SummaryFibrin-based biomaterial preparations can be used as provisional growth matrices for cells important in tissue repair during wound healing in vivo. Their efficacy can be enhanced by including bioactive agents that promote specific cellular responses. This study examined the controlled delivery of the angiogenic growth factors bFGF, VEGF165, and VEGF121 using biomatrix preparations prepared from Fibrin Sealant product components. The growth factors were added prior to formation of the Fibrin Sealant clots, and the release kinetics of the proteins from the clots measured. The results indicated that the proteins were released from the clots more slowly in the order bFGF << VEGF165 < VEGF121. The biologic activity of the growth factors delivered from Fibrin Sealant clots was established by assaying growth stimulation of human microvascular endothelial cells (HMVEC) and angiogenesis in the chicken embryo chorioallantoic membrane (CAM) model of neovascularization. In the latter assay, clots containing bFGF, VEGF165, or V EGF121 all displayed angiogenic activity. However, delivery of either bFGF, VEGF165, or VEGF121 alone resulted in a significant percentage of clots becoming filled with blood, indicating that the newly developing vessels invading the clots were leaky and immature. In contrast, this hemorrhaging behavior did not occur with delivery of combinations, e.g., (VEGF165 + VEGF121) or (VEGF165 + bFGF), indicating that the vessels were more mature than those produced in response to single growth factors. Thus, delivering a combination of growth factors constituted an improvement over the delivery of individual growth factors for enhancing neovascularization.Part of this research was presented at the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society held in Munich, Germany.

Some biomaterials are able to induce ectopic bone formation in muscles of large animals. The osteoinductive potential of macro- micro-porous biphasic calcium phosphate (MBCP) ceramic granules with fibrin glue was evaluated by intramuscular implantation for 6 months in six adult female sheep. The MBCP granules were 1-2 mm in size and were composed of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) in a 60/40 ratio. The fibrin glue was composed of fibrinogen, thrombin and other biological factors. After 6 months of implantation in the dorsal muscles of sheep, the explants were rigid. Histology, back-scattered electron microscopy and micro-computed tomography of the implants indicated that approximately 12% of mineralized bone had formed in between the MBCP granules. The ectopic bone appeared well-mineralized with mature osteocytes and Haversian structures. In addition, the number and thickness of bone trabeculae formed in between the MBCP particles were similar to those measured in trabecular bone in sheep. The overall results therefore confirmed the formation of well-mineralized ectopic bone tissue after intramuscular implantation of MBCP/fibrin glue composites. These bone substitutes exhibiting osteoinductive properties could be used for the reconstruction of large bone defects.

An ageing population implies an increase in bone and dental diseases, which are in turn a source of numerous handicaps. These pathologies are an expensive burden for the European health system. As no specific bioactive materials are efficient enough to cope with this burden, we have to develop an injectable, mouldable, self-hardening bone substitute to support bone tissue reconstruction and augmentation. New, highly bioactive and suitable biomaterials have been developed to replace bone grafts in orthopedic revision and maxillofacial surgery for bone augmentation. These mouldable, self-hardening materials are based on the association of MBCP Biphasic Calcium Phosphate Granules and Tissucol Fibrin Sealant. The in vivo evaluation of ingrowth in relation to the composite was made in an experiment on rabbits. The results indicate that in the presence of fibrin sealant, newly-formed bone developed at a small distance from the surface of the calcium phosphate ceramic. Two different bone apposition processes were identified. Without the fibrin component (MBCP group), bone rested directly on the surface of the granules. This observation is commonly described as osteoconduction in calcium phosphate materials. On the contrary, the presence of the fibrinogen component seemed to modify this standard osteoconduction phenomenon: the newly-formed bone essentially grew at a distance from the surface of the granules, on the fibrillar network, and could be considered as an inductive phenomenon for osteogenic cell differentiation from mesenchymal stem cells.

Calcium phosphate (Ca-P) ceramics are currently used in various types of orthopaedic and maxillofacial applications because of their osteoconductive properties. Fibrin glue is also used in surgery due to its haemostatic, chemotactic and mitogenic properties and also as scaffolds for cell culture and transplantation. In order to adapt to surgical sites, bioceramics are shaped in blocks or granules and preferably in porous forms. Combining these bioceramics with fibrin glue provides a mouldable and self-hardening composite biomaterial. The aim of this work is to study the osteogenic properties of this composite material using two different animal models. The formation of newly formed bone (osteoinduction) and bone healing capacity (osteconduction) have been study in the paravertebral muscles of sheep and in critical sized defects in the femoral condyle of rabbits, respectively. The different implantations sites were filled with composite material associating Ca-P granules and fibrin glue. Ca-P granules of 1-2 mm were composed with 60% of hydroxyapatite and 40% of beta tricalcium phosphate in weight. The fibrin glue was composed of fibrinogen, thrombin and other biological factors. After both intramuscular or intraosseous implantations for 24 weeks and 3, 6, 12 and 24 weeks, samples were analyzed using histology and histomorphometry and mechanical test. In all cases, the newly formed bone was observed in close contact and around the ceramic granules. Depending on method of quantification, 6.7% (with BSEM) or 17% (with micro CT) of bone had formed in the sheep muscles and around 40% in the critical sized bone rabbit defect after 24 weeks. The Ca-P/fibrin material could be used for filling bone cavities in various clinical indications.

Hybrid constructs associating a biodegradable matrix and autologous chondrocytes hold promise for the treatment of articular cartilage defects. In this context, our objective was to investigate the potential use of nasal chondrocytes associated with a fibrin sealant for the treatment of articular cartilage defects. The phenotype of primary nasal chondrocytes (NC) from human (HNC) and rabbit (RNC) origin were characterized by RT-PCR. The ability of constructs associating fibrin sealant and NC to form a cartilaginous tissue in vivo was investigated, firstly in a subcutaneous site in nude mice and secondly in an articular cartilage defect in rabbit. HNC express type II collagen and aggrecan, the two major hallmarks of a chondrocytic phenotype. Furthermore, when injected subcutaneously into nude mice within a fibrin sealant, these chondrocytes were able to form a cartilage-like tissue. Our data indicate that RNC also express type II collagen and aggrecan and maintained their phenotype in three-dimensional culture within a fibrin sealant. Moreover, treatment of rabbit articular cartilage defects with autologous RNC embedded in a fibrin sealant led to the formation of a hyalin-like repair tissue. The use of fibrin sealant containing hybrid autologous NC therefore appears as a promising approach for cell-based therapy of articular cartilage.