binding protein (FABP)4 transcripts were measured by quantitative real-time polymerase chain reaction; FABP4 protein accumulation was evaluated by flow cytometry; and Nile red/4′,6-diamidino-2-phenylindole (DAPI) ratios were measured in fluorescent microplate assay. Skin fibroblasts and MSCs from fat pad, cartilage and umbilical cord were used as controls.ResultsOil red staining indicated considerable heterogeneity between BM donors and individual cells within the same culture. FABP4 transcript levels increased 100- to 5000-fold by day 21, with large donor variability observed. Flow cytometry revealed increasing intra-culture heterogeneity over time; more granular cells accumulated more FABP4 protein and Nile red fluorescence compared with less granular cells. Nile red increase in day-21 MSCs was ∼5- and 4-fold, measured by flow cytometry or microplate assay, respectively. MSC proliferation/apoptosis was accounted through the use of Nile red/DAPI ratios; adipogenesis levels in day-21 BM MSCs increased ∼13-fold, with significant correlations with oil red scoring observed for MSC from other sources.ConclusionsFlow cytometry permits the study of MSC differentiation at the single-cell level and sorting more and less mature cells from mixed cell populations. The microplate assay with the use of the Nile red/DAPI ratio provides rapid quantitative measurements and could be used as a low-cost, high-throughput method to quality-control MSC batches from different tissue sources.

The use of nanoparticles in medicine is ever increasing, and it is important to understand their targeted and non-targeted effects. We have previously shown that nanoparticles can cause DNA damage to cells cultured below a cellular barrier without crossing this barrier. Here, we show that this indirect DNA damage depends on the thickness of the cellular barrier, and it is mediated by signalling through gap junction proteins following the generation of mitochondrial free radicals. Indirect damage was seen across both trophoblast and corneal barriers. Signalling, including cytokine release, occurred only across bilayer and multilayer barriers, but not across monolayer barriers. Indirect toxicity was also observed in mice and using ex vivo explants of the human placenta. If the importance of barrier thickness in signalling is a general feature for all types of barriers, our results may offer a principle with which to limit the adverse effects of nanoparticle exposure and offer new therapeutic approaches.

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.

The purpose of this investigation was to develop a decellularised human dermis suitable for allografting. Samples of human skin were obtained from deceased donors and taken through a series of steps to remove all cellular material. The steps were: chemical removal of the epidermis, disinfection, lysing of cells in hypotonic buffer, a detergent treatment and a nuclease buffer to remove residual nuclear material. Histological preparations of the decellularised dermis produced were then investigated. In addition residual DNA content, structural strength, collagen denaturation, cytotoxicity and in vivo tissue reactivity following implantation in a murine model were examined. For all donors tested there was no change in morphology as viewed by light microscopy. Mean DNA removal was evaluated at 92.1%. There were no significant changes in structural strength or evidence of collagen degradation. The tissue did not appear to be cytotoxic or elicit an immune response when implanted in the mouse model. A decellularised tissue has been developed that would appear to be suitable for a range of surgical procedures.

A new Stratified Approach For Enhanced Reliability (SAFER) pre-clinical simulation testing of joint prostheses has been described in a preceding paper in this volume. The application of SAFER in vitro simulation and testing to metal-on-metal bearings is described in this review paper. The review aims to provide further understanding of the reasons for, and causes of, increased wear in metal-on-metal hips in a proportion of patients. Variation in positioning (mal-positioning) of the head and cup in hip prostheses results in the head contacting the rim of the cup and producing increased wear. Variation in both translational and rotational positioning has been investigated. Variation in translational positioning of the centres of the head and cup, which is not detected on radiographs, is a frequent occurrence clinically and can result in a substantial increase in wear rate. The variation in translational positioning acts synergistically with variation in rotational positioning to produce substantial increases in wear. These recent findings are consistent with the wear mechanisms and formation of stripe wear reported for ceramic-on-ceramic bearings over a decade ago, and provide insight into the reasons for the variation and increases in the wear rate found clinically in metal-on-metal hips in specific patients, which may cause premature failure.

Over the past 20 years self-assembling peptides have been shown to be of value as scaffolds in the field of tissue engineering.1 Self-assembling peptides offer many advantages. Their complex nanostructures can be easily synthesised from simple precursors. It is possible to control their chemical functionality to make them environmentally responsive, for example to pH or temperature. Their biological activity can be modified by the inclusion of bioactive sequences, such as cell adhesion motifs. The development of distinct self-assembling peptide systems with various functionalities will be important for tailoring and optimising biological performance, especially in relation to active scaffolds for tissue engineering and regenerative medicine. A variety of peptides have been rationally designed to spontaneously self-assemble into hierarchical structures in solution and in response to specific physico-chemical environmental triggers.2 Under controlled conditions self-assembling peptides undergo one-dimensional self-assembly forming single molecule thick, micrometer-long beta-sheet nanotapes. Further assembly can be induced such that the nanotapes stack in pairs to form ribbons which further assemble to form fibrils and pairs of fibrils can then entwine to form fibres. Self-assembling peptide hydrogels represent valuable systems for delivering cells embedded within a scaffold. Single component self-assembling systems allow for sol-gel transitions in which the peptide is monomeric, for example within a syringe at room temperature at an injection site, but self-assembles into a gel state at 37 °C upon injection. An alternative approach is to use a binary system in which two separate complementary peptides that cannot self-assemble individually are co-delivered to the injection site and, upon mixing, self-assemble into a gel state. Such systems have been previously documented.2k The Aggeli group have designed over twenty self-assembling peptides to self-assemble under various physico-chemical conditions. In earlier work we exploited the propensity of peptide chains to self-assemble via intermolecular hydrogen bonding into β-sheet structures,2a,b,i and demonstrated that oligopeptides can be designed to self-assemble into micrometer-long β-sheet tapes.2a In order to produce β-sheet tapes in solution, we used the following working criteria: (i) cross-strand attractive forces (hydrophobic, electrostatic, hydrogen-bonding) between side-chains, (ii) lateral recognition between adjacent β-strands to constrain their self-assembly to one dimension, and avoid heterogeneous aggregated β-sheet structures, and (iii) strong adhesion of solvent to the surface of the tapes to control solubility.2a,b,i Based on these criteria, a de novo 11-residue peptide DN1 (later called P11-2; CH3CO-Gln-Gln-Arg-Phe-Gln-Trp-Gln-Phe-Glu-Gln-Gln-NH2) was designed to form β-sheet polymer tapes in water. Subsequently, the P11-family of β-sheet-forming self-assembling peptides were rationally designed. Subtle changes in peptide design can lead to control of mechanical properties, surface chemistry, morphology, bioactivity and responsiveness of the self-assembly of these materials to external chemical triggers (pH, ionic strength).2c,d

Study Design An in-vitro study of the wear rates of the Charite lumbar total disc replacement.