Osteoblastic Wnt1 regulates periosteal bone formation in adult mice

Compelling clinical data together with genetically modified mouse models have demonstrated that Wnt1 is a key Wnt ligand in bone metabolism, regulating both osteoblast activity and osteoclast differentiation. We have previously shown that deletion of Wnt1 in limb mesenchymal cells leads to severe ostepenic bone phenotype and spontaneous fractures very early after birth. However, the function of Wnt1 in mature skeleton remained unknown. To investigate the role of Wnt1 specifically in adult bone metabolism, we generated an osteoblast lineage-targeted inducible Wnt1 knockout mouse model using tetracycline-controlled Osterix-Cre mouse line (Osx-Cre). In this model, the Cre recombinase expression is suppressed by administering doxycycline (Dox) in drinking water. As expected, Wnt1Osx−/− mice without Dox developed spontaneous fractures early by 3 weeks of age due to severe trabecular and cortical osteopenia. Administration of Dox to Wnt1Osx-Dox−/− and control mice until 4 weeks of age suppressed Wnt1 deletion and completely prevented the fractures. Withdrawal of Dox led to deletion in Wnt1 allele but the fracture incidence progressively decreased in Wnt1Osx-Dox−/− mice at 8 or 12 weeks of age (4 and 8 weeks after Dox withdrawal). Interestingly, deletion of Wnt1 at 4 weeks of age resulted only in a modest and transient trabecular osteopenia that was more pronounced in females and was normalized by 12 weeks of age. However, diaphyseal cortical bone mass and cortical thickness in the femurs were significantly decreased in Wnt1Osx-Dox−/− mice of both genders. Mechanisticly, this was due to impaired periosteal bone formation. Based on our data, in addition to its essential role in early skeletal growth, Wnt1 is an important regulator of modeling-based bone formation and cortical thickness in adult mice.</p

Zoledronic acid in vivo increases in vitro proliferation of rat mesenchymal stromal cells

Background and purpose-Bisphosphonates are widely used in the treatment of bone loss, but they might also have positive effects on osteoblastic cells and bone formation. We evaluated the effect of in vivo zoledronic acid (ZA) treatment and possible concomitant effects of ZA and fracture on the ex vivo osteogenic capacity of rat mesenchymal stromal cells (MSCs). Methods-A closed femoral fracture model was used in adult female rats and ZA was administered as a single bolus or as weekly doses up to 8 weeks. Bone marrow MSCs were isolated and cultured for in vitro analyses. Fracture healing was evaluated by radiography, micro -computed tomography (mu CT), and histology. Results-Both bolus and weekly ZA increased fracture-site bone mineral content and volume. MSCs from weekly ZA-treated animals showed increased ex vivo proliferative capacity, while no substantial effect on osteoblastic differentiation was observed. Fracture itself did not have any substantial effect on cell proliferation or differentiation at 8 weeks. Serum biochemical markers showed higher levels of bone formation in animals with fracture than in intact animals, while no difference in bone resorption was observed. Interestingly, ex vivo osteoblastic differentiation of MSCs was found to correlate with in vivo serum bone markers. Interpretation-Our data show that in vivo zoledronic acid treatment can influence ex vivo proliferation of MSCs, indicating that bisphosphonates can have sustainable effects on cells of the osteoblastic lineage. Further research is needed to investigate the mechanisms.Peer reviewe

Angiogenic potential of human mesenchymal stromal cell and circulating mononuclear cell cocultures is reflected in the expression profiles of proangiogenic factors leading to endothelial cell and pericyte differentiation.

Endothelial progenitors found among the peripheral blood (PB) mononuclear cells (MNCs) are interesting cells for their angiogenic properties. Mesenchymal stromal cells (MSCs) in turn can produce proangiogenic factors as well as differentiate into mural pericytes, making MSCs and MNCs an attractive coculture setup for regenerative medicine. In this study, human bone marrow-derived MSCs and PB-derived MNCs were cocultured in basal or osteoblastic medium without exogenously supplied growth factors to demonstrate endothelial cell, pericyte and osteoblastic differentiation. The expression levels of various proangiogenic factors, as well as endothelial cell, pericyte and osteoblast markers in cocultures were determined by quantitative polymerase chain reaction. Immunocytochemistry for vascular endothelial growth factor receptor-1 and α-smooth muscle actin as well as staining for alkaline phosphatase were performed after 10 and 14 days. Messenger ribonucleic acid expression of endothelial cell markers was highly upregulated in both basal and osteoblastic conditions after 5 days of coculture, indicating an endothelial cell differentiation, which was supported by immunocytochemistry for vascular endothelial growth factor receptor-1. Stromal derived factor-1 and vascular endothelial growth factor were highly expressed in MSC-MNC coculture in basal medium but not in osteoblastic medium. On the contrary, the expression levels of bone morphogenetic protein-2 and angiopoietin-1 were significantly higher in osteoblastic medium. Pericyte markers were highly expressed in both cocultures after 5 days. In conclusion, it was demonstrated endothelial cell and pericyte differentiation in MSC-MNC cocultures both in basal and osteoblastic medium indicating a potential for neovascularization for tissue engineering applications

Evidence for the in vivo existence and mobilisation of myeloid angiogenic cells and pericyte-like cells in wound patients after skin grafting

Myeloid angiogenic cells (MACs) and pericyte-like cells, derived from peripheral blood mononuclear cells (MNCs) by in vitro culturing, are suggested as relevant cell types for angiogenesis and tissue repair. However, the in vivo existence and relevance of these cells has so far remained unknown. Our aim was thus to study, if MACs and pericyte-like cells exist in circulation during the wound healing of skin graft patients, and to evaluate the cellular features of wound repair. MNCs were isolated from blood samples of healthy controls (n = 4) and patients with a traumatic full thickness skin defect (n = 4) before skin grafting and on postoperative days 1 and 6. The numbers of circulating CD14+CD45+CD31+CD34− MACs and CD14+CD45+NG2+ pericyte-like cells were assessed by flow cytometry, and gene expression of various pro-angiogenic factors was analysed by qPCR. Wound bed biopsies were taken on postoperative days 6 and 14, and MAC (CD31, CD14 and CD45) and pericyte-related markers (NG2 and PDGFRβ) were histologically studied. MACs and pericyte-like cells were detected in both healthy controls and in patients. Before reconstruction, on average 18% of all circulating MNCs represented MACs and 2% pericyte-like cells in wound patients. Number of MACs significantly increased 1.1−1.7-fold in all patients 1 day after skin grafting (p < 0.01). In addition, histological analysis demonstrated effective vascularization of skin grafts, as well as presence of pericytes, and CD14 and CD45 expressing myeloid cells during wound healing. In conclusion, our data shows, for the first time, the presence and mobilisation of MACs and pericyte-like cells in human circulation.publishedVersionPeer reviewe

Evidence for the in vivo existence and mobilization of myeloid angiogenic cells and pericyte-like cells in wound patients after skin grafting

Myeloid angiogenic cells (MACs) and pericyte-like cells, derived from peripheral blood mononuclear cells (MNCs) by in vitro culturing, are suggested as relevant cell types for angiogenesis and tissue repair. However, the in vivo existence and relevance of these cells has so far remained unknown. Our aim was thus to study, if MACs and pericyte-like cells exist in circulation during the wound healing of skin graft patients, and to evaluate the cellular features of wound repair. MNCs were isolated from blood samples of healthy controls (n = 4) and patients with a traumatic full thickness skin defect (n = 4) before skin grafting and on postoperative days 1 and 6. The numbers of circulating CD14+ CD45+ CD31+ CD34- MACs and CD14+ CD45+ NG2+ pericyte-like cells were assessed by flow cytometry, and gene expression of various pro-angiogenic factors was analysed by qPCR. Wound bed biopsies were taken on postoperative days 6 and 14, and MAC (CD31, CD14 and CD45) and pericyte-related markers (NG2 and PDGFRβ) were histologically studied. MACs and pericyte-like cells were detected in both healthy controls and in patients. Before reconstruction, on average 18% of all circulating MNCs represented MACs and 2% pericyte-like cells in wound patients. Number of MACs significantly increased 1.1-1.7-fold in all patients 1 day after skin grafting (p </p

Mesenchymal cell-derived Wnt1 signaling regulates subchondral bone remodeling but has no effects on the development of growth plate or articular cartilage in mice

Chondrocyte differentiation is a principal progress in endochondral ossification and in the formation of secondary ossification center (SOC) during the long bone development. We have previously reported that targeted deletion of Wnt1 in mesenchymal progenitors (Wnt1Prrx-/-) leads to spontaneous fractures and severe osteopenia in mouse long bones, suggesting that Wnt1 is a key regulator of bone metabolism. However, the effect of Wnt1 on the regulation of cartilage development and chondrocyte differentiation remained unknown. In this study, WNT1 protein expression was observed in lateral superficial cartilage and growth plate pre-hypertrophic chondrocytes in mice. Wnt1 mRNA expression was detected in epiphyseal cartilage from E16.5 to 3 month-old mice. Detailed histological analyses revealed that the average thickness and chondrocyte density of proximal tibial articular cartilage and growth plate were unchanged between Wnt1Prrx-/- and control mice. However, mu CT analysis of tibial epiphyses showed that the subchondral bone mass was reduced in Wnt1Prrx-/- mice compared to control mice, as demonstrated by decreased bone volume, trabecular number, trabecular thickness, and increased trabecular separation in Wnt1Prrx-/- mice. Mechanistically, histomorphometric analyses showed that the reduced subchondral bone mass in Wnt1Prrx-/- mice was due to impaired bone formation and enhanced bone resorption. In vitro, exogenous Wntl inhibited chondrogenesis and chondrocyte hypertrophy in both cell autonomous and juxtacrine manners, while matrix mineralization and the expression of Mmp13, Mmp9 and Opn were induced in a juxtacrine manner. Taken together, mesenchymal cell-derived Wntl is an important regulator of subchondral bone remodeling, although it has no effect on the regulation of growth plate or articular cartilage.</p

Physicochemical and biological characterization of silica-coated alumina particles

ObjectivesA tribochemical silica-coating (TSC) method has been developed to improve the adhesion of dental resin composites to various substrates. The method utilizes airborne-particle abrasion using particles having a silica surface and an alumina core. The impact of the TSC method has been extensively studied but less attention has been paid to the characterization of the silica-modified alumina particles. Due to the role of silicate ions in cell biology, e.g. osteoblast function and bone mineralization, silica-modified alumina particles could also be potentially used as a biomaterial in scaffolds of tissue regeneration. Thus, we carried out detailed physicochemical characterization of the silica-modified alumina particles.MethodsSilica-modified alumina particles (Rocatec, 3 M-ESPE) of an average particle size of 30 µm were studied for the phase composition, spectroscopic properties, surface morphology, dissolution, and the capability to modify the pH of an immersion solution. The control material was alumina without silica modification. Pre-osteoblastic MC3T3-E1 cells were used to assess cell viability in the presence of the particles. Cell viability was tested at 1, 3, 7 and 10 days of culture with various particle quantities. Multivariate ANOVA was used for statistical analyses.ResultsMinor quantities of silica enrichment was verified on the surface of alumina particles and the silica did not evenly cover the alumina surface. In the dissolution test, no change in the pH of the immersion solution was observed in the presence of the particles. Minor quantities of silicate ions were dissolved from the particles to the cell culture medium but no major differences were observed in the viability of pre-osteoblastic cells, whether the cells were cultured with silica-modified or plain alumina particles.SignificanceCharacterization of silica-modified alumina particles demonstrated differences in the particle surface structure compared to control alumina. Dissolution of silica layer in Tris buffer or SBF solution varied from that of cell culture medium: minor quantities of dissolved Si were observed in cell culture test medium. The cell viability test did not shown significant differences between control alumina and its silica-modified counterpart.</p

Structural and elemental characterization of glass and ceramic particles for bone surgery

AbstractObjectiveClinically used bioceramics have been characterized previously with different kinds of methods and comparison of results have proven to be difficult due to varieties of the material properties of interest. Therefore, in this study we compared clinically commonly used bioceramics of hydroxyapatite and carbonate apatite, two bioactive glasses 45S5 and S53P4, and alumina with respect of properties which according to the present knowledge are significant for bone biology.MethodsPhysicochemical properties of the materials were characterized by various methods. Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) was used to analyze the material vibrational features. X-ray Power Diffraction (XRD) was used to characterize the material crystal structure and scanning electron microscopy-energy-dispersive x-ray analysis (SEM-EDXA) was used to evaluate the morphology and size of the materials and to calculate their oxide content. The dissolution behavior of the materials, ion release and pH changes in Tris buffer in a continuous flow-through reaction for 24-hours were determined. The change of the surface of the bioactive glasses by interfacial reaction during the Tris immersion was examined and the thickness of the surface reaction layer of the materials was studied.ResultsSEM examination showed that the particle morphology of BG 45S5, BG S53P4 and alumina particle’s surface was smooth. The surface of HAP was porous, but also CAP showed some surface porosity. An increase in the pH of the immersion solution was observed especially for BG 45S5 and BG S53P4. HAP, CAP and alumina caused only a minor increase in pH. BGs 45S5 and S53P4 showed a rapid initial release of sodium and calcium ions, followed by the release of silicon species. Minor release of sodium ions was registered for HAP, CAP and alumina. Calcium ion release was low but constant over the experimental time while only a minor initial dissolution was measured for HAP.SignificanceThe in vitro study showed differences in the materials’ properties, which are considered to be important for biological suitability and in clinical applications, such as materials tomography, ion release and pH changes.</p

Signature of circulating small non-coding RNAs during early fracture healing in mice

Fracture healing is a complex process with multiple overlapping metabolic and differentiation phases. Small non-coding RNAs are involved in the regulation of fracture healing and their presence in circulation is under current interest due to their obvious value as potential biomarkers. Circulating microRNAs (miRNAs) have been characterized to some extent but the current knowledge on tRNA-derived small RNA fragments (tsRNAs) is relatively scarce, especially in circulation.In this study, the spectrum of circulating miRNAs and tsRNAs was analysed by next generation sequencing to show their differential expression during fracture healing in vivo. Analysed tsRNA fragments included stress-induced translation interfering tRNA fragments (tiRNAs or tRNA halves) and internal tRNA fragments (i-tRF), within the size range of 28–36 bp. To unveil the expression of these non-coding RNAs, genome-wide analysis was performed on two months old C57BL/6 mice on days 1, 5, 7, 10, and 14 (D1, D5, D7, D10, and D14) after a closed tibial fracture.Valine isoacceptor tRNA-derived Val-AAC 5′end and Val-CAC 5′end fragments were the major types of 5′end tiRNAs in circulation, comprising about 65 % of the total counts. Their expression was not affected by fracture. After a fracture, the levels of two 5′end tiRNAs Lys-TTT 5′ and Lys-CTT 5′ were decreased and His-GTG 5′ was increased through D1-D14. The level of miR-451a was decreased on the first post-fracture day (D1), whereas miR-328-3p, miR-133a-3p, miR-375-3p, miR-423-5p, and miR-150-5p were increased post-fracture. These data provide evidence on how fracture healing could provoke systemic metabolic effects and further pinpoint the potential of small non-coding RNAs as biomarkers for tissue regeneration.</p

Bone Marrow Niche: Role of Different Cells in Bone Metastasis

Purpose of ReviewThis report summarizes current knowledge of bone marrow hematopoietic stem cell (HSC) niche, focusing on the identification of niche cells and molecular mechanisms involved in HSC maintenance and bone metastasis.Recent FindingsNovel imaging techniques are greatly improving our understanding of bone marrow niche and latest studies have revealed several complex multicellular regulatory mechanisms of niche function. Especially, the intriguing role of bone marrow macrophages and osteomacs is an emerging topic in the field. It appears that, e.g., macrophage polarization is important for communication with bone marrow stromal cells (BMSCs). Bone marrow is also a favorable environment for disseminated tumor cells and recent data shows that various niche cell types, including endothelial cells and BMSCs, regulate the progression of bone metastasis.SummaryBone marrow niche represents a multicellular system with complex interactions. Emerging data is providing us with a deeper understanding of this fascinating tissue and its role in metastasis.</div