Osteogenesis imperfecta (OI) is a heritable disorder ofconnective tissue associated with fractures, osteopenia, and short stature. 01 results from mutations affecting the proal or proa2 gene of type I collagen. We describe a strain of mice with a nonlethal recessively inherited mutation (oim) that results in phenotypic and biochemical features that simulate moderate to severe human 01. Although imperfect osteogenesis has been previously observed in bovine, feline, and murine species, none of these have duplicated both the biochemical and clinical findings associated with human OI (4-7). The Mov-13 mouse, which has a transcriptional block of the proal(I) collagen gene, has provided a potential model of human 01 type II (8-13). More recently, several transgenic variants of Mov-13 and normal mice have been created as useful models of mild 01 type I or lethal OI type II (tt, 14, 15).In this report we describe a naturally occurring mouse mutation that produces phenotypic and biochemical features similar to those seen in moderate to severe human OI. We have named this mutation osteogenesis imperfecta murine (oim). Homozygous oim mice have osteopenia, fractures, and progressive skeletal deformities. Our data indicate that these mice are deficient in proa2(I) collagen because of a G deletion at nucleotide 3983 of the Cola-2 gene. This mutation results in tissue accumulation of al(I) homotrimeric collagen in the extracellular matrix. Homozygous oim mice should permit the study of type I collagen pathophysiology in a manner not possible in humans. MATERIALS AND METHODSRadiographic and Microscopic Examination. Whole-body radiographs were taken in a Faxitron x-ray machine (34 keV for 1.5 min) using Kodak OM1 film. For light microscopy, excised femurs were fixed in neutral buffered Formalin for 24 hr, decalcified in 10% (wt/vol) EDTA in 0.1 M Tris'HCl buffer, pH 6.9, for 14 days at 4°C, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.Isolation and Culture of Dermal Fibroblasts. Dermis was obtained from the back of 3-to 5-day-old homozygous oim and wild-type pups. The skin was rinsed with iodine then 70o (vol/vol) ethanol, excised, and minced to 1-to 3-mm2 pieces.Explants were grown for 2 weeks in Dulbecco's modified Eagle's medium supplemented with 10%o fetal bovine serum, streptomycin at 100 jug/ml, penicillin at 100 units/ml, and amphotericin B at 0.25 jig/ml. Cultures were assayed in the second passage.Collagen and Procollagen Analysis in Vitro. Fibroblast cultures were grown several days past visual confluence in 10-cm2 dishes. The medium was then supplemented with 150 ,uM sodium ascorbate and 24 hr later the cultures were incubated for 30 min in Dulbecco's modified Eagle's medium plus 1% dialyzed fetal bovine serum, antibiotics, 100 ,uM each nonessential amino acids but no proline, and 150 ,uM sodium ascorbate (starve medium). De novo synthesized proteins were radiolabeled for 2 hr (short-label analysis) or 24 hr (steady-state analysis) with starve medium containing 20 ,uCi (1 Ci = 37 ...
Abstract. A newly defined chick calvariae osteoblast culture system that undergoes a temporal sequence of differentiation of the osteoblast phenotype with subsequent mineralization (Gerstenfeld, L. C., S. Chipman, J. Glowacki, and J. B. Lian. 1987. Dev. Biol. 122:49-60) has been examined for the regulation of collagen synthesis, ultrastructural organization of collagen fibrils, and extracellular matrix mineralization. Collagen gene expression, protein synthesis, processing, and accumulation were studied in this system over a 30-d period. Steady state mRNA levels forpro al(I) and pro ~t2 collagen and total collagen synthesis increased 1.2-and 1.8-fold, respectively, between days 3 and 12. Thereafter, total collagen synthesis decreased 10-fold while mRNA levels decreased 2.5-fold. In contrast to the decreasing protein synthesis after day 12, total accumulated collagen in the cell layers increased sixfold from day 12 to 30. Examination of the kinetics of procollagen processing demonstrated that there was a sixfold increase in the rate of procollagen conversion to ~t chains from days 3 to 30 and the newly synthesized collagen was more efficiently incorporated into the extracellular matrix at later culture times.The macrostructural assembly of collagen and its relationship to culture mineralization were also examined. High voltage electron microscopy demonstrated that culture cell layers were three to four cells thick. Each cell layer was associated with a layer of well developed collagen fibrils orthogonally arranged with respect to adjacent layers. Fibrils had distinct 64-70-nm periodicity typical of type I collagen. Electron opaque areas found principally associated with the deepest layers of the fibrils consisted of calcium and phosphorus determined by electron probe microanalysis and were identified by electron diffraction as a very poorly crystalline hydroxyapatite mineral phase.These data demonstrate for the first time that cultured osteoblasts are capable of assembling their collagen fibrils into a bone-specific macrostructure which mineralizes in a manner similar to that characterized in vivo. Further, this matrix maturation may influence the processing kinetics of the collagen molecule.T HE processes by which mineralization is initiated and regulated in bone and other vertebrate calcifying tissues are incompletely understood. It is known, however, that a prerequisite for mineralization is the synthesis and assembly of an extracellular matrix into which mineral may be deposited. Collagen type I has been shown to be the major extracellular matrix protein of bone. It comprises between 60-70% of its organic components and between 20-30% of its total dry mass (20). Physiologically, type I collagen provides the protein basis for the architecture of bone and the scaffold into which mineral is accumulated (5,20,41). The importance of collagen type I in maintaining structural integrity and proper mineralization of bone has been demonstrated for one form of inherited osteogenesis imperfecta. A specific frame shift m...
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