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Endocrinology Vol. 143, No. 5 1594-1601
Copyright © 2002 by The Endocrine Society

PTH-CALCITONIN-VITAMIN D-BONE

Osteoblastic Response to the Defective Matrix in the Osteogenesis Imperfecta Murine (oim) Mouse

I. Kalajzic, J. Terzic, Z. Rumboldt, K. Mack, A. Naprta, F. Ledgard, G. Gronowicz, S. H. Clark and D. W. Rowe

Department of Genetics and Developmental Biology (I.K., Z.R., K.M., S.H.C., D.W.R.), and Orthopedics (A.N., F.L., G.G.), University of Connecticut Health Center, Farmington, Connecticut 06030; and Department of Physiology (J.T.), University School of Medicine, Split 21000, Croatia

Address all correspondence and requests for reprints to: Dr. David Rowe, Department of Genetics and Developmental Biology, MC 3301 (E-2013), University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06030. E-mail: . rowe{at}neuron.uchc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This work examines the cellular pathophysiology associated with the weakened bone matrix found in a murine model of osteogenesis imperfecta murine (oim). Histomorphometric analysis of oim/oim bone showed significantly diminished bone mass, and the osteoblast and osteoclast histomorphometric parameters were increased in the oim/oim mice, compared with wild-type (+/+) mice. To assess osteoblast activity, a rat Col1a1 promoter linked to the chloramphenicol acetyltransferase reporter transgene was bred into the oim model. At 8 d and 1 month of age, no difference in transgene activity between oim and control mice was observed. However, at 3 months of age, chloramphenicol acetyl transferase activity was elevated in oim/oim;Tg/Tg, compared with +/+;Tg/Tg and oim/+;Tg/Tg. High levels of urinary pyridinoline crosslinks in the oim/oim;Tg/Tg mice were present at all ages, reflecting continuing high bone resorption. Our data portray a state of ineffective osteogenesis in which the mutant mouse never accumulates a normal quantity of bone matrix. However, it is only after the completion of the rapid growth phase that the high activity of the oim/oim osteoblast can compensate for the high rate of bone resorption. This relationship between bone formation and resorption may explain why the severity of osteogenesis imperfecta decreases after puberty is completed. The ability to quantify high bone turnover and advantages of using a transgene that reflects osteoblast lineage activity make this a useful model for studying interventions designed to improve the bone strength in osteogenesis imperfecta.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOGENESIS IMPERFECTA (OI) is a heterogeneous human disease of the skeleton characterized by mild to severe reduction in the quantity of bone matrix that leads to repeated fractures and bone deformity. There is a wide spectrum of clinical severity with the more severe forms (OI type II, III, and IV) resulting from mutations within the type I collagen helix, and many cases of the mild form (OI type I) result from underproduction of normal type I collagen (1, 2). Mutations in both {alpha}1 and {alpha}2 chains of type I collagen, which compose the heterotrimeric collagen molecule, are well documented (3, 4). However, little is known about the response of the osteoblast and osteoclast to a structurally compromised matrix in intact bone.

Because normal bone responds to loading or microfracture by increasing bone resorption and formation (5, 6), we hypothesized that osteoblast and osteoclast activity is affected by the weakened bone structure and initiates a cycle of bone remodeling in the attempt to form a stronger matrix. However, because the collagen is defective, the weakened matrix remains uncorrected. This situation would be reflected by increased osteoblast and osteoclast cellularity in OI bone, high rates of type I collagen synthesis, and elevated excretion of bone-derived collagen cross-links. Most histomorphometric studies in OI bone samples have shown evidence of osteopenic bones with increased bone cell number. A high bone turnover state, especially in severe forms of human OI, has been documented (7, 8).

Attempts to use a gene therapy approach in treatment of OI (9) and advances in pharmacological treatment (10, 11) underline the value of animal homologues of human disorders. In the case of OI, murine models with various levels of disease severity are available (12, 13). Osteogenesis imperfecta murine (oim) model resembles a severe nonlethal recessive form of human OI and provides an opportunity to explore how the bone cells responds to the environment of a weakened matrix in the intact animal (14). It results from a frameshift mutation in the C-terminal propeptide that precludes the pro{alpha}2(I) chain from incorporation into the heterotrimeric collagen molecule. Instead {alpha}1(I) homotrimers are formed, which interfere with the integrity and quantity of the osteoid that accumulates in bone. The homozygous mice are either born with fractures or develop them at an early age. The phenotypic, biochemical, and molecular findings virtually duplicate those seen in a patient with OI type III (15).

We used this murine model to characterize osteoblast and osteoclast activity in OI. To assess the activity of the endogenous Col1a1 gene, we took advantage of a type I collagen promoter (Col1a1) transgene (ColCAT 3.6) (16). This construct consists of 3.6-kb of the Col1a1 promoter ligated to a chloramphenicol acetyl transferase (CAT) reporter gene that has been studied in stably transfected bone cells and the bones of transgenic mice. In all cases, this transgene has been shown to reflect the activity of the endogenous Col1a1 gene in a variety of type I collagen-producing cells (17). The transgene was bred into the oim line, and the CAT activities were measured in +/+;Tg/Tg, oim/+;Tg/Tg, and oim/oim;Tg/Tg mice at different ages. Our results indicated that a state of high bone turnover exists in oim/oim;Tg/Tg mice. However, increased Col3.6 transgene activity associated with high bone formation became apparent only after the phase of rapid somatic growth was completed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Breeding of transgene and selection of genotype
The oim mutation is maintained in the B6C3Fe-a/a (C57BL/6JLe X C3HeB/FeJLe) hybrid background (14). The oim/oim mice were bred with transgenic mice +/+;Tg/Tg (C57BL10/NAW background) in which the CAT transgene is driven by 3.6 kb of ColCAT3.6 (17). The initial offspring oim/+;Tg/+ mice were intercrossed to generate oim/+;Tg/Tg to maintain the mutation in healthy carriers. Experimental mice were obtained from interbreeding the oim/+;Tg/Tg to generate oim/oim;Tg/Tg-affected mice, oim/+;Tg/Tg, or +/+;Tg/Tg mice. The oim/oim;Tg/Tg mice can be distinguished from wild-type by their phenotype (smaller size, lower weight, and presence of deformities owing to numerous fractures). To confirm the oim/oim genotype and to distinguish wild-type and heterozygous mice, DNA was extracted from tail samples and analyzed by a previously described PCR method (18).

Histological analysis of bone
Static histomorphometry was performed on tibiae that were fixed in 4% paraformaldehyde at 4 C, decalcified in 15% EDTA, dehydrated in progressive concentrations of ethanol, cleared in xylene, and embedded in paraffin. The embedded bones were longitudinally sectioned, stained for tartarate-resistant acid phosphatase (TRAP) (19) to visualize osteoclasts, and counterstained with hematoxylin. Histomorphometric analysis was performed in a blinded, nonbiased manner using a computerized image analysis system, BioQuant (R&M Biometrics, Nashville, TN).

To perform dynamic histomorphometry, animals received ip injections of 10 mg/kg of calcein at 12 d and 90 mg/kg of xylenol orange at 2 d before they were killed. The femurs were fixed in 70% ethanol at 4 C, dehydrated in progressive concentrations of ethanol, cleared in xylene, and embedded in methylmethacrylate. The embedded bones were longitudinally sectioned on Jung polycut microtome (Reichert-Jung, Heidelberg, Germany). Sections 5 µm thick were deplasticized and left unstained for evaluation by fluorescence microscopy. Additional sections were deplasticized and stained with modified Masson-Goldner Trichrome with Briebrich Scarlet (Sigma, St. Louis, MO) for analysis of osteoblast and osteoclast parameters. The measurements, terminology, and units used for both static and dynamic histomorphometry were based on the convention of standardized nomenclature (20).

Immunofluorescence
Humeri were fixed in 5% paraformaldehyde, 2% sucrose in 0.1 M sodium cacodylate buffer, pH 7.4, for 4 h at 4 C. Bones were decalcified in 15% EDTA, frozen in liquid nitrogen, stored for 24 h at -20 C, and sectioned on the cryostat. For CAT immunohistochemistry, slides were washed in gelatin and PBS and blocked with 3% normal goat serum for 10 min. Slides were washed in PBS and incubated with 1:50 dilution of a rabbit polyclonal antibody to CAT (5Prime-3Prime, Boulder, CO) for 90 min at room temperature. After a wash in PBS, the sample was exposed to a goat antirabbit CY3-labeled antibody (CY3, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a 1:800 dilution for 1 h. Slides were thoroughly washed in PBS, coverslipped with 2.5% n-propylgallate in 1:1 PBS/glycerol, and photographed.

Measurement of CAT activity
Animals were killed either at 8 d, 1 month, or 3 months. Calvaria and humeri were dissected, cartilage removed, and bone marrow expelled using PBS. The humeri were crushed, and the calvaria were left intact. The bones were placed in separate tubes containing 0.5 ml Tris-Triton extraction buffer (0.25 M Tris, 0.5% Triton X-100). Samples underwent three freeze-thaw cycles followed by incubation at 65 C for 15 min to inactivate endogenous deacetylases and centrifuged for 3 min to remove precipitated protein. The protein concentration of the soluble supernatant was determined using the BCA protein assay kit (Pierce Chemical Co., Rockford, IL) according to the manufacturer’s instructions. CAT activities were measured by a fluor diffusion assay and normalized to total protein content (21). The results were plotted against time, and a linear regression analysis was performed in which the CAT activity in each sample was defined as the slope of the linear regression line. Statistical analysis of the CAT assay data used ANOVA.

RNA isolation and analysis
Animals were killed and the epiphyseal portions of the long bones (tibiae and femurs) were cleaned of attached muscle and the marrow contents flushed using a 25-gauge needle. Samples were frozen in liquid nitrogen and crushed using a custom-fit pestle, suspended in 3 ml Trizol reagent (Life Technologies, Inc., Grand Island, NY) and homogenized with a 5-mm polytron probe (Brinkmann Instruments, Inc., Westbury, NY) for 30 sec. Total RNA was prepared from mouse tissues according to the manufacturer’s instructions. RNA was separated on a 2.2-M formaldehyde/1% agarose gel and transferred onto a nylon membrane (maximum-strength Nytran, Schleicher \|[amp ]\| Schuell, Inc., Keene, NH). Membranes were probed with a 700-bp XhoI-XbaI CAT fragment, a 900-bp PstI fragment of rat Col1a1 (p{alpha}1R2) (22), a 440-bp PstI/EcoRI mouse osteocalcin (OC) fragment (p923) (23), and a 1000-bp EcoRI mouse bone sialoprotein (BSP) fragment (24). Probes were radiolabeled by the random primer method using {alpha}-32P dCTP, 3000 Ci/mmol (NEN Life Science Products, Boston, MA), obtaining a probe-specific activity of approximately 1 x 109 cpm/µg. Filters were hybridized with 3 x 106 cpm/ml labeled probe at 42 C in 50% formamide, 5x sodium chloride, sodium phosphate/EDTA (SSPE) (1x SSPE = 0.149 M NaCl, 10 mM NaH2PO4, 1 mM EDTA, pH 7.4), 1.2x Denhardt’s and 0.5% SDS (25). Filters were washed once in 6x SSPE and 0.5% SDS for 10 min at room temperature, once in 0.1x SSPE and 0.1% SDS for 10 min at 37 C, and once in 0.1x SSPE and 0.1% SDS for 10 min at 65 C. Prior hybridization signals were removed by washing in 0.1x SSPE and 0.1% SDS for 20–30 min at 80 C.

Urinary excretion of deoxypyridinoline (DPD) cross-links
Urine was collected from 1-, 3-, and 5-month-old animals three times a day during a 2-d period and stored at 4 C until the adequate volume from all animals was collected. DPD excretion was determined using the PYRILINKS-D assay (Metra Biosystems, Mountain View, CA). Creatinine concentration in urine was measured and deoxypyridinoline cross-links was expressed relative to creatinine concentration.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Assessment of oim bone by histomorphometric analysis
Histological examination of tibiae from 3-month-old mice revealed a marked reduction in cortical and trabecular bone in the oim/oim;Tg/Tg mice, compared with the +/+;Tg/Tg and oim/+;Tg/Tg (Fig. 1Go, A–C). TRAP staining showed many osteoclasts on the endosteal and trabecular surfaces (Fig. 1DGo, osteoclasts indicated by arrows) in the oim/oim;Tg/Tg mice. Numerous osteoblasts were also found lining the periosteal and endosteal bone surfaces (data not shown). No major differences were observed between the wild-type and heterozygous mice (Fig. 1Go, A and B). Quantitative histomorphometry confirmed these observations. The total bone area per total tissue area and trabecular number were significantly reduced. and the cortical width was less in oim/oim;Tg/Tg animals, compared with oim/+;Tg/Tg and +/+;Tg/Tg mice (Fig. 2Go, A–C). The osteoclast number expressed per bone area as well as the percentage of bone surface covered with osteoblasts (Fig. 2Go, D and E) was increased. Thus, the static histomorphometric analysis suggested rapid bone turnover with increased osteoclast and osteoblast numbers.



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  		Figure 1. Histology of the oim/oim;Tg/Tg mice. Hematoxylin-stained sections of tibiae from 3-month-old mice show a presence of normal trabecular architecture in both +/+;Tg/Tg (A) and oim/+;Tg/Tg mice (B), and some trabecular bone was observed in oim/oim;Tg/Tg mice (C) (trabeculae are indicated by arrows). A high-power image of the trabecular bone in oim/oim;Tg/Tg tibia (D) shows a large number of TRAP-positive cells on the trabecular surface. Bar, 500 µm for A, B, and C. Bar, 100 µm for D.

 


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  		Figure 2. Histomorphometric analysis of tibiae from 3-month-old mice. The percentage of trabecular bone (TA/TTA,%, A), trabecular number (TbN, B), and cortical width (CtWi, C) are significantly lower in oim/oim;Tg/Tg mice, compared with wild-type and oim/+;Tg/Tg mice. Values represent mean ± SEM (n = 6). *, P < 0.01 as determined by one-way ANOVA and post hoc Tukey’s test. The proportion of the bone surface covered with osteoblasts (ObS/BS, %, D) is significantly higher in both oim/oim;Tg/Tg and oim/+;Tg/Tg, compared with +/+;Tg/Tg mice. The osteoclast number expressed per bone area (OcN/Bar, mm2) is increased in oim/oim;Tg/Tg mice, compared with oim/+;Tg/Tg and +/+;Tg/Tg (E) (P < 0.05). Bars show mean ± SEM (n = 3–6). *, P < 0.05, as determined by one-way ANOVA.

 
Dynamic histomorphometry of femurs from 3-month-old mice demonstrated a significant increase in percent of double-labeled surfaces in the oim/oim;Tg/Tg vs. +/+;Tg/Tg mice (31.4 ± 11.7% vs. 8.2 ± 5.7%, Fig. 3Go, A–C) and bone formation rate (BFR) (Fig. 3DGo). In contrast, the mineral apposition rate (1.2 ± 0.4 vs. 0.9 ± 0.3) was not significantly changed in the oim/oim;Tg/Tg vs. +/+;Tg/Tg mice. This suggested that despite the inherent defect in matrix production of the oim/oim;Tg/Tg bone cells, they are still able to deposit matrix, although it does not persist probably owing to rapid removal (see below). Thus, the standard methods for assessing bone formation and resorption indicate a state of high bone turnover in the oim/oim;Tg/Tg mice relative to +/+;Tg/Tg mice.



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  		Figure 3. Bone formation by dual-calcein and xylenol-orange labeling in vivo. Representative images of trabecular bone from femurs of +/+;Tg/Tg (A), oim/+;Tg/Tg (B), and oim/oim;Tg/Tg mice (C) show no difference in the distance between the two labels. However, a higher percentage of the bone surface was double labeled in the oim/oim;Tg/Tg mice, compared with +/+;Tg/Tg and oim/+;Tg/Tg, C vs. A, B. BFR (µ2/day/µ) was significantly higher in oim/oim;Tg/Tg mice (D). *, P < 0.05, as determined by one-way ANOVA.

 
Assessing activity of the osteoblasts by endogenous mRNA and Col1a1 transgene
Analysis of endogenous mRNA markers of osteoblastic activity was performed to determine whether the total effort by the cells within the osteoblast lineage would further explain the histomorphometric results. In addition, a Col1a1 promoter CAT reporter construct was bred into the oim line. This transgene is active in preosteoblasts and osteoblasts (26) and has the advantage of assessing the collagen synthetic activity in cells in a rapid and sensitive manner. Long bones (tibiae and femurs) from litters of CAT-positive mice were examined for expression of bone markers and CAT mRNA. Figure 4AGo shows a representative Northern analysis of the CAT, Col1a1, BSP, and OC hybridization signals from the three genotypes, and the relative levels of the CAT mRNA and bone markers are shown in Fig. 4BGo. A 3-fold increase in CAT mRNA, a 2-fold stronger expression of Col1a1, and 70% of increase in BSP mRNA were observed in oim/oim;Tg/Tg mice, compared with +/+;Tg/Tg mice. OC mRNA expression did not show a significant increase.



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  		Figure 4. Analysis of mRNA in the oim mice. RNA derived from long bones (tibiae and femurs) of 3-month-old mice was analyzed by Northern blot for the expression of the CAT transgene and expression of bone matrix proteins; OC, osteocalcin; COL, {alpha}1-type I collagen; BSP, bone sialoprotein (A). The18S rRNA cDNA was used to assess the loading differences. Densitometry of signals was obtained using a phosphoimager, and values were corrected for the corresponding intensity of the 18S band and expressed as relative values, compared with wild-type mice (B). Number of mice per experimental group is given in parentheses. For statistical analysis, one-way ANOVA test was used, *, P < 0.05.

 
A separate set of experiments was performed to measure CAT enzymatic activity in parallel with the CAT mRNA levels. As shown in Fig. 5AGo, a close relationship between CAT mRNA levels and CAT enzymatic activity was observed in humeri. Subsequently, the CAT enzymatic method was used to assess developmental changes in osteoblastic activity between the oim/oim;Tg/Tg and other genotypes of mice (Fig. 5Go, B and C). Small, statistically insignificant differences in CAT activity were detected between homozygous (oim/oim;Tg/Tg), heterozygous (oim/+;Tg/Tg), and wild-type (+/+;Tg/Tg) mice at 8 d of life in calvaria and humeri. At 1 month of age, oim/oim;Tg/Tg mice showed an 18% increase in calvarial CAT activity over +/+;Tg/Tg animals; however, this difference again was not statistically significant. A similar finding was observed in a sample of humeri confirming that there is no difference in CAT activity between the genotypes at 1 month of age. A fall in CAT activity occurred in both bones of all three groups at 3 months of age. This drop was more than 5-fold in +/+;Tg/Tg and oim/+;Tg/Tg mice. In the case of the oim/oim;Tg/Tg mice, the activity was decreased by less than 3-fold in calvaria and less than 2-fold in humeri. Thus, this represented a higher level of CAT activity in the oim/oim;Tg/Tg, compared with +/+;Tg/Tg mice at 3 months of age. This higher level was more than 2-fold for calvaria and humeri that is statistically significant in both cases (P < 0.01). The differences between oim/+;Tg/Tg and +/+;Tg/Tg mice were not significant. In 5- to 6-month-old mice, the activities in the oim/oim;Tg/Tg humeri were still about 2-fold higher than the +/+;Tg/Tg group (data not shown).



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  		Figure 5. Analysis of CAT enzymatic activity in oim mice. Comparison of CAT enzymatic activity and CAT transgene mRNA expression in humeri reveals a close correlation between these two methods of transgene activity assessment (A). Humeri (B) and calvariae (C) were analyzed for CAT enzymatic activity at the age of 8 d, 1 month, and 3 months (number of mice within each group is given inside a correspondent bar). No difference in CAT activity was detectable between the genotypes (oim/oim;Tg/Tg, oim/+;Tg/Tg, +/+;Tg/Tg) at 8 d or 1 month of age. However, at 3 months of age, significantly higher levels of CAT activity were observed in oim/oim;Tg/Tg mice (*, P < 0.01).

 
Because the Col3.6 promoter drives transgene expression in cells at the preosteoblastic and osteoblastic stages of differentiation, we performed a immunohistochemical staining for the CAT transgene in humeri to visualize the differences in the activity of the osteoblast lineage between oim/oim;Tg/Tg and +/+;Tg/Tg mice. Cells on the periosteal, endosteal, and trabecular bone surface were positive for CAT expression. More cellular regions expressing higher levels of the transgene were observed in the oim/oim;Tg/Tg mice, compared with +/+;Tg/Tg mice (Fig. 6Go, A and B). Negative control (Fig. 6CGo) shows only a background staining within the bone marrow cells.



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  		Figure 6. Localization of CAT transgene in the humeri of oim mice. Humeri of +/+;Tg/Tg (A) and oim/oim;Tg/Tg (B) transgenics were immunostained for CAT to localize the transgene activity. A stronger staining was detected in the endosteal (E) and periosteal (P) regions of oim/oim;Tg/Tg mice, compared with +/+;Tg/Tg. Background staining is shown in (C) with no primary antibody as a control. Bar, 100 µm.

 
Assessing bone turnover by excretion of DPD
To assess the osteoclastic activity, urinary excretion of DPD cross-links was measured and expressed relative to creatinine levels in urine. One-month-old oim/oim;Tg/Tg mice showed significantly increased levels of DPD excretion, compared with +/+;Tg/Tg mice. DPD levels decreased with age in all three genotypes, which complements observation of an overall decrease in previously described data on CAT activity (Fig. 7Go). However, the difference in DPD excretion was already present at 1 month and persisted also at the age of 3 and 5 months (Fig. 7Go).



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  		Figure 7. Excretion of deoxypyridinoline cross-links in urine. Total urinary excretion of deoxypyridinoline cross-links, a marker of bone degradation was measured by ELISA and expressed per urinary creatinine levels. A significant difference between the oim/oim;Tg/Tg and both +/+;Tg/Tg and oim/+;Tg/Tg mice was found at the age of 1, 3, and 5 months. No difference was observed between +/+;Tg/Tg and oim/+;Tg/Tg mice. Data represent the mean ± SD. *, P < 0.01, as determined by one-way ANOVA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The availability of murine homologues of human OI facilitates elucidation of the pathophysiological consequences of the OI matrix on bone as an integrated organ and on the osteoblast lineage within intact bone. The initial goal of this study was to assess the feasibility of using the type I collagen promoter transgene to reflect osteoblastic activity in a normal or OI matrix. Endogenous type I collagen mRNA has been shown to be the most sensitive marker of new bone formation in the rat (27), and our Northern analysis of BSP, OC, and Col1a1 in the oim/oim;Tg/Tg mice appear to confirm that observation.

The activity of the ColCAT3.6 transgene did parallel the content of Col1a1 mRNA in the three groups of mice as it has in a wide variety of other studies, both in cultured cells and transgenic mice (17, 28, 29, 30, 31, 32). In addition, the effect of somatic growth on collagen mRNA content in bone appears to be reflected by the activity of the ColCAT3.6 transgene. Although we did not make a direct comparison between Col1a1 mRNA and ColCAT3.6 activity in normal mouse bones during the first 3 months of life, the decrease in ColCAT3.6 activity that occurred by 13 wk of age in the long bones was similar to the decrease in Col1a1 mRNA levels in rats (33). Both species exhibit a decline in their rate of somatic growth by this age. Thus, the level of CAT activity is probably an accurate reflection of the total Col1a1 mRNA in bone and this affords a rapid, sensitive, and easy assay of the osteogenic activity within intact bone.

The ColCAT3.6 transgene proved to reliably reflect the bone forming activity in the three genotypes (oim/oim;Tg/Tg, oim/+;Tg/Tg, and +/+;Tg/Tg) by detecting higher levels of osteoblastic activity in the bones of the oim/oim;Tg/Tg relative to the +/+;Tg/Tg-type mice. Immunostaining for CAT showed a greater number of strongly positive cells lining the endosteal and periosteal surfaces. It appears that Col1a1 mRNA and CAT levels are a more sensitive marker of new bone formation than is BSP and OC, probably because they are produced by a lower number of cells within the osteoblast lineage. As the immunostaining shows, the entire lineage appears to be activated, further explaining why the ColCAT3.6 transgene is a sensitive and valid marker of cells in the osteoblast lineage recruited to new bone formation.

Based on our histomorphometric data, it would appear that the oim/oim mice resembles human type III OI. Oim/oim;Tg/Tg mice showed a reduced bone mass characterized by a high number of osteoblasts lining the bone surface and increased numbers of osteoclasts. Dynamic histomorphometry showed increased BFR but no differences in mineral apposition rate, compared with +/+;Tg/Tg mice, a similar finding that was reported in studies with children with OI type III (8). Furthermore, increased levels of bone degradation products were observed in oim/oim;Tg/Tg mice, a commonly observed result from patients with OI (34).

Significantly higher CAT levels in bones of the oim/oim;Tg/Tg animals were found. However, this increase was observed only after the rapid phase of growth was completed, suggesting that this stage of murine development utilizes the total synthetic potential of the oim/oim osteoblast lineage. Because the level of bone resorption in the oim/oim;Tg/Tg mice exceeds that of the +/+;Tg/Tg mice at all analyzed time points (1, 3, and 5 months), it is during the rapid phase of somatic growth that the deficit in bone mass is maximal because bone formation cannot increase further. This may explain why fracture frequency is high in young children with OI and falls after puberty (35). Instead of a specific effect of sex hormones on the osteoblast, the maturing effect of the sex hormones on the growth plate limits further linear growth. Once the skeleton is stabilized, the osteogenic activity of the oim/oim cell population can compensate for high bone turnover, leading to a net accumulation of bone matrix. The remodeling may select for a matrix with improved structural properties that can contribute better bone strength (36). Another explanation for the elevated CAT activity at 3 months of age may be continued rapid growth of the oim/oim;Tg/Tg mouse because at this age it is smaller than the oim/+;Tg/Tg and +/+;Tg/Tg mouse. However, at 6 months of age when the oim/oim;Tg/Tg mice are still smaller in size (in fact they never show catch up growth), differences in CAT activity and DPD excretion still persist. This demonstrates that the higher level of CAT in the oim/oim;Tg/Tg mice at 3 months is unlikely owing to a prolonged rapid growth rate but reflects a response to the continued elevation in the rate of bone resorption.

Thus, the physiological consequences of the production of a mutant collagen matrix within bone is a state of high bone turnover driven by the ability of the osteoclast to respond to a defective matrix and initiate a cycle of resorption and new bone formation (37). Because the replacement collagen is no better than what was just removed, the amount of new matrix that accumulates is severely limited. Success of bisphosphonates in reducing fracture frequency and relieving the symptoms of bone pain and diaphoresis relates to the interruption of this cycle (10, 38). This clinical observation suggests that the accumulation of bone matrix, although still defective, is better than no matrix for determining bone strength.

The consequences of the OI mutation at the cellular level are equally damaging. Cultured osteoblastic or fibroblastic cells from mouse and human show a lower proliferation rate and lower cell density than normal control (39). The cause of this observation is unknown but may be a consequence of the collagen-engorged rough endoplasmic reticulum that can be appreciated by electron microscopy studies (40). The level of Col1a1 mRNA is lower in OI osteoblast cultures from murine and human sources either because the number of cells acquiring the high rate of collagen synthesis characteristic of a differentiated osteoblast is reduced or the rate of synthesis per osteoblast is compromised (41). The production and secretion of type I collagen is also impaired with a resulting increase in intracellular degradation of the incompletely assembled collagen molecules (42). The molecular chaperone hsp47 appears to play a crucial role in sensing the malformed collagen molecules and targeting them for destruction (40, 43, 44, 45, 46). Despite these intrinsic proliferative and matrix production defects, histomorphometric analysis shows an increased number of active osteoblasts along the bone surface, increased surfaces undergoing new bone formation and a normal mineral apposition rate. An explanation for the discrepancy between the in vivo and in vitro osteogenic activity may be the effect of mechanical signals for increased bone formation that occur in vivo but are absent in cultured cells. Although the molecular defect does compromise osteoblast activity in both settings, in vivo factors continue to force the osteoblast lineage to produce differentiated osteoblasts and to maintain production of osteoid sufficient to maintain a normal mineral apposition rate. Although the high level of collagen mRNA and increased CAT activity within the oim/oim;Tg/Tg bone probably reflects more osteoblastic cells participating in bone formation, the rate of matrix production per osteoblast is probably low. Although this can not be accurately assessed in intact bone, recent development of a collagen promoter driving green fluorescent protein may provide method for determining the level of collagen mRNA within an individual cell either in cell culture or intact tissue (47).

Thus, OI induces a state of high bone turnover. Clinical studies designed to improve bone mass in OI with agents that stimulate osteoblast number or matrix production (48, 49) would appear to be counterproductive unless they were coupled with steps to reduce the high level of osteoclastic activity. The possibility that the high rate of bone turnover could eventually lead to premature senescence of the osteoblastic lineage may contribute to bone disease in individuals with a mature skeleton and may provide an additional rationale for the use of bisphosphonates to reduce the rate of bone turnover. The use of this promoter-transgene marked oim/oim mouse will be a valuable adjunct for testing these aspects of OI pathogenesis and evaluating pharmacologic and genetic interventions for improving the quantity and quality of bone matrix in OI.


   Footnotes
 
I.K. and J.T. contributed equally to this work.

This work was supported by a grant from the Public Health Service, NIH, NIAMS AR44545. I.K. was supported by a Michael Geisman Fellowship from the Osteogenesis Imperfecta Foundation.

Abbreviations: BFR, Bone formation rate; BSP, bone sialoprotein; CAT, chloramphenicol acetyl transferase; ColCAT 3.6, Col1a1 promoter transgene; Col1a1, type I collagen promoter; DPD, deoxypyridinoline; OC, osteocalcin; OI, osteogenesis imperfecta; oim, osteogenesis imperfecta murine; SSPE, sodium chloride, sodium phosphate/EDTA; TRAP, tartarate-resistant acid phosphatase.

Received September 25, 2001.

Accepted for publication January 23, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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