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Distribution of noncollagenous matrix proteins (NCPS) in the calcified matrix of normal bone: Evidence of anatomical and functional heterogeneity
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Abstracts from Bone Morphology 1992, Lexington, Kentucky
57 DlSTRlBUTlON OF NONCOLLAGENOUS PROTEINS (NCPs) IN THE CALCIFIED
58 MATRIX
MATRIX OF NORMAL BONE: EVIDENCE OF ANATOMICAL AND FUNCTIONAL HETEROGENEITY. BT lnararu BL Clarke, LW Fishefl, LA Fitzpatrick. tNational Institutes oi Health, Bethesda, MD 20892, USA. Mayo Clinic and Mayo Foundation, Rochester, MN 55905, USA.
MICROCRACKS HUMAN
SJsaS. h&i, Dpt. Dpt.
ARTICULAR
CALCIFIED
CARTILAGE
OF
HEADS
F. Kanaya, S. Sale. K. Jbaraki. D. B. Burr*. R.C. Haroff”
Orthopedic Anatomy*.
Loading
lmmunohistological studies indicate that in developing bone most NCPs are limited to osteoblasts, osteoid and osteocytes. However, little is known of the distribution of matrix proteins within the calcified matrix of adult bone. We therefore used immunohistochemistry to examine the anatomical distribution of several NCPs within the calcified matrix of bone from 16 normal males. Undecalcified iliac crest bone biopsies were obtained from healthy male volunteers (ages 20-70; serum and urine chemical indices were normal), embedded in plastic and sections (5 pm) were immunostained using primary antibodies or normal rabbit serum. Osteopontin (OP) and bone sialoprotein (BSP) stained intensely at cement lines and the mineralized matrix immediately adjacent to each haversian canal. Osteocalcin (OC) also stained cement lines, however, a progressive decrease in intensity was observed within each osteon. Osteonectin (ON) did not stain cement lines and within the osteon showed a pattern similar to OC. Decorin was prominent in the matrix surrounding osteocytes, their canaliculi and the matrix adjacent to the haversian canal. Biglycan appeared evenly distributed throughout bone matrix. We hypothesize that the patterns of OP and BSP incorporation may reflect a mechanism by which the attachment of osteoblasts and osteoclasts is promoted at cement lines and haversian canals, respectively. In conclusion, these results suggest that the incorporation of NCPs into matrix may vary depending on the stage of formation of individual bone units.
IN
FEMORAL
Surgery. University Dpt. Pathology**,
within
of the Ryukyus. JAPAN Indiana University. Indiana
the physiologic
range can produce subslanlial load~cycles in cortic@l bone sugis a physiologic event in skeletal aissues. The purposeof lhisstudyis Lo determine whether microcrbcks occur in the calcified cartilage in_riua and represent a physiolqgic event. Materials and Methods Six grossly non-osteoarthrotic femoral heads were fixed. cut in the
microdamige withrela&ly few gesting
Lhat fatigue
mid-frontal plane and 1.5 cm thick slabs were obtainad from each half. 150 mcm thick sections were made from one slab and stained with 1% basic fochsin(conlro1). The other slabs were Block stained with 1% basic fuchsin at first. then 150 mcm thick seQtions were made (experimental). The number of stained and unstained microcracks were counted. Because basic fuchsin permeales every space in the se&on, stained cracks in the experimenlql sections were present before sectioning. whereas unstained cracks were artifacts produced by the sectioning and grinding procdss. Since control sections were stained after sectioning and gridding. slained cracks on lhese sections represent a combination of ciacks presenl before preparation and those caused by lhe preparalian ilself. Results and Discussion The average total crack density (stained and unslainedb of control sections (0.49lmm) did not differ from (hat of experilhental sections (0.47Imm: a.~.). This implies that the bulk stain (technique does not cause more arlifactual cracking through deh).dralion lhan do standard staining techniques. Experimental sections. had significanlly fewer stained cracks (0.19/mm) than the conlrol sections (0.34/mm; pcO.05). This implies that j~u& microcracjks in calcified carlilage can be separated from the artifaclual cracks and are caused by routine physiological evenls. Supported
by
NIH
grant
AR39708
59
60
SYNERGISTIC EFFECT OF 1,25-VITAMIN D AND FLUORIDE ON BONE ALUMINUM ACCUMULATION T.H. It&!, H.G. Sieberth. Dept. of Internal Medicine, RWTH, Aachen, FRG Aluminum (Al) accumulation in bone of patients with renal failure reduces the extent of osteoblastic osteoid surface (Ob.S/OS) and may cause low turnover bone disease. Fluoride (F) has been shown to decrease Al deposition in bone and to antagonize the Al-induced reduction in osteoblast (Ob) number. The present study investigated the effect of combined treatment with F (0.9 mgld) and 1,25(OH)zDs (10 rig/d)) on the deposition of Al in bone in 5/6 nephrectomized rats. Following a parenteral Al load (3.2 mglkgxd, 9 weeks) histomorphometry of lumbar vertebrae revealed a severe adynamic bone disease as evidenced by low Ob.S/OS (12.8*2.6%) and mineral apposition rates [MAR] (O.Olfo.01 pm/d) with little osteoidosis (osteoid volume [OVIBV): 8.4ti.996). Concurrent oral administration of F resulted in a significant increase in Ob.S/OS (88.w4.3%, P
SCANNING ELECTRON MICROSCOPY OF HUMAN LUMBAR VERTEBRAL JAP Jayasingbe. SJ Jones and TBABECULAR BONE SURFACES. A Boyde. Department of Anatomy and Developmental Biology, University College London, London WClE 6BT. UK. to In the present study, SEM was employed characterise trabecular bone surfaces of fourth lumbar vertebral bodies derived from young adult, old and osteoporotic subjects. The objective of the study was to understand the micromorphology of these surfaces and relate them to changes during ageing and in osteoporosis. The specimens used during this study were defatted with chloroform methanol and made superficially anorganic by treating them with hydrogen peroxide to remove the unmineralized organic matrix. prior to Au sputter coating and stereo imaging using both secondary ad backscattered electrons. The basic micro-organisatioln of vertebral trabecular bone is not significantly difflerent from that previously described for other sites. The most interesting results of the present study concern the relative incidence of the different surface activrty states with age and the increasing occurrence of unusually textured in .adult mineral fronts. The rate of mineralizbtion trabecular bone tissue is considerably less than the values found in young growing individuala. The peculiar morphology of the fronts could be regarded as arrested reflecting the reduced mineralization, possibly mineralization rate. but their extent is greater than any estimate of the proportion of double tetra$ycline labelled surfaces. It is concluded that elderly and osteoporotic individuals have, to an extent increasiig with age. a layer of non-mineralizing matrix presenlt at free bone surfaces. There is also evidence for an ihcrease of nonmineralized matrix seams within bone. If Dsteoid is bone matrix which can proceed to mineralization. then this is not such. Whether or not superficial unmitieralizedmatrix can eventually proceed to mineralization. the present study demonstrates that resorption occuirs through the defective "osteoid". and such non-mineralized matrix may be largely removed by resorption rather than diminished by mineralization.
Abstracts from Bone Morphology 1992, Lexington, Kentucky
57 DlSTRlBUTlON OF NONCOLLAGENOUS PROTEINS (NCPs) IN THE CALCIFIED
58 MATRIX
MATRIX OF NORMAL BONE: EVIDENCE OF ANATOMICAL AND FUNCTIONAL HETEROGENEITY. BT lnararu BL Clarke, LW Fishefl, LA Fitzpatrick. tNational Institutes oi Health, Bethesda, MD 20892, USA. Mayo Clinic and Mayo Foundation, Rochester, MN 55905, USA.
MICROCRACKS HUMAN
SJsaS. h&i, Dpt. Dpt.
ARTICULAR
CALCIFIED
CARTILAGE
OF
HEADS
F. Kanaya, S. Sale. K. Jbaraki. D. B. Burr*. R.C. Haroff”
Orthopedic Anatomy*.
Loading
lmmunohistological studies indicate that in developing bone most NCPs are limited to osteoblasts, osteoid and osteocytes. However, little is known of the distribution of matrix proteins within the calcified matrix of adult bone. We therefore used immunohistochemistry to examine the anatomical distribution of several NCPs within the calcified matrix of bone from 16 normal males. Undecalcified iliac crest bone biopsies were obtained from healthy male volunteers (ages 20-70; serum and urine chemical indices were normal), embedded in plastic and sections (5 pm) were immunostained using primary antibodies or normal rabbit serum. Osteopontin (OP) and bone sialoprotein (BSP) stained intensely at cement lines and the mineralized matrix immediately adjacent to each haversian canal. Osteocalcin (OC) also stained cement lines, however, a progressive decrease in intensity was observed within each osteon. Osteonectin (ON) did not stain cement lines and within the osteon showed a pattern similar to OC. Decorin was prominent in the matrix surrounding osteocytes, their canaliculi and the matrix adjacent to the haversian canal. Biglycan appeared evenly distributed throughout bone matrix. We hypothesize that the patterns of OP and BSP incorporation may reflect a mechanism by which the attachment of osteoblasts and osteoclasts is promoted at cement lines and haversian canals, respectively. In conclusion, these results suggest that the incorporation of NCPs into matrix may vary depending on the stage of formation of individual bone units.
IN
FEMORAL
Surgery. University Dpt. Pathology**,
within
of the Ryukyus. JAPAN Indiana University. Indiana
the physiologic
range can produce subslanlial load~cycles in cortic@l bone sugis a physiologic event in skeletal aissues. The purposeof lhisstudyis Lo determine whether microcrbcks occur in the calcified cartilage in_riua and represent a physiolqgic event. Materials and Methods Six grossly non-osteoarthrotic femoral heads were fixed. cut in the
microdamige withrela&ly few gesting
Lhat fatigue
mid-frontal plane and 1.5 cm thick slabs were obtainad from each half. 150 mcm thick sections were made from one slab and stained with 1% basic fochsin(conlro1). The other slabs were Block stained with 1% basic fuchsin at first. then 150 mcm thick seQtions were made (experimental). The number of stained and unstained microcracks were counted. Because basic fuchsin permeales every space in the se&on, stained cracks in the experimenlql sections were present before sectioning. whereas unstained cracks were artifacts produced by the sectioning and grinding procdss. Since control sections were stained after sectioning and gridding. slained cracks on lhese sections represent a combination of ciacks presenl before preparation and those caused by lhe preparalian ilself. Results and Discussion The average total crack density (stained and unslainedb of control sections (0.49lmm) did not differ from (hat of experilhental sections (0.47Imm: a.~.). This implies that the bulk stain (technique does not cause more arlifactual cracking through deh).dralion lhan do standard staining techniques. Experimental sections. had significanlly fewer stained cracks (0.19/mm) than the conlrol sections (0.34/mm; pcO.05). This implies that j~u& microcracjks in calcified carlilage can be separated from the artifaclual cracks and are caused by routine physiological evenls. Supported
by
NIH
grant
AR39708
59
60
SYNERGISTIC EFFECT OF 1,25-VITAMIN D AND FLUORIDE ON BONE ALUMINUM ACCUMULATION T.H. It&!, H.G. Sieberth. Dept. of Internal Medicine, RWTH, Aachen, FRG Aluminum (Al) accumulation in bone of patients with renal failure reduces the extent of osteoblastic osteoid surface (Ob.S/OS) and may cause low turnover bone disease. Fluoride (F) has been shown to decrease Al deposition in bone and to antagonize the Al-induced reduction in osteoblast (Ob) number. The present study investigated the effect of combined treatment with F (0.9 mgld) and 1,25(OH)zDs (10 rig/d)) on the deposition of Al in bone in 5/6 nephrectomized rats. Following a parenteral Al load (3.2 mglkgxd, 9 weeks) histomorphometry of lumbar vertebrae revealed a severe adynamic bone disease as evidenced by low Ob.S/OS (12.8*2.6%) and mineral apposition rates [MAR] (O.Olfo.01 pm/d) with little osteoidosis (osteoid volume [OVIBV): 8.4ti.996). Concurrent oral administration of F resulted in a significant increase in Ob.S/OS (88.w4.3%, P
SCANNING ELECTRON MICROSCOPY OF HUMAN LUMBAR VERTEBRAL JAP Jayasingbe. SJ Jones and TBABECULAR BONE SURFACES. A Boyde. Department of Anatomy and Developmental Biology, University College London, London WClE 6BT. UK. to In the present study, SEM was employed characterise trabecular bone surfaces of fourth lumbar vertebral bodies derived from young adult, old and osteoporotic subjects. The objective of the study was to understand the micromorphology of these surfaces and relate them to changes during ageing and in osteoporosis. The specimens used during this study were defatted with chloroform methanol and made superficially anorganic by treating them with hydrogen peroxide to remove the unmineralized organic matrix. prior to Au sputter coating and stereo imaging using both secondary ad backscattered electrons. The basic micro-organisatioln of vertebral trabecular bone is not significantly difflerent from that previously described for other sites. The most interesting results of the present study concern the relative incidence of the different surface activrty states with age and the increasing occurrence of unusually textured in .adult mineral fronts. The rate of mineralizbtion trabecular bone tissue is considerably less than the values found in young growing individuala. The peculiar morphology of the fronts could be regarded as arrested reflecting the reduced mineralization, possibly mineralization rate. but their extent is greater than any estimate of the proportion of double tetra$ycline labelled surfaces. It is concluded that elderly and osteoporotic individuals have, to an extent increasiig with age. a layer of non-mineralizing matrix presenlt at free bone surfaces. There is also evidence for an ihcrease of nonmineralized matrix seams within bone. If Dsteoid is bone matrix which can proceed to mineralization. then this is not such. Whether or not superficial unmitieralizedmatrix can eventually proceed to mineralization. the present study demonstrates that resorption occuirs through the defective "osteoid". and such non-mineralized matrix may be largely removed by resorption rather than diminished by mineralization.