Objectives. The present study describes a novel technique for revitalising allogenic intrasynovial tendons by combining cell-based therapy and mechanical stimulation in an ex vivo canine model. Methods. Specifically, canine flexor digitorum profundus tendons were used for this study and were divided into the following groups: (1) untreated, unprocessed normal tendon; (2) decellularised tendon; (3) bone marrow stromal cell (BMSC)-seeded tendon; and (4) BMSC-seeded and cyclically stretched tendon. Lateral slits were introduced on the tendon to facilitate cell seeding. Tendons from all four study groups were distracted by a servohydraulic testing machine. Tensile force and displacement data were continuously recorded at a sample rate of 20 Hz until 200 Newton of force was reached. Before testing, the cross-sectional dimensions of each tendon were measured with a digital caliper. Young’s modulus was calculated from the slope of the linear region of the stress-strain curve. The BMSCs were labeled for histological and cell viability evaluation on the decellularized tendon scaffold under a confocal microscope. Gene expression levels of selected extracellular matrix tendon growth factor genes were measured. Results were reported as mean ± SD and data was analyzed with one-way ANOVAs followed by Tukey’s post hoc multiple-comparison test. Results. We observed no significant difference in cross-sectional area or in Young’s modulus among the four study groups. In addition, histological sections showed that the BMSCs were aligned well and viable on the tendon slices after two-week culture in groups three and four. Expression levels of several extracellular matrix tendon growth factors, including collagen type I, collagen type III, and matrix metalloproteinase were significantly higher in group four than in group three (p < 0.05). Conclusion. Lateral slits introduced into de-cellularised tendon is a promising method of delivery of BMSCs without compromising cell viability and tendon mechanical properties. In addition, mechanical stimulation of a cell-seeded tendon can promote cell proliferation and enhance expression of collagen types I and III in vitro. Cite this article: J. H. Wu, A. R. Thoreson, A. Gingery, K. N. An, S. L. Moran, P. C. Amadio, C. Zhao. The revitalisation of flexor tendon allografts with bone marrow stromal cells and mechanical stimulation: An ex vivo model revitalising flexor tendon allografts. Bone Joint Res 2017;6:179–185. DOI: 10.1302/2046-3758.63.BJR-2016-0207.R1

Abstract

Articulating cartilage experiences a multitude of biophysical cues. Due to its primary function in distributing load with near frictionless articulation, it is clear that a major stimulus for cartilage homeostasis and regeneration is the mechanical load it experiences on a daily basis. While these effects are considered when performing in vivo studies, in vitro studies are still largely performed under static conditions. Therefore, an increasing complexity of in vitro culture models is required, with the ultimate aim to recreate the articulating joint as accurately as possible. We have for many years utilized a complex multiaxial load bioreactor capable of applying tightly regulated compression and shear loading protocols. Using this bioreactor, we have been able to demonstrate the mechanical induction of human bone marrow stromal cell (BMSC) chondrogenesis in the absence of exogenous growth factors. Building on previous bioreactor studies that demonstrated the mechanical activation of endogenous TGFβ, and subsequent chondrogenesis of human bone marrow derived MSCs, we have been further increasing the complexity of in vitro models. For example, the addition of high molecular weight hyaluronic acid, a component of synovial fluid, culture medium leads to reduced hypertrophy and increased glycosaminoglycan deposition. The ultimate aim of all of these endeavors is to identify promising materials and therapies during in vitro/ ex vivo studies, therefore reducing the numbers or candidates that are finally tested using in vivo studies. This 3R approach can improve the opportunities for success while leading to more ethically acceptable product development pathways

Despite extensive research aimed at improving surgical outcomes of enthesis injuries, re-tears remain a common problem, as the repairs often lead to fibrovascular scar as opposed to a zonal enthesis. Zonal enthesis formation involves anchoring collagen fibers, synthesizing proteoglycan-rich fibrocartilage, and mineralizing this fibrocartilage [1]. During development, the hedgehog signaling pathway promotes the formation and maturation of fibrocartilage within the zonal tendon-to-bone enthesis [1-4]. However, whether this pathway has a similar role in adult zonal tendon-to-bone repair is not known. Therefore, we developed a murine anterior cruciate ligament (ACL) reconstruction model [5] to better understand the zonal tendon-to-bone repair process and perturb key developmental regulators to determine the extent to which they can promote successful repair in the adult. In doing so, we activated the hedgehog signaling pathway both genetically using transgenic mice and pharmacologically via agonist injections. We demonstrated that both treatments improved the formation of zonal attachments and tunnel integration strength [6]. These improved outcomes were due in part to hedgehog signaling's positive role in proliferation of the bone marrow stromal cell (bMSC) progenitor pool and subsequent fibrocartilage production of bMSC progeny cells that form the attachments. These results suggest that, similar to growth and development, hedgehog signaling promotes the production and maturation of fibrocartilage during tendon-to-bone integration in adults. Lastly, we developed localized drug delivery systems to further improve the treatment of these debilitating injuries in future translational studies. Acknowledgements: This work was supported by NIH R01AR076381, R21AR078429, R00AR067283, F31AR079840, T32AR007132, and P30AR069619, in addition to the McCabe Fund Pilot Award at the University of Pennsylvania

Regeneration of bone defects in elderly patients is limited due to the decreased function of bone forming cells and compromised tissue physiology. Previous studies suggested that the regenerative activity of stem cells from aged tissues can be enhanced by exposure to young systemic and tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells (hiPSCs) can enhance the bone regeneration potential of aged human bone marrow stromal cells (hBMSCs). ECM was engineered from hiPSC-derived mesenchymal-like progenitors (hiPSC-MPs), as well as young (<30 years) and aged (>70 years) hBMSCs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. Three hBMSCs of different ages were cultured on engineered ECMs. Growth and differentiation responses were compared to tissue culture plastic, as well as to collagen and fibronectin coated plates. Decellularized ECMs contained collagens type I and IV, fibronectin, laminin and < 5% residual DNA, suggesting efficient cell elimination. Cultivation of young and aged hBMSCs on the hiPSC-ECM in osteogenic medium significantly increased hBMSC growth and markers of osteogenesis, including collagen deposition, alkaline phosphatase activity, bone sialoprotein expression and matrix mineralization compared to plastic controls and single protein substrates. In aged BMSCs, matrix mineralization was only detected in ECM cultures in osteogenic medium. Comparison of ECMs engineered from hiPSC-MPs and hBMSCs of different ages suggested similar structure, composition and potential to enhance osteogenic responses in aged BMSCs. Engineered ECM induced a higher osteogenic response compared to specific matrix components. Our studies suggest that aged BMSCs osteogenic activity can be enhanced by culture on engineered ECM. hiPSCs represent a scalable cell source, and tissue engineering strategies employing engineered ECM materials could potentially enhance bone regeneration in elderly patients

Objectives. To assess the structure and extracellular matrix molecule expression of osteogenic cell sheets created via culture in medium with both dexamethasone (Dex) and ascorbic acid phosphate (AscP) compared either Dex or AscP alone. Methods. Osteogenic cell sheets were prepared by culturing rat bone marrow stromal cells in a minimal essential medium (MEM), MEM with AscP, MEM with Dex, and MEM with Dex and AscP (Dex/AscP). The cell number and messenger (m)RNA expression were assessed in vitro, and the appearance of the cell sheets was observed after mechanical retrieval using a scraper. β-tricalcium phosphate (β-TCP) was then wrapped with the cell sheets from the four different groups and subcutaneously implanted into rats. Results. After mechanical retrieval, the osteogenic cell sheets from the MEM, MEM with AscP, and MEM with Dex groups appeared to be fragmented or incomplete structures. The cell sheets cultured with Dex/AscP remained intact after mechanical retrieval, without any identifiable tears. Culture with Dex/AscP increased the mRNA and protein expression of extracellular matrix proteins and cell number compared with those of the other three groups. More bridging bone formation was observed after transplantation of the β-TCP scaffold wrapped with cell sheets cultured with Dex/AscP, than in the other groups. Conclusions. These results suggest that culture with Dex/AscP improves the mechanical integrity of the osteogenic cell sheets, allowing retrieval of the confluent cells in a single cell sheet structure. This method may be beneficial when applied in cases of difficult tissue reconstruction, such as nonunion, bone defects, and osteonecrosis. Cite this article: M. Akahane, T. Shimizu, T. Kira, T. Onishi, Y. Uchihara, T. Imamura, Y. Tanaka. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure. Bone Joint Res 2016;5:569–576. DOI: 10.1302/2046-3758.511.BJR-2016-0013.R1

Regeneration of bone defects in elderly patients is limited due to the decreased function of bone forming cells and compromised tissue physiology. Previous studies suggested that the regenerative activity of stem cells from aged tissues can be enhanced by exposure to young systemic and tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells (hiPSCs) can enhance the bone regeneration potential of aged human bone marrow stromal cells (hBMSCs). ECM was engineered from hiPSC-derived mesenchymal-like progenitors (hiPSC-MPs), as well as young (70 years) hBMSCs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. Three hBMSCs of different ages were cultured on engineered ECMs. Growth and differentiation responses were compared to tissue culture plastic controls. Decellularized ECMs contained collagens type I and IV, fibronectin, laminin and < 5% residual DNA. Cultivation of young and aged hBMSCs on the hiPSC-ECM in osteogenic medium significantly increased hBMSC growth and markers of osteogenesis, including collagen deposition, alkaline phosphatase activity, bone sialoprotein expression and matrix mineralization compared to plastic controls. In aged BMSCs, matrix mineralization was only detected in ECM cultures in osteogenic medium. Comparison of ECMs engineered from hiPSC-MPs and hBMSCs of different ages suggested similar structure, composition and potential to enhance osteogenic responses in aged BMSCs. Our studies suggest that aged BMSCs regenerative activity can be enhanced by culture on hiPSC-engineered ECM

Abstract. Objectives. Assess and characterise the suitability of a novel silk reinforced biphasic 3D printed scaffold for osteochondral tissue regeneration. Methods. Biphasic hybrid scaffolds consisted of 3D printed poly(ethylene glycol)-terephthalate-poly(butylene terephthalate)(PEGT/PBT) scaffold frame work (pore size 0.75mm), which has been infilled with a cast and freeze dried porous silk scaffold (5×5×2mm. 3. ), in addition to a seamless silk top layer (1mm). Silk scaffolds alone were used as controls. Both the biphasic and control scaffolds were characterised via uniaxial compression testing (strain rate 0.1mm/min), and the potential biocompatibility of the scaffolds was tested via in vitro culture of seeded bone marrow stromal cells post fabrication. Results. Uniaxial compression testing showed that the biphasic scaffolds (N=4) initially demonstrated similar behaviour on a stress-strain curve to a silk scaffold alone control group (N=6), until a strain of 30% was reached. After 30% strain, load was transitioned from the silk only chondral layer to the 3D printed PEGT/PBT scaffold which resisted further compression and exhibited a significantly greater compressive modulus of 12.6±0.9MPa compared to 0.113±0.01MPa (p<0.001) in the silk scaffold control group. Following 24hours of seeding, no difference was noticed in cell adhesion behaviour under fluorescent microscopy between silk scaffolds and biphasic scaffolds (n=5). Discussion. The use of 3D printing within this novel scaffold provides a solid framework and increases its versatility. The reinforced silk not only provides the secondary Porous structure to the 3D printed scaffold for the bone phase, but also a superficial layer for the cartilage phase. This unique structure has the potential to fill a niche within osteochondral tissue regeneration, especially with the possibility for its use within personalised medicine. Conclusions. These results demonstrate that the novel silk reinforced biphasic 3D printed scaffold is biocompatible and has suitable mechanical properties for osteochondral tissue regeneration. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Summary. Methotrexate chemotherapy (commonly used in treating cancers and rheumatoid arthritis) creates an inflammatory condition in bone, decreasing osteogenesis, enhancing adipogenesis, increasing osteoclastogenesis, leading to bone loss and marrow adiposity; treatment with fish oil or folinic acid counteracts these negative effects and prevents bone loss. Introduction. Chemotherapy with anti-metabolite methotrexate (MTX) is commonly used in treating cancers and rheumatoid arthritis; however it is known to cause bone loss for which currently there are no adjunct preventative treatments. Methods and Materials. Using a rat model, this study investigated the damaging effects in bones caused by daily MTX injections (0.75mg/kg) for 5 consecutive days (mimicking induction phase treatment for childhood leukaemia) and also the potential protective benefits of omega-3 fatty acid-rich fish oil at different doses (0.25, 0.5 or 0.75 mL/100g BW) in comparison to antidote folinic acid (given i.p at 0.75mg/kg 6 hours post MTX, which is clinically used to reduce MTX toxicities in soft tissues). Results. Histological analysis showed that MTX significantly reduced primary spongiosa bone height and metaphyseal trabecular bone volume. MTX also significantly reduced density of osteoblasts at the secondary spongiosa. Ex vivo differentiation assays with bone marrow stromal cell populations of treated rats revealed a significant reduction in osteogenic differentiation but an increase in adipogenesis. Consistently, RT-PCR gene expression study within the stromal cell population revealed a lower expression of osteogenic transcription factors Runx2 and Osx and bone matrix protein osteocalcin but a significantly upregulated adipogenesis-related genes FABP4 and PPARγ, indicating that MTX chemotherapy induces a switch in the differentiation potential towards adipogenesis at the expense of osteogenesis. MTX increased the density of osteoclasts within the metaphyseal bone as revealed by histological analysis and osteoclast precursor cell pool as shown by ex vivo osteoclastogenesis assay with bone marrow samples. Consistently, mRNA expression of proinflammatory and osteoclastogenic cytokines IL-1, IL-6, TNF-α, and the RANKL/OPG ratio were significantly upregulated by MTX. Supplementary treatment with fish oil (0.5mL/100g BW) or folinic acid significantly preserved metaphyseal trabecular bone volume, osteoblast density, and bone marrow stromal cell osteogenic differentiation and suppressed MTX-induced adipogenesis. These supplements also prevented MTX-induced increased osteoclast density, osteoclastogenesis, and expression of proinflammatory and osteoclastogenic cytokines. Conclusion. These results suggest that MTX chemotherapy creates an inflammatory condition in bone resulting in increased osteoclast formation and decreased osteoblast formation thus leading to bone loss, and that supplementary treatment with fish oil at 0.5mL/100g BW or folinic acid counteract these negative effects, helping to conserve bone formation, suppress bone resorption and bone marrow adiposity, and thus prevent bone loss during MTX chemotherapy

Introduction. P-15 (GTPGPQGIAGQRGVV), a fifteen residue synthetic peptide, is a structural analogue of the cell binding domain of Type 1 collagen and creates a biomimetic environment for bone repair when immobilized on anorganic bovine mineral (ABM) scaffolds. ABM-P-15 scaffolds have been shown to enhance bone marrow stromal cell growth and differentiation. This study aimed at evaluating the osteogenic potential of human dental pulp stromal cells (HDPSCs) compared to human bone marrow stromal cells (HBMSCs) in monolayer and on 3D ABM-P-15 scaffolds in vitro and in vivo. Materials and Methods. HDPSCs and HBMSCs were cultured as monolayers in basal or osteogenic media for 3 weeks. Osteogenic differentiation was confirmed using alkaline phosphatase (ALP) staining and ALP specific activity (ALPSA). In addition, the presence and distribution of osteogenic markers including Type 1 collagen, bone sialoprotein (BSP), osteopontin (OPN) and osteocalcin (OCN) was determined by immunohistochemisty. Gene expression for COL1, RUNX2 and OCN was determined using RT-PCR after 1, 3 and 5 weeks in basal culture. For 3D culture, HDPSCs were seeded on ABM scaffolds ± P-15 (CeraPedics LLC) and cultured in basal media for 6 weeks. Cell viability and growth were visualized by confocal and scanning electron microscopy. Osteogenic differentiation was confirmed by ALP staining and ALPSA. For in vivo studies, HDPSCs were injected and sealed in diffusion chambers containing ABM-P-15 or ABM alone which were then implanted intraperitoneally in nude mice for 8 weeks. The retrieved samples were then processed for histology. Results. In monolayers, HDPSCs showed stronger ALP staining compared to HBMSCs in both culture conditions. Type I collagen, BSP and OPN were detected by immunohistochemistry for both HBMSCs and HDPSCs; however, OCN was not detected. RT-PCR indicated an up regulation of all osteogenic markers in both cell types at weeks 1 and 3. At week 5, there was a marked down regulation of COL1 and RUNX2 in HDPSCS compared to HBMSCs. Confocal microscopy and SEM showed ABM-P-15 promoted HDPSCs bridge formation between the scaffold particles. Histological staining and biochemical analysis confirmed that P-15 enhanced HDPSC ALP activity in vitro and fibrillar collagen formation in vivo compared to ABM alone. Discussion and Conclusion. HDPSCs have higher osteogenic capacity compared to HBMSCs. ABM-P-15 enhanced HDPSC ALPSA and collagen formation, suggesting that a combination of ABM-P15 with HDPSCs could be used as an autologous cell based therapy for bone tissue engineering. Acknowledgement: Supported by a University of Leeds studentships and Cerapedics Inc

ONFH with large or lateral-located lesion is challenging due to difficulty of regeneration. We introduce novel tissue engineering technique using ex vivo expanded bone marrow stromal cell seeded on calcium metaphosphate (CMP) scaffold to regenerate dead bone for these challenging cases. Ten millilitres of bone marrow was aspirated from iliac crest and mononuclear cells were collected. These cells were expanded and differentiated to osteoblast-lineage cells using osteogenic media and autologous serum for 2–4 weeks ex vivo. Porous bead-form scaffolds were made of CMP and cells were seeded in a density of million/ml³ into 20 to 30 beads for 1 hour. The necrotic area was curetted and the beads were implanted through core tract in 9 hips (Steinberg IIc in 5 hips and IVc in 4 hips which involved greater than 30% of whole head; JIC classification C1 in 4 hips, and C2 in 5 hips which involved weight bearing area). The tract was blocked with a CMP rod. The age of patients ranged from 16 to 37. Associated factors were; steroid in 4, idiopathic in 3, alcoholic in 1 and traumatic in 1 hip, respectively. Kerboul combined necrotic angle was more than 200° in all hips. We compared preoperative and annual radiographs and MRI images to check dome depression of femoral head and signal change of osteonecrotic area. Follow-up period ranged from 8 to 14 years. Two IIc lesions progressed and were converted to THA at two and six years postoperatively. We could get clinical and radiographic success in 7 hips (78%). Follow-up radiographs and MRI showed partial or nearly complete regeneration of necrotic bone, prevention of collapse, and reduction in necrotic lesion. This can be a good strategy for bone regeneration of unmet need as in a human model

Osteoarthritis (OA) affects millions of people and is the fastest growing cause of disability worldwide. In order to address this burden, early intervention strategies have been proposed. Therapies that utilise bone marrow stromal cells (BM-MSCs) to induce cartilage repair, either as a cell therapy or by endogenous release by drilling or microfracture, have proved promising. However, limitations include fibrotic features of the regenerated cartilage that may affect mechanical properties and therefore the longevity of such a repair. In order to improve this regenerative technique, further research is required to understand the key players in the repair mechanism. An interaction, which may be important, is that between BM-MSCs and the resident chondrocytes. The aim of this study is to understand the interplay between BM-MSC and resident chondrocytesisolated from different zonal locations within the human knee. We compared chondrocytes from three different cartilage areas: chondrocytes from 1) the superficial zone (SZ) and 2) the middle-deep (MDZ) zone of non-weight bearing femoral condyles, and from 3) the osteoarthritic zone (OAZ) of patients undergoing knee replacement. First, we evaluated the influence of different chondrocytes on BM-MSCs monolayer in a transwell co-culture, assessing transcript levels of early chondrogenic markers including Sox9 and Col1. Secondly, in a 3D co-culture system, we evaluated how cartilage chips from the three different zones affect the chondrogenic differentiation of BM-MSC pellets. Results indicated that cells from the SZ induce chondrogenic differentiation of BM-MSCs when co-cultured. In contrast, MDZ and OAZ have a negative effect, compared to control conditions. Our findings suggest that chondrocytes from the SZ, a zone which has been reported to reduce with age and may be lost in advanced OA, is important to direct BM-MSCs differentiation towards the chondrogenic fate. This may be relevant to cartilage repair strategies

Introduction. Cell-based therapy is needed to overcome the lacking intrinsic ability of cartilage to heal. Generating cartilage tissue from human bone marrow-derived stromal cells (MSC) is limited by up-regulation of COL10, ALP and other hypertrophy markers in vitro and calcifying cartilage at heterotopic sites in vivo. MSC hypertrophic differentiation reflects endochondral ossification, unable to maintain a stable hyaline stage, as observed by redifferentiation of articular chondrocytes (AC). Several transcription factors (TF), are held responsible for hypertrophic development. SOX9, the master regulator of chondrogenesis is also, alongside MEF2C, regulating hypertrophic chondrocyte maturation and COL10 expression. RUNX2/3 are terminal markers driving chondrocyte hypertrophy, and skeletogenesis. However, so far regulation of these key fate determining TFs has not been studied thoroughly on mRNA and protein level through chondrogenesis of human MSC. To fill this gap in knowledge, we aim to uncover regulation of SOX9, RUNX2/3, MEF2C and other TFs related to hypertrophy during MSC chondrogenesis in vitro and in comparison to the gold standard AC redifferentiation. Methods. Expression of SOX9, RUNX2/3 and MEF2C was compared before and during 6-week chondrogenic re-/differentiation of human MSC and AC on mRNA level via qRT-PCR and protein level via Western-Blotting. Chondrogenesis was evaluated by histology at d42 and expression of chondrogenic markers like COL2. Hypertrophic development was characterized by ALP activity and expression of hypertrophic markers like COL10. Results. Hypertrophic development, characterized by upregulation of COL10, high COL10/COL2 ratios and ALP activity, was confirmed in MSC and absent in AC. MSC started into differentiation with less SOX9 before induction, while higher RUNX2/3 was observed compared to AC. During MSC chondrogenesis SOX9 and MEF2C steadily increased on mRNA and protein level. Surprisingly, although RUNX2 mRNA level increased in MSC over 42 days, RUNX2 protein remained undetectable. During AC redifferentiation, SOX9 levels remained high on mRNA and protein level while RUNX2/3 and MEF2C remained low. Conclusion. After expansion and before applying chondrogenic stimuli, a chondrogenic priming with more SOX9 and lower RUNX2/3 was found in AC. In contrast osteochondral priming with higher RUNX2/3 and lower SOX9 levels was observed in MSC which could set the stage for endochondral development, leading to hypertrophy. Dynamic regulation of RUNX2/3 and MEF2C at lower SOX9 background levels separated MSC from AC differentiation over 42 days. Adjusting transcription factor levels in MSC could be essential for creating a protocol leading to diminished hypertrophy of MSC during chondrogenesis

Poly-ether-ether-ketone (PEEK) is a biomaterial commonly used for spinal implants and screws. It is often desirable for orthopaedic implants to osseointegrate, but as PEEK is biologically inert this will not occur. The aim of this project was to determine if injection mould nanopatterning can be used to create a make PEEK bioactive and stimulate osteogenesis in vitro. PEEK substrates were fabricated by injection mould nanopatterning to produce near-square (NSQ) nanopatterned PEEK and planar (FLAT) PEEK samples. Atomic force microscopy (AFM) and scanning electron microscopy were used to characterize the surface topography. Human bone marrow stromal cells (hBMSCs) were isolated from patients undergoing primary hip replacement operations and seeded onto the PEEK substrates. After 6 weeks the cells were stained using alizarin red S (ARS) stain (to detect calcium) and the von Kossa technique (to detect phosphate) and analyzed using CellProfiler image analysis software to determine: surface coverage; cell number; and expression of either calcium (ARS stain) or phosphate (von Kossa technique). ARS stain showed calcium expression (quantified relative to the number of cells) was increased on NSQ PEEK compared to FLAT PEEK (not statistically significant) and the surface coverage was similar. Von Kossa staining revealed more surface coverage on FLAT PEEK (69.1% cf. 31.9%), cell number was increased on FLAT PEEK (9803 ± 4066 cf. 4068 ± 1884) and phosphate expression relative to cell number was also increased (seven-fold) on NSQ PEEK (P < 0.05) compared to FLAT PEEK. Although hBMSCs may adhere to NSQ PEEK in smaller numbers, the cells expressed a relatively larger amount of calcium and phosphate. This indicates that the cells adopted a more osteoblastic phenotype and that nanopatterning PEEK induces hBMSC differentiation and stimulates osteogenesis. Injection mould nanopatterning therefore has the potential to improve osseointegration of PEEK implants in vivo

Introduction. Parathyroid hormone-related peptide (PTHrP) has been shown to be an important regulator of bone remodelling1. The aim of this study was to investigate the effect of the N-terminal domain of PTHrP (1–36) on osteogenic and angiogenic gene expression in human osteoblasts (HOB) and human bone marrow stromal cells (hBMSCs). Materials and Methods. Primary hBMSC's and HOBs were cultured in standard or osteogenic media with different concentrations of PTHrP, either continuously for 8, 24, 48 h and 9 days, or with 3 cycles of intermittent exposure (24 h with PTHrP, 24 h without) over 6 days. Cell lysates were then processed for analysis of gene expression. Expression of the osteogenic markers runt-related transcription factor 2 (RUNX-2), alkaline phosphatase (ALP) and Collagen 1, and the angiogenic marker; vascular endothelial growth factor (VEGF), were measured. Results. Exposure to PTHrP for ≤ 48 hours resulted in an upregulation of the angiogenic marker VEGF and the osteogenic markers RUNX-2, ALP and Collagen 1 in both cell types, peaking at a 1 nM PTHrP. Conversely, continuous exposure for 9 days, resulted in a downregulation of all osteogenic and angiogenic gene expression. HOB cells exposed intermittently to PTHrP showed an upregulation in VEGF and ALP, peaking at 10nM PTHrP. Discussion and Conclusion. Continuous exposure for short durations (<48 hours) and intermittent exposures of both HOB cells and BMSC's to PTHrP upregulated both osteogenic and angiogenic gene expression. Continuous exposure to 9 days however had the opposite effect, with a downregulation in gene transcription

Introduction. Enhanced angiogenesis and osteogenesis may provide new strategies for the treatment of osteonecrosis. Methods. Synergistic effects of vascular endothelial growth factor (VEGF) and bone morphogenetic protein - 6 (BMP-6) on in vitro osteogenic differentiation and in vivo ectopic bone formation mediated by a cloned mouse bone marrow stromal cell line, D1, previously isolated from Balb/c mice in our laboratory, were determined. Results. When human VEGF and BMP-6 genes both were expressed in D1 cells, significant increase in alkaline phosphatase activity was observed as compared to D1 cells in control groups. In the in vivo study, D1 cells transfected with hVEGF and hBMP-6 were loaded onto a 3-D PLAGA (polylactic-co-glycolic acid) scaffold and implanted subcutaneously in Balb/c mice. Micro-CT analysis of the retrieved implants clearly indicated the synergistic interaction of VEGF with BMP-6 as greater ectopic bone formation was observed in the VEGF plus BMP-6 group as compared to VEGF or BMP-6 alone. In addition, histology of the implants showed enhanced blood vessel formation with VEGF treatments. Conclusion. This study demonstrated the synergistic interaction of VEGF with BMP-6 during osteogenesis in vitro and in vivo. The results indicated that this novel combination of therapeutic growth factors should be investigated further as a potential treatment of osteonecrosis

Introduction. Osteonecrosis (ON) is a disease that ultimately results in bone collapse. We investigated the correlation between SNPs and osteonecrosis. Methods. In this study, 109 patients with systematic lupus erythematosus (SLE) (21 with and 88 without osteonecrosis) were collected for genotype analysis of 7 genes including VEGF, MTHFR, eNOS, and PAI-1 related to the blood system and BMP2 and PPARγ-2, genes that regulate the differentiation of bone marrow stromal cells. Results. The result of the combined analysis of the susceptible BMP2 (rs3178250) TC genotype, MTHFR (rs1801133) CC genotype and VEGF (rs833069) AA genotype was OR: 0.185, 95 % CI:0.044 - 0.774 (p=0.021). In addition, when the different genotype combinations were analyzed the result for BMP2 (rs3178250) TC, MTHFR (rs1801133)CC, and PPARγ-2 (rs11128596) AA genotype was OR:0.096, 95 % CI:0.044-0.774 (p=0.012); the result for BMP2(rs3178250) TT, VEGF (rs833069) AG, and PPARγ-2 (rs11128596) CA genotype was OR:0.099, 95 % CI:0.016-0.597 (p=0.012); and that of VEGF AA, eNOS 298T GT, and eNOS 27bp tandem repeat 5R5R genotype was OR:0.060, 95 % CI:0.006- 0.588 (p =0.016), respectively. Conclusion. The results of this research provides an important reference to predict corticosteroid-associated osteonecrosis for SLE patients, providing related genotypic molecular epidemiology and possible discussion on mechanisms of pathogenicity for corticosteroid-associated osteonecrosis in SLE patients in Taiwan. The result of this research not only serves as a reference for possible ON risk factors in SLE patients with chronic corticosteroid use, but also forms a basis for treatment and medication in the clinical setting

Introduction. Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily of growth factors and are known to regulate proliferation and expression of the differentiated phenotype of chondrocytes, osteoblasts, and osteoclasts. To investigate the osteoblastic differentiation gene expressions that contribute to BMP-7 dependent ostogenesis, we performed gene expression profiling of BMP-7-treated mouse bone marrow stromal cells. Methods. D1 cells (mouse bone marrow stromal cells) were cultured in osteogenic differentiation medium (ODM) for 3 days, and then treated with BMP-7 for 24 hr. Total RNA was extracted using Trizol, purified using RNeasy columns. Total RNA was amplified and purified using the Ambion Illumina RNA amplification kit to yield biotinylated cRNA. The data analysis up- and down-regulation developmental processes (anterior/posterior patterning, ectoderm development, embryogenesis, gametogenesis, mesoderm development, other development process, and segment specification) genes expression fold. Results. We detected 18 mRNAs (Id2, Igf2, Pparg, S100a10, Foxn3, Tulp3, Mycbp2, Notch3, Ptk7, Lrp4, Tnfrsf11b, Ogn, Cyr61, Mglap, Akp2, Ltbp4, Ibsp, and Thbs1) that were differentially up-regulated after BMP-7 stimulation. 3 mRNAs (Wars, Adss and Trim35) were differentially down-regulated after BMP-7 stimulation. Conclusion. The data indicate that BMP-7 regulate various developmental processes genes expression during osteoblastic differentiation. Though further studies are needed in relation to each expression gene profiles and osteoblastic differentiation, this information may serve as a point of comparison for osteoblastic differentiation of BMP-7. Furthermore, the data should facilitate the informed use of BMP-7 as a therapeutic agent and tissue engineering tool. Acknowledgement. This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. R01-2008-000-10089-0)

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

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