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.

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.

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.

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.

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.