Heritability of articular cartilage regeneration and its association with ear wound healing in mice

Abstract

Objective: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Fig. 1

(A) Anatomical location of full-thickness articular cartilage lesion on the trochlear groove. Arrow indicates site of defect at zero time, lines indicate the direction of serial sagittal sections at 5-micron interval all through the trochlear groove, F = femur, TG = trochlear groove, MM = medial meniscus, LM = lateral meniscus, T = tibia. (B) A sketch of scoring sheet to grade histological sections for articualr cartilage regeneration.

Fig. 2. Time-course of articular cartilage regeneration

(A) A time-course analysis of articular cartilage regeneration in LGXSM-6 and LGXSM-33 lines showed a significant difference in regeneration only at 16-weeks post-surgery. (B–I) Representative sagittal sections of full-thickness articular cartilage lesions form LGXSM-6 (B–E) and LGXSM-33 (F–I) lines stained with toluidine blue for proteoglycan at 4-, 8-, 12- and 16-weeks post-surgery are shown. There was a significant proteoglycan deposition in LGXSM-6 (healer) compared to LGXSM-33 (non-healer) at 16-weeks time-point. At all other time-points, no significant regenerative response was observed. Asterisk (*) indicates statistical significance (p<0.011); arrow indicates site of defect; AC = articular cartilage, SB = subchondral bone, GP = growth plate, BMC = bone marrow cavity, bar (all panels) = 0.1 mm

Fig. 3. Articular cartilage regeneration and ear-wound healing in common inbred strains

(A) Articular cartilage regeneration in common inbred strains showed no healing response in SM/J, DBA/2J, C57BL/6J and DBA/1J strains while it showed better healing ability in MRL/MpJ and LG/J strains. Asterisk (*) with respective p values indicates statistically significant difference from LG/J strain. (B) Correlation between articular cartilage regeneration and ear-wound healing showed that the two phenotypes are strongly correlated in common inbred strains. (C) Representative sagittal sections of full-thickness articular cartilage lesions from common inbred strains namely LG/J, SM/J, MRL/MpJ, DBA/1J, DBA/2J and C57BL/6J showed that only LG/J and MRL/MpJ could show proteoglycan deposition indicating a better regenerative response compared to SM/J, DBA/1J, DBA72J and C57BL/6J strains. Arrow indicates site of defect; AC = articular cartilage, SB = subchondral bone, GP = growth plate, BMC = bone marrow cavity; bar (all panels) = 0.1 mm

Fig. 4. Articular cartilage regeneration and ear-wound healing in recombinant inbred lines

(A) Articular cartilage regeneration in recombinant inbred lines showed no healing response in SM/J, LGXSM-5 and LGXSM-33 strains while intermediate to good healing responses were observed in LGXSM-6, LGXSM-35 and LG/J strains. Asterisk (*) with p given values indicates statistically significant difference from parental strain LG/J. (B) Correlation between knee articular cartilage regeneration and ear-wound healing showed that the two phenotypes are strongly correlated in the recombinant inbred lines. (C) Representative sagittal sections of full-thickness articular cartilage lesions from recombinant inbred lines LGXSM-5, LGXSM-6, LGXSM-33 and LGXSM-35 and parental strains LG/J and SM/J show that except for strains LGXSM-33 and SM/J, all other strains regenerated their articular cartilage and showed a deposition of proteoglycan indicating regenerative response. Arrow indicates site of defect; AC = articular cartilage, SB = subchondral bone, GP = growth plate, BMC = bone marrow cavity; bar (all panels) = 0.1 mm

Fig. 5. Collagen staining

Representative sagittal sections from selected healer and non-healer strains strained with type II collagen show that there was collagen type II positive staining at the site of injury after it has healed. In non-healer strains, there was no collagen type II staining at the injury site. AC = articular cartilage, F = femur