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.

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.

Development and characterization of Polycaprolactone/chitosan-based scaffolds for tissue engineering of various organs: A review.

Research in creating 3D structures mirroring the extracellular matrix (ECM) with accurate environmental cues holds paramount significance in biological applications.Biomaterials that replicate ECM properties-mechanical, physicochemical, and biological-emerge as pivotal tools in mimicking ECM behavior.Incorporating synthetic and natural biomaterials is widely used to produce scaffolds suitable for the intended organs.Polycaprolactone (PCL), a synthetic biomaterial, boasts commendable mechanical properties, albeit with relatively modest biological attributes due to its hydrophobic nature.Chitosan (CTS) exhibits strong biological traits but lacks mechanical resilience for complex tissue regeneration.Notably, both PCL and CTS have demonstrated their application in tissue engineering for diverse types of tissues.Their combination across varying PCL:CTS ratios has increased the likelihood of fabricating scaffolds to address defects in sturdy and pliable tissues.This comprehensive analysis aspires to accentuate their distinct attributes within tissue engineering across different organs.The central focus resides in the role of PCL:CTS-based scaffolds, elucidating their contribution to the evolution of advanced functional 3D frameworks tailored for tissue engineering across diverse organs.Moreover, this discourse delves into the considerations pertinent to each organ.

Bone mineral density, hip bone geometry, and calcaneus trabecular bone texture in obese and normal-weight children.

Our study aimed at comparing bone mineral density (BMD), geometric indices of hip bone strength, and indices of trabecular bone texture at the calcaneus in obese and normal-weight children. Fifty-three obese children (10.3 ± 1.4 yr) and 24 normal-weight children (10.4 ± 1.5 yr) participated in this study. Body composition, bone mineral content, and BMD at whole body (WB), lumbar spine (L2-L4), total forearm, and proximal femur (total hip [TH] and femoral neck [FN]) were measured by dual-energy X-ray absorptiometry (DXA). Bone geometry of the hip was evaluated by the hip structure analysis (HSA) program. DXA scans were analyzed at the FN at its narrowest region and the femoral shaft (FS) by the HSA program. Cross-sectional area (CSA) and section modulus (Z) were measured from hip BMD profiles. Texture analysis was performed on digitized radiographs of the calcaneus to assess trabecular bone microarchitecture, and the result was expressed as Hmean. WB BMD, L2-L4 BMD, TH BMD, and FN BMD were significantly higher in obese children compared with normal-weight peers (p < 0.05). FN Z and FS Z were not significantly different between the 2 groups, whereas Hmean parameter was significantly lower in obese children compared with normal-weight peers (p < 0.001). After adjustment for body weight, obese children displayed lower WB BMD, FN CSA, FN Z, FS CSA, and FS Z compared with normal-weight children. This study suggests that BMD of WB and geometric indices of hip bone strength are not adapted to the increased body weight in obese children.

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) [...].

Tissue Engineering Strategies Applied in Bone Regeneration and Bone Repair.

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

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.

Osteocyte apoptosis and lipid infiltration as mechanisms of alcohol-induced bone loss.

AIMS: We carried out an in vivo study to assess the relationship between increase in adiposity in the marrow and osteocyte apoptosis in the case of alcohol-induced bone loss. METHODS AND RESULTS: After alcohol treatment, the number of apoptotic osteocytes was increased and lipid droplets were accumulated within the osteocytes, the bone marrow and the cortical bone micro-vessels. At last, we found an inverse correlation between bone mineral density and osteocyte apoptosis and strong significant correlations between the osteocyte apoptotic number and lipid droplet accumulation in osteocyte and bone micro-vessels. CONCLUSION: These data show that alcohol-induced bone loss is associated with osteocyte apoptosis and lipid accumulation in the bone tissue. This lipid intoxication, or 'bone steatosis', is correlated with lipid accumulation in bone marrow and blood micro-vessels.

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.

Osteocalcin-insulin relationship in obese children: a role for the skeleton in energy metabolism.

OBJECTIVE: Osteocalcin is a bone-specific protein secreted by osteoblasts and often used as a bone formation biomarker. Rodent studies have reported a hormonal role of osteocalcin on glucose metabolism, increasing insulin secretion and sensitivity and increasing energy expenditure. However, it is unknown whether osteocalcin fulfils the same function in humans. METHODS: We investigated the relationship between serum osteocalcin and insulin concentrations in 27 prepubertal obese children (9-12 years old) randomly divided into two groups, one of which entered a physical training programme, and 16 nonobese control children. Whole body bone mineral density (WB-BMD), serum osteocalcin, circulating insulin and adiponectin were measured at baseline and after 6 months. RESULTS: Trained and untrained obese children had higher WB-BMD than controls at baseline. Trained children also displayed a significant insulin increase and a significant adiponectin decrease while osteocalcin was increased compared to untrained obese children. Significant linear correlations between WB-BMD and adiponectin, delta BMD (variation between baseline and after-training values) and delta adiponectin, insulin and osteocalcin, delta insulin and delta osteocalcin, delta insulin and delta under-carboxylated osteocalcin were found only in trained obese children with no significant relationship in control and untrained obese children. CONCLUSIONS: In trained obese children, correlations indicate that when BMD is increased, osteocalcin is increased and insulin lowered. This suggests that increased BMD is associated with increased energy metabolism and a decreased level of insulin. We thus report statistically significant relationships between the skeleton (osteocalcin) and energy metabolism (insulin), suggesting a regulatory hormonal loop including osteocalcin and insulin.

Airway delivery of mesenchymal stem cells prevents arrested alveolar growth in neonatal lung injury in rats.

RATIONALE: Bronchopulmonary dysplasia (BPD) and emphysema are characterized by arrested alveolar development or loss of alveoli; both are significant global health problems and currently lack effective therapy. Bone marrow-derived mesenchymal stem cells (BMSCs) prevent adult lung injury, but their therapeutic potential in neonatal lung disease is unknown. OBJECTIVES: We hypothesized that intratracheal delivery of BMSCs would prevent alveolar destruction in experimental BPD. METHODS: In vitro, BMSC differentiation and migration were assessed using co-culture assays and a modified Boyden chamber. In vivo, the therapeutic potential of BMSCs was assessed in a chronic hyperoxia-induced model of BPD in newborn rats. MEASUREMENTS AND MAIN RESULTS: In vitro, BMSCs developed immunophenotypic and ultrastructural characteristics of type II alveolar epithelial cells (AEC2) (surfactant protein C expression and lamellar bodies) when co-cultured with lung tissue, but not with culture medium alone or liver. Migration assays revealed preferential attraction of BMSCs toward oxygen-damaged lung versus normal lung. In vivo, chronic hyperoxia in newborn rats led to air space enlargement and loss of lung capillaries, and this was associated with a decrease in circulating and resident lung BMSCs. Intratracheal delivery of BMSCs on Postnatal Day 4 improved survival and exercise tolerance while attenuating alveolar and lung vascular injury and pulmonary hypertension. Engrafted BMSCs coexpressed the AEC2-specific marker surfactant protein C. However, engraftment was disproportionately low for cell replacement to account for the therapeutic benefit, suggesting a paracrine-mediated mechanism. In vitro, BMSC-derived conditioned medium prevented O(2)-induced AEC2 apoptosis, accelerated AEC2 wound healing, and enhanced endothelial cord formation. CONCLUSIONS: BMSCs prevent arrested alveolar and vascular growth in part through paracrine activity. Stem cell-based therapies may offer new therapeutic avenues for lung diseases that currently lack efficient treatments.

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.

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.

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.

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.

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.

Cardiovascular toxicity of minoxidil in the marmoset.

The aim of the experiments was to assess the toxicity of minoxidil, a potent vasodilator, in marmosets. The animals were treated either at escalating doses from 2 to 40 mg/kg, escalating doses from 40 to 200 mg/kg or single doses of 150 mg/kg or 200 mg/kg. ECG recording and echocardiographic examination were conducted before and 1h after treatment. Necropsy and histopathology were performed 24h after the last dose. The treatment with minoxidil induced myocardial necrosis, coronary arteriopathy and degeneration of renal tubules in animals treated with 150 mg/kg or 200 mg/kg. Myocardial necrosis associated with fibrosis in some animals was located mainly in the left and right ventricles (including papillary muscles), but also in the right atrium, left atrium and/or interventricular septum. Arteriopathy was observed in small coronary arteries of the right or left atrium. ECG and echocardiographic examinations showed that in animals treated with 150 mg/kg or 200 mg/kg, there were positive chronotropic and inotropic effects that compensated for the hypotensive effect of the drug and were considered to have played a key role in the pathogenesis of the cardiovascular lesions. The cardiotoxicity of minoxidil in marmosets was similar to that described in dogs, but occurred at much higher doses. In conclusion minoxidil produced cardiovascular toxicity in the marmoset, which was probably due to the marked changes in the cardiac function associated with exaggerated pharmacological effects of the compound. The marmosets were found to be less sensitive than dogs to the cardiotoxicity of minoxidil.

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.

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.