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. Author manuscript; available in PMC: 2022 Dec 29.
Published in final edited form as: Bone. 2022 Aug 24;164:116524. doi: 10.1016/j.bone.2022.116524

Intramembranous Bone Regeneration in Diversity Outbred Mice is Heritable

Meghan M Moran 1,4, Frank C Ko 1,4, Larry D Mesner 2, Gina M Calabrese 2, Basel M Al-Barghouthi 2, Charles R Farber 2,3, D Rick Sumner 1,4
PMCID: PMC9798271  NIHMSID: NIHMS1858536  PMID: 36028119

Abstract

There are over one million cases of failed bone repair in the U.S. annually, resulting in substantial patient morbidity and societal costs. Multiple candidate genes affecting bone traits such as bone mineral density have been identified in human subjects and animal models using genome-wide association studies (GWAS). This approach for understanding the genetic factors affecting bone repair is impractical in human subjects but could be performed in a model organism if there is sufficient variability and heritability in the bone regeneration response. Diversity Outbred (DO) mice, which have significant genetic diversity and have been used to examine multiple intact bone traits, would be an excellent possibility. Thus, we sought to evaluate the phenotypic distribution of bone regeneration, sex effects and heritability of intramembranous bone regeneration on day 7 following femoral marrow ablation in 47 12-week old DO mice (23 males, 24 females). Compared to a previous study using 4 inbred mouse strains, we found similar levels of variability in the amount of regenerated bone (coefficient of variation of 86% v. 88%) with approximately the same degree of heritability (0.42 v. 0.49). There was a trend toward more bone regeneration in males than females. The amount of regenerated bone was either weakly or not correlated with bone mass at intact sites, suggesting that the genetic factors responsible for bone regeneration and intact bone phenotypes are at least partially independent. In conclusion, we demonstrate that DO mice exhibit variation and heritability of intramembranous bone regeneration that will be suitable for future GWAS.

Keywords: bone regeneration, genetics, heritability, DO mouse

Introduction

Bone is a remarkable tissue that normally repairs itself without scarring. However, in approximately 10% of fractures the natural repair mechanism fails [1]. With approximately 12–15 million fractures occurring per year in the U.S. [2], there are likely more than one million cases of failed bone repair each year, leading to substantial patient morbidity and societal costs. In addition to fracture healing, bone regeneration is integral to other situations such as fixation of orthopaedic and dental implants, distraction osteogenesis, spinal fusion, stress fracture repair and healing of bone following tumor resection. Not surprisingly, there has been considerable effort to develop strategies to enhance bone regeneration and to rescue or prevent failed repair, including pharmaceutical and mechanical approaches. The most successful current strategy is to apply exogenous bone morphogenetic protein [3], but concerns about safety have been raised [4], emphasizing the need for a deeper understanding of the biological processes controlling bone repair to aid in the development of novel treatments. We focus on intramembranous bone regeneration because of (i) its importance to implant fixation, both dental and orthopedic, and other situations where this form of bone regeneration plays a critical role [5–8] and (ii) the relative ease of creating a uniform injury with a simple phenotypic readout.

The genetic factors affecting bone repair are poorly understood despite the advancement of genome-wide association studies (GWAS) to understand intact skeletal traits such as bone mineral density (BMD) [9–11]. Unfortunately, the study of bone repair in patients is not readily amenable to GWAS because of the heterogeneous nature of the injuries and lack of a simple, universal readout. This barrier can be overcome by using a model organism with a well-defined injury mechanism coupled with a simple readout such as the mouse marrow ablation model for studying intramembranous bone regeneration [12]. In this model, the bone marrow content is mechanically disrupted, leading to the formation of intramembranous bone in the disturbed marrow cavity [12–14]. The amount of bone formed in response to the injury is easily quantified through micro-computed tomography. Unlike diaphyseal fracture healing models, which undergo both endochondral and intramembranous bone regeneration, bone marrow ablation surgery induces bone regeneration exclusively through the intramembranous pathway [15]. Following marrow ablation, several key biological events, such as inflammation, angiogenesis and growth factor signaling, occur in a characteristic temporal pattern [16, 17]. Previously, we showed in this model using different inbred mouse strains that intramembranous bone regeneration is a heritable trait [18]. Interestingly, the amount of regenerated bone was not correlated with the intact bone phenotypes, suggesting biological processes underlying bone density (i.e., osteoporosis) and bone regeneration are independent.

Knowing that intramembranous bone regeneration is a heritable trait, it should now be possible to identify novel causal genes using a GWAS approach. Such studies have been used in humans, mice and large animal models to identify multiple candidate genes for intact bone phenotypes [19–21]. In mouse models, the Diversity Outbred (DO) outcrossed population has become a valuable resource for these studies because of the high level of genetic variation compared to other reference populations [22]. The DO population is from a multiparental origin, created by “combining” eight genetically diverse inbred founders: A/J, C57BL/6J, 129S1/SvImJ, NZO/H1LtJ, NOD/LtJ, WSB/EiJ, PWK/PhJ and CAST/EiJ [23–25]. The resulting progeny were randomly bred to create DO mice. The DO population has at least 37.8 million single nucleotide polymorphisms (SNPs) and 6.9 million insertion/deletions/structural variants, a level of variation that is roughly three times greater than other existing mouse reference populations [22].

The purpose of the present study was to evaluate the phenotypic distribution of bone regeneration, sex effects and heritability (h), which are critical precursors to a GWAS. Specifically, we sought to determine if the response is heritable and if there is sufficient phenotypic variability in the regenerative response to justify performing a GWAS.

Materials and Methods

Experimental design and surgery:

Our IACUC approved study included 48 DO mice (24 female, 24 male, Jackson Laboratory, #9376). We followed ARRIVE guidelines in this study [26]. Mice were housed 5 mice per cage with separate cages for males and females. All mice were acclimatized to the RUMC animal facility for 7 days prior to surgery, fed standard chow, allowed drinking water ad libitum and maintained on a 12:12 light: dark cycle. Body weights were recorded prior to surgery and again at sacrifice. We performed bilateral femoral marrow ablation surgery, which is a well-accepted means of inducing intramembranous bone regeneration [18, 27, 28]. Briefly, using aseptic technique, a small longitudinal skin incision along the medial knee joint was made followed by a second incision deep to the first at the medial border of the patellar tendon. The patellar tendon was displaced laterally to expose the distal femoral condyles [12, 18]. Using a 23 g bevel edge needle, the intramedullary contents were manually reamed by entering through the intercondylar groove. Then, the marrow cavity was flushed using sterile saline. The entrance hole was sealed with bone wax and the wound was sutured closed. One male mouse died unexpectedly after surgery, leaving a final sample of 24 females and 23 males. At 7 days post-surgery, all mice were euthanized via CO2 inhalation. We chose 7 days because studies in the rat model [29] and mouse model [30] show that inflammation peaks around day 3, bone formation begins by day 5 and remodeling begins around day 10, suggesting that bone volume assessed at day 7 should represent an inherent regenerative response not confounded by subsequent remodeling. Tissues collected at sacrifice included spleen for isolating DNA and genotyping each mouse as well as bilateral hindlimbs (femur and tibia) and lumbar vertebra (L5). The left femurs, tibia and vertebrae were fixed in 10% neutral buffered formalin and changed to 70% EtOH after 3–5 days.

Genotyping and Bone Phenotyping:

The spleen was collected from all mice and frozen at −80°C for isolating DNA and genotyping each mouse (GigaMUGA array, N=~140K SNPs) [21, 31]. The left femur was scanned by micro-computed tomography (Scanco Medical μCT50) at 55 kVp, 200 μA, 600 ms integration with 7.4 μm voxels while submerged in storage solution (70% EtOH) to determine the bone regeneration phenotype. We followed ASBMR guidelines in performing μCT scanning and analysis [32]. The region of interest (ROI) was the marrow cavity between 30%−60% of the total bone length as measured from the distal end of the femur. This space is normally devoid of bone. Thus, bone found in this ROI represents bone formed following marrow ablation. The primary endpoint was the bone volume normalized to the volume of the tissue space (BV/TV)[18, 33]. We used a threshold of 200 mg HA/cm3 to isolate the bone. We also μCT scanned and analyzed intact bones (left tibia and L5 vertebra), using the same scan parameters mentioned above with thresholds of 350 mg HA/cm3 for trabecular bone and 450 mg HA/cm3 for cortical bone. The tibial trabecular ROI started 200 mm distal to the growth plate and extended to 30% of the tibia length and tibial cortical bone was assessed at the midshaft extending 10% of the tibia length. We determined BV/TV for the intact trabecular sites and total area, cortical area, medullary area and cortical thickness for the tibial cortical site. After μCT scanning, the left femurs were decalcified in 20% ethylenediaminetetraacetic for 2 weeks and paraffin embedded. Samples were sectioned at 6μm (Leica RM 2255) and stained using H&E as previously described [34].

Heritability and statistical analyses:

Heritability was calculated using a linear mixed model (“est_herit” function in R/qtl2)[35] for femoral length, body weight at surgery, body weight at sacrifice and all bone traits. We determined the mean and standard deviation of all traits so that we could calculate the coefficient of variation (standard deviation/mean), compared the male and female mice with a student’s t-test, used a paired sample t-test to compare the weight at surgery vs. the weight at sacrifice, and examined correlations among the bone traits using Pearson product moment correlations (SPSS v23). In all cases, we use p < 0.05 to determine statistical significance and report the exact p values.

Results

With the exception of the one mouse that died unexpectedly, all animals survived the surgery and exhibited normal locomotor and grooming behavior post-surgery and were included in the results. The weight at surgery (27.5g ± 6.0g) was slightly greater than the weight at sacrifice (26.6g ± 5.9g, p < 0.001).

The bone regeneration response was variable, as expected using a genetically diverse mouse population (Fig. 1). For the regeneration ROI, the mean and standard deviation for BV/TV were 7.2% and 6.2%, respectively, leading to a coefficient of variation of 86.1%. BV/TV in the regeneration ROI had a broad distribution, ranging from 0.3% to 26.3% (Fig. 1A). There tended to be more bone regeneration in males than females (p = 0.089, Fig. 1B). Mice with little regenerated bone (“low response mice”) had some bone present in the distal portion of the ROI, but no bone present in the proximal portion of the ROI (Fig. 1C). In these mice, the proximal ROI was dominated by hematopoietic cells and evidence of hematoma. Mice that had large amounts of regenerated bone (“high response mice”) had bone more evenly distributed throughout the ROI, with slightly less bone at the proximal third of the ROI (Fig. 1C). Histologically, the proximal ROI in these mice was dominated by unmineralized matrix and fibroblastic-like cells (Fig. 1D).

Fig. 1-.

Fig. 1-

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Bone regeneration at 7 days post-ablation in DO mice. (A) frequency distribution of BV/TV in the ROI outlined in (C) with fitted density curves. (B) box and whisker plot showing a weak trend toward a sex effect. (C) μCT images showing the drill hole used to access the medullary space to perform the marrow ablation with the ROI (red box) for assessing BV/TV (scale bar = 2 mm), 3D renderings of the ROI and corresponding histology (H&E of decalcified specimens, scale bars = 50 μm).

At the intact sites, all measures were sexually dimorphic (Table 1). BV/TV at the regeneration ROI was not correlated with BV/TV of L5 (r = 0.210, p = 0.156, Fig. 2A). However, BV/TV at the regeneration ROI was weakly to moderately correlated with the intact tibial phenotypes, including tibial length (r = 0.321, p = 0.028), cortical area (r = 0.342, p = 0.019), total area (r = 0.324, p = 0.026), and cortical thickness (r = 0.298, p = 0.042), but was not correlated with medullary area (r = 0.252, p = 0.88) (Fig. 2B–3E).

Table 1-.

Mean and standard deviations for intact skeletal traits shown as a function of sex. P-values are from student t-tests (Female, n = 24, male, n = 23). Bone volume/total volume (BV/TV) and the bone mineral density (BMD), which is an estimate of the apparent density, were determined using the same regions of interest.

Sex Mean Standard Deviation p-value
Tibial Length (mm) Female 16.98 0.59 0.003
Male 17.52 0.59
Tibial BV/TV Female 4.1% 2.9% < 0.001
Male 8.8% 3.8%
Tibial BMD (mg HA/cc) Female 35.14 27.68 < 0.001
Male 75.01 34.05
Tibial Cortical Area (mm2) Female 0.566 0.071 < 0.001
Male 0.805 0.070
Tibial Total Area (mm2) Female 0.823 0.121 < 0.001
Male 1.183 0.120
Tibial Medullary Area (mm2) Female 0.256 0.067 < 0.001
Male 0.378 0.070
Tibial Cortical Thickness (mm) Female 0.23 0.02 < 0.001
Male 0.27 0.02
L5 BV/TV Female 14.0% 5.7% 0.002
Male 19.4% 5.4%
L5 BMD (mg HA/cc) Female 114.56 47.70 0.001
Male 159.517 41.42
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Figure 2.

Figure 2.

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Correlations between regenerated BV/TV and intact tibial skeletal traits. Femoral regenerated BV/TV correlated with intact (A) L5 BV/TV, (B) tibial length, (C) tibial cortical area, (D)tibial total area and (E) tibial cortical thickness.

Heritability of the regenerated BV/TV was moderate (h = 0.418, Table 2). Heritability of intact bone traits of the tibia were also mostly moderate (ranging from h = 0.408 to 0.435) while the heritability of BV/TV for L5 was low (h = 0.131). Weight at surgery had low heritability (h = 0.134), but femoral length (h = 0.780) had relatively high heritability.

Table 2.

Heritability of Regenerated and Intact Bone Traits.

Regenerated Bone Traits h
Femur BV/TV 0.418
Intact Bone Traits h
Tibial BV/TV 0.408
Tibial Total Cortical Area 0.411
Tibial Cortical Thickness 0.435
Lumbar Vertebra (L5) BV/TV 0.131
Descriptives h
Femoral Length 0.780
Weight at Surgery 0.134
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Discussion

Heritability of bone regeneration was 0.42 in DO mice when characterized as BV/TV, which is similar to our previous finding of 0.49 for this trait as determined in an experiment involving 4 inbred mouse strains[18]. The coefficient of variation for regenerated BV/TV in DO mice was 86.1%, which is considerably higher than our previously reported values of ~8% to 57% within inbred mouse strains[18], although the overall coefficient of variation across the 4 strains was 88%. We also found either no or only modest correlations between the amount of regenerated bone and intact bone phenotypes, again consistent with our previous study in inbred strains[18], suggesting that the genetic factors responsible for bone regeneration and adult bone phenotypes are at least partially independent. Thus, the present study showed that intramembranous bone regeneration is heritable in the DO, suggesting this population is suitable for use in a future GWAS.

In our previous study of intramembranous bone regeneration in the marrow ablation model, we used inbred mouse strains and showed that the bone regeneration BV/TV had a ~3.5fold range in mean values (4.2% to 15.2%), and an overall mean of 6.8% and standard deviation of 6.0% with a range in individual values varying from less than 1% to 17%[18]. In the present study, we found an overall BV/TV mean of 7.2% and standard deviation of 6.2%, with individual values ranging from less than 1% to 26.3%. We hypothesize that the increased phenotypic variation is a function of increased genetic variation in the DO compared to panels of classical (non-wild-derived) inbred strains. Interestingly, of the four inbred mouse strains we previously surveyed, only C57BL/6 is a DO founder strain.

The primary limitation of the present study was that a small number of mice were used for assessing phenotypic variability and estimating heritability for such a genetically diverse population. Other limitations include that the variability and heritability were only assessed at a single age (10 weeks) and a single timepoint (7 days post-ablation). These limitations do not lesson the importance of the findings with respect to the purpose of the study which was to determine if the regeneration trait was heritable and had sufficient heritability to justify a future GWAS. Based on the data presented herein, it is likely a future GWAS would identify loci influencing intramembranous bone regeneration after marrow ablation. Previous power calculations using other complex bone phenotypes suggest we would have 80% power to detect a single biallelic QTL explaining 4–5% of the trait variance using 1000 DO mice[21,36]. These data are consistent with empirical data from a previous GWAS of complex bone traits in the DO in which we identified 28 genome-wide significant QTL using data from 619 mice[21].

We guarded against inadvertently sampling from a narrow segment of the population by using only one sibling per litter. Even with this small sample, single age and single timepoint, we discovered that there was at least as much variability as observed in a previous study comparing 4 inbred strains, as might be expected since the DO has on the order of 8–10 times more genetic variation than the 4 inbred strains previously studied. In addition, the heritability estimate was consistent with the previous estimate for this trait, supporting the conclusion that the DO population will be suitable for a future GWAS. Thus, the DO population is expected to be useful in identifying the polygenic basis of intramembranous bone regeneration just as it has already proven useful for other complex skeletal traits [37].

Bone regeneration, as is common for most complex traits, is likely regulated by intricate gene networks. In recent years, several approaches to reconstruct molecular networks have been developed and used to identify genes that play central roles in bone regulatory processes[17, 29]. In the present study, we sought to assess the suitability of DO mice for a future GWAS because this population has already been used to identify quantitative trait loci for adult (intact) bone phenotypes[38–40]. The present study indicates that using a GWAS approach in the DO model should prove fruitful for improving the understanding of intramembranous bone regeneration.

Acknowledgements

Research reported in this paper was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award numbers R01AR079179, R01AR077992, R01AR068345, and K01AR077679. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors would like to acknowledge other funding sources (Cohn Research Fellowship, FCK) and the Rush MicroCT/Histology Core.

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