Spinal fusion with recombinant human growth and differentiation factor-5 combined with a mineralized collagen matrix.

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.

Inductive activity of recombinant human growth and differentiation factor-5.

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.

Novel formulation of fibroblast growth factor-2 in a hyaluronan gel accelerates fracture healing in nonhuman primates.

Recent advances in understanding the biology of fracture healing and the availability of specific macromolecules has resulted in the development of novel treatments for injuries to bone. Fibroblast growth factor-2 or basic fibroblast growth factor (4 mg/ml), a potent mitogen, and hyaluronan (20 mg/ml), an extracellular matrix component, were combined into a viscous gel formulation intended for direct, percutaneous injection into fresh fractures. In an experimental primate fracture model, a bilateral 1-mm-gap osteotomy was surgically created in the fibulae of baboons. A single direct administration of this hyaluronan/fibroblast growth factor-2 formulation to the defect site significantly promoted local fracture healing as evidenced by increased callus formation and mechanical strength. Radiographic analysis showed that the callus area was statistically significantly larger at the treated sites than at the untreated sites. Specimens treated with 0.1, 0.25, and 0.75 ml hyaluronan/fibroblast growth factor-2 demonstrated a 48, 50, and 34% greater average load at failure and an 82, 104, and 66% greater energy to failure than the untreated controls, respectively. By histologic analysis, the callus size, periosteal reaction, vascularity, and cellularity were consistently more pronounced in the treated osteotomies than in the untreated controls. These results suggest that hyaluronan/fibroblast growth factor-2 may provide a significant advance in the treatment of fractures.

An osteoconductive collagen/hyaluronate matrix for bone regeneration.

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.

Potential role of fibroblast growth factor in enhancement of fracture healing.

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.

Allograft safety: viral inactivation with bone demineralization.

A study was performed to validate the effectiveness of a bone demineralization process with respect to its inactivation of viruses. The viruses selected for study included human immunodeficiency virus (HIV), duck hepatitis B virus (a model for human hepatitis B), bovine viral diarrheal virus (a model for human hepatitis C), human cytomegalovirus, and human poliovirus (a model for small nonenveloped viruses, e.g., hepatitis A). This study was performed in compliance with Good Laboratory Practice regulations using validation methodology similar to that used to ensure the safety of blood derivatives and other products. Use of the bone demineralization process described in this report resulted in a reduction in infectivity of greater than one million (10(6)) for all viruses and as much as one trillion (10(12)) for the poliovirus.

Effects of a synthetic peptide of a parathyroid hormone-related protein on calcium homeostasis, renal tubular calcium reabsorption, and bone metabolism in vivo and in vitro in rodents.

A synthetic peptide corresponding to the first 34 amino acids of the parathyroid hormone-related protein (PTH-rP) produced by a human tumor associated with hypercalcemia was examined for skeletal and renal effects on calcium metabolism in vivo and in vitro. These effects were compared with those of human parathyroid hormone (1-34), hPTH (1-34). Equal doses of PTH-rP(1-34) and hPTH(1-34) produced equivalent stimulation of adenylate cyclase in vitro in bone cells and kidney cells and tubules. Subcutaneous injection of PTH-rP(1-34) in mice caused a significant dose-related increase in blood ionized calcium similar to that seen with hPTH(1-34) at equivalent doses. Repeated injections of equal doses of both peptides caused sustained hypercalcemia which was significantly greater in PTH-rP(1-34)-treated mice, although each induced comparable increases in histomorphometric indices of osteoclastic bone resorption. PTH-rP(1-34) and hPTH(1-34) also caused similar increases in bone resorption when incubated with fetal rat long bones in organ culture. Infusion of either peptide into thyroparathyroidectomized rats suppressed urinary calcium excretion and increased urinary excretion of cyclic AMP. PTH-rP appears to have similar effects to those of PTH on the skeleton, the kidney, and overall calcium homeostasis.

Elevated serum calcium and osteocalcin levels from calcitriol in preterm infants. A prospective randomized study.

The hypothesis of this study was that pharmacologic doses of calcitriol (1,25-dihydroxyvitamin D3) would result in elevated levels of serum osteocalcin, the major noncollagenous bone protein, and calcium in infants of very low birth weight (less than 1500 g). Twenty-four infants of very low birth weight but of the appropriate weight for gestational age were matched in 250-g weight ranges and randomized into calcitriol treatment and control groups on the first day after birth. Treated infants received 4 micrograms/kg of calcitriol intravenously on entry and on the second and third study days. Controls did not receive calcitriol. Four infants from each group were hypocalcemic (serum calcium level, less than 7.0 mg/dL [less than 1.75 mmol/L]) on entry (five to 20 hours after birth) to the study. Seven infants received calcium replacement; data analyses with and without these infants were similar. Of the remaining 17 infants, eight were in the treatment group and nine were in the control group. Calcitriol significantly increased serum calcium and osteocalcin concentrations on days 2, 3, and 4 after birth compared with the control group. None of eight treated infants manifested hypocalcemia after calcitriol vs eight of nine controls. There were no acute changes in heart rate, respiratory rate, systolic blood pressure, or urinary calcium loss nor were there changes at the infusion site, but the diastolic blood pressure increased with treatment. Although high doses of calcitriol may elevate serum calcium concentrations in infants of very low birth weight, we suggest that the long-term or subtle biologic effects of high doses of calcitriol remain to be studied and that its routine use not be recommended at present.

Estimation of bone turnover evaluated by 47Ca-kinetics. Efficiency of serum bone gamma-carboxyglutamic acid-containing protein, serum alkaline phosphatase, and urinary hydroxyproline excretion.

Bone gamma-carboxyglutamic acid-containing (Gla) protein (BGP, osteocalcin) is a noncollagenous protein of bone present in plasma and removed by the kidney. Plasma BGP has been shown to be elevated in patients with certain bone diseases. The present study evaluates serum BGP (S-BGP), serum alkaline phosphatase (S-AP), and urinary hydroxyproline excretion (U-OHP) in diseases with differing bone turnover rates, and compares the accuracy of these measurements for estimating bone mineralization (m) and resorption (r) rates. S-BGP, S-AP, U-OHP, and creatinine clearance (Clcr) were measured in patients with primary hyperparathyroidism (n = 13), hyperthyroidism (n = 6), and hypothyroidism (n = 6). Bone mineralization and resorption rates were calculated from a 7-d combined calcium balance and 47Ca turnover study. A highly significant correlation (r = 0.69, P less than 0.001) was found between S-BGP and m. Multiple regression analysis disclosed a partial correlation between S-BGP and m when Clcr was taken into account (r = 0.82, P less than 0.001), and between S-BGP and Clcr when m was taken into account (r = -0.62, P less than 0.005). In accordance with this, a stronger correlation (r = 0.89, P less than 0.0001) was found between S-BGP X Clcr and m than between S-BGP and m. A less significant correlation was found between S-AP and m (r = 0.45, P less than 0.05). Furthermore, U-OHP showed a highly significant positive correlation to r (r = 0.78, P less than 0.001). Thus, in the studied disorders of calcium metabolism, individual serum levels of BGP depend on both mineralization rate and renal function. Serum levels of BGP corrected for alterations in renal function are superior to uncorrected S-BGP and to S-AP levels in the estimation of bone mineralization rates.

Constitutive biosynthesis of bone Gla protein in a human osteosarcoma cell line.

A human osteosarcoma cell line derived from cells obtained from a patient with Paget's disease is shown to synthesize and secrete bone Gla protein (BGP); (osteocalcin), a noncollagenous bone matrix protein. Using a human BGP-specific RIA, we show that the human osteosarcoma cells synthesize significant amounts of BGP without any prior induction of BGP synthesis by 1,25-dihydroxyvitamin D. After specific immunoprecipitation of poly-A+ RNA in vitro translation products with antibodies to BGP, we found that BGP is synthesized as a precursor with an apparent mol wt of 13.5K, as demonstrated on 15% sodium dodecyl sulfate-polyacrylamide gels. Finally, pulse labeling of the osteosarcoma cells with [3H]proline reveals that the cells synthesize mature BGP of 12,000 mol wt as well as a higher mol wt precursor (13,500) of the protein.

Vitamin D metabolites regulate osteocalcin synthesis and proliferation of human bone cells in vitro.

The effects of six natural vitamin D metabolites of potential biological and therapeutic interest, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), 25-hydroxyvitamin D3 (25-OH-D3), 24R,25-dihydroxyvitamin D3 (24R,25-(OH)2D3), 1,24R,25-trihydroxyvitamin D3 (1,24R,25-(OH)3D3), 25S,26-dihydroxyvitamin D3 (25S,26-(OH)2D3) and 1,25S,26-trihydroxyvitamin D3 (1,25S,26-(OH)3D3) on cell replication and expression of the osteoblastic phenotype in terms of osteocalcin production were examined in cultured human bone cells. At a dose of 5 X 10(-12) mol/1, 1,25-(OH)2D3 stimulated cell proliferation, whereas at higher doses (5 X 10(-9)-5 X 10(-6) mol/1) cell growth was inhibited in a dose-dependent manner. The same pattern of effects was seen for the other metabolites in a rank order of potency: 1,25-(OH)2D3 greater than 1,25S,26-(OH)3D3 = 1,24R,25-(OH)3D3 greater than 25S,26-(OH)2D3 = 24R,25-(OH)2D3 = 25-OH-D3. Synthesis of osteocalcin was induced by 1,25-(OH)2D3 in doses similar to those required to inhibit cell proliferation. Biphasic responses were observed for some of the metabolites in terms of osteocalcin synthesis, inhibitory effects becoming apparent at 5 X 10(-6) mol/1. The cells did not secrete osteocalcin spontaneously. These results indicate that vitamin D metabolites may regulate growth and expression of differentiated functions of normal human osteoblasts.

Characterization of cells with high alkaline phosphatase activity derived from human bone and marrow: preliminary assessment of their osteogenicity.

Confluent cellular layers are reproducibly obtained (from 21 of 24 specimens) by outgrowth from composite pieces of human trabecular bone and marrow. The cells resemble fibroblasts in terms of morphology, esterase profile, and production of collagen type 1. However, the cells displayed some osteoblastlike features. Both the primary outgrowths and passaged cultures had high alkaline phosphatase activities (37 nmols min-1 X microgram DNA-1) in the range displayed by embryonic osteoblastlike cells. The cellular alkaline phosphatase activity, which showed similarity to the bone isoenzyme on kinetic criteria, was stimulated by 1,25-dihydroxyvitamin D3 but decreased by PTH (1-34). In addition, the cell preparations were shown to increase osteocalcin (bone Gla protein) production in response to 1,25-dihydroxyvitamin D3. The osteogenic potential of the bone and marrow-derived cells has been assessed in an in vivo diffusion chamber assay in which congenitally athymic (nude) mice were used as hosts. None of the 25 chambers examined showed evidence of osteogenesis, although the cells remained viable and fibroblastlike. The alkaline phosphatase activities decreased to less than 1% of the original, high in vitro values. The findings question the hypothesis that bone and marrow-derived cells are osteoblasts or osteoblastlike cells, rather than a mixture of cell lines of the bone and marrow stromal system.

Production of osteocalcin by human bone cells in vitro. Effects of 1,25(OH)2D3, 24,25(OH)2D3, parathyroid hormone, and glucocorticoids.

Cells have been cultured from human bone that possess several characteristics of osteoblasts, including the capacity to produce osteocalcin (bone Gla protein). In these cultures the production of osteocalcin is dependent on 1,25(OH)2D3 but is not affected by 24,25(OH)2D3 either alone or in combination with 1,25(OH)2D3. Two glucocorticoids, prednisolone and deflazacort, reverse the stimulation of osteocalcin synthesis by 1,25(OH)2D3 in a dose-dependent manner (10(-9) - 10(-6)M). Parathyroid hormone also inhibits osteocalcin production in a dose-dependent fashion (0.2-5 IU/ml). These results demonstrate that human bone cell cultures may be of considerable value in investigating the hormonal and pharmacologic regulation of the production of osteocalcin and other bone proteins in vitro.

Laser Raman spectroscopy of calf bone Gla protein.

The Raman spectra of solid calf bone Gla protein in its native state, decarboxylated, with reduced disulfide bond, and as the calcium salt have been obtained. The amide I and III bands are consistent with the presence of alpha-helical, antiparallel beta-sheet, and random-coil regions in all four forms of bone Gla protein. Random coil appears to be the prevailing conformation. The protein conformation in the calcium salt exhibits an increased alpha-helix character compared to the native protein. No significant differences in the backbone conformation are observed among the native, decarboxylated, and reduced forms of bone Gla protein. The Raman band at 504 cm-1, due to the disulfide stretching vibration in native bone Gla protein, is unchanged upon decarboxylation and binding of Ca2+ to the protein, indicating the absence of any changes in the conformation around the disulfide bond in these protein species. The tryptophan and most of the tyrosine residues appear to be 'exposed' rather than 'buried' in the native protein. The environment of at least one of the phenylalanine residues changes when Ca2+ is bound to bone Gla protein. A small change also appears to take place in the environment of at least one of the tyrosine residues upon Ca2+-binding or reduction of the disulfide bond.

Chemotactic activity of the gamma-carboxyglutamic acid containing protein in bone.

We have found that the gamma-carboxyglutamic acid (GLA)-containing protein from bone (BGP, osteocalcin) has chemotactic activity in vitro for a number of cells which are found adjacent to endosteal bone surfaces in vivo. Using the Boyden chamber technique for measuring cell chemotaxis in vitro, we have shown that BGP is chemotactic for cultured human breast cancer cells, human and mouse monocytes, and for cultured rat osteosarcoma cells which have the characteristics of osteoblasts. The migration of these cells in response to BGP is undirectional and not due to spontaneous or random migration. A synthetic peptide (Phe-Tyr-Gly-Pro-Val), which is identical to the carboxy-terminal peptide cleaved from BGP when digested by trypsin, is also chemotactic for the same cells. BGP retains its chemotactic activity after conversion of the gamma-carboxyglutamic acid residues to glutamic acid, indicating that this biological effect requires neither gamma-carboxyglutamate nor the ability of BGP to bind calcium. Since BGP is released from bone during states of increased bone turnover, it is possible that this chemotactic effect of the protein may be a mechanism for recruitment of these cells to sites of active bone remodeling.

Identification of an essential glutamic acid residue in the beta subunit of the adenosine triphosphatase from the thermophilic bacterium PS3.

The TF1-ATPase from the thermophilic bacterium, PS3, is inactivated by dicyclohexylcarbodiimide (DCCD). This inactivation is stimulated by ADP and other specific nucleotides and is inhibited by Mg2+. When the inactivation is carried out with [14C]DCCD, about 2 g atoms of 14C are bound/mol of TF1 when the enzyme is nearly completely inactivated. The isolated subunits from TF1 inactivated with [14C]DCCD contain the following amounts of 14C/mol: alpha, 0.12 g atom; beta, 0.47 g atom; gamma, approximately 0.04 g atom; delta, none; and epsilon, 0.05 g atom. Fractionation of tryptic digests have shown that the 14C bound to the alpha subunit is nonspecifically associated with several peptides, and that the 14C bound to the beta subunit is associated with a single tryptic peptide with the amino acid sequence Ala-Gly-Val-Gly-Glu-Arg, where Glu represents the N-gamma-glutamyl derivative of dicyclohexyl[14C]urea.

Isolation and sequence of the vitamin K-dependent protein from human bone. Undercarboxylation of the first glutamic acid residue.

The gamma-carboxyglutamate-containing protein from human bone has been isolated from proteins released upon demineralization of bone by using a combination of gel filtration and immunoadsorption onto a column of immobilized antibody. The sequence of the protein has been determined by automatic sequence analysis of the whole protein and of peptides isolated from tryptic and Staphylococcus aureus protease digests. The protein consists of 49 amino acids in a single polypeptide chain, contains 2 residues of gamma-carboxyglutamic acid, and a single disulfide bond. There are 4 translational substitutions between the human and calf bone proteins and 23 translational substitutions between the human and swordfish bone proteins. Glu-17 was found to be only 9% gamma-carboxylated, while sequence positions 21 and 24 are fully gamma-carboxylated. The identification of glutamic acid at position 17 represents the first instance where a partially gamma-carboxylated glutamate has been found in a sequence position which is to the NH2-terminal side of a gamma-carboxyglutamate residue. The possible significance of this observation is discussed.

Primary structure of the gamma-carboxyglutamic acid-containing protein from bovine bone.

The amino-acid sequence of the gamma-carboxyglutamic acid-containing protein of bovine bone is presented. The sequence of 43 of the 49 residues was determined automatically on the intact protein and on a tryptic peptide. The remainder was determined by conventional procedures on tryptic and chymotrytic peptides. Residue 9 in the sequence has been identified as 4-hydroxyproline. The protein contains three gamma-carboxyglutamic acid residues, which are located at positions 17, 21, and 24. The role of these unusual amino acids in the binding interaction between the protein and hydroxyapatite crystals is discussed.