A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder of skeletal malformations and progressive extraskeletal ossification. We mapped FOP to chromosome 2q23-24 by linkage analysis and identified an identical heterozygous mutation (617G→A; R206H) in the glycine-serine (GS) activation domain of ACVR1, a BMP type I receptor, in all affected individuals examined. Protein modeling predicts destabilization of the GS domain, consistent with constitutive activation of ACVR1 as the underlying cause of the ectopic chondrogenesis, osteogenesis and joint fusions seen in FOP.

Variation in BMPR1B, TGFRB1 and BMPR2 and control of dizygotic twinning

Genes in the TGF9 signaling pathway play important roles in the regulation of ovarian follicle growth and ovulation rate. Mutations in three genes in this pathway, growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and the bone morphogenetic protein receptor B 1 (BMPRB1), influence dizygotic (DZ) twinning rates in sheep. To date, only variants in GDF9 and BMP15, but not their receptors transforming growth factor ss receptor 1 (TGFBR1), bone morphogenetic protein receptor 2 (BMPR2) and BMPR1B, have been investigated with respect to their roles in human DZ twinning. We screened for rare and novel variants in TGFBR1, BMPR2 and BMPR1B in mothers of dizygotic twins (MODZT) from twin-dense families, and assessed association between genotyped and imputed variants and DZ twinning in another large sample of MODZT. Three novel variants were found: a deep intronic variant in BMPR2, and one intronic and one non-synonymous exonic variant in BMPRB1 which would result in the replacement of glutamine by glutamic acid at amino acid position 294 (p.Gln294Glu). None of these variants were predicted to have major impacts on gene function. However, the p.Gln294Glu variant changes the same amino acid as a sheep BMPR1B functional variant and may have functional consequences. Six BMPR1B variants were marginally associated with DZ twinning in the larger case-control sample, but these were no longer significant once multiple testing was taken into account. Our results suggest that variation in the TGF9 signaling pathway type II receptors has limited effects on DZ twinning rates in humans.

Growth factor-loaded microparticles for tissue engineering: The discrepancies of in vitro characterization assays

Efficient and effective growth factor (GF) delivery is an ongoing challenge for tissue regeneration therapies. The accurate quantification of complex molecules such as GFs, encapsulated in polymeric delivery devices, is equally critical and just as complex as achieving efficient delivery of active GFs. In this study, GFs relevant to bone tissue formation, vascular endothelial growth factor (VEGF) and bone morphogenetic protein 7 (BMP-7), were encapsulated, using the technique of electrospraying, into poly(lactic-co-glycolic acid) microparticles that contained poly(ethylene glycol) and trehalose to assist GF bioactivity. Typical quantification procedures, such as extraction and release assays using saline buffer, generated a significant degree of GF interactions, which impaired accurate assessment by enzyme-linked immunosorbent assay (ELISA). When both dry BMP-7 and VEGF were processed with chloroform, as is the case during the electrospraying process, reduced concentrations of the GFs were detected by ELISA; however, the biological effect on myoblast cells (C2C12) or endothelial cells (HUVECs) was unaffected. When electrosprayed particles containing BMP-7 were cultured with preosteoblasts (MC3T3-E1), significant cell differentiation into osteoblasts was observed up to 3 weeks in culture, as assessed by measuring alkaline phosphatase. In conclusion, this study showed how electrosprayed microparticles ensured efficient delivery of fully active GFs relevant to bone tissue engineering. Critically, it also highlights major discrepancies in quantifying GFs in polymeric microparticle systems when comparing ELISA with cell-based assays.

In situ preparation and protein delivery of silicate/alginate composite microspheres with core-shell structure

It is predicted that with increased life expectancy in the developed world, there will be a greater demand for synthetic materials to repair or regenerate lost, injured or diseased bone (Hench & Thompson 2010). There are still few synthetic materials having true bone inductivity, which limits their application for bone regeneration, especially in large-size bone defects. To solve this problem, growth factors, such as bone morphogenetic proteins (BMPs), have been incorporated into synthetic materials in order to stimulate de novo bone formation in the center of large-size bone defects. The greatest obstacle with this approach is that the rapid diffusion of the protein from the carrier material, leading to a precipitous loss of bioactivity; the result is often insufficient local induction or failure of bone regeneration (Wei et al. 2007). It is critical that the protein is loaded in the carrier material in conditions which maintains its bioactivity (van de Manakker et al. 2009). For this reason, the efficient loading and controlled release of a protein from a synthetic material has remained a significant challenge. The use of microspheres as protein/drug carriers has received considerable attention in recent years (Lee et al. 2010; Pareta & Edirisinghe 2006; Wu & Zreiqat 2010). Compared to macroporous block scaffolds, the chief advantage of microspheres is their superior protein-delivery properties and ability to fill bone defects with irregular and complex shapes and sizes. Upon implantation, the microspheres are easily conformed to the irregular implant site, and the interstices between the particles provide space for both tissue and vascular ingrowth, which are important for effective and functional bone regeneration (Hsu et al. 1999). Alginates are natural polysaccharides and their production does not have the implicit risk of contamination with allo or xeno-proteins or viruses (Xie et al. 2010). Because alginate is generally cytocompatible, it has been used extensively in medicine, including cell therapy and tissue engineering applications (Tampieri et al. 2005; Xie et al. 2010; Xu et al. 2007). Calcium cross-linked alginate hydrogel is considered a promising material as a delivery matrix for drugs and proteins, since its gel microspheres form readily in aqueous solutions at room temperature, eliminating the need for harsh organic solvents, thereby maintaining the bioactivity of proteins in the process of loading into the microspheres (Jay & Saltzman 2009; Kikuchi et al. 1999). In addition, calcium cross-linked alginate hydrogel is degradable under physiological conditions (Kibat PG et al. 1990; Park K et al. 1993), which makes alginate stand out as an attractive candidate material for the protein carrier and bone regeneration (Hosoya et al. 2004; Matsuno et al. 2008; Turco et al. 2009). However, the major disadvantages of alginate microspheres is their low loading efficiency and also rapid release of proteins due to the mesh-like networks of the gel (Halder et al. 2005). Previous studies have shown that a core-shell structure in drug/protein carriers can overcome the issues of limited loading efficiencies and rapid release of drug or protein (Chang et al. 2010; Molvinger et al. 2004; Soppimath et al. 2007). We therefore hypothesized that introducing a core-shell structure into the alginate microspheres could solve the shortcomings of the pure alginate. Calcium silicate (CS) has been tested as a biodegradable biomaterial for bone tissue regeneration. CS is capable of inducing bone-like apatite formation in simulated body fluid (SBF) and its apatite-formation rate in SBF is faster than that of Bioglass® and A-W glass-ceramics (De Aza et al. 2000; Siriphannon et al. 2002). Titanium alloys plasma-spray coated with CS have excellent in vivo bioactivity (Xue et al. 2005) and porous CS scaffolds have enhanced in vivo bone formation ability compared to porous β-tricalcium phosphate ceramics (Xu et al. 2008). In light of the many advantages of this material, we decided to prepare CS/alginate composite microspheres by combining a CS shell with an alginate core to improve their protein delivery and mineralization for potential protein delivery and bone repair applications

Elevated circulating sclerostin concentrations in individuals with high bone mass, with and without LRP5 mutations

CONTEXT: The role and importance of circulating sclerostin is poorly understood. High bone mass (HBM) caused by activating LRP5 mutations has been reported to be associated with increased plasma sclerostin concentrations; whether the same applies to HBM due to other causes is unknown. OBJECTIVE: Our objective was to determine circulating sclerostin concentrations in HBM. DESIGN AND PARTICIPANTS: In this case-control study, 406 HBM index cases were identified by screening dual-energy x-ray absorptiometry (DXA) databases from 4 United Kingdom centers (n = 219 088), excluding significant osteoarthritis/artifact. Controls comprised unaffected relatives and spouses. MAIN MEASURES: Plasma sclerostin; lumbar spine L1, total hip, and total body DXA; and radial and tibial peripheral quantitative computed tomography (subgroup only) were evaluated. RESULTS: Sclerostin concentrations were significantly higher in both LRP5 HBM and non-LRP5 HBM cases compared with controls: mean (SD) 130.1 (61.7) and 88.0 (39.3) vs 66.4 (32.3) pmol/L (both P < .001, which persisted after adjustment for a priori confounders). In combined adjusted analyses of cases and controls, sclerostin concentrations were positively related to all bone parameters found to be increased in HBM cases (ie, L1, total hip, and total body DXA bone mineral density and radial/tibial cortical area, cortical bone mineral density, and trabecular density). Although these relationships were broadly equivalent in HBM cases and controls, there was some evidence that associations between sclerostin and trabecular phenotypes were stronger in HBM cases, particularly for radial trabecular density (interaction P < .01). CONCLUSIONS: Circulating plasma sclerostin concentrations are increased in both LRP5 and non-LRP5 HBM compared with controls. In addition to the general positive relationship between sclerostin and DXA/peripheral quantitative computed tomography parameters, genetic factors predisposing to HBM may contribute to increased sclerostin levels.

Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts

In this study, cell sheets comprising multilayered porcine bone marrow stromal cells (BMSC) were assembled with fully interconnected scaffolds made from medical-grade polycaprolactone–calcium phosphate (mPCL–CaP), for the engineering of structural and functional bone grafts. The BMSC sheets were harvested from culture flasks and wrapped around pre-seeded composite scaffolds. The layered cell sheets integrated well with the scaffold/cell construct and remained viable, with mineralized nodules visible both inside and outside the scaffold for up to 8 weeks culture. Cells within the constructs underwent classical in vitro osteogenic differentiation with the associated elevation of alkaline phosphatase activity and bone-related protein expression. In vivo, two sets of cell-sheet-scaffold/cell constructs were transplanted under the skin of nude rats. The first set of constructs (554mm3) were assembled with BMSC sheets and cultured for 8 weeks before implantation. The second set of constructs (10104mm3) was implanted immediately after assembly with BMSC sheets, with no further in vitro culture. For both groups, neo cortical and well-vascularised cancellous bone were formed within the constructs with up to 40% bone volume. Histological and immunohistochemical examination revealed that neo bone tissue formed from the pool of seeded BMSC and the bone formation followed predominantly an endochondral pathway, with woven bone matrix subsequently maturing into fully mineralized compact bone; exhibiting the histological markers of native bone. These findings demonstrate that large bone tissues similar to native bone can be regenerated utilizing BMSC sheet techniques in conjunction with composite scaffolds whose structures are optimized from a mechanical, nutrient transport and vascularization perspective.

Serum bone formation marker correlation with improved osseointegration in osteoporotic rats treated with simvastatin

Objective: Simvastatin has been shown to enhance osseointegration of pure titanium implants in osteoporotic rats. This study aimed to evaluate the relationship between the serum level of bone formation markers and the osseointegration of pure titanium implants in osteoporotic rats treated with simvastatin. Materials and methods: Fifty-four female Sprague Dawley rats, aged 3 months old, were randomly divided into three groups: Sham-operated group (SHAM; n=18), ovariectomized group (OVX; n=18), and ovariectomized with Simvastatin treatment group (OVX+SIM; n=18). Fifty-six days after ovariectomy, screw-shaped titanium implants were inserted into the tibiae. Simvastatin was administered orally at 5mg/kg each day after the placement of the implant in the OVX+SIM group. The animals were sacrificed at either 28 or 84 days after implantation and the undecalcified tissue sections were processed for histological analysis. Total alkaline phosphatase (ALP), bone specific alkaline phosphatase (BALP) and bone Gla protein (BGP) were measured in all animal sera collected at the time of euthanasia and correlated with the histological assessment of osseointegration. Results: The level of ALP in the OVX group was higher than the SHAM group at day 28, with no differences between the three groups at day 84. The level of BALP in the OVX+SIM group was significantly higher than both OVX and SHAM groups at days 28 and 84. Compared with day 28, the BALP level of all three groups showed a significant decrease at day 84. There were no significant differences in BGP levels between the three groups at day 28, but at day 84 the OVX+SIM group showed significantly higher levels than both the OVX and SHAM groups. There was a significant increase in BGP levels between days 28 and 84 in the OVX+SIM group. The serum bone marker levels correlated with the histological assessment showing reduced osseointegration in the OVX compared to the SHAM group which is subsequently reversed in the OVX+SIM group.

Osteogenic differentiation of murine embryonic stem cells is mediated by fibroblast growth factor receptors

The mechanisms involved in the control of embryonic stem (ES) cell differentiation are yet to be fully elucidated. However, it has become clear that the family of fibroblast growth factors (FGFs) are centrally involved. In this study we examined the role of the FGF receptors (FGFRs 1-4) during osteogenesis in murine ES cells. Single cells were obtained after the formation of embryoid bodies, cultured on gelatin-coated plates, and coaxed to differentiate along the osteogenic lineage. Upregulation of genes was analyzed at both the transcript and protein levels using gene array, relative-quantitative PCR (RQ-PCR), and Western blotting. Deposition of a mineralized matrix was evaluated with Alizarin Red staining. An FGFR1-specific antibody was generated and used to block FGFR1 activity in mES cells during osteogenic differentiation. Upon induction of osteogenic differentiation in mES cells, all four FGFRs were clearly upregulated at both the transcript and protein levels with a number of genes known to be involved in osteogenic differentiation including bone morphogenetic proteins (BMPs), collagen I, and Runx2. Cells were also capable of depositing a mineralized matrix, confirming the commitment of these cells to the osteogenic lineage. When FGFR1 activity was blocked, a reduction in cell proliferation and a coincident upregulation of Runx2 with enhanced mineralization of cultures was observed. These results indicate that FGFRs play critical roles in cell recruitment and differentiation during the process of osteogenesis in mES cells. In particular, the data indicate that FGFR1 plays a pivotal role in osteoblast lineage determination.

Stem cell regulatory gene expression in human adult dental pulp and periodontal ligament cells undergoing odontogenic/osteogenic differentiation

Introduction During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. Methods In this study, we investigated the differential expression of 84 stem cell–related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. Results Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor β (TGF-β)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. Conclusions This study has generated an overview of stem cell–related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-β/BMP, and cadherin signalling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration

Sequential release of BMP-7 and VEGF from the PLGA/AK-Gelatin Composite Scaffolds

Vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMP-7) are key regulators of angiogenesis and osteogenesis during bone regeneration. The aim of this study was to investigate the possibility of realizing sequential release of the two growth factors using a novel composite scaffold. Poly(lactic-co-glycolic acid) (PLGA)-Akermanite (AK) microspheres were used to make the composite scaffold, which was then loaded with BMP-7, followed by embedding in a gelatin hydrogel matrix loaded with VEGF. The release profiles of the growth factors were studied and selected osteogenic related markers of bone marrow stromal cells (BMSCs) were analysed. It was shown that the composite scaffolds exhibited a fast initial burst release of VEGF within the first 3 days and a sustained slow release of BMP-7 over the full period of 20 days. The in vitro proliferation and differentiation of the BMSCs cultured in the osteogenic medium were enhanced by 1 to 2 times, resulting from the additionally and sequentially release of growth factors from the PLGA-AK/gelatin composite scaffolds.

The expression, regulation and function of kallikrein 4 in prostate cancer

Prostate cancer is a significant health problem faced by aging men. Currently, diagnostic strategies for the detection of prostate cancer are either unreliable, yielding high numbers of false positive results, or too invasive to be used widely as screening tests. Furthermore, the current therapeutic strategies for the treatment of the disease carry considerable side effects. Although organ confined prostate cancer can be curable, most detectable clinical symptoms occur in advanced disease when primary tumour cells have metastasised to distant sites - usually lymph nodes and bone. Many growth factors and steroids assist the continued growth and maintenance of prostatic tumour cells. Of these mitogens, androgens are important in the development of the normal prostate but are also required to sustain the growth of prostate cancer cells in the early stage of the disease. Not only are androgens required in the early stage of disease, but also many other growth factors and hormones interact to cause uncontrolled proliferation of malignant cells. The early, androgen sensitive phase of disease is followed by an androgen insensitive phase, whereby androgens are no longer required to stimulate the growth of the tumour cells. Growth factors such as transforming growth factor  and  (TGF/), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factors (IGFs), Vitamin D and thyroid hormone have been suggested to be important at this stage of disease. Interestingly, some of the kallikrein family of genes, including prostate specific antigen (PSA), the current serum diagnostic marker for prostate cancer, are regulated by androgens and many of the aforementioned growth factors. The kallikrein gene family is a group of serine proteases that are involved in a diverse range of physiological processes: regulation of local blood flow, angiogenesis, tissue invasion and mitogenesis. The earliest members of the kallikrein gene family (KLK1-KLK3) have been strongly associated with general disease states, such as hypertension, inflammation, pancreatitis and renal disease, but are also linked to various cancers. Recently, this family was extended to include 15 genes (KLK1-15). Several newer members of the kallikrein family have been implicated in the carcinogenesis and tumour metastasis of hormone-dependent cancers such as prostate, breast, endometrial and ovarian cancer. The aims of this project were to investigate the expression of the newly identified kallikrein, KLK4, in benign and malignant prostate tissues, and prostate cancer cell lines. This thesis has demonstrated the elevated expression of KLK4 mRNA transcripts in malignant prostate tissue compared to benign prostates. Additionally, expression of the full length KLK4 transcript was detected in the androgen dependent prostate cancer cell line, LNCaP. Based on the above finding, the LNCaP cell line was chosen to assess the potential regulation of full length KLK4 by androgen, thyroid hormone and epidermal growth factor. KLK4 mRNA and protein was found to be up-regulated by androgen and a combination of androgen and thyroid hormone. Thyroid hormone alone produced no significant change in KLK4 mRNA or protein over the control. Epidermal growth factor treatment also resulted in elevated expression levels of KLK4 mRNA and protein. To assess the potential functional role(s) of KLK4/hK4 in processes associated with tumour progression, full length KLK4 was transfected into PC-3 cells - a prostate cancer cell line originally derived from a secondary bone lesion. The KLK4/hK4 over-expressing cells were assessed for their proliferation, migration, invasion and attachment properties. The KLK4 over-expressing clones exhibited a marked change in morphology, indicative of a more aggressive phenotype. The KLK4 clones were irregularly shaped with compromised adhesion to the growth surface. In contrast, the control cell lines (parent PC-3 and empty vector clones) retained a rounded morphology with obvious cell to cell adhesion, as well as significant adhesion to their growth surface. The KLK4 clones exhibited significantly greater attachment to Collagen I and IV than native PC-3s and empty vector controls. Over a 12 hour period, in comparison to the control cells, the KLK4 clones displayed an increase in migration towards PC-3 native conditioned media, a 3 fold increase towards conditioned media from an osteoblastic cell line (Saos-2) and no change in migration towards conditioned media from neonatal foreskin fibroblast cells or 20% foetal bovine serum. Furthermore, the increase in migration exhibited by the KLK4 clones was partially blocked by the serine protease inhibitor, aprotinin. The data presented in this thesis suggests that KLK4/hK4 is important in prostate carcinogenesis due to its over-expression in malignant prostate tissues, its regulation by hormones and growth factors associated with prostate disease and the functional consequences of over-expression of KLK4/hK4 in the PC-3 cell line. These results indicate that KLK4/hK4 may play an important role in tumour invasion and bone metastasis via increased attachment to the bone matrix protein, Collagen I, and enhanced migration due to soluble factors produced by osteoblast cells. This suggestion is further supported by the morphological changes displayed by the KLK4 over-expressing cells. Overall, this data suggests that KLK4/hK4 should be further studied to more fully investigate the potential value of KLK4/hK4 as a diagnostic/prognostic biomarker or in therapeutic applications.

Cohesive zone modelling of mineralized collagen fibril arrays in bending

Hard biological materials such as bone possess superior material properties of high stiffness and toughness. Two unique characteristics of bone microstructure are a large aspect ratio of mineralized collagen fibrils (MCF), and an extremely thin and large area of extrafibrillar protein matrix located between the MCF. The objective of this study is to investigate the effects of: (1) MCF aspect ratio, and (2) energy dissipation in extrafibrillar protein matrix on the mechanical behaviour of MCF arrays. In this study, notched specimens of MCF arrays in extrafibrillar protein matrix are subjected to bending. Cohesive zone model was implemented to simulate the failure of extrafibrillar protein matrix. The study reveals that the MCF array with a higher MCF aspect ratio and the MCF array with a higher protein energy dissipation in the interface direction are able to sustain a higher bending force and dissipate higher energy.

Multiphasic construct studied in an ectopic osteochondral defect model

In vivo osteochondral defect models predominantly consist of small animals, such as rabbits. Although they have an advantage of low cost and manageability, their joints are smaller and more easily healed compared with larger animals or humans. We hypothesized that osteochondral cores from large animals can be implanted subcutaneously in rats to create an ectopic osteochondral defect model for routine and high-throughput screening of multiphasic scaffold designs and/or tissue-engineered constructs (TECs). Bovine osteochondral plugs with 4 mm diameter osteochondral defect were fitted with novel multiphasic osteochondral grafts composed of chondrocyte-seeded alginate gels and osteoblast-seeded polycaprolactone scaffolds, prior to being implanted in rats subcutaneously with bone morphogenic protein-7. After 12 weeks of in vivo implantation, histological and micro-computed tomography analyses demonstrated that TECs are susceptible to mineralization. Additionally, there was limited bone formation in the scaffold. These results suggest that the current model requires optimization to facilitate robust bone regeneration and vascular infiltration into the defect site. Taken together, this study provides a proof-of-concept for a high-throughput osteochondral defect model. With further optimization, the presented hybrid in vivo model may address the growing need for a cost-effective way to screen osteochondral repair strategies before moving to large animal preclinical trials.

Survival of rat functional dental pulp cells in vascularized tissue engineering chambers

Regenerative endodontics aims to preserve, repair or regenerate the dental pulp tissue. Dental pulp stem cells, have a potential use in dental tissue generation. However, specific requirements to drive the dental tissue generation are still obscured. We established an in vivo model for studying the survival of dental pulp cells (DPC) and their potential to generate dental pulp tissue. DPC were mixed with collagen scaffold with or without slow release bone morphogenic protein 4 (BMP-4) and fibroblast growth factor 2 (FGF2). The cell suspension was transplanted into a vascularized tissue engineering chamber in the rat groin. Tissue constructs were harvested after 2, 4, 6, and 8 weeks and processed for histomorphological and immunohistochemical analysis. After 2 weeks newly formed tissue with new blood vessel formation were observed inside the chamber. DPC were found around dentin, particularly around the vascular pedicle and also close to the gelatin microspheres. Cell survival, was confirmed up to 8 weeks after transplantation. Dentin Sialophosphoprotein (DSPP) positive matrix production was detected in the chamber, indicating functionality of dental pulp progenitor cells. This study demonstrates the potential of our tissue engineering model to study rat dental pulp cells and their behavior in dental pulp regeneration, for future development of an alternative treatment using these techniques.