- Email: [email protected]
Development of an in vitro mineralization model with growth plate chondrocytes that does not require β-glycerophosphate
Boneand
152
Minerai,
17
(1992) 152-156 Elsevier
This paper was presented at the Fifth International Conference on Cell-Mediated Calcification and MatrixVesicles, held November 16-20,1991, Hilton Head, South Carolina.
Developmentof an in vitro mineralizationmodel withgrowth plate chondrocytesthat require/3-glycerophosphate
Yoshinori Ishikawa and Roy E. Wuthier Department
oJIChemistry
and Biochemistry, University South Carolina, USA
ofSouth Coroliaa, Columbia,
-
Key words: Growth plate chondrocytes;Cell culture;@-Glycerolphosphate;Insulin;Ascorbate; Mineralization: Autofluorescence
Introduction
Many mineralizing cell culture systems so far reported have used high levels (1~ mM) of &glycerophosphate (BGP), a nonphysiological organic phosphate (P,) substrate of alkaline phosphatase (AP), for induction of calcification [l-4]. Recently, however, BGP has been shown to cause ectopic mineralization in cultures of fetal rat parietal cells [5], as well as bone and skin fibroblasts [6]. Establishment of a culture system that utilizes physiological levels of electrolytes, nutrients, hormonal and humoral factors, and produces normal mineral deposition, has been a long sought goal for studying the mechanism of biomineralization, and for elucidating modulators with potential clinical application. In past studies, we analyzed the levels of electrolytes [7] and amino acids [S] in the extracellular fluid (ECF) of growth plate (GP) cartilage. and developed a primary cell culture system for chicken GP chondrocytes that expresses high levels of alkaline phosphatase (ALP) activity and LP-rich vesicles (MV) into the culture medium [9]. We found that addition to these cultures of amino acids at levels found in GP-ECF, plus 50pglml of ascorbate, stimulated formation of MV [lo]. We report here the development of a mineralizing culture system, based on these past findings, that utilizes physiological levels of electrolytes and does not require addition of P,.
153 terials a
S
Chicken GP chondrocytes (5 X 104/cm2)cultures were started in Dulbecco’s Modified Eagle’s Medium (DME ; @a*+1.8 mM, Pi 0.9 mM) with 10% fetal bovine serum (FBS) and ascorbic acid (50 ,ug/ml) as described earlier [lo]. the cells were nearly confluent, the media were changed to DME with 5% FBS, 8 amino acids and ascorbate [lo]. In some exp fig/ml), transferrin (5 ,ug/ml), selenite (5 ng/ml), and 0.5-2.5 phosphates: Na,ti?O, (Pi), pyrophosphate (PPr), a- or P-glycerophosphate (GP), phosphoethanolamint (PEA), or phosphocholine (PC) were also added. Cells were harvested on day 24 or 27 for total protein and alkaline phosphatase (ALP) assay. Insoluble fractions in TMT buffer (0.1 Triton X-100 and 0.5 mM MgCl, in 50 mM Tris pH 7.5) were extracted with 0.1 Cl for Ca*+ and Pi analysis.
es& In this GP chondrocyte system, mineral formation was not limited to the center of multicellular nodules as is often reported [1,3], but was initially found associated with the periphery of the cells, resembling extralacunar mineral formation seen in the growth plate. Phosphate supplementation At 0.5-2.5 mM in the media, Pi caused mineral deposition (Ca*+/Pi = 1.5 -I- 0.2)
equal to or greater than that formed by equivalent levels of the P, tested, (GP, PEA or PC), and generally caused only slight increases in cellular and matrix protein. ALP activity declined proportionate to mineral deposition, as seen earlier during NV mineralization [ll]. PP, (0.5-2.5 mM) in a concen:ration-dependent manner inhibited protein synthesis and AP activity, but caused formation of small amounts of mineral with a Ca/Pi ratio of 1.10 + 0.15, indicating Ca PPi (CPP) formation. When 1 mM PP, was added to media containing either 1 mM Pi or BGP, the Ca*+/Pi ratios of the mineral indicated that CPP also was formed. These results help explain the formation of CPP in pseudogout when elevated PP, is present in synovial fluid. Effects of growth factors and serum
Insulin, transferrin and selenite (ITS) mixture stimulated cell growth and mineralization in a dosage-related manner, in the presence of supplemented Pi (1 mM). Insulin-like growth factors (IGF) I and II stimulated cell growth and mineralization at physiological levels (50-100 nglml), causing loss of ALP activity; both were almost IO-fold more potent than insulin in these effects. Fetal bovine serum (FBS) (l-10%) stimulated protein synthesis and mineral deposition in a dosage-dependent manner. Other sera (cow, calf, goat, pig, and sheep) at 5% level gave equal or better cell growth and mineralization, but failed to support ALP formation.
Mg. 1. Coincidence of autofluorescence with refractile vesicles at early sites of mineralization. Len panels- phase contrast: Right panels- autofluorescence; Upper panels - Day 15culture; LowerpanelsDay 24 culture. Autofluorescence was examined with a Nikon DIAPHOT-TMD inverted microscope usine 495 nm excitation; photographs were made on Kodak Tri-X pan 400 ASA film.
Autofluorescence in mineralizing chondrocytes P\time-course study from Day 9 to 24 of the calcifying chondrocyte cultures re-
vealed the presence of autofluorescent material in small (0.5-2.0 ,um) extracellular vesicular structures and in cells closely associated with mineralization sites (Fig. 1). The appearance of this material preceded the observation of mineralization by phase contrast microscopy. In more heavily mineralized areas the fluorescence were more diffuse. Some cells enclosed within mineralized ‘shells’ had reduced amounts of fluorescence, indicating release or modification of this material. Its punctate pattern of distribution around the mineralizing cells closely coincided with highly refractile bodies seen in phase-contrast, suggesting compartmentalization and association with cell membranes, blebs and vesicles. Scanning electron microscopy observations
The mineralizing chondrocytes showed numerous cell processes, blebs and ruffled membrane surfaces, which in carbon-coated preparations used for EDAX analysis were highly enriched in calcium.
155
Unlike primary osteoblast cultures, our primary cultures of avian growth plate chondrocytes consist purely of chondrocytes, free of fibroblast contamination. This enabled study of isolated chondrocytes free from other cell types. In agreement with others, we have found that increased Pi levels in the medium strongly enhanced the ability of chondrocytes to induce mineral deposition [12]. DMEM does not have a Ca” X Pi ion product sufficient to support normal mineral formation [13]; 10 mM BGP commonly added to induce mineralization, in the presence of ALP activity causes excessive build-up of Pi and ectopic mineral deposition [5,6]. To minimize nonphysiological mineral deposition and to explore the effects of humoral agents, we utilized DMEM as the basal medium; simple increase in the supply of Pi, comparable to that occuring GP-ECF, was sufficient to enable induction of calcification by the chondrocytes. In agreement with earlier studies, we found that insulin and insulin-like growth factor (IGF-I) caused a distinct anabolic effect on the chondrocyte cultures, promoting cell growth, as well as mineral formation [14,15]. I P-I was approximately lo-fold more potent than insulin. The effects on ALP were biphasic, stimulating at low levels and suppressing activity at higher levels, and appeared to be inversely correlated with mineral deposition. Normal collagen synthesis appears to be essential for proper matrix formation and mineralization in growth plate cultures. The importance of ascorbate is well recognized, and this factor is now included in most mineralization media. In past studies we found that ascorbate, in conjunction with elevated levels of 8 amino acids, significantly increased MV formation and cell growth in GP chrondrocyte cultures [lo]. Used with serum-free I-IL-1 medium, ascorbate (50 pglml) was required for normal mineralization [ 161. There appears to be a direct correlation between expression of normal collagen, ALP, and mineralization. We report here the discovery of a natural fluorescent component associated with the induction of mineral deposition in primary GP chondrocyte cultures. Initially, this is associated with the outer membrane of the chondrocytes and refractile vesicles released by the cells. These structures are correlated closely with subsequent mineral formation. In areas of heavy mineralization, the fluorescence was diffuse, possibly adhering to mineral crystallites. It was not removed by demineralization, but was destroyed by oxidization. We frequently noted that cells developed a pericellular ring of mineral deposition in the region containing the fluorescent material. This finding is in close agreement with laser confocal images of intracellular Ca2+ localization using Indo-l as a probe [17]. In mineralizing cultures, and sections of live growth plate cartilage, elevated levels of Ca2+ in the perimeter of chondrocytes were consistently seen at sites of initial mineralization, When observed by scanning electron microscopy, we also observed Ca2+-rich cellular processes and blebs developing at the chondrocyte surface. From these observations, there is little doubt that an intimate relationship exists between the cell processes and cell-mediated extracellular calcification. They indicate that Ca2’ and Pi are preloaded into membrane-derived blebs and vesicles
156 prior to release at th;; calcification front. Using this culture system, Hale et al. [IS] showed that disruption of actin microfilaments stimulates the release of vesicles indistinquishable from MV. Most recently, we have discovered that some of the major proteins of MV are acidic phospholipid-dependent Ca *+-binding proteins, termed annexins 1191.Two of these, annexin V (anchorin CII) and annexin VI, have been found to bind to collagen [20], and are thought to anchor MV to the matrix. These findings help explain how Ca*’ (and Pi) preloaded into the chondrocytes may be transferred to the MV. Thus, this culture system has proven to be a good model for studying chondrocyte mineralization under conditions close to that present in vivo.
Refermses 1 V~tiniinen HK, Morris DC, Anderson HC. Calcification of cartilage matrix in chondrocyte cultures derived from rachitic rat growth plate cartilage. Metab Bone Dis Relat Res 1983;5:87-92. 2 Robey PG, Termine JD. Human bone cells in vitro. Calcif Tissue Intl1985;37:453-460. 3 Bellows CG, Aubin JE, Heersche JNM, Antosz ME. Mineralized bone nodules formed in vitro from enzymatically released bone calvarial cell populations. Calcif Tissue Intl1986;38:143-154. 4 Gerstenfeld LC, Chipman SD, Glowacki J, Lian JB. Expression of differentiated function by mineralizing cultures of chicken osteoblasts. Dev Biol1987;122:49-60. 5 Gronowicz G, Woodiel FN, McCarthy M-B, Raisz LG. In vitro mineralization of fetal rat parietal bones in defined serum-free medium: effect of #&glycerol phosphate. J Bone Min Res 1989;4:313-324. 6 Khouja HI, Bevington A, Kemp GJ, Russell RGG. Calcium and orthophosphate deposits in vitro do not imply osteoblast-mediated mineralizadon: mineralization by beta-glycerolphosphate in the absence of osteoblasts. Bone 1990;11:385-391. 7 Wuthier RE. Electrolytes of isolated epiphyseal chondrocytes, matrix vesicles, and extracellular fluid. Calcif Tissue Res 1977;23:125- 133. 8 Ishikawa Y, Chin JE, Hubbard HL, Wuthier RE. Utilization and formation of amino acids by chicken epiphyseal chondrocytes: comparative studies with cultured cells and native cartilage tissue. J Cell Physiol1985;123:79-88, 9 Wutkier RE, Chin JE, Hale JE, Register TC, Hale LV, Ishikawa Y. Isolation and characterization of ealcium.accumulating matrix vesicles from chondrolcytes of chicken epiphyseal growth plate cartilage in primary culture. J Biol Chem 1985;260:15972- i5979. 10 Ishikawa Y, Chin JE, Schalk EM, Wuthier RE. Effect of amino acids and ascorbic acid on matrix vesicle formation by epiphyseal growth plate chondrocytes in primary culture. In: Ali SY, ed. Cell Mediated Calcification and Matrix Vesicles. Amsterdam: Elsevier Publishers, 1986:231-236. 11 Genge BR, SauerGR, Wu LNY, McLean FM, Wuthier RE. Correlation between loss of alkaline phosphatase activity and accumulation of calcium during matrix vesicle-mediated mineralization. 3 Biol Chem 1988;263:18513-18519. 12 Bingham PJ, Raisz LG. Bone growth in organ culture: effects of phosphate and other nutrients on bone and cartilage. Calcif Tissue Res 1974;14:31-48. 13 Valhmu WB, Wu LNY, Wuthier RE. Effects of CalPi ratio, Ca2” x Pi ion product, and pH of incubation fluid on accumulation of 4SCa2tby matrix vesicles in vitro. Bone Min 1990;8:195-209. 14 Hill DJ, De Sousa D. Insulin is a mitogen for isolated epiphyseal growth plate chondrocytes from the fetal lamb. Endocrin 1990;126:2661-2670. I5 Pfeilschifter J, Oechsner M, Maumann A, Gronwald RGK, Minne HW, Ziegler R. Stimulation of bone matrix apposition in vitro by local growth factors: A comparison of insulin-like growth factor I, platelet derived growth factor, and transforming growth factor,!?. Endocrin 1990;127:69-75.
157 16 Wu LNY, Sauer GR, Genge BR, Wuthier RE. Induction of mineral deposition by primary cultures of chicken growth plate chondrocytes in ascorbate-containing media. J Biol Chem 1989;264:21346-21355. 17 Wu LNY, Wuthier MC, Wuthier RE. Laser confocal cytometric analysis of intracellular Ca2+ in calcifying growth plate chondrocytes using Indo-l AM indicator. Bone Min 1992;7:315(abstract this issue). 18 Hale JE, Wuthier RE. The mechanism of matrix vesicle formation. Studies on the composition of chondrocyte microvilli and on the effects of microfilament-perturbing agents on cellular vesiculation. J Biol Chem 1987;262:1916-1925. 19 Genge BR, Wu LNY, Wuthier RE. Identification of phospholipid-dependent calcium-binding proteins as constituents of matrix vesicles. J Biol Chem 1989;264:10917-10921. 20 Wu LNY, Genge BR, Lloyd GC, Wuthier RF r-‘%Jen-binding proteins in collagenase-released rix vesicle proteins and different types of colmatrix vesicles from cartilage. Interaction betwc lagen. J Biol Chem 1991;266:1195-1203.
152
Minerai,
17
(1992) 152-156 Elsevier
This paper was presented at the Fifth International Conference on Cell-Mediated Calcification and MatrixVesicles, held November 16-20,1991, Hilton Head, South Carolina.
Developmentof an in vitro mineralizationmodel withgrowth plate chondrocytesthat require/3-glycerophosphate
Yoshinori Ishikawa and Roy E. Wuthier Department
oJIChemistry
and Biochemistry, University South Carolina, USA
ofSouth Coroliaa, Columbia,
-
Key words: Growth plate chondrocytes;Cell culture;@-Glycerolphosphate;Insulin;Ascorbate; Mineralization: Autofluorescence
Introduction
Many mineralizing cell culture systems so far reported have used high levels (1~ mM) of &glycerophosphate (BGP), a nonphysiological organic phosphate (P,) substrate of alkaline phosphatase (AP), for induction of calcification [l-4]. Recently, however, BGP has been shown to cause ectopic mineralization in cultures of fetal rat parietal cells [5], as well as bone and skin fibroblasts [6]. Establishment of a culture system that utilizes physiological levels of electrolytes, nutrients, hormonal and humoral factors, and produces normal mineral deposition, has been a long sought goal for studying the mechanism of biomineralization, and for elucidating modulators with potential clinical application. In past studies, we analyzed the levels of electrolytes [7] and amino acids [S] in the extracellular fluid (ECF) of growth plate (GP) cartilage. and developed a primary cell culture system for chicken GP chondrocytes that expresses high levels of alkaline phosphatase (ALP) activity and LP-rich vesicles (MV) into the culture medium [9]. We found that addition to these cultures of amino acids at levels found in GP-ECF, plus 50pglml of ascorbate, stimulated formation of MV [lo]. We report here the development of a mineralizing culture system, based on these past findings, that utilizes physiological levels of electrolytes and does not require addition of P,.
153 terials a
S
Chicken GP chondrocytes (5 X 104/cm2)cultures were started in Dulbecco’s Modified Eagle’s Medium (DME ; @a*+1.8 mM, Pi 0.9 mM) with 10% fetal bovine serum (FBS) and ascorbic acid (50 ,ug/ml) as described earlier [lo]. the cells were nearly confluent, the media were changed to DME with 5% FBS, 8 amino acids and ascorbate [lo]. In some exp fig/ml), transferrin (5 ,ug/ml), selenite (5 ng/ml), and 0.5-2.5 phosphates: Na,ti?O, (Pi), pyrophosphate (PPr), a- or P-glycerophosphate (GP), phosphoethanolamint (PEA), or phosphocholine (PC) were also added. Cells were harvested on day 24 or 27 for total protein and alkaline phosphatase (ALP) assay. Insoluble fractions in TMT buffer (0.1 Triton X-100 and 0.5 mM MgCl, in 50 mM Tris pH 7.5) were extracted with 0.1 Cl for Ca*+ and Pi analysis.
es& In this GP chondrocyte system, mineral formation was not limited to the center of multicellular nodules as is often reported [1,3], but was initially found associated with the periphery of the cells, resembling extralacunar mineral formation seen in the growth plate. Phosphate supplementation At 0.5-2.5 mM in the media, Pi caused mineral deposition (Ca*+/Pi = 1.5 -I- 0.2)
equal to or greater than that formed by equivalent levels of the P, tested, (GP, PEA or PC), and generally caused only slight increases in cellular and matrix protein. ALP activity declined proportionate to mineral deposition, as seen earlier during NV mineralization [ll]. PP, (0.5-2.5 mM) in a concen:ration-dependent manner inhibited protein synthesis and AP activity, but caused formation of small amounts of mineral with a Ca/Pi ratio of 1.10 + 0.15, indicating Ca PPi (CPP) formation. When 1 mM PP, was added to media containing either 1 mM Pi or BGP, the Ca*+/Pi ratios of the mineral indicated that CPP also was formed. These results help explain the formation of CPP in pseudogout when elevated PP, is present in synovial fluid. Effects of growth factors and serum
Insulin, transferrin and selenite (ITS) mixture stimulated cell growth and mineralization in a dosage-related manner, in the presence of supplemented Pi (1 mM). Insulin-like growth factors (IGF) I and II stimulated cell growth and mineralization at physiological levels (50-100 nglml), causing loss of ALP activity; both were almost IO-fold more potent than insulin in these effects. Fetal bovine serum (FBS) (l-10%) stimulated protein synthesis and mineral deposition in a dosage-dependent manner. Other sera (cow, calf, goat, pig, and sheep) at 5% level gave equal or better cell growth and mineralization, but failed to support ALP formation.
Mg. 1. Coincidence of autofluorescence with refractile vesicles at early sites of mineralization. Len panels- phase contrast: Right panels- autofluorescence; Upper panels - Day 15culture; LowerpanelsDay 24 culture. Autofluorescence was examined with a Nikon DIAPHOT-TMD inverted microscope usine 495 nm excitation; photographs were made on Kodak Tri-X pan 400 ASA film.
Autofluorescence in mineralizing chondrocytes P\time-course study from Day 9 to 24 of the calcifying chondrocyte cultures re-
vealed the presence of autofluorescent material in small (0.5-2.0 ,um) extracellular vesicular structures and in cells closely associated with mineralization sites (Fig. 1). The appearance of this material preceded the observation of mineralization by phase contrast microscopy. In more heavily mineralized areas the fluorescence were more diffuse. Some cells enclosed within mineralized ‘shells’ had reduced amounts of fluorescence, indicating release or modification of this material. Its punctate pattern of distribution around the mineralizing cells closely coincided with highly refractile bodies seen in phase-contrast, suggesting compartmentalization and association with cell membranes, blebs and vesicles. Scanning electron microscopy observations
The mineralizing chondrocytes showed numerous cell processes, blebs and ruffled membrane surfaces, which in carbon-coated preparations used for EDAX analysis were highly enriched in calcium.
155
Unlike primary osteoblast cultures, our primary cultures of avian growth plate chondrocytes consist purely of chondrocytes, free of fibroblast contamination. This enabled study of isolated chondrocytes free from other cell types. In agreement with others, we have found that increased Pi levels in the medium strongly enhanced the ability of chondrocytes to induce mineral deposition [12]. DMEM does not have a Ca” X Pi ion product sufficient to support normal mineral formation [13]; 10 mM BGP commonly added to induce mineralization, in the presence of ALP activity causes excessive build-up of Pi and ectopic mineral deposition [5,6]. To minimize nonphysiological mineral deposition and to explore the effects of humoral agents, we utilized DMEM as the basal medium; simple increase in the supply of Pi, comparable to that occuring GP-ECF, was sufficient to enable induction of calcification by the chondrocytes. In agreement with earlier studies, we found that insulin and insulin-like growth factor (IGF-I) caused a distinct anabolic effect on the chondrocyte cultures, promoting cell growth, as well as mineral formation [14,15]. I P-I was approximately lo-fold more potent than insulin. The effects on ALP were biphasic, stimulating at low levels and suppressing activity at higher levels, and appeared to be inversely correlated with mineral deposition. Normal collagen synthesis appears to be essential for proper matrix formation and mineralization in growth plate cultures. The importance of ascorbate is well recognized, and this factor is now included in most mineralization media. In past studies we found that ascorbate, in conjunction with elevated levels of 8 amino acids, significantly increased MV formation and cell growth in GP chrondrocyte cultures [lo]. Used with serum-free I-IL-1 medium, ascorbate (50 pglml) was required for normal mineralization [ 161. There appears to be a direct correlation between expression of normal collagen, ALP, and mineralization. We report here the discovery of a natural fluorescent component associated with the induction of mineral deposition in primary GP chondrocyte cultures. Initially, this is associated with the outer membrane of the chondrocytes and refractile vesicles released by the cells. These structures are correlated closely with subsequent mineral formation. In areas of heavy mineralization, the fluorescence was diffuse, possibly adhering to mineral crystallites. It was not removed by demineralization, but was destroyed by oxidization. We frequently noted that cells developed a pericellular ring of mineral deposition in the region containing the fluorescent material. This finding is in close agreement with laser confocal images of intracellular Ca2+ localization using Indo-l as a probe [17]. In mineralizing cultures, and sections of live growth plate cartilage, elevated levels of Ca2+ in the perimeter of chondrocytes were consistently seen at sites of initial mineralization, When observed by scanning electron microscopy, we also observed Ca2+-rich cellular processes and blebs developing at the chondrocyte surface. From these observations, there is little doubt that an intimate relationship exists between the cell processes and cell-mediated extracellular calcification. They indicate that Ca2’ and Pi are preloaded into membrane-derived blebs and vesicles
156 prior to release at th;; calcification front. Using this culture system, Hale et al. [IS] showed that disruption of actin microfilaments stimulates the release of vesicles indistinquishable from MV. Most recently, we have discovered that some of the major proteins of MV are acidic phospholipid-dependent Ca *+-binding proteins, termed annexins 1191.Two of these, annexin V (anchorin CII) and annexin VI, have been found to bind to collagen [20], and are thought to anchor MV to the matrix. These findings help explain how Ca*’ (and Pi) preloaded into the chondrocytes may be transferred to the MV. Thus, this culture system has proven to be a good model for studying chondrocyte mineralization under conditions close to that present in vivo.
Refermses 1 V~tiniinen HK, Morris DC, Anderson HC. Calcification of cartilage matrix in chondrocyte cultures derived from rachitic rat growth plate cartilage. Metab Bone Dis Relat Res 1983;5:87-92. 2 Robey PG, Termine JD. Human bone cells in vitro. Calcif Tissue Intl1985;37:453-460. 3 Bellows CG, Aubin JE, Heersche JNM, Antosz ME. Mineralized bone nodules formed in vitro from enzymatically released bone calvarial cell populations. Calcif Tissue Intl1986;38:143-154. 4 Gerstenfeld LC, Chipman SD, Glowacki J, Lian JB. Expression of differentiated function by mineralizing cultures of chicken osteoblasts. Dev Biol1987;122:49-60. 5 Gronowicz G, Woodiel FN, McCarthy M-B, Raisz LG. In vitro mineralization of fetal rat parietal bones in defined serum-free medium: effect of #&glycerol phosphate. J Bone Min Res 1989;4:313-324. 6 Khouja HI, Bevington A, Kemp GJ, Russell RGG. Calcium and orthophosphate deposits in vitro do not imply osteoblast-mediated mineralizadon: mineralization by beta-glycerolphosphate in the absence of osteoblasts. Bone 1990;11:385-391. 7 Wuthier RE. Electrolytes of isolated epiphyseal chondrocytes, matrix vesicles, and extracellular fluid. Calcif Tissue Res 1977;23:125- 133. 8 Ishikawa Y, Chin JE, Hubbard HL, Wuthier RE. Utilization and formation of amino acids by chicken epiphyseal chondrocytes: comparative studies with cultured cells and native cartilage tissue. J Cell Physiol1985;123:79-88, 9 Wutkier RE, Chin JE, Hale JE, Register TC, Hale LV, Ishikawa Y. Isolation and characterization of ealcium.accumulating matrix vesicles from chondrolcytes of chicken epiphyseal growth plate cartilage in primary culture. J Biol Chem 1985;260:15972- i5979. 10 Ishikawa Y, Chin JE, Schalk EM, Wuthier RE. Effect of amino acids and ascorbic acid on matrix vesicle formation by epiphyseal growth plate chondrocytes in primary culture. In: Ali SY, ed. Cell Mediated Calcification and Matrix Vesicles. Amsterdam: Elsevier Publishers, 1986:231-236. 11 Genge BR, SauerGR, Wu LNY, McLean FM, Wuthier RE. Correlation between loss of alkaline phosphatase activity and accumulation of calcium during matrix vesicle-mediated mineralization. 3 Biol Chem 1988;263:18513-18519. 12 Bingham PJ, Raisz LG. Bone growth in organ culture: effects of phosphate and other nutrients on bone and cartilage. Calcif Tissue Res 1974;14:31-48. 13 Valhmu WB, Wu LNY, Wuthier RE. Effects of CalPi ratio, Ca2” x Pi ion product, and pH of incubation fluid on accumulation of 4SCa2tby matrix vesicles in vitro. Bone Min 1990;8:195-209. 14 Hill DJ, De Sousa D. Insulin is a mitogen for isolated epiphyseal growth plate chondrocytes from the fetal lamb. Endocrin 1990;126:2661-2670. I5 Pfeilschifter J, Oechsner M, Maumann A, Gronwald RGK, Minne HW, Ziegler R. Stimulation of bone matrix apposition in vitro by local growth factors: A comparison of insulin-like growth factor I, platelet derived growth factor, and transforming growth factor,!?. Endocrin 1990;127:69-75.
157 16 Wu LNY, Sauer GR, Genge BR, Wuthier RE. Induction of mineral deposition by primary cultures of chicken growth plate chondrocytes in ascorbate-containing media. J Biol Chem 1989;264:21346-21355. 17 Wu LNY, Wuthier MC, Wuthier RE. Laser confocal cytometric analysis of intracellular Ca2+ in calcifying growth plate chondrocytes using Indo-l AM indicator. Bone Min 1992;7:315(abstract this issue). 18 Hale JE, Wuthier RE. The mechanism of matrix vesicle formation. Studies on the composition of chondrocyte microvilli and on the effects of microfilament-perturbing agents on cellular vesiculation. J Biol Chem 1987;262:1916-1925. 19 Genge BR, Wu LNY, Wuthier RE. Identification of phospholipid-dependent calcium-binding proteins as constituents of matrix vesicles. J Biol Chem 1989;264:10917-10921. 20 Wu LNY, Genge BR, Lloyd GC, Wuthier RF r-‘%Jen-binding proteins in collagenase-released rix vesicle proteins and different types of colmatrix vesicles from cartilage. Interaction betwc lagen. J Biol Chem 1991;266:1195-1203.