Jean-Christophe Fricain

Physicochemical modulation of chitosan‐based hydrogels induces different biological responses: Interest for tissue engineering

Polysaccharide-based hydrogels are remarkable materials for the development of tissue engineering strategies as they meet several critical requirements for such applications and they may partly mimic the extracellular matrix. Chitosan is widely envisioned as hydrogel in biomedical fields for its bioresorbability, biocompatibility, and fungistatic and bacteriostatic properties. In this study, we report that the modulation of the polymer concentration, the degree of acetylation, the gelation processes [or neutralization routes (NR)] in the preparation of different chitosan-based hydrogels lead to substantially and significantly different biological responses. We show that it is possible to tune the physicochemical characteristics, mechanical properties, and biological responses of such matrices. Physical hydrogels prepared from highly acetylated chitosan were softer, degraded quickly in vivo, and were not suitable for in vitro culture of human mesenchymal stem and progenitor derived endothelial cells. In contrast, for a same chitosan concentration and obtained by the same processing route, a low degree of acetylation chitosan hydrogel provided a more elastic material, better cell adhesion on its surface and tissue regeneration, and restored tissue neo-vascularization as well. This work offers promising and innovative perspectives for the design of hydrogel materials with tunable properties for tissue engineering and regenerative medicine.

In situ printing of mesenchymal stromal cells, by laser-assisted bioprinting, for in vivo bone regeneration applications

Bioprinting has emerged as a novel technological approach with the potential to address unsolved questions in the field of tissue engineering. We have recently shown that Laser Assisted Bioprinting (LAB), due to its unprecedented cell printing resolution and precision, is an attractive tool for the in situ printing of a bone substitute. Here, we show that LAB can be used for the in situ printing of mesenchymal stromal cells, associated with collagen and nano-hydroxyapatite, in order to favor bone regeneration, in a calvaria defect model in mice. Also, by testing different cell printing geometries, we show that different cellular arrangements impact on bone tissue regeneration. This work opens new avenues on the development of novel strategies, using in situ bioprinting, for the building of tissues, from the ground up.

Claudin-16 Deficiency Impairs Tight Junction Function in Ameloblasts, Leading to Abnormal Enamel Formation.

Claudin-16 protein (CLDN16) is a component of tight junctions (TJ) with a restrictive distribution so far demonstrated mainly in the kidney. Here, we demonstrate the expression of CLDN16 also in the tooth germ and show that claudin-16 gene (CLDN16) mutations result in amelogenesis imperfecta (AI) in the 5 studied patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). To investigate the role of CLDN16 in tooth formation, we studied a murine model of FHHNC and showed that CLDN16 deficiency led to altered secretory ameloblast TJ structure, lowering of extracellular pH in the forming enamel matrix, and abnormal enamel matrix protein processing, resulting in an enamel phenotype closely resembling human AI. This study unravels an association of FHHNC owing to CLDN16 mutations with AI, which is directly related to the loss of function of CLDN16 during amelogenesis. Overall, this study indicates for the first time the importance of a TJ protein in tooth formation and underlines the need to establish a specific dental follow-up for these patients.

Pullulan/dextran/nHA Macroporous Composite Beads for Bone Repair in a Femoral Condyle Defect in Rats

The repair of bone defects is of particular interest for orthopedic, oral, maxillofacial, and dental surgery. Bone loss requiring reconstruction is conventionally addressed through bone grafting. Depending on the size and the location of the defect, this method has limits and risks. Biomaterials can offer an alternative and have features supporting bone repair. Here, we propose to evaluate the cellular penetration and bone formation of new macroporous beads based on pullulan/dextran that has been supplemented with nanocrystalline hydroxyapatite in a rat model. Cross-linked beads of 300–500 µm diameters were used in a lateral femoral condyle defect and analyzed by magnetic resonance imaging, micro-computed tomography, and histology in comparison to the empty defects 15, 30, and 70 days after implantation. Inflammation was absent for both conditions. For empty defects, cellularisation and mineralization started from the periphery of the defect. For the defects containing beads, cellular structures filling out the spaces between the scaffolds with increasing interconnectivity and trabecular-like organization were observed over time. The analysis of calcified sections showed increased mineralization over time for both conditions, but was more pronounced for the samples containing beads. Bone Mineral Density and Bone Mineral Content were both significantly higher at day 70 for the beads in comparison to empty defects as well as compared with earlier time points. Analysis of newly formed tissue around the beads showed an increase of osteoid tissue, measured as percentage of the defect surface. This study suggests that the use of beads for the repair of small size defects in bone may be expanded on to meet the clinical need for a ready-to-use fill-up material that can favor bone formation and mineralization, as well as promote vessel ingrowth into the defect site.

Amelogenesis imperfecta in familial hypomagnesaemia and hypercalciuria with nephrocalcinosis caused by CLDN19 gene mutations

Background Amelogenesis imperfecta (AI) is a group of genetic diseases characterised by tooth enamel defects. AI was recently described in patients with familial hypercalciuria and hypomagnesaemia with nephrocalcinosis (FHHNC) caused by CLDN16 mutations. In the kidney, claudin-16 interacts with claudin-19 to control the paracellular passage of calcium and magnesium. FHHNC can be linked to mutations in both genes. Claudin-16 was shown to be expressed during amelogenesis; however, no data are available on claudin-19. Moreover, the enamel phenotype of patients with CLDN19 mutations has never been described. In this study, we describe the clinical and genetic features of nine patients with FHHNC carrying CLDN19 mutations and the claudin-19 expression profile in rat ameloblasts. Methods Six FHHNC Brazilian patients were subjected to mutational analysis. Three additional French patients were recruited for orodental characterisation. The expression profile of claudin-19 was evaluated by RT-qPCR and immunofluorescence using enamel epithelium from rat incisors. Results All patients presented AI at different degrees of severity. Two new likely pathogenic variations in CLDN19 were found: p.Arg200Gln and p.Leu90Arg. RT-qPCR revealed low Cldn19 expression in ameloblasts. Confocal analysis indicated that claudin-19 was immunolocalised at the distal poles of secretory and maturing ameloblasts. Conclusions For the first time, it was demonstrated that AI is associated with FHHNC in patients carrying CLDN19 mutations. The data suggest claudin-19 as an additional determinant in enamel formation. Indeed, the coexistence of hypoplastic and hypomineralised AI in the patients was consistent with claudin-19 expression in both secretory and maturation stages. Additional indirect systemic effects cannot be excluded.

Statins and alveolar bone resorption: a narrative review of preclinical and clinical studies

CONTEXT Tooth extraction and periodontal disease are both common clinical situations associated with alveolar bone loss. OBJECTIVE To analyze the reported results of in vivo studies investigating the effectiveness of statins to reduce alveolar bone resorption. STUDY DESIGN Systematic electronic search of the MEDLINE-PubMed database. RESULTS A total of 21 studies met the inclusion criteria. This review showed that the use of statins reduced significantly alveolar bone resorption observed during periodontal disease and after tooth extraction. Oral administration was effective using high statin concentrations, although local administration using a biodegradable carrier was effective with lower concentrations. It was recently reported that statins were effective to reduce alveolar bone loss as an adjunct to scaling and root planing (SRP) in several clinical trials. Further studies are needed to confirm these promising results.

Impression 3D en médecine régénératrice et ingénierie tissulaire

Additive manufacturing covers a number of fashionable technologies that attract the interest of researchers in biomaterials and tissue engineering. Additive manufacturing applied to regenerative medicine covers two main areas: 3D printing and biofabrication. If 3D printing has penetrated the world of regenerative medicine, bioassembly and bioimprinting are still in their infancy. The objective of this paper is to make a non-exhaustive review of these different complementary aspects of additive manufacturing in restorative and regenerative medicine or for tissue engineering.

In-vitro and in-vivo design and validation of an injectable polysaccharide-hydroxyapatite composite material for sinus floor augmentation

OBJECTIVE Polysaccharide-based composite matrices consisting of natural polysaccharides, pullulan and dextran supplemented with hydroxyapatite (Matrix-HA) have recently been developed. The principal objective of this study was to evaluate the capacities of this composite material to promote new bone formation in a sinus lift model in the sheep. Secondary objectives were to evaluate in vitro properties of the material regarding cell adhesion and proliferation. METHODS In this report, once such composite matrix was prepared as injectable beads after dispersion in a physiological buffer, and evaluated using a large animal model (sheep) for a sinus lift procedure. RESULTS In vitro studies revealed that these microbeads (250-550μm in diameter) allow vascular cell adhesion and proliferation of Endothelial Cells (EC) after 1 and 7 days of culture. In vivo studies were performed in 12 adult sheep, and newly formed tissue was analyzed by Cone Beam Computed Tomography (CBCT scanning electron microscopy (SEM) and by histology 3 and 6 months post-implantation. CBCT analyses at the implantation time revealed the radiolucent properties of these matrices. Quantitative analysis showed an increase of a dense mineralized tissue in the Matrix-HA group up to 3 months of implantation. The mineralized volume over total volume after 6 months reached comparable values to those obtained for Bio-Oss® used as positive control. Histological examination confirmed that the Matrix-HA did not induce any long term inflammatory events, and promoted direct contact between the osteoid tissue and lamellar bone structures and beads. After 6 months, we observed a dense network of osteocytes surrounding both biomaterials as well as a newly vascularized formed tissue in close contact to the biomaterials. SIGNIFICANCE In conclusion, the absence of animal components in Matrix-HA, the osteoconductive property of Matrix-HA in sheep, resulting in a dense bone and vascularized tissue, and the initial radiolucent property to follow graft integration offer great promises of this composite material for clinical use.

Similarities between induced membrane and amniotic membrane: Novelty for bone repair

Previous clinical studies have shown the efficacy of a two-stage surgical procedure - the induced membrane (IM) technique - for reconstruction of large bone defects or bone non-union. The first stage involves radical debridement and insertion of a cement spacer into the bone defect. The second stage, performed weeks to months later, consists of removing the spacer while leaving the foreign body membrane induced by the cement in place, and then filling the cavity with bone autograft. The IM has been shown to (1) act as a protective physical barrier by preventing bone autograft resorption and (2) act as a bioreactor by promoting healing through revascularisation and growth factor secretion, and by concentrating mesenchymal stem cells (MSC) with osteogenic properties. New solutions to reduce this surgical procedure to a single step are being explored, for example by using an IM-like bioactive and protective barrier inserted into the bone defect at the same time as bone graft.

A role for CD154, the CD40 ligand, in granulomatous inflammation

Granulomatous inflammation is a distinctive form of chronic inflammation in which predominant cells include macrophages, epithelioid cells, and multinucleated giant cells. Mechanisms regulating granulomatous inflammation remain ill-understood. CD154, the ligand of CD40, is a key mediator of inflammation. CD154 confers a proinflammatory phenotype to macrophages and controls several macrophagic functions. Here, we studied the contribution of CD154 in a mouse model of toxic liver injury with carbon tetrachloride and a model of absorbable suture graft. In both models, granulomas are triggered in response to endogenous persistent liver calcified necrotic lesions or by grafted sutures. CD154-deficient mice showed delayed clearance of carbon tetrachloride-induced liver calcified necrotic lesions and impaired progression of suture-induced granuloma. In vitro, CD154 stimulated phagocytosis of opsonized erythrocytes by macrophages, suggesting a potential mechanism for the altered granulomatous inflammation in CD154KO mice. These results suggest that CD154 may contribute to the natural history of granulomatous inflammation.