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Emerging evidence suggests that genetic components contribute significantly to cartilage degeneration in osteoarthritis pathophysiology, but little information is available on the genetics of cartilage regeneration. Therefore, this study was undertaken to investigate cartilage regeneration in genetic murine models using common inbred strains and a set of recombinant inbred (RI) lines generated from LG/J (healer of ear wounds) and SM/J (nonhealer) inbred mouse strains.
An acute full-thickness cartilage injury was introduced in the trochlear groove of 8-week-old mice (n = 265) through microsurgery. Mouse knee joints were sagittally sectioned and stained with toluidine blue to evaluate regeneration. For the ear wound phenotype, a bilateral 2-mm through-and-through puncture was created in 6-week-old mice (n = 229), and healing outcomes were measured after 30 days. Broad-sense heritability and genetic correlations were calculated for both phenotypes.
Time-course analysis of the RI mouse lines showed no significant regeneration until 16 weeks after surgery; at that time, the strains could be segregated into 3 categories: good, intermediate, and poor healers. Analysis of heritability (H2) showed that both cartilage regeneration (H2 = 26%; P = 0.006) and ear wound closure (H2 = 53%; P < 0.00001) were significantly heritable. The genetic correlations between the two healing phenotypes for common inbred mouse strains (r = 0.92) and RI mouse lines (r = 0.86) were found to be extremely high.
Our findings indicate that articular cartilage regeneration in mice is heritable, the differences between the mouse lines are due to genetic differences, and a strong genetic correlation between the two phenotypes exists, indicating that they plausibly share a common genetic basis. We therefore surmise that LG/J by SM/J intercross mice can be used to dissect the genetic basis of variation in cartilage regeneration.
Tissue regeneration is a property generally attributed to organisms other than mammals. For example, hydra and planaria can regenerate their entire bodies from a small fraction of their tissue (1, 2). Similarly, vertebrates including newts and salamanders can regenerate limbs or other structures after amputation (3, 4). In contrast, the regenerative capacity in mammals is extremely limited (5) and is generally confined to shedding and regrowth of antlers in deer (6) and moose (7), and ear wound closure in rabbits (8). Despite this limited regeneration in adult mammals, supporting evidence suggests that certain mouse strains possess unusual abilities to regenerate/repair tissue even into adulthood. For instance, MRL/MpJ (9–12) and LG/J (11, 13) mouse strains display closure of 2-mm through-and-through ear holes within 4 weeks.
Articular cartilage is generally thought to have limited regeneration capacity, but there have been sporadic reports of full-thickness articular cartilage healing in rats (14), rabbits (15), dogs (16), and horses (17). Spontaneous regeneration of cartilage is possible only when the defect penetrates through the cartilage extending through the subchondral bone to the bone cavity to presumably access mesenchymal stem cells and recruit them to the site of damage (18, 19). In this case, the newly formed tissue is typically fibrocartilage, which is mechanically weak and degenerates over time (15, 20). In partial-thickness defects, no effective repair response occurs (10, 21), and attempts to restore/repair cartilage injuries have met with unpredictable and varied outcomes (22, 23).
Lack of self-regeneration in articular cartilage, mainly because of its intrinsic avascularity, contributes to cartilage degeneration, predisposing an individual to the development of osteoarthritis (OA), leading to prosthetic joint replacement and disability (22–24). While it is well appreciated that OA is widespread, >50% of individuals are protected against posttraumatic OA despite having cartilage damage (25). The exact cause of this remains unknown but can be attributed to a greater intrinsic ability to regenerate cartilage (26). Our long-term goal is to understand why cartilage can be repaired or regenerated in some people while insult or aging results in degeneration in others.
Recently, two mouse strains, MRL/MpJ and DBA/1, were reported to show regeneration of full-thickness articular cartilage defects in contrast to a failure of healing in age-matched C57BL/6 mice. The regenerated cartilage is hyaline-like in nature and has abundant chondrocytes, an extracellular matrix rich in proteoglycan, persistent type II and type VI collagen neodeposition, and cell proliferation within the repair tissue (10, 21). MRL/MpJ and LG/J, two closely related mouse strains that share 75% of their genomes identical by descent (2), display complete healing of ear wounds, including ear cartilage (9, 13, 27). This raises the possibility that, similar to MRL/MpJ mice, articular cartilage may also heal in LG/J mice. To investigate this possibility, we examined the extent of cartilage regeneration in a set of common inbred mouse strains, including both healers and nonhealers, and in a set of recombinant inbred (RI) lines formed from the intercross of the LG/J (healer) and SM/J (nonhealer) inbred mouse strains. The conceptual starting point for RI lines is that any differential phenotype will be attributed to a restricted set of genes according to the way they have been inherited from parental strains. It is estimated that 30–75% of the variation in OA is due to genetic differences (28, 29). While OA in humans can be studied using cartilage from donors, usually at the time of total joint replacement, it is impossible to directly investigate cartilage repair in humans. Therefore, we took the approach of investigating cartilage regeneration in genetic mouse models.
In this study, we aimed to investigate the heritability of articular cartilage regeneration in common inbred mouse strains and RI mouse lines and to determine the correlation between ear wound healing and articular cartilage regeneration. We found that cartilage regeneration is indeed a heritable trait in the LG/J by SM/J intercross mice, so that this intercross can be used to dissect the genetic basis of variation in articular cartilage regeneration. Furthermore, a strong genetic correlation between the ability to heal ear wounds and the ability to regenerate articular cartilage suggests a common underlying mechanism.
MATERIALS AND METHODS
All procedures were approved by the Animal Studies Committee at Washington University in St. Louis. MRL/MpJ, DBA/1J, DBA/2J, C57BL/6J, LG/J, and SM/J mice were obtained from The Jackson Laboratory. LGXSM RI lines were generated from the F2 intercross of LG/J females with SM/J males. Detailed information on and the history of the RI lines were previously presented (30). We used 9 RI mouse lines, namely, LGXSM-4, LGXSM-5, LGXSM-6, LGXSM-18, LGXSM-19, LGXSM-33, LGXSM-35, LGXSM-46, and LGXSM-48 (Table 1).
Table 1. Distribution of mice according to strain, time point, and phenotype
Time point of assessment of articular cartilage, weeks after surgery
Values are the least squares mean ± SEM articular cartilage score and filled ear wound diameter at the indicated time points.
3.62 ± 0.63
3.43 ± 0.73
2.82 ± 0.69
5.70 ± 0.52
2.54 ± 0.91
3.65 ± 0.73
2.80 ± 0.67
3.26 ± 0.84
1.99 ± 0.36
0.97 ± 0.09
3.70 ± 0.40
0.96 ± 0.10
4.02 ± 1.08
1.24 ± 0.11
3.16 ± 0.52
1.23 ± 0.13
5.71 ± 0.77
1.43 ± 0.08
2.30 ± 0.43
0.89 ± 0.11
2.67 ± 0.40
1.29 ± 0.10
2.84 ± 0.42
0.49 ± 0.10
2.66 ± 1.65
0.86 ± 0.17
2.93 ± 0.36
1.05 ± 0.09
5.45 ± 1.25
1.20 ± 0.13
2.76 ± 0.38
1.42 ± 0.09
2.60 ± 0.40
0.86 ± 0.10
3.34 ± 0.49
1.68 ± 0.12
9.03 ± 1.39
1.18 ± 0.14
2.81 ± 0.35
0.53 ± 0.09
2.23 ± 1.25
0.51 ± 0.13
5.29 ± 0.77
1.19 ± 0.07
5.79 ± 0.95
1.51 ± 0.08
2.22 ± 1.27
0.48 ± 0.11
2.69 ± 1.17
0.54 ± 0.09
4.28 ± 1.42
0.43 ± 0.07
2.27 ± 0.88
0.54 ± 0.07
2.62 ± 0.78
0.55 ± 0.06
Full-thickness cartilage injury (phenotyping).
Bilateral full-thickness articular cartilage defects were created in the knee joints of both hind limbs of 8-week-old mice (n = 265) through microsurgery (10). Briefly, mice were anesthetized by intraperitoneal injection (0.5–1.0 ml/kg body weight) of rodent cocktail comprising ketamine (100 mg/ml), xylazine (20 mg/ml), and acepromazine (10 mg/ml). A small (0.5–1.0 cm) medial parapatellar skin incision was made, the joint capsule was opened, and the patella was luxated laterally to expose the trochlear groove articulating surface. A full-thickness lesion was made by creating a circular defect in the cartilage with a sterile 27-gauge, 0.5-inch needle, using a circular motion until the subchondral bone was reached (Figure 1A). Penetration of the subchondral bone was confirmed by the appearance of a blood droplet following removal of the needle. The joint capsule was closed with 6-0 absorbable polypropylene suture in a simple continuous pattern. Subcutaneous tissue was apposed with a single mattress suture, and VetClose skin glue was applied on the skin to bridge over the closed edges. The mice were fully weight bearing within 2–3 hours of surgery and showed no signs of lameness or systemic effects for the duration of the experiment.
Tissue harvesting and decalcification.
Mice were killed at the indicated time points (Table 1) using CO2 chambers. Both knee joints were dissected, fixed in 10% neutral buffered formalin, and decalcified using 10% formic acid in 5% formaldehyde solution for 48 hours on a rocking platform at 4°C. Following 2 washes with water, the mouse joints were incubated in 0.01M EDTA in phosphate buffered saline (PBS) for 4–6 hours at room temperature. Finally, the samples were washed 4 times with water for 15 minutes each time and subjected to a series of washes with increasing concentrations of ethanol for 30 minutes each time. Samples were paraffin embedded, mounted in paraffin blocks, and sagittally sectioned at 5μ intervals extending through the entire trochlear groove using standard methods. The sections were mounted on polylysine-coated slides (Fisher Scientific) before staining.
Toluidine blue staining.
After dewaxing, every third slide (at 15μ intervals) was subjected to toluidine blue staining. Briefly, slides were immersed for 5 minutes in 0.04% toluidine blue solution prepared in 0.1M sodium acetate (pH 4.0). Then the slides were washed 3 times with tap water, twice with absolute ethanol, and finally twice with xylene before being coverslipped.
Microscopy and histologic scoring.
Stained sections were viewed using 4× and 10× objectives on a brightfield microscope (Eclipse E800; Nikon), and images were captured by a mounted camera (2000R Fast1394; Retiga) and QCapturePro software (QImaging). Three observers (MFR, SH, and EEJ) who were blinded with regard to sample identity each scored 3 selected sections. Specimens were graded by accepted methodology (10) with slight modifications and were assigned a score ranging from 0 (no healing) to 14 (full healing) (Figure 1B). The defect site was either easily recognizable by itself or was identified by a well-defined penetration mark of the needle through the growth plate, which served as a persistent landmark to locate the defect.
Immunofluorescence staining for collagens.
Paraffin sections were baked at 60°C for 2–4 hours followed by deparaffinization in xylene twice for 5 minutes each time. Slides were then rehydrated in descending concentrations of ethanol with final washes with distilled water and PBS. For antigen unmasking, the slides were digested with 1% hyaluronidase in PBS for 30 minutes at 37°C in a customized humidified chamber. After washing with PBS, slides were blocked in 10% normal goat serum in PBS for 1 hour at room temperature. After draining off the blocking buffer, slides were incubated with rabbit polyclonal antibody to type I collagen (ab34710; Abcam) or with antibody IIF (which recognizes the triple- helical domain of type II collagen protein) (31) at 1:200 dilutions in 1.5% goat serum in PBS and incubated at 4°C overnight followed by 3 washes with PBS. The secondary antibodies used were goat anti-rabbit Alexa Fluor 488 (Invitrogen) and goat anti-rat Alexa Fluor 546 (Invitrogen), respectively, at dilutions of 1:250 in 1.5% goat serum in PBS for 30 minutes at room temperature. Finally, the slides were rinsed with distilled water before being sealed with a water-based mount. Sections were viewed using a 4× objective on a fluorescence microscope (Eclipse E800), and images were captured as described above.
Ear wound punch (phenotyping).
A through-and-through hole 2 mm in diameter was created bilaterally in the center of the cartilaginous part of the external ear of 6-week-old mice (n = 229) using a metal ear punch (Fisher Scientific) as previously described (32). The holes were measured 30 days after wounding by using a grid-etched reticle, and the healing area was calculated in millimeters by deducting the diameter of the residual hole from 2 mm.
Postsurgery time course.
All statistical analyses were performed using Systat 12 software. We first examined a postsurgery time series for LGXSM-6 and LGXSM-33 mice, since these two strains have been shown to vary substantially in ear wound healing (27). Variation among strains, sexes, and healing time points was examined by analysis of variance (ANOVA) using the following equation:
where sex, time postsurgery, and strain are considered to be fixed effects. A significant interaction term implies that there is significant variation in the timing of cartilage regeneration between the strains. Since there were no significant sex differences, sex was removed from the model.
We used ANOVA to first test for significant differences between strains at each time point for cartilage regeneration and ear hole size using the following model:.
with sex as a fixed effect and strain as a random effect. Sex was removed from the model because there were no significant sex differences. Since these are fully inbred mouse strains, the broad-sense heritability of the traits was calculated using the following equation:
where the variance among strains (σ) is divided by the sum of the within-strain variance (σ) and between-strain variance (σ). This includes all sources of genetic variation between mouse strains. Genetic correlations were calculated using a similar procedure for the whole between-strain variance/covariance matrices obtained from multivariate ANOVA including both cartilage regeneration and ear wound healing.
Post hoc pairwise significance tests comparing specific mouse strain pairs were performed using Tukey's honest significant difference test to control for multiple comparisons.
To examine the time-course patterns of articular cartilage regeneration in mice, we first selected the RI mouse lines LGXSM-6 and LGXSM-33 and examined them 4, 8, 12, and 16 weeks after surgery. These strains were chosen because they represent ear wound healer and nonhealer mouse strains, respectively, from the intercross of healer (LG/J) and nonhealer (SM/J) parental lines (27). The time-course experiments revealed that there was no significant strain, sex, or strain-by-sex interaction effect on cartilage regeneration at 4, 8, or 12 weeks in these strains. However, when we compared healing at 16 weeks (Figure 2A and Table 1), we found strain effects that were significantly stronger than those found at earlier time points (P = 0.0003) (strain-by-postsurgery time period interaction). At 16 weeks, LGXSM-6 mice regenerated cartilage significantly better, as indicated by a higher cartilage regeneration score, than they had at 12 weeks (P = 0.0002) and also significantly better than LGXSM-33 mice at both 12 weeks (P = 0.0004) and 16 weeks (P = 0.011) postinjury (Figure 2A). LGXSM-33 mice completely failed to regenerate cartilage.
The notable difference in cartilage regeneration observed between LGXSM-6 mice and LGXSM-33 mice was due to the fact that LGXSM-6 mice successfully produced a proteoglycan-positive extracellular matrix at the site of the defect by 16 weeks postsurgery, as indicated by toluidine blue staining (Figures 2B–E), while LGXSM-33 mice never produced such a matrix (Figures 2F–I). Using the procedures described and mice from the LG/J and SM/J intercross, significant healing did not occur until 16 weeks postsurgery. We also found no significant difference in healing between the left and right knees in either mouse strain or at any time point.
Regeneration/repair in common inbred mouse strains.
The parental strains LG/J and SM/J, and several other inbred mouse strains, including MRL/MpJ, DBA/1J, DBA/2J, and C57BL/6J, were evaluated for cartilage regeneration and ear wound healing. There was no significant difference among strains in cartilage regeneration at 12 weeks (P = 0.088; H2 = 0.11). Although MRL/MpJ mice showed evidence of healing (data not shown), they healed significantly better than SM/J mice only (P = 0.015). In contrast, there was highly significant genetic variation in ear wound closure on day 30 (P < 1 × 10−15; H2 = 0.84). The phenotypic correlation between cartilage regeneration and ear wound healing was low at 12 weeks (r = 0.17). We were unable to calculate a genetic correlation because of the lack of genetic variation in articular cartilage regeneration at 12 weeks among these mouse strains.
Sixteen weeks after articular cartilage injury (Figure 3A and Table 1) and 30 days after ear punch, both articular cartilage regeneration and ear hole closure were significantly heritable in the mice, with a heritability of 51% (P < 0.0001) for articular cartilage regeneration and 84% (P < 1 × 10−11) for ear hole closure. The genetic correlation between the two different healing sites was extremely high (r = 0.92, P = 0.005) (Figure 3B), indicating that mouse strains that heal wounds in the ear also regenerate articular cartilage. Post hoc comparisons indicated that LG/J mice had significantly higher cartilage regeneration scores than did C57BL/6J mice (P = 0.0007), DBA/1J mice (P = 0.007), DBA/2J mice (P = 0.0006), and SM/J mice (P = 0.001) (Figure 3A). MRL/MpJ mice had an intermediate level of articular cartilage regeneration, with a cartilage regeneration score that was not significantly lower than that in LG/J mice but not significantly higher than those in the other strains. With regard to ear hole closure, LG/J and MRL/MpJ mice healed significantly better than did C57BL/6J, DBA/1J, DBA/2J, and SM/J mice (P < 0.0001 for all comparisons versus LG/J mice and P < 0.00001 for all comparisons versus MRL/MpJ mice). Again, there was no significant difference in ear wound healing between LG/J mice and MRL/MpJ mice. These findings indicate that LG/J and MRL/MpJ mice are healers for both articular cartilage and ear wounds, while C57BL/6J, DBA/1J, DBA/2J, and SM/J mice are nonhealers.
The defect area in the nonhealer mouse strains showed mostly fibrous tissue, with no cartilage matrix staining with toluidine blue. Both MRL/MpJ and LG/J mice could regenerate cartilage, and the defect site in these mice appeared to be hyaline cartilage, as it showed proteoglycan staining similar to neighboring undisturbed cartilage. It also appeared smooth, of similar thickness compared to adjacent cartilage, and was better integrated with the neighboring cartilage tissue (Figure 3C).
Regeneration/repair in the LGXSM RI mouse lines.
The comparison of the RI mouse lines, including the parental strains, at 12 weeks showed no statistically significant strain (P = 0.26), sex (P = 0.06), or strain-by-sex interaction (P = 0.51) effects on articular cartilage regeneration. The average histologic score for these strains was low at 12 weeks, indicating a uniformly weak healing response (Table 1). Furthermore, these mouse strains failed to produce a proteoglycan-positive extracellular matrix at the site of defect, as indicated by the absence of toluidine blue staining. Also, the defect remained unfilled (no hyaline cartilage) and contained fewer chondrocytes at the wound site (results not shown).
Significant differences between mouse strains were observed at 16 weeks (Figure 4A and Table 1), with a significant strain-by-postinjury time effect on cartilage regeneration (P = 0.0004). By 16 weeks the level of variation between strains had increased, with a broad-sense heritability of 26% (P = 0.006). At 16 weeks, LG/J mice showed the best cartilage regeneration, with significantly higher cartilage regeneration scores than SM/J mice (P = 0.005) and LGXSM-33 mice (P = 0.017), and scores that were higher than those in LGXSM-5 mice at a borderline significance level (P = 0.054) (Figure 4A). None of the other pairwise differences were significant when controlling for multiple comparisons, although after LG/J mice, LGXSM-6 and LGXSM-35 mice showed moderate healing, and LGXSM-5, LGXSM-33, and SM/J mice showed poor healing. The differences between LGXSM-6 and LGXSM-33 mice were not statistically significant in this analysis, although they were statistically significant in the time-course analysis, because in this analysis we adjusted the probability for multiple comparisons. However, taken alone, at 16 weeks these mouse strains showed the same differences they showed in the time-course analysis.
The heritability of 30-day ear wound healing for the LGXSM RI mouse lines was 53% (P < 0.00001). Several strains healed significantly better, as indicated by ear wound healing score, than SM/J, including LG/J (P < 0.01), LGXSM-35 (P = 0.006), LGXSM-5 (P < 0.001), and LGXSM-6 (P < 0.00001). LGXSM-6 healed significantly better than LGXSM-33 (P = 0.0003). The genetic correlation for the RI line set was quite high (r = 0.86, P = 0.014) (Figure 4B), especially given the environmental correlation of −0.12, which is not significantly different from zero.
Histologically, the site of articular cartilage defect in the RI mouse lines showed mostly fibrous tissue with no cartilage matrix staining in nonhealers. In regenerating strains, the injured area had hyaline cartilage, as indicated by proteoglycan staining that was similar to staining in adjacent intact cartilage. The cartilage also appeared relatively smooth, of almost similar thickness compared to adjacent cartilage, and was better integrated with the neighboring cartilage tissue (Figure 4C).
The full-thickness articular cartilage injuries in these mice involved the penetration of the needle through the growth plate. While articular cartilage showed regeneration in healer strains, no significant healing of growth plate cartilage was noted (results not shown). It is known that growth plate does not have the ability to regenerate on its own after an injury. We made the injury in mature mice, so we did not see any regeneration of growth plate in these mice.
There was no type I collagen deposition on the regenerated or nonregenerated defect site or on the native healthy cartilage in the mice; however, bone showed positive signals for type I collagen. On the other hand, healthy cartilage as well as regenerated cartilage in healer mouse strains showed type II collagen deposition. In the nonregenerating strains, the defect site did not stain for this type of collagen as compared to neighboring undamaged cartilage (Figure 5).
There are several reports on cartilage degeneration in OA pathophysiology (see, for example, refs.22–24), but little evidence is available on the genetics of cartilage regeneration. In this study, we demonstrated regeneration of full-thickness articular cartilage lesions in mouse strains, each with a defined genetic composition. While genetic analyses have clearly shown that tissue regeneration is a complex multigenic trait (33), we hypothesize that any differential phenotype between the strains will be attributed to a restricted set of genomic regions.
The present study segregates RI mouse lines into 3 distinct categories of articular cartilage regeneration—good, intermediate, and poor healers—suggesting that rearrangement of the alleles in the RI lines segregated genes that influence regeneration. The broad-sense heritability showed that articular cartilage regeneration is a significantly heritable trait in the mice (P = 0.006). Further, the genetic correlations between the two healing phenotypes (articular cartilage regeneration and ear wound healing) were found to be extremely high, indicating that the two processes share a common genetic basis.
The MRL/MpJ mouse and its close relative, the LG/J mouse, have unique healing and regenerative capabilities, including the complete closure of through-and-through ear puncture wounds with normal tissue architecture without scarring over time, and thus these strains display similar ear wound healing phenotypes (9–13, 34, 35). The MRL/MpJ strain was originally generated from a series of crosses between AKR/J, C3H/HeDi, and C57BL/6J and two final backcrosses to LG/J (2), so that MRL/MpJ and LG/J mice share 75% of their genomes identical by descent (9, 13, 35). We confirmed the earlier ear wound healing results and showed that both MRL/MpJ mice, as demonstrated by Fitzgerald et al (10), and LG/J mice also regenerate articular cartilage over time. This is in contrast to the C57BL/6J and SM/J mouse strains, which are known to be nonhealers with regard to ear wounds (10, 11, 13, 21) and articular cartilage injuries (10, 21). These observations indicate that this exceptional healing ability of LG/J and MRL/MpJ mice is likely due to their shared genetic composition. Furthermore, the high genetic correlation observed among common inbred strains between articular cartilage regeneration and ear hole closure indicate that the same cellular and tissue mechanisms are likely to be responsible for healing in MRL/MpJ and LG/J mice.
Eltawil and colleagues (21) found that DBA/1 mice can regenerate articular cartilage under certain conditions. We were unable to confirm the wound-healing ability of this strain, as it failed to regenerate/repair articular cartilage or ear wounds. There could be multiple reasons these findings were not replicated in the present study. First, we did not follow the same protocol (21). While Eltawil produced a full-thickness cartilage defect void in the patellar groove, they did not pierce the underlying bone. Also, we tested DBA/1J mice (obtained from The Jackson Laboratory) in St. Louis, but the origin of the DBA/1 animals used in London (21) is not clear. The DBA/1OlaHsd strain was separated from DBA/1J more than 55 years ago, while the DBA/1JBomTac strain was separated from DBA/1J nearly 20 years ago. New mutations or even unintended mixing may have occurred over these hundreds of generations. Thus, the reason for the difference between our results and those of Eltawil et al (21) with regard to cartilage regeneration in DBA/1 mice remains unknown and may be attributed to genetic drift or differences in surgical procedure.
The ear wounds of healer mouse strains rapidly re-epithelialize, with regrowth of both hair follicles and elastic cartilage, while in nonhealer strains, including C57BL/6J, 129/SvJ, SM/J, DBA/1J, and DBA/2J mice, scar tissue forms around the edges of the wound (9–13, 35, 36). In addition, increased angiogenesis, cell proliferation, matrix formation, and fibroblast migration occur in tissue undergoing repair in the healer strains compared to the nonhealer strains. The MRL/MpJ mouse strain has also demonstrated a superior microvasculature response to skin wounds (37). Thus, the healing properties of MRL/MpJ mice, and likely LG/J mice, are a result of multiple pathways and not due to a single cause. We believe that the quality of repair in the ear wound in the healer strains examined in this study is similar to what has previously been reported for MRL/MpJ mice (12, 36) and LG/J mice (9), with similar genes involved in the healing process (38). Therefore, in the presence of convincing data on the nature of healed ear tissue, we took these data as exemplary for our study.
Several genetic linkage studies (32, 33, 39) have demonstrated that ear wound healing in mice is a complex genetic trait with contributions from ∼20 quantitative trait loci (QTLs). The large number of QTLs involved in distinguishing healer and nonhealer mouse strains indicates plausible contributions from many genes involved in many different cellular and tissue level processes in the wound healing phenotype (38). Although a strong sexual dimorphism in the rate of ear wound closure in the LGXSM mouse cross exists, the QTLs have largely the same effect in both sexes (40). In our smaller study, we failed to detect sex differences in ear wound healing. Articular cartilage regeneration response in MRL/MpJ mice has been reported to be sexually dimorphic at 12 weeks, albeit no significant sex differences were observed at any earlier time point (10). In the present study, which had a larger sample size and greater statistical power, no sex difference was detected for articular cartilage regeneration.
The specific reason that some mouse strains are healers and others are not remains unknown. However, the healer mouse strains MRL/MpJ, LG/J, and LGXSM-6 have been shown to share some unique molecular features with the classic regenerators with respect to ear wound healing. Healer strains have a distinct cell cycle phenotype, i.e., a G2/M stage arrest and a heightened basal and wound site DNA damage/repair response (27).
The LG/J and SM/J inbred mouse strains differ with regard to a wide variety of phenotypes, including growth (41), obesity and diabetes (42, 43), long bone growth (44–46), skeletal morphology (47), bone biomechanical properties (48), muscle weight (49), and ear wound healing (13). Further, each RI mouse line genome is an independent isogenic line with genetic variance concentrated between lines and eliminated within lines. Previous work related to ear wound healing identified LGXSM-6 as a line that heals ear wounds and LGXSM-33 as a line that fails to heal (27). For all of these phenotypes, we have found that genetic variation is due to many genes of small effect and their interactions with the genetic background. The finding that the LGXSM-5 mouse has a relatively high ear wound healing score and a relatively low cartilage regeneration score shows that the correlation between the traits is <1.00 (r = 0.86) and suggests that there are a few genomic regions with differential effects on the two forms of healing.
We believe that this is the first study to characterize articular cartilage and ear wound healing phenotypes from a genetics viewpoint in a segregating population of mice. The key findings from our study of RI mouse lines are that 1) variation in ear wound healing and articular cartilage regeneration are both heritable, with the differences between RI lines being due to genetic differences; 2) the genetic correlation between ear wound healing and articular cartilage regeneration is very strong, indicating that the two processes likely share a common genetic basis; 3) there is no correlation between ear wound healing and articular cartilage regeneration due to environmental, nongenetic factors; and 4) LGXSM-6 is an RI mouse line that responds to injury in a manner similar to that of LG/J mice, while many other RI lines have intermediate healing phenotypes for both ear wound healing and articular cartilage regeneration.
As a consequence of these findings, it is apparent that the LG/J by SM/J intercross segregates for multiple genetic factors affecting articular cartilage regeneration and that the genetic basis for this difference can be genetically mapped in the intercross population. In addition to the RI lines used in this study, there is also an advanced intercross line (45) in which fine-mapping studies can be performed. The previously mapped QTLs affecting ear wound healing are also likely to affect cartilage regeneration in the knee, so that fine-mapped ear wound healing loci are strong candidates for loci that also affect articular cartilage regeneration.
These findings may also shed light on protection of healer strains against developing OA. In a companion study (50), we have demonstrated that a mouse strain (LGXSM-6) shown to regenerate articular cartilage and heal ear wounds is relatively protected against posttraumatic OA compared to the strain LGXSM-33, which has more extensive OA and does not regenerate articular cartilage or heal ear wounds. Thus, the capacity to regenerate articular cartilage is inversely correlated with the development of posttraumatic OA, as has been provisionally suggested (26).
The phenotypic differences observed among RI mouse lines can be attributed to a restricted set of genes differentially inherited from the parental strains due to random segregation of genomic regions that influence regeneration. Taken together, these data are a unique resource for the study of genes that contribute to tissue healing and cartilage regeneration and thus, perhaps, to protection against OA.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Sandell had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Cheverud, Sandell.
Acquisition of data. Rai, Hashimoto, Johnson, Janiszak, Fitzgerald.
Analysis and interpretation of data. Rai, Hashimoto, Johnson, Janiszak, Heber-Katz, Cheverud, Sandell.
We acknowledge with thanks the important technical support of Crystal Idleburg, John Freeman, Elizabeth DeLassus, Patricia Keller (late), Timothy Morris, and Joseph Futhey.