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Some scanning microscopies of fibrogenesis imperfecta ossium
Bone, 6, 275283 (1985) Printed in the USA. All rights reserved.
8756-3282185 $3.00 + .OO Copyright 0 1985 Pergamon Press Ltd.
Meeting of the Bone and Tooth Society, Oxford, U.K. (March, 1985) ABNORMALLY LOW LYSINE IN INSOLUBLE DENTINE FROM PATIENTS WITH DSTEOGENESIS IMPERFECTA
essential for this action. The results demonstrated that P-C-P’s bound to mineralized matrix can affect its osteoclastic resorptron in two ways. One mechanism IS inhibitron of the mature resorbing osteoclast itself, involves cytotoxrcity and IS an important element in the actionsof EHDP and Cl,MDP. The other mechanism is prevention, rather than Inhibition of osteoclastic resorption. This mechanism is responsrble for the increased potency of APD in the new culture system and may also underlie the high potency of APD relative to EHDP and Cl,MDP in vivo. The action may involve antagonism, like interference with deposition on mineralized matrix of substances that are chemotactic to osteoclast. Replacement of the amino group of APD by a dimethyl-amino group not only increases Its efficacy in both culture systems but also in VIVO,while eliminating specific toxicity.
M.J.O. Francis, J.P. Gage and R. Smith Nuffield Department Orthopaedic
of Orthopaedic
Surgery, Nuffield
Centre, Oxford OX3 7LD
Mature dentrne and bone both contain Type I collagen, therefore the collagen of dentine should be abnormal in patients with osteogenesis imperfecta (01) who have defects in this collagen We have examined the amino acid composition of insoluble dentine (a fraction that normally contains 92-950~ Type I collagen) from the teeth of 59 patients with 01. Compared with control normal teeth, changes were found in the number of lysine, hydroxylysine, proline, hydroxyproline, and glycine residues per 1000 total amino acids in 53 patient samples. The amino acid composition of the insoluble dentine from eight 01 patients showed an unusual pattern. Judged by glycine, the amount of collagen was normal but the number of lysine residues was markedly reduced (17 or less/lOOO; control range 26 ~fr 2.2, mean f 2 SD). Tyrosine was also reduced in all eight samples (3 or less/l 000; control range 4.5 +- 1.5) and hrstidine increased in four (11 or more/l 000; control range 7 2 2). Multiple peaks were observed in the amino acid analyser tracing in the region of the histidine, hydroxylysine, and lysine peaks. None of these peaks was lysinal, the aldehyde oxidation product of lysine. The changes were not constantly associated with dentinogenesis imperfecta, as three patients had clinically normal teeth. These results suggest that dentine is abnormal in the majority of patients with 01. In a subgroup of patients there were striking abnormalities in the amounts of those amino acids involved in the formation of collagen cross-links.
SOMESCANNINGMICROSCOPIESOF FIBROGENESIS IMPERFECTADSSIUM S.A. Rerd, R. Smith*, and B. Boyde Department of Anatomy, University College London and * Nuffield Orthopaedic
Centre, Oxford
Fibrogenesis imperfecta ossium is a rare connective tissue disorder in which patients suffer progressive bone pain and fracturing leading to immobility. It is characterized histologically by the presence of thick osteoid seams that are nonbirefringent when seen in polarized light. The fibrous component of these seams IS a tangled mass of fine fibrils that replaces the normal lamellar organization of much thicker collagen fibrils. We report here the first study of this disease using the scanning electron microscope (SEM) and tandem scanning reflected light microscope (TSRLM). Parts of two transilliac crest biopsies taken from the same patient, a 51 -year-old male Caucasian, were studied. These were taken 12 months apart, and before each one the patient followed a tetracycline double labelling regime. Light microscopic histology revealed a paucity of bone, especially in the second biopsy. Many surfaces were lined by thick osteoid seams and the use of polarized light confirmed that these were nonbirefringent. Fluorescence using the TSRLM (Boyde and Reid, 1984) demonstrated tetracycline fluorescence at many of the surfaces lined with osteord. However, separate labels could never be resolved and it was concluded that mineralization was either proceeding very slowly, or was occurring within a considerable thickness of tissue at any one time. Secondary electron imagining in the SEM of surfaces cleaned to reveal matrix and mineral fronts showed that the pattern of mineralization was abnormal. This was confirmed by backscattered electron (BSE) imaging in the SEM of polished, cut surfaces of plastic embedded material. Combined BSE and cathodoluminescence investigation demon-
DUAL MODESOF ACTIONOF BISPHDSPHDNATESON DSTEDCIASTSIN VITRO P.M. Boonekamp, G.W.G.M. Lowik, L.J.A. van der Wee-Pals, M.L.L. van Wijk-van Lennep, and 0. L.M. Bijvoet Department of Endocnnology, University Hospital, Leiden, The Netherlands The relative potencies of the bisphosphonates Cl ,MDP, EHDP on the one hand and of APD and dimethyl-APD on the other as inhibitors of osteoclastic resorption of mineralized matrix were studied in a classical bone culture system, and in a novel in vitro bone culture system. In the new system, the resorption of calcified cartilage depends entirely upon prior activation of osteoclast precursors and on the subsequent development of functioning osteoclastsfrom these. The potency of APD relative to the two other P-C-P’s was increased considerably in the new system. It was found that binding of ADP tocalcified matrix was 275
Abstracts Fromthe Bone & Tooth Society
276
strated that the nonbirefringent bone matrix sometimes tained a substantially greater mineral density than normal. Supported by Anatomonical land and MRC.
at-
applied to study the relevance of this enzyme in osteoclastic bone resorption.
Society of Great Britain and ire-
Boyde A., Reid S.A. Bone & Tooth Sonety. September 1984, Abstract 13, 1984.
A QUANTITATIVE CYTOCHEMICAL ASSAY FOR ACID PHOSPHATASE ACTIVITY IN OSTEOCLASTS IN THE FOETAL RAT
CALVARIA
D.M. Webber, D.C. Anderson, W.R. Robertson, and I.P. Braidman University Departments of Medicine and Chemical Pathology, Hope Hospital, Eccles Old Road, Salford M6 8HD Traditional histochemical techniques have shown the lysosomal enzyme, acid phosphatase (APase), to be prominent in osteoclasts (0~‘s). Previous attempts to quantify this activity have been limited to subjective estimation of enzyme product in fixed and decalcified specimens, processes known to cause a loss of enzyme activity. Sodium tartrate has also been used to localise APase activity specifically to Oc’s by inhibiting the tartratesensitive form of enzyme thought to be in other cells present in bone (Minkin, 1982). We now report the biochemical validation of a quantitative cytochemical assay for Oc APase activity in unfixed and undecalcified sections of 21day foetal rat calvaria. Tissue, superchilled to -70°C, was sectioned (5-1 Opm) in a cryostat at - 35%. The sections were incubated at 37°C in 0.1 M citrate/citric acid buffer (pH 4.5) 5% polyvinyl alcohol (5% w/w Grade G181140) and naphtholASBI-phosphate substrate (O-5mM) for various incubation times. The reaction was stopped with ice-cold tap water and the insoluble reaction product post-coupled with Fast Garnet GBC salt (1 mglml) in 0.1 M sodium acetate pH 6.2 at 4°C for IO minutes. The resulting red/brown particulate deposite was measured using a Vickers M85A microdensitometer with x 100 objective, wavelength 550nm, spot size 0.2pm and measuring area of 28rm*. Arbitrary machine units were converted to mean absorbance (A) by reference to a standard calibration curve. A comparison of adjacent sections stained with haematoxylin and eosin with those reacted for APase, showed areas strongly positive for enzyme activity to correspond to the presence of 0~‘s. 15-20 such areas were measured per section, and each experimental point was derived from three slides, In preliminary work it became clear that initial velocity rate conditions prevailed only over short time periods (ie < 10 seconds). Thereafter the increase in absorbance was not proportional to time. Using a range of substrate concentrations (0-5mM) it was found that activity was maximal at 3mM (0.4-0.5A). The enzyme was active over the range pH 3-6, with peak absorbance between pH 3.5 and 4.5. We have thus achieved first order enzyme kinetics using 5pm thick sections, a 5second incubation time, a substrate concentration of 3mM and a pH of 4.5. Under these conditions cartilage and periostal cells measured in the same sections as Oc’s, had barely detectable APase activity (IO-fold lower than in the latter). Sodium tartrate (1 O-l OOmM) had no effect on the specific localisation of enzyme activity in Oc’s, although absolute levels were inhibited (- 50% with 1OOmM). In conclusion, a quantitative cytochemical assay for APase activity in Oc’s has been developed in unfixed and undecalcified cryostat sections. It is now being
MInkIn C Cal Tissue Int 34(3):285-90.
1982
INTERLEUKlNl (ILl)lNDUCESOSTEOBLASTlCCELLSTD STlMULATEOSTEOCLASTlCBONERESORPTlON B.M. Thomson and T.J. Chambers Department of Histopathology, London EC7
St Bartholomew’s Hospital,
Osteoclasts were disaggregated from neonatal rat long bone and sedimented onto cortical bone slices. After 24 hours in culture, the majority of osteoclasts excavate the bone surface, and the extent of bone resorption can be quantitated by computer-assisted morphometric and stereophotogrammetric techniques. IL1 had no effect on bone resorption by such disaggregated osteoclasts, but if osteoblastic cells (cells derived from collagenase digestion of neonatal rat calvaria, and cloned rat osteosarcoma cells) were also sedimented onto the bone slices, IL1 caused a dose-dependent stimulation of osteoclastic bone resorption, IL1 did not stimulate osteoclastic bone resorption if osteoblasts shared the same culture volume but were physically distanced from osteoclasts. These results indicate that IL1 stimulates bone resorption, and does so through a primary action on osteoblasts induced by IL1 to transmit a short-range signal, which stimulates osteoclastic bone resorption.
BACTERlALCAPSULARGLYCOPROTEIN:APOTENTBONE RESORBlNGFACTORANDCYTOTOXlCFACTORFOR FIBROBLASTS. ‘M. Wilson, W. Harvey, S. Meghi, A. Scutt, and *S. Kamin Department of Oral & Maxillofacial Surgery and *Department of Clinical Pathology & Immunology, Eastman Dental Hospital, London WClX 8LD Lipopolysaccharide (LPS) from bacterial cell walls is thought to be an important mediator of tissue breakdown in periodontal disease, but bacterial capsules are considered to be important only in adhesion and protection against phagocytosis. We have purified capsular material (CM) from Actinobacillus actinomycetemcomitans and compared it with purified LPS from the same organism in its effects on bone, osteoblasts and glnglval fibroblasts in vitro. Bone resorption was measured by Careleasefrom 5-day-old mouse calvaria over 3 days. Proliferation [3HTdR incorporation and methylene blue uptake], cytotoxicity [lactate dehydrogenase (LDH) release] and lysosomal enzyme release [acid phosphatase (AcP)] were measured in microwell cultures of mouse osteoblast-enriched (OB) cells and human grngival fibroblasts. CM, a glycoprotein, caused bone resorption at 1 nglml and above, 1OO-fold more potent than LPS, and stimulated AcP release from calvaria and isolated OB. CM, but not LPS, Inhibited proliferation in calvaria, OB, and fibroblasts and also suppressed collagen synthesis in a dose-related manner (long-50pglml). LDH release was raised in these cultures by CM but not LPS, indicating some cell lysis. Bacterial capsules can directly influence connectrve tissue destruction, and may thus be involved In the pathogenesis of peridontal disease.
8756-3282185 $3.00 + .OO Copyright 0 1985 Pergamon Press Ltd.
Meeting of the Bone and Tooth Society, Oxford, U.K. (March, 1985) ABNORMALLY LOW LYSINE IN INSOLUBLE DENTINE FROM PATIENTS WITH DSTEOGENESIS IMPERFECTA
essential for this action. The results demonstrated that P-C-P’s bound to mineralized matrix can affect its osteoclastic resorptron in two ways. One mechanism IS inhibitron of the mature resorbing osteoclast itself, involves cytotoxrcity and IS an important element in the actionsof EHDP and Cl,MDP. The other mechanism is prevention, rather than Inhibition of osteoclastic resorption. This mechanism is responsrble for the increased potency of APD in the new culture system and may also underlie the high potency of APD relative to EHDP and Cl,MDP in vivo. The action may involve antagonism, like interference with deposition on mineralized matrix of substances that are chemotactic to osteoclast. Replacement of the amino group of APD by a dimethyl-amino group not only increases Its efficacy in both culture systems but also in VIVO,while eliminating specific toxicity.
M.J.O. Francis, J.P. Gage and R. Smith Nuffield Department Orthopaedic
of Orthopaedic
Surgery, Nuffield
Centre, Oxford OX3 7LD
Mature dentrne and bone both contain Type I collagen, therefore the collagen of dentine should be abnormal in patients with osteogenesis imperfecta (01) who have defects in this collagen We have examined the amino acid composition of insoluble dentine (a fraction that normally contains 92-950~ Type I collagen) from the teeth of 59 patients with 01. Compared with control normal teeth, changes were found in the number of lysine, hydroxylysine, proline, hydroxyproline, and glycine residues per 1000 total amino acids in 53 patient samples. The amino acid composition of the insoluble dentine from eight 01 patients showed an unusual pattern. Judged by glycine, the amount of collagen was normal but the number of lysine residues was markedly reduced (17 or less/lOOO; control range 26 ~fr 2.2, mean f 2 SD). Tyrosine was also reduced in all eight samples (3 or less/l 000; control range 4.5 +- 1.5) and hrstidine increased in four (11 or more/l 000; control range 7 2 2). Multiple peaks were observed in the amino acid analyser tracing in the region of the histidine, hydroxylysine, and lysine peaks. None of these peaks was lysinal, the aldehyde oxidation product of lysine. The changes were not constantly associated with dentinogenesis imperfecta, as three patients had clinically normal teeth. These results suggest that dentine is abnormal in the majority of patients with 01. In a subgroup of patients there were striking abnormalities in the amounts of those amino acids involved in the formation of collagen cross-links.
SOMESCANNINGMICROSCOPIESOF FIBROGENESIS IMPERFECTADSSIUM S.A. Rerd, R. Smith*, and B. Boyde Department of Anatomy, University College London and * Nuffield Orthopaedic
Centre, Oxford
Fibrogenesis imperfecta ossium is a rare connective tissue disorder in which patients suffer progressive bone pain and fracturing leading to immobility. It is characterized histologically by the presence of thick osteoid seams that are nonbirefringent when seen in polarized light. The fibrous component of these seams IS a tangled mass of fine fibrils that replaces the normal lamellar organization of much thicker collagen fibrils. We report here the first study of this disease using the scanning electron microscope (SEM) and tandem scanning reflected light microscope (TSRLM). Parts of two transilliac crest biopsies taken from the same patient, a 51 -year-old male Caucasian, were studied. These were taken 12 months apart, and before each one the patient followed a tetracycline double labelling regime. Light microscopic histology revealed a paucity of bone, especially in the second biopsy. Many surfaces were lined by thick osteoid seams and the use of polarized light confirmed that these were nonbirefringent. Fluorescence using the TSRLM (Boyde and Reid, 1984) demonstrated tetracycline fluorescence at many of the surfaces lined with osteord. However, separate labels could never be resolved and it was concluded that mineralization was either proceeding very slowly, or was occurring within a considerable thickness of tissue at any one time. Secondary electron imagining in the SEM of surfaces cleaned to reveal matrix and mineral fronts showed that the pattern of mineralization was abnormal. This was confirmed by backscattered electron (BSE) imaging in the SEM of polished, cut surfaces of plastic embedded material. Combined BSE and cathodoluminescence investigation demon-
DUAL MODESOF ACTIONOF BISPHDSPHDNATESON DSTEDCIASTSIN VITRO P.M. Boonekamp, G.W.G.M. Lowik, L.J.A. van der Wee-Pals, M.L.L. van Wijk-van Lennep, and 0. L.M. Bijvoet Department of Endocnnology, University Hospital, Leiden, The Netherlands The relative potencies of the bisphosphonates Cl ,MDP, EHDP on the one hand and of APD and dimethyl-APD on the other as inhibitors of osteoclastic resorption of mineralized matrix were studied in a classical bone culture system, and in a novel in vitro bone culture system. In the new system, the resorption of calcified cartilage depends entirely upon prior activation of osteoclast precursors and on the subsequent development of functioning osteoclastsfrom these. The potency of APD relative to the two other P-C-P’s was increased considerably in the new system. It was found that binding of ADP tocalcified matrix was 275
Abstracts Fromthe Bone & Tooth Society
276
strated that the nonbirefringent bone matrix sometimes tained a substantially greater mineral density than normal. Supported by Anatomonical land and MRC.
at-
applied to study the relevance of this enzyme in osteoclastic bone resorption.
Society of Great Britain and ire-
Boyde A., Reid S.A. Bone & Tooth Sonety. September 1984, Abstract 13, 1984.
A QUANTITATIVE CYTOCHEMICAL ASSAY FOR ACID PHOSPHATASE ACTIVITY IN OSTEOCLASTS IN THE FOETAL RAT
CALVARIA
D.M. Webber, D.C. Anderson, W.R. Robertson, and I.P. Braidman University Departments of Medicine and Chemical Pathology, Hope Hospital, Eccles Old Road, Salford M6 8HD Traditional histochemical techniques have shown the lysosomal enzyme, acid phosphatase (APase), to be prominent in osteoclasts (0~‘s). Previous attempts to quantify this activity have been limited to subjective estimation of enzyme product in fixed and decalcified specimens, processes known to cause a loss of enzyme activity. Sodium tartrate has also been used to localise APase activity specifically to Oc’s by inhibiting the tartratesensitive form of enzyme thought to be in other cells present in bone (Minkin, 1982). We now report the biochemical validation of a quantitative cytochemical assay for Oc APase activity in unfixed and undecalcified sections of 21day foetal rat calvaria. Tissue, superchilled to -70°C, was sectioned (5-1 Opm) in a cryostat at - 35%. The sections were incubated at 37°C in 0.1 M citrate/citric acid buffer (pH 4.5) 5% polyvinyl alcohol (5% w/w Grade G181140) and naphtholASBI-phosphate substrate (O-5mM) for various incubation times. The reaction was stopped with ice-cold tap water and the insoluble reaction product post-coupled with Fast Garnet GBC salt (1 mglml) in 0.1 M sodium acetate pH 6.2 at 4°C for IO minutes. The resulting red/brown particulate deposite was measured using a Vickers M85A microdensitometer with x 100 objective, wavelength 550nm, spot size 0.2pm and measuring area of 28rm*. Arbitrary machine units were converted to mean absorbance (A) by reference to a standard calibration curve. A comparison of adjacent sections stained with haematoxylin and eosin with those reacted for APase, showed areas strongly positive for enzyme activity to correspond to the presence of 0~‘s. 15-20 such areas were measured per section, and each experimental point was derived from three slides, In preliminary work it became clear that initial velocity rate conditions prevailed only over short time periods (ie < 10 seconds). Thereafter the increase in absorbance was not proportional to time. Using a range of substrate concentrations (0-5mM) it was found that activity was maximal at 3mM (0.4-0.5A). The enzyme was active over the range pH 3-6, with peak absorbance between pH 3.5 and 4.5. We have thus achieved first order enzyme kinetics using 5pm thick sections, a 5second incubation time, a substrate concentration of 3mM and a pH of 4.5. Under these conditions cartilage and periostal cells measured in the same sections as Oc’s, had barely detectable APase activity (IO-fold lower than in the latter). Sodium tartrate (1 O-l OOmM) had no effect on the specific localisation of enzyme activity in Oc’s, although absolute levels were inhibited (- 50% with 1OOmM). In conclusion, a quantitative cytochemical assay for APase activity in Oc’s has been developed in unfixed and undecalcified cryostat sections. It is now being
MInkIn C Cal Tissue Int 34(3):285-90.
1982
INTERLEUKlNl (ILl)lNDUCESOSTEOBLASTlCCELLSTD STlMULATEOSTEOCLASTlCBONERESORPTlON B.M. Thomson and T.J. Chambers Department of Histopathology, London EC7
St Bartholomew’s Hospital,
Osteoclasts were disaggregated from neonatal rat long bone and sedimented onto cortical bone slices. After 24 hours in culture, the majority of osteoclasts excavate the bone surface, and the extent of bone resorption can be quantitated by computer-assisted morphometric and stereophotogrammetric techniques. IL1 had no effect on bone resorption by such disaggregated osteoclasts, but if osteoblastic cells (cells derived from collagenase digestion of neonatal rat calvaria, and cloned rat osteosarcoma cells) were also sedimented onto the bone slices, IL1 caused a dose-dependent stimulation of osteoclastic bone resorption, IL1 did not stimulate osteoclastic bone resorption if osteoblasts shared the same culture volume but were physically distanced from osteoclasts. These results indicate that IL1 stimulates bone resorption, and does so through a primary action on osteoblasts induced by IL1 to transmit a short-range signal, which stimulates osteoclastic bone resorption.
BACTERlALCAPSULARGLYCOPROTEIN:APOTENTBONE RESORBlNGFACTORANDCYTOTOXlCFACTORFOR FIBROBLASTS. ‘M. Wilson, W. Harvey, S. Meghi, A. Scutt, and *S. Kamin Department of Oral & Maxillofacial Surgery and *Department of Clinical Pathology & Immunology, Eastman Dental Hospital, London WClX 8LD Lipopolysaccharide (LPS) from bacterial cell walls is thought to be an important mediator of tissue breakdown in periodontal disease, but bacterial capsules are considered to be important only in adhesion and protection against phagocytosis. We have purified capsular material (CM) from Actinobacillus actinomycetemcomitans and compared it with purified LPS from the same organism in its effects on bone, osteoblasts and glnglval fibroblasts in vitro. Bone resorption was measured by Careleasefrom 5-day-old mouse calvaria over 3 days. Proliferation [3HTdR incorporation and methylene blue uptake], cytotoxicity [lactate dehydrogenase (LDH) release] and lysosomal enzyme release [acid phosphatase (AcP)] were measured in microwell cultures of mouse osteoblast-enriched (OB) cells and human grngival fibroblasts. CM, a glycoprotein, caused bone resorption at 1 nglml and above, 1OO-fold more potent than LPS, and stimulated AcP release from calvaria and isolated OB. CM, but not LPS, Inhibited proliferation in calvaria, OB, and fibroblasts and also suppressed collagen synthesis in a dose-related manner (long-50pglml). LDH release was raised in these cultures by CM but not LPS, indicating some cell lysis. Bacterial capsules can directly influence connectrve tissue destruction, and may thus be involved In the pathogenesis of peridontal disease.