Vasorin as an actor of bone turnover?

Bone diseases are increasing with aging populations and it is important to identify clues to develop innovative treatments. Vasn, which encodes vasorin (Vasn), a transmembrane protein involved in the pathophysiology of several organs, is expressed during the development in intramembranous and endochondral ossification zones. Here, we studied the impact of Vasn deletion on the osteoblast and osteoclast dialog through a cell Coculture model. In addition, we explored the bone phenotype of Vasn KO mice, either constitutive or tamoxifen-inducible, or with an osteoclast-specific deletion. First, we show that both osteoblasts and osteoclasts express Vasn. Second, we report that, in both KO mouse models but not in osteoclast-targeted KO mice, Vasn deficiency was associated with an osteopenic bone phenotype, due to an imbalance in favor of osteoclastic resorption. Finally, through the Coculture experiments, we identify a dysregulation of the Wnt/ß-catenin pathway together with an increase in RANKL release by osteoblasts, which led to an enhanced osteoclast activity. This study unravels a direct role of Vasn in bone turnover, introducing a new biomarker or potential therapeutic target for bone pathologies.

Allogenic Stem Cells Carried by Porous Silicon Scaffolds for Active Bone Regeneration In Vivo.

To date, bone regeneration techniques use many biomaterials for bone grafting with limited efficiencies. For this purpose, tissue engineering combining biomaterials and stem cells is an important avenue of development to improve bone regeneration. Among potentially usable non-toxic and bioresorbable scaffolds, porous silicon (pSi) is an interesting biomaterial for bone engineering. The possibility of modifying its surface can allow a better cellular adhesion as well as a control of its rate of resorption. Moreover, release of silicic acid upon resorption of its nanostructure has been previously proved to enhance stem cell osteodifferentiation by inducing calcium phosphate formation. In the present study, we used a rat tail model to experiment bone tissue engineering with a critical size defect. Two groups with five rats per group of male Wistar rats were used. In each rat, four vertebrae were used for biomaterial implantation. Randomized bone defects were filled with pSi particles alone or pSi particles carrying dental pulp stem cells (DPSC). Regeneration was evaluated in comparison to empty defect and defects filled with xenogenic bone substitute (Bio-Oss®). Fluorescence microscopy and SEM evaluations showed adhesion of DPSCs on pSi particles with cells exhibiting distribution throughout the biomaterial. Histological analyzes revealed the formation of a collagen network when the defects were filled with pSi, unlike the positive control using Bio-Oss®. Overall bone formation was objectivated with µCT analysis and showed a higher bone mineral density with pSi particles combining DPSC. Immunohistochemical assays confirmed the increased expression of bone markers (osteocalcin) when pSi particles carried DPSC. Surprisingly, no grafted cells remained in the regenerated area after one month of healing, even though the grafting of DPSC clearly increased bone regeneration for both bone marker expression and overall bone formation objectivated with µCT. In conclusion, our results show that the association of pSi with DPSCs in vivo leads to greater bone formation, compared to a pSi graft without DPSCs. Our results highlight the paracrine role of grafted stem cells by recruitment and stimulation of endogenous cells.

Tissue Engineering Strategies Applied in Bone Regeneration and Bone Repair.

Bone regeneration and repair present significant challenges in the field of regenerative medicine [...].

Cell-Biomaterial Interactions.

In animals, the extracellular matrix (ECM) forms a three-dimensional network occupying the intercellular spaces (interstitial matrix) or serving as physical and biochemical support for cells and tissues (basement membrane) [...].

Osteogenic Effect of Fisetin Doping in Bioactive Glass/Poly(caprolactone) Hybrid Scaffolds.

Treating large bone defects or fragile patients may require enhancing the bone regeneration rate to overcome a weak contribution from the body. This work investigates the osteogenic potential of nutrient fisetin, a flavonoid found in fruits and vegetables, as a doping agent inside the structure of a SiO2-CaO bioactive glass-poly(caprolactone) (BG-PCL) hybrid scaffold. Embedded in the full mass of the BG-PCL hybrid during one-pot synthesis, we demonstrate fisetin to be delivered sustainably; the release follows a first-order kinetics with active fisetin concentration being delivered for more than 1 month (36 days). The biological effect of BG-PCL-fisetin-doped scaffolds (BG-PCL-Fis) has been highlighted by in vitro and in vivo studies. A positive impact is demonstrated on the adhesion and the differentiation of rat primary osteoblasts, without an adverse cytotoxic effect. Implantation in critical-size mouse calvaria defects shows bone remodeling characteristics and remarkable enhancement of bone regeneration for fisetin-doped scaffolds, with the regenerated bone volume being twofold that of nondoped scaffolds and fourfold that of a commercial trabecular bovine bone substitute. Such highly bioactive materials could stand as competitive alternative strategies involving biomaterials loaded with growth factors, the use of the latter being the subject of growing concerns.

Monosodium Glutamate Supplementation Improves Bone Status in Mice Under Moderate Protein Restriction.

Adequate protein intake during development is critical to ensure optimal bone gain and to attain a higher peak bone mass later. Using a mild protein restriction model in Balb/C mice consuming 6% of their total energy intake as soy protein (LP-SOY)-for which we observed a significantly lower femoral cortical thickness, bone volume, trabecular number, and thickness reduction-we evaluated the effects of monosodium glutamate (MSG) supplementation at different concentrations (0.5, 1, 5, 10, and 20 g/kg of diet) on bone characteristics in LP-SOY-fed mice. After 6 and 12 weeks, LP-SOY-fed mice had lower BMD and reduced body weight related to lower lean mass, which was associated with a reduced IGF-1 level. The negative effect of the LP-SOY diet on BMD correlated with impaired bone formation. MSG supplementation, at 5, 10, and 20 g/kg of diet, and PTH injection, used as a positive control, were able to improve BMD and to increase osteoblast activity markers (P1NP and osteocalcin), as well as glutamine plasma concentration. An analysis of bone microarchitecture found that cortical bone was less sensitive to protein restriction than trabecular bone, and that MSG ingestion was able to preserve bone quality through an increase of collagen synthesis, although it did not allow normal bone growth. Our study reinforces the view that glutamate can act as a functional amino acid for bone physiology and support clinical investigation of glutamate supplementation in adults characterized by poor bone status, notably as a result of insufficient protein intake. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Human Enriched Serum Following Hydrolysed Collagen Absorption Modulates Bone Cell Activity: from Bedside to Bench and Vice Versa.

Collagen proteins are crucial components of the bone matrix. Since collagen-derived products are widely used in the food and supplement industry, one may raise the question whether collagen-enriched diets can provide benefits for the skeleton. In this study, we designed an innovative approach to investigate this question taking into account the metabolites that are formed by the digestive tract and appear in the circulation after ingestion of hydrolysed collagen. Blood samples collected in clinical and pre-clinical trials following ingestion and absorption of hydrolysed collagen were processed and applied on bone-related primary cell cultures. This original ex vivo methodology revealed that hydrolysed collagen-enriched serum had a direct impact on the behaviour of cells from both human and mouse origin that was not observed with controls (bovine serum albumin or hydrolysed casein-enriched serum). These ex vivo findings were fully in line with in vivo results obtained from a mouse model of post-menopausal osteoporosis. A significant reduction of bone loss was observed in mice supplemented with hydrolysed collagen compared to a control protein. Both the modulation of osteoblast and osteoclast activity observed upon incubation with human or mouse serum ex vivo and the attenuation of bone loss in vivo, clearly indicates that the benefits of hydrolysed collagen for osteoporosis prevention go beyond the effect of a simple protein supplementation.

Priming Dental Pulp Stem Cells from Human Exfoliated Deciduous Teeth with Fibroblast Growth Factor-2 Enhances Mineralization Within Tissue-Engineered Constructs Implanted in Craniofacial Bone Defects.

The craniofacial area is prone to trauma or pathologies often resulting in large bone damages. One potential treatment option is the grafting of a tissue-engineered construct seeded with adult mesenchymal stem cells (MSCs). The dental pulp appears as a relevant source of MSCs, as dental pulp stem cells display strong osteogenic properties and are efficient at bone formation and repair. Fibroblast growth factor-2 (FGF-2) and/or hypoxia primings were shown to boost the angiogenesis potential of dental pulp stem cells from human exfoliated deciduous teeth (SHED). Based on these findings, we hypothesized here that these primings would also improve bone formation in the context of craniofacial bone repair. We found that both hypoxic and FGF-2 primings enhanced SHED proliferation and osteogenic differentiation into plastically compressed collagen hydrogels, with a much stronger effect observed with the FGF-2 priming. After implantation in immunodeficient mice, the tissue-engineered constructs seeded with FGF-2 primed SHED mediated faster intramembranous bone formation into critical size calvarial defects than the other groups (no priming and hypoxia priming). The results of this study highlight the interest of FGF-2 priming in tissue engineering for craniofacial bone repair. Stem Cells Translational Medicine 2019;8:844&857.

Optimized Bioactive Glass: the Quest for the Bony Graft.

Technological advances have provided surgeons with a wide range of biomaterials. Yet improvements are still to be made, especially for large bone defect treatment. Biomaterial scaffolds represent a promising alternative to autologous bone grafts but in spite of the numerous studies carried out on this subject, no biomaterial scaffold is yet completely satisfying. Bioactive glass (BAG) presents many qualifying characteristics but they are brittle and their combination with a plastic polymer appears essential to overcome this drawback. Recent advances have allowed the synthesis of organic-inorganic hybrid scaffolds combining the osteogenic properties of BAG and the plastic characteristics of polymers. Such biomaterials can now be obtained at room temperature allowing organic doping of the glass/polymer network for a homogeneous delivery of the doping agent. Despite these new avenues, further studies are required to highlight the biological properties of these materials and particularly their behavior once implanted in vivo. This review focuses on BAG with a particular interest in their combination with polymers to form organic-inorganic hybrids for the design of innovative graft strategies.

Mouse Wnt1-CRE-RosaTomato Dental Pulp Stem Cells Directly Contribute to the Calvarial Bone Regeneration Process.

Stem cells endowed with skeletogenic potentials seeded in specific scaffolds are considered attractive tissue engineering strategies for treating large bone defects. In the context of craniofacial bone, mesenchymal stromal/stem cells derived from the dental pulp (DPSCs) have demonstrated significant osteogenic properties. Their neural crest embryonic origin further makes them a potential accessible therapeutic tool to repair craniofacial bone. The stem cells' direct involvement in the repair process versus a paracrine effect is however still discussed. To clarify this question, we have followed the fate of fluorescent murine DPSCs derived from PN3 Wnt1-CRE- RosaTomato mouse molar (T-mDPSCs) during the repair process of calvaria bone defects. Two symmetrical critical defects created on each parietal region were filled with (a) dense collagen scaffolds seeded with T-mDPSCs, (b) noncellularized scaffolds, or (c) no scaffold. Mice were imaged over a 3-month period by microcomputed tomography to evaluate the extent of repair and by biphotonic microscopy to track T-mDPSCs. Histological and immunocytochemical analyses were performed in parallel to characterize the nature of the repaired tissue. We show that T-mDPSCs are present up to 3 months postimplantation in the healing defect and that they rapidly differentiate in chondrocyte-like cells expressing all the expected characteristic markers. T-mDPSCs further maturate into hypertrophic chondrocytes and likely signal to host progenitors that form new bone tissue. This demonstrates that implanted T-mDPSCs are able to survive in the defect microenvironment and to participate directly in repair via an endochondral bone ossification-like process. Stem Cells 2019;37:701-711.

Bioactive Glass/Polycaprolactone Hybrid with a Dual Cortical/Trabecular Structure for Bone Regeneration.

Organic-inorganic hybrid biomaterials stand as a promise for combining bone bonding and bone mineral-forming ability, stimulation of osteogenic cells, and adequate mechanical properties. Bioactive glass (BG)-polycaprolactone (PCL) hybrids are of special interest as they gather the ability of BG to enhance osteoblast-mediated bone formation with the slow degradation rate and the toughness of PCL. In this study, BG-PCL hybrids were synthesized in the form of scaffold, owing to a dual cortical/trabecular structure mimicking the bone architecture. Their biological potential was evaluated both in vitro using rat primary osteoblasts (RPO) and in vivo in a mice model of critical-size calvarial defects. BG-PCL scaffolds were compared to Lubboc (BTB), a commercial purified bovine xenograft widely used in orthopedics and periodontal procedures and known for its efficiency. BG-PCL hybrids were found to facilitate RPO adhesion at their surface and to enhance RPO differentiation when compared to BTB. An in vivo micro-CT study demonstrates a higher bone ingrowth with BG-PCL scaffolds and a complete chemical conversion of the remaining BG-PCL after 3 months of implantation, while histological data show the vascularization of BG-PCL scaffolds and confirm the well-advanced bone regeneration with ongoing remodeling. Finally, we evidence the complete chemical conversion of the remaining BG-PCL into a bone-like mineral.

From Vascular Smooth Muscle Cells to Folliculogenesis: What About Vasorin?

First described in 1988, vasorin (VASN) is a transmembrane glycoprotein expressed during early mouse development, and with a less extent, in various organs and tissues (e.g., kidney, aorta, and brain) postnatally. Vasn KO mice die after 3 weeks of life from unknown cause(s). No human disease has been associated with variants of this gene so far, but VASN seems to be a potential biomarker for nephropathies and tumorigenesis. Its interactions with the TGF-ß and Notch1 pathways offer the most serious assumptions regarding VASN functions. In this review, we will describe current knowledge about this glycoprotein and discuss its implication in various organ pathophysiology.

Early angiogenesis detected by PET imaging with 64Cu-NODAGA-RGD is predictive of bone critical defect repair.

Therapies using stem cells may be applicable to all fields of regenerative medicine, including craniomaxillofacial surgery. Dental pulp stem cells (DPSCs) have demonstrated in vitro and in vivo osteogenic and proangiogenic properties. The aim of the study was to evaluate whether early angiogenesis investigated by nuclear imaging can predict bone formation within a mouse critical bone defect. Two symmetrical calvarial critical-sized defects were created. Defects were left empty or filled with i) DPSC-containing dense collagen scaffold, ii) 5% hypoxia-primed DPSC-containing dense collagen scaffold, iii) acellular dense collagen scaffold, or iv) left empty. Early angiogenesis assessed by PET using 64Cu-NODAGA-RGD as a tracer was found to be correlated with bone formation determined by micro-CT within the defects from day 30, and to be correlated to the late calcium apposition observed at day 90 using 18F-Na PET. These results suggest that nuclear imaging of angiogenesis, a technique applicable in clinical practice, is a promising approach for early prediction of bone grafting outcome, thus potentially allowing to anticipate alternative regenerative strategies. STATEMENT OF SIGNIFICANCE: Bone defects are a major concern in medicine. As life expectancy increases, the number of bone lesions grows, and occurring complications lead to a delay or even lack of consolidation. Therefore, to be able to predict healing or the absence of scarring at early times would be very interesting. This would not "waste time" for the patient. We report here that early nuclear imaging of angiogenesis, using 64Cu-NODAGA-RGD as a tracer, associated with nuclear imaging of mineralization, using 18F-Na as a tracer, is correlated to late bone healing objectivized by classical histology and microtomography. This nuclear imaging represents a promising approach for early prediction of bone grafting outcome in clinical practice, thus potentially allowing to anticipate alternative regenerative strategies.

Phosphorylated and Non-phosphorylated Leucine Rich Amelogenin Peptide Differentially Affect Ameloblast Mineralization.

The Leucine Rich Amelogenin Peptide (LRAP) is a product of alternative splicing of the amelogenin gene. As full length amelogenin, LRAP has been shown, in precipitation experiments, to regulate hydroxyapatite (HAP) crystal formation depending on its phosphorylation status. However, very few studies have questioned the impact of its phosphorylation status on enamel mineralization in biological models. Therefore, we have analyzed the effect of phosphorylated (+P) or non-phosphorylated (-P) LRAP on enamel formation in ameloblast-like cell lines and ex vivo cultures of murine postnatal day 1 molar germs. To this end, the mineral formed was analyzed by micro-computed tomography, Field Emission Scanning Electron Microscopy, Transmission Electron Microscopy, Selected Area Electon Diffraction imaging. Amelogenin gene transcription was evaluated by qPCR analysis. Our data show that, in both cells and germ cultures, LRAP is able to induce an up-regulation of amelogenin transcription independently of its phosphorylation status. Mineral formation is promoted by LRAP(+P) in all models, while LRAP(-P) essentially affects HAP crystal formation through an increase in crystal length and organization in ameloblast-like cells. Altogether, these data suggest a differential effect of LRAP depending on its phosphorylation status and on the ameloblast stage at the time of treatment. Therefore, LRAP isoforms can be envisioned as potential candidates for treatment of enamel lesions or defects and their action should be further evaluated in pathological models.

Strategies Developed to Induce, Direct, and Potentiate Bone Healing.

Bone exhibits a great ability for endogenous self-healing. Nevertheless, impaired bone regeneration and healing is on the rise due to population aging, increasing incidence of bone trauma and the clinical need for the development of alternative options to autologous bone grafts. Current strategies, including several biomolecules, cellular therapies, biomaterials, and different permutations of these, are now developed to facilitate the vascularization and the engraftment of the constructs, to recreate ultimately a bone tissue with the same properties and characteristics of the native bone. In this review, we browse the existing strategies that are currently developed, using biomolecules, cells and biomaterials, to induce, direct and potentiate bone healing after injury and further discuss the biological processes associated with this repair.

GPR40 mediates potential positive effects of a saturated fatty acid enriched diet on bone.

SCOPE: The stimulation of the free fatty acid receptor G-protein coupled receptor (GPR) 40 by GW9508 prevents bone loss by inhibiting osteoclast activity, both in vitro and in vivo. Here, we questioned whether the stimulation of the GPR40 receptor by dietary fatty acids may lead to the same beneficial effect on bone. METHODS AND RESULTS: We investigated (i) the impact of a fatty acid enriched diet (high-fat diet [HFD]) on bone health in C57/BL6 female mice depending on (ii) the estrogen status (ovariectomy) and (iii) the genotype (GPR40+/+ or GPR40-/- ). Bone mineral density (BMD), body composition, weight, inflammation and bone remodeling parameters were monitored. HFD decreased BMD in HFD-SH-GPR40+/+ mice but OVX failed to further impact BMD in HFD-OVX-GPR40+/+ mice, while additional bone loss was observed in HFD-OVX-GPR40-/- animals. These data suggest that when stimulated by fatty acid enriched diets GPR40 contributes to counteract ovariectomy-induced bone alteration. The sparing effect is supported by the modulation of both the osteoprotegerin/receptor activator of nuclear factor kappa-B ligand (OPG/RANKL) ratio in blood stream and the expression level of inflammatory markers in adipose tissues. Bone preservation by GPR40 stimulation is dependent on the presence of long-chain saturated fatty acids. CONCLUSION: GPR40 contributes to counter ovariectomy-induced bone loss in a context of saturated fatty acid enrichment.

Accelerated craniofacial bone regeneration through dense collagen gel scaffolds seeded with dental pulp stem cells.

Therapies using mesenchymal stem cell (MSC) seeded scaffolds may be applicable to various fields of regenerative medicine, including craniomaxillofacial surgery. Plastic compression of collagen scaffolds seeded with MSC has been shown to enhance the osteogenic differentiation of MSC as it increases the collagen fibrillary density. The aim of the present study was to evaluate the osteogenic effects of dense collagen gel scaffolds seeded with mesenchymal dental pulp stem cells (DPSC) on bone regeneration in a rat critical-size calvarial defect model. Two symmetrical full-thickness defects were created (5 mm diameter) and filled with either a rat DPSC-containing dense collagen gel scaffold (n = 15), or an acellular scaffold (n = 15). Animals were imaged in vivo by microcomputer tomography (Micro-CT) once a week during 5 weeks, whereas some animals were sacrificed each week for histology and histomorphometry analysis. Bone mineral density and bone micro-architectural parameters were significantly increased when DPSC-seeded scaffolds were used. Histological and histomorphometrical data also revealed significant increases in fibrous connective and mineralized tissue volume when DPSC-seeded scaffolds were used, associated with expression of type I collagen, osteoblast-associated alkaline phosphatase and osteoclastic-related tartrate-resistant acid phosphatase. Results demonstrate the potential of DPSC-loaded-dense collagen gel scaffolds to benefit of bone healing process.

A Non-Destructive Method for Distinguishing Reindeer Antler (Rangifer tarandus) from Red Deer Antler (Cervus elaphus) Using X-Ray Micro-Tomography Coupled with SVM Classifiers.

Over the last decade, biomedical 3D-imaging tools have gained widespread use in the analysis of prehistoric bone artefacts. While initial attempts to characterise the major categories used in osseous industry (i.e. bone, antler, and dentine/ivory) have been successful, the taxonomic determination of prehistoric artefacts remains to be investigated. The distinction between reindeer and red deer antler can be challenging, particularly in cases of anthropic and/or taphonomic modifications. In addition to the range of destructive physicochemical identification methods available (mass spectrometry, isotopic ratio, and DNA analysis), X-ray micro-tomography (micro-CT) provides convincing non-destructive 3D images and analyses. This paper presents the experimental protocol (sample scans, image processing, and statistical analysis) we have developed in order to identify modern and archaeological antler collections (from Isturitz, France). This original method is based on bone microstructure analysis combined with advanced statistical support vector machine (SVM) classifiers. A combination of six microarchitecture biomarkers (bone volume fraction, trabecular number, trabecular separation, trabecular thickness, trabecular bone pattern factor, and structure model index) were screened using micro-CT in order to characterise internal alveolar structure. Overall, reindeer alveoli presented a tighter mesh than red deer alveoli, and statistical analysis allowed us to distinguish archaeological antler by species with an accuracy of 96%, regardless of anatomical location on the antler. In conclusion, micro-CT combined with SVM classifiers proves to be a promising additional non-destructive method for antler identification, suitable for archaeological artefacts whose degree of human modification and cultural heritage or scientific value has previously made it impossible (tools, ornaments, etc.).

Priming Dental Pulp Stem Cells With Fibroblast Growth Factor-2 Increases Angiogenesis of Implanted Tissue-Engineered Constructs Through Hepatocyte Growth Factor and Vascular Endothelial Growth Factor Secretion.

Tissue engineering strategies based on implanting cellularized biomaterials are promising therapeutic approaches for the reconstruction of large tissue defects. A major hurdle for the reliable establishment of such therapeutic approaches is the lack of rapid blood perfusion of the tissue construct to provide oxygen and nutrients. Numerous sources of mesenchymal stem cells (MSCs) displaying angiogenic potential have been characterized in the past years, including the adult dental pulp. Establishment of efficient strategies for improving angiogenesis in tissue constructs is nevertheless still an important challenge. Hypoxia was proposed as a priming treatment owing to its capacity to enhance the angiogenic potential of stem cells through vascular endothelial growth factor (VEGF) release. The present study aimed to characterize additional key factors regulating the angiogenic capacity of such MSCs, namely, dental pulp stem cells derived from deciduous teeth (SHED). We identified fibroblast growth factor-2 (FGF-2) as a potent inducer of the release of VEGF and hepatocyte growth factor (HGF) by SHED. We found that FGF-2 limited hypoxia-induced downregulation of HGF release. Using three-dimensional culture models of angiogenesis, we demonstrated that VEGF and HGF were both responsible for the high angiogenic potential of SHED through direct targeting of endothelial cells. In addition, FGF-2 treatment increased the fraction of Stro-1+/CD146+ progenitor cells. We then applied in vitro FGF-2 priming to SHED before encapsulation in hydrogels and in vivo subcutaneous implantation. Our results showed that FGF-2 priming is more efficient than hypoxia at increasing SHED-induced vascularization compared with nonprimed controls. Altogether, these data demonstrate that FGF-2 priming enhances the angiogenic potential of SHED through the secretion of both HGF and VEGF.