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Keywords:

  • mechanotransduction;
  • histomorphometry;
  • congenic;
  • loading;
  • adaptation

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The degree to which bone tissue responds to mechanical loading events is partially under genetic control. We assess the contribution of three genetic loci (QTLs linked to bone geometry and strength)—located on mouse Chrs. 1, 8, and 13—to mechanically stimulated bone formation, through in vivo skeletal loading of congenic strains. Bone size was not consistently associated with mechano-responsiveness, indicating that the genetic regulation of mechanotransduction is a complex process that involves a number of genes and is sex-specific.

Introduction: We showed previously that C57BL/6J (B6) mice are more responsive to mechanical stimulation than C3H/HeJ (C3H) mice and that B6 mice harboring a 40-Mb region of distal C3H Chromosome (Chr.) 4 are more responsive to mechanical stimulation than are fully B6 mice. Here, we assess the contribution of three more genetic loci—located on mouse Chrs. 1, 8, and 13—to mechanically stimulated bone formation.

Materials and Methods: Three congenic mouse strains were created in which a region of mouse Chr. 1 (∼64 cM; 150 Mb), Chr. 8 (∼45 cM; 86 Mb), or Chr. 13 (∼24 cM; 42 Mb) was moved from C3H stock to a B6 background through selective breeding over nine generations. The regions moved to the B6 background correspond to three of several quantitative trait loci (QTLs) identified for bone size and strength. The resulting congenic mice were 99% B6, with the remaining genomic DNA comprised of the Chr. 1, 8, or 13 QTLs of interest. Male and female congenic (1T, 8T, and 13B) and B6 control mice were subjected to in vivo loading of the right ulna at one of three different load magnitudes. A separate set of animals from each group had strain gauges applied at the ulnar midshaft to estimate strain at each loading level. Loading was conducted once per day for 3 days (60 cycles/d; 2 Hz). Fluorochrome labels were injected intraperitoneally 4 and 11 days after loading began. Using quantitative histomorphometry, bone formation rates were measured in loaded (right) and control (left) ulnas.

Results: All male congenic mice exhibited significantly reduced mechano-responsiveness compared with male B6 controls, but the same comparison among females yielded no difference from controls, with the exception of the 1T congenics, which showed increased responsiveness to loading. Among the congenic strains, smaller bone size was not consistently associated with reduced mechano-responsiveness.

Conclusions: Our results indicate that the genetic regulation of mechanotransduction is a complex process that involves a number of genes and is sex-specific. Our data might explain why different individuals can engage in similar exercise protocols yet experience different results in terms of bone mass accrual.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Adequate bone mass and strength are key factors in the prevention of osteoporotic fracture. The genetic makeup of an individual plays a major role in determining whether osteoporotic fracture will occur; roughly 50–70% of the variability in peak bone mass and BMD—two factors strongly linked to osteoporotic fracture risk—can be explained by heredity.(1–3)

Numerous genetic linkage studies have been conducted in mice and rats, aimed at identifying regions of the genome that are responsible for controlling bone mass and strength.(4–7) One of our earlier studies in this arena involved identifying quantitative trait loci (QTLs; chromosomal regions throughout the genome) that were associated with BMD in an F2 population derived from the intercross of low BMD (C57BL/6J [B6]) and high BMD (CAST/EiJ CAST]) mouse strains.(8) Several loci were revealed, located on chromosomes (Chrs.) 1, 5, 13, and 15, that were significantly associated with BMD. Subsequent studies, using the same QTL approach, have been conducted in a large population of F2 hybrids generated from a B6 and C3H/HeJ (C3H) cross. These studies have yielded a number of QTLs associated with BMD(9) and with bone geometric properties and strength.(10) Three QTLs were found (on Chrs. 4, 8, and 13) that exhibited a strong association with both femoral structure and strength.

Once a QTL is identified, a frequently used procedure for verifying its effect on the phenotype is to create a congenic strain. Congenic strains are created by moving a chromosomal region containing a QTL from one donor strain to a recipient strain through a series of backcrosses, making sure the QTL is present at each backcross generation. For example, we have generated congenic mouse strains for several of the bone size and strength QTLs identified earlier, in which the Chrs. 1, 8, and 13 bone size/strength QTLs (among others) were moved from C3H onto a pure B6 background.(11) These mice exhibit significant alterations in bone size and strength compared with the B6 controls, confirming our initial observations from QTL analysis.

A fundamental question in the genetic regulation of bone size and strength is to ask how the implicated genes exert their influence on bone size and strength. One attractive hypothesis holds that those genes affecting bone size, shape, and strength might modulate the efficiency with which bone tissue responds to mechanical stimulation. Greater responsiveness to mechanical events would generate larger, stronger bones despite similar mechanical loading environments (e.g., normal cage activity) during growth. We recently tested this hypothesis by measuring the mechano-responsiveness of the Chr. 4 congenic mouse (designated 4T), which has greater bone size and strength than the B6 controls, yet the only difference between these two mice is a ∼40-cM region of Chr. 4 transferred from C3H into the B6 mice. We observed increased responsiveness to mechanical stimulation in female 4T mice beyond that observed in the B6 controls.(12)

In this study, we extended our mechanotransduction experiments to include several other congenic mouse strains that harbor other bone strength QTLs from C3H, namely the Chrs. 8 (designated 8T) and 13 (designated 13B) strains. In addition, we also tested the Chr. 1 congenic mouse (designated 1T) because of a previously observed sex-specific effect on bone structure, where males exhibited reduced geometric properties of the femur, but this trend was reversed in females.(11) We used the rodent ulna loading model(13) to induce mechanical stimulation in male and female mice from each of the 1T, 8T, and 13B strains and measured the bone formation response through fluorochrome-labeled ulnar tissue sections. We hypothesized that the osteogenic response to mechanical stimulation would be proportional to the baseline bone size phenotype of each congenic. We found that bone size was proportional to responsiveness for some, but not all, of the congenic strains, indicating that the genetic regulation of bone mechano-responsiveness is complex.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Animals

Virgin male and female mice from each of the congenic strains B6.C3H-1T (1T), B6.C3H-8T (8T), B6.C3H-13B (13B), and C57BL/6J (B6) were produced at The Jackson Laboratory as described below. These mice were shipped to Indiana University at 13 wk of age. The animals were housed at the animal care facility for 5 wk (acclimation period) before the experiment began. NIH-31 (Harlan Teklad, Madison, WI, USA) mouse chow and water were provided ad libitum during the acclimation and loading periods. All procedures performed in the experiments were in accordance with the Institutional Animal Care and Use Committee guidelines.

Generation of congenic strains

Each of three congenic strains was developed by transfer of a specific chromosomal region from the C3H strain to the B6 strain background by repeated backcrossing. The regions were on Chrs. 1, 8, and 13, based on femoral polar moment of inertia QTLs described previously.(11) Transfer of the donor region was accomplished by first producing (B6 × C3H) N1F1 offspring and backcrossing a N1F1 mouse to a recipient B6 strain mouse to obtain N2F1 progeny. Tail tip DNA samples, extracted from female and male offspring by standard NaOH digestion methods, were genotyped to find carriers of the desired chromosomal region. These carriers were backcrossed to new B6 mice to generate N3F1 progeny for genotyping. This backcross followed by genotyping of progeny for carriers was conducted for nine cycles. At the N9F1 generation, their genomes were estimated to be 99.8% B6 in composition (except for the specific segment donated from the C3H strain; Fig. 1B). N9F1 mice were intercrossed to produce N9F2 progeny that were genotyped and known to be homozygous for C3H or B6 alleles within the QTL regions. Mice that were genotypically heterozygous (b6/c3) for the congenic regions were not retained for these studies. The subpopulation of B6 mice used to backcross the congenic lines were used as B6 controls for all experiments described. These mice showed no measurable differences from N9F2 mice that are homozygous for the B6 QTLs (unpublished data).

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Figure Figure 1. (A) QTLs for bone size and strength phenotypes were previously identified on mouse Chrs. 1, 8, and 13 using the microsatellites indicated. (B) The congenic strains (B6.C3H-1T, B6.C3H-8T, and B6.C3H-13B) were developed by transfer of each QTL from the C3H strain to the B6 strain background by repeated backcrossing. Transfer of the donor region was accomplished by first producing (B6 × C3H) N1F1 offspring and then backcrossing an N1F1 mouse to a recipient B6 strain mouse to obtain N2F1 progeny. C3H QTL carriers were backcrossed to new B6 mice to generate N3F1 progeny for genotyping. At the N9F1 generation, their genomes are estimated to be 99.4% B6 in composition (except for the specific segment from either Chr. 1, Chr. 8, or Chr. 13 donated from the C3H strain). N9F1 mice were intercrossed to produce N9F2 progeny. Mice that were genotypically heterozygous (b6/c3) for the congenic regions were not retained for these studies.

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Genetic analyses

Genomic DNA was prepared by digestion of 1-mm tail tips in 0.5 ml of 50 μM NaOH for 10 min at 95°C, and pH was adjusted to 8.0 with 0.05 mM Tris-HCl. Genotyping of individual mouse DNA was accomplished by PCR using oligonucleotide primer pairs from Research Genetics (Birmingham, AL, USA). These primer pairs amplify simple CA repeated sequences of anonymous genomic DNA that are of different length and through gel electrophoresis can uniquely discriminate between B6 and C3H genomes. The 1T QTL comprised a region of ∼64 cM spanning D1Mit282 to D1Mit406. The 8T QTL comprised a region of ∼45 cM, spanning D8Mit4 to D8Mit167. The 13B QTL comprised a region of ∼24 cM, spanning D13Mit147 to D13Mit77. Details of standard PCR reaction conditions have been described previously.(8) PCR products from B6, C3H, and their F1 hybrids were used as electrophoretic standards in every gel to identify the genotypes of N9F2 mice (i.e., homozygous B6 [b6/b6] or C3H [c3/c3] and heterozygous [b6/c3]).

In situ ulnar strain measurements and intact forearm mechanical properties

When the animals reached 18 wk of age, five mice from each strain were chosen at random, anesthetized, and killed by cervical dislocation. Immediately after death, the right forearm was minimally dissected to expose the medial surface of the midshaft ulna. A single element strain gauge (EA-06–015DJ-120; Measurements Group, Raleigh, NC, USA) was bonded to the exposed medial ulnar surface at midshaft. Once fitted with a strain gauge, the forearm was loaded in cyclic axial compression using an electromagnetic actuator with feedback control (Fig. 2). Using a 2-Hz haversine waveform, the forearms were loaded at 1.17, 1.40, 1.62, 1.85, and 2.07 N, during which peak-to-peak voltage output from the strain gauge was measured on a digital oscilloscope. Voltage measurements were converted to strain using a calibration factor derived from measured and calculated (using beam theory) strains collected from an aluminum cantilever.

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Figure Figure 2. Schematic diagram of the mouse ulna loading model, developed after a model originally proposed by Torrance et al.(13) The right distal forelimb is fixed between upper and lower aluminum cups (shown in hemisection), which are fixed to the loading platens and actuator. When force is applied to the upper platen, the pre-existing mediolateral curvature of the ulnar diaphysis becomes accentuated and translates most of the axial load into a bending moment, which is maximal near the midshaft. Animals were administered 60 load cycles/d (2 Hz) for 3 consecutive days and labeled with calcein green and alizarin complexone (red) 5 and 9 days (respectively) after the first load.

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In vivo ulnar loading

The remaining animals in each of the three congenic strains were divided randomly into three load magnitude groups for in vivo loading. Under isofluorane-induced anesthesia, the right forearm of each mouse was loaded 60 cycles/d for 3 consecutive days using a 2-Hz haversine waveform. The electromagnetic actuator used for strain gauge measurements was used for in vivo loading (Fig. 2). The left forearms were not loaded and served as an internal control for loading effects. All mice were allowed normal cage activity between loading bouts. Intraperitoneal injections of calcein (25 mg/kg body mass; Sigma Chemical, St Louis, MO, USA) and alizarin complexone (35 mg/kg body mass; Sigma Chemical) were administered 4 and 11 days after the first load day, respectively. All animals were killed 18 days after the first load day.

Tissue processing, histomorphometry, ulnar geometry, and strain calculations

The right and left ulna were removed, cleaned of soft tissue, measured for total length, fixed in 10% neutral buffered formalin for 48 h, dehydrated in graded alcohols, cleared in xylene, and embedded in methyl methacrylate (Aldrich Chemical, Milwaukee, WI, USA). Using a diamond-embedded wire saw (Histo-saw; Delaware Diamond Knives, Wilmington, DE, USA), transverse thick sections (∼70 μm) were removed from the ulnar midshaft. The wafers were ground to a final thickness of ∼20 μm and were mounted unstained on standard microscope slides.

One section per limb was read on a Nikon Optiphot fluorescence microscope (Nikon, Garden City, NJ, USA). The following primary data were collected from the periosteal surface at ×425 magnification using the Bioquant digitizing system (R&M Biometrics, Nashville, TN, USA): total perimeter (B.Pm); single label perimeter (sL.Pm); double label perimeter, measured along the first label (dL.Pm); and double label area (dL.Ar). From these primary data, the following derived quantities were calculated: mineralizing surface, (MS/BS = [½ sL.Pm + dL.Pm]/B.Pm × 100; %), mineral apposition rate (MAR = dL.Ar/dL.Pm/4 d; μm/d), and bone formation rate (BFR/BS = MAR × MS/BS × 3.65; μm3/μm2/yr).(14) Mineralizing surface (MS) reflects the percentage of the bone surface (BS) that was actively incorporating mineral into the matrix during the labeling period. Because mineralization normally occurs in the wake of new bone formation, MS/BS reveals the fraction of pre-existing bone surface that was engaged in new bone formation. The MAR reflects the rate at which new bone was deposited in the radial direction. The BFR is an overall measure of new bone formation, combining the percentage of surface actively forming new bone (MS/BS) with the radial rate of that formation (MAR). All of the derived quantities (measured from 2D tissue sections) were converted into 3D units using standard stereological techniques.(14) To control for individual differences in systemic factors, left ulna (nonloaded control) values were subtracted from right ulna values; this procedure results in a new set of relative (r) values for each variable (e.g., rBFR/BS).

The strain gauged ulna removed from the calibration animals were scanned in the transverse plane through the center of the strain gauge (still attached at midshaft) on a desktop μCT (μCT-20; Scanco Medical, Bassersdorf, Switzerland) using 7-μm voxel size. The midshaft tomograph slices were imported into Scion Image, wherein IMIN and the maximum section diameter in the IMIN plane (Se.Dm; mm) were calculated. From the geometric measurements and mechanical strain recordings, strain was estimated for the medial periosteal surface of the histological sections (initial bone surface) from animals loaded in vivo:

  • equation image

where ϵPM is the calculated strain on the medial periosteal surface resulting from load F, cPM is the distance from the IMIN neutral plane to the outer most fiber on the medial periosteal surface (2/3 Se.Dm),(15) and k is a constant that represents the ratio of moment arm to elastic modulus. k was calculated separately for each mouse strain and sex as the slope of a regression of section modulus (IMIN/cPM) on to mechanical strain.

Statistical methods

Differences between bone formation parameters in the loaded (right) and nonloaded (left) ulnas were tested using Student's t-test for paired variates. Dose responses to different load magnitudes and mechanical strain within mouse strains were tested for significance with least squares regression. Differences in slope and x-intercept (e.g., bone formation versus mechanical strain, bone formation versus peak load) between congenic and control lines were tested for significance by analysis of covariance (ANCOVA). For all tests, α = 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Within each sex, body mass was statistically similar among strains, with the exception of the 1T males and females, which were significantly heavier than sex-matched B6 controls (Table 1). Ulnar lengths also were similar among strains, although the 8T males exhibited significantly shorter (∼1.4% difference, p < 0.05) ulnas than B6 control males. Comparison of right (loaded) and left (nonloaded) ulnas within each sex and mouse strain revealed that loading had no significant effect on ulnar length for any of the congenic or control strains.

Table Table 1.. Summary of Body Mass, Ulnar Bone Lengths, and Midshaft Ulnar Geometric Properties in Nonleaded Ulna From Three Congenic Strains of Mice
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Midshaft geometric properties in the nonloaded (left) ulna showed considerable variation among congenic strains. Among males, the 1T and 8T ulna exhibited significantly lower IMIN values (10% and 12%, respectively), whereas the 13B males exhibited significantly greater (9%) geometric properties (Table 1). A different pattern emerged from the female ulnar midshafts; the 1T and 13B females exhibited significantly greater IMIN (11% each) compared with B6 females, whereas the 8T ulna yielded 8% lesser IMIN, which approached statistical significance (p = 0.085).

Mechanical loading of the right ulna induced a significant increase in relative bone formation rate (rBFR/BS), relative mineralizing surface (rMS/BS), and relative mineral apposition rate (rMAR) among all load groups, within both sexes in each congenic strain. Exceptions for this finding included rMS/BS in the low load 8T male group and rMAR in the low load 1T female and 8T male groups, which did not differ significantly from zero. Consistent with previous findings, the vast majority of load-induced bone formation was localized to the medial and lateral quadrants of the periosteal surface, as indicated by the strong calcein and alizarin labeling in those regions (Fig. 3). The microstructural organization of the newly formed bone tissue—assessed using polarized light microscopy—was consistently lamellar. Within both mouse strains, no differences in left (nonloaded) ulna MS/BS, MAR, or BFR/BS were detected among load magnitude groups (p > 0.60), indicating that the degree of bone formation stimulated in the loaded ulna had no detectable systemic effects on nonloaded bones.

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Figure Figure 3. At 20 weeks of age, the periosteal surface of the mouse ulna is largely quiescent (left). Axial loading activates the formerly quiescent medial and lateral periosteal surfaces in C57BL/6J (B6), B6.C3H-1T (1T), B6.C3H-8T (8T), and B6.C3H-13B (13B) strains, as shown by double labeling (calcein and alizarin). Note the lack of response in the caudal and cranial periosteal surfaces, which straddle the neutral bending axis and consequently are subjected to very low strains. Conversely, bone formation is greatly enhanced on the medial and lateral periosteal surfaces, where bending strains are greatest in the axial loading model. The sections shown are from the high load magnitude groups of male mice.

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The periosteal rBFR/BS response was dose-specific (in terms of peak strain magnitude) among all congenic and control strains tested (p < 0.01 for all strains/sexes). The 1T congenic strain exhibited a sex-specific response to mechanical stimulation (Fig. 4A): males exhibited a significant decrease in responsiveness compared with B6 controls, characterized by a greater osteogenic threshold (12% increase, p < 0.05), with no difference in the amount of new bone formed per unit strain (rBFR/BS versus strain slope). Conversely, 1T females exhibited an increase in responsiveness, characterized by a significant increase (46% increase, p < 0.05) in rBFR/BS per unit strain, with no detectable difference in the osteogenic threshold (i.e., the minimum strain needed to elicit an osteogenic response; Fig. 4B). Similar to the 1T males, the 8T and 13B males exhibited significant reductions in mechano-responsiveness compared with B6 males. The pattern of impaired response was also the same as for 1T males: increased osteogenic threshold (17–19% increase in threshold, p < 0.05) with no significant difference in rBFR/BS per unit strain (Figs. 4C and 4E). 8T and 13B females, on the other hand, showed no significant difference from B6 females in mechanical response. In both of these congenic strains, the slopes and intercepts were not significantly different from those of the B6 controls (Figs. 4D and 4F).

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Figure Figure 4. (A, C, and E) All three male congenic lines exhibited significantly impaired mechanical responsiveness, manifested as greater osteogenic thresholds (greater strain needed to induce mechanotransduction), compared with male B6 controls. (B) Among female congenic lines, 1T mice exhibited enhanced responsiveness to mechanical loading, as indicated by a greater increase in new bone formation per unit mechanical strain. The osteogenic threshold, however, was not different from B6. (D and F) The 8T and 13 B female congenic mice exhibited a similar bone formation response to loading as was expressed by the female B6 controls.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Our main objective in this study was to determine whether QTLs controlling bone size and strength regulate skeletal mechano-responsiveness. Specifically, we tested whether C3H genomic loci that impart enhanced size and strength in the B6 skeleton would increase the osteogenic response to loading, and conversely, whether C3H loci associated with reduced bone size and strength in the B6 skeleton would reduce the loading response. Our results indicated that, among male mice, the C3H QTLs were deleterious to mechanical responsiveness, regardless of which QTL was tested and regardless of the baseline bone size/strength phenotype in each congenic. Among female mice, two of the three C3H loci (8T and 13B), which correspond to reduced and enhanced bone size strength phenotypes, respectively, resulted in no change in responsiveness. However, the 1T QTL, which produced a larger and stronger female skeleton, also produced an enhanced response to loading. Taken together, our data from all three congenic strains in both sexes suggested that QTLs contributing to bone size and strength are not per se associated with the degree of responsiveness to mechanical stimulation (Table 2). These results are consistent with some previous studies. For example, DBA/2 mice exhibit a small, more mechanically frail skeleton than C3H mice, yet they exhibit significantly greater responsiveness to mechanical stimulation than C3H mice and roughly equivalent responsiveness to the larger-boned B6 mice.(15) In addition, the degree of bone loss induced by tail suspension appears to be similar in C3H and BALB mice, despite their pre-existing differences in bone size and shape.(16)

Table Table 2.. Summary of Bone Size and Mechanical Responsiveness Changes in Donor (C3H/HeJ) and Congenic Strains, Relative to the Control (C57BL/6) Strain
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Our findings in this study have similarities with a recent report of genetic variation in mechano-responsiveness in B6 and C3H mice.(17) In this study, a population of B6 × C3H F2 mice were exposed to in vivo tibial loading over a period of 2 wk, followed by bone mass and size measurements in the loaded and nonloaded tibias using pQCT. Subsequent interval mapping identified QTLs for mechano-responsiveness on Chrs. 1, 3, 8, 9, 17, and 18. In addition, interactions between QTLs were found on Chrs. 1, 3, 8, and 13. It is interesting that the observed QTLs on Chrs. 1, 8, and 13 overlap with the QTLs studied in this study. However, there are also inconsistencies between our findings and the study of Kesavan et al. For instance, they studied female mice and, in our hands, female mice from Chr. 8 and 13 congenic lines showed no significant differences in ulnar mechano-responsiveness (whereas mechano-responsiveness in these lines was significantly less in male mice). At the Chr. 1 QTL, Kesavan et al. observed decreased mechano-responsiveness in mice carrying C3H alleles, whereas we observed increased mechano-responsiveness in congenic Chr. 1 female mice. These inconsistencies in findings could be the result of differing study designs. For instance, Kesavan et al. used four-point bending of the tibia with pQCT measurements as endpoints, whereas we used axial loading of the ulna with histomorphometry measurements as endpoints. Nevertheless, the study from Kesavan et al. and our current observations strongly suggest that mechano-responsiveness of bone is regulated by several genetic loci.

In previous experiments, we tested the mechano-responsiveness of female 4T congenic mice using the same ulnar model and loading protocol described herein. The 4T females exhibited more advantageous geometric properties in their cortices and had improved mechanical properties. In that study, we found that by replacing a ∼40-cM portion of the female B6 Chr. 4 with the same region from C3H, we improved the responsiveness to loading beyond that observed in an already highly mechanosensitive mouse strain (B6). That observation provided fodder for the hypotheses we put forth in this communication (i.e., mechanotransduction genes are linked to, or correspond to, size/strength genes as a mechanism of action). Recall, however, that the improvement in mechano-responsiveness occurred among the 4T females despite the fact that the Chr. 4 QTL derived from a less mechanosensitive mouse strain (C3H female). Thus, the regulation of mechano-responsiveness, although clearly under some degree of genetic control (as we showed above), is a complex trait.

The sex-specific response in the congenic strains was particularly noteworthy. For example, the 1T males were less responsive than B6 male controls, but the 1T females were more responsive than B6 female controls. These trends correspond to the sex-specific effect of the 1T QTL on bone size and strength reported earlier.(11) Conversely, the 13B males exhibit enhanced geometric and biomechanical properties, yet their osteogenic response to loading was reduced. It would be interesting to test these strains of mice for sex-specific loading effects before puberty, when the sex hormones are largely absent. Doing so might provide some insight into whether the onset of sex hormone production and signaling is responsible for the observed sex-specific differences in mechanotransduction.

It should be noted that the QTLs we studied are large regions that contain up to 70 cM of sequence. The possibility of these QTLs housing multiple subloci, each with its own action on the responses we studied, can have potentially confounding effects. For example, we previously reported an analysis of eight sublines derived from the B6.C3H-1T congenic region, which yielded two BMD QTLs—one located between 36.9 and 49.7 cM and the other located between 73.2 and 100.0 cM.(18) These data indicate that more than one gene is responsible for the BMD effects of the 1T QTL. Whereas the same might be true of the mechano-responsiveness effects of the 1T QTL, our data suggested that at least one of the smaller loci affects mechanotransduction. The existence of multiple subloci on the other QTLs studied (e.g., Chrs. 8 and 13) are presently unknown, but analogous subloci could exist at those sites as well. Creation of congenic lines harboring only the subloci would better resolve the potential confounding effects of multiple subloci within a large QTL and provide greater focus in the identification of the individual gene/genes responsible for the phenotype captured by the QTL.

The mechanical strains we applied during exogenous loading are in or near the physiological range. Lee et al.(19) found that peak ulnar strains in live mice reached ∼1700 μϵ during locomotion and ∼2600 μϵ during a drop from a 20-cm height. The ulnar axial loading model that we used generates mediolateral bending about the IMIN plane at midshaft, and consequently, the medial and lateral periosteal surfaces are subjected to the greatest strains during loading. New bone formation in all of the mouse strains was localized predominantly to the medial and lateral periosteal surfaces, signifying that the lamellar bone response we are measuring is an adaptive response aimed at improving the geometric properties of the tissue, particularly in the high strain regions. Thus, all strains were capable of a clear adaptive response, but the degree of their responsiveness per unit mechanical strain was variable across QTLs and sex.

The ability of mechanical loading (through physical activity) to enhance bone gain, retard bone loss, and improve bone strength has prompted the Centers for Disease Control and the American College of Sports Medicine to recommend that all adults “accumulate 30 minutes or more of moderate intensity exercise on most, preferably all, days of the week.”(20) Although exercise is, in general, an effective means to stimulate bone formation and inhibit bone loss, a number of studies have shown that some individuals respond robustly to exercise, whereas others respond in a meager fashion. A similar phenomenon seems to exist in the murine skeleton—we showed previously that different strains of mice exhibit significantly different anabolic responses to the same mechanical stimulus. Dissecting out the regions of the genome responsible for mechano-responsiveness, using genome-wide screens followed by the creation of congenic lines, have led to significant and unexpected findings. However, the genes controlling bone strength/structure seem incapable of fully explaining the mechano-responsiveness of the tissue.

In summary, the collection of in vivo mechanotransduction experiments we describe in this communication suggests that certain regions of the mouse genome that exhibit significant linkage to bone size and strength are associated with the tissue's ability to respond to mechanical stimulation, whereas other regions of the genome that also have significant linkage to bone size and strength seem to be ineffective in modulating mechano-responsiveness. These data suggest that a large and complex set of genes control the degree to which bone tissue can respond to mechanical stimulation. Defining the specific genes (and their interactions) that modulate mechanotransduction efficiency could ultimately be used to improve bone mass and reduce the fracture burden on the population.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The authors thank Keith Condon and Diana Jacob for assistance with tissue processing. This work was supported by NIH Grants AR47879, AR43730, AR43618, and T32, AR07581.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References