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Immunolocalization of choline phospholipids in mineralized tissues
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BIOCHEMICAL MARKERS AS A VALUABLE ALTERNATIVE TO BONE WINERAL CONTENT DETERMINATION G. Reina (a), L. ktoro lb), P. Bettica (al, P. Villani (a), A. Rubinacci (cl, G.L. fjoro (cl, B. de Bernard-m and A. Pecile (al (a) Dept. of Pharmacology, Cheno;herapy and #edical Toxicology, Univ. of f:ilano, Italy; (b) Dept. of Biochenistry, Biophysics and Chenistry of liacronolecules, liniv. of Triesee, Italy; (c) 0r;hopaedic Clinic, SCientific Inst., 5. Raffaele Hosp., Univ. of Flilano, Italy. Ain of this study is to show how physical deterninatiOnS of bone mineral ConCent can be effectively substituted by nore practical ;neasurenen:s of biochemical ;;larkersof bone catabolism in urines, This statement, based on the results of a new physical model developed by ;he authors, was proved by measuring urinary excretion of galactosyl-hydroxylysine and hydroxyproline in growins rats. Indeed, the developed model predicts a linear relationship be:ween bone mineral content and biochemical markers excreted with urines and thus suggests the possibility of substituting bone mineral conten; determinations with those of ;;larkers by a simple calibra:ion of the parameters of the i;lodel. These parameters, depending only on the marker enployed and on the aninal species involved, can be easily precalculated standardized method of thus producing a possible
ZALACTOSYL 3RINARY
HYDROXYLYSINE AND EXCRETION AND IN VIVO "ONTENT OF GROWING RATS. ‘P. Villani, “*L.Moro.***A.Rubinacci,**~GL.Moro **@.de Bernard. “Dep. pharmacology Orthop. IMilano) ,**Dep. Biochem.
and ***Cllnlc Trieste, Italy This study was sought to establish the rates of galactosy1 hydroxylyslne (GHYLI and hydroxyproline (OH-PROL) urinary excretion and the changes in bone mineral content (BMC) a~ a function of age and’sex in the rat. 5 male (Ml and 5 female (F) rats 49 days old were studled for 4.5 months. 24 hrs urine samples were collected and BMC of the proximal metaphysis of the hind limb was measured a t. different days during the experiment. GHYL excretion was 112596 umoles/g creatinine (CR) (M) and 94.227.1 umoles/ CR (FI at the beginning of the experiment and 3 decreased three fold by the end of It, with no different ’ age-course in the two sexes. OH-PROL was 14.8i1.6 mmoles/g CR (M) and 15.321 mmoles/g CR (8) initially and dlminlshed five fold by the end of the experlrent wrth no difference for the sex. FOP both GHYL and OH-PROL 86% to 83% of the decrement took place ln the first 63 days, when the rats can be consldered adult [age 98 days) . BMC that was 91.7t6.6 mg/cm (M) and 96.63;3 mg/cm (F) at the beginning increased twice by the end of the experiment. Al so for BMC 68% of the maximal change took place in the first 63 days. In conclusion: 11 the excretion of GHYL and OH-PROL decreases wrth age; 2) BMC increases with 3) the maximal changes of the parameters age; occur in Lhe first 3 months of the rat life.
analysis.
The experimental data obeained in growingi rats conpletely confirm the resulCs predicted by the model and urge us on further studies on bone metabolism by application of similar nodelling approaches. The proposed model and its application to galactosyl-hydroxylysine and hydroxyproline urinary excretion will be discussed. 243 IMMlJNOLOCALIZATIONOF
HYOROXYPROLINE BONE ;.IINERAL *p. Bettlca, *A.pcclle .I
244
CHOLINE PHOSPHOLIPIDS IN MINERALIZED TISSUES. T. Tsuji. k$ P. 3lrk. J. V. Ruth. Institut de Biologie Midicale. Facult6 de MBdecine. Universit6 Louis Pasteur. Strasbourg. France. INSEAM CJF 88-08. Mineralization in various tissues such as cartilage. bone and dentine includes the potential calcification of numerous independent. spatially distinct intra- and ex.,laccllular tissue components. Calcifiable phospholipids are enriched in calcifying cells and tissues and seem to be involved in mineral deposition. Tissue distribution and subcellular localization of phospholipids in calcifying tissues were examined using a monoclonal antibody against choline phospholipids (MCZZ-33F):Mark et. al submitted. 3 day old Swiss mouse demineralized condyle cartilage. mandibular bone and tooth germ were subjected for indirect immunofluorescence microscope and immunoelectron microscope studies. Cellular lysosomes and matrix vesicles present in calcifying tissues strongly reacted with MC22-33FMAb. Positively reacting lysosomes showed membrane-like structure and frequency of matrix vesicles increased with mineral deposition in extracellular matrix. This study using MC22-33F MAb offers an interesting alternative to histochcmical lipids stains for investing fatty metamorphosis and extracellular lipid deposition in ion under in calcificat matrix vesicles involved dcvclopmental conditions.
IN VW0 AND IN VITRCJ OSTEOGENIC EFFECT OF NACRE POWDER IN THE PRESENCE OF HUMAN BONE AND BONE CELLS. E. LowP.. 8. Vidal".. G. Camprasse.. S. Cambrass?..C. SilveO. *Museum National d’Hisloire Naturelle, Physiologic; CNRS URA 90; “HBpital Necker Enfants Malades, CNI?S URA SS3,Paris, France. Wh;n nacreous shell produced by Ihc marine oyster PinctaduMaxima, used as a biomaterial in oral surgery, is implanted in human bone, new bone formation occurs, resulting in a tight welding of the bone lo Ihe nacrc. To furlher characterize Ihe osbzogcnic effect of nacre, a biomincralized calcium carbonate under pure aragonie form (bman laser spcc~roscopy), we evaluated Ihe capacity of nacre powder lo induce bone formation and mineralization in uivo and in vitro in the presence of human osrcoblas,s. Nacre powder was used as a rcplaning biomalcrial to repair maxillary bone defects in 6 patients. Rapid stimulation of maxillary canccllous bone L .nation was obtained in all cases. Undccalcificd sections of a control bone biopsy obtained 6 months after surgery in one patient showed inlcnse oslcoblastic activily, rcsuhing in an important development ofwoven bone. This new bone was in intimate contact with the biomatcrial. No fibrous tissue could be observed at Ihe tight smooth interfaces between new bone and thebiomatcrial,and there was no cvidcnccofinllammatoryor foreignbody-type reactions. No ostcoclasts were seen at Ihc time of evaluation. New bone appeared clcctrodcnsc on microradiographs, demonstrating In one palicnt, compact jaw bone destroyed following mineralization. drastic extended infection was successfully repaired following the nacre powder rcplaning. In this patient, compact bone, cnrichcdin ramified ostcocytes and organized into havcrsicn system, was observed on a biopsic obtained 12 months aflcr surgery. The effect of nacrc powder Was also tested in vitro in confluent cultures of human osteoblasls, in the absence of chemical inducers (e.g, P-glycerophosphalc or ascorbic acid). 3 weeks aflcr fhc addition of nacre powder 10 the ostcoblasts, tridimcnsionnal mineralized nodules devcloppcd, and a dccrcasc in alkaline phosphatasc aclivity was concomitantly dcmonstralcd. E.M observation of Clw nodules demonstralcd findings typical of those described in Ihc courseof bono matrix mineralization in uivu. No evidence of loxiciCy WJS observed. ln conclusion, “acre powder induced bone formlion in UI’UO and inf&o. TIC availability of this biomatcrial should have imporlant applications in oral, maxilla-facial and orthopedic surgery, and facilMes studies evaluating the cellular cvcnls rcquircd fur mineralized process.
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242
BIOCHEMICAL MARKERS AS A VALUABLE ALTERNATIVE TO BONE WINERAL CONTENT DETERMINATION G. Reina (a), L. ktoro lb), P. Bettica (al, P. Villani (a), A. Rubinacci (cl, G.L. fjoro (cl, B. de Bernard-m and A. Pecile (al (a) Dept. of Pharmacology, Cheno;herapy and #edical Toxicology, Univ. of f:ilano, Italy; (b) Dept. of Biochenistry, Biophysics and Chenistry of liacronolecules, liniv. of Triesee, Italy; (c) 0r;hopaedic Clinic, SCientific Inst., 5. Raffaele Hosp., Univ. of Flilano, Italy. Ain of this study is to show how physical deterninatiOnS of bone mineral ConCent can be effectively substituted by nore practical ;neasurenen:s of biochemical ;;larkersof bone catabolism in urines, This statement, based on the results of a new physical model developed by ;he authors, was proved by measuring urinary excretion of galactosyl-hydroxylysine and hydroxyproline in growins rats. Indeed, the developed model predicts a linear relationship be:ween bone mineral content and biochemical markers excreted with urines and thus suggests the possibility of substituting bone mineral conten; determinations with those of ;;larkers by a simple calibra:ion of the parameters of the i;lodel. These parameters, depending only on the marker enployed and on the aninal species involved, can be easily precalculated standardized method of thus producing a possible
ZALACTOSYL 3RINARY
HYDROXYLYSINE AND EXCRETION AND IN VIVO "ONTENT OF GROWING RATS. ‘P. Villani, “*L.Moro.***A.Rubinacci,**~GL.Moro **@.de Bernard. “Dep. pharmacology Orthop. IMilano) ,**Dep. Biochem.
and ***Cllnlc Trieste, Italy This study was sought to establish the rates of galactosy1 hydroxylyslne (GHYLI and hydroxyproline (OH-PROL) urinary excretion and the changes in bone mineral content (BMC) a~ a function of age and’sex in the rat. 5 male (Ml and 5 female (F) rats 49 days old were studled for 4.5 months. 24 hrs urine samples were collected and BMC of the proximal metaphysis of the hind limb was measured a t. different days during the experiment. GHYL excretion was 112596 umoles/g creatinine (CR) (M) and 94.227.1 umoles/ CR (FI at the beginning of the experiment and 3 decreased three fold by the end of It, with no different ’ age-course in the two sexes. OH-PROL was 14.8i1.6 mmoles/g CR (M) and 15.321 mmoles/g CR (8) initially and dlminlshed five fold by the end of the experlrent wrth no difference for the sex. FOP both GHYL and OH-PROL 86% to 83% of the decrement took place ln the first 63 days, when the rats can be consldered adult [age 98 days) . BMC that was 91.7t6.6 mg/cm (M) and 96.63;3 mg/cm (F) at the beginning increased twice by the end of the experiment. Al so for BMC 68% of the maximal change took place in the first 63 days. In conclusion: 11 the excretion of GHYL and OH-PROL decreases wrth age; 2) BMC increases with 3) the maximal changes of the parameters age; occur in Lhe first 3 months of the rat life.
analysis.
The experimental data obeained in growingi rats conpletely confirm the resulCs predicted by the model and urge us on further studies on bone metabolism by application of similar nodelling approaches. The proposed model and its application to galactosyl-hydroxylysine and hydroxyproline urinary excretion will be discussed. 243 IMMlJNOLOCALIZATIONOF
HYOROXYPROLINE BONE ;.IINERAL *p. Bettlca, *A.pcclle .I
244
CHOLINE PHOSPHOLIPIDS IN MINERALIZED TISSUES. T. Tsuji. k$ P. 3lrk. J. V. Ruth. Institut de Biologie Midicale. Facult6 de MBdecine. Universit6 Louis Pasteur. Strasbourg. France. INSEAM CJF 88-08. Mineralization in various tissues such as cartilage. bone and dentine includes the potential calcification of numerous independent. spatially distinct intra- and ex.,laccllular tissue components. Calcifiable phospholipids are enriched in calcifying cells and tissues and seem to be involved in mineral deposition. Tissue distribution and subcellular localization of phospholipids in calcifying tissues were examined using a monoclonal antibody against choline phospholipids (MCZZ-33F):Mark et. al submitted. 3 day old Swiss mouse demineralized condyle cartilage. mandibular bone and tooth germ were subjected for indirect immunofluorescence microscope and immunoelectron microscope studies. Cellular lysosomes and matrix vesicles present in calcifying tissues strongly reacted with MC22-33FMAb. Positively reacting lysosomes showed membrane-like structure and frequency of matrix vesicles increased with mineral deposition in extracellular matrix. This study using MC22-33F MAb offers an interesting alternative to histochcmical lipids stains for investing fatty metamorphosis and extracellular lipid deposition in ion under in calcificat matrix vesicles involved dcvclopmental conditions.
IN VW0 AND IN VITRCJ OSTEOGENIC EFFECT OF NACRE POWDER IN THE PRESENCE OF HUMAN BONE AND BONE CELLS. E. LowP.. 8. Vidal".. G. Camprasse.. S. Cambrass?..C. SilveO. *Museum National d’Hisloire Naturelle, Physiologic; CNRS URA 90; “HBpital Necker Enfants Malades, CNI?S URA SS3,Paris, France. Wh;n nacreous shell produced by Ihc marine oyster PinctaduMaxima, used as a biomaterial in oral surgery, is implanted in human bone, new bone formation occurs, resulting in a tight welding of the bone lo Ihe nacrc. To furlher characterize Ihe osbzogcnic effect of nacre, a biomincralized calcium carbonate under pure aragonie form (bman laser spcc~roscopy), we evaluated Ihe capacity of nacre powder lo induce bone formation and mineralization in uivo and in vitro in the presence of human osrcoblas,s. Nacre powder was used as a rcplaning biomalcrial to repair maxillary bone defects in 6 patients. Rapid stimulation of maxillary canccllous bone L .nation was obtained in all cases. Undccalcificd sections of a control bone biopsy obtained 6 months after surgery in one patient showed inlcnse oslcoblastic activily, rcsuhing in an important development ofwoven bone. This new bone was in intimate contact with the biomatcrial. No fibrous tissue could be observed at Ihe tight smooth interfaces between new bone and thebiomatcrial,and there was no cvidcnccofinllammatoryor foreignbody-type reactions. No ostcoclasts were seen at Ihc time of evaluation. New bone appeared clcctrodcnsc on microradiographs, demonstrating In one palicnt, compact jaw bone destroyed following mineralization. drastic extended infection was successfully repaired following the nacre powder rcplaning. In this patient, compact bone, cnrichcdin ramified ostcocytes and organized into havcrsicn system, was observed on a biopsic obtained 12 months aflcr surgery. The effect of nacrc powder Was also tested in vitro in confluent cultures of human osteoblasls, in the absence of chemical inducers (e.g, P-glycerophosphalc or ascorbic acid). 3 weeks aflcr fhc addition of nacre powder 10 the ostcoblasts, tridimcnsionnal mineralized nodules devcloppcd, and a dccrcasc in alkaline phosphatasc aclivity was concomitantly dcmonstralcd. E.M observation of Clw nodules demonstralcd findings typical of those described in Ihc courseof bono matrix mineralization in uivu. No evidence of loxiciCy WJS observed. ln conclusion, “acre powder induced bone formlion in UI’UO and inf&o. TIC availability of this biomatcrial should have imporlant applications in oral, maxilla-facial and orthopedic surgery, and facilMes studies evaluating the cellular cvcnls rcquircd fur mineralized process.
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