Osteoblastic Activity in the OIM Mouse

I. Kalajzic., J. Terzic., K. Mack., D. Visnjic., A. Naprta., G. Gronowicz., S.H. Clark., D. Rowe.
Dept.of Genetics and Developmental biology, Medicine and Orthopedics,
University of Conn. Health Center, Farmington CT06030

Osteogenesis imperfecta (OI) is heterogenous in its genetic expression and clinical manifestation, creating a problem for developing common treatment protocols. However, there are mouse models of OI that are genetically uniform and are phenotypically similar to human OI. These models are useful for studying the disease. The osteogenesis imperfecta murine (OIM) model resembles severe type III human OI. It results from a frameshift mutation in the C-terminal propeptide leading to accumulation of an a1(I) trimer collagen molecule. Our goal was to investigate the osteoblastic activity in this model of OI.

Static histomorphometry of oim/oim bone showed a significantly decreased cortical width, trabecular area and perimeter along with a significant reduction in total bone volume. Osteoclast number/bone surface was significantly higher in the oim/oim compared to wild type and heterozygous mice. Dynamic histomorphometry demonstrated a significant increase in double labeled surfaces/total bone surface, percent mineralized perimeter, mineral apposition rate and bone formation rate in oim/oim vs. +/+. The oim/+ differed from oim/oim only in bone formation rate which was intermediate between +/+ and oim/oim. This data suggested a high rate of bone turnover in the oim/oim mice.

To assess osteoblastic activity in OI bone, a transgene carrying the 3.6 kb promoter region of the rat Type I collagen gene (Col3.6) linked to the choramphenical acetyltransferase (CAT) reporter gene was bred into a murine model of OI (OIM/ColCAT3.6). Previous work has determined a strong correlation of this transgene activity with endogenous Col1A1 mRNA levels in cultured cells and intact bone. Humeri and calvariae were analyzed for CAT enzymatic activity and mRNA levels were measured in samples from marrow flushed femurs and tibias. Unexpectedly, no difference in CAT activity was detectable between the genotypes (oim/oim, oim/+, +/+) at 7 days or 1 month of age. However at 3 months of age when rapid growth diminishes, a major drop in CAT activity was found in all three genotypes, but the fall was less in oim/oim mice (50%) than in other two groups (80%). Expressed as a change from the +/+, both CAT activity and mRNA levels in oim/oim was 4-fold and oim/+ was 2-fold greater than in +/+ mice. RNA levels for type I collagen (Col1A1), bone sialoprotein (BSP) and osteocalcin (OC) were also 2-fold higher in oim/oim mice while oim/+ was indistinguishable from +/+. CAT immunofluorescence microscopy of humeri from the three genotypes revealed staining of osteoblasts at both the periosteal and endosteal surfaces. However the oim/oim mice had more staining of cortical osteocytes and osteoblasts. By 5 months of age, CAT mRNA and protein levels were still 2 fold higher in oim/oim while mRNA for ColA1, BSP and OC was not increased significantly. No increase was present in the OIM/+ mice.

To assess the osteoclastic activity in OI bone, urinary pyridinoline crosslinks (DPD) excretion was measured at 1, 3 and 5 months of age. DPD levels were increased approximately 2-fold in oim/oim while excretion was not significantly increased in oim/+ mice. These data correlated with the higher number of osteoclasts seen in oim/oim mice by histological methods and is consistent with a high turnover of the oim bone matrix.

Marrow stromal fibroblast (MSF) culture derived from these mice also demonstrated differences due to age and genotype. At 3 mo, equal numbers of alkaline phosphatase (AP+) colonies were obtained from each genotype, but at 5 mo colonies were slower to develop and fewer (AP+) colonies were seen in oim/oim than in +/+ or oim/+. The mRNA levels for ColA1, BSP and OC were also decreased in oim/oim MSF cultures derived from 5 month old mice. The MSF cultures from heterozygous mice varied in results, ranging between oim/oim and +/+. These findings suggest that with advancing age the regenerative capacity of OI bone cell is compromised perhaps secondary to remodeling activity. Furthermore, the defect in collagen production previously noted in bone and in fibroblast cells also affects other genes uniquely produced by the osteoblast.

Thus the physiological picture that emerges from this study is a bone cell with intrinsic difficulty in matrix production and cell proliferation that is required to make matrix at a greater rate than a normal bone cell. During periods of maximal linear growth the OI bone cell can not increase output further. It is only after growth has slowed, that bone has sufficient reserve to remodel the abnormal bone. This may explain why fractures and poor linear growth is prominent in severe OI and why fracture rates fall after puberty.

The use of osteoblast-specific transgene marker is a very sensitive tool for measuring osteoblast activity. It will be of therapeutic value in evaluating the response to an intervention designed to increase bone strength in OI.

Reference: Proceedings of the 7th International Conference on Osteogenesis Imperfecta. Montreal, Canada, 1999.