Alexander Aarvold

Skeletal stem cells and bone regeneration: translational strategies from bench to clinic.

Abstract Clinical imperatives for new bone to replace or restore the function of traumatized or bone lost as a consequence of age or disease has led to the need for therapies or procedures to generate bone for skeletal applications. Tissue regeneration promises to deliver specifiable replacement tissues and the prospect of efficacious alternative therapies for orthopaedic applications such as non-union fractures, healing of critical sized segmental defects and regeneration of articular cartilage in degenerative joint diseases. In this paper we review the current understanding of the continuum of cell development from skeletal stem cells, osteoprogenitors through to mature osteoblasts and the role of the matrix microenvironment, vasculature and factors that control their fate and plasticity in skeletal regeneration. Critically, this review addresses in vitro and in vivo models to investigate laboratory and clinical based strategies for the development of new technologies for skeletal repair and the key translational points to clinical success. The application of developmental paradigms of musculoskeletal tissue formation specifically, understanding developmental biology of bone formation particularly in the adult context of injury and disease will, we propose, offer new insights into skeletal cell biology and tissue regeneration allowing for the critical integration of stem cell science, tissue engineering and clinical applications. Such interdisciplinary, iterative approaches will be critical in taking patient aspirations to clinical reality.

A comparison of polymer and polymer–hydroxyapatite composite tissue engineered scaffolds for use in bone regeneration. An in vitro and in vivo study

Previous in vitro work demonstrated porous PLA and PLGA both had the mechanical strength and sustained the excellent skeletal stem cell (SSC) growth required of an osteogenic bonegraft substitute, for use in impaction bone grafting. The purpose of this investigation was to assess the effects of the addition of hydroxyapatite (HA) to the scaffolds before clinical translation. PLA, PLA+10% HA, PLGA, and PLGA+10% HA were milled and impacted into discs before undergoing a standardized shear test. Cellular compatibility analysis followed 14 days incubation with human skeletal stems cells (SSC). The best two performing polymers were taken forward for in vivo analysis. SSC seeded polymer discs were implanted subcutaneously in mice. All polymers had superior mechanical shear strength compared with allograft (p < 0.01). Excellent SSC survival was demonstrated on all polymers, but the PLA polymers showed enhanced osteoblastic activity (ALP assay p < 0.01) and collagen-1 formation. In vivo analysis was performed on PLA and PLA+10% HA. MicroCT analysis revealed increased bone formation on the PLA HA (p < 0.01), and excellent neo-vessel formation in both samples. Histology confirmed evidence of de novo bone formation. PLA HA showed both enhanced osteoinductive and osteogenic capacity. This polymer composite has been selected for scaled-up experimentation before clinical translation.

Enhancing the osteogenic efficacy of human bone marrow aspirate: concentrating osteoprogenitors using wave-assisted filtration

BACKGROUND Recent approaches have sought to harness the potential of stem cells to regenerate bone that is lost as a consequence of trauma or disease. Bone marrow aspirate (BMA) provides an autologous source of osteoprogenitors for such applications. However, previous studies indicated that the concentration of osteoprogenitors present in BMA is less than required for robust bone regeneration. We provide further evidence for the importance of BMA enrichment for skeletal tissue engineering strategies using a novel acoustic wave-facilitated filtration strategy to concentrate BMA for osteoprogenitors, clinically applicable for intraoperative orthopedic use. METHODS Femoral BMA from 15 patients of an elderly cohort was concentrated for the nucleated cell fraction against erythrocytes and excess plasma volume via size exclusion filtration facilitated by acoustic agitation. The effect of aspirate concentration was assessed by assays for colony formation, flow cytometry, multilineage differentiation and scaffold seeding efficiency. RESULTS BMA was filtered to achieve a mean 4.2-fold reduction in volume with a corresponding enrichment of viable and functional osteoprogenitors, indicated by flow cytometry and assays for colony formation. Enhanced osteogenic and chondrogenic differentiation was observed using concentrated aspirate and enhanced cell-seeding efficiency onto allogeneic bone graft as an effect of osteoprogenitor concentration relative specifically to the concentration of erythrocytes in the aspirate. CONCLUSIONS These studies provide evidence for the importance of BMA nucleated cell concentration for both cell differentiation and cell seeding efficiency and demonstrate the potential of this approach for intraoperative application to enhance bone healing.

Supercritical CO2 fluid-foaming of polymers to increase porosity: a method to improve the mechanical and biocompatibility characteristics for use as a potential alternative to allografts in impaction bone grafting?

Disease transmission, availability and cost of allografts have resulted in significant efforts to find an alternative for use in impaction bone grafting (IBG). Recent studies identified two polymers with both structural strength and biocompatibility characteristics as potential replacements. The aim of this study was to assess whether increasing the polymer porosity further enhanced the mechanical and cellular compatibility characteristics for use as an osteogenic biomaterial alternative to allografts in IBG. Solid and porous poly(DL-lactide) (P(DL)LA) and poly(DL-lactide-co-glycolide) (P(DL)LGA) scaffolds were produced via melt processing and supercritical CO(2) foaming, and the differences characterized using scanning electron microscopy (SEM). Mechanical testing included milling and impaction, with comparisons made using a shear testing rig as well as a novel agitation test for 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 skeletal stem cells. SEM showed excellent porosity throughout both of the supercritical-foam-produced polymer scaffolds, with pores between 50 and 200 μm. Shear testing showed that the porous polymers exceeded the shear strength of allograft controls (P<0.001). Agitation testing showed greater cohesion between the particles of the porous polymers (P<0.05). Cellular studies showed increased cell number, viability, and osteogenic differentiation on the porous polymers compared to solid block polymers (P<0.05). The use of supercritical CO(2) to generate porous polymeric biodegradable scaffolds significantly improves the cellular compatibility and cohesion observed compared to non-porous counterparts, without substantial loss of mechanical shear strength. These improved characteristics are critical for clinical translation as a potential osteogenic composite for use in IBG.

From bench to clinic and back: skeletal stem cells and impaction bone grafting for regeneration of bone defects

Tissue engineering offers enormous potential for bone regeneration. Despite extensive in vitro and in vivo work, few strategies translate into clinical practice. This paper describes the combination of skeletal stem cells (SSCs) and impaction bone grafting (IBG) for the treatment of patients with bone defects associated with avascular necrosis of the femoral head. SSCs and milled allograft were impacted into necrotic bone in the femoral heads of four patients. Three patients remained asymptomatic at 22–44 month follow‐up, but one patient has required total hip replacement (both hips). This has allowed retrieval of the femoral heads, which were analysed structurally and functionally by μCT, histology and mechanical testing. A central channel of impacted bone was found in the femoral heads, which displayed a mature trabecular micro‐architecture. The impacted bone was denser than the surrounding trabecular bone, as strong in compression and with histological micro‐architecture comparable to that of trabecular bone. Analysis of the retrieved femoral head samples has demonstrated that this tissue‐engineering strategy regenerates bone that is both structurally and functionally analogous to normal trabecular bone. SSCs, together with IBG, have proved an effective treatment for avascular necrosis of the femoral head and offer significant potential for the broader spectrum of bone defects. Copyright

A tissue engineering strategy for the treatment of avascular necrosis of the femoral head

Background & purpose Skeletal stem cells (SSCs) and impaction bone grafting (IBG) can be combined to produce a mechanically stable living bone composite. This novel strategy has been translated to the treatment of avascular necrosis of the femoral head. Surgical technique, clinical follow-up and retrieval analysis data of this translational case series is presented. Methods SSCs and milled allograft were impacted into necrotic bone in five femoral heads of four patients. Cell viability was confirmed by parallel in vitro culture of the cell-graft constructs. Patient follow-up was by serial clinical and radiological examination. Tissue engineered bone was retrieved from two retrieved femoral heads and was analysed by histology, microcomputed tomography (μCT) and mechanical testing. Results Three patients remain asymptomatic at 22- to 44-month follow-up. One patient (both hips) required total hip replacement due to widespread residual necrosis. Retrieved tissue engineered bone demonstrated a mature trabecular micro-architecture histologically and on μCT. Bone density and axial compression strength were comparable to trabecular bone. Conclusions Clinical follow-up shows this to be an effective new treatment for focal early stage avascular necrosis of the femoral head. Unique retrieval analysis of clinically translated tissue engineered bone has demonstrated regeneration of tissue that is both structurally and functionally analogous to normal trabecular bone.

Taking tissue engineering principles into theatre: retrieval analysis from a clinically translated case

AIM Tissue engineering has enormous potential for the regeneration of bone defects. Approximately 4 years ago we reported on a 62 year old patient who underwent treatment of a benign cyst in the proximal femur by impaction bone grafting supplemented with autologous bone marrow. The cyst and symptoms subsequently recurred and this patient has now required a total hip replacement. This has provided a rare opportunity for ex vivo analysis of clinically applied tissue engineered bone. MATERIALS & METHODS The femoral head was retrieved at surgery and the structural and functional characteristics of the tissue engineered bone were analyzed by micro-computed tomography, histology and mechanical testing. RESULTS The impacted bone demonstrated a trabecular structure that contained islands of nonincorporated graft. The graft was denser than the patients trabecular bone with comparable strength. The cyst material had penetrated along the channel of bone and an increased number of osteoclasts were observed. DISCUSSION This study has provided detailed ex vivo analysis of retrieved human tissue engineered bone and possible reasons for the observed construct failure are discussed in this article. The impacted bone displayed some evidence of remodeled trabecular structure, although the bone marrow aspirate that was initially combined with the allograft contained a relatively low concentration of osteoprogenitor cells. Cellular augmentation was insufficient to overcome the osteoclastic process associated with renewed cyst formation. Concentration or culture expansion of osteoprogenitor cells from aspirated bone marrow is recommended for biological augmentation of bone graft.

Large animal in vivo evaluation of a binary blend polymer scaffold for skeletal tissue-engineering strategies; translational issues

Binary blend polymers offer the opportunity to combine different desirable properties into a single scaffold, to enhance function within the field of tissue engineering. Previous in vitro and murine in vivo analysis identified a polymer blend of poly(l‐lactic acid)–poly(ε‐caprolactone) (PLLA:PCL 20:80) to have characteristics desirable for bone regeneration. Polymer scaffolds in combination with marrow‐derived skeletal stem cells (SSCs) were implanted into mid‐shaft ovine 3.5 cm tibial defects, and indices of bone regeneration were compared to groups implanted with scaffolds alone and with empty defects after 12 weeks, including micro‐CT, mechanical testing and histological analysis. The critical nature of the defect was confirmed via all modalities. Both the scaffold and scaffold/SSC groups showed enhanced quantitative bone regeneration; however, this was only found to be significant in the scaffold/SSCs group (p = 0.04) and complete defect bridging was not achieved in any group. The mechanical strength was significantly less than that of contralateral control tibiae (p < 0.01) and would not be appropriate for full functional loading in a clinical setting. This study explored the hypothesis that cell therapy would enhance bone formation in a critical‐sized defect compared to scaffold alone, using an external fixation construct, to bridge the scale‐up gap between small animal studies and potential clinical translation. The model has proved a successful critical defect and analytical techniques have been found to be both valid and reproducible. Further work is required with both scaffold production techniques and cellular protocols in order to successfully scale‐up this stem cell/binary blend polymer scaffold.

The effect of porosity of a biphasic ceramic scaffold on human skeletal stem cell growth and differentiation in vivo.

Skeletal stem cell (SSC) growth on a novel porous HA/TCP scaffold has been investigated in vivo. The effect of porosity on osteogenic differentiation was assessed by comparing two groups of scaffolds with differing porosity but controlled pore size. Histology, microCT, scanning electron microscopy, and biochemical analysis were used to assess SSC proliferation and differentiation. The 45 pores per inch (ppi) scaffold demonstrated a greater increase in density than the 30 ppi scaffold following in vivo culture, and a reduction in dimensions of the pores and channels of the higher porosity scaffold was observed, indicating generation of new tissue within the pores. All scaffolds supported SSC proliferation but the higher scaffold porosity augmented osteogenic differentiation. ALP specific activity was enhanced on the 45 ppi scaffold compared to the 30 ppi scaffold. These studies demonstrate the importance of porosity in scaffold design and impact therein for tissue engineering application.

Tantalum trabecular metal - addition of human skeletal cells to enhance bone implant interface strength and clinical application.

The osteo‐regenerative properties of allograft have recently been enhanced by addition of autogenous human bone marrow stromal cells (HBMSCs). Limitations in the use of allograft have prompted the investigation of tantalum trabecular metal (TTM) as a potential alternative. TTM is already in widespread orthopaedic use, although in applications where there is poor initial stability, or when TTM is used in conjunction with bone grafting, initial implant loading may need to be limited. The aim of this study was to evaluate the osteo‐regenerative potential of TTM with HBMSCs, in direct comparison to human allograft and autograft. HBMSCs were cultured on blocks of TTM, allograft or autograft in basal and osteogenic media. Molecular profiling, confocal and scanning electron microscopy (SEM) and biochemical assays were used to characterize cell adherence, proliferation and phenotype. Mechanical testing was used to define the tensile characteristics of the constructs. HBMSCs displayed adherence and proliferation throughout TTM, evidenced by immunocytochemistry and SEM, with significant cellular ingrowth and matrix production through TTM. In contrast to cells cultured with allograft, cell proliferation assays showed significantly higher activity with TTM (p < 0.001), although molecular profiling confirmed no significant difference in expression of osteogenic genes. In contrast to acellular constructs, mechanical testing of cell–TTM constructs showed enhanced tensile characteristics, which compared favourably to cell–allograft constructs. These studies demonstrated the ability of TTM to support HBMSC growth and osteogenic differentiation comparable to allograft. Thus, TTM represents an alternative to allograft for osteo‐regenerative strategies, extending its clinical applications as a substitute for allograft. Copyright