Matrix Gla protein binding to hydroxyapatite is dependent on the ionic environment: calcium enhances binding affinity but phosphate and magnesium decrease affinity.

Matrix Gla protein (MGP) is an inhibitor of mineralization found in bone, cartilage, developing tissues, smooth muscle, and atherosclerotic plaques. MGP interaction with hydroxyapatite (HA) has been inferred by its function, but has never been measured directly. In this study, the influence of MGP antibody (x-MGP) binding, plasmin digestion, and various ions, including calcium and phosphate, on (125)I-labeled MGP-HA binding was examined. Nonlinear regression analysis of MGP binding yielded K(a) (association constant; approximately 8.0 x 10(4) M(-1)) and B(max) (maximum specific bound fraction of MGP; approximately 0.53). Anti-MGP antiserum reduced K(a) to less than half of control (0.33% x-MGP). Plasmin-digested MGP decreased HA binding parameters by almost a third, showing that protein binding and limited proteolysis greatly affected HA binding. The presence of free calcium ions significantly increased binding in a dose-dependent manner, with approximately 1 mmol/L calcium increasing K(a) by a factor of 2. Phosphate ions decreased binding significantly in a dose-dependent fashion, with approximately 1 mmol/L PO(4) decreasing K(a) by a third. Magnesium at approximately 1 mmol/L decreased K(a) significantly by half, but the effect was not dose-dependent. Carbonate, sulfate, and sodium ions had no significant effect on binding. MGP binding to HA is sensitive to protein binding, limited proteolysis, and the surrounding ionic environment.

Correlations between osteocalcin content, degree of mineralization, and mechanical properties of C. carpio rib bone.

Osteocalcin is one of the most abundant noncollagenous proteins in bone. It is strongly associated with the mineral phase of bone, and has long been associated as a marker of bone turnover. However, its relationship to bone composition, strength, and structure is unclear. Carp rib bone is an excellent model for the study, because osteocalcin represents almost 60% of the total extractable noncollagenous proteins found in it. Because of the abundance of osteocalcin relative to other extractable proteins, any changes in the properties of carp rib bone would be more likely influenced by the osteocalcin concentration. To test the hypotheses that the concentration of osteocalcin is reflected in other properties of bone, the correlations between the osteocalcin concentration and the mineral content, microstructural properties, and physical characteristics of the bone mineral crystals were determined utilizing radioimmunoassay (RIA), spectrophotometry, nanoindentation, and small-angle X-ray scattering (SAXS) techniques, respectively. Osteocalcin concentration was found to be correlated to the molar Ca/P ratio and inversely correlated to the elastic modulus and hardness in the longitudinal plane. This study provides evidence for a putative relationship between the concentration of osteocalcin and the microstructural mechanical properties of bone. Correlations were also found between the mechanical properties in the longitudinal plane and both the phosphate content and the molar Ca/P ratio. However, no relationships could be identified between osteocalcin concentration and several parameters of bone crystals, as determined by SAXS.

The distribution of osteocalcin, degree of mineralization, and mechanical properties along the length of Cyprinus carpio rib bone.

This study examined the spatial distribution of selected biochemical and mechanical properties along the length of carp rib bone. Carp rib bone was chosen because of its unusually high osteocalcin content relative to other extractable proteins. The amount of osteocalcin was significantly lower (p<0.01) at the most distal section, relative to all other sections. The amount of phosphate (p<0.05) and the elastic modulus in the longitudinal plane (p<0.0001) were found to be significantly higher in the most distal section, relative to the most proximal section. There was no significant difference in the calcium distribution, molar Ca/P ratio, or elastic modulus in the transverse plane. It was speculated that the distal section contains less mature bone. The methods illustrate the potential usefulness of nanoindentation to characterize the mechanical properties of bone, relative to its biochemical composition.

A plasmin-derived hexapeptide from the carboxyl end of osteocalcin counteracts oxytocin-mediated growth inhibition [corrected] of osteosarcoma cells.

We have previously described the presence of the functional plasminogen activator system on the surfaces of bone neoplastic cells and the fact that plasmin specifically cleaves bone matrix protein osteocalcin (OC). The cleavage of OC to NH2-midterminal (1-44) and COOH-terminal RFYGPV hexapeptide (44-49) proceeds with detachment of both products from bone mineral. Because the sequence of OC-derived hexapeptide (HP) is nearly identical to the E2 region of the oxytocin receptor (OTR), we set out to ascertain whether the HP interferes with the osteosarcoma (OS)-associated oxytocin (OT) system. We documented the presence and functional activity of OTRs in several OS cells by means of (a) OT-mediated inhibition of OS growth; (b) expression of OTR mRNA by means of reverse transcription-PCR; (c) immunofluorescence staining with IF3 monoclonal antibody specific for human OTR; and (d) saturation binding and Scatchard analysis of OT binding to the receptors of isolated membranes or intact OS cells. Although we could not demonstrate direct binding of HP to OT, the presence of HP in cultures of OS cells antagonizes the inhibitory effect of OT on these cells. Additionally, in competitive binding assays, the HP effectively competes with binding of OT to its cognate receptors. The results indicate the existence of an OTR/OT system in tumor cells of bone origin. Suggested plasminogen activator-OC-OTR/OT interactions may have an effect on the regulation of cell proliferation within the bone tissue as well as properties of the extracellular matrix surrounding the tumor foci in the bone.

Osteopontin expression in spontaneously developed neointima in fowl (Gallus gallus).

Fowl show spontaneous elevation of blood pressure and neointimal plaque formation in the abdominal aorta at young ages. A similar neointima can be induced by a balloon-catheter-induced endothelium injury to the fowl aorta. Both spontaneously developed and injury-induced vascular lesions exhibit subendothelial hyperplasia consisting of neointimal cells with a synthetic phenotype and abundant extracellular matrix. The role of the extracellular matrix in the formation of neointima is not known. In this study, we investigated whether osteopontin, an adhesive glycoprotein present in the extracellular matrix, is expressed in aortic smooth muscle tissue of the fowl abdominal aorta, in spontaneously developed neointimal plaques and in the aortic smooth muscle underlying neointimal plaques. Crude protein extracted from isolated aortic smooth muscle tissues and neointimal plaques was fractionated by SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting with rabbit anti-fowl osteopontin (provided by Dr L. C. Gerstenfeld, Boston University) or anti-&agr; smooth muscle actin antibodies. The anti-fowl osteopontin antibody predominantly recognized a 66-70 kDa protein band in neointimal plaques that co-migrated with the osteopontin phosphoprotein from chick bone. In contrast, intact aortic smooth muscle and the smooth muscle underlying neointimal plaques equally expressed three proteins (66-70 kDa, approximately 50 kDa and approximately 43 kDa) recognized by the anti-osteopontin antibody. Anti-&agr; smooth muscle actin antibody recognized a 43 kDa protein band, and the expression of &agr; smooth muscle actin was higher in aortic smooth muscle than in neointimal plaques. Osteopontin mRNA expression was examined using reverse transcription-polymerase chain reaction (RT-PCR) of total RNA from vascular tissues with specific primers constructed on the basis of the reported fowl osteopontin nucleotide sequence. The PCR products from intact aortic smooth muscle and neointimal plaques correspond to the product from recombinant plasmid cDNA (a gift from Dr L. C. Gerstenfeld) transcribed in vitro. These results suggest that osteopontin is synthesized in intact aortic smooth muscle and neointimal plaques in fowl and that unmetabolized approximately 66 kDa osteopontin protein is a predominant form in the neointima, indicating that osteopontin protein may be actively synthesized in the neointima.

Plasmin-mediated proteolysis of osteocalcin.

Plasmin cleaves osteocalcin at a site within its carboxyl end, thus creating an N-midterminal 1-43 and a short C-terminal 44-49 peptides. The products of the cleavage were identified by matrix assisted laser desorption ionization time of flight mass spectrophotometry and by reversed phase high performance liquid chromatography followed by N-terminal sequence determination. When separated by sodium dodecyl sulfide-polyacrylamide gel electrophoresis in the presence of reducing agents, large (LF; N-midterminal) and a small molecular weight (SF; C-terminal) fragments can be identified. The major cleavage site involves arg43-arg44 amino acid residues, and the resulting 44-49 C-terminal fragment appears as a slow migrating band on native gels (SFnat). Elevated levels of calcium ion inhibit the plasmin-mediated lysis of osteocalcin. Plasmin-mediated cleavage of osteocalcin occurs both in solution and when bound to hydroxyapatite. Both osteocalcin cleavage products detach from the hydroxyapatite substrate. Diisopropyl fluorophosphate-inhibited plasmin does not displace osteocalcin from the hydroxyapatite surface. Previously, the C-terminal pentapeptide has been shown to be chemotactic for bone cells while bone particles lacking osteocalcin were resistant to bone resorption. We therefore hypothesize that the plasmin-mediated digestion of free and hydroxyapatite-bound osteocalcin could play a role in the regulation of bone remodeling.

Matrix Gla protein gene expression is elevated during postnatal development.

Matrix Gla protein (MGP) is a vitamin K-dependent extracellular matrix protein with a wide tissue distribution. Developmental expression of the MGP gene is characterized by competitive RT-PCR in kidney and calvaria. High levels of MGP mRNA were observed in kidneys and calvaria from 19-day-old embryos to 1-month-old rats. There was a peak in MGP mRNA at 7 days in both tissues. MGP mRNA expression was very low or undetectable in 3-, 5- and 7- month-old kidneys. Similar observations were seen in lung, heart and spleen. However, in connective tissues like calvaria, tibia and trachea, low levels of MGP mRNA are maintained throughout life. Kidney MGP protein was present from birth to 15 days, with the highest MGP protein level at 7 days. Calvarial MGP protein was present throughout development and maturation but peaked at 7 days. The highest MGP protein levels were coincident with peak levels of MGP mRNA. Thus, MGP protein level correlated with mRNA level during rat development. In situ hybridization revealed that MGP staining was most intense in the straight tubules of the developing kidney medulla at 7 days. Staining was absent in stromal cells and in mature nephrons. Taken together, our finding of high MGP mRNA and its intense in situ staining during the postnatal growth phase prove that increased MGP synthesis occurs at a specific time and place during development and maturation.

Quantitation of matrix Gla protein mRNA by competitive polymerase chain reaction using glyceraldehyde-3-phosphate dehydrogenase as an internal control.

Matrix Gla (gamma-carboxyglutamic acid) protein (MGP) is a vitamin-K-dependent extracellular matrix protein. A method was developed to quantitate MGP mRNA based on competitive polymerase chain reaction following reverse transcription (competitive RT-PCR). The MGP cDNA was coamplified with a mutant MGP cDNA (competitor). The ratio of MGP to competitor after the PCR reaction was compared to standards to determine the amount of MGP mRNA in RT samples. MGP mRNA in as little as 3.125 ng total RNA was accurately quantitated and was far more sensitive than RNA hybridization methods. To control for variations due to sample preparation, a second competitive RT-PCR was developed to measure the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA from the same sample as an internal control. Thus, the amount of MGP is normalized to the amount of the housekeeping gene GAPDH. The accuracy, sensitivity and ease of this new method enables rapid mRNA quantitation without blotting, hybridization or autoradiography. The method is particularly advantageous for MGP mRNA measurement from a small amount of sample. Using this assay, we established that MGP mRNA increases approx. fivefold with co-treatment of retinoic and ascorbic acids.

Presence of messenger ribonucleic acid encoding osteocalcin, a marker of bone turnover, in bone marrow megakaryocytes and peripheral blood platelets.

Osteocalcin (Oc), an abundant gamma-carboxylated protein (mol wt, 5800) of bone matrix, is used as a serum marker of bone turnover because it is considered to be uniquely synthesized by the osteoblast. Our finding of Oc messenger RNA (mRNA) in rat tibial diaphyseal marrow led us to investigate the cellular origins of Oc mRNA in peripheral blood and bone marrow. In anticoagulated blood, Oc mRNA was detected in total RNA prepared from buffy coat cells (BCC). Fractionation of rat and human blood showed that platelets contain Oc mRNA identical to that found in bone cells. In rat bone marrow, Oc mRNA is highly enriched in the platelet-producing megakaryocyte population. Depending upon the RIA used, immunoreactive Oc was either undetectable or present at very low levels in platelets and megakaryocytes, suggesting that synthesis of Oc by these cells may be under strong translational regulation. In addition, Oc levels were higher in serum vs. plasma obtained from the same blood, suggesting that Oc may be released by platelets during blood clotting. Interestingly, the magnitude of this difference was greater in female rats. Injection of 1,25-dihydroxyvitamin D3 dose-dependently increased plasma Oc, but did not cause correlative changes in steady state levels of Oc mRNA in BCC. During rat growth, plasma Oc was maximal, whereas Oc mRNA levels in BBC were low. This relationship was reversed during aging. A correlation between Oc mRNA levels in BCC and rat age suggests a developmental regulation of Oc mRNA levels in platelets. These data indicate that Oc mRNA is not restricted to cells on mineralizing surfaces, but is also found in megakaryocytes and peripheral blood platelets, which possibly contribute to the Oc levels in blood and the regulation of bone turnover.

Isolation and characterization of the reaction product of 4-diazobenzenesulfonic acid and gamma-carboxyglutamic acid: modification of the assay for measurement of beta-carboxyaspartic acid.

gamma-Carboxyglutamic acid (Gla)-containing proteins are extracellular proteins with enhanced cation or mineral-binding properties. Discovery of new Gla-containing proteins is facilitated by methods that decrease the number of steps and time involved in assaying for Gla. Reaction of 4-diazobenzenesulfonic acid (DBS) and Gla or Gla-containing proteins produces an intensely red-colored product. This method has been used to identify Gla-containing proteins in crude extracts of proteins. The reaction product of Gla and DBS has been purified by reversed-phase HPLC and characterized by uv-visible spectroscopy and electrospray ionization (ESI) mass spectrometry (MS). The red-colored product exhibits an absorption maximum at 530 nm. The ESI-MS data of the colored derivative of Gla are consistent with replacement of the two gamma-carboxyl groups in Gla with two DBS groups. DBS also reacts with another malonic acid derivative, beta-carboxyaspartic acid (Asa). The optimum conditions for colorimetric assay of Asa were established, and the potential of this reaction as an assay for Asa and Asa-containing proteins was studied.

Effect of aging and dietary restriction on matrix Gla protein and other components of rat tracheal cartilage.

Age-related changes in connective tissues can alter their functions of elasticity, compressibility and support. Matrix Gla protein (MGP) is a vitamin K-dependent connective tissue component of unknown function. We have purified bovine MGP, and developed a specific radioimmunoassay for it. Since it is found in highest concentration in cartilage, we have developed quantitative extraction methods for MGP, and examined the age-related changes of MGP relative to other components found in the cartilage matrix. The ratio of hydroxyproline to MGP increases with age, while the ratio of glycosaminoglycan to MGP is constant. No effect is seen for MGP in the dietary restricted rat with prolonged lifespan, while both hydroxyproline and glycosaminoglycan contents of tracheal cartilage are significantly increased by dietary restriction (p < or = .05). These data show that MGP and glycosaminoglycan concentration are relatively constant in rats from 6 to 30 months of age, while hydroxyproline concentration increases with age.

Rapid and sensitive method of quantitation of bone gla protein mRNA using competitive polymerase chain reaction.

A method for sensitive quantitation of bone gla protein (BGP, osteocalcin) mRNA has been developed using competitive polymerase chain reaction after reverse transcription (competitive RT-PCR). The complementary DNA (cDNA) were transcribed from sample RNA was co-amplified in a PCR with a known amount of mutant BGP cDNA (competitor) using the identical oligonucleotide primers. The mutant cDNA with its unique restriction site allowed quantitation of sample and mutant PCR products after densitometric analysis of ethidium bromide-stained agarose gels. A linear relationship between initial sample BGP amount and the ratio of BGP to mutant BGP band intensity was obtained and used to make a standard curve to determine the initial BGP mRNA of unknown samples. These standard curves were made with known amounts of recombinant BGP cDNA. The competitive RT-PCR for BGP allows measurement of twofold differences in 1 and 2 micrograms total RNA and requires at least 10 times less sample RNA than usual Northern blotting. Moreover, heteroduplexes with one BGP strand and one mutant BGP strand formed as a result of high PCR cycles were quantifiable. This provided the advantages of rapid quantitation from ethidium bromide-stained gels without blotting, hybridization, or autoradiography. Multiple samples could be assayed for greater confidence in the results. The sensitivity, accuracy, and ease of the assay will facilitate analysis of BGP mRNA from a small amount of sample. The assay has been used to confirm the BGP mRNA changes with hormonal treatment in cultured cells and the age-related changes in whole tibia in vivo.

Discovery of bone gamma-carboxyglutamic acid protein in mineralized scales. The abundance and structure of Lepomis macrochirus bone gamma-carboxyglutamic acid protein.

The mineralized scale of the freshwater sunfish Lepomis macrochirus (bluegill) contains a Gla protein. The protein was identified in extracts of scale by a new colorimetric assay for Gla-containing proteins. The protein was purified by gel filtration chromatography followed by reversed phase high performance liquid chromatography (HPLC). Several tests establish the identity of scale Gla protein and bone Gla protein (BGP). First, the proteins exhibit identical mobilities on electrophoresis and by reversed phase HPLC. Second, they have identical amino-terminal amino acid sequences. Finally, identical peptides are generated by proteolytic digestion. The 45-residue amino acid sequence of the bone Gla protein from L. macrochirus has a high sequence homology with swordfish, as well as homology to mammalian bone Gla protein. The BGP of bluegill shares with swordfish BGP a truncated NH2 terminus and an extended COOH terminus. These features may be unique to fish, as they have not been observed in terrestrial vertebrates. The bluegill BGP is the first vitamin K-dependent protein to contain a non-gamma-carboxylated residue to the NH2-terminal side of all of its Gla residues. In all other vitamin K-dependent proteins, Gla always appears to the NH2-terminal side of the first Glu. The implications of this result are discussed. The bluegill rib bone is curiously enriched in BGP, as are other mineralized tissues of this species. One hypothesis is that this may be due to the acellular nature of the bone in this species. The abundance of BGP in the bones of this fish may provide clues to the unknown function of this bone protein.

Evaluation of the usefulness of serum phosphatases and osteocalcin as serum markers in a calcium depletion-repletion rat model.

The present study examined the usefulness of the serum alkaline phosphatase (ALP) activity, osteocalcin, and tartrate-resistant acid phosphatase (TrACP) activity as bone turnover markers in a calcium depletion/repletion rat model. Weanling rats were fed calcium-deficient diet for 4 weeks, followed by 2 weeks of dietary calcium repletion. Serum phosphatases and osteocalcin were determined and compared with those of corresponding age-matched, pair-weighted controls. Rats were sacrificed at the end of each phase of the study, and bone phosphatase activities in tibiae and vertebrae were measured. During calcium depletion, rats developed hypocalcemia and lost significant bone calcium, which were reversed with dietary calcium repletion. During depletion when previously published histologic studies indicated a suppressed bone formation and stimulated bone resorption, serum ALP activity and osteocalcin levels were significantly elevated and serum TrACP activity reduced; at the same time, the bone ALP and TrACP activities were increased. Because the serum level of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) which has been shown to stimulate the synthesis of skeletal ALP and of osteocalcin, was also significantly increased during depletion, the increased serum ALP and osteocalcin level could be indirect consequences of the hypocalcemia-mediated elevation in 1,25(OH)2D3 level. These effects were reversed upon calcium repletion, during which previously published histologic studies demonstrated a stimulated bone formation and a suppressed resorption in these rats. In conclusion, although there is increasing evidence for the usefulness of these serum proteins as markers of bone metabolism in humans, a great deal more work is required before we can understand the significance of these assays. Until such is accomplished, these assays should not be assumed to be validated.

The effect of food restriction and germ-free environment on age-related changes in bone matrix.

Studies were performed to determine the effects of food restriction or a germ-free environment on age-related changes in bone. Four groups of male Lobund-Wistar rats were examined at 6 months, 18 months, and 30 months of age. Conventional-free-fed rats were housed in routine laboratory cages and fed ad libitum. Conventional-restricted diet rats were fed 12 grams a day, which becomes restrictive at 8 weeks of age. Germ-free rats were maintained using gnotobiotic procedures and were free of pathogens. The germ-free rats were maintained on either the full-fed or restricted diet regimens. Serum bone Gla protein and matrix concentrations of calcium, magnesium, and hydroxyproline (reflecting collagen content) and bone Gla protein were not significantly different between the four treatment groups. All of these parameters except hydroxyproline, however, showed age-related declines in all four treatment groups. We conclude that prolongation of rat life span by dietary restriction and/or a germ-free environment did not alter the measured parameters in each age group, and did not alter or slow the age-related changes in the bone matrix.

A colorimetric assay specific for gamma-carboxyglutamic acid-containing proteins: its utility in protein purification procedures.

A colorimetric method for the detection of gamma-carboxyglutamic acid (Gla)-containing proteins after reaction with 4-diazobenzenesulfonic acid is presented. Proteins can be visualized after electroblotting from polyacrylamide gels onto membrane supports, after dot-blotting onto membranes, or in solution as a red colored product with an absorbance maximum at 530 nm. The method is specific since other proteins without gamma-carboxyglutamic acid do not form a red color. The presence of other proteins does not inhibit or affect color production by gamma-carboxyglutamic acid-containing proteins. Application of the method for staining a Western blot of a crude extract of bone resulted in staining of only the gamma-carboxyglutamic acid-containing proteins. The usefulness of the method was verified when a second gamma-carboxyglutamic acid-containing protein, prothrombin, also resulted in red color production. A linear color response is seen up to 17 microM for the gamma-carboxyglutamic acid-containing protein bone Gla protein and up to 27 microM for the amino acid. The detection limit is down to 1 microgram of bone Gla protein or 0.17 nmol of the protein on electroblots or dot blots. The simplicity of the method allows rapid screening for gamma-carboxyglutamic acid-containing proteins or allows monitoring of purifications of these proteins in chromatographic or electrophoretic separations.

Cells isolated from fetal rat calvaria by isopycnic separation express different collagen phenotypes.

Calvaria from 20-21 day old fetuses were obtained under sterile conditions and the endo- and exoperiosteum stripped off. Cells were dispersed by sequential collagenase-DNase treatment and suspended in 0.5% low Tm agarose in the presence of DMEM supplement with 10% FCS. After 4-5 days of incubation some 30% of these cells showed active synthesis of metachromatic extracellular matrix. Cells from skin, muscle and periosteum failed to show metachromatic matrix positive colonies to a comparable extent. The phenotypic expression of these cells was determined by analysis of collagen types. Eleven day old cultures were incubated in the presence of [3H]-proline plus beta-aminopropionitrile and ascorbic acid and the collagen extracted analyzed by polyacrylamide electrophoresis of their intact chains or CNBr-derived peptides. The results show that anchorage independence is a requirement for calvaria cells to express type II collagen. Type I collagen was preferentially expressed in monolayer culture or when pre-attached to a substrate before being cultured in agarose. Type II collagen was the predominant collagen when cells were cultured in agarose. Further characterization of cell populations was achieved by isopycnic centrifugation in a percoll gradient. Cell fractions were tested for their collagen phenotype when cultured in agarose. Cells recovered from densities 1.04 g/ml or higher synthesized type II collagen, while cells with densities lower than 1.04 g/ml synthesized mainly type I collagen. Isopycnic centrifugation appears to be a novel method for separation of phenotypically different cells from a heterogeneous population in fetal calvaria. The high density cell fractions may represent a mixture of pre-chondrocytes as well as pluripotential cells.

Cellular events associated with the induction of bone by demineralized bone.

Implantation of demineralized bone (DB) in the form of powder or intact segments in extra skeletal sites stimulates new bone formation. Urist and co-workers presented substantial evidence that there is a noncollagenous protein that has the ability to induce bone formation. One aim of this study was to trace the process of bone formation when DB, in the form of perforated rectangular plates, is implanted subcutaneously in 2-month-old rats. A second objective was to determine whether cartilage cells play a role in the formation of bone in this model. Various DB plates with 0.25 mm diameter holes were implanted subcutaneously for 1-4 weeks in rats. One week after implantation, DB plates were covered by vascularized connective tissue that invaded the perforations. Aggregates of chondrocytes were observed within the holes and on periosteal surfaces in only a few specimens. Further cartilage proliferation was not observed, and by the 2nd week there was no evidence of endochondral bone formation. Where these cartilage-like cells were present, a thin layer of mineral was deposited around them; resorption and fibrous tissue infiltration followed. This aborted form of endochondral calcification was not followed spatially by bone formation. Patent vascularized channels were invaded by alkaline phosphatase-positive mononuclear cells and fibroblasts, and became enlarged by the enzymatic action of macrophages. The next step involved the calcification of DB plates adjacent to the wide spaces. Osteoclasts now appeared leading to the resorption of this recalcified matrix. The eroded and now enlarged lacunar surfaces were lined by newly formed bone and osteoblasts. This process continued so that, at the end of 4 weeks following implantation, the original DB plates were replaced by trabecular bone. Biochemical data on calcium and alkaline phosphatase levels in the implants paralleled the morphological observations.