A new boronate ester-based crosslinking strategy allows the design of nonswelling and long-term stable dynamic covalent hydrogels.

Dynamic hydrogels are viscoelastic materials that can be designed to be self-healing, malleable, and injectable, making them particularly interesting for a variety of biomedical applications. To design dynamic hydrogels, dynamic covalent crosslinking reactions are attracting increasing attention. However, dynamic covalent hydrogels tend to swell, and often lack stability. Boronate ester-based hydrogels, which result from the dynamic covalent reaction between a phenylboronic acid (PBA) derivative and a diol, are based on stable precursors, and can therefore address these limitations. Yet, boronate ester formation hardly occurs at physiological pH. To produce dynamic covalent hydrogels at physiological pH, we performed a molecular screening of PBA derivatives in association with a variety of diols, using hyaluronic acid as a polymer of interest. The combination of Wulff-type PBA (wPBA) and glucamine stood out as a unique couple to obtain the desired hydrogels. We showed that optimized wPBA/glucamine hydrogels are minimally- to non-swelling, stable long term (over months), tunable in terms of mechanical properties, and cytocompatible. We further characterized their viscoelastic and self-healing properties, highlighting their potential for biomedical applications.

Development and characterization of a humanized mouse model of osteoarthritis.

OBJECTIVE: In light of the role of immune cells in OA pathogenesis, the development of sophisticated animal models closely mimicking the immune dysregulation during the disease development and progression could be instrumental for the preclinical evaluation of novel treatments. Among these models, immunologically humanized mice may represent a relevant system, particularly for testing immune-interacting DMOADs or cell therapies before their transfer to the clinic. Our objective, therefore, was to develop an experimental model of OA by destabilization of the medial meniscus (DMM) in humanized mice. METHOD: Irradiated 5-week-old NOD/LtSz-scid IL2Rγnull (NSG) mice were humanized by intravenous injection of CD34+ human hematopoietic stem cells. The engraftment efficiency was evaluated by flow cytometry 17 weeks after the humanization procedure. Humanized and non-humanized NSG mice underwent DMM or sham surgery and OA development was assessed 1, 6, and 12 weeks after the surgery. RESULTS: 120 days after the humanization, human T and B lymphocytes, macrophages and NK cells, were present in the blood and spleen of the humanized NSG mice. The DMM surgery induced articular cartilage and meniscal alterations associated with an increase in OA and the meniscal score. Moreover, the surgery triggered an inflammatory response that was sustained at a low grade in the DMM group. CONCLUSIONS: Our study shows for the first time the feasibility of inducing OA by DMM in humanized mice. This novel OA model could constitute a useful tool to bridge the gap between the preclinical and clinical evaluation of immune interacting DMOADs and cell-based therapies.

Correlation between magnetic resonance, X-ray imaging alterations and histological changes in an ovine model of age-related disc degeneration.

Sheep are one of the many animal models used to investigate the pathophysiology of disc degeneration and the regenerative strategies for intervertebral disc (IVD) disease. To date, few studies have thoroughly explored ageing of ovine lumbar IVDs. Hence, the objective of the present study was to concomitantly assess the development of spontaneous age-related lumbar IVD degeneration in sheep using X-ray, magnetic resonance imaging (MRI) as well as histological analyses. 8 young ewes (< 48 months old) and 4 skeletally mature ewes (> 48 months old) were included. Disc height, Pfirrmann and modified Pfirrmann grades as well as T2-wsi and T2 times were assessed by X-ray and MRI. The modified Boos score was also determined using histology sections. Pfirrmann (2 to 3) and modified Pfirrmann (2 to 4) grades as well as Boos scores (7 to 13) gradually increased with ageing, while T2-weighted signal intensity (1.18 to 0.75), T2 relaxation time (114.36 to 70.65 ms) and disc height (4.1 to 3.2 mm) decreased significantly. All the imaging modalities strongly correlated with the histology (p < 0.0001). The present study described the suitability of sheep as a model of age-related IVD degeneration by correlation of histological tissue alterations with the changes observed using X-ray and MRI. Given the structural similarities with humans, the study demonstrated that sheep warrant being considered as a pertinent animal model to investigate IVD regenerative strategies without induction of degeneration.

Identification of TGFß signatures in six murine models mimicking different osteoarthritis clinical phenotypes.

OBJECTIVE: TGFß is a key player in cartilage homeostasis and OA pathology. However, few data are available on the role of TGFß signalling in the different OA phenotypes. Here, we analysed the TGFß pathway by transcriptomic analysis in six mouse models of OA. METHOD: We have brought together seven expert laboratories in OA pathophysiology and, used inter-laboratories standard operating procedures and quality controls to increase experimental reproducibility and decrease bias. As none of the available OA models covers the complexity and heterogeneity of the human disease, we used six different murine models of knee OA: from post-traumatic/mechanical models (meniscectomy (MNX), MNX and hypergravity (HG-MNX), MNX and high fat diet (HF-MNX), MNX and seipin knock-out (SP-MNX)) to aging-related OA and inflammatory OA (collagenase-induced OA (CIOA)). Four controls (MNX-sham, young, SP-sham, CIOA-sham) were added. OsteoArthritis Research Society International (OARSI)-based scoring of femoral condyles and ribonucleic acid (RNA) extraction from tibial plateau samples were done by single operators as well as the transcriptomic analysis of the TGFß family pathway by Custom TaqMan® Array Microfluidic Cards. RESULTS: The transcriptomic analysis revealed specific gene signatures in each of the six models; however, no gene was deregulated in all six OA models. Of interest, we found that the combinatorial Gdf5-Cd36-Ltbp4 signature might discriminate distinct subgroups of OA: Cd36 upregulation is a hallmark of MNX-related OA while Gdf5 and Ltbp4 upregulation is related to MNX-induced OA and CIOA. CONCLUSION: These findings stress the OA animal model heterogeneity and the need of caution when extrapolating results from one model to another.

Degenerative lumbar disc disease: in vivo data support the rationale for the selection of appropriate animal models.

Since low-back pain is increasing in ageing populations, current research efforts are focused on obtaining a better understanding of the pathophysiology of intervertebral disc degeneration and on developing new therapeutic strategies. This requires adequate and clinically relevant models of the disease process. Ex vivo models can provide insights into isolated aspects of the degenerative/regenerative processes involved; although, ultimately, in vivo models are needed for preclinical translational studies. Such models have been developed in numerous animal species with significant variations in size and disc physiology and their number is considerable. Importantly, the choice of the model has to be tailored to the aim of the study. Given the number of available options, it is important to have a good understanding of the various models of disc degeneration and to be fully aware of their advantages and limitations. After comparing the anatomy and histology of intervertebral discs in animals and humans, the present study provides an overview of the different models of in vivo disc degeneration. It also provides a comprehensive guide with suggested criteria to select the most appropriate animal model in a question-driven manner.

Comparing "intra operative" tissue engineering strategies for the repair of craniofacial bone defects.

BACKGROUND: In craniofacial reconstruction, the gold standard procedure for bone regeneration is the autologous bone graft (BG). However, this procedure requiring bone harvesting is a source of morbidity. Bone substitutes, such as biphasic calcium phosphate (BCP), represent an interesting alternative but are not sufficient for bone healing in hypoplastic conditions. In such conditions, osteoprogenitors are essential to provide osteoinduction. Previous studies have shown that BCP associated with total bone marrow (TBM) provides same bone reconstruction as bone graft in a rat model of calvaria defect. Furthermore, adipose tissue stromal vascular fraction (SVF) seems to be another promising source of osteoprogenitor cells that can be used intra-operatively. This study aimed to combine, intra-operative BCP-based bone tissue engineering strategies with TBM or SVF from human sources. METHODS: 5 mm critical-size calvaria defects were performed in 18 nude rat. The defects were filled with intra-operative bone tissue engineering procedures: human BG, human TBM + BCP, human SVF + BCP and, rat TBM + BCP. Animals were sacrificed 8 weeks after implantation and calvaria were processed for histological and radiological examinations. Implanted cells were labelled with a fluorochrome. RESULTS: Micro-CT analysis revealed partial repair of bone defect. Only hBG significantly succeeded in healing the defect (43.1%). However, low rate of newly formed bone tissue was observed in all tissue engineering conditions (hTBM, hSVF, ratTBM). DISCUSSION: The lack of bone formation observed in this study could possibly be attributed to the model. CONCLUSION: This study combined with a literature analysis show the stringency of the nude rat calvaria model in term of bone regeneration.

Clinical relevance of 3D gait analysis in patients with haemophilia.

Haemophilia is characterized by a congenital deficiency of clotting factor VIII or IX. One of the consequences of haemophilia is joint bleedings. Repetitive haemathroses induce cartilage damage and chronic synovitis leading to joint deterioration, and to definitive haemophilic arthropathy which is source of walking disability. Three-dimension gait analysis (3DGA) appears particularly relevant in the case of haemophilia because it allows an evaluation of several joints in weight-bearing situations. The purpose of this study was to review the interest and the contribution of 3DGA in the management of patients with haemophilia. The greatest interest of gait analysis would be to detect early walking changes with a non-invasive and well-tolerated examination, especially in paediatric population. In adulthood, this technic may be also useful to help detect walking worsening in patients known to have already arthropathy. However, it takes time to realize and needs expensive equipment, which limits its possibility of routine use. Although generalizations of these results remain difficult, especially to compare patients with haemophilia to normal population. Indeed, in the studies, patient groups are small and usually heterogeneous in terms of age and target joints. It certainly results of the rarity of the disease. So, it could be interesting to perform a study with a larger cohort in order to allow subgroup analysis, helping to define clearly the place of 3DGA in the strategy of haemophilia evaluation.

Assessing glucose and oxygen diffusion in hydrogels for the rational design of 3D stem cell scaffolds in regenerative medicine.

Hydrogels are attractive biomaterials for replicating cellular microenvironments, but attention needs to be given to hydrogels diffusion properties. A large body of literature shows the promise of hydrogels as 3D culture models, cell expansion systems, cell delivery vehicles, and tissue constructs. Surprisingly, literature seems to have overlooked the important effects of nutrient diffusion on the viability of hydrogel-encapsulated cells. In this paper, we present the methods and results of an investigation into glucose and oxygen diffusion into a silated-hydroxypropylmethylcellulose (Si-HPMC) hydrogel. Using both an implantable glucose sensor and implantable oxygen sensor, we continuously monitored core glucose concentration and oxygen concentration at the centre of hydrogels. We demonstrated that we could tune molecular transport in Si-HPMC hydrogel by changing the polymer concentration. Specifically, the oxygen diffusion coefficient was found to significantly decrease from 3.4 × 10-10 to 2.4 × 10-10  m2  s-1 as the polymer concentration increased from 1% to 4% (w/v). Moreover, it was revealed during in vitro culture of cellularized hydrogels that oxygen depletion occurred before glucose depletion, suggesting oxygen diffusion is the major limiting factor for cell survival. Insight was also gained into the mechanism of action by which oxygen and glucose diffuse. Indeed, a direct correlation was found between the average polymer crosslinking node size and glucose parameters, and this correlation was not observed for oxygen. Overall, these experiments provide useful insights for the analysis of nutrient transport and gas exchange in hydrogels and for the development of future cellular microenvironments based on Si-HPMC or similar polysaccharide hydrogels.

Expression of Phosphate Transporters during Dental Mineralization.

The importance of phosphate (Pi) as an essential component of hydroxyapatite crystals suggests a key role for membrane proteins controlling Pi uptake during mineralization in the tooth. To clarify the involvement of the currently known Pi transporters (Slc17a1, Slc34a1, Slc34a2, Slc34a3, Slc20a1, Slc20a2, and Xpr1) during tooth development and mineralization, we determined their spatiotemporal expression in murine tooth germs from embryonic day 14.5 to postnatal day 15 and in human dental samples from Nolla stages 6 to 9. Using real-time polymerase chain reaction, in situ hybridization, immunohistochemistry, and X-gal staining, we showed that the expression of Slc17a1, Slc34a1, and Slc34a3 in tooth germs from C57BL/6 mice were very low. In contrast, Slc34a2, Slc20a1, Slc20a2, and Xpr1 were highly expressed, mostly during the postnatal stages. The expression of Slc20a2 was 2- to 10-fold higher than the other transporters. Comparable results were obtained in human tooth germs. In mice, Slc34a2 and Slc20a1 were predominantly expressed in ameloblasts but not odontoblasts, while Slc20a2 was detected neither in ameloblasts nor in odontoblasts. Rather, Slc20a2 was highly expressed in the stratum intermedium and the subodontoblastic cell layer. Although Slc20a2 knockout mice did not show enamel defects, mutant mice showed a disrupted dentin mineralization, displaying unmerged calcospherites at the mineralization front. This latter phenotypical finding raises the possibility that Slc20a2 may play an indirect role in regulating the extracellular Pi availability for mineralizing cells rather than a direct role in mediating Pi transport through mineralizing plasma cell membranes. By documenting the spatiotemporal expression of Pi transporters in the tooth, our data support the possibility that the currently known Pi transporters may be dispensable for the initiation of dental mineralization and may rather be involved later during the tooth mineralization scheme.

Enriching a cellulose hydrogel with a biologically active marine exopolysaccharide for cell-based cartilage engineering.

The development of biologically and mechanically competent hydrogels is a prerequisite in cartilage engineering. We recently demonstrated that a marine exopolysaccharide, GY785, stimulates the in vitro chondrogenesis of adipose stromal cells. In the present study, we thus hypothesized that enriching our silated hydroxypropyl methylcellulose hydrogel (Si-HPMC) with GY785 might offer new prospects in the development of scaffolds for cartilage regeneration. The interaction properties of GY785 with growth factors was tested by surface plasmon resonance (SPR). The biocompatibility of Si-HPMC/GY785 towards rabbit articular chondrocytes (RACs) and its ability to maintain and recover a chondrocytic phenotype were then evaluated in vitro by MTS assay, cell counting and qRT-PCR. Finally, we evaluated the potential of Si-HPMC/GY785 associated with RACs to form cartilaginous tissue in vivo by transplantation into the subcutis of nude mice for 3 weeks. Our SPR data indicated that GY785 was able to physically interact with BMP-2 and TGFß. Our analyses also showed that three-dimensionally (3D)-cultured RACs into Si-HPMC/GY785 strongly expressed type II collagen (COL2) and aggrecan transcripts when compared to Si-HPMC alone. In addition, RACs also produced large amounts of extracellular matrix (ECM) containing glycosaminoglycans (GAG) and COL2. When dedifferentiated RACs were replaced in 3D in Si-HPMC/GY785, the expressions of COL2 and aggrecan transcripts were recovered and that of type I collagen decreased. Immunohistological analyses of Si-HPMC/GY785 constructs transplanted into nude mice revealed the production of a cartilage-like extracellular matrix (ECM) containing high amounts of GAG and COL2. These results indicate that GY785-enriched Si-HPMC appears to be a promising hydrogel for cartilage tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.

Cartilage tissue engineering: From biomaterials and stem cells to osteoarthritis treatments.

Articular cartilage is a non-vascularized and poorly cellularized connective tissue that is frequently damaged as a result of trauma and degenerative joint diseases such as osteoarthrtis. Because of the absence of vascularization, articular cartilage has low capacity for spontaneous repair. Today, and despite a large number of preclinical data, no therapy capable of restoring the healthy structure and function of damaged articular cartilage is clinically available. Tissue-engineering strategies involving the combination of cells, scaffolding biomaterials and bioactive agents have been of interest notably for the repair of damaged articular cartilage. During the last 30 years, cartilage tissue engineering has evolved from the treatment of focal lesions of articular cartilage to the development of strategies targeting the osteoarthritis process. In this review, we focus on the different aspects of tissue engineering applied to cartilage engineering. We first discuss cells, biomaterials and biological or environmental factors instrumental to the development of cartilage tissue engineering, then review the potential development of cartilage engineering strategies targeting new emerging pathogenic mechanisms of osteoarthritis.

Vascular imaging with contrast agent in hard and soft tissues using microcomputed-tomography.

Vascularization is essential for many tissues and is a main requisite for various tissue-engineering strategies. Different techniques are used for highlighting vasculature, in vivo and ex vivo, in 2-D or 3-D including histological staining, immunohistochemistry, radiography, angiography, microscopy, computed tomography (CT) or micro-CT, both stand-alone and synchrotron system. Vascularization can be studied with or without a contrast agent. This paper presents the results obtained with the latest Skyscan micro-CT (Skyscan 1272, Bruker, Belgium) following barium sulphate injection replacing the bloodstream in comparison with results obtained with a Skyscan In Vivo 1076. Different hard and soft tissues were perfused with contrast agent and were harvested. Samples were analysed using both forms of micro-CT, and improved results were shown using this new micro-CT. This study highlights the vasculature using micro-CT methods. The results obtained with the Skyscan 1272 are clearly defined compared to results obtained with Skyscan 1076. In particular, this instrument highlights the high number of small vessels, which were not seen before at lower resolution. This new micro-CT opens broader possibilities in detection and characterization of the 3-D vascular tree to assess vascular tissue engineering strategies.

Direct comparison of current cell-based and cell-free approaches towards the repair of craniofacial bone defects - A preclinical study.

For craniofacial bone defect repair, several alternatives to bone graft (BG) exist, including the combination of biphasic calcium phosphate (BCP) biomaterials with total bone marrow (TBM) and bone marrow-derived mesenchymal stromal cells (MSCs), or the use of growth factors like recombinant human bone morphogenic protein-2 (RhBMP-2) and various scaffolds. Therefore, clinicians might be unsure as to which approach will offer their patients the most benefit. Here, we aimed to compare different clinically relevant bone tissue engineering methods in an "all-in-one" study in rat calvarial defects. TBM, and MSCs committed or not, and cultured in two- or three-dimensions were mixed with BCP and implanted in bilateral parietal bone defects in rats. RhBMP-2 and BG were used as positive controls. After 7 weeks, significant de novo bone formation was observed in rhBMP-2 and BG groups, and in a lesser amount, when BCP biomaterials were mixed with TBM or committed MSCs cultured in three-dimensions. Due to the efficacy and safety of the TBM/BCP combination approach, we recommend this one-step procedure for further clinical investigation. STATEMENT OF SIGNIFICANCE: For craniofacial repair, total bone marrow (BM) and BM mesenchymal stem cell (MSC)-based regenerative medicine have shown to be promising in alternative to bone grafting (BG). Therefore, clinicians might be unsure as to which approach will offer the most benefit. Here, BM and MSCs committed or not were mixed with calcium phosphate ceramics (CaP) and implanted in bone defects in rats. RhBMP-2 and BG were used as positive controls. After 7 weeks, significant bone formation was observed in rhBMP-2 and BG groups, and when CaP were mixed with BM or committed MSCs. Since the BM-based procedure does not require bone harvest or cell culture, but provides de novo bone formation, we recommend consideration of this strategy for craniofacial applications.

Age-related changes in the articular cartilage of the stifle joint in non-working and working German Shepherd dogs.

The aims of this study were to define age-related histological changes in the articular cartilage of the stifle joint in non-chondrodystrophic dogs and to determine whether physical activity has a positive impact on preservation of cartilage structure during ageing. Twenty-eight German shepherd dogs were included in the study. These dogs had no evidence of joint inflammation as defined by clinical assessment, radiology and synovial fluid analysis (specifically absence of synovial fluid serum amyloid A). The dogs were grouped as young working (n » 4), young non-working (n » 5), aged working (n » 13) and aged non-working (n » 6) animals. Gross changes in the stifle joints were recorded and biopsy samples of femoral and tibial articular cartilage were evaluated for thickness; chondrocyte number, density, surface area and morphology; isogenous group morphology; tidemark integrity; subchondral bone structure; presence of proteoglycans/ glycosaminoglycans; and expression of type I, II and X collagens. The major age-related changes, not related to type of physical activity, included elevated chondrocyte density and thinning of tibial cartilage and increased chondrocyte surface area in the superficial and intermediate zone of the femoral cartilage. There was also expression of type X collagen in the femoral and tibial calcified and non-calcified cartilage; however, type X collagen was not detected in the superficial zone of old working dogs. Therefore, ageing, with or without physical activity, leads to slight cartilage degeneration, while physical activity modulates the synthesis of type X collagen in the superficial cartilage zone, partially preserving the structure of hyaline cartilage.

Evaluation of new bone formation in irradiated areas using association of mesenchymal stem cells and total fresh bone marrow mixed with calcium phosphate scaffold.

The consequences of the treatment of the squamous cell carcinomas of the upper aerodigestive tract (bone removal and external radiation therapy) are constant. Tissue engineering using biphasic calcium phosphate (BCP) and mesenchymal stem cells (MSC) is considered as a promising alternative. We previously demonstrated the efficacy of BCP and total fresh bone marrow (TBM) in regenerating irradiated bone defect. The aim of this study was to know if adding MSC to BCP + TBM mixture could improve the bone formation in irradiated bone defects. Twenty-four Lewis 1A rats received a single dose of 20 Gy to the hind limbs. MSC were sampled from non-irradiated donors and amplified in proliferative, and a part in osteogenic, medium. 3 weeks after, defects were created on femurs and tibias, which were filled with BCP alone, BCP + TBM, BCP + TBM + uncommitted MSC, or BCP + TBM + committed MSC. 3 weeks after, samples were removed and prepared for qualitative and quantitative analysis. The rate of bone ingrowth was significantly higher after implantation of BCP + TBM mixture. The adding of a high concentration of MSC, committed or not, didn't improve the bone regeneration. The association BCP + TBM remains the most efficient material for bone substitution in irradiated areas.

Osteoinduction of biphasic calcium phosphate scaffolds in a nude mouse model.

Bioceramics combined with isolated stem cells, or with total bone marrow, constitute the main strategies under consideration in the field of bone tissue engineering. In the present preclinical study, two biphasic calcium phosphate scaffolds currently on the market, MBCP® and MBCP+®, with different hydroxyapatite/ß-tricalcium phosphate ratio, were implanted ectopically in a nude mouse model. These scaffolds were supplemented either with human mesenchymal stromal cells, or with human total bone marrow, or rat total bone marrow. Biomaterials alone were found to have potentially low, but non-zero, osteoinductive properties, while biomaterials associated with total bone marrow consistently improved osteoinduction in comparison with high concentrations of isolated human stromal cells.

Controlling the biological function of calcium phosphate bone substitutes with drugs.

There is a growing interest in bone tissue engineering for bone repair after traumatic, surgical or pathological injury, such as osteolytic tumor or osteoporosis. In this regard, calcium phosphate (CaP) bone substitutes have been used extensively as bone-targeting drug-delivery systems. This localized approach improves the osteogenic potential of bone substitutes by delivering bone growth factors, thus extending their biofunctionality to any pathological context, including infection, irradiation, tumor and osteoporosis. This review briefly describes the physical and chemical processes implicated in the preparation of drug-delivering CaPs. It also describes the impact of these processes on the intrinsic properties of CaPs, especially in terms of the drug-release profile. In addition, this review focuses on the potential influence of drugs on the resorption rate of CaPs. Interestingly, by modulating the resorption parameters of CaP biomaterials, it should be possible to control the release of bone-stimulating ions, such as inorganic phosphate, in the vicinity of bone cells. Finally, recent in vitro and in vivo evaluations are extensively reported.

Health claims assessment in the field of joint and cartilage: a consensus viewpoint of the Group for the Respect of Ethics and Excellence in Science.

INTRODUCTION: In 2006, the European Parliament and Council issued a regulation (No. 1924/2006) for the nutrition and health claims made on foods, including food supplements. According to the regulation, the use of nutrition and health claims shall only be permitted if the substance in respect of which the claim is made has been shown to have a beneficial nutritional or physiological effect. In the field of joint and cartilage health, there is no clear scientific-based definition of the nature of such a beneficial nutritional or physiological effect. The objective of this paper is to scientifically define the possible content of health claims related to joint and cartilage health and to provide scientific guidelines for the design of clinical studies which need to be adopted to substantiate such health claims. METHODS: Literature review up to September 2011 followed by a consensus expert discussion organized by the Group for the Respect of Ethics and Excellence in Science (GREES). RESULTS: In line with the general principles of the PASSCLAIM and the Codex recommendations, the GREES identified four acceptable health claims related to joint and cartilage health based on the effects on discomfort, joint and cartilage structural integrity or risk factors for joint and cartilage diseases. The GREES considers that randomized controlled trials on a relevant outcome is the best design to assess health claims. Moreover, animal studies could also be of interest to substantiate some health claims, to assess the clinical relevance of endpoints used in human studies or to extrapolate data obtained in patients to the target (apparently) healthy population. CONCLUSION: According to the methodology and biomarkers used in the study and whether or not additional animal studies are provided to support the claim, various health claims can be acceptable in the field of joint and cartilage health.

In vivo experimental imaging of osteochondral defects and their healing using (99m)Tc-NTP 15-5 radiotracer.

PURPOSE: A rabbit model of osteochondral defects (OD) and spontaneous healing was longitudinally followed over 12 weeks, by in vivo joint scintigraphy using (99m)Tc-NTP 15-5, and histology. METHODS: We used two models, one with one OD (OD1 group) in the femoral condyle of one knee and the other with two ODs (OD2 group) in the femoral condyle of one knee, with the contralateral knees serving as the reference. A serial longitudinal imaging study was performed with the scintigraphic ratio (SR, operated knee uptake/contralateral knee uptake) determined at each time-point. RESULTS: ODs were imaged as radioactive defects. The SR was decreased with respective to controls, with values of 0.73 ± 0.08 and 0.65 ± 0.07 in the OD1 and OD2 groups, respectively, at 4 weeks after surgery. Histology of both OD groups revealed the presence of repair tissue characterized by a small amount of sulphated glycosaminoglycans and collagen. CONCLUSION: (99m)Tc-NTP 15-5 imaging provided quantitative criteria useful for in vivo evaluation of cartilage trauma and healing.

Molecular effects of gallium on osteoclastic differentiation of mouse and human monocytes.

We had previously reported that gallium (Ga) inhibited both the differentiation and resorbing activity of osteoclasts in a dose-dependent manner. To provide new insights into Ga impact on osteoclastogenesis, we investigated here the molecular mechanisms of Ga action on osteoclastic differentiation of monocytes upon Rankl treatment. We first observed that Ga treatment inhibited the expression of Rankl-induced early differentiation marker genes, while the same treatment performed subsequently did not modify the expression of late differentiation marker genes. Focusing on the early stages of osteoclast differentiation, we observed that Ga considerably disturbed both the initial induction as well as the autoamplification step of Nfatc1 gene. We next demonstrated that Ga strongly up-regulated the expression of Traf6, p62 and Cyld genes, and we observed concomitantly an inhibition of IκB degradation and a blockade of NFκB nuclear translocation, which regulates the initial induction of Nfatc1 gene expression. In addition, Ga inhibited c-Fos gene expression, and subsequently the auto-amplification stage of Nfatc1 gene expression. Lastly, considering calcium signaling, we observed upon Ga treatment an inhibition of calcium-induced Creb phosphorylation, as well as a blockade of gadolinium-induced calcium entry through TRPV-5 calcium channels. We identify for the first time Traf6, p62, Cyld, IκB, NFκB, c-Fos, and the calcium-induced Creb phosphorylation as molecular targets of Ga, this tremendously impacting the expression of the master transcription factor Nfatc1. In addition, our results strongly suggest that the TRPV-5 calcium channel, which is located within the plasma membrane, is a target of Ga action on human osteoclast progenitor cells.