Mechanisms of bone induction by decalcified bone matrix in rats of various ages

Abstracts from the Calcified Tissues Workshop dentate nucleus 17.3, reticular formation 15.2, thalamus anterior 5.3, hypothalamus 4.5, caudate nucleus 3.0, pituitary 2.3, cerebellum 1 .O. Binding of hormones to specific receptors on the cells is the first step in biological interaction between the hormones and the cells. The occurence of PTH specific binding sites in brain and pituitary suggest, that PTH may be excert an autonomic-regulative role in the Ca-homeostasis in central nervous system and pituitary. 1 Balabanova et al. Klin Wochenschr, 63.419 (1985) 2. Segre et al. J. BIOI Chem. 193:265 (1951)

SEPARATION, PURIFICATION AND CHARACTERIZATION OF ENAMEL PROTEINS, USING HPLC, 20 ELECTROPHORESIS, LIMITED PROTEOLYSIS AND SPECIFIC POLYCLONAL ANTIBODIES D. Deutsch, M. Haisraeli, A. Palmon, A. Arad, A. Alayoff Dental Research Unit, Hebrew University, Hadassah School of Dental Medicine, Jerusalem, Israel Amelogenin and enamelin proteins were extracted from forming, bovine enamel according to the method of Termine et al, 1980. Each protein fraction was subjected to IO-18% SDS slab-gel electrophoresrs followed by Coomassie and silver staining (Sammons et al., 1984), to 2D electrophoresis (Ofarrell 1975) and to HPLC separation (size exclusion and reverse-phase columns). Samples from amelogenin and enamelin fractions as well as individual polypeptides separated by electrophoresis and then electroeluted or individual protein fractions obtained after HPLC separation were each subjected to amino-acid analysi and SDS gel electrophoresis and reacted with specific antibodies against amelogenin or enamelin (Deutsch et al., J. Dent. Res. 64 1985). Silver staining of SDS gel after electrophoresis revealed over 20 specific protein bands, and the changes which occur in the distribution of these proteins at the different stages of development. Further purification of enamelin from contaminating amelogenins and vice-versa was achieved by HPLC using reverse-phase columns. The separation of amelogenin fraction on HPLC size exclusion column revealed about 12 different peaks, some of which have now been identified as amelogenin proteins. The separation of enamelin fraction revealed 6 peaks, 2 peaks being identified as enamelin. Standard molecular weight markers reveal that aggregation often occurred during amelogenin and enamelin separation on the HPLC. Separation of amelogenin and enamelins using isoelectric focussing revealed over 14 distinct protein bands which are now being further characterized using SDS gel, specific antibodies and limited proteolysis. Similar studies are now being carried out on developing human enamel. Supported by research grant 2 R01 DE05780-04. NIH

DISTRIBUTION AND CONCENTRATION OF CALCIUM, PHOSPHORUS AND FLUORIDE IN DEVELOPING HUMAN AND BOVINE ENAMEL FROM ENAMEL SURFACE TO DENTlNOENAMEL JUNCTION D. Deutsch, D. Levrel, Z. Yoeli Dental Research Unit, Hebrew University, Hadassah School of Dental Medicine, Jerusalem, Israel

To study enamel development and mineralisation across the width of enamel (DEJ-Enamel Surface dimension) and the corresponding distribution of fluoride in developing human and bovine teeth, a microdissection technique recently described by Deutsch et al (1984), which allows precise measurements of ion concentrations on a tissue volume basis across this dimension, was used. The results show that generally in the forming stage the concentration of mineral ions Ca and P increase from enamel surface (ES) to Dentino-Enamel Junction (DEJ). In some instances in the very young enamel adjacent to the cervical margin, Ca and P concentrations were high at ES, decreased towards the enamel interior and rose again towards the DEJ. The transition from forming to maturing enamel was characterised by a change in the distribution of mineral ions (Ca and P) and by an increase in mineral concentration. The Ca/P ratio (w/w) of enamel did not change significantly, neither in the width dimension nor In the longitudinal dimension across the different stages of development, and was on average 1.999 2 0.098, indicating that hydroxyapatite is the predominant mineral in developing enamel. The fluoride results show that the gradient in fluoride concentration from enamel surface to DEJ, characteristic of fully mature enamel of the unerupted and erupted tooth, begins to be established at the late formation-early maturation stage, which in some of the deciduous teeth only appears between 8 and 9 months of fetal age and in others only after birth (Deutsch and Pe’er, J. Dent Res 61, 1543- 1551, 1982). These results are important in understanding both the mechanism of dental fluorosis and the timing of fluoride supplementation in an attempt to increase the resistance of enamel to dental caries. Supported by Research Grant 2 ROI DE05780-04 NIH.

MECHANISMS OF BONE INDUCTION BY DECALCIFIED BONE MATRIX IN RATS OF VARIOUS AGES S. Bernick, W. Paule, D. Ertl, M.E. Nimnr Depts. of Anatomy, Biochemistry and Medicine, Univ. of So. Calif., USC School of Medicine and Orthopaedic Hospital of Los Angeles, Los Angeles, CA, USA The purpose of this study was to evaluate the efficacy of decalcified bone to induce osteogenesis when implanted in a non-osteogenic environment (subcutaneously). Rats, two and ten months of age (Long Evans, ACI, Fisher and Wistar), were used. In some instances holes (0.3 mm in diameter) were made prior to decalcification. Bone implants including the surrounding connective tissue were removed 1, 2, 3 and 4 weeks post operative and placed in 10% formalin or in an AFA solution. Following decalcification the specimens were prepared for nitrocellulose embedding in the routine manner. Alternate cut section (12-14 in thickness) were stained with H&E PAS-hematoxylin Alcian blue-PAS, and silver nitrate impregnation. Frozen sections were used for demonstration of acid and alkaline phosphatases. After one week a developing penosteum covered all the surfaces of the implant. Connective tissue elements and blood vessels from this periosteum invaded the “holes”. A few specimens contained aggregates of large chondrogenic-like cells adjacent to the periosteal surface of the decalcified bone as well as in the “holes”. There were no indications of further cartilage for-

Abstracts

from the Calcified

Tissues Workshop

mation or endochondral bone development. By the second week post operative connective tissue elements including macrophages, fibroblasts and blood vessels appeared to invade the patent spaces of the nutrient canals, lacunae and canaliculi, Acid phosphatase positive macrophages were seen within the patent spaces leading to enlarged openings. The openings were then filled with fibroblasts and blood capillaries. With time, round cells resembling osteoblasts (alkaline phosphatase positive) line the surfaces and new bone was deposited. Detectable amounts of calcium begin to appear after 2 weeks. After 4 weeks the Fisher and Long Evans strains showed significantly more calcium (-100 mg Ca+ gm of dry weight in the younger rats) than the Wistar. There was a tendency of bones with drilled holes to calcify more readily (5-15% more) probably as a result of the larger surface area. Older animals showed significantly less calcium accumulation in all the strains evaluated. In all animals at the edges of the holes and spaces there were osteoclasts reflecting remineralization of the implanted matrix. After three to four weeks trabecular bone replacing the decalcified bone matrix was still present and the cavities within the bone trabeculae became filled with marrow cells and fat cells. There was no indication though that the new bone was derived from endochondral bone formation. Supported by NIH Grant AG02577-04.

Ca*+-DEPENDENT INCORPORATION OF POLYAMINES IN THE RENAL BRUSH BORDER MEMBRANE: EFFECTS ON TRANSPORT A. Elgavish, R.W. Wallace, E. Meezan Department of Pharmacology. University of Alabama Birmingham, Birmingham, AL, USA

at

The in vitro effects of polyamines on characteristic properties of isolated renal brush-border membrane vesicles (BB) were investigated to determine whether polyamines have a regulatory role on membrane transport processes. Concomitantly, the effect of small polycationic proteins was also studied. Membranes were incubated for 1 hr, at 37°C with various concentrations of polycations. Following incubation, Na+ gradient-dependent D-glucose transport and Na+/H+ antiport were assayed. The polyamines putrescine, spermidine and spermine were found to stimulate, while lysozyme (MW14,400) and cytochrome C (MW 12,380) inhibited D-glucose uptake in a dose dependent manner. Diffusional L-glucose uptake was not altered, indicating that the polyamines and the small proteins affected the active D-glucose transport, rather than inducing nonspecific changes in membrane lipid properties. The amiloride-sensitive Na+/H+ exchange was slightly inhibited by polyamines and was stimulated by small proteins. The polyamine effects could not be explained solely by the polycationic properties of these agents since the polycationic polypeptides had opposite effects. Spermine was shown to be incorporated into a trichloroacetic acid-insoluble fraction of the membrane proteins by a Ca2+-independent and a Ca 2+-dependent process, the latter possibly as a result of transglutaminase activity present in the isolated membrane. Using SDS-polyacrylamide gel electrophoresis in conjunction with fluorography, [3H]-spermine was shown to be incorporated in several membrane proteins, mainly the 57 KDa, 74KDa and 100 KDa, a heavy M.W. band (~200 KDa) and a low M.W. band (
Our results suggest that polyamine effects may be due to a covalent modification of membrane proteins, possibly via a Caz+-dependent transglutaminase-mediated crosslinking of membrane proteins.

THE EFFECT OF pH, SERUM AND FRESH MEDIUM SUPPLY ON EMBRYONIC RAT CHONDROCYTES Hans Gerber, Isabel Hoerler, Hansjorg Hoch Laboratory

for Experimental

Surgery, Davos-Platz,

Chondrocytes grown in the standard (pH 7.3-7.4; 10% Fetal Calf Serum FCS; 25% fresh medium supply every 2nd day) showed after one week polygonal and fibroblastlike cells and small aggregates with round cells (cartilaginous nodules CN). As time progressed new CN appeared and increased; the remaining space was occupied by monolayered polygonal and fibroblastlike cells, some filled with regular vacuoles by day 28. With 1% FCS cell proliferation was inhibited regardless of pH. From pH 7.0-7.9 and with decreasing FCS (10%-5%-3%-l%) an increasing ratio of round cell shape versus fibroblastlike or polygonal flat spread forms was observed by day 7; at day 28 higher amounts of CN were observed. Regardless of pH, increasing FCS-amounts favoured the appearance of vacuolated cells with time. 8% fresh medium every 4th day favoured differentiation at beginning of culture; after reaching confluency the CN were stagnant in size. 50% of fresh medium every day favoured cell proliferation at beginning of culture but not differentiation; after confluency random CN appeared and increased in size and thickness until day 28 and the monolayered areas displayed predominantly vacuolated cells. In conclusion cell shape of the chondrocyte was influenced in the different conditions of pH, serum and fresh medium supply. A round cell shape encouraged chondrocyte differentiation i.e. development of cartilaginous nodules. The role of vacuolated cells remains unclear.

SINGLE ORAL HIGH DOSE VITAMIN D, PROPHYLAXIS IN THE ELDERLY Y. Weisman, R. Schen, Z. Eisenberg, lchilov Hospital,

N. Amarilio, A. Harell

Tel Aviv, Israel

A poor vitamin D status is common in the elderly during the winter months. Since it is possible that hypovitaminosis D may be a cause of senile osteopenia, a simple method of prophylaxis would be useful. The single oral, high dose method, was tested in two old age homes and the efficacy of vitamin D3 (100000 units) was compared with that of 25-hydroxyvitamin D, (25-OH-DJ (10 Kg/Kg). The trials showed that 25-OHD, caused a higher peak value in the serum 25OHD levels in the second week (51.4 * 4.8 ng/ml) than did vitamin D3 (35.4 & 2.3 ng/,l). However, follow-up after 4-5 months showed that in those patients who received a single oral dose of 25-0HD3, the serum 25-OHD levels had returned to the baseline low values (9.3 2 0.9) whereas in those platients who had had oral vitamin DJ, the serum 25-OHD levels still remained significantly raised (20.3 -t 2.6 ng/ml) compared with the baseline values and were within normal limits. It is concluded that the single, oral high dose method, using vitamin D3, is