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The reconstruction of bones following tumor excision and radiotherapy remains a challenge. Our previous study, performed using polysaccharide-based microbeads that contain hydroxyapatite, found that these have osteoconductivity and osteoinductive properties. New formulations of composite microbeads containing HA particles doped with strontium (Sr) at 8 or 50% were developed to improve their biological performance and were evaluated in ectopic sites. In the current research, we characterized the materials by phase-contrast microscopy, laser dynamic scattering particle size-measurements and phosphorus content, before their implantation into two different preclinical bone defect models in rats: the femoral condyle and the segmental bone. Eight weeks after the implantation in the femoral condyle, the histology and immunohistochemistry analyses showed that Sr-doped matrices at both 8% and 50% stimulate bone formation and vascularization. A more complex preclinical model of the irradiation procedure was then developed in rats within a critical-size bone segmental defect. In the non-irradiated sites, no significant differences between the non-doped and Sr-doped microbeads were observed in the bone regeneration. Interestingly, the Sr-doped microbeads at the 8% level of substitution outperformed the vascularization process by increasing new vessel formation in the irradiated sites. These results showed that the inclusion of strontium in the matrix-stimulated vascularization in a critical-size model of bone tissue regeneration after irradiation.

Microbeads consisting of pullulan and dextran supplemented with hydroxyapatite have recently been developed for bone tissue engineering applications. Here, we evaluate the bone formation in two different preclinical models after injection of microbeads reconstituted with either saline buffer or autologous blood. Addition of saline solution or autologous blood to dried microbeads packaged into syringes allowed an easy injection. In the first rat bone defect model performed in the femoral condyle, microcomputed tomography performed after 30 and 60 days revealed an important mineralization process occurring around and within the core of the microbeads in both conditions. Bone volume/total volume measurements revealed no significant differences between the saline solution and the autologous blood groups. Histologically, osteoid tissue was evidenced around and in contact of the microbeads in both conditions. Using the sinus lift model performed in sheep, cone beam computed tomography revealed an important mineralization inside the sinus cavity for both groups after 3 months of implantation. Representative Masson trichrome staining images showed that bone formation occurs at the periphery and inside the microbeads in both conditions. Quantitative evaluation of the new bone formation displayed no significant differences between groups. In conclusion, reconstitution of microbeads with autologous blood did not enhance the regenerative capacity of these microbeads compared to the saline buffer group. This study is of particular interest for clinical applications in oral and maxillofacial surgery.

International audience; In this article, we first developed a new medium to culture together primary human osteoblastic, osteoclastic, and endothelial cells (ECs) chosen to represent the three major bone cell tissues. Indeed, no study has been conducted on primary human cells and on the phenotype/activity retention of these three primary human cell types. Thus, we established an original triculture model with osteoblastic, osteoclastic, and ECs, where not only both cell phenotype and cell activity were maintained but also cell culture homeostasis. These promising results will permit further investigations to create in vitro conditions to mimic the bone microenvironment and analyze cell interactions in ex vivo studies.

International audience; L'approche globale multi-échelle du vivant est mise en oeuvre par les industries de la santé pour lever les verrous qu'elles rencontrent actuellement dans la découverte et le développement de nouvelles thérapies, ainsi que pour la médecine régénérative dans sa démarche de reconstruction d'organes. Un des goulets est celui de la prédictibilité limitée qu'offrent les systèmes animaux modèles dans les différents domaines du développement du médicament, de la médecine de précision, de la pharmaco génomique et de la médecine régénérative. Cette faible prédictibilité est une des explications d'un taux d'attrition important de molécules en développement, entre autres. Les systèmes modèles actuels sont mis en question et l'utilisation potentielle de substituts tissulaires, ou organoïdes, en complément des modèles animaux couramment utilisés est désormais largement prise en compte dans l'évaluation des candidats

International audience; Despite significant progress in the field of biomaterials for bone repair, the lack of attention to the vascular and nervous networks within bone implants could be one of the main reasons for the delayed or impaired recovery of bone defects. The design of innovative biomaterials should improve the host capacity of healing to restore a functional tissue, taking into account that the nerve systems closely interact with blood vessels in the bone tissue. The aim of this work is to develop a cell-free and growth factor-free hydrogel capable to promote angiogenesis and innervation. To this end, we have used elastin-like polypeptides (ELPs), poly(ethylene glycol) (PEG) and increasing concentrations of the adhesion peptide IKVAV (25% (w/w) representing 1.7 mM and 50% (w/w) representing 4.1 mM) to formulate and produce hydrogels. When characterized in vitro, hydrogels have fine-tunable rheological properties, microporous structure and are biocompatible. At the biological level, 50% IKVAV composition up-regulated Runx2, Osx, Spp1, Vegfa and Bmp2 in mesenchymal stromal cells and Tek in endothelial cells, and sustained the formation of long neurites in sensory neurons. When implanted subcutaneously in mice, hydrogels induced no signals of major inflammation and the 50% IKVAV composition induced higher vessel density and formation of nervous terminations in the peripheral tissue. This novel composite has important features for tissue engineering, showing higher osteogenic, angiogenic and innervation potential in vitro, being not inflammatory in vivo, and inducing angiogenesis and innervation subcutaneously. STATEMENT OF SIGNIFICANCE: One of the main limitations in the field of tissue engineering remains the sufficient vascularization and innervation during tissue repair. In this scope, the development of advanced biomaterials that can support these processes is of crucial importance. Here, we formulated different compositions of Elastin-like polypeptide-based hydrogels bearing the IKVAV adhesion sequence. These compositions showed controlled mechanical properties, and were degradable in vitro. Additionally, we could identify in vitro a composition capable to promote neurite formation and to modulate endothelial and mesenchymal stromal cells gene expression, in view of angiogenesis and osteogenesis, respectively. When tested in vivo, it showed no signs of major inflammation and induced the formation of a highly vascularized and innervated neotissue. In this sense, our approach represents a potential advance in the development of new strategies to promote tissue regeneration, taking into account both angiogenesis and innervation.

International audience; Elastin-like polypeptides (ELPs) are biocompatible-engineered polypeptides, with promising interest in tissue engineering due to their intrinsic biological and physical properties, and their ease of production. The IKVAV (Ile-Lys-Val-Ala-Val) laminin-1 sequence has been shown to sustain neuron attachment and growth. In this study, the IKVAV adhesion sequence, or a scrambled VKAIV sequence, were incorporated by genetic engineering in the structure of an ELP, expressed in Escherichia coli and purified. The transition temperatures of the ELP-IKVAV and ELP-VKAIV were determined to be 23 °C. Although the phase transition was fully reversible for ELP-VKAIV, we observed an irreversible aggregation for ELP-IKVAV. The corresponding aggregates shared some characteristics with amyloid-like polypeptides. The two ELPs were then reacted with functionalized polyethylene glycol (PEG) to form hydrogels. These hydrogels were characterized for rheological properties, tested with cultures of rat primary sensory neurons, and implanted subcutaneously in mice for 4 weeks. Sensory neurons cultured on high IKVAV concentration hydrogels (20%) formed longer neurite than those of neurons grown on hydrogels containing the scrambled IKVAV sequence. Finally, in vivo evaluation showed the absence of detectable inflammation. In conclusion, this functionalized ELP-IKVAV biomaterial shows interesting properties for tissue engineering requiring neurotization.

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.

To discriminate the most important physicochemical parameters for bone reconstruction, the inflammatory potential of seven nanoporous hydroxyapatite powders synthesized by hard or soft templating was evaluated both in vitro and in vivo. Aim: To discriminate the most important physicochemical parameters for bone reconstruction, the inflammatory potential of seven nanoporous hydroxyapatite powders synthesized by hard or soft templating was evaluated both in vitro and in vivo. Materials & methods: After physical and chemical characterization of the powders, we studied the production of inflammatory mediators by human primary monocytes after 4 and 24 h in contact with powders, and the host response after 2 weeks implantation in a mouse critical size defect model. Results:In vitro results highlighted increases in the secretion of TNF-α, IL-1, -8, -10 and proMMP-2 and -9 and decreases in the secretion of IL-6 only for powders prepared by hard templating. In vivo observations confirmed an extensive inflammatory tissue reaction and a strong resorption for the most inflammatory powder in vitro. Conclusion: These findings highlight that the most critical physicochemical parameters for these nanoporous hydroxyapatite are, the crystallinity that controls dissolution potential, the specific surface area and the size and shape of crystallites. journal article research support, non-u.s. gov't 2015 imported

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; Introduction: Standard care for malignant tumors arising next to a bone structure is surgical removal with safety margins, followed by external beam radiotherapy (EBRT). Complete tumor removal can result in large bone defects. A two-step bone reconstruction technique using the induced membrane (IM) technique has proven its efficacy to bridge gap nonunion. During the first step, a spacer is placed in the bone gap. The spacer then is removed and the IM around it is filled with autologous cancellous bone graft. However, the feasibility of this technique with the addition of adjuvant EBRT between the two reconstruction steps has not yet been studied. Polymethyl methacrylate (PMMA) used to be the standard spacer material for the first step. Silicone spacers could replace them owing to their good behavior when submitted to EBRT and their easier removal from the surgical site during the second step. The aim of this study was to evaluate the influence of EBRT on the histological and biochemical properties of IM induced using PMMA or silicone as spacer.Materials and methods: The analyses were performed on PMMA- or silicone-IM with and without EBRT in a 6-mm bilateral femoral defect in 32 rats. Thickness and vessel content were measured in both groups. Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) content in lysates of the crushed membranes were measured by enzyme immunoassay. Finally, alkaline phosphatase activity was analyzed in human bone marrow stromal cell cultures in contact with the same lysates.Results: EBRT did not change the histological structure of the cellular internal layer or the fibrous outer layer. The nature of the spacer only influenced IM thickness, PMMA-IM with external radiotherapy being significantly thicker. EBRT decreased the vascular density of IM but was less effective on VEGF/BMP2 production. In vitro, IM could have an osteoinductive potential on human bone marrow stem cells.Conclusion: EBRT did not modify the histological properties of IMs but decreased their vascular density. VEGF and BMP2 production within IMs was not affected by EBRT. Silicone spacers are able to induce membranes with similar histological characteristics to PMMA-IM.

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.

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.

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.

Tissue engineering of large organs is currently limited by the lack of potent vascularizationin vitro. Tissue-engineered bone grafts can be prevascularizedin vitrousing endothelial cells (ECs). The microvascular network architecture could be controlled by printing ECs following a specific pattern. Using laser-assisted bioprinting, we investigated the effect of distance between printed cell islets and the influence of coprinted mesenchymal cells on migration. When printed alone, ECs spread out evenly on the collagen hydrogel, regardless of the distance between cell islets. However, when printed in coculture with mesenchymal cells by laser-assisted bioprinting, they remained in the printed area. Therefore, the presence of mesenchymal cell is mandatory in order to create a pattern that will be conserved over time. This work describes an interesting approach to study cell migration that could be reproduced to study the effect of trophic factors.

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.

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.

Layer-by-layer biofabrication represents a novel strategy to create three-dimensional living structures with a controlled internal architecture, using cell micromanipulation technologies. Laser assisted bioprinting (LAB) is an effective printing method for patterning cells, biomolecules, and biomaterials in two dimensions. "Biopapers,'' made of thin polymer scaffolds, may be appropriate to achieve three-dimensional constructs and to reinforce mechanical properties of printed materials. The aim of this work was to evaluate the effect of the tridimensional organization of cells and biomaterials on cell proliferation in vitro and in vivo. The experimental LAB setup was comprised of an infrared laser, focused onto a glass ribbon coated with an absorbing layer of gold. The cell bioink was made of MG63 cells (50 millions cells/mL in culture medium and 1% alginate), transduced with Luciferase gene for tracking and quantification. The printing substrate was a 100-mm-thick polycaprolacton (PCL) electrospun scaffold. The building sequence comprised sequential layers of cells and PCL scaffolds stacked using two different tridimensional arrangements, which were compared in this study (layer-by-layer vs. seeding on a single locus of the scaffolds). Then the cell-seeded materials were cultured in vitro or implanted in vivo in NOD-SCID mice. The qualitative follow-up involved scanning electron microscopy (SEM) observations, live-dead assays, and histology. The cell amount was quantified by photon imager during 21 days in vitro and 2 months in vivo. Live-dead assay and SEM revealed that the cells survived after printing and spread onto PCL membranes. Circle-shaped patterns were maintained in vitro during the first week but they were no longer observable after 2 weeks, due to cell proliferation. Luciferase tracking displayed that the cell amount was increased in vitro and in vivo when the materials and the cells where stacked layer by layer. Histological sections of the in vivo samples revealed a thicker fibrous tissue in the layer-by-layer samples. We have demonstrated in this study that PCL electrospun biopapers can act as a shock-absorbing mattress for cell printing and could further support cell proliferation. The layer-by-layer printing provided an appropriate 3D environment for cell survival and enhanced cell proliferation in vitro and in vivo.

Laser-assisted bioprinting is one among several technologies that are being developed in the recent and growing field of bioprinting. Bioprinting is defined as the use of computeraided transfer processes for patterning and assembling living and non-living materials with a prescribed 2D or 3D organization in order to produce bio-engineered structures serving in regenerative medicine, pharmacology, and basic cell biology studies. We describe the physical parameters that need to be tuned for laser-assisted bioprinting of materials and cells, with high throughput and controlled printing resolution. We present its applications for printing cells and tissue-relevant biomaterials, both in vitro and in vivo. Finally, we discuss how this technique may help in reproducing the local cell micro-environment and dealing with tissue complexity and heterogeneity for fabricating functional tissue-engineered 3D constructs.

Free Article on the editor website; International audience; For bone tissue engineering, human Adipose Derived Stem Cells (hADSCs) are proposed to be associated with a scaffold for promoting bone regeneration. After implantation, cellularised scaffolds require a non-invasive method for monitoring their fate in vivo. The purpose of this study was to use Magnetic Resonance Imaging (MRI)-based tracking of these cells, labelled with magnetic agents for in vivo longitudinal assessment. hADSCs were isolated from adipose tissue and labelled with USPIO-rhodamine (Ultrasmall SuperParamagnetic Iron Oxide). USPIO internalisation, absence of toxicity towards hADSCs, and osteogenic differentiation of the labelled cells were evaluated in standard culture conditions. Labelled cells were then seeded within a 3D porous polysaccharide-based scaffold and imaged in vitro using fluorescence microscopy and MRI. Cellularised scaffolds were implanted subcutaneously in nude mice and MRI analyses were performed from 1 to 28 d after implantation. In vitro, no effect of USPIO labelling on cell viability and osteogenic differentiation was found. USPIO were efficiently internalised by hADSCs and generated a high T2* contrast. In vivo MRI revealed that hADSCs remain detectable until 28 d after implantation and could migrate from the scaffold and colonise the area around it. These data suggested that this scaffold might behave as a cell carrier capable of both holding a cell fraction and delivering cells to the site of implantation. In addition, the present findings evidenced that MRI is a reliable technique to validate cell-seeding procedures in 3D porous scaffolds, and to assess the fate of hADSCs transplanted in vivo.

L’ingenierie tissulaire osseuse a pour objectif general de repousser les limites des methodes conventionnelles de regeneration osseuse en utilisant les progres des connaissances dans le domaine de la biologie, de l’ingenierie, de la physique ou encore de la robotique. Quatre elements sont associes dans cette demarche d’ingenierie : un materiau support pour la croissance tissulaire (scaffold), des cellules autologues, des facteurs de croissance et une periode de maturation in vitro ou in vivo . Dans la sphere buccale et maxillo-faciale, il s’agit de repondre au probleme de la vascularisation des greffons de grande taille sur un terrain fragilise et de remplacer les autogreffes osseuses qui peuvent provoquer une morbidite importante du site donneur.L’objectif de cet article est une mise au point sur les applications cliniques humaines de l’ingenierie tissulaire osseuse pour la cavite buccale et la face. La methode utilisee comporte une recherche bibliographique sur Pubmed et Medline avec des mots cles specifiques. De plus, des criteres d’inclusion et d’exclusion des articles recueillis ont ete definis. Quarante-huit publications internationales ont ete retenues et classees en fonction de leur domaine d’application. Il s’agissait de reconstruction mandibulaire (19 articles), de reconstruction osseuse dans le traitement des fentes faciales (6 articles) ou de regeneration osseuse pre-implantaire (23 articles). L’analyse de ces publications a montre qu’il s’agissait essentiellement d’etudes de faible puissance, a l’exception de 6 etudes prospectives randomisees dans le domaine de la regeneration osseuse pre-implantaire. L’association d’un scaffold a des facteurs de croissance (BMPs) semble constituer la combinaison qui donne les meilleurs resultats. Pour la reconstruction mandibulaire, la methode de prevascularisation in vivo des produits d’ingenierie tissulaire est une technique complexe mais prometteuse pour les patients en echec therapeutique. L’ingenierie tissulaire osseuse a montre son efficacite dans la chirurgie pre-implantaire. Elle permettra peut-etre bientot d’envisager des techniques moins invasives.

Over this decade, cell printing strategy has emerged as one of the promising approaches to organize cells in two and three dimensional engineered tissues. High resolution and high speed organization of cells are some of the key requirements for the successful fabrication of cell-containing two or three dimensional constructs. So far, none of the available cell printing technologies has shown an ability to concomitantly print cells at a cell-level resolution and at a kHz range speed. We have studied the effect of the viscosity of the bioink, laser energy, and laser printing speed on the resolution of cell printing. Accordingly, we demonstrate that a laser assisted cell printer can deposit cells with a microscale resolution, at a speed of 5 kHz and with computer assisted geometric control. We have successfully implemented such a cell printing precision to print miniaturized tissue like layouts with de novo high cell density and micro scale organization.

Matrix proteins of the SIBLING family interact with bone cells and with bone mineral and are thus in a key position to regulate bone development, remodeling and repair. Within this family, bone sialoprotein (BSP) is highly expressed by osteoblasts, hypertrophic chondrocytes and osteoclasts. We recently reported that mice lacking BSP (BSP−/−) have very low trabecular bone turnover. In the present study, we set up an experimental model of bone repair by drilling a 1 mm diameter hole in the cortical bone of femurs in both BSP−/− and +/+ mice. A non-invasive MRI imaging and bone quantification procedure was designed to follow bone regeneration, and these data were extended by μCT imaging and histomorphometry on undecalcified sections for analysis at cellular level. These combined approaches revealed that the repair process as reflected in defect-refilling in the cortical area was significantly delayed in BSP−/− mice compared to +/+ mice. Concomitantly, histomorphometry showed that formation, mineralization and remodeling of repair (primary) bone in the medulla were delayed in BSP−/− mice, with lower osteoid and osteoclast surfaces at day 15. In conclusion, the absence of BSP delays bone repair at least in part by impairing both new bone formation and osteoclast activity.

RGD peptides are used in biomaterials science for surface modifications with a view to elicit selective cellular responses. Our objective is to replace peptides by small peptidomimetics acting similarly. We designed novel molecules targeting alpha(v)beta(3) integrin and featuring spacer-arms (for surface grafting), which do not disturb the biological activity, from (l) N-(3-(trifluoromethyl)benzenesulfonyl) tyrosine used as scaffold. Various Arg-mimics were fixed on the phenol function, and the ortho position was used for the coupling of OEG spacers. All peptidomimetics were active in the nM range in a binding test toward human alpha(v)beta(3) integrin (IC(50) = 0.1 to 1.7 nM) and selective versus platelet integrin alpha(IIb)beta(3). Selected compounds revealed excellent ability to inhibit bone cells adhesion on vitronectin. Modeling and docking studies were performed for comparing the most active RGD peptidomimetic to cilengitide, i.e., cyclo-[RGDfN(Me)V]-. Lastly, the adhesion of endothelial cells on a cultivation support grafted with RGD peptidomimetics was significantly improved.

There have been extensive research efforts to develop new strategies for bone tissue engineering. These have mainly focused on vascularization during the development and repair of bone. It has been hypothesized that pre-seeding a scaffold with endothelial cells could improve angiogenesis and bone regeneration through a complex dialogue between endothelial cells and bone-forming cells. Here, we focus on the paracrine signals secreted by both cell types and the effects they elicit. We discuss the other modes of cell-to-cell communication that could explain their cell coupling and reciprocal interactions. Endothelial cell-derived tube formation in a scaffold and the dialogue between endothelial cells and mesenchymal stem cells provide promising means of generating vascular bone tissue-engineered constructs.

We examined the hypothesis that human mesenchymal stem cells detect physiological mechanical signals. Human bone marrow stromal cells (HBMSCs) were exposed to fluid shear stress of 12 dynes/cm2 and analysed for their ability to express osteoblast-specific markers and associated signalling pathways. HBMSCs showed a significant increase in alkaline phosphatase (ALP) gene expression and a marked decrease in type I collagen, while no effect on Cbfa1/Runx2 was detected. This regulation is related to p38 and ERK1/2 activation, although the use of specific inhibitors to these two MAP kinases suggests that ALP mRNA induction is especially dependent on p38 activity, while type I collagen downregulation is ERK1/2-dependent. Interestingly, the expression of connexin43, which is involved in cell-to-cell communication of osteoblastic cells through gap junction formation, and its distribution through the cells, were modified by fluid flow (FF). HBMSCs are sensitive to shear stress and it appears essential to take their responsiveness into consideration before associating these regenerative cells with a bioactive biomaterial in a new bone tissue-engineering strategy. Copyright © 2009 John Wiley & Sons, Ltd.

Induced Membranes Technique was first described to enhance bone reconstruction of large osseous defects. Previous in vitro studies established their osteoinductive potential, due to the presence of opteoblasts precursors and to high amounts of growth factors contained within. The purpose of this study was to test in vivo the osteoinductive properties of induced membranes on a macroporous HA-TCP in a nonosseous subcutaneous site. Subcutaneous-induced membranes were obtained in 21 rabbits; 1 month later, the membranes were filled with a biphasic calcium phosphate material composed of 75% hydroxyapatite (HA) and 25% β-tricalcium phosphate associated or not with autograft. Histological and immunohistochemical studies were performed on membrane biopsies. Undecalcified and decalcified sections were qualitatively and quantitatively analyzed. 45Ca uptake was observed and quantified on the sections using microimager analysis. Dense vascularity was found in the induced membranes. New bone formation was detected in the HA-TCP + autograft samples and increased significantly from 3 to 6 months (p

Titanium-based biomaterials for endosseous implants have found widespread applications in the orthopedic, maxillofacial, and dental domains. Indeed, the surface characteristics such as their chemical modification control considerably the cellular response and, subsequently, the quality and the quantity of new-formed bone around the implant. In this study, human osteoprogenitor (HOP) cell adhesion on different titanium surfaces functionalized with hydroxyapatite (HA), type I collagen, or Arg-Gly-Asp (RGD)-containing peptides is investigated by the quartz crystal resonators and by confocal laser scanning microscopy (CLSM) for the imaging of focal contact formation. Data obtained by quartz crystal resonator technique revealed that RGD-containing peptides alone increase HOP cell adhesion in early time period of culture. Moreover, association of RGD-containing peptides with either type I collagen or with HA layers induces an additive effect on HOP cell adhesion compared to Ti-Coll or Ti-HA. CLSM shows both the area of focal contact by cell unit and the cytoskeleton network organization to differ according to the surfaces. Interestingly, association of RGD-containing peptides with HA layers induces an additive effect on focal contact formation on HOP cells compared to Ti-HA alone. These data confirm that an RGD peptide effect occurs in the early time of culture, which is beneficial for osteoblast to spreading, differentiation, and survival.

The aim of this study was to evaluate the in vivo effects of the grafting of cyclo- DfKRG on the early phase of integration of biomaterials made with macroporous hydroxyapatite/b-tricalcium phosphate colonized with autologous bone marrow stromal cells. Non-grafted (-RGD) and grafted (+RGD) biomaterials were implanted in both femoral condyles of rabbits for 2 and 4 weeks. Two weeks after implantation, the global bone formation was higher with +RGD samples (p=0.02). There remained no significant difference 4 weeks after implantation. In conclusion, grafting of cyclo-DfKRG peptide on materials could promote cell colonization and improve osteoconduction during the first stage of the bone cicatrization process.

NMR microscopy is usually achieved at ultra-high-static magnetic field (9.4 Tesla) with the use of home-built coils adapted to the size of the sample. In that way, well-resolved images can be obtained with voxel size smaller than 50 m. The purpose of this article is to show that high-resolution imaging can be realized at lower field strength (4.7 Tesla) using 5- and 10-mm probes initially dedicated to vertical magnets. These coils are easily adaptable to horizontal imaging systems. Excellent quality and filling factors permitted isotro- pic resolutions (50 m) on a biomaterial and on mouse femoris. This study also demonstrates that spoiled-gradient echo sequence (FLASH) with low reception bandwidth or with the use of gadolinium are particularly effective for 3D MR microscopy. © 2005 Wiley Periodicals, Inc.

Despite the controversy about the ideal properties of bone replacement materials, it seems that the concept of tissue engineered constructs, materials pre-colonized with autologous bone cells before implantation gathers most of favorable opinions. Cellulosic materials possess osteoconductive properties but have no intrinsic osteoinductive capacity. Then, one solution to develop bone substitutes with osteogenic properties would be to associate biomaterials with osteoprogenitors cells and the biomaterial should support cellular adhesion and induce tissue ingrowth. In a first part of this paper, we describe a new method to immobilize bioactive molecules onto cellulose. Second part of this paper is dedicated to the characterization by X-ray photoelectron spectroscopy (XPS) and static contact angle measurements of cellulose surfaces functionalized with RGD peptides. Finally, the influence of these RGD-containing peptides on osteoprogenitor cells adhesion and proliferation is discussed. These data suggest that, by controlling the preparation of the raw material, bone substitutes with intrinsic osteoinductive property can be developed from cellulose.

Background Atomic force microscopy (AFM) can be used to visualize the cell morphology in an aqueous environment and in real time. It also allows the investigation of mechanical properties such as cell compliance as a function of cell attachment. This study characterized and evaluated osteoblast adhesion by AFM. Methods Human bone marrow stromal cells were cultured on two types of surface to induce weak and strong cellular adhesions. Results Cells were considered as spreading if they had a flattened and lengthened shape and a cytoskeletal organization in the submembrane cytosolic region. Cell detachment demonstrated different adhesion states between adherent cells to be distinguished. The stability of the cytoskeletal fibers indicated that cells were adherent. The elastic modulus was estimated by two complementary approaches. The values deduced were between 3 × 102 and 2 × 105 Nm−2 according to the state of cell adhesion and the approaches used to measure this elastic modulus. Conclusions Although the results were qualitative, a relation may be deduced between the elasticity of living cells as demonstrated by cytoskeletal organization and the state of cell adhesion. The technique could be used to determine the adhesion state of an adherent osteoblast observed under AFM. Cytometry Part A 54A:36–47, 2003. © 2003 Wiley-Liss, Inc.

Surface modification of a biodegradable polymer, poly(L-lactic acid) (PLLA), with an extracellular matrix molecule, collagen, has been carried out by covalent bonding to PLLA via gamma irradiation grafting with poly(acrylic acid) as a coupling agent. The grafting yield increased with the increase of gamma absorbed dose. A grafting yield of more than 7% for collagen type I grafting was obtained at 21 kGy dose. The grafting yield was dose dependent. X-ray photoelectron spectroscopy confirmed the presence of proteins on the grafted PLLA. Swelling experiments revealed a crosslinked structure in the grafted polymer, whereas in comparison non-treated PLLA is linear. Localisation of collagen using immunostaining showed high levels of collagen on the grafted PLLA, confirming that the grafted collagen in the PLLA was still biologically active. The presence of collagen epitopes on the surface, i.e. having reactivity of the antibody against collagen on the surface of the grafted PLLA, demonstrated that irradiation may alter the molecular weight of collagen but not the availability of active binding sites. These results indicate that the single-step procedure of grafting by gamma irradiation could provide a simple but efficient technique to modify the biocompatibity of a scaffold for tissue engineering.

International audience; Spherulites are multilamellar vesicles consisting of concentric shells that can encapsulate small organic molecules or macromolecules. We investigate the possibility of targeting neutral spherulites to adherent culture cells by functionalizing their surface with RGD-containing ligands. The strength and specificity of association of RGD spherulites with several cell lines (EAhy 926 endothelial cell line, human umbilical vein endothelial cell (HUVEC) and human osteoprogenitor (HOP) primary cells) was studied, and the molecular interaction of RGD spherulites with the EAhy 926 cell surface was investigated. We show that, after binding to cells, spherulites are internalized.

B-type natriuretic peptide (BNP) is a hormone mainly secreted by cardiac ventricle myocytes and which is increased in cardiac diseases. Moreover, BNP expression has been shown in various cell/tissue types. Six different human endothelial cell (EC) culture models arising from macro and microcirculation either primary cultures or cell lines were cultured and screened for BNP presence and secretion. All cell types expressed BNP mRNA while only the ECs arising from bone marrow stromal compartment secreted high amounts of BNP protein. This report is the first to identify ECs as a new source of BNP. However, BNP secretion is limited to a particular EC type.

In the search for new compounds that might, once incorporated into biomaterials, stimulate the natural processes of bone regeneration, a new series of silicon-containing alkyl nucleobase analogues has been synthesized. An active hypoxanthine transport process in human osteoblasts was demonstrated, with an apparent Michaelis constant of 2.3 microM and a maximum possible rate of 0.47 pmol s(-1) x 10(6) cell. The synthesized analogues were tested for toxicity in human osteoblasts. Nontoxic analogues were tested in competition transport studies with [(14)C]hypoxanthine. Two of them were found to inhibit the active transport of hypoxanthine in human osteoblasts, with IC(50) values of 6.5 and 11.6 microM.

Bone development and remodeling depend on complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly vascular endothelial cells that may be pivotal members of a complex interactive communication network in bone. Our aim was to investigate the interaction between human umbilical vein endothelial cells (HUVEC) and human bone marrow stromal cells (HBMSC). Cell differentiation analysis performed with different cell culture models revealed that alkaline phosphatase activity and type I collagen synthesis were increased only by the direct contact of HUVEC with HBMSC. This “juxtacrine signaling” could involve a number of different heterotypic connexions that require adhesion molecules or gap junctions. A dye coupling assay with Lucifer yellow demonstrated a functional coupling between HUVEC and HBMSC. Immunocytochemistry revealed that connexin43 (Cx43), a specific gap junction protein, is expressed not only in HBMSC but also in the endothelial cell network and that these two cell types can communicate via a gap junctional channel constituted at least by Cx43. Moreover, functional inhibition of the gap junction by 18α-glycyrrhetinic acid treatment or inhibition of Cx43 synthesis with oligodeoxyribonucleotide antisense decreased the effect of HUVEC cocultures on HBMSC differentiation. This stimulation could be mediated by the intercellular diffusion of signaling molecules that permeate the junctional channel.

Cell adhesion directly influences cell growth, differentiation and migration as well as morphogenesis, integrity and repair. The extracellular matrix (ECM) elaborated by osteoblast cells constitutes a regulator of the cell adhesion process and then of the related phenomenon. These regulatory effects of ECM are mediated through integrins and some of them are able to bind RGD sequences. The aim of this study was to determine the role of the sequence and the structure of RGD-containing peptides (linear and cyclic) as well as their role in the cell adhesion process. Cell adhesion assays onto ECM proteins coated surfaces were performed using a range of linear and cyclic RGD-containing peptides. We showed a different human osteoprogenitor cell adhesion according to the coating for ECM proteins and for RGD-peptides. Inhibition assays using peptides showed different responses depending on the coated protein. Depending on the amino-acid sequence and the structure of the peptides (cyclic/linear), we observed 100% inhibition of cell adhesion onto vitronectin. These results suggest the importance of sequence, structure and conformation of the peptide, which may play a crucial function in the ligand/receptor interaction and/or in the stability of the interaction.

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.

The effect of surface roughness of the titanium alloy Ti–6Al–4V (Ti alloy) on the short- and long-term response of human bone marrow cells in vitro and on protein adsorption was investigated. Three different values in a narrow range of surface roughness were used for the substrata (Rα: 0.320, 0.490 and 0.874 μm). Cell attachment, cell proliferation and differentiation (alkaline phosphatase specific activity) were determined past various incubation periods. The protein adsorption of bovine serum albumin and fibronectin, from single protein solutions, on rough and smooth Ti alloy surfaces was examined with two methods, X-ray photoelectron spectroscopy (XPS) and radiolabeling. Cell attachment and proliferation were surface roughness sensitive and increased as the roughness of Ti alloy increased. No statistically significant difference was observed in the expression of ALP activity on all three Ti alloy surfaces and culture plastic. Both methods, XPS and protein radiolabeling, showed that human serum albumin was adsorbed preferentially onto the smooth substratum. XPS technique showed that the rough substratum bound a higher amount of total protein (from culture medium supplied with 10% serum) and fibronectin (10-fold) than did the smooth one. The cell attachment may be explained by the differential adsorption of the two proteins onto smooth and rough Ti alloy surfaces.

The purpose of this work was to develop qualitative methods for in situ analysis of bone formation in an osteoconductive hydroxyapatite matrix (ENDOBON), loaded with human bone marrow cells (HBMSC) implanted subcutaneously in athymic mice. Samples were taken before implantation (T0), 1, 2, 4 and 6 weeks after implantation. Bone-biomaterial interaction were investigated on undecalcified sections by histological, cytochemical, immunological and molecular biology methodologies. Histological observations were performed in order to observe inflammatory cells, vessels, newly formed bone, woven and lamellar bone. Enzymohistochemistry was carried out to detect positive tartrate resistant acid phosphatase activity (TRAP+). Immunohistochemistry using antibodies against type I collagen and osteocalcin permitted us to characterize the content of the matrix elaborated within the implant. Moreover, in situ hybridization was carried out to discriminate, the implanted human cells from the murine cells, and to evaluate the function of these human cells in osteogenesis. Results demonstrated an early formation of lamellar bone only in the pores of the studied HAP loaded with HBMSC. This bone contained a matrix showing positive reaction for type I collagen and osteocalcin. In situ hybridization identified some of these cells as human cells. At 6 weeks, examination of histological results showed persistance of lamellar bone in the implants. We only found TRAP+ activity in the materials loaded with human bone marrow cells. Molecular hybridization no longer revealed positive cells for the human DNA probe. All these results indicate that the various evaluation techniques performed on undecalcified sections, permit us to evaluate the response of human bone marrow cells in HAP implanted into mice.

Endothelial cell (EC) seeding of small caliber vascular grafts prior to their implantation has proved to significantly improve long-term patency in humans. We have previously demonstrated that a monolayer of EC could be obtained on type I collagen-coated knitted ultrathin polyster grafts (InterVascular, La Ciotat, France). Thus, the aim of the present work was to understand the nature of cell adhesion mechanisms involved in the cell /biomaterial interface, using HemaCarotid (InterVascular) patches made of type I collagen-coated knitted ultrathin polyster (type I collagen is used to coat patches to attain low permeability). By means of quantitative attachment tests, adhesion blocking assays, RT-PCR for the expression of beta1 integrin mRNA, indirect immunofluorescence with antivinculin antibody, we were able to show that EC are able to adhere to such surfaces by the means (non-unique) of cell surface receptors of the beta1 integrin group. However, the latter are probably downregulated at the cell/biomaterial interface.

The main problem for macroporous structures used as bone substitutes is their lower resistances when compared to that of cancellous bone. The present investigation aimed to improve the strength of ceramics with 65% porosities based on beta-TCP. The initial mixtures were rendered plastic by addition of non-ionic carbohydrate binders. Macropores were created using substances which were eliminated by heat. Mechanical tests indicated that the resistance of the ceramics depended more on the quantity than the nature of the binders. Porosity measurements were done with a mercury porosimeter, and cellular biocompatibility was evaluated by performing cellular attachment tests and observing the proliferation of differentiated cells.

The two crystalline forms of CaCO3, aragonite (from natural coral) and calcite (from natural limestone), have been used with success as bone graft substitutes. However, natural coral transformed into calcite by heating has never been tested. The objective of this work was to study the proliferation and alkaline phosphatase, osteonectin, and osteocalcin expression of human bone marrow cells cultured on CaCO3 crystallized both in the aragonite form (natural coral) and in the calcite form (natural coral modified by heating). The methods used to characterize calcite obtained from the coral were volumic porosimetry, scanning electron microscopy (SEM) and X-ray diffraction. Cell colonization of the material was assessed by SEM performed on days 1, 7, 20, and 30 and [3H]thymidine incorporation was performed on days 3, 7, 12, 18, 25, and 32. Phenotypic expression was assessed by using in situ cytochemistry (alkaline phosphatase), immunocytochemistry (osteonectin and osteocalcin), and hybridization (osteocalcin, β-actin, and alkaline phosphatase mRNA). Results showed the transformation of aragonite into calcite after heating, the conservation of macroporosity, and a modification of the surface. Calcite appeared to have a smoother and more uniform surface than aragonite crystals. As for [3H]thymidine there was an increase incorporation from days 3 to 18, a stabilization from days 18 to 25, and a decrease from days 25 to 32. After 20 days of culture, immunological studies using monoclonal antibodies to osteocalcin, osteonectin, cytochemical analysis of alkaline phosphatase activity, and in situ hybridization using osteocalcin, β-actin, and alkaline phosphatase cDNA indicated that the cells had not lost their osteoblastic phenotype. These experiments demonstrate that coral crystallized in the aragonite or calcite form present a similar degree of specific cytocompatibility. © 1998 John Wiley & Sons, Inc. J. Biomed Mater Res, 42, 96–102, 1998.

BMP3 and TGF beta 1 were found to induce opposite effects on human bone marrow stromal cells both in cell proliferation and cell differentiation and on calcium deposition onto the extracellular matrix. Moreover, BMP3 may exert, in part, the inhibitory effect of TGF beta 1 through decreasing the affinity of TGF-beta for its receptors which are now identified in these human bone cells.