Hervé Petite

Tissue-engineered bone regeneration.

Bone lesions above a critical size become scarred rather than regenerated, leading to nonunion. We have attempted to obtain a greater degree of regeneration by using a resorbable scaffold with regeneration-competent cells to recreate an embryonic environment in injured adult tissues, and thus improve clinical outcome. We have used a combination of a coral scaffold with in vitro-expanded marrow stromal cells (MSC) to increase osteogenesis more than that obtained with the scaffold alone or the scaffold plus fresh bone marrow. The efficiency of the various combinations was assessed in a large segmental defect model in sheep. The tissue-engineered artificial bone underwent morphogenesis leading to complete recorticalization and the formation of a medullary canal with mature lamellar cortical bone in the most favorable cases. Clinical union never occurred when the defects were left empty or filled with the scaffold alone. In contrast, clinical union was obtained in three out of seven operated limbs when the defects were filled with the tissue-engineered bone.

A biodegradable fibrin scaffold for mesenchymal stem cell transplantation.

A potential therapy to enhance healing of bone tissue is to deliver isolated mesenchymal stem cells (MSCs) to the site of a lesion to promote bone formation. A key issue within this technology is the development of an injectable system for the delivery of MSCs. Fibrin gel exploits the final stage of the coagulation cascade in which fibrinogen molecules are cleaved by thrombin, convert into fibrin monomers and assembled into fibrils, eventually forming fibers in a three-dimensional network. This gel could have many advantages as a cell delivery vehicle in terms of biocompatibility, biodegradation and hemostasis. The objective of this study was to explore the possibility of using fibrin gel as a delivery system for human MSCs (HMSCs). To this end we have determined the optimal fibrinogen concentrations and thrombin activity for loading HMSCs in vitro into the resultant fibrin gels to obtain cell proliferation. We found that a concentration of 18 mg/ml of fibrinogen and a thrombin activity of 100 IU/ml was optimal for producing fibrin scaffolds that would allow good HMSCs spreading and proliferation. In these conditions, cells were able to proliferate and expressed alkaline phosphatase, a bone marker, in vitro. When implanted in vivo, HMSCs were able to migrate out of the fibrin gel and invade a calcium carbonate based ceramic scaffold suggesting that fibrin gel could serve as a delivery system for HMSCs.

Fibroblast growth factor receptor 2 promotes osteogenic differentiation in mesenchymal cells via ERK1/2 and protein kinase C signaling.

Mesenchymal stem cells (MSCs) are able to differentiate into several lineages including osteoblasts. The signaling mechanisms involved in the osteogenic differentiation of MSCs are however not fully understood. We investigated the role of fibroblast growth factor receptor 2 (FGFR2) in osteoblast committment and differentiation of murine mesenchymal C3H10T1/2 cells stably transfected with wild type (WT) or activated FGFR2 due to Apert S252W genetic mutation (MT). WT FGFR2 slightly increased, whereas MT FGFR2 strongly increased, FGFR2 tyrosine phosphorylation, indicating activation of the receptor. WT and MT FGFR2 increased C3H10T1/2 cell proliferation but not survival. Both WT and MT FGFR2 increased early and late osteoblast gene expression and matrix mineralization. Forced expression of WT and MT FGFR2 also increased osteoblast gene expression in MC3T3-E1 calvaria osteoblasts. In both cell types, MT FGFR2 was more effective than WT FGFR2. In contrast, WT and MT FGFR2 decreased adipocyte differentiation of C3H10T1/2 cells. WT and MT FGFR2 induced ERK1/2 but not JNK or PI3K/AKT phosphorylation. MT, but not WT, also increased protein kinase C (PKC) activity. Pharmacological inhibition of ERK1/2 prevented cell proliferation induced by WT and MT FGFR2. Using dominant-negative ERK and PKCα vectors, we demonstrated that WT and MT FGFR2 promoted osteoblast gene expression through ERK1/2 and PKCα signaling, respectively. This study identifies FGFR2 as a novel regulatory molecule that promotes osteogenic differentiation in murine MSCs. The promoting effect of WT and MT FGFR2 is mediated by ERK1/2 and PKCα pathways that play essential and distinct roles in FGFR2-induced osteogenic differentiation of mesenchymal cells.

CURRENT TRENDS IN THE ENHANCEMENT OF FRACTURE HEALING

Repair of fractures involves a sequence of dynamic events which ultimately restores the integrity of the bone and its biomechanical properties. In some cases healing is compromised, leading to delayed union or nonunion. It is estimated that 10% of the fractures which occur annually will require further surgical procedures because of impaired healing. The preferred management of nonunion and bone defects is by autologous cancellous bone grafting, because this provides the essential elements for bone formation, namely living osteogenic cells, bone-inductive proteins which stimulate cell proliferation and differentiation, and a scaffold of apatite which supports the ingrowth of newly formed bone. The supply of suitable bone, however, is limited and its harvest results in additional morbidity to the donor site, leading to pain, haematoma, or infection. For more than 30 years investigators have been developing alternative treatments, by physical or biological methods, aimed at providing the benefits of bone grafting without the complications related to harvest of an autograft. The physical strategy includes the use of mechanical stimulation, electromagnetic fields, and low-intensity ultrasound. The biological approach involves the use of osteoconductive biomaterials, growth factors which stimulate tissue repair (including bone morphogenetic proteins), and osteocompetent cells. We will review the preclinical and clinical data on each therapeutic approach and discuss potential areas of development in the field of bone healing.

Experimental vertebroplasty using osteoconductive granular material.

STUDY DESIGNnOsteoporotic human cadaveric thoracic vertebral bodies and vertebral bodies from mature sheep were used as model systems to assess coral resorption and new bone formation after injection of coral granules.nnnOBJECTIVEnTo evaluate the use of natural coral exoskeleton, an osteoconductive material, for the filling of vertebral bodies.nnnSUMMARY OF BACKGROUND DATAnPercutaneous injection of polymethylmetacrylate (PMMA) is often proposed for prophylactically stabilizing osteoporotic vertebral bodies at risk for fracture or augmentation of vertebral bodies that have already fractured. Recently, the possibility of using osteoconductive materials in granular formulation was assessed in pilot studies.nnnMETHODSnAs a first step, the possibility of injecting coral granules percutaneously within osteoporotic human cadaveric thoracic vertebral bodies was assessed. As a second step, cavities were drilled into vertebral bodies of 10 mature ewes and were either left empty (control group) or filled with coral alone (CC) or coral supplemented with fibrin sealant (CC+FS). Quantitative evaluation of coral resorption and new bone formation was made 2 months and 4 months after implantation.nnnRESULTSnThe distribution of coral granules injected into human cadaveric thoracic vertebral bodies was homogenous as assayed radiographically. In the experimental animal model, osteogenesis was increased in cavities filled with coral in comparison with cavities left empty at both 2 months and 4 months (P < 0.005 and P < 0.02, respectively). Surprisingly, supplementation of coral with a fibrin sealant had no positive influence on osteogenesis (P < 0.0008 at 2 months; P < 0.002 at 4 months). In addition, it led to an increase in coral resorption by as soon as 2 months (P < 0.0008).nnnCONCLUSIONnThese results demonstrate the osteoconductivity of coral in granular form for vertebral filling. Interestingly, interconnectivity between adjacent bone trabeculae and newly formed bone was restored; however, its mechanical significance remains to be determined. Further investigations are needed to evaluate the efficacy of coral in osteopenic animals and in relieving pain.

Addition of fibrin sealant to ceramic promotes bone repair: Long‐term study in rabbit femoral defect model

Despite their impact on the healing of soft connective tissue, fibrin sealants have not been shown conclusively to have an important role in the healing of bone defects. We report the positive influence of fibrin sealants on repair of cancellous bone cavities filled with a porous, resorbable ceramic. We studied two fibrin sealants: Autocolle and Tissucol. Autocolle is enriched in platelet factors during its preparation. Tissucol is a commercially available fibrin glue prepared from pooled human plasma that has no enrichment in platelet factors. Cavities 10 mm in depth and 5 mm in diameter were drilled in lateral condyles of 45 New Zealand rabbits. These defects were filled with either coral granules or a mixture of fibrin sealant (Autocolle or Tissucol) and coral granules or left empty. At 1 month addition of a fibrin sealant (Autocolle or Tissucol) to the coral led to a significant increase in bone formation in comparison to coral alone. At 2 months significant fibrin sealant mediated enhancement of bone repair was observed with Autocolle only. At 6 months bone formation was similar to the adult bone amount in nonoperated animals, whatever the initial material. Control cavities, on the other hand, were invaded with fibrous tissue only at each time period.

Osteogenesis with coral is increased by BMP and BMC in a rat cranioplasty

Autologous bone marrow cells (BMC), bone morphogenetic protein (BMP) and natural coral exoskeleton (CC) were used to enhance the repair of large skull bone defects in a craniotomy model. Nine millimeter calvarial defects were created in adult rats and were either left empty (control defects) or implanted with CC alone, CC-BMC, CC-BMP, or CC-BMC-BMP. After 1 or 2 months, osteogenesis was insufficient to allow union when defects were left empty or filled with CC. Addition of BMC alone to CC had no positive influence on osteogenesis at any time and increased CC resorption at 2 months (0.1 +/- 0.1 mm2 versus 0.5 +/- 0.3 mm2). In contrast addition of BM P or BM P/BMC to CC led to a significant increase in osteogenesis and allowed bone union after 1 month. At 2 months, the combination of CC-BM P-BMC was the most potent activator of osteogenesis. Filling a defect with CC-BMP-BMC resulted in significantly increased bone surface area (11 +/- 2.7 mm2) in comparison to filling a defect with CC-BMP (7.0 +/- 1.4 mm2), CC-BMC (3.5 +/- 1.1 mm2) or CC (4.5 +/- 0.4 mm2). CC resorption was significantly decreased in the presence of BMP with or without BMC at both times. These data are in accordance with the presence of progenitor cells in bone marrow that are inducible by BMP to the osteogenic pathway in a cranial site. The increase in material resorption in defects filled with CC-BMC could suggest that cells from the granulocyte-macrophage lineage survived the grafting procedure and were still active after 2 months.

Cytocompatibility of calf pericardium treated by glutaraldehyde and by the acyl azide methods in an organotypic culture model

Glutaraldehyde (GTA) is used to cross-link collagen-based biomaterials, but these materials are often cytotoxic. In order to overcome this problem, we have proposed the use of the acyl azide methods with either hydrazine or diphenylphosphoryl azide (DPPA) as reagents. In this paper we determine the cytocompatibility of acyl azide- and GTA-treated pericardium in vitro, by an organotypic chick aorta culture technique developed for the evaluation of the propensity of vascular cells (both endothelial and smooth muscle cells) to migrate and grow on the surface of biomaterials. We first examined pericardium stabilization as a function of GTA concentration and time, so that we could minimize residual GTA molecules in the material. Treatment for 72 h with 0.05% GTA was optimal for thermal stabilization of the pericardium with a denaturation temperature (Td) of 86.8 degrees C, providing similar results to treatment with 0.6% GTA for 4 h (Td = 85.1 degrees C). Pericardium treated in this way was, however, poorly cytocompatible with little vascular cell migration and growth when compared with tissues treated by the acyl azide methods. The best results were obtained with 0.5% DPPA; treated tissues showed a high level of cross-linking (Td = 82.4 degrees C) and three-fold increases in cell growth and migration over those in a non-toxic control.

In vivo tracking of bone marrow fibroblasts with fluorescent carbocyanine dye.

Recent advances in the field of tissue engineering have culminated in new tissue substitutes that combine a biomaterial and precursor cells. The effectiveness of these materials is generally assessed in animals, but few studies explore the fate of the transplanted cells in vivo, despite its paramount importance for understanding the function of the engineered tissues. Current methods that use reporter genes or chimeric animals are not always well suited to solving tissue-engineering problems. We therefore developed a new method for irreversible labeling of cells to track their fate in vivo. We used a fluorescent lipophilic probe, CM-Dil, that avidly binds to the cell membrane. Human bone marrow stromal fibroblasts could be labeled with 20 microM CM-Dil in 30 min. The CM-Dil was not cytotoxic and did not affect cell proliferation in vitro. Cells could be monitored for up to 30 days when placed in a coral scaffold and implanted intramuscularly or in a bony site. However, the fluorescence intensity decreased roughly in parallel with the number of cell divisions. This fact needs to be taken into account during the design and interpretation of experiments. We believe that this technique is also of interest for other cell types.

Coral grafting supplemented with bone marrow

Limited success in regenerating large bone defects has been achieved by bridging them with osteoconductive materials. These substitutes lack the osteogenic and osteoinductive properties of bone autograft. A direct approach would be to stimulate osteogenesis in these biomaterials by the addition of fresh bone-marrow cells (BMC). We therefore created osteoperiosteal gaps 2 cm wide in the ulna of adult rabbits and either bridged them with coral alone (CC), coral supplemented with BMC, or left them empty. Coral was chosen as a scaffold because of its good biocompatibility and resorbability. In osteoperiosteal gaps bridged with coral only, the coral was invaded chiefly by fibrous tissue. It was insufficient to produce union after two months. In defects filled with coral and BMC an increase in osteogenesis was observed and the bone surface area was significantly higher compared with defects filled with coral alone. Bony union occurred in six out of six defects filled with coral and BMC after two months. An increase in the resorption of coral was also observed, suggesting that resorbing cells or their progenitors were present in bone marrow and survived the grafting procedure. Our findings have shown that supplementation of coral with BMC increased both the resorption of material and osteogenesis in defects of a clinical significance.