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. Aims. To develop a novel strategy to enrich skeletal stem cells (SSCs) from human BMA, clinically applicable for intra-operative orthopaedic use. Methods. Iliac crest BMA was purchased from commercial suppliers and femoral canal BMA was obtained with informed consent from older patients undergoing total hip replacement. 5 to 40ml of BMA was processed to obtain 2–8 fold volume reductions. SSC function was assessed by assays for fibroblastic colony-forming units (CFU-F). Cell viability and seeding efficiency of processed and unprocessed aspirates applied to allograft was assessed. Results. 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, functional SSCs as confirmed by flow cytometry, CFU-F assays and histological analysis. The SSC enrichment of bone marrow aspirate significantly enhanced cell seeding efficiency onto allograft confirming the utility of this approach for application to bone regeneration. Conclusion. The ability to rapidly enrich BMA demonstrates the potential of this strategy for intra-operative application to enhance bone healing. The development of this device offers immediate potential for clinical application to reduce morbidity in many scenarios associated with local bone stock loss. Further analysis in vivo is ongoing prior to clinical tests

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. Aim. To use tissue engineering strategies to enhance the reconstructive properties of tantalum, as an alternative to allograft. Methods. Human bone marrow stromal cells (5×10. 5. cells/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 detail cell adherence, proliferation and phenotype. Results. Cells displayed extensive adherence and proliferation throughout trabecular tantalum. Samples cultured in osteogenic conditions showed abundant matrix production. Electron microscopy confirmed significant cellular growth through tantalum to a depth of 5mm. In contrast to cells cultured with allograft in both basal and osteogenic conditions, cell proliferation and biochemical assays showed significantly higher activity with tantalum than allograft. Furthermore, 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. Conclusions. These studies demonstrate trabecular tantalum supports cell growth and osteogenic differentiation at least as well as allograft. Trabecular tantalum represents a good alternative to allograft for tissue engineering osteoregenerative strategies in the context of lost bone stock. Further mechanical testing and in vivo studies are on-going

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. Aim. To investigate the effect of nanotopographical cues on adult skeletal stem cell (SSC) fate, phenotype and function within in-vitro environments. Materials and methods. Adult human skeleltal stem cells (SSCs) were immunoselected and enriched using STRO-1 antibody and cultured on tissue culture plastic (TCP) and titanium-coated nanotopgraphical substrates (illustrated in Figure 1). Following culture, metabolic activity of SSCs on TCP and Ti substrates was compared. Subsequently, osteoinductive potential was analysed under basal and osteogenic conditions (four groups: TCP in basal media, TCP in osteogenic media, Ti planar substrates basal and Ti pillar substrates basal). Results. At 7 days, cell metabolic activity was significantly enhanced on Ti substrates, specifically on Ti pillars of defined height in comparison to TCP (Figure 2). Following culture on defined topographies for 21 days, expression of the bone matrix protein, osteopontin, on Ti pillars was significantly enhanced when compared to TCP or Ti planar (Figure 3). Conclusion. We demonstrate the ability of discrete raised nanopillars to modualte adult SSC populations in the absence of any chemical cues. These results indicate the potential of discrete and defined nanopillar constructs to stimulate SSC function, an effect not observed on planar Ti constructs. These findings herald exciting opportunities to improve the bioactivity of implant design and, ultimately, osseointegration with clinical implications therein

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 CO. 2. fluid foaming (SCF) enhanced the mechanical and cellular compatibility characteristics for use as an osteogenic alternative to allograft in IBG. Methods. High molecular weight PLA scaffolds were produced via traditional (solid block) and SCF (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 SSC's. Results. SEM showed increased porosity of the SCF produced PLA scaffolds, with pores between 50–100µm. Shear testing showed the SCF polymer exceeded the shear strength of allograft controls (P< 0.001). Agitation testing showed greater cohesion between the particles of the SCF polymer (P< 0.05). Cellular studies showed increased cell number, viability and osteogenic differentiation on the SCF polymer compared to traditional polymer (P< 0.05) and allograft (P< 0.001). Conclusions. The use of supercritical C0. 2. 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 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. Methods. 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. Results. 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. Conclusions. 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