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International audience; Layer-by-layer (LBL) BioAssembly method was developed to enhance the control of cell distribution within 3D scaffolds for tissue engineering applications. The objective of this study was to evaluate in vivo the development of blood vessels within LBL bioassembled membranes seeded with human primary cells, and to compare it to cellularized massive scaffolds. Poly(lactic) acid (PLA) membranes fabricated by fused deposition modeling were seeded with monocultures of human bone marrow stromal cells or with cocultures of these cells and endothelial progenitor cells. Then, four cellularized membranes were assembled in LBL constructs. Early osteoblastic and endothelial cell differentiation markers, alkaline phosphatase, and von Willebrand's factor, were expressed in all layers of assemblies in homogenous manner. The same kind of LBL assemblies as well as cellularized massive scaffolds was implanted subcutaneously in mice. Human cells were observed in all scaffolds seeded with cells, but not in the inner parts of massive scaffolds. There were significantly more blood vessels observed in LBL bioassemblies seeded with cocultures compared to all other samples. LBL bioassembly of PLA membranes seeded with a coculture of human cells is an efficient method to obtain homogenous cell distribution and blood vessel formation within the entire volume of a 3D composite scaffold.

Thanks to its biological properties, the human amniotic membrane (HAM) combined with a bone substitute could be a single-step surgical alternative to the two-step Masquelet induced membrane (IM) technique for regeneration of critical bone defects. However, no study has directly compared these two membranes. We first designed a 3D-printed scaffold using calcium phosphate cement (CPC). We assessed its suitability in vitro to support human bone marrow mesenchymal stromal cells (hBMSCs) attachment and osteodifferentiation. We then performed a rat femoral critical size defect to compare the two-step IM technique with a single-step approach using the HAM. Five conditions were compared. Group 1 was left empty. Group 2 received the CPC scaffold loaded with rh-BMP2 (CPC/BMP2). Group 3 and 4 received the CPC/BMP2 scaffold covered with lyophilized or decellularized/lyophilized HAM. Group 5 underwent a two- step induced membrane procedure with insertion of a polymethylmethacrylate (PMMA) spacer followed by, after 4 weeks, its replacement with the CPC/BMP2 scaffold wrapped in the IM. Micro-CT and histomorphometric analysis were performed after six weeks. Results showed that the CPC scaffold supported the proliferation and osteodifferentiation of hBMSCs in vitro. In vivo, the CPC/BMP2 scaffold very efficiently induced bone formation and led to satisfactory healing of the femoral defect, in a single-step, without autograft or the need for any membrane covering. In this study, there was no difference between the two-step induced membrane procedure and a single step approach. However, the results indicated that none of the tested membranes further enhanced bone healing compared to the CPC/BMP2 group.

Chitosan hydrogel and adipose derived stem cells (ADCS) have been reported as the optimal partnership for colorectal tissue engineering. In that field, the aim of the current experiment was to assess the interest of seeding ADSC on chitosan hydrogel patches in an in vivo comparative study and on a tube intended replace a colonic segment in an in vivo feasibility study. In the comparative study, a 2 × 3 cm colonic wall defect was performed in 20 swine and repaired by suturing a chitosan hydrogel patch: acellular matrix (group A, n = 10) versus matrix seeded with autologous stromal vascular fraction (SVF) (group B, n = 10). In the feasibility study, a circular colonic section was performed and a 2-cm-length chitosan hydrogel tube (seeded with autologous SVF) was implanted between the two edges of the colon in 3 pigs. Graft areas were explanted at 8 weeks for examinations. Endpoints were technical feasibility, fibrosis ratio, and smooth muscle layer regeneration. A complete coverage of the patched area was observed with an ad integrum regeneration of the colonic wall including smooth muscle cells layer around a thin fibrosis scare. Fibrosis ratio was significantly lower group B: 13% versus 55% (p = 0.013). Segmental colonic replacement appeared accurate. Our data confirmed in a large animal model the healing effect of chitosan on colorectal tissue. The very low rate of the fibrosis ratio in the cellularized group emphasizes inflammatory control effect of the chitosan hydrogel and SVF association. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 460-467, 2018.

International audience; Hydroxyapatite coatings have been currently used on hip prostheses for their ability to promote faster osseointe- gration and bone growth. Nevertheless, post-operative infections remain a recurring problem. To overcome this issue, doping with antibacterial elements has become a new trend. In this work, hydroxyapatite coatings elab- orated by radio-frequency suspension plasma spraying (rf-SPS) were doped with silver and strontium. Several doping strategies were explored thanks to the versatility offered by SPS compared with conventional spraying. First way: calcium phosphate doped powders were synthesized by coprecipitation and then dispersed into water before plasma spraying; second way: undoped powder was dispersed into aqueous medium in which nitrates or nanoparticles of the dopant(s) were respectively dissolved/dispersed. XRD revealed a high level of crystallinity ratio (ISO 13 779) and hydroxyapatite proportion for most of the coatings, with the presence of Ag/Ag 2 O nanopar- ticles whatever the doping route. SEM-EDS and STEM have demonstrated a more homogeneous distribution of the strontium within the coating made from the doped powder. Adherence of the coatings was estimated via a 3-point bending test, while bacteriological tests with S. aureus and proliferation of mesenchymal stem cells (hMSC) were performed. The results indicated a preferential incorporation of strontium into the secondary phases, showed efficient bactericidal properties, excellent mechanical properties in comparison with an APS reference coating, and no evidence of cytotoxic effect. This opens the way of a new type of coatings with a finer structure and a higher homogeneity through a better control of physicochemical properties using a suspension as the precursor.

International audience; Human amniotic membrane (hAM) is considered as an attractive biological scaffold for tissue engineering. For this application, hAM has been mainly processed using cryopreservation, lyophilization and/or decellularization. However, no study has formally compared the influence of these treatments on hAM properties. The aim of this study was to develop a new decellularization-preservation process of hAM, and to compare it with other conventional treatments (fresh, cryopreserved and lyophilized). The hAM was decellularized (D-hAM) using an enzymatic method followed by a detergent decellularization method, and was then lyophilized and gamma-sterilized. Decellularization was assessed using DNA staining and quantification. D-hAM was compared to fresh (F-hAM), cryopreserved (C-hAM) and lyophilized/gamma-sterilized (L-hAM) hAM. Their cytotoxicity on human bone marrow mesenchymal stem cells (hBMSCs) and their biocompatibility in a rat subcutaneous model were also evaluated. The protocol was effective as judged by the absence of nuclei staining and the residual DNA lower than 50 ng/mg. Histological staining showed a disruption of the D-hAM architecture, and its thickness was 84% lower than fresh hAM (p < 0.001). Despite this, the labeling of type IV and type V collagen, elastin and laminin were preserved on D-hAM. Maximal force before rupture of D-hAM was 92% higher than C-hAM and L-hAM (p < 0.01), and D-hAM was 37% more stretchable than F-hAM (p < 0.05). None of the four hAM were cytotoxic, and D-hAM was the most suitable scaffold for hBMSCs proliferation. Finally, D-hAM was well integrated in vivo. In conclusion, this new hAM decellularization process appears promising for tissue engineering applications.

The development of non-cellularized injectable suspensions of viscous chitosan (CHI) solutions (1.7&ndash, 3.3% (w/w)), filled with cellulose nanofibers (CNF) (0.02&ndash, 0.6% (w/w)) of the type nanofibrillated cellulose, was proposed for viscosupplementation of the intervertebral disc nucleus pulposus tissue. The achievement of CNF/CHI formulations which can gel in situ at the disc injection site constitutes a minimally-invasive approach to restore damaged/degenerated discs. We studied physico-chemical aspects of the sol and gel states of the CNF/CHI formulations, including the rheological behavior in relation to injectability (sol state) and fiber mechanical reinforcement (gel state). CNF-CHI interactions could be evidenced by a double flow behavior due to the relaxation of the CHI polymer chains and those interacting with the CNFs. At high shear rates resembling the injection conditions with needles commonly used in surgical treatments, both the reference CHI viscous solutions and those filled with CNFs exhibited similar rheological behavior. The neutralization of the flowing and weakly acidic CNF/CHI suspensions yielded composite hydrogels in which the nanofibers reinforced the CHI matrix. We performed evaluations in relation to the biomedical application, such as the effect of the intradiscal injection of the CNF/CHI formulation in pig and rabbit spine models on disc biomechanics. We showed that the injectable formulations became hydrogels in situ after intradiscal gelation, due to CHI neutralization occurring in contact with the body fluids. No leakage of the injectate through the injection canal was observed and the gelled formulation restored the disc height and loss of mechanical properties, which is commonly related to disc degeneration.

Tissue engineering is a promising alternative to autografts, allografts, or biomaterials to address the treatment of severe and large bone lesions. Classically, tissue engineering products associate a scaffold and cells and are implanted or injected into the lesion. These cells must be embedded in an appropriate biocompatible scaffold, which offers a favourable environment for their survival and differentiation. Here, we designed a composite hydrogel composed of collagen I, an extracellular matrix protein widely used in several therapeutic applications, which we associated with a physical hydrogel generated from a synthetic small amphiphilic molecule. This composite showed improved mechanical and biological characteristics as compared with gels obtained from each separate compound. Incorporation of the physical hydrogel prevented shrinkage of collagen and cell diffusion out of the gel and yielded a gel with a higher elastic modulus than those of gels obtained with each component alone. The composite hydrogel allowed cell adhesion and proliferation in vitro and long-term cell survival in vivo. Moreover, it promoted the differentiation of human adipose-derived stem cells in the absence of any osteogenic factors. In vivo, cells embedded in the composite gel and injected subcutaneously in immunodeficient mice produced lamellar osteoid tissue and differentiated into osteoblasts. This study points this new composite hydrogel as a promising scaffold for bone tissue engineering applications.

Current approaches in bone tissue engineering have shown limited success, mostly owing to insufficient vascularization of the construct. A common approach consists of co-culture of endothelial cells and osteoblastic cells. This strategy uses cells from different sources and differentiation states, thus increasing the complexity upstream of a clinical application. The source of reparative cells is paramount for the success of bone tissue engineering applications. In this context, stem cells obtained from human bone marrow hold much promise. Here, we analyzed the potential of human whole bone marrow cells directly expanded in a three-dimensional (3D) polymer matrix and focused on the further characterization of this heterogeneous population and on their ability to promote angiogenesis and osteogenesis, both in vitro and in vivo, in a subcutaneous model. Cellular aggregates were formed within 24 h and over the 12-day culture period expressed endothelial and bone-specific markers and a specific junctional protein. Ectopic implantation of the tissue-engineered constructs revealed osteoid tissue and vessel formation both at the periphery and within the implant. This work sheds light on the potential clinical use of human whole bone marrow for bone regeneration strategies, focusing on a simplified approach to develop a direct 3D culture without two-dimensional isolation or expansion.

International audience; Because cell interactions play a fundamental role for cell differentiation, we investigated the expression of Pannexin 1 and Pannexin 3 in human bone marrow mesenchymal stromal cells (HBMSCs) in a three-dimensional (3D) microenvironment provided by a polysaccharide-based macroporous scaffold. The pannexin (Panx) family consists of three members, Panx1, Panx2, and Panx3. The roles of Panx large-pore ion and metabolite channels are recognized in many physiological and pathophysiological scenarios, but the role of these proteins in human physiological processes is still under investigation. Our study demonstrates that HBMSCs cultured within 3D scaffolds have induced Panx1 and Panx3 expression, compared with two-dimensional culture and that the Panx3 gene expression profile correlates with those of bone markers on mesenchymal stromal cells culture into the 3D scaffold. We showed that Panx1 is involved in the HBMSCs 3D cell-cell organization, as acting on the size of cellular aggregates, demonstrated by the use of Probenecid and the mimetic peptide 10panx1 as specific inhibitors. Inhibition of Panx3 using siRNA strategy shows to reduce the expression of osteocalcin as osteoblast-specific marker by HBMSCs cultured in 3D conditions, suggesting a role of this Panx in osteogenesis. Moreover, we evaluated Panx1 and Panx3 expression within the cellularized scaffolds upon subcutaneous implantation in NOG (NOD/Shi-scid/IL-2Rγnull ) mice, where we could observe a more intense expression in the constructs than in the surrounding tissues in vivo. This study provides new insights on the expression of pannexins in HBMSCs on a 3D microenvironment during the osteogenic differentiation, in vitro and in vivo.

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.

The conventional tissue engineering is based on seeding of macroporous scaffold on its surface (“top–down” approach). The main limitation is poor cell viability in the middle of the scaffold due to poor diffusion of oxygen and nutrients and insufficient vascularization. Layer-by-Layer (LBL) bioassembly is based on “bottom–up” approach, which considers assembly of small cellularized blocks. The aim of this work was to evaluate proliferation and differentiation of human bone marrow stromal cells (HBMSCs) and endothelial progenitor cells (EPCs) in two and three dimensions (2D, 3D) using a LBL assembly of polylactic acid (PLA) scaffolds fabricated by 3D printing. 2D experiments have shown maintain of cell viability on PLA, especially when a co-cuture system was used, as well as adequate morphology of seeded cells. Early osteoblastic and endothelial differentiations were observed and cell proliferation was increased after 7 days of culture. In 3D, cell migration was observed between layers of LBL constructs, as well as an osteoblastic differentiation. These results indicate that LBL assembly of PLA layers could be suitable for BTE, in order to promote homogenous cell distribution inside the scaffold and gene expression specific to the cells implanted in the case of co-culture system.

Insufficient angiogenesis remains a major hurdle in current bone tissue engineering strategies. An extensive body of work has focused on the use of angiogenic factors or endothelial progenitor cells. However, these approaches are inherently complex, in terms of regulatory and methodologic implementation, and present a high cost. We have recently demonstrate the potential of electrospun poly(lactic acid) (PLA) fiber-based membranes, containing calcium phosphate (CaP) ormoglass particles, to elicit angiogenesis in vivo , in a subcutaneous model in mice. Here we have devised an injectable composite, containing CaP glass-ceramic particles, dispersed within a (Hydroxypropyl)methyl cellulose (HPMC) matrix, with the capacity to release calcium in a more sustained fashion. We show that by tuning the release of calcium in vivo , in a rat bone defect model, we could improve both bone formation and increase angiogenesis. The bone regeneration kinetics was dependent on the Ca 2+ release rate, with the faster Ca 2+ release composite gel showing improved bone repair at 3 weeks, in relation to control. In the same line, improved angiogenesis could be observed for the same gel formulation at 6 weeks post implantation. This methodology allows to integrate two fundamental processes for bone tissue regeneration while using a simple, cost effective, and safe approach. Statement of Significance In current bone tissue engineering approaches the achievement of sufficient angiogenesis, during tissue regeneration, is a major limitation in order to attain full tissue functionality. Recently, we have shown that calcium ions, released by the degradation of calcium phosphate ormoglasses (CaP), are effective angiogenic promoters, in both in vitro and in a subcutaneous implantation model. Here, we devised an injectable composite, containing CaP glass-ceramic particles, dispersed within a HPMC matrix, enabling the release of calcium in a more sustained fashion. We show that by tuning the release of calcium in vivo , in a rat bone defect model, we could improve both bone formation and increase angiogenesis. This simple and cost effective approach holds great promise to translate to the clinics.

International audience; The conventional tissue engineering is based on seeding of macroporous scaffold on its surface ("top-down" approach). The main limitation is poor cell viability in the middle of the scaffold due to poor diffusion of oxygen and nutrients and insufficient vascularization. Layer-by-Layer (LBL) bioassembly is based on "bottom-up" approach, which considers assembly of small cellularized blocks. The aim of this work was to evaluate proliferation and differentiation of human bone marrow stromal cells (HBMSCs) and endothelial progenitor cells (EPCs) in two and three dimensions (2D, 3D) using a LBL assembly of polylactic acid (PLA) scaffolds fabricated by 3D printing. 2D experiments have shown maintain of cell viability on PLA, especially when a co-cuture system was used, as well as adequate morphology of seeded cells. Early osteoblastic and endothelial differentiations were observed and cell proliferation was increased after 7 days of culture. In 3D, cell migration was observed between layers of LBL constructs, as well as an osteoblastic differentiation. These results indicate that LBL assembly of PLA layers could be suitable for BTE, in order to promote homogenous cell distribution inside the scaffold and gene expression specific to the cells implanted in the case of co-culture system.

International audience; Aim: To develop a biological scaffold that could be moulded to reproduce the geometry of a gutta-percha point with precision and allow the differentiation of mesenchymal stem cells into osteoblasts to be used as a regenerative endodontic material.Methodology: A collagen/alginate composite scaffold was cast into a sodium alginate mould to produce a gutta-percha point-like cone. Prior to gelation, the cone was seeded with human stem cells from the apical papilla (SCAPs) to evaluate cell/scaffold interactions. The reconstructed tissue was characterized after 8 days in culture. Elastic modulus, tissue compaction and cell differentiation were assessed. Student t-tests and the Mann-Whitney U test were performed.Results: The fabrication method developed enabled the shape of a gutta-percha point to be mimicked with great accuracy and reproducibility (P = 0.31). Stem cells seeded into this composite scaffold were able to spread, survive and proliferate (P < 0.001). Moreover, they were able to differentiate into osteoblasts and produce calcified osseous extracellular matrix (P < 0.001). The construct showed no significant contraction after 8 days, preserving its shape and tip diameter (P = 0.58).Conclusions: The composite scaffold could present substantial benefits compared to synthetic materials. It could provide a favourable healing environment in the root canal conducive for regenerative endodontics and is therefore appropriate to be evaluated in vivo in further studies.

Chitosan hydrogel and adipose derived stem cells (ADCS) have been reported as the optimal partnership for colorectal tissue engineering. In that field, the aim of the current experiment was to assess the interest of seeding ADSC on chitosan hydrogel patches in an in vivo comparative study and on a tube intended replace a colonic segment in an in vivo feasibility study. In the comparative study, a 2 × 3 cm colonic wall defect was performed in 20 swine and repaired by suturing a chitosan hydrogel patch: acellular matrix (group A, n = 10) versus matrix seeded with autologous stromal vascular fraction (SVF) (group B, n = 10). In the feasibility study, a circular colonic section was performed and a 2-cm-length chitosan hydrogel tube (seeded with autologous SVF) was implanted between the two edges of the colon in 3 pigs. Graft areas were explanted at 8 weeks for examinations. Endpoints were technical feasibility, fibrosis ratio, and smooth muscle layer regeneration. A complete coverage of the patched area was observed with an ad integrum regeneration of the colonic wall including smooth muscle cells layer around a thin fibrosis scare. Fibrosis ratio was significantly lower group B: 13% versus 55% (p = 0.013). Segmental colonic replacement appeared accurate. Our data confirmed in a large animal model the healing effect of chitosan on colorectal tissue. The very low rate of the fibrosis ratio in the cellularized group emphasizes inflammatory control effect of the chitosan hydrogel and SVF association. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 460-467, 2018.

Fibre-shaped materials are useful for creating different functional three-dimensional (3D) structures that could mimic complex tissues. Several methods (e.g. extrusion, laminar flow or electrospinning) have been proposed for building hydrogel microfibres, with distinctive cell types and with different degrees of complexity. However, these methods require numerous protocol adaptations in order to achieve fibre fabricating and lack the ability to control microfibre alignment. Here, we present a simple method for the production of microfibers, based on a core shell approach, composed of calcium alginate and type I collagen. The process presented here allows the removal of the calcium alginate shell, after only 24 hours of culture, leading to stable and reproducible fibre shaped cellular constructs. With time of culture cells show to distribute preferentially to the surface of the fibre and display a uniform cellular orientation. Moreover, when cultured inside the fibres, murine bone marrow mesenchymal stem cells show the capacity to differentiate towards the osteoblastic lineage, under non-osteoinductive culture conditions. This work establishes a novel method for cellular fibre fabrication that due to its inherent simplicity can be easily upscaled and applied to other cell types.

Association of the bone-forming osteoblasts (OBs) and vascular endothelial cells (ECs) into a biomaterial composite provides a live bone graft substitute that can repair the bone defect when implanted. An intimate functional relationship exists between these cell types. This communication is crucial to the coordinated cell behaviour necessary for bone development and remodelling. Previous studies have shown that direct co-culture of primary human osteoprogenitors (HOPs) with primary human umbilical vein endothelial cells (HUVECs) stimulates HOPs differentiation and induces tubular-like networks. The present work aims to test the use of human bone marrow stromal cells (HBMSCs) co-cultured with human endothelial progenitor cells in order to assess whether progenitor-derived ECs (PDECs) could support osteoblastic differentiation as mature ECs do. Indeed, data generated from the literature by different laboratories considering these co-culture systems appear difficult to compare. Monocultures of HUVECs, HOPs, HBMSCs (in a non-orientated lineage), PDECs (from cord blood) were used as controls and four combinations of co-cultures were undertaken: HBMSCs-PDECs, HBMSCs-HUVECs, HOPs-PDECs, HOPs-HUVECs with ECs (mature or progenitor) for 6 h to 7 days. At the end of the chosen co-culture time, intracellular alkaline phosphatase (ALP) activity was detected in HOPs and HBMSCs and quantified in cell extracts. Quantitative real-time polymerase chain reaction (qPCR) of ALP was performed over time and vascular endothelial growth factor (VEGF) was measured. After 21 days, calcium deposition was observed, comparing mono- and co-cultures. We confirm that ECs induce osteoblastic differentiation of mesenchymal stem cells in vitro. Moreover, HUVECs can be replaced by PDECs, the latter being of great interest in tissue engineering.

International audience; Aims: The need for small caliber vessels to treat cardiovascular diseases has grown. However, synthetic polymers perform poorly in small-diameter applications. Chitosan hydrogels can provide a novel biological scaffold for vascular engineering. The goal of this study was to explore host cell and tissue behavior at the interface with chitosan-based scaffolds in vitro and in vivo.Methods and results: in vitro, we assessed the ability of endothelial cells lining chitosan hydrogels to produce tissue factor (TF), thrombomodulin (TM) and nitric oxide. We showed that endothelial cells behave as a native endothelium since under stimulation, TF and TM expression increased and decreased, respectively. Endothelial cells seeded on chitosan produced nitric oxide, but no change was observed under stimulation. After in vivo subcutaneous implantation of chitosan hydrogels in rats, macrophage activation phenotypes, playing a crucial role in biomaterial/tissue, were explored by immunohistochemistry. Our results suggested a balance between pro- and anti-inflammatory signals since we observed an inflammatory response in favor of macrophage M2 phenotype.Conclusion: in vitro exploration of endothelial cell response at the interface with chitosan hydrogel showed a functional endothelium and in vivo exploration of tissue response revealed a biointegration of chitosan hydrogels.

International audience; In current bone tissue engineering strategies the achievement of sufficient angiogenesis during tissue regeneration is still a major limitation in order to attain full functionality. Several strategies have been described to tackle this problem, mainly by the use of angiogenic factors or endothelial progenitor cells. However, when facing a clinical scenario these approaches are inherently complex and present a high cost. As such, more cost effective alternatives are awaited. Here, we demonstrate the potential of electrospun poly(lactic acid) (PLA) fiber-based membranes, containing calcium phosphate ormoglass (CaP) particles, to elicit angiogenesis in vivo, in a subcutaneous model in mice. We show that the current approach elicited the local expression of angiogenic factors, associated to a chemotactic effect on macrophages, and sustained angiogenesis into the biomaterial. As both PLA and CaP are currently accepted for clinical application these off-the-shelf novel membranes have great potential for guided bone regeneration applications.Statement of significance: In current bone tissue engineering approaches the achievement of sufficient angiogenesis, during tissue regeneration, is a major limitation in order to attain full tissue functionality. Recently, our group has found that calcium ions released by the degradation of calcium phosphate ormoglasses (CaP) are effective angiogenic promoters. Based on this, in this work we successfully produced hybrid fibrous mats with different contents of CaP nanoparticles and thus with different calcium ion release rates, using an ormoglass - poly(lactic acid) blend approach. We show that these matrices, upon implantation in a subcutaneous site, could elicit the local expression of angiogenic factors, associated to a chemotactic effect on macrophages, and sustained angiogenesis into the biomaterial, in a CaP dose dependent manner. This off-the-shelf cost effective approach presents great potential to translate to the clinics.

International audience; Objective. Tissue engineering may provide new operative tools for colorectal surgery in elective indications. The aim of this study was to define a suitable bioscaffold for colorectal tissue engineering.Methods. We compared 2 bioscaffolds with in vitro and in vivo experiments: porcine small intestinal submucosa (SIS) versus chitosan hydrogel matrix. We assessed nontoxicity of the scaffold in vitro by using human adipose-derived stem cells (hADSC). In vivo, a 1 X 2-cm colonic wall defect was created in 16 rabbits. Animals were divided randomly into 2 groups according to the graft used, SIS or chitosan hydrogel. Graft area was explanted at 4 and 8 weeks. The end points of in vivo experiments were technical feasibility, behavior of the scaffold, in situ putative inflammatory effect, and the quality of tissue regeneration, in particular smooth muscle layer regeneration.Results. In vitro, hADSC attachment and proliferation occurred on both scaffolds without a substantial difference. After proliferation, hADSCs kept their mesenchymal stem cell characteristics. In vivo, one animal died in each group. Eight weeks after implantation, the chitosan scaffold allowed better wound healing compared with the SIS scaffold, with more effective control of inflammatory activity and an integral regeneration of the colonic wall including the smooth muscle cell layer.Conclusion. The outcomes of in vitro experiments did not differ greatly between the 2 groups. Macroscopic and histologic findings, however, revealed better wound healing of the colonic wall in the chitosan group suggesting that the chitosan hydrogel could serve as a better scaffold for colorectal tissue engineering.

Purpose In order to track location and distribution of endothelial cells (ECs) within scaffolds in vitro, we chose lentiPGK-TdTomato transduction of human endothelial progenitor cells (EPCs) isolated and differentiated from cord blood. Because transduction could have a functional impact on cell behavior, we checked different parameters for qualification of labeled- EPCs as well as their use for potential applications in the context of vascular and bone tissue engineering. Methods After isolation and expansion, EPCs were classically characterized then transduced with the lentiviral vector containing the TdTomato protein gene under the control of the phosphoglycerate kinase (PGK) promoter. Conventional karyotyping, differentiation capacity, viability, proliferation assays were performed with labeled and unlabeled EPCs. Scaffolds and co-cultures were explored with labeled EPCs, in static or shear stress conditions. Results Our results show that cell labeling did not affect cell adhesion nor induce cell death. Cell labeling did not induce more chromosomal aberrations. Phenotypical characterization was not affected. In the context of tissue engineering applications, labeled EPCs maintained their ability to line 2D or 3D scaffolds, withstand physiological arterial shear stress, and form tubular networks in co-cultures with human osteoblast progenitor cells. Conclusions It is possible to label human EPCs with TdTomato without affecting their behavior by the transduction procedure. This creates an important tool for numerous applications. Our results provide a qualification of labeled EPCs in comparison with unlabeled ones for vascular and bone tissue engineering.

International audience; The introduction of silver, either in the liquid phase (as silver nitrate solution: Ag(L)) or in the solid phase (as silver phosphate salt: Ag(S)) of calcium carbonate–calcium phosphate (CaCO3–CaP) bone cement, its influence on the composition of the set cement (C-Ag(L)and C-Ag(S) cements with a Ca/Ag atomic ratio equal to 10.3) and its biological properties were investigated. The fine characterisation of the chemical setting of silver-doped and reference cements was performed using FTIR spectroscopy. We showed that the formation of apatite was enhanced from the first hours of maturation of C-Ag(L) cement in comparison with the reference cement, whereas a longer period of maturation (about 10 h) was required to observe this increase for C-Ag(S) cement, although in both cases, silver was present in the set cements mainly as silver phosphate. The role of silver nitrate on the setting chemical reaction is discussed and a chemical scheme is proposed. Antibacterial activity tests (S. aureus and S. epidermidis) and in vitro cytotoxicity tests (human bone marrow stromal cells (HBMSC)) showed that silver-loaded CaCO3–CaP cements had antibacterial properties (anti-adhesion and anti-biofilm formation) without a toxic effect on HBMSC cells, making C-Ag(S) cement a promising candidate for the prevention of bone implant-associated infections.

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.

Several studies have reported the benefits of mesenchymal stem cells (MSCs) for bone tissue engineering. However, vascularization remains one of the main obstacles that must be overcome to reconstruct large bone defects. In vitro prevascularization of the three-dimensional (3-D) constructs using co-cultures of human progenitor-derived endothelial cells (PDECs) with human bone marrow mesenchymal stem cells (HBMSCs) appeared as a potential strategy. However, the crosstalk between the two lineages has been studied in two-dimensional (2-D), but remains unknown in 3-D. The aim of this study is to investigate the cell interactions between PDECs and HBMSCs in a porous matrix composed of polysaccharides. This biodegradable scaffold promotes cell interactions by inducing multicellular aggregates composed of HBMSCs surrounded by PDECs. Cell aggregation contributes to the formation of junctional proteins composed of Connexin43 (Cx43) and VE-cadherin, and an activation of osteoblastic differentiation of HBMSCs stimulated by the presence of PDECs. Inhibition of Cx43 by mimetic peptide 43GAP27 induced a decrease in mRNA levels of Cx43 and all the bone-specific markers. Finally, subcutaneous implantations for 3 and 8 weeks in NOG mice revealed an increase in osteoid formation with the tissue-engineered constructs seeded with HBMSCs/PDECs compared with those loaded with HBMSCs alone. Taking together, these results demonstrate that this 3-D microenvironment favored cell communication, osteogenesis and bone formation.

Research in bone tissue engineering is focused on the development of alternatives to allogenic and autologous bone grafts that can stimulate bone healing. Here, we present scaffolds composed of the natural hydrophilic polysaccharides pullulan and dextran, supplemented or not with nanocrystalline hydroxyapatite particles (nHA). In vitro studies revealed that these matrices induced the formation of multicellular aggregates and expression of early and late bone specific markers with human bone marrow stromal cells in medium deprived of osteoinductive factors. In absence of any seeded cells, heterotopic implantation in mice and goat, revealed that only the composite macroporous scaffold (Matrix + nHA) (i) retained subcutaneously local growth factors, including Bone Morphogenetic Protein 2 (BMP2) and VEGF165, (ii) induced the deposition of a biological apatite layer, (iii) favored the formation of a dense mineralized tissue subcutaneously in mice, as well osteoid tissue after intramuscular implantation in goat. The composite scaffold was thereafter implanted in orthotopic preclinical models of critical size defects, in small and large animals, in three different bony sites, i.e. the femoral condyle of rat, a transversal mandibular defect and a tibial osteotomy in goat. The Matrix + nHA induced a highly mineralized tissue in the three models whatever the site of implantation, as well as osteoid tissue and bone tissue regeneration in direct contact to the matrix. We therefore propose this composite matrix as a material for stimulating bone cell differentiation of host mesenchymal stem cells and bone formation for orthopedic and maxillofacial surgical applications.

This study aims to evaluate in vitro the release properties and biological behavior of original compositions of strontium (Sr)-loaded bone mineral cements. Strontium was introduced into vaterite CaCO3-dicalcium phosphate dihydrate cement via two routes: as SrCO3 in the solid phase (SrS cements), and as SrCl2 dissolved in the liquid phase (SrL cements), leading to different cement composi- tions after setting. Complementary analytical techniques implemented to thoroughly investigate the release/dissolu- tion mechanism of Sr-loaded cements at pH 7.4 and 37 ! C during 3 weeks revealed a sustained release of Sr and a centripetal dissolution of the more soluble phase (vaterite) limited by a diffusion process. In all cases, the initial burst of the Ca and Sr release (highest for the SrL cements) that occurred over 48 h did not have a significant effect on the expression of bone markers (alkaline phosphatase, osteocal- cin), the levels of which remained overexpressed after 15 days of culture with human osteoprogenitor (HOP) cells. At the same time, proliferation of HOP cells was significantly higher on SrS cements. Interestingly, this study shows that we can optimize the sustained release of Sr 2" , the cement biodegradation and biological activity by controlling the route of introduction of strontium in the cement paste.

International audience; With the development of minimally invasive surgical techniques, there is a growing interest in the research and development of injectable biomaterials especially for orthopedic applications. In a view to enhance the overall surgery benefits for the patient, the BIOSINJECT project aims at preparing a new generation of mineral-organic composites for bone regeneration exhibiting bioactivity, therapeutic activity and easiness of use to broaden the application domains of the actual bone mineral cements and propose an alternative strategy with regard to their poor resorbability, injectability difficulties and risk of infection. First, a physical-chemical study demonstrated the feasibility of self-setting injectable composites associating calcium carbonate-calcium phosphate cement and polysaccharides (tailor-made or commercial polymer) in the presence or not of an antibacterial agent within the composite formulation. Then, bone cell response and antimicrobial activity of the composite have been evaluated in vitro. Finally, in order to evaluate resorption rate and bone tissue response an animal study has been performed and the histological analysis is still in progress. These multidisciplinary and complementary studies led to promising results in a view of the industrial development of such composite for dental and orthopaedic applications.