Florian Boukhechba

Human Primary Osteocyte Differentiation in a 3D Culture System

Studies on primary osteocytes, which compose >90–95% of bone cells, embedded throughout the mineralized matrix, are a major challenge because of their difficult accessibility and the very rare models available in vitro. We engineered a 3D culture method of primary human osteoblast differentiation into osteocytes. These 3D‐differentiated osteocytes were compared with 2D‐cultured cells and with human microdissected cortical osteocytes obtained from bone cryosections. Human primary osteoblasts were seeded either within the interspace of calibrated biphasic calcium phosphate particles or on plastic culture dishes and cultured for 4 wk in the absence of differentiation factors. Osteocyte differentiation was assessed by histological and immunohistological analysis after paraffin embedding of culture after various times, as well as by quantitative RT‐PCR analysis of a panel of osteoblast and osteocyte markers after nucleic acid extraction. Histological analysis showed, after only 1 wk, the presence of an osteoid matrix including many lacunae in which the cells were individually embedded, exhibiting characteristics of osteocyte‐like cells. Real‐time PCR expression of a set of bone‐related genes confirmed their osteocyte phenotype. Comparison with plastic‐cultured cells and mature osteocytes microdissected from human cortical bone allowed to assess their maturation stage as osteoid‐osteocytes. This model of primary osteocyte differentiation is a new tool to gain insights into the biology of osteocytes. It should be a suitable method to study the osteoblast‐osteocyte differentiation pathway, the osteocyte interaction with the other bone cells, and orchestration of bone remodeling transmitted by mechanical loading and shear stress. It should be used in important cancer research areas such as the cross‐talk of osteocytes with tumor cells in bone metastasis, because it has been recently shown that gene expression in osteocytes is strongly affected by cancer cells of different origin. It could also be a very efficient tool for drug testing and bone tissue engineering applications.

Oxytocin Reverses Ovariectomy-Induced Osteopenia and Body Fat Gain

Osteoporosis and overweight/obesity constitute major worldwide public health burdens that are associated with aging. A high proportion of women develop osteoporosis and increased intraabdominal adiposity after menopause. which leads to bone fractures and metabolic disorders. There is no efficient treatment without major side effects for these 2 diseases. We previously showed that the administration of oxytocin (OT) normalizes ovariectomy-induced osteopenia and bone marrow adiposity in mice. Ovariectomized mice, used as an animal model mimicking menopause, were treated with OT or vehicle. Trabecular bone parameters and fat mass were analyzed using micro-computed tomography. Herein, we show that this effect on trabecular bone parameters was mediated through the restoration of osteoblast/osteoclast cross talk via the receptor activator of nuclear factor-κB ligand /osteoprotegerin axis. Moreover, the daily administration of OT normalized body weight and intraabdominal fat depots in ovariectomized mice. Intraabdominal fat mass is more sensitive to OT that sc fat depots, and this inhibitory effect is mediated through inhibition of adipocyte precursors differentiation with a tendency to lower adipocyte size. OT treatment did not affect food intake, locomotors activity, or energy expenditure, but it did promote a shift in fuel utilization favoring lipid oxidation. In addition, the decrease in fat mass resulted from the inhibition of the adipose precursors differentiation. Thus, OT constitutes an effective strategy for targeting osteopenia, overweight, and fat mass redistribution without any detrimental effects in a mouse model mimicking the menopause.

Differentiation and activity of human preosteoclasts on chitosan enriched calcium phosphate cement

Chitosan associated to various scaffolds has been shown to promote growth and mineral rich matrix deposition by osteoblasts in vitro, whereas its influence on osteoclast differentiation, which plays also a central role in bone remodeling, has never been described. The purpose of this study was to investigate the differentiation and activity of human preosteoclastic cells on calcium phosphate cement containing 2% chitosan (Cementek/chitosan) compared to the Cementek alone. Human primary osteoclast precursors were cultured directly on both biomaterials in the presence of rhM-CSF and rhRANK-L for 7 days. Using LIVE/DEAD fluorescent assay, tartrate-resistant acid phosphatase staining, scanning electron microscopy and quantitative RT-PCR, we demonstrated that incorporation of chitosan to Cementek does not affect the proliferation and adhesion of preosteoclasts but inhibits the formation of TRACP positive cells and prevents the osteoclastic resorption of the composite biomaterial compared to Cementek alone. This inhibitory effect of chitosan on osteoclast resorption activity should have important implications on bone formation and bone remodeling after in vivo implantation. Indeed, based on the positive results obtained in vivo by several investigators, one can suggest that this property of chitosan can be beneficial for bone regeneration.

Design and properties of novel gallium-doped injectable apatitic cements

UNLABELLED Different possible options were investigated to combine an apatitic calcium phosphate cement with gallium ions, known as bone resorption inhibitors. Gallium can be either chemisorbed onto calcium-deficient apatite or inserted in the structure of β-tricalcium phosphate, and addition of these gallium-doped components into the cement formulation did not significantly affect the main properties of the biomaterial, in terms of injectability and setting time. Under in vitro conditions, the amount of gallium released from the resulting cement pellets was found to be low, but increased in the presence of osteoclastic cells. When implanted in rabbit bone critical defects, a remodeling process of the gallium-doped implant started and an excellent bone interface was observed. STATEMENT OF SIGNIFICANCE The integration of drugs and materials is a growing force in the medical industry. The incorporation of pharmaceutical products not only promises to expand the therapeutic scope of biomaterials technology but to design a new generation of true combination products whose therapeutic value stem equally from both the structural attributes of the material and the intrinsic therapy of the drug. In this context, for the first time an injectable calcium phosphate cement containing gallium was designed with properties suitable for practical application as a local delivery system, implantable by minimally invasive surgery. This important and original paper reports the design and in-depth chemical and physical characterization of this groundbreaking technology.

Adaptive immune response inhibits ectopic mature bone formation induced by BMSCs/BCP/plasma composite in immune-competent mice.

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.

Gallium, a promising candidate to disrupt the vicious cycle driving osteolytic metastases.

Bone metastases of breast cancer typically lead to a severe osteolysis due to an excessive osteoclastic activity. On the other hand, the semi-metallic element gallium (Ga) displays an inhibitory action on osteoclasts, and therefore on bone resorption, as well as antitumour properties. Thus, we explored in vitro Ga effects on osteoclastogenesis in an aggressive bone metastatic environment based on the culture of pre-osteoclast RAW 264.7 cells with conditioned medium from metastatic breast tumour cells, i.e. the breast tumour cell line model MDA-MB-231 and its bone-seeking clone MDA-231BO. We first observed that Ga dose-dependently inhibited the tumour cells-induced osteoclastic differentiation of RAW 264.7 cells. To mimic a more aggressive environment where pro-tumourigenic factors are released from bone matrix due to osteoclastic resorption, metastatic breast tumour cells were stimulated with TGF-β, a mayor cytokine in bone metastasis vicious cycle. In these conditions, we observed that Ga still inhibited cancer cells-driven osteoclastogenesis. Lastly, we evidenced that Ga affected directly and strongly the proliferation/viability of both cancer cell lines, as well as the expression of major osteolytic factors in MDA-231BO cells. With the exception of two small scale clinical studies from 1980s, this is the first time that antitumour properties of Ga have been specifically studied in the context of bone metastases. Our data strongly suggest that, through its action against the vicious cycle involving bone cells and tumour cells, Ga represents a relevant and promising candidate for the local treatment of bone metastases in patients with breast cancer.

Effect of G-CSF on the osteoinductive property of a BCP/blood clot composite

We previously reported that blood clot combined with biphasic calcium phosphate microparticles constitute a biomaterial (BRB) that can repair a bone critical defect in rat and induces subcutaneous bone formation in mice. The granulocyte colony-stimulating factor (G-CSF) is the agent most commonly used in human to enrich blood with hematopoietic stem and progenitor cells (HSPCs) as well as granulocytes (GCs). Moreover, recent data also suggest that it can mobilize mesenchymal stem cells (MSCs). Here, we asked whether the osteoinductive properties of the BRB could be further enhanced by G-CSF, either by replacing normal blood by G-CSF-mobilized blood (BRBe) or by treating the recipient animals with G-CSF. The experiments performed in C57BL/6 mice showed that G-CSF induces a marked increase of circulating HPCs and GCs, but not of MSCs. BRBe prepared with G-CSF-enriched blood induced a slight but significant decrease of subcutaneous bone formation compared to BRB prepared with normal blood. Additional injection of G-CSF to the recipient mice had no significant effect on the bone formation induced by BRB or BRBe. Altogether these results indicate that, in this model of ectopic implantation, cell mobilization induced by G-CSF has a negative effect on the osteoinductive property of this blood/BCP composite.

Green laser light irradiation enhances differentiation and matrix mineralization of osteogenic cells

BACKGROUND AND OBJECTIVE Low level laser therapy (LLLT) in both infrared and visible light is a therapeutic tool ever more proposed in clinical practice in different fields. The effect of near infrared LLLT has been described in a growing number of scientific publications related to bone tissue healing, both in vitro and in vivo. More recently, green visible light using potassium-titanyl-phosphate KTiOPO4 (KTP, 532 nm) laser has been proposed in dermatology, urology, oral and maxillofacial surgery but has never been tested on bone tissue. The aim of the present work was to perform a preliminary in vitro study to analyze the effects of KTP laser, on the osteogenic differentiation of bone marrow stromal cells (BMSCs). MATERIALS AND METHODS Using a power meter the first step of this study aimed to evaluate the real power emitted by the KTP laser device and the amount of energy absorbed by culture medium and plastic in order to calculate the appropriate irradiation parameters for cultured cells. Primary bone marrow stromal cells prepared from C57BL/6 mice were cultured and induced to differentiate in the osteogenic lineage in the presence or in the absence of KTP LLLT at a fluence of 4 J/cm(2) three times a week. Specific staining of the cells and the extracellular matrix, microscopic analysis as well as quantitative RT-PCR were used to assess cell proliferation and differentiation. RESULTS We show here that KTP LLLT enhances the osteogenic differentiation of bone marrow stromal cells and the mineralization of their extracellular matrix. CONCLUSION Our results highlight that this LLLT experimental protocol with green light (KTP, 532 nm) at 4 J/cm(2) has a positive effect on the osteogenic differentiation of murine bone marrow stromal cells. These preliminary results could be used as a basis to further investigate the effect of this KTP laser protocol on bone tissue engineering models in vivo and in vitro.

Gallium enhances reconstructive properties of a calcium phosphate bone biomaterial

Calcium phosphate (CaP)‐based biomaterials are commonly used in bone reconstructive surgery to replace the damaged tissue, and can also serve as vectors for local drug delivery. Due to its inhibitory action on osteoclasts, the semi‐metallic element gallium (Ga) is used for the systemic treatment of disorders associated with accelerated bone resorption. As it was demonstrated that Ga could be incorporated in the structure of CaP biomaterials, we investigated the biological properties of Ga‐loaded CaP biomaterials. Culturing bone cells on Ga–CaP, we observed a decrease in osteoclast number and a downregulation of late osteoclastic markers expression, while Ga–CaP upregulated the expression of osteoblastic marker genes involved in the maturation of bone matrix. We next investigated in vivo bone reconstructive properties of different Ga‐loaded biomaterials using a murine bone defect healing model. All implanted biomaterials showed a good osseointegration into the surrounding host tissue, accompanied by a successful bone ingrowth and bone marrow reconstruction, as evidenced by histological analysis. Moreover, quantitative micro‐computed tomography analysis of implants revealed that Ga enhanced total defect filling. Lastly, we took advantage for the first time of a particular mode of non‐linear microscopy (second harmonic generation) to quantify in vivo bone tissue reconstruction within a CaP bone substitute. By doing so, we showed that Ga exerted a positive impact on mature organized collagen synthesis. As a whole, our data support the hypothesis that Ga represents an attractive additive to CaP biomaterials for bone reconstructive surgery. Copyright

Design and properties of a novel radiopaque injectable apatitic calcium phosphate cement, suitable for image-guided implantation: DESIGN AND PROPERTIES OF A NOVEL RADIOPAQUE INJECTABLE APATITIC CPC

An injectable purely apatitic calcium phosphate cement (CPC) was successfully combined to a water-soluble radiopaque agent (i.e., Xenetix® ), to result in an optimized composition that was found to be as satisfactory as poly(methyl methacrylate) (PMMA) formulations used for vertebroplasty, in terms of radiopacity, texture and injectability. For that purpose, the Xenetix dosage in the cement paste was optimized by injection of the radiopaque CPC in human cadaveric vertebrae under classical PMMA vertebroplasty conditions, performed by interventional radiologists familiar with this surgical procedure. When present in the cement paste up to 70 mg I mL-1 , Xenetix did not influence the injectability, cohesion, and setting time of the resulting composite. After hardening of the material, the same observation was made regarding the microstructure, mechanical strength and alpha-tricalcium phosphate to calcium deficient apatite transformation rate. Upon implantation in bone in a small animal model (rat), the biocompatibility of the Xenetix-containing CPC was evidenced. Moreover, an almost quantitative release of the contrast agent was found to occur rapidly, on the basis of in vitro static and dynamic quantitative studies simulating in vivo implantation.