RESUMEN
The availability of recombinant osteoinductive growth factors and new osteoconductive matrices offers an alternative to the use of autogenous bone (autograft) for grafting indications. This study evaluates the bone-forming activity of a mineralized collagen matrix combined with recombinant human growth and differentiation factor-5 in a rabbit posterolateral spinal fusion model. The activity of three distinct matrix-growth factor formulations is assessed by radiographic, histologic, and mechanical strength methods. Results show that the radiographic density, histologic quality, and mechanical strength of fusion at 12 weeks post-treatment rank consistently within the treatment groups. Optimal formulations are shown to perform similar to autograft in both the rate and strength of fusion. Fusion rates as high as 80% are observed within specific matrix/growth factor formulations. The average biomechanical strength of treated motion segments in the most efficacious formulation is 82% higher than that obtained with autograft, although this difference is not statistically significant. The fusion mass formed in response to matrix/growth factor formulations is composed of normal trabecular bone with a thin outer cortical plate and modest hematopoietic bone marrow. These results demonstrate that the combination of a mineralized collagen matrix with recombinant human growth and differentiation factor-5 maximizes the inherent conductive and inductive properties of each component, respectively, to provide an effective alternative to autograft for bone grafting procedures.
Asunto(s)
Proteínas Morfogenéticas Óseas , Colágeno/administración & dosificación , Sustancias de Crecimiento/administración & dosificación , Osteogénesis/efectos de los fármacos , Proteínas Recombinantes/administración & dosificación , Fusión Vertebral/métodos , Animales , Trasplante Óseo , Bovinos , Fuerza Compresiva , Curación de Fractura/efectos de los fármacos , Factor 5 de Diferenciación de Crecimiento , Vértebras Lumbares/efectos de los fármacos , Vértebras Lumbares/cirugía , Masculino , Ensayo de Materiales , ConejosRESUMEN
Growth and differentiation factor-5 (GDF-5) is a divergent member of the transforming growth factor-beta/bone morphogenetic protein (BMP) superfamily that is required for proper skeletal patterning and development in the vertebrate limb. Based on the homology of GDF-5 with other bone-inducing BMP family members, the inductive activity of a recombinant form of human GDF-5 (rhGDF-5) was evaluated in a series of in vitro assays and in vivo bone-formation models. The in vitro response to rhGDF-5 resulted in the formation of chondrogenic nodules in fetal rat calvarial cells cultured in the context of collagen or collagen/hyaluronate extracellular matrices. Matrices loaded with rhGDF-5 induced ectopic cartilaginous and osseous tissue when implanted in subcutaneous or intramuscular sites. In non-human primate long-bone-defect and spinal-fusion models, rhGDF-5 combined with a mineralized collagen matrix induced bone formation in a manner equivalent to autogenous bone. These results highlight the unique potential of rhGDF-5 in a wide variety of orthopaedic applications.
Asunto(s)
Implantes Absorbibles , Proteínas Morfogenéticas Óseas , Sustancias de Crecimiento/metabolismo , Sustancias de Crecimiento/fisiología , Proteínas Recombinantes/metabolismo , Animales , Desarrollo Óseo , Trasplante Óseo/métodos , Bovinos , Células Cultivadas , Colágeno/metabolismo , Colágeno/uso terapéutico , Femenino , Factor 5 de Diferenciación de Crecimiento , Sustancias de Crecimiento/uso terapéutico , Humanos , Masculino , Papio , Radiografía , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/uso terapéutico , Columna Vertebral/diagnóstico por imagen , Factores de TiempoRESUMEN
A new type of collagen-hyaluronate (COL/HA) matrix was synthesized by cross-linking collagen fibers with modified hyaluronate polymers bearing active formyl groups. The resulting matrix is a three-dimensional scaffold consisting of interconnected pores with an average size of 40 microm and a high pore volume/surface area ratio. The covalent nature of the bond between the collagen fibers and the modified hyaluronate was demonstrated by extended elution with phosphate buffered saline and by extraction in increasing ionic gradients. The fraction of covalently bound hyaluronate in the matrix ranged from 5 to 25 w%. The total hyaluronate content of the COL/HA matrix affected both the in vitro non-enzymatic and enzymatic degradation as well as the in vivo turnover. When implanted in cranial defects in rats, the COL/HA matrix demonstrated good biocompatibility and exhibited greater osteoconductive potential than matrices composed of either cross-linked collagen or cross-linked hyaluronate alone.
Asunto(s)
Regeneración Ósea , Sustitutos de Huesos/química , Colágeno/química , Ácido Hialurónico/química , Osteogénesis , Hueso Parietal/lesiones , Animales , Bovinos , Colágeno/ultraestructura , Colagenasas/metabolismo , Ácido Hialurónico/ultraestructura , Hialuronoglucosaminidasa/metabolismo , Hidrólisis , Masculino , Microscopía Electrónica de Rastreo , Concentración Osmolar , Hueso Parietal/fisiología , Hueso Parietal/cirugía , Ratas , Ratas Sprague-DawleyRESUMEN
Fibroblast growth factors are present in significant amounts in bone and several studies have suggested that they may be involved in normal fracture healing. It is well established that fibroblast growth factors have mitogenic and angiogenic activity on mesoderm and neuroectoderm derived cells. Of particular interest as a member of the fibroblast growth factor family, basic fibroblast growth factor stimulates mitogenesis, chemotaxis, differentiation, and angiogenesis. It also plays an important role in the development of vascular, nervous, and skeletal systems, promotes the maintenance and survival of certain tissues, and stimulates wound healing and tissue repair. Animal studies have shown that the direct injection of fibroblast growth factor into fresh fractures stimulates callus formation, which provides mechanical stability to the fracture, accelerates healing, and restores competence. The matrix used to present the fibroblast growth factor at the fracture site plays a critical role in the effectiveness of the treatment. The evaluation of injectable basic fibroblast growth factor in a sodium hyaluronate gel for its effectiveness in stimulating fracture healing is described. When applied directly into a freshly created fracture in the rabbit fibula, a single injection of the basic fibroblast growth factor and hyaluronan results in the stimulation of callus formation, increased bone formation, and earlier restoration of mechanical strength at the fracture site. The hyaluronan gel serves as a reservoir that sequesters the basic fibroblast growth factor at the injection site for the length of time necessary to create an environment conducive to fracture healing. It is concluded that basic fibroblast growth factor and sodium hyaluronate act synergistically to accelerate fracture healing and that the combination is suitable for clinical evaluation as a therapy in fracture treatment.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/uso terapéutico , Curación de Fractura/efectos de los fármacos , Fracturas Óseas/tratamiento farmacológico , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/uso terapéutico , Inductores de la Angiogénesis/uso terapéutico , Animales , Callo Óseo/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Preparaciones de Acción Retardada , Combinación de Medicamentos , Ectodermo/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/administración & dosificación , Geles , Ácido Hialurónico/administración & dosificación , Ácido Hialurónico/uso terapéutico , Inyecciones , Masculino , Mesodermo/efectos de los fármacos , Mitógenos/uso terapéutico , Neovascularización Fisiológica/efectos de los fármacos , Conejos , Ratas , Ratas Sprague-Dawley , Estrés Mecánico , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Transforming growth factor beta is a multifunctional protein with known actions on bone and bone cells. The ability of TGF-beta to stimulate osteogenic parameters in vitro and osteogenesis adjacent to injection sites in vivo is well established. The purpose of this study was to determine if systemic administration of recombinant TGF-beta 2 (rTGF-beta 2) could stimulate bone formation in rats of different ages. Juvenile (25-day-old) and adult (160-day-old) rats were treated daily for 5 days and 14 days, respectively, with rTGF-beta 2 given by subcutaneous injection. Bone formation was measured in cancellous bone of the lumbar vertebrae in juvenile rats and the femoral epiphysis in adult rats. Endochondral bone growth rates were measured in the distal femurs from both juvenile and adult rats using histomorphometric methods. Systemic administration of rTGF-beta 2 resulted in substantial increases in bone formation rates (both surface and volume referent) in both juvenile and adult rats. In the juvenile rats, rTGF-beta 2 increased the percent double labeled surface and the mineral appositional rate. In the adult rats, TGF-beta 2 treatment increased the double labeled surface and also endochondral (longitudinal) growth parameters without changing the number of osteoclasts or the number of osteoclast nuclei per cell. These results demonstrate that short-term systemic administration of rTGF-beta 2 substantially increases cancellous bone formation rate in rats.
Asunto(s)
Envejecimiento/fisiología , Desarrollo Óseo/efectos de los fármacos , Fémur/crecimiento & desarrollo , Vértebras Lumbares/crecimiento & desarrollo , Factor de Crecimiento Transformador beta/farmacología , Animales , Fémur/efectos de los fármacos , Vértebras Lumbares/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacologíaRESUMEN
We previously identified a novel glycoprotein in an osteoinductive fraction from bovine bone (Bentz et al.: Amino acid sequence of bovine osteoinductive factor. J. Biol. Chem. 265: 5024-5029, 1990). We now find that this fraction also contained small amounts of bone morphogenetic protein-2 and 3 (BMP-2 + 3) previously identified by others (see Wozney, J.M.: Bone morphogenetic proteins. Prog. Growth Factor Res. 1: 267-280, 1990). Separation of BMP-2 + 3 from the glycoprotein was achieved with a modified reversed phase-high pressure liquid chromatographic procedure. When assayed in the rat subcutis using a collagen-ceramic carrier, the osteoinductive activity was found in the subfraction containing BMP-2 + 3. This activity was potentiated and the ratio of cartilage to bone was increased by transforming growth factor-beta 2. The glycoprotein, originally called osteoinductive factor, has been renamed osteoglycin. In its precursor form, osteoglycin is a member of the leucine-rich family of proteins showing the characteristic 24-residue internal homology. Its biological function is unknown.
Asunto(s)
Huesos/química , Osteogénesis/efectos de los fármacos , Proteínas/farmacología , Factor de Crecimiento Transformador beta/farmacología , Secuencia de Aminoácidos , Animales , Proteínas Morfogenéticas Óseas , Bovinos , Fraccionamiento Químico , Cromatografía Líquida de Alta Presión , Clonación Molecular , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Sustancias de Crecimiento/farmacología , Datos de Secuencia Molecular , Proteínas/genéticaRESUMEN
The complete amino acid sequence of bovine osteoinductive factor (OIF) was determined by automated Edman degradation of S-pyridylethylated bovine OIF and selected fragments. Cleavage with endoproteinase Lys-C, endoproteinase Glu-C, or endoproteinase Asp-N established all fragments in an unambiguous sequence. Bovine OIF contains 105 residues with a calculated molecular weight of 12,055. It is a single chain polypeptide containing two intramolecularly linked cysteines at residues 62 and 95. Two asparagine-linked glycosylation sites at positions 52 and 65 were found by comparing sequence data and peptide profiles of native and deglycosylated OIF fragments. The amino acid sequence of OIF has no homology to other reported proteins.
Asunto(s)
Huesos/metabolismo , Glicoproteínas , Secuencia de Aminoácidos , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Glicoproteínas/aislamiento & purificación , Datos de Secuencia Molecular , Fragmentos de Péptidos/aislamiento & purificación , Péptido Hidrolasas , Conformación ProteicaRESUMEN
A unique protein that promotes ectopic osteoinduction in the rat has been isolated and characterized. Osteoinductive factor (OIF) was extracted from the organic matrix of bovine bone with 4 M guanidine HCl and purified by gel filtration, ion-exchange chromatography, affinity chromatography, and reversed phase high performance liquid chromatography. OIF is a glycoprotein with an apparent molecular mass of 22-28 kDa based on sodium dodecyl sulfate gel electrophoresis. Enzymatic or chemical deglycosylation of OIF reduces its mass to about 12 kDa with apparent loss of activity. OIF activity in the model used is substantially increased by addition of transforming growth factor (TGF)-beta 1 or TGF-beta 2, suggesting an important role for TGF-beta 1 and -2 in bone regeneration and repair. The N-terminal sequence of OIF has no homology to other reported proteins.
Asunto(s)
Desarrollo Óseo , Huesos/fisiología , Glicoproteínas/aislamiento & purificación , Sustancias de Crecimiento/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Desarrollo Óseo/efectos de los fármacos , Huesos/efectos de los fármacos , Bovinos , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Sustancias de Crecimiento/farmacología , Péptidos y Proteínas de Señalización Intercelular , Masculino , Datos de Secuencia Molecular , Peso Molecular , Técnicas de Cultivo de Órganos , Ratas , Ratas Endogámicas , Factores de Crecimiento Transformadores/farmacologíaRESUMEN
Transforming growth factor beta (TGF-beta) has been shown to induce chondrogenesis by embryonic rat mesenchymal cells (Seyedin et al., J. Biol. Chem., 261: 5693, 1986). Here we report the effects of bovine TGF-beta on the phenotypic expression of differentiated primary rat osteoblastic and chondroblastic cells. Culture of rat calvarial osteoblasts with TGF-beta resulted in a dose and time-dependent decrease in alkaline phosphatase activity. Levels of alkaline phosphatase were reduced to less than 10% of control values by 0.4 nM TGF-beta. The decrease became apparent after 24 hours and reached a maximum by 72 hours. Similarly, treatment of chondroblasts with 0.4 nM TGF-beta resulted in decreased production of cartilage-specific macromolecules: type II collagen and cartilage proteoglycan. Both cell types exhibited dramatic changes in cell shape after treatment with TGF-beta. Modulation of these differentiated markers by TGF-beta could be mimicked, in part, by addition of fibronectin. Addition of dihydrocytochalasin B blocked the inhibition of phenotypic expression by TGF-beta. These results indicate that TGF-beta inhibits phenotypic expression by osteoblasts and chondroblasts in vitro and suggest that this activity of TGF-beta may be mediated through interactions between the extracellular matrix and cytoskeletal elements.
Asunto(s)
Cartílago/citología , Osteoblastos/efectos de los fármacos , Péptidos/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Cartílago/efectos de los fármacos , Cartílago/enzimología , Cartílago/ultraestructura , Células Cultivadas , Colágeno/biosíntesis , Colágeno/genética , Citocalasina B/análogos & derivados , Citocalasina B/farmacología , Citoesqueleto/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Fibronectinas/farmacología , Osteoblastos/citología , Osteoblastos/enzimología , Osteoblastos/ultraestructura , Péptidos/antagonistas & inhibidores , Fenotipo , Biosíntesis de Proteínas , Proteoglicanos/biosíntesis , ARN Mensajero/análisis , Ratas , Factores de Crecimiento TransformadoresRESUMEN
Cartilage-inducing factors-A (CIF-A) and -B (CIF-B), purified from bovine bone on the basis of their ability to induce the cartilage phenotype in vitro, are proteins with molecular weights of 26,000 composed of two apparently identical disulfide-linked chains. CIF-A is apparently identical to TGF-beta from human platelets (Seyedin S. M., Thompson, A. Y., Bentz, H., Rosen, D. M., McPherson, J. M., Conti, A., Siegel, N. R., Galluppi, G. R., and Piez, K. A. (1986) J. Biol. Chem. 261, 5693-5695). We have now found that, like CIF-A and TGF-beta, CIF-B induces anchorage-independent proliferation of NRK-49F cells when these cells are simultaneously treated with epidermal growth factor. Furthermore, CIF-B competes with CIF-A for the same cell membrane receptors in NRK-49F cells. Partial amino acid sequencing reveals that CIF-B is a distinct molecule with extensive homology to CIF-A/TGF-beta. These results show that CIF-B and TGF-beta are structurally and functionally similar molecules, but differ more from each other than does TGF-beta from different species.
Asunto(s)
Cartílago/efectos de los fármacos , Péptidos/farmacología , Proteínas/análisis , Factor de Crecimiento Transformador beta , Secuencia de Aminoácidos , Animales , Bovinos , Línea Celular , Colágeno/genética , Ensayo de Unidades Formadoras de Colonias , Relación Dosis-Respuesta a Droga , Riñón/efectos de los fármacos , Proteínas/farmacología , ARN Mensajero/análisis , Ratas , Factor de Crecimiento Transformador beta2 , Factores de Crecimiento TransformadoresRESUMEN
The role of cell shape in chondrogenesis was studied by using rat mesenchymal cells cultured with cartilage-inducing factor (CIF). Here we report that enhanced expression of chondroblastic markers by induced cells was attained by culturing cells in monolayer in the presence of dihydrocytochalasin B (DHCB). This effect was optimal at 3 microM DHCB and was apparent after 3 days in culture. Mesenchymal cells cultured with DHCB alone exhibited no detectable increase in cartilage proteoglycan synthesis, whereas cells cultured with 3 microM DHCB and 0.1 nM CIF showed a 4-5 fold increase in proteoglycan synthesis. When cells were cultured with CIF alone on plastic, only small increases in proteoglycan synthesis were observed. Cells cultured with CIF in monolayer and then transferred to a permissive environment (either agarose or cultured with DHCB) showed enhanced synthesis of chondroblastic proteins. These results suggest that expression, but not induction, of a chondroblastic phenotype by CIF is inhibited by growth in monolayer. The altering of cell shape with DHCB releases that inhibition.
Asunto(s)
Cartílago/embriología , Diferenciación Celular/efectos de los fármacos , Citocalasina B/análogos & derivados , Mesodermo/citología , Proteínas/farmacología , Animales , Cartílago/citología , Células Cultivadas , Citocalasina B/farmacología , Ensayo de Inmunoadsorción Enzimática , Sueros Inmunes/inmunología , Técnicas para Inmunoenzimas , Mesodermo/efectos de los fármacos , Mesodermo/fisiología , Fenotipo , Proteoglicanos/biosíntesis , Conejos/inmunología , Ratas , Ratas Endogámicas/embriologíaRESUMEN
Comparison of the sequence of the N-terminal 30 amino acids of cartilage-inducing factor-A (CIF-A) from bovine demineralized bone with the corresponding sequence of human transforming growth factor-beta (TGF-beta) revealed 100% identity. Furthermore, CIF-A stimulated normal rat kidney fibroblasts to become anchorage-independent and form colonies in soft agar (in the presence of epidermal growth factor) in a manner similar to TGF-beta. Similarly, TGF-beta from human platelets induced rat muscle mesenchymal cells to differentiate and synthesize cartilage-specific macromolecules in a manner equivalent to CIF-A. These data show that CIF-A and TGF-beta are closely related or identical molecules and that these factors may be involved in cell differentiation including cartilage formation as the first step in endochondral bone formation.
Asunto(s)
Sustancias de Crecimiento , Proteínas , Secuencia de Aminoácidos , Animales , Huesos , Bovinos , División Celular/efectos de los fármacos , Línea Celular , Cromatografía Líquida de Alta Presión , Proteínas/aislamiento & purificación , Proteínas/farmacologíaRESUMEN
Two naturally occurring peptides that induce chondrogenesis in culture have been purified to apparent homogeneity. These cartilage-inducing factors (CIF-A and CIF-B) were isolated from bovine demineralized bone by dissociative extraction, gel filtration, cation-exchange chromatography, and reversed-phase HPLC. CIF-A and CIF-B at concentrations of 1-10 ng/ml each induce embryonic rat mesenchymal cells in culture to assume a cartilage morphology and synthesize cartilage-specific proteoglycan and type II collagen. The amino acid compositions of CIF-A and CIF-B are similar but not identical. Both factors have an apparent Mr of 26,000, as determined by NaDodSO4/PAGE. In the presence of 2-mercaptoethanol, both are converted to species of about one-half that Mr, indicating that they are dimers of identical or very similar chains.
Asunto(s)
Huesos/análisis , Cartílago/crecimiento & desarrollo , Sustancias de Crecimiento/aislamiento & purificación , Proteínas/aislamiento & purificación , Animales , Bovinos , Células Cultivadas , Colágeno/biosíntesis , Proteínas/farmacología , Proteoglicanos/biosíntesis , RatasRESUMEN
An in vitro model has been developed to study chondrogenic induction, proliferation and differentiation. Embryonic rat mesenchymal cells isolated from muscle and embedded in agarose were treated with a partially purified extract from bovine demineralized bone powder. Treated cells proliferated and synthesized matrix similar to differentiated chondrogenic cells in a dose-dependent manner. By employing an enzyme-linked immunosorbent assay (ELISA), cartilage-specific proteoglycan and type II collagen synthesis were quantitated. Of the cells tested, only embryonic mesenchymal cells from muscle responded to bone extract. Proteoglycan synthesis was sensitive to type of medium and cell density.
Asunto(s)
Cartílago/embriología , Diferenciación Celular , Inducción Embrionaria , Animales , Sangre , Huesos , Cartílago/citología , Cartílago/metabolismo , Recuento de Células , Células Cultivadas , Colágeno/biosíntesis , Medios de Cultivo , ADN/biosíntesis , Músculos/citología , Músculos/embriología , Músculos/metabolismo , Proteoglicanos/biosíntesis , Ratas , Ratas Endogámicas , Sefarosa , Extractos de Tejidos/farmacologíaRESUMEN
An in vitro system has been developed to study the onset of chondrogenesis. Embryonic rat muscle mesenchymal cells, when treated in suspension culture with an extract of bovine bone matrix, synthesized cartilage-specific proteoglycan and type II collagen. The synthesis of these two macromolecules was assayed by the enzyme-linked immunosorbent assay inhibition technique. Further evidence of chondrogenesis was demonstrated by morphological changes of treated cells when cultured in firm agarose and stained for metachromatic matrix. Even with crude bone matrix extracts, the assay was sensitive at the microgram level and significant differences in cartilage macromolecules compared with controls were observed in 2-3 d. In vivo the same extract induced first cartilage and then bone.