Nathalie Rochet

Reverse Shoulder Arthroplasty Combined with a Modified Latissimus Dorsi and Teres Major Tendon Transfer for Shoulder Pseudoparalysis Associated with Dropping Arm

AbstractAlthough a reverse shoulder arthroplasty (RSA) can restore active elevation in the cuff deficient shoulder, it cannot restore active external rotation when both the infraspinatus and teres minor muscles are absent or atrophied. We hypothesized that a latissimus dorsi and teres major (LD/TM) transfer with a concomitant RSA would restore shoulder function and activities of daily living (ADLs). We prospectively followed 11 consecutive patients (mean age, 70xa0years) with a combined loss of active elevation and external rotation (shoulder pseudoparalysis and dropping arm) who underwent this procedure. All had severe cuff tear arthropathy (Hamada Stage 3, 4, or 5) and severe atrophy or fatty infiltration of infraspinatus and teres minor on preoperative MRI or CT-scan. The combined procedure was performed through a single deltopectoral approach in the same session. Postoperatively, mean active elevation increased from 70° to 148° (+78°) and external rotation from −18° to 18° (+36°). The Constant score, subjective assessment and ADLs improved. The combination of a RSA and LD/TM transfer restored both active elevation and external rotation in this selected subgroup of patients with a cuff deficient shoulder and absent or atrophied infraspinatus and teres minor.n Level of Evidence: Level IV, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence.

Effects of low-level laser therapy on proliferation and differentiation of murine bone marrow cells into osteoblasts and osteoclasts.

Low‐Level Laser Therapy (LLLT) has been suggested to improve bone tissue healing. The cellular and molecular mechanisms involved in this effect are still unclear but bone cell proliferation and differentiation alteration have been proposed. The aim of the present study was to investigate, in vitro, the effect of LLLT on bone cell proliferation, osteoblastic and osteoclastic differentiation, both involved in bone remodeling and regeneration.

Activation of hedgehog signaling inhibits osteoblast differentiation of human mesenchymal stem cells.

Mesenchymal stem cells within the bone are responsible for the generation of osteoblasts, chondrocytes, and adipocytes. In rodents, Indian hedgehog has been shown to play a role in osteoblast differentiation. However, evidence for a direct function of hedgehog (Hh) in human osteoblastic differentiation is missing. Using different models of human mesenchymal stem cells we show that Hh signaling decreases during osteoblast differentiation. This is associated with a decrease in Smoothened expression, a key partner that triggers Hh signaling, and in the number of cells displaying a primary cilium, an organelle necessary for Hh signaling. Remarkably, treatment of human mesenchymal stem cells with sonic hedgehog or two molecules able to activate Hh signaling inhibits osteoblast differentiation. This inhibition is visualized through a decrease in mineralization and in the expression of osteoblastic genes. In particular, activation of Hh signaling induces a decrease in Runx2 expression, a key transcriptional factor controlling the early stage of osteoblast differentiation. Consistently, the activation of Hh signaling during the first days of differentiation is sufficient to inhibit osteoblast differentiation, whereas differentiated osteoblasts are not affected by Hh signaling. In summary, we show here, using various inducers of Hh signaling and mesenchymal stem cells of two different origins, that Hh signaling inhibits human osteoblast differentiation, in sharp contrast to what has been described in rodent cells. This species difference should be taken into account for screening for pro‐osteogenic molecules. STEM CELLS 2009;27:703–713

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.

Establishment, characterisation and partial cytokine expression profile of a new human osteosarcoma cell line (CAL 72)

Permanent human osteosarcoma cell lines are important tools for the study of bone cancer. As representative of an osteoblastic phenotype, they partly reflect their normal osteoblastic counterparts and, thus, may represent appropriate models to investigate the mechanisms involved in bone remodelling and in haematopoietic differentiation. In the present work, we describe a new human cell line, CAL 72, obtained from an osteosarcoma of the knee of a 10‐year‐old boy. These cells grow in continuous culture, and karyotypic analysis has revealed clonal abnormalities in number and structure, especially loss of chromosome Y. These cells exhibit morphological, immuno‐histochemical and molecular characteristics of the osteoblastic lineage. Using RT‐PCR, we have shown that the CAL 72 cell line expresses high levels of mRNA coding for several cytokines, such as G‐CSF, GM‐CSF, IL‐1β and IL‐6. In view of this expression profile, the CAL 72 phenotype appears to be closer to normal primary osteoblasts than other reported osteosarcomas. Moreover, these cells express mRNA for both HGF and its receptor c‐MET, suggesting that this autocrine loop might contribute to the invasiveness of the tumour from which CAL 72 originated. Int. J. Cancer82:282–285, 1999.

Evidence for a p23 caspase-cleaved form of p27[KIP1] involved in G1 growth arrest.

p27[KIP1] (p27) is a cyclin dependent kinase inhibitor, involved in the negative regulation of G1 progression in response to a number of anti-proliferative signals. In this study we show, in growing mouse hybridoma (7TD1) and human myeloma (U266) cell lines, that p27 is highly expressed but slightly upregulated when cells are arrested, regardless to the phases of the cell cycle. In contrast, the specific blockade of these cells in early G1 phase reveals the induction of a protein of 23u2009kDa (p23) specifically recognized by polyclonal anti-p27 antibodies raised against the NH2 terminal part of p27 but not by anti-p21[CIP1] antibodies. Experiments using caspase inhibitors strongly suggest that p23 results from the proteolysis of p27 by a `caspase-3-like protease. This cleavage leads to the cytosolic sequestration of p23 but does not alter its binding properties to CDK2 and CDK4 kinases. Indeed, p23 associated in vivo with high molecular weight complexes and coprecipitated with CDK2 and CDK4. We demonstrate by transfection experiments in SaOS-2 cells that p23 induces a G1 phase growth arrest by inhibition of cyclin/CDK2 activity. In summary we describe here a caspase-cleaved form of p27, induced in absence of detectable apoptosis and likely involved in cell cycle regulation.

Postnatal Soluble FGFR3 Therapy Rescues Achondroplasia Symptoms and Restores Bone Growth in Mice

A recombinant soluble fibroblast growth factor receptor 3 (FGFR3) restored normal skeletal growth and prevented disease-related complications in a mouse model of achondroplasia. Receptor Decoy Restores Bone Growth Achondroplasia is a rare disease where bone growth is stunted and cartilage does not form correctly. It is caused by a mutation in the gene that encodes fibroblast growth factor receptor 3 (FGFR3), which leads to overactive receptor signaling. Yet, despite knowing the cause, a treatment has not been discovered. In an innovative approach, Garcia and colleagues used receptor “decoys” to prevent the FGF ligand from binding its mutated receptor, thus interrupting the signaling cascade and restoring bone growth in mice. Normal mice or mice with the mutation in the gene encoding FGFR (Fgfr3ach/+) were treated with recombinant, soluble FGFR3 (sFGFR3) or a vehicle control for 3 weeks. More than 60% of Fgfr3ach/+ mice that were left untreated died during the treatment period, whereas only 12% of sFGFR3-treated mice died from achondroplasia-related complications, such as respiratory distress or paraplegia. In the surviving Fgfr3ach/+ animals, those treated with sFGFR3 had normal body and tail lengths, normal rib cage development, and decreased spinal and skull deformities—all similar to their healthy, wild-type counterparts. Untreated transgenic animals suffered from the defects common to achondroplasia: shortened stature, abnormal rib cage structure, and spinal compression, leading to paraplegia and bladder dysfunction. The sFGFR3 therapy was not toxic to the animals and did not affect reproduction (in fact, by increasing pelvis size in treated transgenic females, litter sizes were normal). Additional preclinical studies will be needed to see if this is a viable long-term treatment for achondroplasia, but with a long half-life and promising early outcomes in animals, this FGFR decoy may be a viable postnatal treatment for translation. Achondroplasia is a rare genetic disease characterized by abnormal bone development, resulting in short stature. It is caused by a single point mutation in the gene coding for fibroblast growth factor receptor 3 (FGFR3), which leads to prolonged activation upon ligand binding. To prevent excessive intracellular signaling and rescue the symptoms of achondroplasia, we have developed a recombinant protein therapeutic approach using a soluble form of human FGFR3 (sFGFR3), which acts as a decoy receptor and prevents FGF from binding to mutant FGFR3. sFGFR3 was injected subcutaneously to newborn Fgfr3ach/+ mice—the mouse model of achondroplasia—twice per week throughout the growth period during 3 weeks. Effective maturation of growth plate chondrocytes was restored in bones of treated mice, with a dose-dependent enhancement of skeletal growth in Fgfr3ach/+ mice. This resulted in normal stature and a significant decrease in mortality and associated complications, without any evidence of toxicity. These results describe a new approach for restoring bone growth and suggest that sFGFR3 could be a potential therapy for children with achondroplasia and related disorders.

Characterization of a Novel Bipotent Hematopoietic Progenitor Population in Normal and Osteopetrotic Mice

Several reports indicate that osteoclasts and B‐lymphocytes share a common progenitor. This study focuses on the characterization of this bipotent progenitor from the bone marrow of the osteopetrotic oc/oc mouse, where the bipotent progenitor population is amplified, and of normal mice.

Early G1 growth arrest of hybridoma b cells by DMSO involves cyclin D2 inhibition and p21|CIP1| induction

Dimethylsulfoxide (DMSO) was shown to inhibit the proliferation of several B cell lines including Raji, Daudi, and SKW6-CL4 but the mechanisms involved in this growth arrest are still unclear. We show that in 7TD1 mouse hybridoma cells a DMSO-induced reversible G1 arrest involves inactivation of Rb kinases, cyclin D2/CDK4 and cyclin E/CDK2. This occurs by at least three distinct mechanisms. Inhibition of cyclin D2 neosynthesis leads to a dramatic decrease of cyclinD2/CDK4 complexes. This in turn enables the redistribution of p27[KIP1] from cyclin D2/CDK4 to cyclin E/CDK2 complexes. In addition, the simultaneous accumulation of p21[CIP1] entails increasing association with cyclin D3/CDK4 and cyclin E/CDK2. Thus, p21[CIP1] and p27[KIP1], act in concert to inhibit cyclin E/CDK2 activity which, together with CDK4 inactivation, confers a G1-phase arrest.

Modification of gene expression induced in human osteogenic and osteosarcoma cells by culture on a biphasic calcium phosphate bone substitute

Bone hybrids made of bioceramics seeded with mesenchymal or osteoblastic cells are very promising alternatives to autologous bone graft. Along this line, the development of in vitro models, dedicated to analyze the influence of these biomaterials on osteogenic cells, will help to improve the performance of these bone substitutes. In the present work we analyzed the effects of a macroporous biphasic calcium phosphate ceramic (BCP, Triosite) on three different human osteosarcoma cell lines and on human primary osteogenic cells and compared this culture substratum to traditional culture on plastic. We showed that all these osteoblastic cells adhere and proliferate on the trabecular BCP blocks, with a different spatial organization for osteosarcoma cells compared to normal osteogenic cells. We also demonstrated that osteoblastic marker genes such as Cbfa1, type I collagen, osteonectin, osteopontin, and osteocalcin were expressed at similar levels by these cells cultured on either substratum, suggesting that adhesion to BCP does maintain the osteoblastic phenotype of these cells. Next, we provided the first evidence of differences of cytokine expression profiles revealed on this Ca-P ceramic as compared to expression in classical culture. These modifications affected the expression of cytokines such as TGF-beta1, G-CSF, and IL-3 and were quantitatively different between osteosarcoma cells and normal osteogenic cells. Given the role of these cytokines in bone biology and in hematopoiesis, these results obtained in vitro suggest that the BCP ceramic studied here could stimulate osteogenesis in vivo by activating cellular processes during bone formation and healing. This study highlights the notion that the nature of the culture substratum must be taken into account when studying bone cell biology in vitro. Owing to the nature and spatial organization of the BCP, our hypothesis is that culture on BCP is closer to the physiological situation than culture on plastic.