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.

[Intra-articular distal radial fracture with lunate fossa rotated, about 4 cases: interest of surgical procedure by volar medial approach]. / Fracture articulaire du radius distal avec fragment de fossette lunarienne retourné, à propos de 4 cas : intérêt du traitement chirurgical par voie antéro-médiale.

INTRODUCTION: Intra-articular distal radial fractures in young subjects occur in severe trauma. Articular reduction needs to be anatomical. We report four cases with the particularity of having a 90° or 180° rotated lunate fossa. Our goal is to bring out the positive aspects of surgical procedure by volar medial approach and to assess long-term functionnal and radiological results. MATERIAL AND METHODS: Our study focused on four men whose average age was 27 (age range from 19 to 43). The fractures were type IV according to Melone's classification. The associated lesions included: one fracture of the base of the ulnar styloid, one fracture of the distal quarter of the ulnar diaphysis and one scapho-lunate diastasis. We used a volar medial approach between the flexors tendons and the ulnar bundle in order to pin the fragment of lunate fossa. The rest of the radial epiphysis was pinned after a 5mm skin incision. In two cases, this pinning was complemented with a brachial-antebrachial-palmar cast and in the other two cases with an external fixator. RESULTS: The follow-up period averaged 68.8 (18 to 115) months, all the patients were clinically examined through antero-posterior, lateral and dynamic X-rays. The objective results assessed according to Green and O'Brien's criteria, later modified by Cooney, were as follows: two very good, one good, one average. The X-rays showed consolidated fractures. According to Knirk and Jupiter's classification of arthritis, we had three grades 0, one of which showed a subchondral sclerosis of the lunate fossa, and one grade 3. DISCUSSION AND CONCLUSION: Imaging with simple radiographs is not sufficient and needs to be complemented with CT scan. Our approach allows for direct access to the fragment of the lunate fossa and easier visualization of the distal radioulnar, compared to Henry's approach, thereby avoiding excessive traction of the median nerve. TYPE D'ÉTUDE: Niveau IV.

Biofunctionality of MBCP ceramic granules (TricOs) plus fibrin sealant (Tisseel) versus MBCP ceramic granules as a filler of large periprosthetic bone defects: an investigative ovine study.

We aimed to quantify bone colonization toward an untreated titanium implant with primary stability following filling of the defect with micromacroporous biphasic calcium phosphate (MBCP) granules (TricOs) or MBCP granules mixed with fibrin sealant (Tisseel). Medial arthrotomy was performed on the knees of 20 sheep to create a bone defect (16 mm deep; 10 mm diameter), followed by anchorage of a titanium screw. Defects were filled with TricOs or TricOs-Tisseel granules, a perforated MBCP washer, a titanium washer and titanium screw. Sheep were euthanized at 3, 6, 12 and 26 weeks. From Week 12 onwards, the percentage of bone in contact with the 8 mm anchorage part of the screw increased in both groups, confirming its primary stability. At 26 weeks, whereas bone colonization was similar in both groups, biodegradation of ceramic was more rapid in the TricOs-Tisseel group (P = 0.0422). The centripetal nature of bone colonization was evident. Bone contact with the titanium implant surface was negligible. In conclusion, the use of a model that reproduces a large metaphyseal bone defect around a titanium implant with primary stability, filled with a mixture of either TricOs ceramic granules or TricOs granules mixed with Tisseel fibrin sealant, suggests that the addition of fibrin to TricOs enhances bone filling surgical technology.

Radiation effects on bone healing and reconstruction: interpretation of the literature.

OBJECTIVE: Reconstructing irradiated mandibles with biomaterials is still a challenge but little investigated. We collected data that could help us understand studies in the field of regeneration with biomaterials and irradiated bone. STUDY DESIGN: Systematic review of the literature. RESULTS: Delay and duration of radiation delivery and total equivalent dose are the most variable parameters in the various studies, resulting in confusion when interpreting the literature. Most reproducible experiments show that radiation reduces osteogenic cell numbers, alters cytokine capacity, and delays and damages bone remodeling. Interindividual variations and how such changes become irreversible lesions are still uncertain. In the case of regeneration using biomaterials, most studies have addressed the question of reconstruction in previously irradiated bone. The results show that osseointegration is often possible, although the failure rate is higher. The sooner the implantation takes place after the end of the radiation, the higher the likelihood of failure. Few studies have focused on primary reconstruction followed by early irradiation, and most of the currently available engineering models would be altered by radiation. Good outcomes have been obtained with bone morphogenetic protein and with total bone marrow transplanation. CONCLUSION: This review points out the difficulties in achieving reproducible experiments and interpreting literature in this underinvestigated field.

Developments in injectable multiphasic biomaterials. The performance of microporous biphasic calcium phosphate granules and hydrogels.

Calcium phosphate bioceramic granules associated with hydrosoluble polymers were developed as bone substitutes for various maxillofacial and orthopaedic applications. These injectable bone substitutes, support and regenerate bone tissue and resorb after implantation. The efficiency of these multiphasic materials is due to the osteogenic and osteoconductive properties of the microporous biphasic calcium phosphate. The associated hydrosoluble polymers are considered as carriers in order to achieve the rheological properties of injectable bone substitutes (IBS). In this study, we used 2 semi synthetic hydrosoluble polymers of polysaccharidic origin. The hydroxy propyl methyl cellulose (HPMC), with and without silane, was combined with microporous BCP granules. The presence of silane induced considerable gelation of the suspension. The 2 IBS used (without gelation, IBS1, with gelation, IBS2) were implanted in critical size femoral epiphysis defects in rabbits. No foreign body reactions were observed in either sample. However, because of the higher density from gelation, cell colonisation followed by bone tissue ingrowth was delayed over time with IBS2 compared to the IBS1 without gelation. The results showed resorption of the BCP granule and bone ingrowth at the expense of both IBS with different kinetics. This study demonstrates that the hydrogel cannot be considered merely as a carrier. The gelation process delayed cell and tissue colonisation by slow degradation of the HPMC Si, compared to the faster release of HPMC with IBS1, in turn inducing faster permeability and spaces for tissue ingrowth between the BCP granules.

[Mandibular reconstruction for the treatment of oral carcinomas: state of the art and perspectives]. / Reconstruction mandibulaire en cancérologie: état actuel et perspectives.

INTRODUCTION: Mandibular reconstruction in head and neck oncology uses a number of techniques whose results are inconsistent and whose indications do not benefit from consensus. MATERIALS AND METHODS: A review of the literature allowed us to assemble the available knowledge on current mandibular reconstruction techniques, their functional results, and the research perspectives. RESULTS AND DISCUSSION: Marginal resections lead to dental rehabilitation problems, which can be palliated by alveolar enhancement techniques but whose results have not been validated in cases subjected to irradiation. Reconstruction of segmental substance loss is warranted by the repercussions on the vital prognosis when it is anterior and on the quality of life when it is posterior. The ideal means of reconstruction is the free fibular flap, which is limited by cost, morbidity of the donor site, and selection of the patient's surgical team. For these reasons, it may be necessary to turn to pedicled osteo-myocutaneous flaps, abandoned because of their reputedly very high failure rate, but few have been reported in the literature. CONCLUSION: Poor functional results of mandibular reconstruction plates make this a last-resort solution. Tissue engineering is currently the most promising line of research. It runs counter to the principles of oncology itself because postoperative radiotherapy reduces the osteoinduction potential of the biomaterials proposed.

Effects of high doses of ionising radiation on bone in rats: a new model for evaluation of bone engineering.

The purpose of this study was to assess the effects of high doses of ionising radiation on the histology and healing of bone in an experimental model of 12 inbred rats. Ten of the rats had external irradiation of a single dose of 30 or 45 Gy on the hind limbs, which is equivalent to 2 or 3 times the routine doses used for treatment in humans. Three weeks later, two bony defects were created on their left sides, and the animals were killed 12 or 18 weeks after irradiation. Decalcified bony specimens were studied with light microscopy for qualitative analysis. Thirty Gy irradiation induced medullar oedema or fibro-oedema and normal or fibrous healing of the defects. Forty-five Gy induced medullar oedema or fibro-oedema and depletion in bone marrow. In addition, pathological healing of the defects was obvious and characterised by oedema, fibrosis, and necrosis. In this study high doses of ionising radiation modified the histology of bone, particularly into fibro-oedema, and delayed healing. This new animal model could be used to evaluate the capacities of tissue-engineered materials to repair bony defects after irradiation and osteoradionecrosis.

Clinical, radiological and histological evaluation of biphasic calcium phosphate bioceramic wedges filling medial high tibial valgisation osteotomies.

We report clinical, radiological and histological findings following high tibial valgisation osteotomy (HTVO) using micro-macroporous biphasic calcium phosphate wedges fixed with a plate and locking screws. From 1999 to 2002, 43 knees were operated on and studied prospectively. All underwent clinical and radiological follow-up at days 1, 90, and 365 to evaluate consolidation and bone substitute interfaces. Additionally, biopsies were taken for histology at least 1 year after implantation from 10 patients who requested plate removal. Radiologically, consolidation was observed in 98% of cases. At 1 year, correction was unchanged in 95% of cases. Histological analysis revealed considerable MBCP resorption and bone ingrowth, both into the pores and replacing the bioceramic material. Polarised light microscopy confirmed normal bony architecture with trabecular and/or dense lamellar bone growth at the expense of the wedge implants. X-ray and micro-CT scan revealed a well organised and mineralised structure in the newly-formed bone. This study shows that using MBCP wedges in combination with orientable locking screws and a plate is a simple, safe and fast surgical technique for HTVO. The is the first study to examine the results by histological analysis, which confirmed good outcomes.

Nasal chondrocytes and fibrin sealant for cartilage tissue engineering.

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.

Biphasic calcium phosphate: a comparative study of interconnected porosity in two ceramics.

Interconnection, one of the main structural features of macroporous calcium-phosphate ceramics, contributes to the biological and physicochemical properties of bone substitutes. As no satisfactory method exists for evaluating this feature, analysis was performed to determine the permeability, tortuosity, and equivalent diameter of interconnecting channels, that is the parameters that appear to be representative of the way pores are linked. The testing of two ceramics with similar porosity levels revealed important differences in all three interconnection parameters. One ceramic showed poor permeability, corresponding to a small equivalent diameter for interconnecting channels in conjunction with a high tortuosity factor, while the other displayed high permeability, a large diameter for interconnecting channels, and a low tortuosity factor. The methodology used, which can be applied to the quantification of interconnection in all calcium-phosphate ceramics, constitutes the first step in a complete study of the role of this feature in cellular colonization of the ceramic, matrix dissolution, and drug release from the calcium-phosphate matrix.

Engineering cartilage with human nasal chondrocytes and a silanized hydroxypropyl methylcellulose hydrogel.

Tissue engineering strategies, based on developing three-dimensional scaffolds capable of transferring autologous chondrogenic cells, holds promise for the restoration of damaged cartilage. In this study, the authors aimed at determining whether a recently developed silanized hydroxypropyl methylcellulose (Si-HPMC) hydrogel can be a suitable scaffold for human nasal chondrocytes (HNC)-based cartilage engineering. Methyltetrazolium salt assay and cell counting experiments first revealed that Si-HPMC enabled the proliferation of HNC. Cell tracker green staining further demonstrated that HNC were able to form nodular structures in this three-dimensional scaffold. HNC phenotype was then assessed by RT-PCR analysis of type II collagen and aggrecan expression as well as alcian blue staining of extracellular matrix. Our data indicated that Si-HPMC allowed the maintenance and the recovery of a chondrocytic phenotype. The ability of constructs HNC/Si-HPMC to form a cartilaginous tissue in vivo was finally investigated after 3 weeks of implantation in subcutaneous pockets of nude mice. Histological examination of the engineered constructs revealed the formation of a cartilage-like tissue with an extracellular matrix containing glycosaminoglycans and type II collagen. The whole of these results demonstrate that Si-HPMC hydrogel associated to HNC is a convenient approach for cartilage tissue engineering.

Injectable bone substitute to preserve alveolar ridge resorption after tooth extraction: a study in dog.

The aim of the present study was to assess the efficacy of a ready-to-use injectable bone substitute on the prevention of alveolar ridge resorption after tooth extraction. Maxillary and mandibular premolars were extracted from 3 Beagle dogs with preservation of alveolar bone. Thereafter, distal sockets were filled with an injectable bone substitute (IBS), obtained by combining a polymer solution and granules of a biphasic calcium phosphate (BCP) ceramic. As a control, the mesial sockets were left unfilled. After a 3 months healing period, specimens were removed and prepared for histomorphometric evaluation with image analysis. Histomorphometric study allowed to measure the mean and the maximal heights of alveolar crest modifications. Results always showed an alveolar bone resorption in unfilled sockets. Resorption in filled maxillary sites was significantly lower than in control sites. Interestingly, an alveolar ridge augmentation was measured in mandibular filled sockets including 30% of newly-formed bone. It was concluded that an injectable bone substitute composed of a polymeric carrier and calcium phosphate can significantly increase alveolar ridge preservation after tooth extraction.

[Experimental evaluation of microscan assessment of bone fusion]. / Etude expérimentale de la microtomographie X dans l'évaluation de la fusion osseuse.

PURPOSE OF THE STUDY: Certain confirmation of bone fusion remains difficult to obtain after arthrodesis despite progress in imaging techniques. Microscanning enables both qualitative and quantitative analysis of the bone microarchitecture. The purpose of this study was to evaluate this technique using a cervical arthrodesis with an intersomatic cage on an animal model and to validate results with histological analysis and electron scan microscopy (SEM). MATERIAL AND METHODS: C3-C4 discectomy was performed in 8 goats divided into two groups. In group 1 (3 animals), PEEK cages were inserted without bone graft. In group 2 (5 goats) the same cage was inserted and filled with an autologous iliac graft. The animals were sacrificed at six months. The instrumented levels were analyzed with a microscan. Histological slides were obtained and SEM performed. RESULTS: Nonunion was observed in the three animals with an empty cage (group 1) while only one animal in group 2 presented nonunion. Histology and SEM confirmed the diagnosis established with the microscan which also enabled a 3D analysis of the sample and study of the trabecular architecture of the intersomatic graft. DISCUSSION: The microscan enabled a micrometric analysis of the sample. This is the only technique enabling 3D analysis (slices can be obtained in the three planes for 3D reconstruction) for both qualitative and quantitative assessment. Analysis of the trabecular microstructure constitutes a major progress in evaluating the mechanical value of the fusion. The sample is not destroyed and can be studied further with other biomechanical techniques. CONCLUSION: Microscanning is an important technical advancement for the analysis of bone fusion. Future applications will undoubtedly be numerous (follow-up after arthrodesis, analysis of the mechanical quality of a graft). In vivo applications will probably be adapted soon.

Cartilage and bone tissue engineering using hydrogels.

Tissue engineering is an emerging field of regenerative medicine which holds promise for the restoration of tissues and organs affected by chronic diseases, age-linked degeneration, congenital deformity and trauma. During the past decade, tissue engineering has evolved from the use of naked biomaterials, which may just replace small area of damaged tissue, to the use of controlled three-dimensional scaffolds in which cells can be seeded before implantation. These cellularized constructs aims at being functionally equal to the unaffected tissue and could make possible the regeneration of large tissue defects. Among the recently developed scaffolds for tissue engineering, polymeric hydrogels have proven satisfactory in cartilage and bone repair. Major technological progress and advances in basic knowledge (physiology and developmental biology) are today necessary to bring this proof of concept to clinical reality. The present review focuses on the recent advances in hydrogel-based tissue engineered constructs potentially utilizable in bone and cartilage regenerative medicine.

Evaluation of an injectable bone substitute (betaTCP/hydroxyapatite/hydroxy-propyl-methyl-cellulose) in severely osteopenic and aged rats.

The use of injectable biomaterials is of interest in osteoporotic patients to locally restore bone mass in sites at risk of fracture. An injectable bone substitute (IBS1 made of betaTCP/hydroxyapatite as a calcium phosphate substitute and hydroxy-propyl-methyl-cellulose as a polymer carrier) was used in a severely osteopenic rat model obtained by combining orchidectomy (ORX) and disuse (paralysis induced by botulinum toxin - BTX). Fifty-six aged male rats were randomized into three groups: 18 were SHAM operated; 38 were ORX and BTX injected in the right hindlimb; they constituted the OP (osteoporotic) group. One month after ORX-BTX surgery, 20 of these OP rats received a IBS1 injection in the right femur (OP-IBS1 rats). Animals were studied at the time of IBS1 injection 1 month post ORX-BTX (M1), 1 month (M2) and 2 months (M3) after IBS1 injection. Bone mass (BV/TV) and microarchitectural parameters were measured by microCT. BV/TV was decreased after ORX-BTX; ORX and BTX had cumulative effects on bone loss (differences maximized on the right femur). BV/TV (combining the volume of both bone and material in OP-IBS1 rats) was elevated at M1 but decreased at M2. Marked bone formation was found onto the biomaterial granules but bone had a woven texture. A marked increase in the number of nonosteoclastic TRAcP+ cells was found in the implanted area. IBS1 induced new bone formation shortly after implantation but both IBS1 and woven bone were resorbed without inducing lamellar bone. Biomaterial trials must be conducted with long-term implantation periods, in aged osteoporotic animals.

A silanized hydroxypropyl methylcellulose hydrogel for the three-dimensional culture of chondrocytes.

Articular cartilage has limited intrinsic repair capacity. In order to promote cartilage repair, the amplification and transfer of autologous chondrocytes using three-dimensional scaffolds have been proposed. We have developed an injectable and self-setting hydrogel consisting of hydroxypropyl methylcellulose grafted with silanol groups (Si-HPMC). The aim of the present work is to assess both the in vitro cytocompatibility of this hydrogel and its ability to maintain a chondrocyte-specific phenotype. Primary chondrocytes isolated from rabbit articular cartilage (RAC) and two human chondrocytic cell lines (SW1353 and C28/I2) were cultured into the hydrogel. Methyl tetrazolium salt (MTS) assay and cell counting indicated that Si-HPMC hydrogel did not affect respectively chondrocyte viability and proliferation. Fluorescent microscopic observations of RAC and C28/I2 chondrocytes double-labeled with cell tracker green and ethidium homodimer-1 revealed that chondrocytes proliferated within Si-HPMC. Phenotypic analysis (RT-PCR and Alcian blue staining) indicates that chondrocytes, when three-dimensionnally cultured within Si-HPMC, expressed transcripts encoding type II collagen and aggrecan and produced sulfated glycosaminoglycans. These results show that Si-HPMC allows the growth of differentiated chondrocytes. Si-HPMC therefore appears as a potential scaffold for three-dimensional amplification and transfer of chondrocytes in cartilage tissue engineering.

Small-animal models for testing macroporous ceramic bone substitutes.

The aim of this study was to compare the bone colonization of a macroporous biphasic calcium phosphate (MBCP) ceramic in different sites (femur, tibia, and calvaria) in two animal species (rats and rabbits). A critical size defect model was used in all cases with implantation for 21 days. Bone colonization in the empty and MBCP-filled defects was measured with the use of backscattered electron microscopy (BSEM). In the empty cavities, bone healing remained on the edges, and did not bridge the critical size defects. Bone growth was observed in all the implantation sites in rats (approximately 13.6-36.6% of the total defect area, with ceramic ranging from 46.1 to 51.9%). The bone colonization appeared statistically higher in the femur of rabbits (48.5%) than in the tibia (12.6%) and calvaria (22.9%) sites. This slightly higher degree of bone healing was related to differences in the bone architecture of the implantation sites. Concerning the comparison between animal species, bone colonization appeared greater in rabbits than in rats for the femoral site (48.5% vs. 29.6%). For the other two sites (the tibia and calvaria), there was no statistically significant difference. The increased bone ingrowth observed in rabbit femurs might be due to the large bone surface area in contact with the MBCP ceramics. The femoral epiphysis of rabbits is therefore a favorable model for testing the bone-bonding capacity of materials, but a comparison with other implantation sites is subject to bias. This study shows that well-conducted and fully validated models with the use of small animals are essential in the development of new bone substitutes.

A comparative study of biphasic calcium phosphate ceramics for human mesenchymal stem-cell-induced bone formation.

For the repair of bone defects, a tissue engineering approach would be to combine cells capable of osteogenic (i.e. bone-forming) activity with an appropriate scaffolding material to stimulate bone regeneration and repair. Human mesenchymal stem cells (hMSCs), when combined with hydroxyapatite/beta-tricalcium phosphate (HA/TCP) ceramic scaffolds of the composition 60% HA/40% TCP (in weight %), have been shown to induce bone formation in large, long bone defects. However, full repair or function of the long bone could be limited due to the poor remodeling of the HA/TCP material. We conducted a study designed to determine the optimum ratio of HA to TCP that promoted hMSC induced bone formation yet be fully degradable. In a mouse ectopic model, by altering the composition of HA/TCP to 20% HA/80% TCP, hMSC bone induction occurred at the fastest rate in vivo over the other formulations of the more stable 100% HA, HA/TCP (76/24, 63/37, 56/44), and the fully degradable, 100% TCP. In vitro studies also demonstrated that 20/80 HA/TCP stimulated the osteogenic differentiation of hMSCs as determined by the expression of osteocalcin.

A review of bioceramics and fibrin sealant.

This review focuses on bone substitute composites made by mixing ceramic biomaterials with fibrin sealants. Different biomaterials such as coral, bone-derived materials, bioactive glass ceramics, and synthetic calcium phosphate have been mixed with fibrin sealant, resulting in a combination of the biological properties of the two components. This type of association has not produced identical results in all studies. In the past for some, the addition of fibrin sealant to the biomaterial failed to produce any significant, positive effect on osteointegration, whereas others found a positive impact on bone colonization. Despite the negative biological effects reported previously, bioceramic-fibrin composites have been widely used in various types of bone surgery because they are easy to manipulate. In particular, the intra-operative preparation of these composites makes it possible to add bone growth factors or autologous osteoprogenitor cells prior to bone reconstruction. The bone growth factors and autologous osteoprogenitor cells associated with the bioceramic-fibrin composites should provide surgeons with tissue engineered grafts with enhanced osteointegrative properties. This review discusses both the advantages and disadvantages, as well as the future perspectives, of using bioceramic-fibrin composites in various clinical indications.

Development of an odontoblast in vitro model to study dentin mineralization.

The aim of the present work was to characterize the odontoblastic proliferation, differentiation, and matrix mineralization in culture of the recently established M2H4 rat cell line. Proliferation was assessed by cell counts, differentiation by RT-PCR analysis, and mineralization by alizarin red staining, atomic absorption spectrometry, and FTIR microspectroscopy. The results showed that M2H4 cell behavior closely mimics in vivo odontoblast differentiation, with, in particular, temporally regulated expression of DMP-1 and DSPP. Moreover, the mineral phase formed by M2H4 cells was similar to that in dentin from rat incisors. Finally, because in mice, transforming growth factor (TGF)-beta1 over-expression in vivo leads to an hypomineralization similar to that observed in dentinogenesis imperfecta type II, effects of TGF-beta1 on mineralization in M2H4 cell culture were studied. Treatment with TGF-beta1 dramatically reduced mineralization, whereas positive control treatment with bone morphogenetic protein-4 enhanced it, suggesting that M2H4 cell line is a promising tool to explore the mineralization mechanisms in physiopathologic conditions.