Development and characterisation of a decellularised bovine osteochondral biomaterial for cartilage repair.

It is proposed that an acellular natural osteochondral scaffold will provide a successful repair material for the early intervention treatment of cartilage lesions, to prevent or slow the progression of cartilage deterioration to osteoarthritis. Here, we investigated the efficacy of methods for the decellularisation of bovine osteochondral plugs. The plugs were subject to four freeze/thaw cycles followed by two cycles of washes in hypotonic solution and low concentration (0.1% w/v) sodium dodecyl sulphate with protease inhibitors. Plugs were treated with nuclease (DNase and RNase) treatment followed by sterilization in peracetic acid. Full tissue decellularisation was achieved as confirmed by histological analysis and DNA quantification, however the resultant acellular matrix had reduced glycosaminoglycan content which led to an increased percent deformation of cartilage. Furthermore, the acellular scaffold was not reproducibly biocompatible. Additional terminal washes were included in the process to improve biocompatibility, however, this led to visible structural damage to the cartilage. This damage was found to be minimised by reducing the cut edge to cartilage area ratio through decellularisation of larger cuts of osteochondral tissue.

Assay validation for the assessment of adipogenesis of multipotential stromal cells--a direct comparison of four different methods.

BACKGROUND AIMS: Mesenchymal 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. METHODS: Four 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. RESULTS: Oil 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. CONCLUSIONS: Flow 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.

Investigation of the suitability of decellularized porcine pericardium in mitral valve reconstruction.

BACKGROUND AND AIM OF THE STUDY: Autologous and glutaraldehyde-treated xenogeneic and homogeneic pericardium has been used extensively in mitral valve repair, but there are a number of limitations associated with its use. These include calcification, limited durability and lack of in vivo regeneration with glutaraldehyde-treated xenografts, as well as the sacrifice of the patient's own pericardium in the case of repair with autologous pericardium. The study aim was to investigate the suitability of decellularized porcine pericardium for heterotopic repair of the mitral valve leaflets, and its potential to regenerate through endogenous cell repopulation in vivo, or in vitro cell seeding prior to implantation. METHODS: Fresh porcine anterior and posterior mitral valve leaflets, together with fresh and decellularized porcine pericardium, were tested histologically, biochemically and biomechanically to investigate potential similarities and differences between the different types of tissue. Subsequently, the decellularized pericardial scaffolds were tested both in terms of biocompatibility, using contact and extract cytotoxicity assays, and in terms of regenerative capacity through porcine mesenchymal stem cell (pMSC) seeding. RESULTS: Histological examination of fresh pericardium and leaflets showed the typical trilaminar and quadlaminar structures of the two tissues, respectively. No cell remnants were observed in the decellularized pericardium, whereas the histoarchitecture of the collagen, elastin and glycosaminoglycan (GAG) matrix appeared well preserved. Significant differences were found in the GAG and hydroxyproline contents and the biomechanics between the leaflet and the pericardial groups. No indication of cytotoxicity was observed with the decellularized pericardial scaffolds. The optimum cell seeding density of pMSCs was 1 x 10(5) cells per cm2, which represented the lowest density at which the cells were capable of repopulating the scaffold by migrating through its full thickness. CONCLUSION: Porcine mitral valve leaflets and porcine fresh/decellularized pericardium shared similar histoarchitectures, but had different biochemical compositions and biomechanics. Decellularized pericardium was shown to be an optimum material for cell repopulation, delivering the necessary biological and biomechanical cues to seeded or migrating cells, and representing a plausible scaffold option for the regeneration of the mitral leaflets in vitro or in vivo, respectively.

Wear of surface-engineered metal-on-metal bearings for hip prostheses under adverse conditions with the head loading on the rim of the cup.

Clinical studies have found high wear rates, elevated ion levels and high revision rates of large-diameter metal-on-metal surface replacement bearings in some patients, which have been associated with edge loading of the head on the rim of the cup. We have simulated increased wear and ion levels in metal-on-metal bearings in vitro by introducing variations in translational and rotational positioning of the components, which reproduces stripe wear on the femoral head, cup rim wear and clinically relevant large as well as small wear particles. There is interest in technologies such as surface engineering, which might reduce metal wear and the release of wear particles and ions. Reduced wear with surface-engineered surface replacements compared to metal-on-metal controls has been reported under standard walking conditions with correctly aligned and concentric components. In this in vitro study, the wear of chromium nitride surface-engineered metal-on-metal bearings under conditions of microseparation associated with translational and rotational malpositioning of the components was investigated and the results were compared with a previously reported study of metal-on-metal bearings under the same conditions. Simulations were conducted using our unique hip simulation microseparation methodologies, which reproduce accelerated wear in metal-on-metal bearings and have previously been clinically validated with ceramic-on-ceramic bearings. Four of the six surface-engineered bearings had evidence of head contact on the rim of the cup, which produced stripe wear on the femoral head. Four of the six surface-engineered bearings (two without stripe and two with stripe wear) had lower wear than the previously reported high wearing metal-on-metal bearings. At 2 million cycles, two of the surface-engineered bearings had substantially increased wear rates, four times higher than the high wear rates previously reported for metal-on-metal bearings under the same conditions. There was wear through and cohesive failure of the thick atomic emission physical vapour deposition (AEPVD) chromium nitride (CrN) coating. At this point, the study was stopped to investigate the failure mode. This study highlights the need to pre-clinically investigate the tribology of new bearings under a wide set of clinical conditions as demonstrated by our stratified approach for enhanced reliability (SAFER) simulation methods. In adopting this SAFER approach to pre-clinical simulation testing of new bearings, it is important to communicate the failures as well as successes of technologies arising from the research, in order that the wider community can benefit from the analysis of the pre-clinical failure modes.

Development of a decellularised dermis.

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.

Comparison of human and animal femoral head chondral properties and geometries.

Investigations into tissue-preserving orthopaedic treatments should consider the tribology of articular cartilage; where simulations using animal joints are a predominant choice. However, very few studies have investigated the differences between human and animal cartilage. The aim of the present study was to characterise the differences in geometry and mechanical properties of human, porcine, bovine and ovine articular cartilage. Creep indentation was performed on osteochondral plugs taken from the superior region of femoral heads of all these species. Cartilage thickness was measured via the resistive force change of a needle descending through cartilage and bone. A biphasic finite element model was used to derive equilibrium elastic modulus and permeability. Results showed that human cartilage was significantly thicker than all other species tested. A positive correlation was found between femoral head diameter and cartilage thickness when comparing between species of quadrupeds. Human cartilage had the largest equilibrium elastic modulus, which was significant when comparing against porcine and bovine. However, porcine cartilage had significantly lower permeability. Significant differences in geometry and mechanical properties of articular cartilage were found between all species tested. It is necessary to consider these variations when choosing animal tissue to represent human.

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.

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.

Prediction of oxygen distribution in aortic valve leaflet considering diffusion and convection.

BACKGROUND: Oxygen supply and transport is an important consideration in the development of tissue engineered constructs. Previous studies from our group have focused on the effect of tissue thickness on the oxygen diffusion within a three-dimensional aortic valve leaflet model, and highlighted the necessity for additional transport mechanisms such as oxygen convection. The aims of this study were to investigate the effect of interstitial fluid flow within the aortic valve leaflet, induced by the cyclic loading of the leaflet, on oxygen transport. MATERIALS & METHODS: Indentation testing and finite element modelings were employed to derive the biphasic properties of the leaflet tissue. The biphasic properties were subsequently used in the computational modeling of oxygen convection in the leaflet, which was based on the effective interstitial fluid velocity and the tissue deformation. Subsequently, the oxygen profile was predicted within the valve leaflet model by solving the diffusion and convection equation simultaneously utilizing the finite difference method. RESULTS: The compression modulus (E) and hydraulic permeability were determined by adapting a finite element model to the experimental indentation test on valvular tissue, E = 0.05MPa, and k =2.0 mm4/Ns. Finite element model of oxygen convection in valvular tissue incorporating the predicted biphasic properties was developed and the interstitial fluid flow rate was calculated falling in range of 0.025-0.25 mm/s depending on the tissue depth. Oxygen distribution within valvular tissue was predicted using one-dimensional oxygen diffusion model taking into consider the interstitial fluid effect. It was found that convection did enhance the oxygen transport in valvular tissue by up to 68% increase in the minimum oxygen tension within the tissue, depending on the strain level of the tissue as reaction of the magnitude and frequencies of the cardiac loading. CONCLUSIONS: The effective interstitial fluid velocity was found to play an important role in enhancing the oxygen transport within the valve leaflet. Such an understanding is important in the development of valvular tissue engineered constructs.

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.

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.

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.

The genotoxicity of physiological concentrations of chromium (Cr(III) and Cr(VI)) and cobalt (Co(II)): an in vitro study.

Humans are exposed to chromium and cobalt in industry, from the environment and after joint replacement surgery from the CoCr alloy in the implant. In this study we have investigated whether Cr(III), Cr(VI), Co(II) and Cr in combination with Co could induce chromosome aberrations in human fibroblasts in vitro at the same concentrations that have been found in the peripheral blood of exposed humans. We used 24 colour M-FISH as a sensitive way to detect translocations and aneuploidy and examined the effects of a 24-h exposure and its consequences up to 30 days after the exposure in order to record genomic instability and/or repair. At these physiological doses the metals induced predominantly numerical rather than structural aberrations. Co was the least reactive and Cr(VI) especially in combination with Co the most. All metals at the highest physiological doses caused simple (gain or loss of 3 or less chromosomes) and complex (more than 49 chromosomes) aneuploidy. All metals at the lowest physiological dose caused a significant increase of total aberrations. Cr(VI) was much more effective than Cr(III) in causing chromosome fragments, which were only induced at the highest doses. There was a slow resolution of aneuploidy with time after exposure. This involved a reduction in the proportion of aneuploid cells and a reduction of the number of chromosomes within cells showing complex aneuploidy. We conclude that these metal ions can cause chromosome aberrations at physiological concentrations and that their main effect is aneugenic.

2009 Knee Society Presidential Guest Lecture: Polyethylene wear in total knees.

Knee arthroplasties in young and active patients place a substantial increase in the lifetime tribological demand and potential for wear-induced osteolysis. Polyethylene materials have advanced in recent years, reducing the potential for oxidative degradation and delamination failure. It is timely to consider tribological design variables and their potential to reduce surface wear and the long-term risk of osteolysis. The influence of reduced cross shear in rotating platform mobile-bearing knee designs and reduced surface wear area in low conforming fixed-bearing knees has been investigated. A reduction in cross shear substantially reduced wear in both multidirectional pin-on-plate studies and in rotating platform mobile-bearing designs in knee simulator studies. A reduction in bearing surface contact area substantially reduced surface wear in multidirectional pin-on-plate simulations and in low conforming fixed-bearing knee designs in knee simulator studies. This offers potential for a paradigm shift in knee design predicated by enhanced mechanical properties of new polymer materials. We describe two distinct low-wearing tribological design solutions: (1) a rotating platform design solution with reduced cross shear provides reduced wear with conformity and intrinsic stability; and (2) a low conformity fixed bearing with reduced surface area, provides reduced wear, but has less intrinsic stability and requires good soft tissue function.

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.

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.

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.