Osteoarthritis (OA) is a leading cause of joint deformity and functional limitation. An imbalance of anabolic and catabolic activity results in destruction of the extracellular matrix of articular cartilage. There is evidence to support the role of DNA methylation in the pathogenesis of OA, but the effect of other epigenetic modifiers is yet to be described. This study looks at the effect of novel epigenetic modulators, PFI-1, a bromodomain inhibitor, and SGC707, a histone methytransferase inhibitor, and their effects on gene expression in the pathogenesis of OA. Chondrocytes were extracted from OA femoral heads (n=6), cultured and incubated. Samples were treated with media alone (control), interleukin 1-beta (IL-1β) plus oncostatin M (OSM) alone, or in combination with increasing concentrations of PFI-1 or SGC707. Levels of expression of iNOS, COX2, IL8, IL1B, matrix metalloproteinase-13 (MMP13), RUNX2 and COL9A1 were measured using qRT-PCR, and expressed relative to GAPDH. PFI-1 (0.5 and 5µM) suppressed expression of catabolic genes in OA chondrocytes, at basal levels and when co-stimulated with IL-1β+OSM. Catabolic gene expression decreased (iNOS, COX2, IL-8, IL-1β and MMP), and RUNX2 expression was also supressed. There was no effect on expression of the anabolic gene COL9A1. SGC707 (0.1 and 1µM) did not induce a reduction in expression of all the catabolic genes. This study has demonstrated that PFI-1 has a potent protective effect against cartilage degradation, by modulating the expression of catabolic genes in OA chondrocytes. This further validates the role of epigenetics in OA, with implications for therapeutic interventions in the future.

Advances in our understanding of skeletal stem cells and their role in bone development and repair, offer the potential to open new frontiers in bone regeneration. However, the ability to harness these cells to replace or restore the function of traumatised or lost skeletal tissue as a consequence of age or disease remains a significant challenge. We have developed protocols for the isolation, expansion and translational application of skeletal cell populations with cues from developmental biology informed by in vitro and ex vivo models as well as, nanoscale architecture and biomimetic niche development informing our skeletal tissue engineering approaches. We demonstrate the importance of biomimetic cues and delivery strategies to directly modulate differentiation of human adult skeletal cells and, central to clinical application, translational studies to examine the efficacy of skeletal stem and cell populations in innovative scaffold compositions for orthopaedics. While a number of challenges remain multidisciplinary approaches that integrate developmental and engineering processes as well as cell, molecular and clinical techniques for skeletal tissue engineering offer significant promise. Harnessing such approaches across the hard tissue interface will ultimately improve the quality of life of an increasing ageing population.

Background

Following endosteal uncemented orthopaedic device implantation, the initial implant/bone interface retains spaces and deficiencies further exacerbated by pressure necrosis and resultant bone resorption. This implant-bone space requires native bone infill through the process of de novo osteogenesis. New appositional bone formation on the implant surface is known as contact osteogenesis and is generated by osteogenic cells, including skeletal stem cells (SSCs), colonising the implant surface and depositing the extracellular bone matrix. Surface nanotopographies provide physical cues capable of triggering SSC differentiation into osteoblasts, thus inducing contact osteogenesis, translated clinically into enhanced osseointegration and attainment of secondary stability. The current study has investigated the in vitro and in vivo effects of unique nanotopographical pillar substrates on SSC phenotype and function.

Background

In 2012, the National Joint Registry recorded 86,488 primary total hip replacements (THR) and 9,678 revisions (1). To date aseptic loosening remains the most common cause of revision in hip and knee arthroplasty, accounting for 40% and 32% of all cases respectively and emphasising the need to optimise osseointegration in order to reduce revisions. Clinically, osseointegration results in asymptomatic stable durable fixation of orthopaedic implants. Osseointegration is a complex process involving a number of distinct mechanisms affected by the implant surface topography, which is defined by surface orientation and surface roughness. Micro- and nano-topography levels have discrete effects on implant osseointegration and yet the role on cell function and subsequent bone implant function is unknown. Nanotopography such as collagen banding is a critical component influencing the SSC niche in vivo and has been shown to influence a range of cell behaviours in vitro (2,3). We have used unique fabricated nanotopographical pillar substrates to examine the function of human bone stem cells on titanium surfaces.

Recently, the osteoregenerative properties of allograft have been enhanced by addition of autogenous skeletal stem cells to treat orthopaedic conditions characterised by lost bone stock. There are multiple disadvantages to allograft, and trabecular tantalum represents a potential alternative. This metal is widely used, although in applications where there is poor initial stability, or when it is used in conjunction with bone grafting, loading may need to be limited until sound integration has occurred. Strategies to speed up implant incorporation to surrounding bone are therefore required. This may improve patient outcomes, extending the clinical applications of tantalum as a substitute for allograft.

Background

Impaction bone grafting with milled human allograft is the gold standard for replacing lost bone stock during revision hip surgery. Problems surrounding the use of allograft include cost, availability, disease transmission and stem subsidence (usually due to shear failure of the surrounding allograft). Aims. To investigate various polymers for use as substitute allograft. The ideal graft would be a composite with similar mechanical characteristics as allograft, and with the ability to form de novo bone.

Background

Replacing bone lost as a consequence of trauma or disease is a major challenge in the treatment of musculoskeletal disorders. Tissue engineering strategies seek to harness the potential of stem cells to regenerate lost or damaged tissue. Bone marrow aspirate (BMA) provides a promising autologous source of skeletal stem cells (SSCs) however, previous studies have demonstrated that the concentration of SSCs required for robust tissue regeneration is below levels present in iliac crest BMA, emphasising the need for cell enrichment strategies prior to clinical application.

Background

Skeletal stem cells (SSCs) have been used for the treatment of osteonecrosis of the femoral head to prevent subsequent collapse. In isolation SSCs do not provide structural support but an innovative case series in Southampton, UK, has used SSCs in combination with impaction bone grafting (IBG) to improve both the biological and mechanical environment and to regenerate new bone at the necrotic site.

Aims

Disease transmission, availability and economic costs of allograft have resulted in significant efforts into finding an allograft alternative for use in impaction bone grafting (IBG). Biotechnology offers the combination of skeletal stem cells (SSC) with biodegradable polymers as a potential solution. Recently polymers have been identified with both structural strength and SSC compatibility that offer the potential for clinical translation.

The aim of this study was to assess whether increasing the porosity of one such polymer via super critical CO₂ fluid foaming (SCF) enhanced the mechanical and cellular compatibility characteristics for use as an osteogenic alternative to allograft in IBG.

Aims

Impaction bone grafting with milled human allograft is the gold standard for replacing lost bone stock during revision hip surgery. Problems surrounding the use of allograft include cost, availability, disease transmission and stem subsidence (usually due to shear failure of the surrounding allograft).

The aim of this study was to investigate various polymers for use as substitute allograft. The ideal graft would be a composite with similar mechanical characteristics as allograft, and with the ability to form de novo bone.

The osteo-regenerative properties of allograft have recently been enhanced by addition of autogenous skeletal stem cells to treat orthopaedic conditions characterised by lost bone stock. There are however, multiple disadvantages to allograft, including cost, availability, consistency and potential for disease transmission, and trabecular tantalum represents a potential alternative. Tantalum is already in widespread orthopaedic use, although in applications where there is poor initial implant stability, or when tantalum is used in conjunction with bone grafting, loading may need to be limited until sound integration has occurred. Development of enhanced bone-implant integration strategies will improve patient outcomes, extending the clinical applications of tantalum as a substitute for allograft.

The aim of this study was to examine the osteoconductive potential of trabecular tantalum in comparison to human allograft to determine its potential as an alternative to allograft.

Human bone marrow stromal cells (500,000 cells per ml) were cultured on blocks of trabecular tantalum or allograft for 28 days in basal and osteogenic media. Molecular profiling, confocal and scanning electron microscopy, as well as live-dead staining and biochemical assays were used to characterise cell adherence, proliferation and phenotype.

Cells displayed extensive adherence and proliferation throughout trabecular tantalum evidenced by CellTracker immunocytochemistry and SEM. Tantalum-cell constructs cultured in osteogenic conditions displayed extensive matrix production. Electron microscopy confirmed significant cellular growth through the tantalum to a depth of 5mm. In contrast to cells cultured with allograft in both basal and osteogenic conditions, cell proliferation assays showed significantly higher activity with tantalum than with allograft (P<0.01). Alkaline phosphatase (ALP) assay and molecular profiling confirmed no significant difference in expression of ALP, Runx-2, Col-1 and Sox-9 between cells cultured on tantalum and allograft.

These studies demonstrate the ability of trabecular tantalum to support skeletal cell growth and osteogenic differentiation comparable to allograft. Trabecular tantalum represents a good alternative to allograft for tissue engineering osteo-regenerative strategies in the context of lost bone stock. Such clinical scenarios will become increasingly common given the ageing demographic, the projected rates of revision arthroplasty requiring bone stock replacement and the limitations of allograft. Further mechanical testing and in vivo studies are on-going.

Recent approaches have sought to harness the potential of stem cells to regenerate bone lost as a consequence of trauma or disease. Bone marrow aspirate (BMA) provides an autologous source of skeletal stem cells (SSCs) for such applications, however previous studies have demonstrated that the concentration of SSCs present in iliac crest BMA is below that required for robust bone regeneration. Here we present a novel acoustic-facilitated filtration strategy to concentrate BMA for SSCs, clinically applicable for intra-operative orthopaedic use.

The aim of this study was to demonstrate the efficacy of this strategy in concentrating SSCs from iliac crest bone marrow, as well as femoral canal BMA from older patients.

Iliac crest BMA (Lonza, Rockville, MD, USA) and femoral canal BMA was obtained with informed consent from older patients during total hip replacement. 5 to 40ml of BMA was processed via the acoustically-aided exclusion filtration process to obtain 2-8 fold volume reductions. SSC concentration and function was assessed by flow-cytometry, assays for fibroblastic colony-forming units (CFU-F) and multi-lineage differentiation along chondrogenic, osteogenic and adipogenic pathways examined. Seeding efficiency of enriched and unprocessed BMA (normalised to cell number) onto allograft was assessed.

Iliac crest BMA from 15 patients was enriched for SSCs in a processing time of only 15 minutes. Femoral BMA from 15 patients in the elderly cohort was concentrated up to 5-fold with a corresponding enrichment of viable and functional SSCs, confirmed by flow cytometry and assays for CFU-F. Enhanced osteogenic (P<0.05) and chondrogenic (P<0.001) differentiation was observed using concentrated aspirate, as evidenced by biochemical assay and semi-quantitative histological analysis. Furthermore, enhanced cell seeding efficiency onto allograft was seen as an effect of SSC concentration per ml of aspirate (P<0.001), confirming the utility of this approach for application to bone regeneration.

The ability to rapidly enrich BMA demonstrates potential for intra-operative application to enhance bone healing and offers immediate capacity for clinical application to treat many scenarios associated with local bone stock loss. Further in vivo analysis is ongoing prior to clinical tests.

Background

Skeletal stem cells can be combined with human allograft, and impacted to produce a mechanically stable living bone composite. This strategy has been used for the treatment of femoral head avascular necrosis, and has been translated to four patients, of which three remain asymptomatic at up to three year follow-up. In one patient collapse occurred in both hips due to widely distributed and advanced AVN disease, necessitating bilateral hip arthroplasty. However this has provided the opportunity to retrieve the femoral heads and analyse human tissue engineered bone.

Despite the development of skeletal or mesenchymal stem cell (MSC) constructs aimed at creating viable cartilage and bone, few studies have examined the effects of cytokines present in rheumatoid arthritis (RA) and osteoarthritis (OA) synovial tissues, or inhibition of these, on such constructs. This work addresses these issues using both in vitro and in vivo approaches and examines potential ways of overcoming the effects of cytokines on the integrity of cartilage and bone constructs.

Synovial samples were obtained from RA or OA (n=10) patients undergoing elective hip or knee arthroplasty at Southampton General Hospital. Full ethical approval was obtained. Control bone marrow-derived stromal cells were obtained from patients undergoing emergency fractured neck of femur repair, cultured in basal, osteogenic (ascorbate and dexamethasone) and chondrogenic (transforming growth factor beta (TGFbeta3)) conditions. Differentiation towards bone and cartilage was assessed using alkaline phosphatase (ALP) staining, ALP and DNA biochemical assays and analysis of osteogenic/chondrogenic gene expression using real time polymerase chain reaction (rt-PCR). Exogenous interleukin-1 (IL-1) (10ng/mL), tumour necrosis factor alpha (TNFalpha) (10ng/mL) or interleukin-6 (IL-6) (100ng/mL) was added and effects on differentiation noted. RA and OA synovial samples were digested, cultured for 48 hours then centrifuged to produce supernatants. Cytokine profiles were determined using ELISA. These supernatants were then added to MSCs and their effects on differentiation assessed.

Mesenchymal cultures in osteogenic media with IL-1 showed an additive osteogenic effect on biochemical assays. TNF exerted a less marked and IL-6 no apparent effect on osteogenic differentiation. ALP expression by rt-PCR correlated with these findings. Addition of supernatants to mesenchymal cultures produced a marked osteogenic profile that was IL-1 and TNFalpha concentration dependent, correlating with lower supernatant dilutions on initial ELISA analysis.

Preliminary studies indicate that exogenous IL-1 and TNFalpha modulate the osteogenic phenotype in MSCs in vitro. OA and RA synovial supernatants affect skeletal cell differentiation. Variations in cytokine profiles between supernatants require analysis for potential confounders. A larger study is underway to investigate these effects, the effects of cytokines on skeletal cell differentiation on commercially available scaffolds both in vitro and in an in vivo murine model of bone formation.

Disease transmission, availability and economic costs of allograft have resulted in significant efforts into finding an allograft alternative for use in impaction bone grafting (IBG). Biotechnology offers the combination of skeletal stem cells (SSC) with biodegradable polymers as a potential solution. Recently polymers have been identified with both structural strength and SSC compatibility that offer the potential for clinical translation.

The aim of this study was to assess whether increasing the porosity of one such polymer via super critical CO2 dissolution (SCD) enhanced the mechanical and cellular compatibility characteristics for use as an osteogenic alternative to allograft in IBG.

High molecular weight PLA scaffolds were produced via traditional (solid block) and SCD (porous) techniques, and the differences characterised using scanning electron microscopy (SEM). The polymers were milled, impacted, and mechanical comparison between traditional vs SCD created scaffolds and allograft controls was made using a custom shear testing rig, as well as a novel agitation test to assess cohesion. Cellular compatibility tests for cell number, viability and osteogenic differentiation using WST-1 assays, fluorostaining and ALP assays were determined following 14 day culture with SSCs.

SEM showed increased porosity of the SCD produced PLA scaffolds, with pores between 50-100 micrometres. Shear testing showed the SCD polymer exceeded the shear strength of allograft controls (P<0.001). Agitation testing showed greater cohesion between the particles of the SCD polymer (P<0.05). Cellular studies showed increased cell number, viability and osteogenic differentiation on the SCD polymer compared to traditional polymer (P<0.05) and allograft (P<0.001).

The use of supercritical C02 to generate PLA scaffolds significantly improves the cellular compatibility and cohesion compared to traditional non-porous PLA, without substantial loss of mechanical shear strength. The improved characteristics are critical for clinical translation as a potential osteogenic composite for use in impaction bone grafting.

Impaction bone grafting with milled human allograft is the gold standard for replacing lost bone stock during revision hip surgery. Problems surrounding the use of allograft include cost, availability, disease transmission and stem subsidence (usually due to shear failure of the surrounding allograft).

The aim of this study was to investigate various polymers for use as substitute allograft. The ideal graft would be a composite with similar mechanical characteristics as allograft, and with the ability to form de novo bone.

High and low molecular weight (MW) forms of three different polymers (polylactic acid (PLA), poly (lactic co-glycolic) acid (PLGA) and polycaprolactone (PCL)) were milled, impacted into discs, and then tested in a custom built shear testing rig, and compared to allograft.

A second stage of the experiment involved the addition of skeletal stem cells (SSC) to each of the milled polymers, impaction, 8 days incubation, and then tests for cell viability and number, via fluorostaining and biochemical (WST-1) assays.

The shear strengths of both high/ low MW PLA, and high/low MW PLGA were significantly higher than those of milled allograft (P<0.001, P<0.001, P<0.005 and P<0.005) but high and low MW PCL was poor to impact, and had significantly lower shear strengths (P<0.005, P<0.001). Fluorostaining showed good cell survival on high MW PLA, high MW PCL and high MW PLGA. These findings were confirmed with WST-1 assays.

High MW PLA as well as high MW PLGA performed well both in mechanical testing and cell compatibility studies. These two polymers are good contenders to produce a living composite for use as substitute human allograft in impaction bone grafting, and are currently being optimised for this use via the investigation of different production techniques and in-vivo studies.

AIM

Avascular necrosis (AVN) of the femoral head is a potentially debilitating disease of the hip in young adults. Impaction bone grafting (IBG) of morcellised fresh frozen allograft is used in a number of orthopaedic conditions. This study has examined the potential of skeletal stem cells (SSC) to augment the mechanical properties of impacted bone graft and we translate these findings into clinical practice.

Background: Intrauterine protein restriction in rodent models is associated with low bone mass which persists into adulthood. This study examined how early nutritional compromise affects the mechanical and structural properties of spinal tissues in sheep throughout the lifecourse.

Methods: Lumbar spines were removed from 19 sheep; 5 control animals and 14 that received a restricted diet in-utero. Eight animals (2 control/6 diet) were sacrificed at a mean age of 2.7 years and eleven at a mean age of 4.4 yrs. Two motion segments from each spine were tested on a hydraulically-controlled materials testing machine to determine their mechanical properties. Vertebral bodies were assessed for a number of structural parameters including cortical thickness and area, and regional trabecular density.

Results: Younger animals in the diet group showed a 25% reduction in forward bending stiffness (p< 0.05) and a 32% reduction in extension strength (p< 0.05) compared to controls of the same age. Furthermore, these young animals showed a 25% reduction in the thickness of the anterior cortex (p< 0.001) and an 18% reduction in the thickness of the superior cortex (p< 0.02). In older animals, no differences were observed in any of the mechanical parameters examined between diet and control groups, although animals in the diet group showed an average increase in cortical thickness of 14%, across all regions (p< 0.01).

Conclusions: These results suggest that early nutritional challenge can have detrimental effects on the mechanical and structural properties of spinal tissues in young animals but that adaptation occurs over the lifecourse to compensate for these differences in older animals.

Conflicts of Interest: None

Source of Funding: None

Introduction: One of the main factors in the success of impaction bone grafting (IBG) in revision hip surgery is its ability to resist shear and to form a stable construct. Bone marrow contains multipotent skeletal stem cells and we propose that in combination with allograft will produce a living composite with biological and mechanical potential. In this study we looked at whether coating of the allograft with type 1 collagen followed by seeding with human bone marrow stromal cells (hBMSC) would enhance the grafts mechanical and biological properties.

Methods: A control group of plain allograft and three experimental groups where used to determine the effects that collagen and hBMSC have on IBG. The samples where impacted in standardised fashion previously validated to replicate femoral IBG, and cultured in vitro for 2 weeks. The samples then underwent mechanical shear testing and biochemical analysis for DNA content and Osteogenic activity.

Results: Collagen coating of the allograft prior to seeding with hBMSC significantly enhanced the mechanical properties of the construct compared to the ‘gold standard’ of plain allograft with a 22% increase in shear strength (p=0.002). The collagen coated group also showed increased osteogenic differentiation of the stromal cells (Alkaline Phospatase specific activity: 124 +/− 18.6 vs 54.6 +/− 9.6 nM pNPP/Hr/ngDNA p= < 0.01).

Discussion: This study has shown a role in the improvement of the biomechanical properties of IBG by coating with collagen and seeding with hBMSC. Collagen coating of IBG is a simple process and translation of the technique into the theatre setting feasible. The improvement in shear strength and cohesion could lead to earlier weight bearing for the patients and allow quicker recovery. The therapeutic implications of such composites auger well for orthopaedic applications. We are currently strengthening the above findings with an in vivo study.

Introduction: Revision hip surgery is predicted to rise significantly over the coming decades. There is therefore likely to be an increasing need to overcome the large bone loss and cavitatory defects encountered in failed primary hip replacements. Impaction bone grafting (IBG) is a recognised technique for replacing lost bone stock. Achieving optimal graft impaction is a difficult surgical skill with a significant learning curve, balancing the need to achieve sufficient compaction to provide primary stability versus the need to keep impaction forces to a minimum to prevent iatrogenic fracture. In this study we have developed a revision acetabular model to test the hypothesis that the use of vibration and drainage with a new custom made perforated tamp could reduce the peak stresses imparted to the acetabulum during the impaction process and also improve the reliability and reproducibility of the impaction technique

Methods: Composite Sawbone hemi Pelvis models were used, with identical contained cavitatory defects created (Paprosky Type 2a). A strain gauge was attached to the medial wall of each hemi pelvis. A custom set of IBG tamps were made, and coupled a pneumatic hammer used to generate the vibrations. A standard impaction technique was used for the control group and the new vibration impaction for the experimental group. The cavity was progressively filled with morsellised allograft in 6 set steps for both groups with strain gauge readings taken during all impaction to monitor peak stresses. A standard Exeter Contemporary cup was then cemented into the graft bed for both groups. The models were mechanically loaded according to the protocol developed by Westphal et al at the angle of the joint reaction force during heel strike for a total of 50 000 cycles. 3D assessment of any micro motion post mechanical testing and degree of graft compaction was done with high resolution micro CT.

Results: Vibration impaction lead to a significant reduction in the peak stresses during the impaction process throughout the 6 steps (e.g. Step 1: 34.6 vs 110.8 MPa p=0.03). There was also far less variability in the peak stresses in the vibration group compared to standard impaction both in sequential impactions by the same surgeon and between different surgeons. One medial wall fracture occurred in the control group only. There was no difference in the degree of graft compaction or in the subsidence of the implant post cyclical loading.

Conclusion: Impaction bone grafting can be a difficult surgical skill with a significant learning curve. We believe that this new technique of applying vibration coupled with drainage to the IBG process in the acetabulum can reduce the risk of intraoperative fracture whilst achieving good graft compaction and implant stability. This technique therefore has the potential to widen the ‘safety margins’ of IBG and reduce the learning curve allowing more widespread adoption of the technique for replacing lost bone stock.

Introduction: Impaction bone grafting (IBG) using fresh frozen morsellised allograft is considered by many as the method of choice for replacing lost bone stock encountered during revision hip surgery. Bone marrow contains multipotent skeletal stem cells which have the potential to differentiate down a number of different cell lineages including osteoblasts, chondrocytes and adipocytes. In IBG it is desirable for as many as possible to go on to form bone rather than fibrous tissue to form a solid osseous construct. Whilst it is possible to push cells down the osteogenic lineage in vitro, some of these methods (e.g. the addition of Dexamethasone) are not translatable to clinical practice due to undesirable side effects. In this study we test the hypothesis that by coating the allograft with type 1 Collagen prior to seeding with human bone marrow stromal cells (hBMSC), the cellular adhesion and proliferation down an osteogenic lineage can be increased, leading to improved mechanical and biological properties of the IBG composite.

Methods: A control group of plain allograft and three experimental groups where used to determine the effects that collagen and hBMSC have on IBG (both individually and in combination). The samples where impacted in standardised fashion previously validated to replicate Femoral IBG, and cultured in vitro for 2 weeks. The samples then underwent mechanical shear testing giving a family of stress strain curves for each group, from which a Mohr coulomb failure curve can be plotted. Using the Mohr Coulomb failure equation τ = σ tanΦ + c, the shear strength (τ), Internal friction angle (tanΦ) and inter particulate cohesion (c) can then be calculated. Biochemical analysis was also performed for DNA content and Osteogenic activity.

Results: Mechanical shear testing demonstrated a significant improvement (p=0.002) in the grafts ability to resist shear with the coating of Collagen and seeding with hBMSC (245 vs 299 kPa) as well as improved cohesion between the bone graft particles (46 vs 144 kPa). Regression analysis of the shear strength showed a linear increase with compressive stress (R2 > 0.98) for all groups, indicating that the grafts satisfied the Mohr Coulomb failure law. In the two groups seeded with cells, the collagen coated group also showed increased osteogenic cell activity compared to the plain allograft.

Conclusion: This study has shown a role in the improvement of the mechanical and biological properties of IBG coated with type 1 Collagen and seeded with hBMSC. Collagen coating of IBG is a facile process and translation of the technique into the theatre setting feasible. The improvement in shear strength and cohesion could lead to earlier weight bearing for the patients and allow quicker recovery. The therapeutic implications of such composites auger well for orthopaedic applications. We are currently strengthening the above findings with an in vivo study.

Introduction: Impaction bone grafting (IBG) for revision hip surgery can be a difficult surgical skill with a fine line between construct failure from insufficient compaction and intraoperative fracture from high impaction forces. Following on from our experience in the femur, in this study we used an acetabular model to test the hypothesis that the use of vibration for IBG could reduce the peak stresses thus reducing the intraoperative fracture risk and also improve the reliability and reproducibility of the impaction technique.

Methods: Revision hemi pelvis models were made (Pra-prosky Type 2a). A standard impaction technique was used for the control group, and the impactor tamps were coupled with a pneumatic hammer for the vibration group. The cavity was filled in 6 set steps with strain gauge readings taken throughout. The pelvis construct was then mechanically loaded. Graft compaction and micro motion post mechanical testing was assessed with micro CT.

Results: Vibration impaction led to a significant reduction (p=0.03) in the peak stresses during the impaction process. There was also significantly less variability in peak stresses for the vibration group compared to standard, both in sequential impactions by the same surgeon and between different surgeons. One medial wall fracture occurred in the control group only, similar to fractures encountered in the clinical situation. There was no significant difference in the degree of graft compaction or in the subsidence of the cup.

Discussion: We believe that this new technique of applying vibration to the IBG process can reduce the risk of intraoperative fracture whilst achieving good graft compaction and implant stability. This technique therefore has the potential to widen the ‘safety margins’ of IBG and reduce the learning curve allowing more widespread adoption of the technique for replacing lost bone stock.

Introduction: One of the main factors in the success of impaction bone grafting (IBG) in revision hip surgery is its ability to resist shear and to form a stable construct. Bone marrow contains multipotent skeletal stem cells and we propose that in combination with allograft will produce a living composite with biological and mechanical potential. In this study we looked at whether coating of the allograft with type 1 collagen followed by seeding with human bone marrow stromal cells (hBMSC) would enhance the grafts mechanical and biological properties.

Methods: A control group of plain allograft and three experimental groups where used to determine the effects that collagen and hBMSC have on IBG. The samples where impacted in standardised fashion previously validated to replicate femoral IBG, and cultured in vitro for 2 weeks. The samples then underwent mechanical shear testing and biochemical analysis for DNA content and Osteogenic activity.

Results: In isolation, both Collagen coating and seeding with hBMSC significantly enhanced the mechanical properties of the construct compared to the ‘gold standard’ of plain allograft. This was further enhanced (p=0.002) when the two processes are combined both with shear strength (245 vs. 299 kPa) and cohesion between the graft particles (46 vs. 144 kPa). The collagen coated group also showed increased osteogenic cell proliferation.

Discussion: This study has shown a role in the improvement of the mechanical properties of IBG coated with collagen and seeded with hBMSC. Collagen coating of IBG is a simple process and translation of the technique into the theatre setting feasible. The improvement in shear strength and cohesion could lead to earlier weight bearing for the patients and allow quicker recovery. The therapeutic implications of such composites auger well for orthopaedic applications. We are currently strengthening the above findings with an in vivo study.

Regenerative medicine provides the hope for many intractable diseases as a treatment option and the area is currently the subject of intense investigation in academia and industry. Human bone marrow stromal cells (HBMSCs) possess the ability to differentiate into a variety of cell types of the stromal lineage including cells of the osteogenic and chondrogenic lineages. However, the process of in vitro differentiation is usually inefficient, difficult to reproduce in many cases and, to date, unable to produce homogenous cell populations, which is critical for tissue engineering. Epigenetic regulation of gene expression is recognized as a key mechanism governing cell determination, commitment, and differentiation as well as maintenance of those states. The main components of epigenetic control are DNA methylation and histone acetylation. During development, the epigenetic status changes as cells differentiate along specific lineages. We reasoned that epigenetic modifiers might direct the differentiation pathway of HBMSCs towards either osteogenic or chondrogenic lineage. HBMSCs were serum-starved for 24 hours to synchronise the cell cycle, then treated on three consecutive days either with the DNA demethylating agent 5-Aza-deoxycytidine (5-Aza-dC) 1?M, or the histone deacetylase inhibitor Trichostatin A (TSA) 100 nM or a combination of both. After confluency, the cells were grown in pellet culture for 21 days to facilitate formation of an extracellular matrix. 5-Aza-dC increased the amount of osteoid in the pellet by at least 5 fold compared with controls as assessed by histochemistry, whereas TSA enhanced formation of a cartilage matrix. The differentiation was further enhanced by culturing the pellets in osteogenic or chondrogenic media. These studies suggest that loss of DNA methylation stimulates osteogenic differentiation, whereas inhibition of histone deacetylation favours chondrogenesis. Epigenetic changes thus play an important role in HBMSCs differentiation and offer new approaches in skeletal tissue engineering programs. The challenge will be to define the crucial genes in which loss of DNA methylation has taken place or how changes in histone acetylation (and other histone modifications) affect lineage differentiation.

Quantification and 3D visualization of new vessel networks in vivo remains a major unresolved issue in tissue engineering constructs. This study has examined the potential of combining the use of a radio opaque dye and micro-CT to visualize and quantify microvascular networks in 3D in vivo. We have applied this technique to the study of neoangiogenesis in a bone impaction graft model in vivo as proof of concept. Tissue engineered constructs were created with natural (morsellised allograft) and synthetic grafts (Poly Lactic Acid, PLA)

Culture expanded human bone marrow stromal cells (HBMSC) labeled with a fluorescent probe (Cell Tracker Green, CTG) to measure cell viability, were seeded onto prepared scaffolds (morsellised allograft or PLA) and impacted with a force equivalent to a standard femoral impaction (474J/m2). The impacted HBMSC / scaffolds and scaffolds alone were contained within capsules and implanted subcutaneously into severely compromised immunodeficient mice. Radiopaque dye was infused into all vessels via cardiac cannulation prior to removal of implants. Micro CT imaging and immunohistochemistry was performed in all samples.

Cell survival was evident by abundant fluorescent staining. The average number of blood vessels penetrating the capsules were 16.33 in the allograft / HBMSC constructs compared to 3.5 (p=0.001) in the allograft alone samples and 32.67 in the PLA / HBMSC constructs compared to 7.67 (p=0.001) in the PLA alone samples. The average total vessel volume within the capsules was 0.43mm3 in the allograft / HBMSC constructs compared to 0.04mm3 (p=0.05) in the allograft alone samples and 1.19mm3 in the PLA / HBMSC constructs compared to 0.12mm3 (p=0.004) in the PLA alone samples. Extensive staining for Type 1 Collagen, new matrix and Von Willebrand factor in living tissue engineered constructs confirmed osteogenic cell phenotype, and new blood vessel formation respectively.

In summary, these studies demonstrate, HBMSC combined with either morsellised allograft or PLA can survive the forces of femoral impaction, differentiate along the osteogenic lineage and promote neovascularisation in vivo. Successful combined neovascularisation and bone formation in impacted tissue engineered constructs in vivo augers well for their potential use in IBG.

This novel technique utilising contrast enhanced 3D reconstructions in combination with immunohistochemistry enables quantification of neovascularisation and new bone formation in impacted tissue engineered constructs with widespread experimental application in regenerative medicine and tissue engineering analysis.

Impaction bone grafting with morsellised allograft is a recognized technique to reconstitute loss of bone stock often encountered during revision hip surgery. Concerns over disease transmission, high costs and limited supply has led to interest in synthetic grafts. Poly (lactic acid) (PLA) grafts are attractive to the tissue engineering community as a consequence of their biocompatibility, ease of processing into three-dimensional structures, their established safety as suture materials and the versatility that they offer for producing chemically defined substrates for bone graft matrices. This study set out to examine the potential of PLA scaffolds augmented with human bone marrow stromal cells in impaction bone grafting (IBG).

Methods: In vitro and in vivo studies were performed on impacted morsellised PLA seeded with human bone marrow stromal cells (HBMSC) and compared to PLA alone. In vitro samples were incubated under osteogenic conditions and in vivo samples were implanted subcutaneously into severely compromised immunodeficient mice, both for 4 weeks. In vitro samples were analysed for cell viability, DNA content, specific alkaline phosphatase activity, immunohistochemistry and mechanical shear testing using a cam shear tester. In vivo samples were analysed for cell viability, immunohistochemistry and evidence of neovascularisation and new bone formation using contrast enhance micro computer tomography.

Results: HBMSC survival post impaction, as evidenced by cell tracker green staining, was seen throughout the samples in vitro and in vivo. In vitro there was a significant increase in DNA content (P< 0.001) and specific alkaline phosphatase activity (P< 0.001) in PLA / HBMSC samples compared to impacted PLA alone. Mechanical shear testing of in vitro PLA / HBMSC samples demonstrated a significant increase in shear strength and interparticulate cohesion compared to PLA alone. Immunohistochemistry for type I collagen, osteocalcin, confirmed cell differentiation along the osteogenic lineage in vitro and in vivo. In vivo studies showed a significant increase in blood vessel number and volume penetrating the PLA / HBMSC constructs (32.6 vessels, 1.19mm3, p=0.02, p=0.004) compared to PLA alone (7.6vessels, 0.12mm3). There was a significant relative increase in new bone formation in the PLA / hBMSC constructs (0.47mm3) compared to PLA alone (0mm3), further confirmed with positive staining for osteoid using Goldners Trichrome.

Conclusion: HBMSC seeded onto PLA can withstand the forces of femoral impaction and continue to differentiate and proliferate along the osteogenic lineage. Furthermore PLA / hBMSC constructs in vitro offer a mechanical advantage over PLA alone and in vivo induce neovascularisation and new bone formation. From both a biological and mechanical perspective these studies have demonstrated that PLA is a suitable and beneficial bone graft extender for use in IBG.

Background: The use of fresh morsellised allograft in impaction bone grafting for revision hip surgery remains the gold standard. Bone marrow contains osteogenic progenitor cells that arise from multipotent mesenchymal stem cells and we propose that in combination with allograft will produce a living composite with biological and mechanical potential. This study aimed to determine if human bone marrow stromal cells (HBMSC) seeded onto highly washed morsellised allograft could survive the impaction process, differentiate and proliferate along the osteogenic lineage and confer biomechanical advantage in comparison to impacted allograft alone

Methods: HBMSC were isolated and culture expanded in vitro under osteogenic conditions. Cells were seeded onto prepared morsellised allograft and impacted with a force equivalent to a standard femoral impaction (474J/m2). Samples were incubated for either two or four week periods under osteogenic conditions and analysed for cell viability, histology, immunohistochemistry, and biochemical analysis of cell number and osteogenic enzyme activity. Mechanical shear testing, using a Cam shear tester was performed, under three physiological compressive stresses (50N, 150N, 250N) from which the shear strength, internal friction angle and particle interlocking values were derived.

Results: Cell viability of HBMSC post impaction, was confirmed with cell tracker green staining, a marker of viable cells, and observed throughout all samples. There was a significant increase in DNA content and specific alkaline phosphatase activity compared to impacted seeded allograft samples. Immunohistochemical staining for type I collagen confirmed cell differentiation along the osteogenic lineage. Mechanical shear testing demonstrated a statistical significant increase in shear strength and interparticulate cohesion in the allograft/hBMSC group over allograft alone at 2 and 4 week intervals (p< 0.001).

Conclusion: HBMSC seeded onto allograft resulted in the formation of a living composite capable of withstanding the forces equivalent to a standard femoral impaction. HBMSC under osteogenic conditions were observed to differentiate and proliferate along the osteogenic lineage. In addition, an allograft/HBMSC living composite confers a biomechanical advantage over allograft alone These changes resulting in enhancement of biological and mechanical properties of bone graft within impaction bone grafting have implications for translation and future change in orthopaedic practice in an increasing ageing population.

Cartilage and bone degeneration are major healthcare problems affecting millions of individuals worldwide. Elucidation of the processes modulating the cell-matrix interactions involved in cartilage or bone formation offer tremendous potential in the development of clinically relevant strategies for cartilage and bone regeneration. We have therefore adopted an ex vivo tissue engineering approach to investigate chondrogenesis and osteogenesis using a mix human mesenchymal progenitor populations encapsulated in biomineralised polysac-charide templates with or without the addition of type-I collagen.

Alginate/chitosan polysaccharide capsules containing 2.5mg/ml type-I collagen and TGF-beta-3 were encapsulated with human bone marrow cells (HBMC), articular chondrocytes or a co-culture at a ratio of 2:1 respectively and placed in a rotating (Synthecon) biore-actor or held in static 2D culture conditions for 28 days, to determine whether the presence of type-I collagen within the alginate could promote the synthesis of an extracellular matrix.

Constructs were stained with alcian blue, sirius red and von Kossa. In bioreactor samples encapsulated with HBMC and type-I collagen, viable cells were present within lacunae, surrounded by a matrix of proteo-glycans and fibrous collagen, which was mineralized. Immunohistochemistry and polarised light microscopy indicated an organised collagenous matrix with extensive expression of type I collagen and bone sialoprotein with small regions of type II collagen. Type X collagen was also expressed indicating the presence of hypertrophic chondrocytes. Within the static HBMC groups, smaller areas of matrix were generated with decreased expression of type-I and type-II collagen. Co-culture bioreactor samples also demonstrated regions of new mineralised bone matrix; however these were less prominent than in the HBMC only groups. No matrix formation was observed in chondrocyte cultures although the cells remained viable as assessed by live/dead staining. Biochemical analysis indicated significantly increased (p< 0.05) DNA in all bioreactor samples in comparison with static constructs and significantly increased protein in HBMC bioreactor constructs in comparison with other cell types.

These studies outline a unique tissue engineering approach, utilizing individual and mixed human mesen-chymal progenitor populations coupled with innovative polysaccharide templates containing type I collagen and bioreactor systems to promote chondrogenic and osteo-genic differentiation.

The use of fresh morsellised allograft in impaction bone grafting for revision hip surgery remains the gold standard. Bone marrow contains osteogenic progenitor cells that arise from multipotent mesenchymal stem cells and we propose that in combination with allograft will produce a living composite with biological and mechanical potential. This study aimed to determine if human bone marrow stromal cells (HBMSC) seeded onto highly washed morsellised allograft could survive the impaction process, differentiate and proliferate along the osteogenic lineage and confer biomechanical advantage in comparison to impacted allograft alone. Future work into the development of a bioreactor is planned for the potential safe translation of such a technique into clinical practice.

Methods: HBMSC were isolated and culture expanded in vitro under osteogenic conditions. Cells were seeded onto prepared morsellised allograft and impacted with a force equivalent to a standard femoral impaction (474J/m2). Samples were incubated for either two or four week periods under osteogenic conditions and analysed for cell viability, histology, immunocytochemistry, and biochemical analysis of cell number and osteogenic enzyme activity. Mechanical shear testing, using a Cam shear tester was performed, under three physiological compressive stresses (50N, 150N, 250N) from which the shear strength, internal friction angle and particle interlocking values were derived.

Results: HBMSC survival post impaction, as evidenced by cell tracker green staining, was seen throughout the samples. There was a significant increase in DNA content (P< 0.05) and specific alkaline phosphatase activity (P< 0.05) compared to impacted seeded allograft samples. Immunocytochemistry staining for type I collagen confirmed cell differentiation along the osteogenic lineage. There was no statistical difference in the shear strength, internal friction angle and particulate cohesion between the two groups at 2 and 4 weeks.

Conclusion: HBMSC seeded onto allograft resulted in the formation of a living composite capable of withstanding the forces equivalent to a standard femoral impaction and, under osteogenic conditions, differentiate and proliferate along the osteogenic lineage. In addition, there was no reduction in aggregate shear strength and longer term studies are warranted to examine the biomechanical advantage of a living composite. The therapeutic implications of such composites auger well for orthopaedic applications.

Introduction: Bone is unique with a vast potential for regeneration from cells with stem cell characteristics. With an increasing aging population, clinical imperatives to augment and facilitate tissue repair have highlighted the therapeutic potential of harnessing mes-enchymal populations from bone. We describe laboratory and clinical findings from two clinical cases, where different proximal femoral conditions (AVN, bone cyst) were treated with impacted allograft augmented with marrow-derived allogeneic progenitor cells.

Methods: Marrow was aspirated from the posterior superior iliac crest and seeded onto prepared washed morsellised allograft. The seeded graft was left for 40 minutes to allow adherence of the marrow-derived osteoprogenitor cells prior to impaction into the defect. Samples of the impacted graft were taken for in-vitro analysis of cell viability, histology and biochemical analysis of cell number and osteogenic enzyme activity. The total force imparted during impaction was calculated using a load cell, with three independent surgeons performing a laboratory simulation of the impaction technique.

Results: Both patients made a rapid clinical recovery after an overnight stay. Imaging confirmed filling of the defects with increased density on plain radiographs suggesting good impaction of the graft composite. Immu-nohistochemical staining of graft samples demonstrated that a living composite graft with osteogenic activity had been introduced into the defects as evidenced by cell tracker green viability and alkaline phosphatase (osteogenic marker) expression and specific activity. The average peak forces during impaction were 0.7kN corresponding to average peak stresses within the graft of 8.3MPa. Similar forces were seen between operators.

Discussion: Replacement of bone loss remains a major challenge in orthopaedic practice. Although allograft remains the gold standard where large volumes preclude autograft, allograft has little osteoinductive potential. We demonstrate that marrow-derived cells can adhere to highly washed morsellised allograft, survive the impaction process, and are of the osteoblastic phenotype creating a living composite. Thus we conclude, impacted allograft seeded with autologous marrow cells allows the delivery of a biologically active scaffold for the treatment of bone deficiency. In addition this is a novel straightforward technique, surgeon independent and with applications in a number of orthopaedic scenarios.

Introduction: Intrauterine protein restriction in rats is associated with low bone mass which persists with development through to adulthood. However, such adverse effects are not only restricted to bone. Intervertebral discs are the largest avascular structures in the body, and are particularly sensitive to their nutritional environment. We have examined the hypothesis that changes in the intervertebral disc (or ligaments), as a result of early nutritional compromise, affect the spine’s mechanical properties.

Material and methods: Lumbar spines were removed from 8 sheep (6 male, 2 female: mean age 2.7 yrs) that had received different diets early in their development: two animals received a control diet, three received low protein in utero (IU), and three received low protein both in utero and postnatally (PN). Fifteen motion segments (consisting of two vertebrae and the intervening disc and ligaments) were dissected from the spines and tested on a hydraulically-controlled materials testing machine. Compressive stiffness and bending stiffness were measured before and after creep loading, in both flexion and extension. Reflective markers attached to the specimens were tracked during loading, enabling intervertebral angles to be calculated. Bending moment-angular rotation curves were used to calculate bending stiffness. Repeated measures ANOVA was used to test for differences in stiffness with posture and creep, and between the dietary groups.

Results: Compressive stiffness increased after creep loading (p=0.002) but was unaffected by posture or dietary group. In contrast, bending stiffness was unaffected by creep but differed significantly between groups and with posture. When compared to controls, bending stiffness in the IU group was reduced by 35% in flexion and 26% in extension (p< 0.02). In the PN group, reductions of 28% in flexion and 15% in extension were observed (p=0.056).

Discussion: These results indicate that early protein restriction can affect the mechanical properties of the spine. These effects were evident in bending but not in compression, and tended to be greater in flexion than extension. These preliminary findings suggest that early protein restriction may affect the composition and mechanical function of the annulus fibrosus and the intervertebral ligaments which are the structures most involved in resisting flexion movements.

Introduction: Epidemiology suggests that an intrauterine nutrient restriction increases the likelihood of osteoporosis in later life, possibly due to differences in bone structure and strength. We hypothesise that, in an ovine model, early nutritional compromise reduces vertebral cancellous bone density and cortical thickness, and thereby reduces vertebral compressive strength.

Materials and methods: Lumbar spines were dissected from 8 sheep (6 male, 2 female: mean age 2.7 yrs). Spines were divided into different groups, based on the early diet of the sheep: group CC received a control diet, group IU received low protein in utero, and group PN received low protein both in utero and postnatally. Fifteen motion segments (consisting of two vertebrae and the intervening disc and ligaments) were prepared from the spines, and compressed to failure using a hydraulically-controlled materials testing machine to obtain yield strength. 1mm-thick bone slices were taken from the mid-sagittal and para-sagittal regions of each vertebral body and micro-radiographed. Digital images of the micro-radiographs were analysed to obtain the cancellous bone density in anterior and posterior regions, and the cortical thickness in the anterior, posterior, superior and inferior regions. Repeated measures ANOVA was used to test for differences in parameters at the different locations, and between the groups.

Results: The anterior cortex was 28% thinner for the IU group, and 23% thinner for the PN group compared to controls (both p< 0.001). In the PN group, the superior cortex was also 18% thinner than controls (p< 0.02). There was no significant difference between cancellous bone density in either region. Yield strength was 16% lower in the IU group compared to controls, but this did not reach significance.

Discussion: In the nutritionally compromised groups, cortical thickness was lower in regions of the vertebral body where fractures often occur in elderly people. However, the reduction in cortical thickness is not accompanied by a significant reduction in compressive strength in the sheep model. These findings suggest that the well-maintained cancellous bone protects the vertebra from fracture.

Background: Impaction bone-grafting in revision hip surgery generates high forces that may be transmitted through the graft to the femoral cortex, generating high surface strains and a concomitant risk of femoral fracture. Concern of inducing fracture may lead to under-compaction of the graft, with subsequent risk of implant migration. Vibration is commonly used in civil engineering applications to increase aggregate compressive and shear strengths. We have therefore examined the hypotheses that vibration-assisted graft compaction would (a) increase graft compaction compared with the standard femoral impaction grafting technique and subsequently reduce prosthesis migration and (b) reduce femoral hoop strains in the production of graft of a given density and mechanical properties.

Method: Physiological composite femurs were adapted to represent femurs encountered in revision hip surgery by widening of the internal diameter and thinning of the outer shell. In the control group, revision with the standard Exeter technique was simulated using highly washed morcellised bone graft from fresh-frozen human femoral heads. In the study group, vibration-assisted graft compaction was used. The femurs were mounted on a 5kN capacity load cell to measure the total force imparted during graft impaction. Strain gauges placed at the medial calcar and midshaft, measured hoop strains generated during the impaction process. On completion of graft impaction, an Exeter stem was cemented in place. Implant subsidence under physiological cyclic loading (5x 105 cycles) and graft density using micro CT were measured after compaction.

Results: There were no significant differences between the two groups in the peak forces (3.8-4.1kN) imparted during the impaction process. Similar peak hoop strains were observed in the both groups (1.2-1.4%). However a greater graft density was seen in the vibration group with minimal implant subsidence under cyclic loading.

Conclusion: The use of vibration during the impaction process allowed improved graft compaction to be achieved without increasing hoop strains in the femoral cortex. This has implications in preventing failure from under impaction without increasing the risk of fracture. Furthermore, this analysis is applicable to the study of novel synthetic grafts / mixtures in the impaction process for orthopaedic application.

Cartilage is a realistic target for tissue engineering given the avascular nature and cellular composition of the tissue. Much of the work in this field has been largely empirical, indicating the need for alternative approaches to the design of cartilage formation protocols. Given the heterogeneity associated with human mesenchymal populations, continuous cell lines may offer an alternative to model and simplify cartilage generation protocols. We therefore exploited the potential of the murine chondrocytic ATDC5 cell line to, i) delineate the process of chondrocyte differentiation in monolayer culture and three-dimensional micromass pellet culture systems, and ii) model cartilage formation utilising appropriate scaffold and bioreactor (perfused and rotating) technologies. Monolayer cultures of ATDC5 cells over a 28-day period in presence of insulin demonstrated various stages of chondrocyte differentiation- proliferative, pre-hypertrophic, hypertrophic and finally, mineralisation of cartilaginous nodules. This was confirmed by gene and protein expression, by qPCR and Western blotting respectively, of chondrogenic differentiation markers- Sox-9, Bcl-2, Type II and X collagens. Pellet cultures of ATDC5 cells under chondrogenic conditions (10 ng/ml TGF-beta3, 1X ITS {insulin, transferrin, selenium}, 10 nanomolar dexamethasone, 100 micromolar ascorbate-2-phosphate) illustrated a gradual progression from an aggregation of cells at day 7, to initiation of matrix synthesis at day 14, followed by formation of well-defined cartilaginous structures at day 21. Chondrogenic differentiation at day 21 was evident by numerous proliferative/ pre-hypertrophic chondrocytes, staining for Sox-9, Aggrecan, Type II collagen and PCNA, lodged in distinct lacunae embedded in cartilaginous matrix of proteogly-cans and Type II collagen. Inclusion of TGF-beta3 in the chondrogenic medium during pellet culture beyond 21 days maintained the pre-hypertrophic phenotype, even at day 28. In contrast, removal of TGF-beta3, addition of 50 nanomolar thyroxine and reduction of dexa-methasone to 1 nanomolar in the chondrogenic medium stimulated hypertrophy at day 28, evident by down-regulation of Sox-9 expression. ATDC5 cells cultured on Polyglycolic acid fleece in the rotating bioreactor or encapsulated in chitosan /alginate and cultured in the perfused bioreactor for 21 days, formed cartilaginous explants reminiscent of hyaline cartilage. Thus, ATDC5 cells constitute an ideal cell line to elucidate the steps of chondrocyte differentiation and cartilage formation.

The ability to generate replacement human tissues on demand is a major clinical need. Indeed the paucity of techniques in reconstructive surgery and trauma emphasize the urgent requirement for alternative strategies for the formation of new tissues and organs. The idea of biomimesis is to abstract good design principles and optimizations from nature and incorporate them in the construction of synthetic materials and structures. Direct appropriation of natural inorganic skeletons is also biomimetic since their unique properties inform us on ways to generate functional, optimized scaffolds.

A number of well characterized natural skeletons were investigated as potential scaffolds for tissue regeneration using mesenchymal stem cell populations. Marine sponges, sea urchin skeletons and nacre were found to possess unique functional properties that supported human cell attachment, growth and proliferation and provided organic/ inorganic extracellular matrix analogues for guided tissue regeneration.

A good understanding of the processses involved in biomineralisation and the emergence of complex inorganic forms has inspired synthetic strategies for the formation of biological analogues (organised inorganic materials with biological form). We have developed two functional examples of biological structures generated using biomimetic materials chemistry with applications for human tissue regeneration. Mineralised biopoly-saccharide microcapsules provided enclosed micro-environments with an appropriate physical structure and physiological milieu, for the support of the initial stages of tissue regeneration combined with a capacity to deliver human cells, plasmid DNA and controlled release of biological factors such as cytokines. Calcium carbonate porous microspheres analogous to microscopic coccolithophore shells provided a template for tissue formation and a mechanism for the delivery of DNA and functional biological factors. These biomi-metic structures have considerable potential as scaffolds for skeletal repair and regeneration, particularly when combined with inductive and stimulatory biological factors (cytokines, morphogens, signal molecules) and plasmid DNA carrying with them chemical cues that modulate and direct permanent tissue formation complimentary with the host.

Idiopathic osteoarthritis (OA) is a complex, late-onset disease whose causes are still unknown. In spite of tremendous efforts, the search for the genes pre-disposing towards osteoarthritis has so far met with little success. We hypothesize that epigenetic changes play a major role in the pathology of OA. Epigenetics refers to stable, heritable, but potentially reversible modifications of gene expression that do not involve mutations in the DNA sequence, for example DNA methylation or histone modification. Epigenetic changes are gene and cell-type specific, may arise sporadically with increasing age or be provoked by environmental factors. To investigate whether epigenetic changes are significant factors in OA, we examined the DNA methylation status of the promoter regions of three genes that are expressed by OA, but not by normal, articular chondrocytes, namely MMP-3 (stromelysin-1), MMP-9 (gelatinase B) and MMP-13 (collagenase3). We hypothesized that these genes are silenced in normal chondrocytes by methylation of the cytosines of CpG dinucleotides in the respective promoter regions, but that abnormal expression is associated with a de-methylation, leading to eunsilencing f of gene expression. Cartilage was obtained from the femoral heads of 16 OA and 10 femoral neck fracture (#NOF) patients, which served as controls due to the inverse relationship between osteoporosis and OA. The cartilage was milled in a freezer mill with liquid nitrogen, DNA was extracted with a Qiagen kit, digested with methylation sensitive restriction enzymes, followed by PCR amplification. These enzymes will cut at their specific cleavage sites only if the CpGs is not methylated and thus allow us to determine methylation status of specific CpG sites.

Results. Less than 5% of the chondrocytes in superficial layer from #NOF cartilage expressed degradative enzymes, whereas all cloned chondrocytes from advanced-stage OA cartilage were immunopositive. The overall % of CpG demethylation in the promoters of control patients (whose chondrocytes did not express the enzymes) was 20.1%, whereas 48.6% of CpG sites were demethylated in degradative chondrocytes of OA patients (p< 0.001). For MMP-13, the increase in demethylation between control and OA was from 4 ^..20%; for MMP-9 from 47 ^..81% and for MMP-3 from 30 ^..57%. However, not all available CpG sites were equally demethylated. Some sites were uniformly methylated in both OA and controls, others were demethylated even in controls. However, there was at least one crucial site for each degradative enzyme, where the differences in the degree of methylation were greatest and statistically different. These sites were at −110 for MMP-13; −36 for MMP-9; −635 for MMP-3. There was no relation between the % demethylation and the patient fs age and no apparent difference between males and females.

Conclusions: We have demonstrated an association between abnormal gene expression of MMP-3, MMP-9 and MMP-13 and promoter DNA demethylation. This epigenetic dysregulation of genes appeared to be clonally inherited by daughter cells and may be typical for osteoarthritis and other complex, late-onset diseases.

Clonal chondrocytes of osteoarthritic (OA) cartilage express an aberrant set of genes. We hypothesize that this aberrant gene expression may be due to clonally inherited epigenetic changes, defined as altered gene expression without changes in genetic sequence. The major epigenetic changes are due to altered DNA methylations in crucial parts of the promoter region. If the cytosines of CpG dinucleotides are methylated, the gene will be silenced, even if the right transcription factors are present. Similarly, de-methylations may activate previously silenced genes. Our aims were to provide ‘proof-of-concept’ data by examining the methylation status of genes in OA vs non-OA chondrocytes. Articular cartilage was obtained a) from the cartilage of fracture-neck-of-femur (#NOF) patients and b) from or around the eroded regions of OA samples. The former was full thickness cartilage, the latter was partially degraded cartilage, which contained mostly clonal chondrocytes as confirmed by histology. The cartilage samples were ground in a freezer mill (Glen Creston, UK) and DNA was extracted with a Qiagen DNeasy maxi kit. To assess DNA methylation status, the genomic DNA was treated overnight with methylation-sensitive restriction enzymes. Cleavage of selected sites was detected by PCR amplifications with primer pairs designed to bracket selected promoter regions. Loss of the PCR band after digestion with the enzymes indicated absence of methylations, whereas presence of the band indicated methylated cytosine. We selected MMP-9 as one of genes that is activated in OA. Transcription of mmp-9 is regulated by a 670 bp sequence at the 5′-end flanking region, which contains 6 CpGs and a further 21 CpGs within the 1.5 kb region further upstream. A PCR primer pair was designed to bracket a 350bp sequence upstream from the transcription start site of mmp-9, which contained four of the six potential methylation sites, cleaved by the methylation-sensitive enzymes AciI and HhaI. DNA from 9 OA patients, 5 #NOF patients and 1 rheumatoid arthritic (RA) patient were digested with HhaI or AciI and examined for the presence or absence of PCR bands. In all patients, digestion with HhaI abolished the PCR band, indicating that the HhaI site was never methylated in either #NOF or OA patients. However, a remarkable difference was found after digestion with AciI: in 8/9 OA patients, the PCR band was no longer detectable, while in 4/5 #NOF patients the PCR band was still present. This suggested that all three AciI cleavage sites were methylated in the majority of chondrocytes from #NOF patients, while at least one of the three AciI cleavage sites was unmethylated in OA patients. Interestingly, the PCR band was present in the RA patient, suggesting methylation of the AciI cleavage sites. The present study provides the first ‘proof-of-concept’ data that suggest epigenetic changes may play a role in the etiology of osteoarthritis. Clearly further work is required to establish the generality of the present findings and whether de-methylations are also found in the promoter regions of other genes that are aberrantly expressed in OA.

Introduction: The ability to generate bone for skeletal repair, replacement or restoration is a major clinical need. Indeed the paucity of techniques in reconstructive surgery and trauma emphasise the need for alternative bone formation strategies. Natural biological ceramic structures possess arrangements of structural elements that govern and optimise tissue function, nutrition and organisation. The aim of this study was to fabricate biomineral microporous shells with highly complex forms and to examine their ability to interact with human osteoprogenitor cells as cell and growth factor delivery vehicles.

Methods: Microporous vaterite shells were generated using a synthetic in-solution mineralisation technique in which mineral is spontaneously deposited around vesicular templates (Walsh and Mann 1999)* Porous and textured self-organising hollow microspheres (5–20 _m) were generated expressing controlled and uniform shapes. These micropores puncture the surface at high densities and are interconnected throughout the sphere. Primary human bone marrow cells labelled with Cell Tracker Green and ethidium homodimer-1 fluorescent labels and osteoprogenitors transfected with an adenoviral vector expressing Green Fluorescent Protein (AdGFP) were cultured with vaterite shells over three weeks.

Results: Cell biocompatibility of these biomimetic spheres was confirmed by confocal fluorescence and light microscopy in primary human bone marrow cultures labelled with CTG and bone marrow cultures transfected with AdGFP. At three weeks microspheres were encapsulated and integrated with osteoprogenitor cells. Histological analysis confirmed expression of alkaline phosphatase, extracellular matrix synthesis and the capacity for extensive mineralisation. Examination by SEM, fluorescent and light microscopy showed that the growth of osteoprogenitors transfected with AdGFP and microspheres in pellet culture showed vaterite spheres were encapsulated and integrated within the osteoprogenitor cell matrix indicating the potential of growth factor delivery. To determine the potential of the spheres to encapsulate selected proteins, microporous spheres were incubated with bovine haemoglobin. FITC microscopic evidence showed haemoglobin could be entrapped inside the spheres and between the biomineral crystal plates during self-assembly.

Discussion and Conclusion: These studies demonstrate the development of facile techniques for the generation of porous microsphere sponges that are biocompatible, possess the ability to aid mineralisation and for the delivery of cell and growth factors. These calcium carbonate structures provide a material with widespread application in a range of tissue engineering applications including skeletal tissue regeneration.

Introduction: The clinical need for a biodegradable material with broad application is evidenced by the fact that tissue loss as a result of injury or disease provides reduced quality of life for many at significant socio-economic cost. The development of simple biodegradable materials, with broad applicability and tissue/ cell specificity has to date proved elusive. Natural biopolymers such as alginate and chitosan are structural biomaterials of increasing significance to tissue repair and regeneration due to their potential for fabrication, design and efficient, environmentally benign synthesis. We describe the development of innovative microcapsule scaffolds based on chitosan and alginate that can be tailored to a range of cell types for a variety of tissues.

Methods: Semi-permeable polysaccharide microcapsules were produced by a one-step method, in which the deposition of a semi-permeable alginate/chitosan membrane around droplets of sodium alginate was coupled with in-situ precipitation of amorphous calcium phosphate as described by Leveque et al (2002)*. A variety of human cell types including mesenchymal stem cells, osteoprogenitors selected using the STRO-1 antibody by magnetically activated cell separation (MACS), osteoprogenitors transfected with adenovirus expressing Green Fluorescent Protein (GFP) and chondrocytes were mixed with sodium alginate and encapsulated within alginate/chitosan and calcium phosphate.

Results: Hybrid spheres (750–10,000um) were generated encapsulating primary human osteoprogenitor cells, STRO-1 selected osteoprogenitors and AdGFP transfected osteoprogenitors. Encapsulated cells remain viable inside the polysaccharide microcapsules for 2 weeks as shown by positive alkaline phosphatase staining of encapsulated cells. Cells expressing GFP were observed within microspheres indicating the e ability to deliver cells/factors as well as the potential for gene therapy. Encapsulation and delivery of active BMP-2 was confirmed using the promyoblast cell line C2C12 known to be exquisitely sensitive to BMP-2. Nucleation of calcium phosphate occurred within the polysaccharide membrane and could be controlled by the phosphate concentration in the alginate droplets to produce hybrid microcapsules with enhanced mechanical strength. Thin walled capsules were shown to split and degrade in culture within 2–4 days releasing viable osteoprogenitor cells indicating the ability to manipulate the mechanical integrity and to programme degradation of the microspheres. Finally we have shown that aggregation of the microspheres into extended frameworks can be achieved using a designed droplet/vapour aerosol system resulting in foams of aggregated beads.

Discussion and Conclusion: A variety of human skeletal cells have been encapsulated within polysaccharide/ calcium phosphate microspheres and extended frameworks with specifiable dimensions. These composite scaffolds offer stable mechanical and chemical biomimetic environments conducive to normal cell function. Natural polysaccharides are also highly amenable to complexation with a range of bioactive molecules and consequently offer tremendous potential in tissue engineering and regeneration of hard and soft tissues.

The use of designer scaffolds to deliver biologically active osteogenic growth factors such as recombinant human bone morphogenetic protein-2 (rhBMP-2) to the sites of tissue regeneration in for example orthopaedics, has tremendous therapeutic implications. The aims of this study were to generate biomimetic biodegradable porous osteogenic scaffolds using a supercritical fluid process to encapsulate rhBMP-2, and to examine the ability of the scaffolds to promote human osteoprogenitor differentiation and bone formation in vitro and in vivo.

The rhBMP-2 encapsulated in Poly(-lactic acid) (PLA) scaffolds (100ng/mg PLA) were generated using an innovative supercritical fluid mixing method. The bioactivity of rhBMP-2 encapsulated PLA scaffolds were confirmed by induction of the C2C12 promyoblast cell line into the osteogenic lineage as detected by alkaline phosphatase expression. No induction of alkaline phosphatase-positive cells was observed using blank scaffolds. BMP-2 released from encapsulated constructs promoted adhesion, migration, expansion and differentiation of human osteoprogenitor cells on 3-D scaffolds. Enhanced matrix synthesis and cell differentiation on growth factor encapsulated scaffolds was observed following culture of human osteoprogenitors on explants of chick femoral bone wedge defects in an ex vivo model of bone formation developed using the chick chorioallantoic membrane model. In vivo studies using diffusion chamber implantation and subcutaneous implantation of human osteoprogenitors on rhBMP-2 encapsulated scaffolds showed morphologic evidence of new bone matrix and cartilage formation in athymic mice as assessed by x-ray analysis, immunocytochemistry and birefringence. These studies provide evidence of controlled release of BMP-2 from biodegradable polymer scaffolds initiating new bone formation in vivo.

The generation of 3-D biomimetic structures incorporating osteoinductive factors such as BMP-2 indicates their potential for de novo bone formation that exploits cell-matrix interactions and, significantly, realistic delivery protocols for growth factors in musculo-skeletal tissue engineering.

The formation of biomimetic environments using scaffolds containing cell recognition sequence and osteo-inductive factors in combination with bone cells offers tremendous potential for bone and cartilage regeneration. In tissues, collagen forms the scaffold by mediating the flux of chemical and mechanical stimuli. Recently, a synthetic 15-residue peptide P-15, related biologically to the active domain of type I collagen, has been found to promote attachment and the osteoblast phenotype of human dermal fibroblasts and periodontal ligament fibroblasts on particulate anorganic bone mineral (ABM). The aim of this study was to exam the ability of the collagen peptide, P-15, to promote human osteoprogenitor attachment, proliferation and differentiation on cell culture surfaces and 3-D scaffolds.

Selected human bone marrow cells were cultured on particulate microporous anorganic bone mineral (‘pure ‘ hydroxyapatite based on x-ray diffraction standard JCPDS9-432) phase and polygalactin vicryl mesh adsorbed with or without P-15 in basal or osteogenic conditions. Cell adhesion, spreading and patterning were examined by light and confocal microscopy following incorporation of cell tracker green and ethidium homodimer fluorescent labels. Osteoprogenitor proliferation and differentiation was assessed by DNA content and alkaline phosphatase specific activity. Growth and differentiation on 3-D ABM structures were examined by confocal and scanning electron microscopy (SEM).

P-15 promoted human osteoprogenitor cell attachment and patterning on particulate bovine anorganic bone mineral phase and polygalactin vicryl mesh over 5–24 hours compared to culture on ABM and vicryl mesh alone as observed by photomicroscopy. Increased alkaline phosphatase specific activity was enhanced following culture on P-15 adsorbed matrices as recognized by enhanced expression of alkaline phosphatase, type I collagen, osteocalcin and cfba-1. The presence of mineralised bone matrix and extensive cell ingrowth and cellular bridging between 3-D ABM matrices and polygalactin vicryl mesh adsorbed with P-15 was observed by confocal microscopy and alizarin red staining. SEM confirmed the 3-D structure of newly formed cell constructs and cellular ingrowth on and between the P-15 modified inorganic bone mineral materials. Negligible cell growth was observed on ABM alone or polygalactin vicryl mesh alone.

These observations demonstrate that the synthetic 15-residue collagen peptide, P-15, when adsorbed to ABM or polygalactin vicryl mesh, can stimulate human osteoprogenitor attachment and spreading. They also demonstrated that P-15 coupled 3-D matrices stimulate human osteoprogenitor differentiation and materialisation. The studies indicate that a synthetic analogue of collagen provides a biomimetic environment supportive for cell differentiation and tissue regeneration and indicate a potential for the use of extracellular matrix cue in the development of biomimetic environments for bone tissue engineering.

Ex vivo gene transfer of osteogenic factors into multipotential stem cells offers potentially important therapeutic implications in a variety of musculoskeletal diseases. One possible approach is the development of a cellular vehicle, namely bone morphogenetic protein (BMP)-producing bone marrow cells, created using adenoviral gene transfer. These transduced cells provide local delivery of BMP for bone formation. The aims of this study were to study the feasibility of gene transfer to human bone osteoprogenitor cells, using adenoviral vectors. Specifically, the aims were to study the efficacy of transduction with an adenoviral vector expressing BMP-2 and then to determine the ability of the transduced cells to produce active BMP-2 and to generate bone ex vivo.

Primary human bone marrow osteoprogenitor cells were expanded in culture and infected with AxCALacZ, a replication-deficient adenoviral vector carrying the E. coli lacZ gene, with a range of multiplicity of infection (MOI) of 6.25 to100. Transduced cells showed positive staining for β-galactosidase using X-Gal with an efficiency close to 100%. Uninfected cells showed no β-galactosidase activity. Efficiency was independent from MOI, however cells infected at the lower MOIs expressed lower levels of β-galactosidase. Following confirmation that primary bone marrow cells could be infected by adenoviral constructs, additional osteoprogenitors were infected with AxCAOBMP-2, a vector carrying the human BMP-2 gene, at a multiplicity of infection of 10–20. In order to determine BMP-2 activity, conditioned media from bone marrow cells expressing BMP-2 was added to promyoblast C₂C₁₂ cells. The promyoblast C₂C₁₂ cells are exquisitely sensitive to BMP-2 with induction of alkaline phosphatase activity (ED₅₀ 20 nM) in a dose-dependant manner. Alkaline phosphatase activity was induced following culture with conditioned media from BMP-2 expressing cells, in a dose dependant manner, confirming successful secretion of active BMP-2. Immunohistochemical staining for alka- line phosphatase in C₂C₁₂ cells also confirmed the bio-chemical observations. Media from uninfected control human bone marrow cells failed to produce a similar effect. The concentration of BMP-2 in the media was estimated to be 5–10 nM/10⁷ cells.

To examine whether adenoviral transfection affected the osteoblast phenotype and their ability to mineralise in vitro, adenovirally-transduced bone marrow cells expressing BMP-2 were seeded onto poly(-lactic acid co÷glycolic acid) (75:25) porous scaffolds (provided by K. Shakesheff and S. Howdle; Nottingham University) and cultured for up to 6 weeks. Expression of alkaline phosphatase activity, type I collagen formation, as well as the synthesis of osteoblast stimulating factor-1 confirmed bone cell differentiation and maintenance of the osteoblast phenotype in extended culture for up to 6 weeks.

These results indicate the ability to deliver active BMP-2 using human bone marrow osteoprogenitor cells following adenoviral infection. The maintenance of osteoblast phenotype in extended culture and generation of mineralised 3-D scaffolds containing such constructs offers a realistic approach to tissue engineer bone for orthopaedic applications.

The growth plates of rapidly growing animals have been studied extensively. Nevertheless, several questions remain unanswered, partly because many events happen simultaneously, especially at the vascular front. Terminal chondrocytes are thought to undergo programmed cell death, but the fate of the cell remnants remains unclear. Are the dying cells released into the vascular space and phagocytosed by macrophages, as one would expect for apoptosis? Or are the cells eliminated prior to opening of the lacunae, leaving empty lacunae? Do all terminal chondrocytes die or do some become bone-forming cells? Rodents maintain a growth plate into old age, long after longitudinal growth has ceased. These stationary growth plates have several features not found in the growth plates of rapidly growing animals and closer study of these features may provide answers to the above questions. Femurs and tibiae from 4–16 week-old and 62–80 week-old rats were decalcified, processed into paraffin, and the morphological changes were documented.

Between 4–16 weeks, the heights of the growth plates decreased due to loss of the large hypertrophic chondrocytes, but the various zones were still present. In the aged rats, the growth plates were identifiable as a narrow cartilaginous band with some short columns of inactive cells. The vascular front was irregular, the narrow spicules of primary spongiosa were absent and the much thicker spicules, which are normally seen in secondary spongiosa, directly abutted to the cartilage. Horizontal apposition of bone matrix onto the cartilage edge was frequently present. In addition, the following features were noted. 1) Acellular areas: Nearly all growth plates contained regions of cartilage from which all cells and their lacunae had disappeared. In some cases, these acellular regions stretched from the reserve zone to the vascular front and even persisted as a relatively wide core within the spicules of spongiosa, indicating increased resistance of acellular cartilage to resorption. The absence of cells or cell debris was consistent with an autophagic mode of cell death and subsequent collapse of the lacunae. 2) Remodelling within the growth plate; in some growth plates, large regions of growth plate cartilage had been resorbed and new bone had been laid down in a pattern similar to the remodelling of cortical bone. This suggested that the normal resistance of cartilage to vascular invasion had been lost locally, but was maintained in adjacent non-remodelled regions. 3) Trans-differentiation of chondrocytes to bone-forming cells; extensive new medullary bone formation was noted in the diaphysis of approximately 30% of the aged rats, suggesting that they had received an (unknown) osteogenic stimulus. In these rats, bone matrix was identifiable inside chondrocytic lacunae, and spreading beyond the confines of the lacunae, thus directly replacing growth plate cartilage with bone matrix.

The results suggest that i) chondrocytes are capable of self-elimination, perhaps by a mechanism similar to the autophagic cell death that occurs during insect metamorphosis; ii) resorption of cartilage and vascular invasion requires the presence of the viable chondrocytes; and iii) chondrocytes have the capacity to transdifferentiate to bone-forming cells, but only do so when receiving an increased osteogenic stimulus.