- Email: [email protected]
Osteoinduction by perforated demineralized bone matrix xenograft (PDBMX)
Abstracts
from the Calcified
at 16 days, but the treated cells had sixfold greater amounts. This phenomenon may be due to the binding of BGP to the hydroxyapatite formed in vitro. PROTEOGLYCANSASMOLECULARMARKERSOF EMBRYONIC CARTILAGE AND BONE FORMATION Arnold I. Caplan,
David A. Carrino, Michael Weitzhandler
Biology Department, Case Western Reserve University, Cleveland, Ohio, USA Proteoglycans (PGs) are complex macromolecules composed of a protein core to which one or more polysaccharide chains are attached. The chondroitin sulfate (CS) PGs, together with type II collagen, are particularly abundant in cartilage and are directly responsible for the resiliency of articular cartilage. The articular cartilage CS-PG has a distinctive chemistry that seems to be programatitally modulated during the differentiation of mesenchymal cells into first chondrocytes through maturation and senescent expression. Thus, the relative “age” of a chondrocyte or the presence of its mesenchymal progenitor cell can be estimated by chemically characterizing the CS-PGs they synthesize. In embryonic development of bone in the chick, a core of embryonic cartilage is replaced by marrow and bone. During this process, the chondrocyte further differentiates into an unusual phenotypic variant referred to as a hypertrophic chondrocyte. This cell is not a terminal cell but rather makes a unique spectrum of macromolecules including type X collagen and a distinctive CS-PG. Likewise, osteoblasts are a unique phenotype which make type I collagen and a distinct CS-PG. As an aside, embryonic muscle and muscle-connective tissue also make phenotype-distinctive CS-PGs. At the minimum, the chemical characterization of the CS-PGs made by a group of cells can be used as a distinctive marker for the presence of certain cells or the stage of development. More importantly, these phenotype CS-PGs contribute to the unique extracellular fabric in which the complex cellular and molecular events of morphogenesis is taking place. By isolating and characterizing this class of complex macromolecules, we have gained a better understanding of the molecular transitions involved in embryonic cartilage and bone formation. Supported by grants from NIH.
CELLULARANDMOLECULARASPECTSOF EMBRYONIC BONE FORMATION Arnold I. Caplan, David Michael Weitzhandler
389
Tissues Workshop
G. Pechak, Mary J. Kujawa,
Biology Department, Case Western Reserve University, Cleveland, Ohio, USA. It is essential to understand the details of embryonic bone formation so that a better understanding of both abnormal events and bone repair can be properly accessed. We have used modern technologies to reevaluate the cellular and molecular aspects of bone formation. These studies were prompted by our success in microdissection and cell culturing of first bone tibia1 osteoblasts and their companion cartilage core hypertrophic chondrocytes. The osteoblasts made bone matrix in culture while the chondrocytes synthesized macromolecules specific for hypertrophic chondrocytes including type X collagen and a hypertrophic cartilage specific proteoglycan. We deter-
mined that osteogenesis was completely separated from the cartilaqe core and occurred in a periosteal layer. Importantly, cartilage does not provide’ a scaffolding onto which this bone is built. In fact, greater than 90% of the length of the day 16 to 18 embryonic chick tibia is formed by the conversion of mesenchymal cells into osteoblasts in the periosteum. Moreover, cartilage is not replaced by bone but rather by the marrow space. There exists an unusual and interesting interplay between cartilage and bone with the production and dispersal of chondrocalcin, a 35,000 dalton protein, by chondrocytes and its deposition in newly mineralizing bone matrix. However, the tissue culture studies strongly suggest that osteogenesis can take place in the absence of this interplay. Based on our observations and those of others, we speculate that osteoprogenitor cells become committed prior to chondrogenesis and that these osteogenic cells may, indeed, determine the shape and boundaries of the cartilage core which eventually develops and is replaced by marrow. Supported by grants for NIH. MARROW DERIVED FACTORS REGULATING CELL PROLIFERATION AND ACTIVITY D. Gazit, A. Shteyer,
BONE
I. Bab
Hebrew University-Hadassah Jerusalem, Israel
School of Dental Medicine,
The following experiments were undertaken to test the hypothesis that healing marrow elaborates a factor or factors that affect(s) growth and differentiation of osteogenic cells. Bone marrow was removed from rat tibiae. After IO days the healing tissue (HBM) was separated from the marrow space. Conditioned culture medium was prepared by an overnight incubation with this tissue. Control medium was conditioned with normal marrow (NM) from untreated animals Different dilutions of the conditioned media were added to 24 h cultures of osteoblastic osteosarcoma cells (ROS 17/2). The addition of medium conditioned with HBM to serum-free ROS cell cultures at dilutions l/200 and l/100 resulted in stimulation of 3[H]-thymidine incorporation into ROS cell DNA after 24 h. This was associated with a marked increase in cell number after 48 h at dilutions l/100-1/25. The stimulatory effect of HBM was serum-independent and the combined effect of serum and this conditioned medium was additive. At dilution l/200 HBM elicited a two-fold increase in alkaline phosphatase activity, which was followed by inhibition of at the lower dilutions NM had a marked inhibitory effect on ROS cell proliferation only in the absence of serum. It is concluded that factors with inhibitory and stimulatory effects on osteogenic cells are produced by normal and healing marrow, respectively. These factors activities may be part of an osteogenic regulatory mechanism.
OSTEOINDUCTION BY PERFORATED DEMINERALIZED BONE MATRIX XENOGRAFT (PDBMX) E. Gendler University of Southern California Orthopaedic Histology Lab Los Angeles, CA, USA
Hospital,
It has been previously reported, using rat and rabbit models, that creation of multiple artificial perforations in al-
Abstracts
logenic demineralized bone matrix signtfrcantly increased I& ability to induce chondro and osteogenesis after subcutaneous implantation. Each perforation became a center of osteoinduction, with subsequent centrifugal resorption of the matrix and its replacement by new bone. In the current study cross-species implantation of perforated decalcified bone matrix xenograft (PDBMX) was performed Onemonth-old Long-Evans rats recerved implants subcutaneously in the thoracic area. The implants consisted of rectangular plates (5 x 20 mm) that had been prepared from long bones of New Zealand rabbits. Multiple perforations 0.35 mm in diameter were created in the bone with a highspeed drill. The bone was then demineralized In 0.6N HCI. After various intervals, animals were sacrificed and the implants examined histologically and histochemically. PDBMX retained its osteoinductive potential in thus crossspecies experiment. Mesenchymal cells ingrowing into perforations underwent transformation into chondrocytes. Subsequently, cartilage became resorbed by invading capillaries, and endochondral osteogenesis occurred. On the periphery of perforatrons xenograft underwent active resorption by multiple osteoclasts, with srmultaneous apposition of new bone which gradually replaced the whole implant. The space within the new ossicle became filled with myelord tissue.
permeability of the membranes to calcium ions. The results herein suggest the involvement of calcium Ions In both the effects of 1,25(OH)2D, on HL-60 cells: the immediate effect on cell membranes and the long-term effect on cell proliferation and differentratron.
CALCIUM DEPRIVATION INDUCED METABOLIC INACTIVATION OF VITAMIN D M R Clements,
R. Levy, I Nathan, C. Charmovitz, S. Shany Clinical Bochemistry Unit, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheua, lsrael It has previously been shown that the active metabolite of vitamin D, 1,25dihydroxyvitamin D, (1,25(OH),D,), suppresses proliferation and induces differentiation of leukemic cells to monocytes. In the present work, the role of calcium ions rn the effect of 1,25(OH),D, on HL-60 cells was studied. Lowering the calcium levels (by means of EGTA or addrtion of the calcrum channel blocker verapamil) during the treatment with 1,25(OH),D, caused an enhanced inhibition of cell growth and a reduction in the number of differentiated cells. Cell growth and differentiation processes could be restored by the addition of calcium ions to a physiological level, even in the presence of EGTA. The increased inhibitron of proliferatron caused by verapamil in the presence of 1,25(OH)2D, is not a result of an increased uptake of 1,25(0H),D3, since verapamil did not affect its uptake. These results demonstrate the necessity of calcium ions in the effect of 1,25(OH)*D3 on the proliferation and differentiation processes of HL-60 cells. In addition, 1 ,25(OH),D3 had immediate and direct effects on the HL-60 cell membrane. Incubation of HL-60 cells with 1,25(OH)2D3 even for 2 hours caused an increase in calcium efflux. This phenomenon was found to be unrelated to new protein synthesis, since it was also found in the presence of RNA and protern synthesis inhibitors. Addition of 1,25(OH),D, to the cells for 4 hours caused changes in the phospholipid composition In the outer membranes The amount of phosphatidylcholine increased and the amount of phosphatidylethanolamine decreased The ratto PC/PE changed from 1.2 to 1 5. The alteratrons in the phospholrprd composition in the membranes induced by 1,25(OH),D, may be responsible for the changes in the
L. Johnson,
D.R Fraser
Dunn Nutritional Laborafory, Universtty of Cambncfge and Meckal Research Council, Cambridge, England Rats were fed vitamin D-deficient diets containing either 0.02% calcium ( - Ca) or 0.64% calcium ( + Ca). Adequate and defined vitamin D status was attained by subcut. Injection of 5 kg D, and the 25(OH)D pool was labelled by intracardial injection of 0.3 FCi 3H-25(OH)D3. The elrmination half-life (tlR) of 3H-25(OH)D, in plasma of -Ca rats (8-10 days) was 40% faster than that of +Ca controls (13- 15 days), the difference berng quantitatively accounted for by the appearance of excess 3H as highly polar metabolites in faeces of -Ca rats. Plasma analysis after 4 weeks on the diets gave the following values (means IT s.e.m.; n = 6 per group): [Ca]mmoM
THE INVOLVEMENT OF CALCIUM IONS IN THE EFFECT OF 1,25-DIHYDROXYVITAMIN D ON HL-60 CELLS
from the Calclfled Tissues Workshop
+Ca 251 t 0 10 Ca248?007
[POJmmolll
[PS(OH)D]ngiml
183 t- 016 2732010
134i14 79 t 10
[1,25(OH)2D]pglml 780~56 z 44
388 7
a
Varying dietary [Cal down to 0.02% caused proportional acceleration in t,,, of 3H-25(OH)D3. Similar accelerated t,,z values could be produced by feeding the +Ca dret with additional PO., (1,2%P) or phytic acid (5.1%). The accelerated loss of plasma 25(OH)D on the -Ca diet cannot be explained merely by the Increased production of 1,25(OH),D. When the 25(OH)D pool was labelled with 3H-5,6-trans-25(OH)D3, which cannot be converted to 1,25(OH),D, the - Ca diet still caused the same reduction rn the tl, of 3H. Furthermore, the t,,* of 3H-1,25(OH),D, on +Ca diet (34 6 +- 12h) was unchanged by feeding -Ca diet (35.0 & 0.6h). The alternative explanation is that hepatic Inactivation of 25(OH)D IS enhanced in calcium deprivation. The normal [Cal and elevated [1,25(OH),D] in plasma of rats on -Ca diet indicate a marked degree of secondary hyperparathyroidrsm. Parathyroidectomy of rats on the high-PO, diet abolrshes the effect on t,,> of 3H-25(OH)D,. A sjmllar effect of parathyroidectomy on t,,? of 3H-25(OH)D, has been found in patients with hyperparathyroidism. It IS therefore postulated that elevated parathyroid secretion leads to enhanced hepatic inactivation of 25(OH)D and ultimately, with low vitamin D supply, to vrtamin D deficiency.
EFFECTS OF PARATHYROID HORMONE (PTH) AND VITAMIN D ON NA+,K+-ATPASE ACTIVITY IN RAT TUBULE SEGMENTS Elstern. D., W.J Czaczkes Hadassah Medical Center, Jerusalem,
israel
As an Initial step towards understanding the interdrgrtatrng influences of PTH and 1,25 dihydroxycholecalciferol (vitamrn D) on transport of sodium (Na) in renal tubules,
from the Calcified
at 16 days, but the treated cells had sixfold greater amounts. This phenomenon may be due to the binding of BGP to the hydroxyapatite formed in vitro. PROTEOGLYCANSASMOLECULARMARKERSOF EMBRYONIC CARTILAGE AND BONE FORMATION Arnold I. Caplan,
David A. Carrino, Michael Weitzhandler
Biology Department, Case Western Reserve University, Cleveland, Ohio, USA Proteoglycans (PGs) are complex macromolecules composed of a protein core to which one or more polysaccharide chains are attached. The chondroitin sulfate (CS) PGs, together with type II collagen, are particularly abundant in cartilage and are directly responsible for the resiliency of articular cartilage. The articular cartilage CS-PG has a distinctive chemistry that seems to be programatitally modulated during the differentiation of mesenchymal cells into first chondrocytes through maturation and senescent expression. Thus, the relative “age” of a chondrocyte or the presence of its mesenchymal progenitor cell can be estimated by chemically characterizing the CS-PGs they synthesize. In embryonic development of bone in the chick, a core of embryonic cartilage is replaced by marrow and bone. During this process, the chondrocyte further differentiates into an unusual phenotypic variant referred to as a hypertrophic chondrocyte. This cell is not a terminal cell but rather makes a unique spectrum of macromolecules including type X collagen and a distinctive CS-PG. Likewise, osteoblasts are a unique phenotype which make type I collagen and a distinct CS-PG. As an aside, embryonic muscle and muscle-connective tissue also make phenotype-distinctive CS-PGs. At the minimum, the chemical characterization of the CS-PGs made by a group of cells can be used as a distinctive marker for the presence of certain cells or the stage of development. More importantly, these phenotype CS-PGs contribute to the unique extracellular fabric in which the complex cellular and molecular events of morphogenesis is taking place. By isolating and characterizing this class of complex macromolecules, we have gained a better understanding of the molecular transitions involved in embryonic cartilage and bone formation. Supported by grants from NIH.
CELLULARANDMOLECULARASPECTSOF EMBRYONIC BONE FORMATION Arnold I. Caplan, David Michael Weitzhandler
389
Tissues Workshop
G. Pechak, Mary J. Kujawa,
Biology Department, Case Western Reserve University, Cleveland, Ohio, USA. It is essential to understand the details of embryonic bone formation so that a better understanding of both abnormal events and bone repair can be properly accessed. We have used modern technologies to reevaluate the cellular and molecular aspects of bone formation. These studies were prompted by our success in microdissection and cell culturing of first bone tibia1 osteoblasts and their companion cartilage core hypertrophic chondrocytes. The osteoblasts made bone matrix in culture while the chondrocytes synthesized macromolecules specific for hypertrophic chondrocytes including type X collagen and a hypertrophic cartilage specific proteoglycan. We deter-
mined that osteogenesis was completely separated from the cartilaqe core and occurred in a periosteal layer. Importantly, cartilage does not provide’ a scaffolding onto which this bone is built. In fact, greater than 90% of the length of the day 16 to 18 embryonic chick tibia is formed by the conversion of mesenchymal cells into osteoblasts in the periosteum. Moreover, cartilage is not replaced by bone but rather by the marrow space. There exists an unusual and interesting interplay between cartilage and bone with the production and dispersal of chondrocalcin, a 35,000 dalton protein, by chondrocytes and its deposition in newly mineralizing bone matrix. However, the tissue culture studies strongly suggest that osteogenesis can take place in the absence of this interplay. Based on our observations and those of others, we speculate that osteoprogenitor cells become committed prior to chondrogenesis and that these osteogenic cells may, indeed, determine the shape and boundaries of the cartilage core which eventually develops and is replaced by marrow. Supported by grants for NIH. MARROW DERIVED FACTORS REGULATING CELL PROLIFERATION AND ACTIVITY D. Gazit, A. Shteyer,
BONE
I. Bab
Hebrew University-Hadassah Jerusalem, Israel
School of Dental Medicine,
The following experiments were undertaken to test the hypothesis that healing marrow elaborates a factor or factors that affect(s) growth and differentiation of osteogenic cells. Bone marrow was removed from rat tibiae. After IO days the healing tissue (HBM) was separated from the marrow space. Conditioned culture medium was prepared by an overnight incubation with this tissue. Control medium was conditioned with normal marrow (NM) from untreated animals Different dilutions of the conditioned media were added to 24 h cultures of osteoblastic osteosarcoma cells (ROS 17/2). The addition of medium conditioned with HBM to serum-free ROS cell cultures at dilutions l/200 and l/100 resulted in stimulation of 3[H]-thymidine incorporation into ROS cell DNA after 24 h. This was associated with a marked increase in cell number after 48 h at dilutions l/100-1/25. The stimulatory effect of HBM was serum-independent and the combined effect of serum and this conditioned medium was additive. At dilution l/200 HBM elicited a two-fold increase in alkaline phosphatase activity, which was followed by inhibition of at the lower dilutions NM had a marked inhibitory effect on ROS cell proliferation only in the absence of serum. It is concluded that factors with inhibitory and stimulatory effects on osteogenic cells are produced by normal and healing marrow, respectively. These factors activities may be part of an osteogenic regulatory mechanism.
OSTEOINDUCTION BY PERFORATED DEMINERALIZED BONE MATRIX XENOGRAFT (PDBMX) E. Gendler University of Southern California Orthopaedic Histology Lab Los Angeles, CA, USA
Hospital,
It has been previously reported, using rat and rabbit models, that creation of multiple artificial perforations in al-
Abstracts
logenic demineralized bone matrix signtfrcantly increased I& ability to induce chondro and osteogenesis after subcutaneous implantation. Each perforation became a center of osteoinduction, with subsequent centrifugal resorption of the matrix and its replacement by new bone. In the current study cross-species implantation of perforated decalcified bone matrix xenograft (PDBMX) was performed Onemonth-old Long-Evans rats recerved implants subcutaneously in the thoracic area. The implants consisted of rectangular plates (5 x 20 mm) that had been prepared from long bones of New Zealand rabbits. Multiple perforations 0.35 mm in diameter were created in the bone with a highspeed drill. The bone was then demineralized In 0.6N HCI. After various intervals, animals were sacrificed and the implants examined histologically and histochemically. PDBMX retained its osteoinductive potential in thus crossspecies experiment. Mesenchymal cells ingrowing into perforations underwent transformation into chondrocytes. Subsequently, cartilage became resorbed by invading capillaries, and endochondral osteogenesis occurred. On the periphery of perforatrons xenograft underwent active resorption by multiple osteoclasts, with srmultaneous apposition of new bone which gradually replaced the whole implant. The space within the new ossicle became filled with myelord tissue.
permeability of the membranes to calcium ions. The results herein suggest the involvement of calcium Ions In both the effects of 1,25(OH)2D, on HL-60 cells: the immediate effect on cell membranes and the long-term effect on cell proliferation and differentratron.
CALCIUM DEPRIVATION INDUCED METABOLIC INACTIVATION OF VITAMIN D M R Clements,
R. Levy, I Nathan, C. Charmovitz, S. Shany Clinical Bochemistry Unit, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheua, lsrael It has previously been shown that the active metabolite of vitamin D, 1,25dihydroxyvitamin D, (1,25(OH),D,), suppresses proliferation and induces differentiation of leukemic cells to monocytes. In the present work, the role of calcium ions rn the effect of 1,25(OH),D, on HL-60 cells was studied. Lowering the calcium levels (by means of EGTA or addrtion of the calcrum channel blocker verapamil) during the treatment with 1,25(OH),D, caused an enhanced inhibition of cell growth and a reduction in the number of differentiated cells. Cell growth and differentiation processes could be restored by the addition of calcium ions to a physiological level, even in the presence of EGTA. The increased inhibitron of proliferatron caused by verapamil in the presence of 1,25(OH)2D, is not a result of an increased uptake of 1,25(0H),D3, since verapamil did not affect its uptake. These results demonstrate the necessity of calcium ions in the effect of 1,25(OH)*D3 on the proliferation and differentiation processes of HL-60 cells. In addition, 1 ,25(OH),D3 had immediate and direct effects on the HL-60 cell membrane. Incubation of HL-60 cells with 1,25(OH)2D3 even for 2 hours caused an increase in calcium efflux. This phenomenon was found to be unrelated to new protein synthesis, since it was also found in the presence of RNA and protern synthesis inhibitors. Addition of 1,25(OH),D, to the cells for 4 hours caused changes in the phospholipid composition In the outer membranes The amount of phosphatidylcholine increased and the amount of phosphatidylethanolamine decreased The ratto PC/PE changed from 1.2 to 1 5. The alteratrons in the phospholrprd composition in the membranes induced by 1,25(OH),D, may be responsible for the changes in the
L. Johnson,
D.R Fraser
Dunn Nutritional Laborafory, Universtty of Cambncfge and Meckal Research Council, Cambridge, England Rats were fed vitamin D-deficient diets containing either 0.02% calcium ( - Ca) or 0.64% calcium ( + Ca). Adequate and defined vitamin D status was attained by subcut. Injection of 5 kg D, and the 25(OH)D pool was labelled by intracardial injection of 0.3 FCi 3H-25(OH)D3. The elrmination half-life (tlR) of 3H-25(OH)D, in plasma of -Ca rats (8-10 days) was 40% faster than that of +Ca controls (13- 15 days), the difference berng quantitatively accounted for by the appearance of excess 3H as highly polar metabolites in faeces of -Ca rats. Plasma analysis after 4 weeks on the diets gave the following values (means IT s.e.m.; n = 6 per group): [Ca]mmoM
THE INVOLVEMENT OF CALCIUM IONS IN THE EFFECT OF 1,25-DIHYDROXYVITAMIN D ON HL-60 CELLS
from the Calclfled Tissues Workshop
+Ca 251 t 0 10 Ca248?007
[POJmmolll
[PS(OH)D]ngiml
183 t- 016 2732010
134i14 79 t 10
[1,25(OH)2D]pglml 780~56 z 44
388 7
a
Varying dietary [Cal down to 0.02% caused proportional acceleration in t,,, of 3H-25(OH)D3. Similar accelerated t,,z values could be produced by feeding the +Ca dret with additional PO., (1,2%P) or phytic acid (5.1%). The accelerated loss of plasma 25(OH)D on the -Ca diet cannot be explained merely by the Increased production of 1,25(OH),D. When the 25(OH)D pool was labelled with 3H-5,6-trans-25(OH)D3, which cannot be converted to 1,25(OH),D, the - Ca diet still caused the same reduction rn the tl, of 3H. Furthermore, the t,,* of 3H-1,25(OH),D, on +Ca diet (34 6 +- 12h) was unchanged by feeding -Ca diet (35.0 & 0.6h). The alternative explanation is that hepatic Inactivation of 25(OH)D IS enhanced in calcium deprivation. The normal [Cal and elevated [1,25(OH),D] in plasma of rats on -Ca diet indicate a marked degree of secondary hyperparathyroidrsm. Parathyroidectomy of rats on the high-PO, diet abolrshes the effect on t,,> of 3H-25(OH)D,. A sjmllar effect of parathyroidectomy on t,,? of 3H-25(OH)D, has been found in patients with hyperparathyroidism. It IS therefore postulated that elevated parathyroid secretion leads to enhanced hepatic inactivation of 25(OH)D and ultimately, with low vitamin D supply, to vrtamin D deficiency.
EFFECTS OF PARATHYROID HORMONE (PTH) AND VITAMIN D ON NA+,K+-ATPASE ACTIVITY IN RAT TUBULE SEGMENTS Elstern. D., W.J Czaczkes Hadassah Medical Center, Jerusalem,
israel
As an Initial step towards understanding the interdrgrtatrng influences of PTH and 1,25 dihydroxycholecalciferol (vitamrn D) on transport of sodium (Na) in renal tubules,