Requirement of vitamin C for cartilage calcification in a differentiating chick limb-bud mesenchymal cell culture

Boskey, Adele L.; Stiner, Dalina; Doty, Stephen B.; Binderman, Itzhak

doi:10.1016/8756-3282(91)90076-u

Cited by 36 publications

(42 citation statements)

References 36 publications

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“…Mineralizing cultures were also supplemented with 3 mM inorganic phosphate from day 2 onward, making the total inorganic phosphate content of the medium 4 mM; non-mineralizing cultures received no additional inorganic phosphate, their inorganic phosphate content was 1 mM. The mineralizing conditions were previously shown to be optimal for cell maturation and mineral deposition in the absence of dystrophic or artifactual mineralization [13].…”

Section: Materials and Methods Cell Culturementioning

confidence: 99%

“…Chick mesenchymal cells were isolated from stage 21-24 [28] fertilized White Leg Horn eggs (Truslow Farms, Chestertown, MD) as described in detail elsewhere [13]. In brief, the eggs were maintained in a humidified incubator at 37°C.…”

Section: Materials and Methods Cell Culturementioning

confidence: 99%

“…To alter cell differentiation, and prevent aggrecan synthesis, 10 28 -10 210 M retinoic acid (Sigma Chemicals, St. Louis, MO) was added to mineralizing and non-mineralizing cultures either on days 2, 5, or 7, or from day 5 until the conclusion of the experiment. At these initial times mesenchymal differentiation into chondrocytes had commenced, but hypertrophic chondrocytes had not formed [13]. Control mineralizing cultures for these experiments received ethanol, the carrier for retinoic acid.…”

Section: Modification Of Aggrecan Synthesis and Contentmentioning

confidence: 99%

“…Whether this change is essential for growth plate calcification or coincident to this process is in debate. Some solution studies indicate that aggrecan modification (decreases in size, desulfation) facilitates cell-free in vitro calcification [3][4][5][6][7], while others suggest that it is the change in local phosphate concentration, in the absence of proteoglycan modification, which facilitates calcification [8,9].Mesenchymal cells, plated in a micromass culture [10], differentiate into chondroblasts and chondrocytes [11,12], and can form a mineralized matrix when the media is supplemented with inorganic or organic phosphate [12][13][14][15]. The mineralized matrix resembles that of the calcified cartilage of 17-day-old chicks [15].…”

mentioning

confidence: 98%

“…The mineralized matrix resembles that of the calcified cartilage of 17-day-old chicks [15]. Aggrecan core protein expression, along with the synthesis of types II and X collagen, occurs in a time-dependent and reproducible fashion [13]. This mineralizing system is therefore appropriate for studying the effect of proteoglycan modification on the calcification process.…”

mentioning

confidence: 98%

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Effects of proteoglycan modification on mineral formation in a differentiating chick limb-bud mesenchymal cell culture system

et al. 1997

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In the presence of 4 mM inorganic phosphate, differentiating chick limb-bud mesenchymal cells plated in micromass cultures form a mineralized matrix resembling that of chick calcified cartilage. To test the hypothesis that cartilage proteoglycans are inhibitors of cell mediated mineralization, the synthesis, content, and turnover of proteoglycans were altered in this system, and the extent of mineralization and properties of the mineral crystals examined. In all cases where the proteoglycan synthesis or proteoglycans present were modified to provide fewer or smaller molecules, mineralization was enhanced. Specifically, when proteoglycan synthesis was blocked by treatment with 10(-10) M retinoic acid, extensive mineral deposition occurred on a matrix devoid of both proteoglycans and cartilage nodules. The crystals, which formed rapidly, were relatively large in size based on analysis by X-ray diffraction or FT-1R microspectroscopy, and were more abundant than in controls. When 2.5 or 5 mM xylosides were used to cause the synthesis of smaller proteoglycans, the extent of mineral accretion was also increased relative to controls; however, the matrix was less affected, and the extent of mineral deposition and the size of the crystals were not as markedly altered as in the case of retinoic acid. Modification of existing proteoglycans by either chondroinase ABC or hyaluronidase treatment similarly resulted in increased mineral accretion (based on 45Ca uptake or total Ca uptake) relative to cultures in which the proteoglycan content was not manipulated. Crystals were more abundant and larger than in control mineralizing cultures. In contrast, when proteoglycan degradation by metalloproteases was inhibited by metal chelation with o-phenanthroline, the Ca accretion at early time points was increased, but as mineralization progressed, Ca accumulation decreased. These data provide evidence that in this culture system, proteoglycans are inhibitors of mineralization.

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Section: Materials and Methods Cell Culturementioning

confidence: 99%

Section: Materials and Methods Cell Culturementioning

confidence: 99%

Section: Modification Of Aggrecan Synthesis and Contentmentioning

confidence: 99%

mentioning

confidence: 98%

mentioning

confidence: 98%

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Effects of proteoglycan modification on mineral formation in a differentiating chick limb-bud mesenchymal cell culture system

et al. 1997

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Type I collagen influences cartilage calcification: An immunoblocking study in differentiating chick limb-bud mesenchymal cell cultures

et al. 2000

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Chick limb-bud mesenchymal cells, plated in high-density micro-mass culture, differentiate and form a matrix resembling chick epiphyseal cartilage. In the presence of 4 mM inorganic phosphate or 2.5 mM beta-glycerophosphate mineral deposits upon this matrix forming a mineralized tissue that, based on electron microscopy, x-ray diffraction and Fourier Transform Infrared microspectoscopy, is like that of chick calcified cartilage. In this culture system the initial mineral deposits are found on the periphery of the chondrocyte nodules. During differentiation of the cells in the high-density micro-mass cultures there is a switch from expression of type I collagen to type II, and then to type X collagen. However, type I collagen persists in the matrix. Because there is some debate about whether type I collagen influences cartilage calcification, an immunoblocking technique was used to determine the importance of type I collagen on the mineralization process in this system. Studies using nonspecific goat anti-chick IgG demonstrated that 1-100 ng/ml antibody added with the media after the cartilage nodules had developed (day 7) had no effect on the accumulation of mineral in the cultures. Nonspecific antibody added before day 7 blocked development of the cultures. Parallel solution based cell-free studies showed that IgG did not have a strong affinity for apatite crystals, and had no significant effect on apatite crystal growth. Type I collagen antibodies (1-200 ng/ml) added to cultures one time on day 9 (before mineralization started), or on day 11 (at the start of mineralization), slightly inhibited the accumulation of mineral. There was a statistically significant decrease in mineral accretion with 100 or 200 ng/ml collagen antibody addition continuously after these times. Fab' fragments of nonspecific and type I collagen antibodies had effects parallel to those of the intact antibodies, indicating that the decreased mineralization was not attributable to the presence of the larger, bulkier antibodies. The altered accumulation of mineral was not associated with cell death in the presence of antibody (demonstrated by fluorescent labeling of DNA) or with increased apoptosis (TUNEL-stain). In the immunoblocked cultures, EM analysis demonstrated that mineral continued to deposit on collagen fibrils, but there appeared to be fewer deposits. The data demonstrate that type I collagen is important for the mineralization of these cultures.

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Octacalcium phosphate combined with collagen orthotopically enhances bone regeneration

Sasaki²,

et al. 2006

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Octacalcium phosphate (OCP) is resorbable bone regenerative material, but its brittleness makes it difficult to maintain its shape without restraint. We have engineered a scaffold constructed of synthetic OCP and porcine collagen sponge (OCP/Collagen) and investigated whether OCP/Collagen composite could improve bone regeneration. To examine this hypothesis, bone regeneration by the implantation of OCP/Collagen was compared with those by OCP and collagen. Radiographic and histological examination was performed and the percentage of newly formed bone (n-Bone%) in the defect was determined by a histomorphometrical analysis. OCP/Collagen, OCP, or collagen was implanted into the critical-sized defects in rat crania and fixed at 2, 4, or 8 weeks after implantation. OCP/Collagen improved the handling performance than the granules of OCP, and synergistically enhanced the bone regeneration beyond expectation, which were composed of bone nucleation by OCP and cell infiltration by collagen. Histomorphometrical analysis showed that n-Bone% +/- standard error treated with OCP/Collagen (48.4 +/- 5.14) was significantly higher than those with OCP (27.6 +/- 4.04) or collagen (27.4 +/- 5.69) in week 8. The present study suggests that the combination OCP with collagen elicited the synergistic effect for bone regeneration.

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Requirement of vitamin C for cartilage calcification in a differentiating chick limb-bud mesenchymal cell culture

Cited by 36 publications

References 36 publications

Effects of proteoglycan modification on mineral formation in a differentiating chick limb-bud mesenchymal cell culture system

Effects of proteoglycan modification on mineral formation in a differentiating chick limb-bud mesenchymal cell culture system

Type I collagen influences cartilage calcification: An immunoblocking study in differentiating chick limb-bud mesenchymal cell cultures

Octacalcium phosphate combined with collagen orthotopically enhances bone regeneration

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