Using the present experimental protocol, we concluded that an 808 nm wavelength infrared LLLT does not alter murine bone progenitor cell proliferation and differentiation. Moreover our results confirm the necessary use of a powermeter to fix LLLT protocol parameters.

Particulate forms of biphasic calcium phosphate (BCP) biomaterials below 500  μm are promising bone substitutes that provide with interconnected open porosity allowing free circulation of fluids and cells. Dispersion of the particles in the surrounding tissues at the time of implantation is a major drawback preventing from an easy use. We have asked whether blood clot could be a convenient natural hydrogel for handling BCP microparticles, and we hypothesized that blood clot might also confer osteoinductive properties to these particles. We show here that blood clotted around BCP microparticles constitutes a cohesive, moldable, and adaptable biomaterial that can be easily implanted in subcutaneous sites but also inserted and maintained in segmental bone defects, conversely to BCP microparticles alone. Moreover, implantation in bony and ectopic sites revealed that this composite biomaterial has osteogenic properties. It is able to repair a 6-mm critical femoral defect in rat and induced woven bone formation after subcutaneous implantation. Parameters such as particle size and loading into the clot are critical for its osteogenic properties. In conclusion, this blood/BCP microparticle composite is a moldable and osteoinductive biomaterial that could be used for bone defect filling in dental and orthopedic surgery.

A combination of autologous bone marrow stromal cells (BMSCs) and biomaterials is a strategy largely developed in bone tissue engineering, and subcutaneous implantation in rodents or large animals is often a first step to evaluate the potential of new biomaterials. This study aimed at investigating the influence of the immune status of the recipient animal on BMSCs-induced bone formation. BMSCs prepared from C57BL/6 mice, composed of a mixture of mesenchymal stromal and monocytic cells, were combined with a biomaterial that consisted of biphasic calcium phosphate (BCP) particles and plasma clot. This composite was implanted subcutaneously either in syngenic C57BL/6 immune-competent mice or in T-lymphocyte-deficient Nude (Nude) mice. Using histology, immunohistochemistry, and histomorphometry, we show here that this BMSC/BCP/plasma clot composite implanted in Nude mice induces the formation of mature lamellar bone associated to hematopoietic areas and numerous vessels. Comparatively, implantation in C57BL/6 results in the formation of woven bone without hematopoietic tissue, a lower number of new vessels, and numerous multinucleated giant cells (MNGCs). In situ hybridization, which enabled to follow the fate of the BMSCs, revealed that BMSCs implanted in Nude mice survived longer than BMSCs implanted in C57BL/6 mice. Quantitative expression analysis of 280 genes in the implants indicated that the differences between C57BL/6 and Nude implants corresponded almost exclusively to genes related to the immune response. Gene expression profile in C57BL/6 implants was consistent with a mild chronic inflammation reaction characterized by Th1, Th2, and cytotoxic T-lymphocyte activation. In the implants retrieved from T-deficient Nude mice, Mmp14, Il6st, and Tgfbr3 genes were over-expressed, suggesting their putative role in bone regeneration and hematopoiesis. In conclusion, we show here that the T-mediated inflammatory microenvironment is detrimental to BMSCs-induced bone formation and shortens the survival of implanted cells. Conversely, the lack of T-lymphocyte reaction in T-deficient animals is beneficial to BMSCs-induced mature bone formation. This should be taken into account when evaluating cell/biomaterial composites in these models.

Bone marrow stromal cells (BMSCs) have been demonstrated to induce bone formation when associated to osteoconductive biomaterials and implanted in vivo. Nevertheless, their role in bone reconstruction is not fully understood and rare studies have been conducted to follow their destiny after implantation in syngenic models. The aim of the present work was to use sensitive and quantitative methods to track donor and recipient cells after implantation of BMSCs in a syngenic model of ectopic bone formation. Using polymerase chain reaction (PCR) amplification of the Sex determining Region Y (Sry) gene and in situ hybridization of the Y chromosome in parallel to histological analysis, we have quantified within the implants the survival of the donor cells and the colonization by the recipient cells. The putative migration of the BMSCs in peripheral organs was also analyzed. We show here that grafted cells do not survive more than 3 weeks after implantation and might migrate in peripheral lymphoid organs. These cells are responsible for the attraction of host cells within the implants, leading to the centripetal colonization of the biomaterial by new bone.

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Les cellules souches mésenchymateuses adultes ont suscité un engouement important dans le milieu de la recherche biomédicale (Bianco et al. 2008). A ne pas confondre avec les cellules souches embryonnaires dont l'usage est prohibé en France pour des raisons éthiques, les cellules souches mésenchymateuses adultes ont fait l'objet de nombreuses études in vitro et in vivo qui ont permis de caractériser leurs propriétés uniques de prolifération et de différentiation, et donc potentiellement de régénération tissulaire. Grâce à une exploration systématique des différents tissus mésenchymateux adultes, il a été possible d'isoler ces cellules souches mésenchymateuses à partir de la moelle osseuse, de tissu adipeux, de sang périphérique ou même de la pulpe dentaire (Cancedda et al. 2007). Ces cellules ont un très fort taux de prolifération et sont capables de se différencier en plusieurs types cellulaires tels que les myoblastes, chondroblastes, ostéoblastes ou encore adipocytes. Ces propriétés en font des candidats très intéressants pour la mise en place d'une thérapie cellulaire de certaines pathologies dégénératives, comme l'ostéonécrose des maxillaires, ou pour l'ingénierie tissulaire dans le domaine des pertes de substances osseuses acquises ou congénitales. Au travers d'une revue de la littérature (Schimming et al. 2004, Pradel et al 2006, Pradel et al. 2007), nous rapporterons des études précliniques et cliniques utilisant les cellules souches mésenchymateuses adultes dans la sphère maxillo-faciale. Nous comparerons ces résultats aux données obtenues avec des modèles d'ingénierie tissulaire développés au laboratoire (Elabd et al. 2007, Trojani et al. 2008). Malgré de nombreux progrès, cette approche thérapeutique est encore souvent jugée trop lourde et trop coûteuse en raison des nombreux intervenants, du plateau technique ou des substances pharmacologiques nécessaires, ce qui limite malheureusement les retombées cliniques. Par ailleurs l'effet biologique de ces cellules souches mésenchymateuses après implantation, semblerait lié à l'expression in situ de nombreux facteurs paracrines. Plusieurs équipes essaient de les identifier, afin de pouvoir stimuler directement la réparation tissulaire et de simplifier les applications cliniques chez l'homme.