Cells Isolated From Fetal Rat Calvaria by Isopycnic Separation Express Different Collagen Phenotypes

J. E. Villanueva, Satoru Nishimoto, M. E. Nimni

Research output: Contribution to journalArticle

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Abstract

Calvaria from 20–21 day old fetuses were obtained under sterile conditions and the endo- and exoperiosteum stripped off. Cells were dispersed by sequential collagenase-DNase treatment and suspended in 0.5% low Tm agarose in the presence of DMEM supplement with 10% FCS. After 4–5 days of incubation some 30% of these cells showed active synthesis of metachromatic extracellular matrix. Cells from skin, muscle and periosteum failed to show metachromatic matrix positive colonies to a comparable extent. The phenotypic expression of these cells was determined by analysis of collagen types. Eleven day old cultures were incubated in the presence of [3H]-proline plus β-aminopropionitrile and ascorbic acid and the collagen extracted analyzed by polyacrylamide electrophoresis of their intact chains or CNBr-derived peptides. The results show that anchorage independence is a requirement for calvaria cells to express type II collagen. Type I collagen was preferentially expressed in monolayer culture or when pre-attached to a substrate before being cultured in agarose. Type II collagen was the predominant collagen when cells were cultured in agarose. Further characterization of cell populations was achieved by isopycnic centrifugation in a percoll gradient. Cell fractions were tested for their collagen phenotype when cultured in agarose. Cells recovered from densities 1.04 g/ml or higher synthesized type II collagen, while cells with densities lower than 1.04 g/ml synthesized mainly type I collagen. Isopycnic centrifugation appears to be a novel method for separation of phenotypically different cells from a heterogeneous population in fetal calvaria. The high density cell fractions may represent a mixture of pre-chondrocytes as well as pluripotential cells.

Original languageEnglish (US)
Pages (from-to)40-48
Number of pages9
JournalMatrix
Volume9
Issue number1
DOIs
StatePublished - Jan 1 1989

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Skull
Collagen
Phenotype
Sepharose
Collagen Type II
Isopycnic Centrifugation
Cell Count
Collagen Type I
Aminopropionitrile
Periosteum
Deoxyribonucleases
Collagenases
Chondrocytes
Proline
Muscle Cells
Population
Ascorbic Acid
Extracellular Matrix
Electrophoresis
Cultured Cells

All Science Journal Classification (ASJC) codes

  • Rheumatology

Cite this

Cells Isolated From Fetal Rat Calvaria by Isopycnic Separation Express Different Collagen Phenotypes. / Villanueva, J. E.; Nishimoto, Satoru; Nimni, M. E.

In: Matrix, Vol. 9, No. 1, 01.01.1989, p. 40-48.

Research output: Contribution to journalArticle

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