Expression Of Bone Matrix Proteins Research Articles

Hydroxyapatite Nanoparticles Promote the Development of Bone Microtissues for Accelerated Bone Regeneration by Activating the FAK/Akt Pathway.

Scaffold-free bone microtissues differentiated from mesenchymal stem cell (MSC) spheroids offer great potential for bottom-up bone tissue engineering as a direct supply of cells and osteogenic signals. Many biomaterials or biomolecules have been incorporated into bone microtissues to enhance their osteogenic abilities, but these materials are far from clinical approval. Here, we aimed to incorporate hydroxyapatite (HAP) nanoparticles, an essential component of bone matrix, into MSC spheroids to instruct their osteogenic differentiation into bone microtissues and further self-organization into bone organoids with a trabecular structure. Furthermore, the biological interaction between HAP nanoparticles and MSCs and the potential molecular mechanisms in the bone development of MSC spheroids were investigated by both in vitro and in vivo studies. As a result, improved cell viability and osteogenic abilities were observed for the MSC spheroids incorporated with HAP nanoparticles at a concentration of 30 μg/mL. HAP nanoparticles could promote the sequential expression of osteogenic markers (Runx2, Osterix, Sclerostin), promote the expression of bone matrix proteins (OPN, OCN, and Collagen I), promote the mineralization of the bone matrix, and thus promote the bone development of MSC spheroids. The differentiated bone microtissues could further self-organize into linear, lamellar, and spatial bone organoids with trabecular structures. More importantly, adding FAK or Akt inhibitors could decrease the level of HAP-induced osteogenic differentiation of bone microtissues. Finally, excellent new bone regeneration was achieved after injecting bone microtissues into cranial bone defect models, which could also be eliminated by the Akt inhibitor. In conclusion, HAP nanoparticles could promote the development of bone microtissues by promoting the osteogenic differentiation of MSCs and the formation and mineralization of the bone matrix via the FAK/Akt pathway. The bone microtissues could act as individual ossification centers and self-organize into macroscale bone organoids, and in this meaning, the bone microtissues could be called microscale bone organoids. Furthermore, the bone microtissues revealed excellent clinical perspectives for injectable cellular therapies for bone defects.

TempEasy 3D Hydrogel Coculture System Provides Mechanistic Insights into Prostate Cancer Bone Metastasis.

Patients diagnosed with advanced prostate cancer (PCa) often experience incurable bone metastases; however, a lack of relevant experimental models has hampered the study of disease mechanisms and the development of therapeutic strategies. In this study, we employed the recently established Temperature-based Easy-separable (TempEasy) 3D cell coculture system to investigate PCa bone metastasis. Through coculturing PCa and bone cells for 7 days, our results showed a reduction in PCa cell proliferation, an increase in neovascularization, and an enhanced metastasis potential when cocultured with bone cells. Additionally, we observed increased cell proliferation, higher stemness, and decreased bone matrix protein expression in bone cells when cocultured with PCa cells. Furthermore, we demonstrated that the stiffness of the extracellular matrix had a negligible impact on molecular responses in both primary (PCa cells) and distant malignant (bone cells) sites. The TempEasy 3D hydrogel coculture system is an easy-to-use and versatile coculture system that provides valuable insights into the mechanisms of cell-cell communication and interaction in cancer metastasis.

Chromatin accessibility and epigenetic deoxyribose nucleic acid (DNA) modifications in chronic kidney disease (CKD) osteoblasts: a study of bone and osteoblasts from pediatric patients with CKD.

Maturation defects are intrinsic features of osteoblast lineage cells in CKD patients. These defects persist ex vivo, suggesting that CKD induces epigenetic changes in bone cells. To gain insights into which signaling pathways contribute to CKD-mediated, epigenetically driven, impairments in osteoblast maturation, we characterized RNA expression and DNA methylation patterns by RNA-Seq and MethylationEpic in primary osteoblasts from nine adolescent and young adult dialysis patients with end-stage kidney disease and three healthy references. ATAC-Seq was also performed on a subset of osteoblasts. Bone matrix protein expression was extracted from the iliac crest and evaluated by proteomics. Gene set enrichment analysis was used to establish signaling pathways consistently altered in chromatin accessibility, DNA methylation, and RNA expression patterns. Single genes were suppressed in primary osteoblasts using shRNA and mineralization characterized in vitro. The effect of nuclear factor of activated T cells (NFAT) signaling suppression was also assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) incorporation. We found that signaling pathways critical for osteoblast differentiation were strongly downregulated in CKD osteoblasts. Gene set enrichment analysis identified highly significant methylation changes, differential chromatin accessibility, and altered RNA expression in NFAT signaling targets. NFAT inhibition reduced osteoblast proliferation. Combined analysis of osteoblast RNA expression and whole bone matrix composition identified 13 potential ligand-receptor pairs. In summary, epigenetic changes in CKD osteoblasts associate with altered expression of multiple osteoblast genes and signaling pathways. An increase in NFAT signaling may play a role in impaired CKD osteoblast maturation. Epigenetic changes also associate with an altered bone matrix, which may contribute to bone fragility. Further studies are necessary to elucidate the pathways affected by these genetic alterations since elucidating these pathways will be vital to correcting the underlying biology of bone disease in the CKD population.

Enhancing the effectiveness of Alkaline Phosphatase and bone matrix proteins by tunable metal-organic composite for accelerated mineralization

The irregular expression of bone matrix proteins occurring during the mineralization of bone regeneration results in various deformities which poses a major concern of orthopedic reconstruction. The limitations of the existing reconstruction practice paved a way for the development of a metal-organic composite [TQ-Sr-Fe] with Metal ions strontium [Sr] and iron [Fe] and a biomolecule Thymoquinone [TQ] in an attempt to enhance the bone mineralization due to their positive significance in osteoblast differentiation, proliferation and maturation. TQ-Sr-Fe was synthesized by in-situ coprecipitation and subjected to various characterization to determine their nature, compatibility and osteogenic efficiency. The crystallographic and electron microscopy analysis reveals sheet like structure of the composite. The negative cytotoxicity of TQ-Sr-Fe in the MG 63 cell line signified their biocompatibility. Cell adhesion and proliferation rate affirmed osteoconductive and osteoinductive nature of the composites and it was further supported by the gene expression of osteoblastic differentiation. The sequential expression of bone matrix proteins such as OCN, SPARC, COL 1, and Alkaline Phosphatase elevate the calcium deposition of MG-63 osteoblast like cells and initiates mineralization compared to control. Thus, the metal-organic composite TQ-Sr-Fe would make a suitable composite for accelerating mineralization process which would leads to faster bone regeneration.

Osseous ingrowth in allogeneic bone blocks applied for vertical bone augmentation: a preclinical randomised controlled study.

The aim of the present study was the qualitative and quantitative evaluation of osseous graft consolidation using allogeneic bone blocks for vertical bone augmentation in an animal model. Standardised allogeneic and autologous bone blocks were fixed on the frontal skull of 20 adult female pigs and covered with a resorbable collagen membrane. Animals were sacrificed after 2 and 6 months. Specimens were histologically and histomorphometrically analysed focusing on the amount of vital bone, residual bone substitute material and connective tissue. Furthermore, the amount of expression of bone matrix proteins (collagen type I and osteocalcin) and de novo vessel formation (von Willebrand factor) were quantified by immunohistochemistry. Significantly more allogeneic bone blocks failed for both evaluation time points (p < 0.05). Allogeneic blocks showed significantly less vital bone with more connective tissue formation compared to autologous bone blocks. Increased vessel formation could be detected for both evaluation time points in the contact area of autologous bone with local bone. The expression of collagen type I and osteocalcin was significantly lower in the allogeneic bone graft. Allogeneic cancellous bone blocks showed a significantly higher failure rate compared to autologous bone blocks. Allogeneic bone blocks seemed to negatively affect bone formation or negatively influence the host in the long term, and increased connective tissue formation and block loss should be anticipated. In order to maintain patient safety and treatment success clinicians should be persuaded to make a conscious choice of the applied biomaterials with regard to their components and structure.

Deficiency of stress-associated gene Nupr1 increases bone volume by attenuating differentiation of osteoclasts and enhancing differentiation of osteoblasts.

Nuclear protein 1 (NUPR1) is a multifunctional stress-induced protein involved in regulating tumorigenesis, apoptosis, and autophagy. Bone homeostasis is maintained by bone-resorbing osteoclasts and bone-forming osteoblasts and osteocytes. We aimed to determine the role of NUPR1 in bone metabolism. Using microcomputed tomography, we found that mice lacking Nupr1 exhibited increased bone volume. Histologic analysis showed that Nupr1 deficiency decreased osteoclast numbers but increased osteoblast numbers and osteoid formation. In vitro culture of bone marrow macrophages showed that receptor activator of NF-κB ligand-induced osteoclastogenesis was down-regulated in Nupr1-deficient mice. In contrast, primary osteoblasts from Nupr1-deficient mice revealed that proliferation of osteoblasts and expression of bone matrix proteins were markedly enhanced. In addition, expression of autophagy-related genes, formation of autophagosomes, and cell survival were up-regulated in Nupr1-deficient osteoblasts. In contract, deletion of Nupr1 reduced the formation of osteocyte cellular projection, which is an indicator of mature osteocytes. Importantly, we found that the expression of sclerostin (Sost), an inhibitor of bone formation, was down-regulated in the osteoblasts and osteocytes of Nupr1-deficient mice. Conversely, Nupr1 overexpression enhanced Sost expression in primary osteoblasts. Collectively, these results indicate that Nupr1 deficiency increases bone volume by attenuating production of Sost and osteoclastogenesis and enhancing differentiation of osteoblasts.-Shiraki, M., Xu, X., Iovanna, J. L., Kukita, T., Hirata, H., Kamohara, A., Kubota, Y., Miyamoto, H., Mawatari, M., Kukita, A. Deficiency of stress-associated gene Nupr1 increases bone volume by attenuating differentiation of osteoclasts and enhancing differentiation of osteoblasts.

Calcification and Thrombosis as Mediators of Bioprosthetic Valve Deterioration

Calcific aortic valve disease is the most common indication for surgical aortic valve replacement (SAVR) and transcatheter aortic valve replacement (TAVR) of diseased valves worldwide. 1 For years,...

L-Carnitine Reduces Oxidative Stress and Promotes Cells Differentiation and Bone Matrix Proteins Expression in Human Osteoblast-Like Cells.

Bone fragility and associated fracture risk are major problems in aging. Oxidative stress and mitochondrial dysfunction play a key role in the development of bone fragility. Mitochondrial dysfunction is closely associated with excessive production of reactive oxygen species (ROS). L-Carnitine (L-C), a fundamental cofactor in lipid metabolism, has an important antioxidant property. Several studies have shown how L-C enhances osteoblastic proliferation and activity. In the current study, we investigated the potential effects of L-C on mitochondrial activity, ROS production, and gene expression involved in osteoblastic differentiation using osteoblast-like cells (hOBs) derived from elderly patients. The effect of 5mM L-C treatment on mitochondrial activity and L-C antioxidant activity was studied by ROS production evaluation and cell-based antioxidant activity assay. The possible effects of L-C on hOBs differentiation were assessed by analyzing gene and protein expression by Real Time PCR and western blotting, respectively. L-C enhanced mitochondrial activity and improved antioxidant defense of hOBs. Furthermore, L-C increased the phosphorylation of Ca2+/calmodulin-dependent protein kinase II. Additionally, L-C induced the phosphorylation of ERK1/2 and AKT and the main kinases involved in osteoblastic differentiation and upregulated the expression of osteogenic related genes, RUNX2, osterix (OSX), bone sialoprotein (BSP), and osteopontin (OPN) as well as OPN protein synthesis, suggesting that L-C exerts a positive modulation of key osteogenic factors. In conclusion, L-C supplementation could represent a possible adjuvant in the treatment of bone fragility, counteracting oxidative phenomena and promoting bone quality maintenance.

L-Quebrachitol Promotes the Proliferation, Differentiation, and Mineralization of MC3T3-E1 Cells: Involvement of the BMP-2/Runx2/MAPK/Wnt/β-Catenin Signaling Pathway.

Osteoporosis is widely recognized as a major health problem caused by an inappropriate rate of bone resorption compared to bone formation. Previously we showed that d-pinitol inhibits osteoclastogenesis but has no effect on osteoblastogenesis. However, the effect on osteoblast differentiation of its isomer, l-quebrachitol, has not yet been reported. The purpose of this study was, therefore, to investigate whether l-quebrachitol promotes the osteoblastogenesis of pre-osteoblastic MC3T3-E1 cells. Moreover, the molecular mechanism of action of l-quebrachitol was further explored. Here, it is shown for the first time that l-quebrachitol significantly promotes proliferation and cell DNA synthesis. It also enhances mineralization accompanied by increases in mRNA expression of bone matrix proteins including alkaline phosphatase (ALP), collagen type I (ColI), osteocalcin (OCN), and osteopontin (OPN). In addition, l-quebrachitol upregulates the mRNA and protein expression of bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor-2 (Runx2), while down-regulating the receptor activator of the nuclear factor-κB ligand (RANKL) mRNA level. Moreover, the expression of regulatory genes associated with the mitogen-activated protein kinase (MAPK) and wingless-type MMTV integration site (Wnt)/β-catenin signaling pathways are also upregulated. These findings indicate that l-quebrachitol may promote osteoblastogenesis by triggering the BMP-2-response as well as the Runx2, MAPK, and Wnt/β-catenin signaling pathway.

Evidence of Osteogenic Regulation in Calcific Porcine Aortic Valves.

Chemically cross-linked animal tissues, such as porcine aortic valves (PAVs) have many documented advantages over mechanical valves. However, calcification is the major underlying pathologic process that results in bioprosthetic valve failure. Recently, several reports described the expression of noncollagenous bone matrix proteins in bioprosthetic valves and suggested an actively regulated process of tissue repair. Thirty-one explanted PAVs with evidence of calcification were collected and examined for the protein expression implicated in myofibroblast activation, osteoblast differentiation, and bone matrix deposition by using immunohistochemistry. The mean duration that PAVs were implanted was 11.5 ± 5.6 years, ranging from 12 months to 28 years. Pearson correlation analysis showed a significant relationship between the duration and valvular calcification (r = 0.3818, P = .034). The number of vimentin-positive mesenchymal cells in explanted PAVs was significantly lower than that of unused PAVs (P < .01). However, increased expression of α-smooth muscle actin (α-SMA) (P < .01), proliferating cell nuclear antigen (PCNA, P < .01), Cbfa1/Runx2 (P < .01), osterix (P = .0126), bone sialoprotein (BSP, P < .01), osteocalcin (P < .01), and osteopontin (P < .01) was found in explanted PAVs. Immunohistochemical staining of alkaline phosphatase (ALP) and osteocalcin was negative in the unused PAVs. In explanted PAVs, the expression level of these 2 proteins was also significantly increased. Our results support the view that PAV calcification is an actively regulated process with osteogenic signaling activation.

Expression of Non-collagenous Bone Matrix Proteins in Osteoblasts Stimulated by Mechanical Stretching in the Cranial Suture of Neonatal Mice.

We investigated the influence of mechanical stretching on the genetic expression pattern of non-collagenous bone matrix proteins in osteoblasts. The cranial sutures of neonatal mice were subjected to ex vivo mechanical stretching. In the non-stretched control group, as osteoblast differentiation progressed, the successive genetic expression of bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN) was detected using in situ hybridization, in that order. In the stretched group, the sutures were widened, and after 24 hr of cultivation, a large number of osteoblasts and abundant new osteoid were observed on the borders of the parietal bones. All new osteoblasts expressed BSP and some of them expressed OPN, but very few of them expressed OCN. After 48 hr, more extensive presence of osteoid was noted on the borders of the parietal bones, and this osteoid was partially mineralized; all osteoblasts on the osteoid surface expressed BSP, and more osteoblasts expressed OPN than those after 24 hr cultivation. Surprisingly, many of the osteoblasts that did not express OPN, expressed OCN. This suggests that when osteoblast differentiation is stimulated by mechanical stress, the genetic expression pattern of non-collagenous proteins in the newly differentiated osteoblasts is affected.

Effects of strength training on osteogenic differentiation and bone strength in aging female Wistar rats

The effects of strength training (ST) on the mechanical bone strength and osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs) from adult, aged and exercised aged rats were determined. The exercised aged animals displayed higher values of areal bone mineral density, compression test, alkaline phosphatase activity (ALP) and biological mineralization, while oil red O staining for adipocytes was lower. ST increased gene expression of runt-related transcription factor 2 (Runx2), osterix (Osx) as well as bone matrix protein expression, and reduced expression of peroxisome proliferator-activated receptor gamma (Pparγ). The production of pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) was lower in BMSCs of the aged exercised group. The ST practice was able to improve the bone mechanical properties in aged female rats, increasing the potential for osteogenic differentiation of BMSCs, reducing the adipogenic differentiation and pro-inflammatory cytokine level. In summary, the data achieved in this study showed that strength training triggers physiological responses that result in changes in the bone microenvironment and bring benefits to biomechanical parameters of bone tissue, which could reduce the risk of fractures during senescent.

Expression of SPARC and the osteopontin-like protein during skeletal development in the cichlid fish Oreochromis mossambicus.

Bones are mainly composed of calcium hydroxyapatite and a proteinous matrix. In this study, we focus on the bone matrix proteins, the fish osteopontin orthologous protein (osteopontin-like protein; OP-L) and SPARC, because the current knowledge regarding their expression is fragmentary or contradictory. We first provide a comprehensive and detailed description of skeletal development in the cichlid fish Oreochromis mossambicus. Following this, we analyzed the expression pattern of OP-L and SPARC in detail during development. OP-L expression was only found in tissues that undergo ossification (i.e., developing bones and teeth). Furthermore, we show that there is a fundamental difference in cartilage formation of the splanchnocranium and all other cartilages, concerning SPARC expression. Significantly, we show that the initial calcification of cranial bones occurs simultaneously with the expression of OP-L and SPARC in the osteoblast-like cells, which appear early in development. The difference in SPARC expression during chondrogenesis of the splanchnocranium is likely based on its different evolutionary history compared with the dermatocranium and chondrocranium. Moreover, our results suggest a co-occurrence of the initial calcium deposition and bone matrix protein expression during osteogenesis. Overall, this study enhances our understanding of fish skeletal development and evolution.

The effects of bone pâté on human osteoblasts cell cultures.

The aim of the present study was to evaluate the effect of bone pate on human osteoblast differentiation by measuring cell viability, alkaline phosphatase activity and expression of the transcription factors and of the major components of the extracellular matrix. Although bone paté has been used in ear surgery for many years and when placed in contact with mastoid and external auditory canal bone become viable, the cellular mechanisms that lead to its osteointegration have never been described. Bone paté taken from four patients subjected to mastoidectomy and affected by middle ear and mastoid cholesteatoma was placed in contact with osteoblast-like cell cultures. Four experimental conditions were obtained: cell cultures treated with bone patè, with bone paté mixed with fibrin glue, with fibrin glue and untreated. After 24h, the viability of the cells was evaluated; after 1week, alkaline phosphatase activity and the expression of transcription factors and bone matrix proteins were assessed by quantitative polymerase chain reaction. After 24h osteoblasts showed increased viability when treated with bone paté (19% increase) and bone pate mixed with fibrin glue (34% increase). After 1week, the number of alkaline phosphatase positive cells increased by 97 and 94% in cultures treated with bone paté alone and bone pate mixed with fibrin glue. Treatment with bone patè upregulated transcription factors and components of the extracellular matrix. The present data show that bone paté has a high osteoinductive potential on human osteoblasts, enhancing their activity.

Epigenetically Modified Bone Marrow Stromal Cells in Silk Scaffolds Promote Craniofacial Bone Repair and Wound Healing.

Epigenetic regulation of gene expression is a central mechanism that governs cell stemness, determination, commitment, and differentiation. It has been recently found that PHF8, a major H4K20/H3K9 demethylase, plays a critical role in craniofacial and bone development. In this study, we hypothesize that PHF8 promotes osteoblastogenesis by epigenetically regulating the expression of a nuclear matrix protein, special AT-rich sequence-binding protein 2 (SATB2) that plays pivotal roles in skeletal patterning and osteoblast differentiation. Our results showed that expression levels of PHF8 and SATB2 in preosteoblasts and bone marrow stromal cells (BMSCs) increased simultaneously during osteogenic induction. Overexpressing PHF8 in these cells upregulated the expression of SATB2, Runx2, osterix, and bone matrix proteins. Conversely, knockdown of PHF8 reduced the expression of these genes. Furthermore, ChIP assays confirmed that PHF8 specifically bound to the transcription start site (TSS) of the SATB2 promoter, and the expression of H3K9me1 at the TSS region of SATB2 decreased in PHF8 overexpressed group. Implantation of the BMSCs overexpressing PHF8 with silk protein scaffolds promoted bone regeneration in critical-sized defects in mouse calvaria. Taken together, our results demonstrated that PHF8 epigenetically modulates SATB2 activity, triggering BMSCs osteogenic differentiation and facilitating bone formation and regeneration in biodegradable silk scaffolds.

Effect of low-level laser therapy on bone repair: a randomized controlled experimental study.

The aim of this study was to investigate the effect of low-level laser therapy (LLLT) on bone repair in femoral fractures. Sixty adult Wistar rats were randomly assigned into one of two groups: group A (ostectomy + LLLT) or group B (ostectomy + sham laser). An experimental model of complete bone fracture was surgically created by removing a 2-mm fragment from the middle third of the femoral shaft. Data were analyzed on days 8, 13, and 18 after the fracture (subgroups 1, 2, and 3). Samples were assessed for changes in inflammatory infiltration; trabecular bone matrix, periosteal, and new bone formations; and changes in the expression of particular osteogenic-related proteins (osteocalcin, osteopontin, and osteonectin). Microscopic analysis revealed a significant decrease in inflammatory infiltration, intense trabecular bone matrix and periosteal formation, and an increase in newly formed bone after laser irradiation. We also found an increase in the expression of bone matrix proteins with LLLT, with a significant difference measured for osteocalcin in the LLLT group at day 8 (p = 0.007). We show that LLLT plays an important role in augmenting bone tissue formation, which is relevant to fracture healing. LLLT may therefore be indicated as an adjunct therapeutic tool in clinical practice for the treatment or recovery of nonunion injuries.

First titanium dental implants with white surfaces: Preparation and in vitro tests

ObjectivesTo evaluate first white titanium surfaces developed for improvement of existing clinically used titanium-based implants Ticer. MethodsThe anodic plasma-electrochemical oxidation in aqueous solutions of sodium hydroxide and calcium dihydrogen phospate was used to prepare three novel anodic conversion layers with white titanium oxide surfaces. The surfaces have been characterized by the means of scanning electron microscopy, surface microanalysis and X-ray diffraction. In vitro studies were conducted on primary human osteoblast cells using novel surfaces (M1–M3) as well as commercially pure titanium (Ti cp), Ticer and SS (subtracted surface). An indirect toxicity test using MTT and SRB assays has been carried out. Furthermore, immunohistochemical analysis of cell proliferation, morphology, and expression of non-collagenous bone matrix proteins (sialoprotein, BSP, and osteocalcin, OC) were performed. ResultsThe basic morphology of the surfaces shows clusters in a size of 100μm of knob-like structures. The coatings are composed of rutile and monoclinic sodium titanates. Novel white surfaces (M1–M3) induced proliferation rates, morphological changes and influenced the expression of OC and BSP similarly to Ticer. On the other hand, Ti cp and SS exhibited different in vitro behavior. SignificanceThe novel surfaces expressed similar in vitro behavior as Ticer, successfully used in clinical practice. Furthermore, due to their white color they are also promising from the esthetic point of view. The results described herein open the door toward a new generation of white titanium dental implants.

Bone response to biomimetic implants delivering BMP-2 and VEGF: An immunohistochemical study

This animal study evaluated bone healing around titanium implant surfaces biomimetically coated with bone morphogenic protein-2 (BMP-2) and/or vascular endothelial growth factor (VEGF) by examining bone matrix proteins and mineralisation. Five different implant surfaces were established: acid-etched surface (AE), biomimetic calcium phosphate surface (CAP), BMP-2 loaded CAP surface, VEGF loaded CAP surface and dual BMP-2 + VEGF loaded CAP surface. The implants were inserted into calvariae of adult domestic pigs. For the comparison of osteoconductive capacity of each surface, bone mineral density and expression of bone matrix proteins (collagen I, BMP-2/4, osteocalcin and osteopontin) inside defined chambers around the implant were assessed using light microscopy and microradiography and immunohistochemical analysis at 1, 2 and 4 weeks. In the both groups delivering BMP-2, the bone mineral density was significantly enhanced after 2 weeks and the highest value was measured for the group BMP + VEGF. In the group VEGF, collagen I and BMP-2/4 expressions were significantly up-regulated at the first and second weeks. The percentage of BMP-2/4 positive cells in the group BMP + VEGF was significantly enhanced compared with the groups AE and CAP at the second week. Although the highest osteocalcin and osteopontin expression values were observed for the group BMP + VEGF after 2 weeks, no statistically significant difference in osteocalcin and osteopontin expressions was found between all groups at any time. It was concluded that combined delivery of BMP-2 and VEGF favoured bone mineralisation and expression of important bone matrix proteins that might explain synergistic interaction between both growth factors.

Low osteocalcin/collagen type I bone gene expression ratio is associated with hip fragility fractures

IntroductionOsteocalcin (OC) is the most abundant non-collagenous bone protein and is determinant for bone mineralization.We aimed to compare OC bone expression and serum factors related to its carboxylation in hip fragility fracture and osteoarthritis patients. We also aimed to identify which of these factors were associated with worse mechanical behavior and with the hip fracture event. MethodsIn this case-control study, fragility fracture patients submitted to hip replacement surgery were evaluated and compared to a group of osteoarthritis patients submitted to the same procedure. Fasting blood samples were collected to assess apolipoproteinE (apoE) levels, total OC and undercarboxylated osteocalcin (ucOC), vitamin K, LDL cholesterol, triglycerides and bone turnover markers. The frequency of the apoε4 isoform was determined.Femoral epiphyses were collected and trabecular bone cylinders drilled in order to perform compression mechanical tests. Gene expression of bone matrix components was assessed by quantitative RT-PCR analysis. Results64 patients, 25 submitted to hip replacement surgery due to fragility fracture and 39 due to osteoarthritis, were evaluated. Bone OC/collagen expression (OC/COL1A1) ratio was significantly lower in hip fracture compared to osteoarthritis patients (p<0.017) adjusted for age, gender and body mass index. Moreover, OC/COL1A1 expression ratio was associated with the hip fracture event (OR ~0; p=0.003) independently of the group assigned, or the clinical characteristics. Apoε4 isoform was more frequent in the hip fracture group (p=0.029). ucOC levels were higher in the fracture group although not significantly (p=0.058). No differences were found regarding total OC (p=0.602), apoE (p=0.467) and Vitamin K (p=0.371).In hip fracture patients, multivariate analysis, adjusted for clinical characteristics, serum factors related to OC metabolism and gene expression of bone matrix proteins showed that low OC/COL1A1 expression ratio was significantly associated with worse trabecular strength (β=0.607; p=0.013) and stiffness (β=0.693; p=0.003). No association was found between ucOC and bone mechanics. Moreover, in osteoarthritis patients, the multivariate analysis revealed that serum total OC was negatively associated with strength (β=−0.411; p=0.030) and stiffness (β=−0.487; p=0.009). ConclusionWe demonstrated that low bone OC/COL1A1 expression ratio was an independent predictor of worse trabecular mechanical behavior and of the hip fracture event. These findings suggest that in hip fracture patients the imbalance of bone OC/COL1A1 expression ratio reflects disturbances in osteoblast activity leading to bone fragility.

Osteomimicry of Mammary Adenocarcinoma Cells In Vitro; Increased Expression of Bone Matrix Proteins and Proliferation within a 3D Collagen Environment

Bone is the most common site of metastasis for breast cancer, however the reasons for this remain unclear. We hypothesise that under certain conditions mammary cells possess osteomimetic capabilities that may allow them to adapt to, and flourish within, the bone microenvironment. Mammary cells are known to calcify within breast tissue and we have recently reported a novel in vitro model of mammary mineralization using murine mammary adenocarcinoma 4T1 cells. In this study, the osteomimetic properties of the mammary adenocarcinoma cell line and the conditions required to induce mineralization were characterized extensively. It was found that exogenous organic phosphate and inorganic phosphate induce mineralization in a dose dependent manner in 4T1 cells. Ascorbic acid and dexamethasone alone have no effect. 4T1 cells also show enhanced mineralization in response to bone morphogenetic protein 2 in the presence of phosphate supplemented media. The expression of several bone matrix proteins were monitored throughout the process of mineralization and increased expression of collagen type 1 and bone sialoprotein were detected, as determined by real-time RT-PCR. In addition, we have shown for the first time that 3D collagen glycosaminoglycan scaffolds, bioengineered to represent the bone microenvironment, are capable of supporting the growth and mineralization of 4T1 adenocarcinoma cells. These 3D scaffolds represent a novel model system for the study of mammary mineralization and bone metastasis. This work demonstrates that mammary cells are capable of osteomimicry, which may ultimately contribute to their ability to preferentially metastasize to, survive within and colonize the bone microenvironment.