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. 2015 Nov;74(11):2076-83.
doi: 10.1136/annrheumdis-2014-205601. Epub 2014 Jul 10.

Dietary Fatty Acid Content Regulates Wound Repair and the Pathogenesis of Osteoarthritis Following Joint Injury

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Free PMC article

Dietary Fatty Acid Content Regulates Wound Repair and the Pathogenesis of Osteoarthritis Following Joint Injury

Chia-Lung Wu et al. Ann Rheum Dis. .
Free PMC article

Abstract

Objective: The mechanisms linking obesity and osteoarthritis (OA) are not fully understood and have been generally attributed to increased weight, rather than metabolic or inflammatory factors. Here, we examined the influence of fatty acids, adipokines, and body weight on OA following joint injury in an obese mouse model.

Methods: Mice were fed high-fat diets rich in various fatty acids (FA) including saturated FAs (SFAs), ω-6 polyunsaturated FAs (PUFAs), and ω-3 PUFAs. OA was induced by destabilising the medial meniscus. Wound healing was evaluated using an ear punch. OA, synovitis and wound healing were determined histologically, while bone changes were measured using microCT. Activity levels and serum cytokines were measured at various time-points. Multivariate models were performed to elucidate the associations of dietary, metabolic and mechanical factors with OA and wound healing.

Results: Using weight-matched mice and multivariate models, we found that OA was significantly associated with dietary fatty acid content and serum adipokine levels, but not with body weight. Furthermore, spontaneous activity of the mice was independent of OA development. Small amounts of ω-3 PUFAs (8% by kcal) in a high-fat diet were sufficient to mitigate injury-induced OA, decreasing leptin and resistin levels. ω-3 PUFAs significantly enhanced wound repair, SFAs or ω-6 PUFAs independently increased OA severity, heterotopic ossification and scar tissue formation.

Conclusions: Our results indicate that with obesity, dietary FA content regulates wound healing and OA severity following joint injury, independent of body weight, supporting the need for further studies of dietary FA supplements as a potential therapeutic approach for OA.

Figures

Figure 1
Figure 1
(A) The influence of diets on body weight over time. (B) To precisely describe the effect of weight on knee joint over time, the areas under weight curve of different diets were calculated for the period from 4 to 28 weeks (AUC4-28wk) and from 17 to 28 weeks (AUC17-28wk), respectively. The Control mice had lower AUC4-28wk values as compared to the mice fed high-fat diets. (C and D) SFA and ω-6 mice had increased percentages of body fat, but decreased body BMD relative to Control mice at 28 weeks of age. (E) The ω-6 mice had significantly increased percentages of fat in the inguinal and epididymal depots relative to body weight compared to Control mice. (F) SFA and ω-6 mice showed increased F4/80+ macrophage infiltration (red arrowheads) into adipose tissues (Sub: subcutaneous fat; Vis: visceral fat). Infiltrated macrophages into the visceral fat pads in SFA and ω-6 mice also showed “crown-like” structure (green square). Scale bar = 100 μm. (G) Analysis of F4/80, CD11c. IL-6, TNF-α, and MCP-1 gene expression in visceral adipose tissue in the mice at 28 weeks of age. For gene expression, n = 4 mice/diet. For other figures, n = 11–14 mice/diet. Different letters are significantly different, p < 0.05, from each other. (A, C and D) Statistical significance was determined by two-way repeated measures ANOVA using age and diet as factors. (B, E and G) Statistical significance was determined by one-way ANOVA using diet as factor. ANOVA was then followed by Tukey’s post-hoc test. All data are presented as mean ± SEM.
Figure 2
Figure 2
(A) 3D reconstruction of MicroCT of limbs at 28 weeks of age. Right (non-operated) joint from SFA mice showed intact bone structure (F = femur, T = tibia, P = patella; black arrows = partially calcified menisci). Left (DMM) joints of ω-6 and SFA mice had increased heterotopic ossification (white arrows) relative to the other groups. (B) Cancellous bone fraction (bone volume/total volume, BV/TV) for femoral condyle (FC), and cancellous and cortical BV/TV for tibial epiphysis (TP). ω-3 mice had significantly lower BV/TV in FC and TP as compared to the mice fed other diets. (C) Bone mineral density (BMD) of FC and TP. All the mice showed significantly decreased BMD after DMM surgery and the ω-3 mice also showed relatively low BMD to the mice fed other diets. (D and E) BV of TP and heterotopic ossification of DMM joints. All the mice had significantly increased BV in TP after DMM. ω-3 mice exhibited low heterotopic BV among the mice fed other high-fat diets. n = 11–14 mice/diet. * p < 0.05 versus corresponding right (non-operated) joints. Different letters are significantly different, p < 0.05, from each other. (B–D) Statistical significance was determined by two-way repeated measures ANOVA using right (non-operated) joints as the contralateral control. (E) The line inside the box represents the median of each diet group and the length of the box indicates the interquartile range. Statistical significance was determined by Kruskal-Wallis test with Mann-Whitney U test and Holm–Bonferroni correction. Except for (E), all data are presented as mean ± SEM.
Figure 3
Figure 3
(A) Safranin-O (glycosaminoglycans) and fast green (bone and tendon) histology for the DMM-operated joint (F = femur, M = meniscus, T = tibia). Severe cartilage loss (yellow arrowheads) was found in ω-6 and SFA mice. The DMM-operated joints from Control and ω-3 mice had significantly lower OA scores compared to those from ω-6 mice and SFA mice. (B) Accumulative counts of osteophyte diseases-stages of the DMM-operated joint. The ω-6 and SFA mice had more mature osteophytes relative to Control and ω-3 mice. (C) OA severity was positively correlated with osteophyte disease stages. (D) H&E histology of the medial femoral condyle of DMM-operated joints (S = synovium). Thickened synovium from ω-6 and SFA mice with high density of infiltrated cells was observed (black arrows). The DMM-operated joints from ω-3 mice had significantly lower synovial inflammation than those from ω-6 mice. (E) F4/80+ macrophage IHC (red arrowheads) of each quadrant of the DMM-operated joint and its quantification (medial femoral condyle, MF; medial tibial plateau, MT; lateral femoral condyle, LF; lateral tibial plateau, LT). The ω-6 mice also exhibited high macrophage scores at medial side of the joint. Levels of serum (F) insulin, (G) leptin, (H) adiponectin, and (I) resistin at various time-points; (J) PGE2 and (K) the Active/Total TGF-β1 ratio were measured at 28 weeks of age. n = 11–14 mice/diet. * p < 0.05 for regression analysis. # p < 0.05, versus all the other diets. Different letters are significantly different, p < 0.05, from each other. (A and D) Statistical significance was determined by two-way repeated measures ANOVA using right (non-operated) joints as the contralateral control. (B) Statistical significance was determined by Kruskal-Wallis H Test, p = 0.07. (F–I) Statistical significance was determined by two-way repeated measures ANOVA using age and diets as factors, while (E, J and K) statistical significance was determined by one-way ANOVA using diet as the factor. ANOVA was then followed by Tukey’s post-hoc test. All data are presented as mean ± SEM.
Figure 4
Figure 4
Diets supplemented with different types of FAs did not significantly affect (A) spontaneous locomotion activity, (B) rotarod performance, (C) forelimb and (D) hind-limb grip strength prior to and post-surgery. For nociception, diet significantly influenced (E) the tail flick but not (F) the hot plate latency. However, if mice fed the 60% kcal high-fat diets were pooled together and were compared to the mice fed the 10% kcal Control low-fat diet, high-fat feeding significantly decreased rotarod performance and forelimb grip strength (main effect, p < 0.05). Nonetheless, no effects on spontaneous locomotion activity were observed. n = 11–14 mice/diet. * p < 0.05, versus ω-6 mice. # p < 0.05, high-fat feeding versus age-matched low-fat feeding. For 6 and 14 weeks of age, statistical significance was determined by two-way repeated measures ANOVA using age and diet as factors. For 24 weeks of age, statistical significance was determined by one-way ANOVA using diet as factor. ANOVA was then followed by Fisher’s post-hoc test. All data are presented as mean ± SEM.
Figure 5
Figure 5
The ω-3 mice demonstrated enhanced ear wound healing capacity. (A) Representative images of ear hole (white arrows) at 12 weeks post-wounding. (B) The ω-3 mice had the smallest wound area as compared to the mice fed other diets. (C) H&E stained images showed that ω-3 mice had a thickened epithelium (yellow arrowheads). Black arrows indicated the wound edge of each sample. (D) ω-3 mice had significantly thicker epithelia as compared to other mice. (E) Masson’s Trichrome indicated that all mice exhibited healing features such as regeneration of sebaceous glands (yellow arrowheads) and new cartilage islands (green arrowheads); however, only the ω-3 mice had several new cartilage condensations. (F) No difference in cell proliferation marker (Ki-67) among the diets was observed. (G) Picrosirius red staining indicated that the ω-3 mice had less deposition of collagen type I fibers in the wound area, suggesting less scar formation. (H) Wound healing capacity exhibited a trend towards a negative relationship with OA severity in the more severe ear wounding (1.5-mm punch) model, but not in the less severe (1.0-mm punch) model. n = 11–14 mice/diet. (A) The scale bar = 5 mm, and for others images the scale bar = 100 μm. (B) Statistical significance was determined by two-way repeated measures ANOVA. * p < 0.05, versus all the other groups. (D) Statistical significance was determined by one-way ANOVA. * p < 0.05 and # p < 0.01, versus all the other groups. All data are presented as mean ± SEM.

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