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.

Nodule formation and calcification of mandibular condylar cartilage cell cultures mimic in vivo ultrastructure.

Mandibular condylar cartilage (MCC) of growing mammals contains four layers of cells which display a series of increasingly differentiated phenotypes and which culminate in a terminally differentiated cell that produces a calcified matrix. In this study, MCC cells were placed into culture in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 50 micrograms/ml ascorbic acid. After 12 h in culture, transmission electron microscopy revealed the presence of multiple cell types that underwent differentiation with additional time in culture. By 7 days, fusiform-shaped cells were seen that contained numerous actin-like cytofilaments and micropinocytotic vesicles characteristic of myofibroblasts. Chondroblast-like cells were also observed. By 10 days, without addition of beta-glycerophosphate or dexamethasone, these cellular events culminated in the formation of mineralized nodules containing matrix vesicles. The nodular surface at day 13 consisted of two or more layers of myofibroblast-like cells, while the deeper zones of the nodule contained cells displaying a morphology typical of calcified hypertrophic chondroblasts. These ultrastructural observations are consistent with the hypothesis that cells from the MCC are capable of recapitulating in culture the maturational events seen in vivo. This cell culture model may be useful for investigating cell-mediated cartilage calcification without the addition of exogenous phosphate or other regulatory factors.

Nongenomic regulation of chondrocyte membrane fluidity by 1,25-(OH)2D3 and 24,25-(OH)2D3 is dependent on cell maturation.

1,25-(OH)2D3 and 24,25-(OH)2D3 regulate rat costochondral chondrocyte cultures in a metabolite-specific manner; 1,25-(OH)2D3 targets primarily growth zone cells (GC) and 24,25-(OH)2D3 targets primarily resting zone cells (RC). Some of the effects are nongenomic, since incubation of isolated membrane fractions with the metabolites results in regulation of enzyme activities comparable to that seen in culture. This study examined whether changes in membrane fluidity might be one mechanism involved in the nongenomic regulatory pathway. Chondrocyte cultures were incubated with the vitamin D metabolites and changes in plasma membrane fluidity monitored using the fluorophore, TMA-DPH, which is specific for membranes exposed to external fluids. Isolated matrix vesicles were also incubated directly with the metabolites and anisotropy of the membrane, as well as alkaline phosphatase-specific activity, determined. 1,25-(OH)2D3 caused a rapid and constant increase in alkaline phosphatase-specific activity in GC matrix vesicles; 24,25-(OH)2D3 caused an increase in RC matrix vesicle enzyme activity that was both dose- and time-dependent. Matrix vesicles produced by GC had a lower degree of fluidity than their parent plasma membranes or RC plasma membranes and matrix vesicles. Fluidity of the GC membrane fractions was increased by 1,25-(OH)2D3 in a dose- and time-dependent manner. 1,25-(OH)2D3 had no effect on the fluidity of the RC membranes. 24,25-(OH)2D3 caused a decrease in fluidity in GC at later time points. This metabolite caused an increase in fluidity of RC plasma membranes that returned to normal levels by 6 h; however, the increase induced in the matrix vesicles remained elevated throughout the 24-h experimental period.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of matrix vesicle enzyme activity and phosphatidylserine content by ceramic implant materials during endosteal bone healing.

This study examined effects of bone bonding and nonbonding implants on parameters associated with matrix vesicle-mediated primary bone formation, matrix vesicle alkaline phosphatase and phospholipase A2 specific activities, and phosphatidylserine content. Tibia marrow ablation followed by implantation of KG-Cera, Mina 13 (bonding), KGy-213, or M 8/1 (nonbonding) was used as the experimental model. Postsurgery, matrix vesicle-enriched microsomes (MVEM) were isolated from implanted and contralateral limbs. MVEM alkaline phosphatase and phospholipase A2 were stimulated adjacent to bonding implants with similar, though reduced, effects contralaterally. Alkaline phosphatase exhibited slight stimulation in nonbonding tissue; phospholipase A2 was inhibited or unchanged in treated and contralateral limbs. Phosphatidylserine content of MVEM was differentially affected by the implant materials. Thus, MVEM are modulated by implant materials locally and systemically. The data demonstrate that the model is a biologically relevant diagnostic for assessing the tissue/implant interface, primary calcification is affected by implant materials, and implant-specific effects are detected in the contralateral unimplanted limb.

1,25-(OH)2D3 and 24,25-(OH)2D3 regulation of arachidonic acid turnover in chondrocyte cultures is cell maturation-specific and may involve direct effects on phospholipase A2.

Previous studies have shown that 1,25-(OH)2D3 stimulates phospholipase A2 (PA2) activity in growth zone chondrocytes (GC), but has no effect on the resting zone chondrocyte (RC) enzyme activity. 24,25-(OH)2D3 inhibits the RC enzyme but has no effect on the GC. This study examined whether the vitamin D metabolites affect arachidonic acid turnover in their contra-target cell populations. Incorporation and release of [14C]arachidonate was measured at various times following addition of hormone. Acylation and reacylation were measured independently by incubating with p-chloromercuribenzoate. The results demonstrated that 1,25-(OH)2D3 has no effect on arachidonic acid turnover in RC, but stimulates turnover in GC. In contrast, 24,25-(OH)2D3 stimulates arachidonic acid turnover in RC, but inhibits both incorporation and release in GC. To determine whether direct interaction with PA2 is one mechanism by which 1,25-(OH)2D3 and 24,25-(OH)2D3 regulate arachidonic acid release, snake venom (Niger niger) PA2 was incubated with the vitamin D metabolites. Enzyme specific activity was inhibited by 24,25-(OH)2D3 and stimulated by 1,25-(OH)2D3 in a time- and dose-dependent manner. These results suggest that at least part of the direct effect of vitamin D3 metabolites on cell membranes may be related to changes in PA2 activity. The regulation is related to the stage of differentiation of the target cell population. Changes in fatty acid acylation and reacylation may be one mode of vitamin D3 action in cartilage.

Effects of combining transforming growth factor beta and 1,25-dihydroxyvitamin D3 on differentiation of a human osteosarcoma (MG-63).

Transforming growth factor beta (TGF beta) and 1,25-dihydroxyvitamin D3 (1,25D3), when added simultaneously to a human osteosarcoma cell line, MG-63, induce alkaline phosphatase activity 40-70-fold over basal levels, 6-7-fold over 1,25D3 treatment alone, and 15-20-fold over TGF beta treatment alone. TGF beta and 1,25D3 synergistically increased alkaline phosphatase specific activity in both matrix vesicles and plasma membrane isolated from the cultures, but the specific activity was greater in and targeted to the matrix vesicle fraction. Inhibitor and cleavage studies proved that the enzymatic activity was liver/bone/kidney alkaline phosphatase. Preincubation of MG-63 cells with TGF beta for 30 min before addition of 1,25D3 was sufficient for maximal induction of enzyme activity. Messenger RNA for liver/bone/kidney alkaline phosphatase was increased 2.1-fold with TGF beta, 1.7-fold with 1,25D3, and 4.8-fold with the combination at 72 h. Human alkaline phosphatase protein as detected by radioimmunoassay was stimulated only 6.3-fold over control levels with the combination. This combination of factors was tested for their effect on production of three other osteoblast cell proteins: collagen type I, osteocalcin, and fibronectin. TGF beta inhibited 1,25D3-induced osteocalcin production, whereas both factors were additive for fibronectin and collagen type I production. TGF beta appears to modulate the differentiation effects of 1,25D3 on this human osteoblast-like cell and thereby retain the cell in a non-fully differentiated state.

Initial effects of partially purified bone morphogenetic protein on the expression of glycosaminoglycan, collagen, and alkaline phosphatase in nonunion cell cultures.

Bone morphogenetic protein (BMP) stimulates mesenchymal cells to differentiate, resulting in de novo endochondral ossification in vivo. The response of fibrocartilage and periosteal cells from human and canine nonunion tissues to partially purified BMP was examined in culture. Cells derived from neonatal rat muscle explants were used for comparison. Alkaline phosphatase activity and expression of alkaline phosphatase and Types I and II collagen mRNAs were compared to that of rat chondrocytes. Synthesis of Type II collagen by the muscle cells was verified by enzyme-linked immunosorbent assay (ELISA). Addition of BMP to the muscle cell and nonunion cell cultures resulted in a dose-dependent decrease in cell number. There was a decrease in matrix vesicle and plasma membrane alkaline phosphatase activity concomitant with an increase in mRNA levels for alkaline phosphatase and collagen genes. Synthesis of immunoreactive Type II collagen increased. These data indicate that neonatal rat muscle cells and nonunion cells may respond in a similar fashion to BMP. Bone morphogenetic protein stimulated hyaluronic acid synthesis at three days, but chondroitin sulfate synthesis did not increase until ten days exposure to BMP. These data, together with those summarized above, suggest that more than three days may be required for complete expression of the chondrocyte phenotype typical of endochondral ossification.

Regulation of matrix vesicle phospholipid metabolism is cell maturation-dependent.

We have developed a chondrocyte culture model for assessing the regulation of matrix vesicles at two different stages of chondrogenic maturation. These chondrocytes, resting zone (RC) and growth zone (GC), retain their phenotypic markers in culture, including production of matrix vesicles with distinctive lipid compositions and enzyme activities. Isolated matrix vesicles incubated in vitro with 1,25-(OH)2D3 (1,25) or 24,25-(OH)2D3 (24,25) respond differentially. 1,25 stimulates phospholipase A2 (PA2) in GC vesicles, but not on those from RC. 24,25 inhibits PA2 in RC vesicles, but has no effect on GC. PA2 activity is required for fatty acid turnover and is the rate-limiting step in prostaglandin production. Plasma membrane phospholipids are more susceptible to the release of arachidonic acid by PA2 than are matrix vesicle phospholipids. Matrix vesicles are distinct from the plasma membrane in terms of lipid composition and arachidonic acid incorporation. 1,25 and 24,25 stimulate arachidonic acid turnover in their target cells, but by different mechanisms. 1,25 has no effect on arachidonic acid turnover in RC; however, 24,25 inhibits turnover in RC and GC. 1,25 and 24,25 also affect isolated matrix vesicle membrane fluidity. These results suggest that vitamin D metabolites modulate PA2 activity, change the composition of membrane phospholipids by altering fatty acid composition, and affect calcium transport. The effects are mediated by altering membrane fluidity and is dependent on the stage of cell differentiation.

Stimulation of matrix vesicle enzyme activity in osteoblast-like cells by 1,25(OH)2D3 and transforming growth factor beta (TGF beta).

After demonstrating the presence of matrix vesicles in three osteosarcoma cell lines, MG-63, ROS 17/2.8 and MC-3T3-E1, we sought to determine whether two major enzymes localized to matrix vesicles, alkaline phosphatase and phospholipase A2, could be regulated by 1,25(OH)2D3 and/or TGF beta. Intravesicular calcification is probably dependent on these two enzymes. Alkaline phosphatase is essential for hydrolysis of phosphate-containing substrates and phospholipase A2 hydrolyzes diacylphosphatides in a calcium-mediated manner at lipid-aqueous interfaces leading to changes in membrane fluidity and possibly breakdown of the matrix vesicle. The 1,25(OH)2D3 induced increase of alkaline phosphatase in bone cells is localized to the matrix vesicle. TGF beta also increased alkaline phosphatase activity in two of the cell lines, MG-63 and ROS 17/2.8 but to a greater degree than 1,25(OH)2D3. Matrix vesicle alkaline phosphatase activity exhibited a greater response than that in the plasma membrane. TGF beta increased phospholipase A2 activity in both matrix vesicles and plasma membranes, therefore, no targeting was observed with respect to this enzyme. When TGF beta was combined with 1,25(OH)2D3, 1,25(OH)2D3 had no effect on phospholipase A2 and did not interfere with TGF beta stimulation of phospholipase A2 activity. When 1,25(OH)2D3 and TGF beta were combined, a tremendous synergy was observed in alkaline phosphatase specific activity in both plasma membranes and matrix vesicles with targeting to matrix vesicles. Therefore, TGF beta not only plays an important role in matrix formation and differentiation, but works in conjunction with 1,25(OH)2D3 to greatly potentiate the effects seen with 1,25(OH)2D3 alone.

The effect of bone injury on extracellular matrix vesicle proliferation and mineral formation.

Removal of tibial bone marrow in rats is followed by primary bone formation, resorption and marrow restitution. The first week of healing is characterized by partially calcified trabeculae. After 2 weeks, a higher degree of calcification and partial resorption are observed. The third week is characterized by massive resorption of the trabeculae, which are replaced in the fourth week by new bone marrow tissue. This model was used to study primary calcification. Transmission electron micrographs of the young bone revealed osteoblasts, matrix vesicles and calcified fronts. The different vesicular types were defined as 'empty', 'amorphous', 'crystal', and 'rupture'. The vesicles were studied on days 3, 6, 8, 12, 14, 18, 21, 23 and 28 after injury. The mean diameters of most vesicles ranged between 100.3 and 121.9 nm, and their mean distance from the calcified front was less than 976.6 nm. Vesicular density, calculated as number per 10 m2, increased on the eighth day and decreased from the fourteenth day onwards. Highest diameter values were recorded on the sixth day, and decreased onward. Vesicular distance from the calcified front decreased continuously. Distribution of vesicle number, diameter, and distance in each class showed that numbers of empty and amorphous vesicles decreased and of crystal and rupture increased throughout the experiment. Distances from the calcified front and vesicular diameters varied as follows: 'rupture', 'crystal', amorphous', and 'empty', the 'rupture' type being the closest to the front and of the largest diameter. The results confirm the hypothesis that the cell is responsible for the secretion of electron lucent vesicles that accumulate Ca and Pi to form amorphous calcium phosphate complexes that convert to hydroxyapatite. Crystal growth is followed by membrane rupture.

Matrix vesicles contain metalloproteinases that degrade proteoglycans.

This study explored whether extracellular matrix processing enzymes are present in matrix vesicles produced by rat costochondral resting zone and growth zone chondrocytes in culture. It was found that there was a differential distribution of enzyme activities related to the cartilage zone from which the cells were isolated. There was a 3-fold enrichment of total and active acid metalloproteinase in growth zone chondrocyte (GC) matrix vesicles whereas no enrichment in enzyme activity was observed in resting zone chondrocyte (RC) matrix vesicles. Total and active neutral metalloproteinase were similarly enriched 2-fold in GC matrix vesicles. TIMP, plasminogen activator and beta-glucuronidase activities were highest in the plasma membranes of both cell types. No collagenase, lysozyme, or hyaluronidase activity was found. The data indicate that matrix vesicles are selectively enriched in enzymes that degrade proteoglycans. The highest concentrations of these enzymes are found in matrix vesicles produced by growth zone chondrocytes, suggesting that this may be a mechanism by which the more differentiated cell modulates the matrix for calcification.

Studies of matrix vesicle-induced mineralization in a gelatin gel.

Matrix vesicles isolated from fourth-passage cultures of chondrocytes were tested for their ability to induce hydroxyapatite formation in a gelatin gel in order to gain insight into the function of matrix vesicles in in situ mineralization. These matrix vesicles did not appear to be hydroxyapatite nucleators per se since the extent of mineral accumulation in the gel diffusion system was not altered by the presence of matrix vesicles alone, and in the vesicle containing gels, mineral crystals were formed whether associated with vesicles or not. In gels with these matrix vesicles and beta-glycerophosphate, despite the presence of alkaline phosphatase activity, there was no increase in mineral deposition. This suggested that in the gel system these culture-derived vesicles did not increase local phosphate concentrations. However, when known inhibitors of mineral crystal formation and growth (proteoglycan aggregates [4 mg/ml], or ATP [1 mM], or both proteoglycan and ATP) were included in the gel, more mineral was deposited in gels with the vesicles than in comparable gels without vesicles, indicating that enzymes within these vesicles were functioning to remove the inhibition. These data support the suggestion that one function of the extracellular matrix vesicles is to transport enzymes for matrix modification.

Cell maturation-specific autocrine/paracrine regulation of matrix vesicles.

Matrix vesicles are extracellular organelles produced with distinctive phospholipid composition and enzyme activity. They are produced by cells which typically calcify their extracellular matrix and their characteristics are cell-maturation dependent. Regulation of matrix vesicle structure and function occurs at the genomic and non-genomic levels. By following alkaline phosphatase gene transcription, protein concentration, and enzyme specific activity, we have shown that steroid hormones and growth factors exhibit a regulatory influence over gene transcription, protein synthesis, and matrix vesicle activity. Matrix vesicles respond to peptide hormones, other matrix proteins, like alpha 2-HS-glycoprotein, and autocoid mediators as well. Matrix vesicle metabolism can be directly affected by vitamin D metabolites, even in the absence of cells. The results indicate that 1,25-(OH)2D3(1,25D) or 24,25-(OH)2D3(24,25D) produced by the cells in culture can modulate matrix vesicle activity, and suggest that calcifying cells can modulate events in the matrix via autocrine/paracrine stimulation or inhibition of the matrix vesicles. 1,25D and 24,25D regulate matrix vesicle phospholipase A2 activity, fatty acid turnover, arachidonic acid release, PGE2 production and membrane fluidity, which act on the matrix vesicle to alter enzyme activity. Since vitamin D metabolite production is sensitive to both hormones and growth factors, there is potential for fine tuning matrix vesicle behavior.

Epithelial cell lines that induce bone formation in vivo produce alkaline phosphatase-enriched matrix vesicles in culture.

Hypertrophic chondrocytes and osteoblasts produce alkaline phosphatase (ALPase)-enriched matrix vesicles in vivo and in vitro and, along with certain epithelial cell lines and osteoblast precursors, induce bone when implanted in mesenchymal tissues. This study examined whether ALPase-enriched matrix vesicle production in vitro was a general property of cells that induce bone in vivo. Epithelial cell lines FL, WISH, and OK 16; connective tissue cell lines HEPM 1 and HEPM 2; neonatal rat muscle cells; rat costochondral chondrocytes; and human fibroblasts were implanted intramuscularly into nude mice. The FL and WISH cells produced tumors and induced large islands of bone with focal areas of cartilage immediately adjacent to the tumors. The chondrocytes formed cartilage nodules but did not induce bone, indicating that the ability of the cells to form a solid mass was not an a priori requirement for bone formation. No other cell type produced tumors or nodules or induced bone formation, although connective tissue cells have been shown to induce chondrogenesis in vitro and osteogenesis in vivo. Only matrix vesicles from normal chondrocytes, FL, WISH, and OK16 cultures exhibited enriched ALPase-specific activity. Matrix vesicles from FL and WISH cultures exhibited ALPase specific activities similar to those isolated from osteoblast or chondrocyte cultures. These data suggest that the ability to produce ALPase-enriched matrix vesicles in culture may be associated with the ability of cells to induce bone or cartilage in vivo.

Matrix vesicles are enriched in metalloproteinases that degrade proteoglycans.

This study examined the presence of extracellular matrix processing enzymes in matrix vesicles produced by rat costochondral resting zone and growth zone chondrocytes in culture. Optimum procedures for the extraction of each enzyme activity were determined. Enzyme activity associated with chondrocyte plasma membrane microsomes was used for comparison. There was a differential distribution of the enzyme activities related to the cartilage zone from which the cells were isolated. Acid and neutral metalloproteinase (TIMP), plasminogen activator, and beta-glucuronidase were highest in the growth zone chondrocyte (GC) membrane fractions when compared with matrix vesicles and plasma membranes isolated from resting zone chondrocyte (RC) cultures. There was a threefold enrichment of total and active acid metalloproteinase in GC matrix vesicles, whereas no enrichment in enzyme activity was observed in RC matrix vesicles. Total and active neutral metalloproteinase were similarly enriched twofold in GC matrix vesicles. TIMP, plasminogen activator, and beta-glucuronidase activities were highest in the plasma membranes of both cell types. No collagenase, lysozyme, or hyaluronidase activity was found in any of the membrane fractions. The data indicate that matrix vesicles are selectively enriched in enzymes which degrade proteoglycans. The highest concentrations of these enzymes are found in matrix vesicles produced by growth zone chondrocytes, suggesting that this may be a mechanism by which the more differentiated cell modulates the matrix for calcification.

Regulation of prostaglandin E2 production by vitamin D metabolites in growth zone and resting zone chondrocyte cultures is dependent on cell maturation.

The production of PGE2 by chondrocytes and its regulation by vitamin D metabolites was examined in this study as a function of cell maturation. Costochondral chondrocytes, derived from the resting zone and growth zone cartilage, were grown in culture to fourth passage. At confluence, they were exposed to 10(-8)-10(-11)M 1,25-(OH)2D3 or to 10(-7)-10(-10)M 24,25-(OH)2D3 for either five minutes or 3, 6, 12, or 24 hours. Indomethacin (10(-7)M) was added to one-half of the cultures to block the production of PGE2. The amount of PGE2 released into the media was determined by radioimmunoassay. Both growth zone and resting zone cells produced PGE2 in a time-dependent manner; PGE2 concentration was greater in the resting zone cell cultures. 1,25-(OH)2D3 stimulated PGE2 production by growth zone cells in a dose-dependent manner, significant at 10(-8)-10(-10)M. This effect was observed at 3 hours and remained elevated during the 24 hours of culture. 1,25-(OH)2D3 had no effect on PGE2 production by resting zone cells. However, 24,25-(OH)2D3 (10(-7)-10(-8)M) inhibited PGE2 production from 3-24 hours. No effect was noted when 24,25-(OH)2D3 was added to growth zone cells. Indomethacin reduced PGE2 production to baseline values in all groups examined. The results indicate that chondrocytes in culture produce PGE2. Production is regulated by vitamin D3 metabolites and is cell maturation-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro studies on the regulation of endochondral ossification by vitamin D.

The research described in this article has focused on the complex autocrine, paracrine, and endocrine regulation of endochondral ossification using vitamin D metabolites and TGF-beta as models. By comparing results from a number of laboratories utilizing a diverse array of in vivo and in vitro systems, a coherent picture is beginning to emerge. Vitamin D metabolites influence cell differentiation and maturation and have direct effects on cell function. Differentiation of the mesenchymal cells into chondroblasts is regulated by both 1,25-(OH)2D3 and 24,25-(OH)2D3, as well as by TGF-beta. The resting zone chondrocytes respond primarily to 24,25-(OH)2D3 in terms of matrix synthesis and matrix vesicle biochemistry. They synthesize both metabolites and other factors that stabilize matrix vesicle enzymes like AHSG. In addition to the paracrine role these factors may play in regulating the matrix, it is possible that they may influence the cells in the growth plate itself. Growth zone chondrocytes also synthesize both metabolites, but respond primarily to 1,25-(OH)2D3 for the parameters measured in the studies described. These cells also synthesize TGF-beta which further increases alkaline phosphatase activity, perhaps via an autocrine stimulation of the cell. While cells from the calcified zone have not yet been studied directly in culture, it is likely that they respond to paracrine signals from the avascular cartilage as well as to serum-derived factors. How the signals are transferred among the cells is unknown. Certainly one can postulate information flow in both upward and downward directions. The signal transduction mechanisms for the factors at the cellular level are complex. While it is known that 1,25-(OH)2D3 stimulates gene transcription and stabilization of mRNA for proteins like alkaline phosphatase, its nongenomic effects are only beginning to emerge. Membrane effects of this metabolite have been shown in intestine and kidney in conjunction with studies on Ca flux. It is becoming increasingly evident that other steroid hormones may operate in similar ways. Studies with the rat costochondral chondrocytes are the first to show that there are specific membrane effects for at least two vitamin D metabolites and that membrane enzymes, including those involved in phospholipid metabolism, can be differentially regulated by them. Furthermore, these experiments have provided for the first time a clear hypothesis for how cells can regulate events in the extracellular matrix after the matrix vesicles are produced and incorporated into the matrix.

Effect of glass ceramic and titanium implants on primary calcification during rat tibial bone healing.

The effect of bone bonding (KG Cera, Mina 13, and titanium) and nonbone bonding (KGy-213, M 8/1) implants on primary calcification in endosteal bone was examined by comparing changes in the morphometry of matrix vesicles to those occurring during normal bone healing following ablation of rat tibial marrow. The concentration of matrix vesicles, their diameter, and their distance from the calcification front were determined using computerized cytomorphometry at the transmission electron microscopic level. The results demonstrated that bone bonding materials supported an increase in matrix vesicle concentration when compared with control bone at 6 and 14 days postimplantation. At 14 days, there were fewer matrix vesicles in the bone adjacent to the nonbonding implants. Though matrix vesicle diameter decreased in the control bone between 6 and 14 days, it increased in all of the experimental samples. Diameters were significantly greater in the bone bonding samples at 14 days and significantly lower in the nonbonding samples at 6 days. Distance from the calcification front decreased between 6 and 14 days in all groups except in bone adjacent to the KGy-213 implants. In bone adjacent to the bone bonding implants, distance from the calcification front was comparable to or further than that of control bone; in the nonbonding samples it was closer to the calcification front. These results demonstrate that production and maturation of matrix vesicles is influenced in a differential manner by the presence of implant materials.

Inhibition of 1,25-(OH)2D3- and 24,25-(OH)2D3-dependent stimulation of alkaline phosphatase activity by A23187 suggests a role for calcium in the mechanism of vitamin D regulation of chondrocyte cultures.

This study used the ionophore, A23187, to examine the hypothesis that the regulation of alkaline phosphatase and phospholipase A2 activity by vitamin D3 metabolites in cartilage cells is mediated by changes in calcium influx. Confluent, fourth-passage cultures of growth zone and resting zone chondrocytes from the costochondral cartilage of 125 g rats were incubated with 0.01-10 microM A23187. Specific activities of alkaline phosphatase and phospholipase A2 were measured in the cell layer and in isolated plasma membranes and matrix vesicles. There was an inhibition of alkaline phosphatase specific activity at 0.1 microM A23187 in resting zone cells and at 0.1 and 1 microM in growth zone chondrocytes. At these concentrations of ionophore, the 45Ca content of the chondrocytes was shown to increase. Both the plasma membrane and matrix vesicle enzyme activities were inhibited. There was no effect of ionophore on matrix vesicle or plasma membrane phospholipase A2 in either cell type. In contrast, alkaline phosphatase activity is stimulated when growth zone chondrocytes are incubated with 1,25-(OH)2D3 and in resting zone cells incubated with 24,25-(OH)2D3. Phospholipase A2 activity is differentially affected depending on the metabolite used and the cell examined. Addition of ionophore to cultures preincubated with 1,25-(OH)2D3 or 24,25-(OH)2D3 blocked the stimulation of alkaline phosphatase by the vitamin D3 metabolites in a dose-dependent manner. The effects of ionophore were not due to a direct effect on the membrane enzymes since enzyme activity is isolated membranes incubated with A23187 in vitro was unaffected. These results suggest a role for calcium in the action of vitamin D metabolites on chondrocyte membrane enzyme activity but indicate that mechanisms other than merely Ca2+ influx per se are involved.

Matrix vesicle enzyme activity in endosteal bone following implantation of bonding and non-bonding implant materials.

The effect of bone bonding (KGy-Cera) and non-bone bonding (KGy-213) implant materials on primary mineralization was examined in endosteal bone repair following marrow ablation. Comparisons were made to determine implant effect on concentration and biochemical parameters of matrix vesicles, as contrasted to vesicles in normal bone healing. Matrix vesicle number was determined by high-resolution computerized morphometric analysis, and implant effect on the specific activity of alkaline phosphatase and phospholipase A2 was measured. Bone responses differed according to the composition of the implant material. The bone bonding implant in this study stimulated matrix vesicle formation, alkaline phosphatase specific activity, and, to a lesser extent, phospholipase A2 activity. The effect of the non-bonding implant on healing bone was of suppression of enzyme specific activities and reduced matrix vesicle production. The results indicate that the bone bonding implant material (KGy-Cera) promotes the initiation of primary mineralization, whereas failure of the KGy-213 to bond may be related to toxic materials that leach from the implant and inhibit the normal sequence of events in the mineralization cascade. The results also demonstrate that the implant materials alter the healing process distal to the injury site. Changes observed in the contralateral control limb mimic the changes observed in the injured limb, but at lower magnitude.