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Bone allografts primed with bFGF enhance human bone cell proliferation in vitro
560
Bone Vol, 17, No. 6 December 1995:557-596
Abstracts
9
11
BONE A L L O G R A F T S P R I M E D W I T H b F G F E N H A N C E H U M A N B O N E CELL PROLIFERATION IN V I T R O
NITRIC OXIDE DEPENDENT AND INDEPENDENT INDUCTION OF PROSTAGLANDIN SYNTHESIS IN OSTEOBLASTS
A. Brink, A. Battmarm, C. FOlsch 1 and A. Schulz. Institute of Pathology, IDeparlment o f Surgery, Justus-Liebig-University, Langhansstr. 10, D-35385 Giessen, Germany Osteointegration o f bone implants is o f major importance for the clinical use o f bone graft materials. Priming o f these materials with growth factors might result in a faster integration o f the bone graR into the skeleton and therefore in an accelerated healing process. We evaluated the effect o f two different hydroxyapatite based bone implants loaded with basic fibroblast growth factor CoFGF) on normal human bone cells in vitro. Normal human bone cells (femoral heads) were isolated according to the method described by Robey and Termine (1985). The cells were exposed either to bFGF or implant materials alone or after priming o f the materials with bFGF. Cell proliferation was assessed using a formazan (MTT) assay. Both, bFGF as well as the tested materials showed a significant increase in bone cell proliferation o f about 50% vs untreated control. Priming o f the implant materials with bFGF prior to exposure to the cells increased proliferation up to 140% vs control. In conclusion, the combination o f bone implants and bFGF showed a significant increase in bone cell proliferation in vitro. An enhanced osteointegration o f primed implants into the human skeleton might be achieved using the combination tested in these experiments. Robey PG, Termine JD (1985) Human bone cells in culture. Calcif Tissue Int 37(5):453-460
LDK Buttery, MVJ Htlkkanen. A O'Donnell'. JM Polak and FI Hu~hes*, Dept. Histochemistry, Royal Postgraduate Medical School, London WI20NN and Dept. Periodontology, London Hospital Medical College, London E1 2AD, U.K.
10
12
IL8 AND IL6 MAY MEDIATE THE RESPONSE TO T3 IN HUMAN OSTEOBLAST-LIKE CELLS A. Siddioi (1.2). J.M. Burrin (1). J P Monson (2). DF. Wood (2). Departments of Clinical Biochemistry (1) and Endocrinology (2), London Hospital Medical College, Turner Street, London El 2AD, UK.
INCREASED LAMELLAR NUMBER PER BONE STRUCTURE UNIT IN MYELODYSPLASTIC DISORDERS WITH
Thyrotoxicosis increasesbone turnover in favour of net bone resorption,which ifuncontrolled may lead to osteoporosis. Osteoblasts are thought to mediate thyroid hormone actions on osteoclaststhrough the releaseof soluble factorssuch as cytokines. W e have thereforeexamined the effectsofT3 on the human ostcoblastlccelllinesM G 63 and SaOs 2, which differin their degree of differentiation.Cells were culturedin duplicateon three occasions for 3, 6, 8, 12 and 24 brs in T3-depleted serum in the absence or presence ofT3 (l, I0 and 100 nM). Intcdeukin IB was used as a positivecontrol.Media were analysed for ILg, IL6, IL4 and T N F a by sensitiveimmunoassays. M G 63 ceilsproduced IL8 and IL6 both constitutivelyand on stimulation.The ILg response was time dependent with 24hr levelssignificantlyhigher when compared to untreated control (T3 InM I08 ± 2.7 vs control 38.9 ± 9.1 pg/ml, P<0.05) but was not dose-dependant (T3 I0 n M lI l ± 3.2 pg/ml; P<0.05). The IL6 response was dose dependent (control 205 ± 82.6 vs T3 InM 113 ± 34, n.s.;T3 10 n M 256 ± 72.2, n.s.; T3 I00 n M 293 ± 82.6 pg/ml, P<0.01). SaOs 2 cellsproduced IL8 and IL6 constitutivelybut showed no response to T3. IL4 and TNF~z were not detected eitherbasally or on stimulationfrom either cell line.W e conclude thatIL8 and IL6 may mediate osteoblastosteodast interactionin response to T3 and that thisresponse may depend on the degree of differentiationof the osteoblast.
Prostaglandins (PGs) have powerful effects on bone matrix resorption and deposition. Their function includes initial suppression of bone resorption but the long-term effect is to increase the over-all activity of bone resorption. The major source of PGs in bone are the cells of osteoblast lineage. PG synthesis is highly regulated by cytokines which exert their effects on the synthesis and activity of the inducible isoform of cyclooxygenase (COX-2). Recent evidence by us and others has demonstrated the production of nitric oxide (NO) by osteoblasts and osteoclasts in response to eytokine stimulation, and suggest a role for NO in mediating cytokine effects on osteoblast and osteoclast function. In this study, we have investigated the possibility that cytokine-induced NO production by osteoblasts might exert its functions partly by activating the synthesis of COX-2. Primary osteoblastic cultures from neonatal rat calvariae were stimulated with eytokines for 48 hours in the presence or absence of L-NAME, an inhibitor of NO-synthase (NOS) activity, and PG~ production was measured by enzyme immunoassay. IL-I strongly stimulated PGE2 production in a dose-dependent manner but this was not inhibited by L-NAME. TNF-u stimulated PGE2 production was only partially inhibited by L-NAME whereas IFN-7 induced PGE2 synthesis was almost totally inhibited by L-NAME. Immunostalning and Western blots of primary osteoblasts with specific antibodies for the inducible NOS (iNOS) and COX-2 isoforms showed induction of both enzymes after stimulation with IL-I, TNF-c~ or IFN-3' alone or in combination. Addition of L-NAME did not have any effects on IL-1 stimulated COX-2 production but was shown to reduce TNF-u and diminish IFN-3' stimulated COX-2 expression. These results demonstrate that NO can induce COX-2 and PG synthesis in osteoblasts and suggest the presence of NO-dependent and NO-independentpathways of PG production in these cells. The data provide further support for the importance of NO as a mediator of cytokine-inducedeffects in bone metabolism given that the effects of TNF-c~ and IFN-7 on COX-2 expression are either partially or totally mediated by NO.
OSTEOSCLEROSlS. O. (~HAPPARD. N. IFRAH, S. FRAN(~OIS,M BOASSON, MF BASLE LHEA - Lab. Histologie Embryologie Angers - 49045-Angers Cddex France. It is now recognized that bone marrow microenvironment is essential for both hematopoietic and bone cell precursors differentiation and activities. Malignant proliferations are able to interact with bone cells: e.g. bone remodeling is known to be markedly affected in hematological disorders of the B cell lineage (i.e. myeloma mad lymphomas). Bone sclerosis and bone marrow fibrosis are common features in the evolution of myelodysplastic disorders. Platelet Derived Growth Factor (PDGF), released by an increased number of cells of the megakaryocyte lineage in these diseases, has been recognized as one of the most potent stimulating factor to explain the marrow fibrosis. Five patients having a myeloid metaplasia associated with osteosclerosis were included in the study. They were biopsied at the ilium and the bone core was processed undecalcified. Bone morphometry was done on a Leica Quantimet Q570 image analyzer with lab-made softwares. An increased bone volume (BV/TV) was noted that could be related to increased trabecular thickness (Tb.Th) and number (Tb.N). An increased connectivity of the trabecular network was evidenced with decreased Inter Connectivity Index, increased Node number and Node-to-Node strut number, decreased star volume of the bone marrow ( V ~ s ~ ) and increased star volume of the trabeculae ( V~b)The wall thickness (W.Th) of the bone packets (BSU) was measured under polarized light and the mean number of lamella per BSU was determined. W.Th was found to be drastically increased due to an abnormally high number of lamella per BSU. In myelodysplastic diseases, a prolonged formation period could be observed in relation with an altered cytokine release by tumor cells. PDGF and other cytokines produced by megakaryocytic cells may increase the osteoblastic life span or/and activity.
Bone Vol, 17, No. 6 December 1995:557-596
Abstracts
9
11
BONE A L L O G R A F T S P R I M E D W I T H b F G F E N H A N C E H U M A N B O N E CELL PROLIFERATION IN V I T R O
NITRIC OXIDE DEPENDENT AND INDEPENDENT INDUCTION OF PROSTAGLANDIN SYNTHESIS IN OSTEOBLASTS
A. Brink, A. Battmarm, C. FOlsch 1 and A. Schulz. Institute of Pathology, IDeparlment o f Surgery, Justus-Liebig-University, Langhansstr. 10, D-35385 Giessen, Germany Osteointegration o f bone implants is o f major importance for the clinical use o f bone graft materials. Priming o f these materials with growth factors might result in a faster integration o f the bone graR into the skeleton and therefore in an accelerated healing process. We evaluated the effect o f two different hydroxyapatite based bone implants loaded with basic fibroblast growth factor CoFGF) on normal human bone cells in vitro. Normal human bone cells (femoral heads) were isolated according to the method described by Robey and Termine (1985). The cells were exposed either to bFGF or implant materials alone or after priming o f the materials with bFGF. Cell proliferation was assessed using a formazan (MTT) assay. Both, bFGF as well as the tested materials showed a significant increase in bone cell proliferation o f about 50% vs untreated control. Priming o f the implant materials with bFGF prior to exposure to the cells increased proliferation up to 140% vs control. In conclusion, the combination o f bone implants and bFGF showed a significant increase in bone cell proliferation in vitro. An enhanced osteointegration o f primed implants into the human skeleton might be achieved using the combination tested in these experiments. Robey PG, Termine JD (1985) Human bone cells in culture. Calcif Tissue Int 37(5):453-460
LDK Buttery, MVJ Htlkkanen. A O'Donnell'. JM Polak and FI Hu~hes*, Dept. Histochemistry, Royal Postgraduate Medical School, London WI20NN and Dept. Periodontology, London Hospital Medical College, London E1 2AD, U.K.
10
12
IL8 AND IL6 MAY MEDIATE THE RESPONSE TO T3 IN HUMAN OSTEOBLAST-LIKE CELLS A. Siddioi (1.2). J.M. Burrin (1). J P Monson (2). DF. Wood (2). Departments of Clinical Biochemistry (1) and Endocrinology (2), London Hospital Medical College, Turner Street, London El 2AD, UK.
INCREASED LAMELLAR NUMBER PER BONE STRUCTURE UNIT IN MYELODYSPLASTIC DISORDERS WITH
Thyrotoxicosis increasesbone turnover in favour of net bone resorption,which ifuncontrolled may lead to osteoporosis. Osteoblasts are thought to mediate thyroid hormone actions on osteoclaststhrough the releaseof soluble factorssuch as cytokines. W e have thereforeexamined the effectsofT3 on the human ostcoblastlccelllinesM G 63 and SaOs 2, which differin their degree of differentiation.Cells were culturedin duplicateon three occasions for 3, 6, 8, 12 and 24 brs in T3-depleted serum in the absence or presence ofT3 (l, I0 and 100 nM). Intcdeukin IB was used as a positivecontrol.Media were analysed for ILg, IL6, IL4 and T N F a by sensitiveimmunoassays. M G 63 ceilsproduced IL8 and IL6 both constitutivelyand on stimulation.The ILg response was time dependent with 24hr levelssignificantlyhigher when compared to untreated control (T3 InM I08 ± 2.7 vs control 38.9 ± 9.1 pg/ml, P<0.05) but was not dose-dependant (T3 I0 n M lI l ± 3.2 pg/ml; P<0.05). The IL6 response was dose dependent (control 205 ± 82.6 vs T3 InM 113 ± 34, n.s.;T3 10 n M 256 ± 72.2, n.s.; T3 I00 n M 293 ± 82.6 pg/ml, P<0.01). SaOs 2 cellsproduced IL8 and IL6 constitutivelybut showed no response to T3. IL4 and TNF~z were not detected eitherbasally or on stimulationfrom either cell line.W e conclude thatIL8 and IL6 may mediate osteoblastosteodast interactionin response to T3 and that thisresponse may depend on the degree of differentiationof the osteoblast.
Prostaglandins (PGs) have powerful effects on bone matrix resorption and deposition. Their function includes initial suppression of bone resorption but the long-term effect is to increase the over-all activity of bone resorption. The major source of PGs in bone are the cells of osteoblast lineage. PG synthesis is highly regulated by cytokines which exert their effects on the synthesis and activity of the inducible isoform of cyclooxygenase (COX-2). Recent evidence by us and others has demonstrated the production of nitric oxide (NO) by osteoblasts and osteoclasts in response to eytokine stimulation, and suggest a role for NO in mediating cytokine effects on osteoblast and osteoclast function. In this study, we have investigated the possibility that cytokine-induced NO production by osteoblasts might exert its functions partly by activating the synthesis of COX-2. Primary osteoblastic cultures from neonatal rat calvariae were stimulated with eytokines for 48 hours in the presence or absence of L-NAME, an inhibitor of NO-synthase (NOS) activity, and PG~ production was measured by enzyme immunoassay. IL-I strongly stimulated PGE2 production in a dose-dependent manner but this was not inhibited by L-NAME. TNF-u stimulated PGE2 production was only partially inhibited by L-NAME whereas IFN-7 induced PGE2 synthesis was almost totally inhibited by L-NAME. Immunostalning and Western blots of primary osteoblasts with specific antibodies for the inducible NOS (iNOS) and COX-2 isoforms showed induction of both enzymes after stimulation with IL-I, TNF-c~ or IFN-3' alone or in combination. Addition of L-NAME did not have any effects on IL-1 stimulated COX-2 production but was shown to reduce TNF-u and diminish IFN-3' stimulated COX-2 expression. These results demonstrate that NO can induce COX-2 and PG synthesis in osteoblasts and suggest the presence of NO-dependent and NO-independentpathways of PG production in these cells. The data provide further support for the importance of NO as a mediator of cytokine-inducedeffects in bone metabolism given that the effects of TNF-c~ and IFN-7 on COX-2 expression are either partially or totally mediated by NO.
OSTEOSCLEROSlS. O. (~HAPPARD. N. IFRAH, S. FRAN(~OIS,M BOASSON, MF BASLE LHEA - Lab. Histologie Embryologie Angers - 49045-Angers Cddex France. It is now recognized that bone marrow microenvironment is essential for both hematopoietic and bone cell precursors differentiation and activities. Malignant proliferations are able to interact with bone cells: e.g. bone remodeling is known to be markedly affected in hematological disorders of the B cell lineage (i.e. myeloma mad lymphomas). Bone sclerosis and bone marrow fibrosis are common features in the evolution of myelodysplastic disorders. Platelet Derived Growth Factor (PDGF), released by an increased number of cells of the megakaryocyte lineage in these diseases, has been recognized as one of the most potent stimulating factor to explain the marrow fibrosis. Five patients having a myeloid metaplasia associated with osteosclerosis were included in the study. They were biopsied at the ilium and the bone core was processed undecalcified. Bone morphometry was done on a Leica Quantimet Q570 image analyzer with lab-made softwares. An increased bone volume (BV/TV) was noted that could be related to increased trabecular thickness (Tb.Th) and number (Tb.N). An increased connectivity of the trabecular network was evidenced with decreased Inter Connectivity Index, increased Node number and Node-to-Node strut number, decreased star volume of the bone marrow ( V ~ s ~ ) and increased star volume of the trabeculae ( V~b)The wall thickness (W.Th) of the bone packets (BSU) was measured under polarized light and the mean number of lamella per BSU was determined. W.Th was found to be drastically increased due to an abnormally high number of lamella per BSU. In myelodysplastic diseases, a prolonged formation period could be observed in relation with an altered cytokine release by tumor cells. PDGF and other cytokines produced by megakaryocytic cells may increase the osteoblastic life span or/and activity.