Spontaneous Akt2 deficiency in a colony of NOD mice exhibiting early diabetes.

Diabetes constitutes a major public health problem, with dramatic consequences for patients. Both genetic and environmental factors were shown to contribute to the different forms of the disease. The monogenic forms, found both in humans and in animal models, specially help to decipher the role of key genes in the physiopathology of the disease. Here, we describe the phenotype of early diabetes in a colony of NOD mice, with spontaneous invalidation of Akt2, that we called HYP. The HYP mice were characterised by a strong and chronic hyperglycaemia, beginning around the age of one month, especially in male mice. The phenotype was not the consequence of the acceleration of the autoimmune response, inherent to the NOD background. Interestingly, in HYP mice, we observed hyperinsulinemia before hyperglycaemia occurred. We did not find any difference in the pancreas' architecture of the NOD and HYP mice (islets' size and staining for insulin and glucagon) but we detected a lower insulin content in the pancreas of HYP mice compared to NOD mice. These results give new insights about the role played by Akt2 in glucose homeostasis and argue for the ß cell failure being the primary event in the course of diabetes.

PiT2 deficiency prevents increase of bone marrow adipose tissue during skeletal maturation but not in OVX-induced osteoporosis.

The common cellular origin between bone marrow adipocytes (BMAds) and osteoblasts contributes to the intimate link between bone marrow adipose tissue (BMAT) and skeletal health. An imbalance between the differentiation ability of BMSCs towards one of the two lineages occurs in conditions like aging or osteoporosis, where bone mass is decreased. Recently, we showed that the sodium-phosphate co-transporter PiT2/SLC20A2 is an important determinant for bone mineralization, strength and quality. Since bone mass is reduced in homozygous mutant mice, we investigated in this study whether the BMAT was also affected in PiT2-/- mice by assessing the effect of the absence of PiT2 on BMAT volume between 3 and 16 weeks, as well as in an ovariectomy-induced bone loss model. Here we show that the absence of PiT2 in juveniles leads to an increase in the BMAT that does not originate from an increased adipogenic differentiation of bone marrow stromal cells. We show that although PiT2-/- mice have higher BMAT volume than control PiT2+/+ mice at 3 weeks of age, BMAT volume do not increase from 3 to 16 weeks of age, leading to a lower BMAT volume in 16-week-old PiT2-/- compared to PiT2+/+ mice. In contrast, the absence of PiT2 does not prevent the increase in BMAT volume in a model of ovariectomy-induced bone loss. Our data identify SLC20a2/PiT2 as a novel gene essential for the maintenance of the BMAd pool in adult mice, involving mechanisms of action that remain to be elucidated, but which appear to be independent of the balance between osteoblastic and adipogenic differentiation of BMSCs.

Microgels based on Infernan, a glycosaminoglycan-mimetic bacterial exopolysaccharide, as BMP-2 delivery systems.

Bone Morphogenetic Protein (BMP-2) is an osteoinductive growth factor clinically used for bone regeneration. Tuneable sustained strategies for BMP-2 delivery are intensely developed to avoid severe complications related to supraphysiological doses applied. To address this issue, we investigated the ability of the bacterial exopolysaccharide (EPS) called Infernan produced by the deep-sea hydrothermal vent bacterium Alteromonas infernus, exhibiting both glycosaminoglycan-mimetic and physical gelling properties, to efficiently bind and release the bioactive BMP-2. Two delivery systems were designed based on BMP-2 retention in either single or complex EPS-based microgels, both manufactured using a microfluidic approach. BMP-2 release kinetics were highly influenced by the ionic strength, affecting both microgel stability and growth factor/EPS binding, appearing essential for BMP-2 bioactivity. The osteogenic activity of human bone-marrow derived mesenchymal stem cells studied in vitro emphasized that Infernan microgels constitute a promising platform for BMP-2 delivery for further in vivo bone repair.

IL-36γ is a pivotal inflammatory player in periodontitis-associated bone loss.

Periodontitis is a prevalent chronic inflammatory disease due to the host response (IL-1ß, IL-6, TNF-α and IL-17A) to oral bacteria such as Porphyromonas gingivalis. The newer members of the IL-1 family, IL-36s (IL-36α/IL-36ß/IL-36γ/IL-36Ra/IL-38) are known to be involved in host defense against P. gingivalis in oral epithelial cells (OECs) and are considered as key inflammatory mediators in chronic diseases. The aim of this study was to investigate the potential role of IL-36s in periodontitis. We showed here that IL-36γ mRNA gingival expression is higher in periodontitis patients, whereas IL-36ß and IL-36Ra mRNA expression are lower compared to healthy controls. Interestingly, the elevated IL-36γ expression in patients is positively correlated with the RANKL/OPG ratio, an index of bone resorption. In vitro, IL-36γ expression was induced through TLR2 activation in primary OECs infected with P. gingivalis but not in gingival fibroblasts, the most widespread cell type in gingival connective tissue. In OECs, recombinant IL-36γ enhanced the expression of inflammatory cytokines (IL-1ß, IL-6, TNF-α and IL-36γ), of TLR2 and importantly, the RANKL/OPG ratio. These findings suggest that IL-36γ could be a pivotal inflammatory player in periodontitis by perpetuating gingival inflammation and its associated alveolar bone resorption and could be a relevant therapeutic target.

Slc20a2, Encoding the Phosphate Transporter PiT2, Is an Important Genetic Determinant of Bone Quality and Strength.

Osteoporosis is characterized by low bone mineral density (BMD) and fragility fracture and affects over 200 million people worldwide. Bone quality describes the material properties that contribute to strength independently of BMD, and its quantitative analysis is a major priority in osteoporosis research. Tissue mineralization is a fundamental process requiring calcium and phosphate transporters. Here we identify impaired bone quality and strength in Slc20a2-/- mice lacking the phosphate transporter SLC20A2. Juveniles had abnormal endochondral and intramembranous ossification, decreased mineral accrual, and short stature. Adults exhibited only small reductions in bone mass and mineralization but a profound impairment of bone strength. Bone quality was severely impaired in Slc20a2-/- mice: yield load (-2.3 SD), maximum load (-1.7 SD), and stiffness (-2.7 SD) were all below values predicted from their bone mineral content as determined in a cohort of 320 wild-type controls. These studies identify Slc20a2 as a physiological regulator of tissue mineralization and highlight its critical role in the determination of bone quality and strength. © 2019 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

PiT1/Slc20a1 Is Required for Endoplasmic Reticulum Homeostasis, Chondrocyte Survival, and Skeletal Development.

During skeletal mineralization, the sodium-phosphate co-transporter PiT1Slc20a1 is assumed to meet the phosphate requirements of bone-forming cells, although evidence is missing. Here, we used a conditional gene deletion approach to determine the role of PiT1 in growth plate chondrocytes. We show that PiT1 ablation shortly after birth generates a rapid and massive cell death in the center of the growth plate, together with an uncompensated endoplasmic reticulum (ER) stress, characterized by morphological changes and increased Chop, Atf4, and Bip expression. PiT1 expression in chondrocytes was not found at the cell membrane but co-localized with the ER marker ERp46, and was upregulated by the unfolded protein response cascade. In addition, we identified the protein disulfide isomerase (Pdi) ER chaperone as a PiT1 binding partner and showed that PiT1 ablation impaired Pdi reductase activity. The ER stress induced by PiT1 deficiency in chondrocytes was associated with intracellular retention of aggrecan and vascular endothelial growth factor A (Vegf-A), which was rescued by overexpressing a phosphate transport-deficient mutant of PiT1. Our data thus reveal a novel, Pi-transport independent function of PiT1, as a critical modulator of ER homeostasis and chondrocyte survival during endochondral ossification. © 2018 American Society for Bone and Mineral Research.

Evaluation of a hydrogel membrane on bone regeneration in furcation periodontal defects in dogs.

The aim of the study was to evaluate bone regeneration using a canine model with surgically created periodontal defects filled for 12 weeks using a stratified biomaterial consisting in a biphasic calcium phosphate (BCP) covered with a crosslinking hydrogel acting as polymer membrane of silated hydroxypropyl methylcellulose (Si-HPMC) as the tested new concept. Bilateral, critical-sized, defects were surgically created at the mandibular premolar teeth of six adult beagle dogs. The defects were randomly allocated and: (i) left empty for spontaneous healing or filled with: (ii) BCP and a collagen membrane; (iii) BCP and hydrogel Si-HPMC membrane. At 12 weeks, the experimental conditions resulted in significantly enhanced bone regeneration in the test BCP/Si-HPMC group. Within the limits of this study, we suggest that the hydrogel Si-HPMC may act as an occlusive barrier to protect bone area from soft connective tissue invasion and then effectively contribute to enhance bone regeneration.

Phosphate-dependent FGF23 secretion is modulated by PiT2/Slc20a2.

OBJECTIVE: The canonical role of the bone-derived fibroblast growth factor 23 (Fgf23) is to regulate the serum inorganic phosphate (Pi) level. As part of a feedback loop, serum Pi levels control Fgf23 secretion through undefined mechanisms. We recently showed in vitro that the two high-affinity Na+-Pi co-transporters PiT1/Slc20a1 and PiT2/Slc20a2 were required for mediating Pi-dependent signaling. Here, we addressed the contribution of PiT1 and PiT2 to the regulation of Fgf23 secretion. METHODS: To this aim, we used PiT2 KO and DMP1Cre; PiT1lox/lox fed Pi-modified diets, as well as ex vivo isolated long bone shafts. Fgf23 secretion and expression of Pi homeostasis-related genes were assessed. RESULTS: In vivo, PiT2 KO mice responded inappropriately to low-Pi diets, displaying abnormally normal serum levels of intact Fgf23. Despite the high iFgf23 level, serum Pi levels remained unaffected, an effect that may relate to lower αKlotho expression in the kidney. Moreover, consistent with a role of PiT2 as a possible endocrine Pi sensor, the iFGF23/cFGF23 ratios were suppressed in PiT2 KO mice, irrespective of the Pi loads. While deletion of PiT1 in osteocytes using the DMP1-Cre mice was inefficient, adenovirus-mediated deletion of PiT1 in isolated long bone shafts suggested that PiT1 does not contribute to Pi-dependent regulation of Fgf23 secretion. In contrast, using isolated bone shafts from PiT2 KO mice, we showed that PiT2 was necessary for the appropriate Pi-dependent secretion of Fgf23, independently from possible endocrine regulatory loops. CONCLUSIONS: Our data provide initial mechanistic insights underlying the Pi-dependent regulation of Fgf23 secretion in identifying PiT2 as a potential player in this process, at least in high Pi conditions. Targeting PiT2, therefore, could improve excess FGF23 in hyperphosphatemic conditions such as chronic kidney disease.

Adhesion, proliferation and osteogenic differentiation of human MSCs cultured under perfusion with a marine oxygen carrier on an allogenic bone substitute.

Tissue engineering strategies have been developed to optimize osseointegration in dental implant surgery. One of the major problems is the non-homogeneous spatial cell distribution in the scaffold, as well as subsequent matrix production. Insufficient nutrient and oxygen supplies inside the scaffold are factors in this phenomenon. To mediate this gradient formation, we have implemented a perfusion culture method to seed human bone marrow mesenchymal stem cells (MSCs) into three-dimensional (3-D)-allogenic bone scaffolds in combination with a marine haemoglobin, HEMOXCell®, for oxygen delivery. Cell culture was performed under static and perfusion conditions, with standard and osteogenic media, with and without HEMOXCell®. The cell seeding efficiency, as well as MSC/scaffold cytocompatibly were assessed using viability and proliferation assays. Scaffolds' cellularization and extracellular matrix (ECM) formation were analyzed using scanning electron microscopy and histological staining. Cell differentiation was investigated with osteogenic biomarkers gene expression analysis. The perfusion culture was observed to significantly promote MSC proliferation and differentiation throughout the scaffolds, especially when using the induction medium w/HEMOXCell®. Our data suggest that perfusion culture of MSC into allogenic bone substitute with HEMOXCell® as a natural oxygen carrier is promising for tissue engineering applications to oxygenate hypoxic areas and to promote cellular proliferation.

Phosphate (Pi)-regulated heterodimerization of the high-affinity sodium-dependent Pi transporters PiT1/Slc20a1 and PiT2/Slc20a2 underlies extracellular Pi sensing independently of Pi uptake.

Extracellular phosphate (Pi) can act as a signaling molecule that directly alters gene expression and cellular physiology. The ability of cells or organisms to detect changes in extracellular Pi levels implies the existence of a Pi-sensing mechanism that signals to the body or individual cell. However, unlike in prokaryotes, yeasts, and plants, the molecular players involved in Pi sensing in mammals remain unknown. In this study, we investigated the involvement of the high-affinity, sodium-dependent Pi transporters PiT1 and PiT2 in mediating Pi signaling in skeletal cells. We found that deletion of PiT1 or PiT2 blunted the Pi-dependent ERK1/2-mediated phosphorylation and subsequent gene up-regulation of the mineralization inhibitors matrix Gla protein and osteopontin. This result suggested that both PiTs are necessary for Pi signaling. Moreover, the ERK1/2 phosphorylation could be rescued by overexpressing Pi transport-deficient PiT mutants. Using cross-linking and bioluminescence resonance energy transfer approaches, we found that PiT1 and PiT2 form high-abundance homodimers and Pi-regulated low-abundance heterodimers. Interestingly, in the absence of sodium-dependent Pi transport activity, the PiT1-PiT2 heterodimerization was still regulated by extracellular Pi levels. Of note, when two putative Pi-binding residues, Ser-128 (in PiT1) and Ser-113 (in PiT2), were substituted with alanine, the PiT1-PiT2 heterodimerization was no longer regulated by extracellular Pi These observations suggested that Pi binding rather than Pi uptake may be the key factor in mediating Pi signaling through the PiT proteins. Taken together, these results demonstrate that Pi-regulated PiT1-PiT2 heterodimerization mediates Pi sensing independently of Pi uptake.

Interleukin-33 and RANK-L Interplay in the Alveolar Bone Loss Associated to Periodontitis.

INTRODUCTION: Chronic Periodontitis (CP) is an inflammatory disease of bacterial origin that results in alveolar bone destruction. Porphyromonas gingivalis (Pg), one of the main periopathogens, initiates an inflammatory cascade by host immune cells thereby increasing recruitment and activity of osteoclasts, the bone resorbing cells, through enhanced production of the crucial osteoclastogenic factor, RANK-L. Antibodies directed against some cytokines (IL-1ß, IL-6 and TNF-α) failed to exhibit convincing therapeutic effect in CP. It has been suggested that IL-33, could be of interest in CP. OBJECTIVE: the present study aims to analyze whether and how IL-33 and RANK-L and/or their interplay are involved in the bone destruction associated to CP. MATERIAL AND METHODS: mRNAs and protein expressions of IL-33 and RANK-L were analyzed in healthy and CP human gingival samples by immunohistochemistry (IHC) and RT-qPCR. Murine experimental periodontitis (EP) was induced using Pg infected ligature and Pg free ligature around the first maxillary molar. Alveolar bone loss was recorded by µCT. Mouse gingival explants were stimulated for 24 hours with IL-33 and RANK-L mRNA expression investigated by RT-qPCR. Human oral epithelial cells were infected by Pg for 6, 12; 24 hours and IL-33 and RANK-L mRNA expressions were analyzed by RT-qPCR. RESULTS: IL-33 is overexpressed in gingival epithelial cells in human affected by CP as in the murine EP. In human as in murine gingival cells, RANK-L was independently induced by Pg and IL-33. We also showed that the Pg-dependent RANK-L expression in gingival epithelial cells occured earlier than that of IL-33. CONCLUSION: Our results evidence that IL-33 overexpression in gingival epithelial cells is associated with CP and may trigger RANK-L expression in addition to a direct effect of Pg. Finally, IL-33 may act as an extracellular alarmin (danger signal) showing proinflammatory properties in CP perpetuating bone resorption induced by Pg infection.

A simple and effective approach to prepare injectable macroporous calcium phosphate cement for bone repair: Syringe-foaming using a viscous hydrophilic polymeric solution.

In this study, we propose a simple and effective strategy to prepare injectable macroporous calcium phosphate cements (CPCs) by syringe-foaming via hydrophilic viscous polymeric solution, such as using silanized-hydroxypropyl methylcellulose (Si-HPMC) as a foaming agent. The Si-HPMC foamed CPCs demonstrate excellent handling properties such as injectability and cohesion. After hardening the foamed CPCs possess hierarchical macropores and their mechanical properties (Young's modulus and compressive strength) are comparable to those of cancellous bone. Moreover, a preliminary in vivo study in the distal femoral sites of rabbits was conducted to evaluate the biofunctionality of this injectable macroporous CPC. The evidence of newly formed bone in the central zone of implantation site indicates the feasibility and effectiveness of this foaming strategy that will have to be optimized by further extensive animal experiments. STATEMENT OF SIGNIFICANCE: A major challenge in the design of biomaterial-based injectable bone substitutes is the development of cohesive, macroporous and self-setting calcium phosphate cement (CPC) that enables rapid cell invasion with adequate initial mechanical properties without the use of complex processing and additives. Thus, we propose a simple and effective strategy to prepare injectable macroporous CPCs through syringe-foaming using a hydrophilic viscous polymeric solution (silanized-hydroxypropyl methylcellulose, Si-HPMC) as a foaming agent, that simultaneously meets all the aforementioned aims. Evidence from our in vivo studies shows the existence of newly formed bone within the implantation site, indicating the feasibility and effectiveness of this foaming strategy, which could be used in various CPC systems using other hydrophilic viscous polymeric solutions.

Development of mandibular osteoradionecrosis in rats: Importance of dental extraction.

OBJECTIVES: To develop an animal model of mandibular osteoradionecrosis (ORN) using a high-energy radiation source (as used in human therapeutics) and to assess the role of tooth extraction on ORN development. MATERIALS AND METHODS (STUDY DESIGN): Ten animals were irradiated with a single 35- or 50-Gy dose. Three weeks later, the second left mandibular molar was extracted from three animals in each group. Nine weeks after irradiation, the animals were euthanized, with an injection of contrast agent in the bloodstream to highlight vascularization. Mandibles were harvested and studied using micro-CT, histology, tartrate-resistant acid phosphatase activity and scanning electron microscopy. RESULTS: This study demonstrates that a single 50-Gy dose associated with molar extraction is necessary for ORN development. In these conditions, absence of healing of the mucosa and bone, dental effects, fibrosis, an increase in osteoclast activity and a decrease in vascularization were observed. We also determined that molar extraction increases the impact of the cellular effects of radiation. CONCLUSION: The mandibular ORN animal model was validated after 50-Gy irradiation and molar extraction. The results of this study therefore support an animal ORN model and tissue engineering strategies will now be developed to regenerate bone for patients with head and neck cancer.

Role of the stromal vascular fraction from adipose tissue in association with a phosphocalcic scaffold in bone regeneration in an irradiated area.

BACKGROUND: Osteoradionecrosis of the jaw is a major side-effect of radiotherapy used in the treatment of squamous cell carcinomas of the upper aerodigestive tract. The standard reconstruction procedure is a free flap transfer of autogenous bone. A new approach using a tissue engineering strategy has shown that total bone marrow (TBM) associated with biphasic calcium phosphate (BCP) is the best combination for bone regeneration in an irradiated area. Recently, the stromal vascular fraction from adipose tissue (SVF) was described as an alternative to TBM for promoting new bone formation. The aim of this study was to identify the capacity of a freshly isolated SVF to induce new bone formation in an irradiated area. METHODS: Four weeks after irradiation of the hind limbs of 15 rats, bone defects were created and filled with either SVF or TBM with and without BCP. RESULTS: Three weeks after the implantations, analysis showed that the BCP-TBM mixture improved new bone formation after radiation (p < 0.05). The BCP-SVF association induced significant neoangiogenesis but failed to enhance new bone formation. CONCLUSION: The BCP-SVF mixture was insufficient to enhance new bone formation in the irradiated area, suggesting that the role of the environment might be crucial for ossification.

Development of a cyclosporin-A-induced immune tolerant rat model to test marrow allograft cell type effects on bone repair.

Bone repair is an important concept in tissue engineering, and the ability to repair bone in hypotrophic conditions such as that of irradiated bone, represents a challenge for this field. Previous studies have shown that a combination of bone marrow and (BCP) was effective to repair irradiated bone. However, the origin and role played by each cell type in bone healing still remains unclear. In order to track the grafted cells, the development of an animal model that is immunotolerant to an allograft of bone marrow would be useful. Furthermore, because the immune system interacts with bone turnover, it is of critical importance to demonstrate that immunosuppressive drugs do not interfere with bone repair. After a preliminary study of immunotolerance, cyclosporin-A was chosen to be used in immunosuppressive therapy. Ten rats were included to observe qualitative and quantitative bone repair 8 days and 6 weeks after the creation of bone defects. The defects were filled with an allograft of bone marrow alone or in association with BCP under immunosuppressive treatment (cyclosporin-A). The results showed that there was no significant interaction of cyclosporin-A with osseous regeneration. The use of this new immunotolerant rat model of bone marrow allograft in future studies will provide insight on how the cells within the bone marrow graft contribute to bone healing, especially in irradiated conditions.

Notchless is required for axial skeleton formation in mice.

Maintenance of cell survival is essential for proper embryonic development. In the mouse, Notchless homolog 1 (Drosophila) (Nle1) is instrumental for survival of cells of the inner cell mass upon implantation. Here, we analyze the function of Nle1 after implantation using the Meox2(tm1(cre)Sor) mouse that expresses the Cre recombinase specifically in the epiblast at E5.5. First, we find that NLE1 function is required in epiblast cells, as Nle1-deficient cells are rapidly eliminated. In this report, we also show that the Meox2(Cre) transgene is active in specific tissues during organogenesis. In particular, we detect high Cre expression in the vertebral column, ribs, limbs and tailbud. We took advantage of this dynamic expression profile to analyze the effects of inducing mosaic deletion of Nle1 in the embryo. We show that Nle1 deletion in this context, results in severe developmental anomalies leading to lethality at birth. Mutant embryos display multiple developmental defects in particular during axial skeletal formation. We also provide evidence that axial defects are due to an increase in apoptotic cell death in the somite at E9.5. These data demonstrate an essential role for Nle1 during organogenesis and in particular during axial development.

Giant canine with dentine anomalies in oculo-facio-cardio-dental syndrome.

Radiculomegaly affecting incisors, canines or premolars is a rare radiological finding (Maden et al., 2010) but is pathognomomic of a rare x-linked dominant syndrome called oculo-facio-cardio-dental syndrome (OFCDS). As this syndrome includes cardiac malformations and can lead to blindness due to congenital glaucoma, oral and maxillofacial surgeons should be aware of the somatic anomalies potentially associated with radiculomegaly. We report a typical case of OFCDS and provide the first description of the microscopic dental anomalies associated with this syndrome.

The in vitro and in vivo effects of a low-molecular-weight fucoidan on the osteogenic capacity of human adipose-derived stromal cells.

Human adipose-derived stromal cells (hASCs) may hold potential for bone tissue engineering. Osteogenic differentiation of these cells is crucial to bone formation. Low-molecular-weight fucoidan (LMWF) is a sulfated polysaccharide that potentiates several growth factors, including pro-angiogenic growth factors. To investigate whether hASC preconditioning with LMWF promoted bone repair, we compared the effects of LMWF and low-molecular-weight heparin on hASC phenotype and osteogenic differentiation. LMWF did not modify the stem-cell phenotype of hASCs but enhanced their osteogenic differentiation (formation of calcium deposits, increased activity and expression of alkaline phosphatase, and increased expression of osteopontin and runt-related transcription factor 2). However, when hASCs were exposed to LMWF before their adhesion to biphasic calcium phosphate particles and implantation in a bone-growth mouse model, no bone formation was apparent after 5 or 8 weeks, probably due to cell death. In conclusion, LMWF may hold promise for enhancing the osteogenic differentiation of hASCs before their implantation. However, concomitant vascularization would be required to enhance bone formation.

Determining a clinically relevant strategy for bone tissue engineering: an "all-in-one" study in nude mice.

PURPOSE: Autologous bone grafting (BG) remains the standard reconstruction strategy for large craniofacial defects. Calcium phosphate (CaP) biomaterials, such as biphasic calcium phosphate (BCP), do not yield consistent results when used alone and must then be combined with cells through bone tissue engineering (BTE). In this context, total bone marrow (TBM) and bone marrow-derived mesenchymal stem cells (MSC) are the primary sources of cellular material used with biomaterials. However, several other BTE strategies exist, including the use of growth factors, various scaffolds, and MSC isolated from different tissues. Thus, clinicians might be unsure as to which method offers patients the most benefit. For this reason, the aim of this study was to compare eight clinically relevant BTE methods in an "all-in-one" study. METHODS: We used a transgenic rat strain expressing green fluorescent protein (GFP), from which BG, TBM, and MSC were harvested. Progenitor cells were then mixed with CaP materials and implanted subcutaneously into nude mice. After eight weeks, bone formation was evaluated by histology and scanning electron microscopy, and GFP-expressing cells were tracked with photon fluorescence microscopy. RESULTS/CONCLUSIONS: Bone formation was observed in only four groups. These included CaP materials mixed with BG or TBM, in which abundant de novo bone was formed, and BCP mixed with committed cells grown in two- and three-dimensions, which yielded limited bone formation. Fluorescence microscopy revealed that only the TBM and BG groups were positive for GFP expressing-cells, suggesting that these donor cells were still present in the host and contributed to the formation of bone. Since the TBM-based procedure does not require bone harvest or cell culture techniques, but provides abundant de novo bone formation, we recommend consideration of this strategy for clinical applications.

Mice with hypomorphic expression of the sodium-phosphate cotransporter PiT1/Slc20a1 have an unexpected normal bone mineralization.

The formation of hydroxyapatite crystals and their insertion into collagen fibrils of the matrix are essential steps for bone mineralization. As phosphate is a main structural component of apatite crystals, its uptake by skeletal cells is critical and must be controlled by specialized membrane proteins. In mammals, in vitro studies have suggested that the high-affinity sodium-phosphate cotransporter PiT1 could play this role. In vivo, PiT1 expression was detected in hypertrophic chondrocytes of murine metatarsals, but its implication in bone physiology is not yet deciphered. As the complete deletion of PiT1 results in embryonic lethality at E12.5, we took advantage of a mouse model bearing two copies of PiT1 hypomorphic alleles to study the effect of a low expression of PiT1 on bone mineralization in vivo. In this report, we show that a 85% down-regulation of PiT1 in long bones resulted in a slight (6%) but significant reduction of femur length in young mice (15- and 30-day-old). However, despite a defect in alcian blue / alizarin red S and Von Kossa staining of hypomorphic 1-day-old mice, using X-rays micro-computed tomography, energy dispersive X-ray spectroscopy and histological staining techniques we could not detect differences between hypomorphic and wild-type mice of 15- to 300-days old. Interestingly, the expression of PiT2, the paralog of PiT1, was increased 2-fold in bone of PiT1 hypomorphic mice accounting for a normal phosphate uptake in mutant cells. Whether this may contribute to the absence of bone mineralization defects remains to be further deciphered.