Diminished Chondrogenesis and Enhanced Osteoclastogenesis in Leptin-Deficient Diabetic Mice (ob/ob) Impair Pathologic, Trauma-Induced Heterotopic Ossification

Abstract

Diabetic trauma patients exhibit delayed postsurgical wound, bony healing, and dysregulated bone development. However, the impact of diabetes on the pathologic development of ectopic bone or heterotopic ossification (HO) following trauma is unknown. In this study, we use leptin-deficient mice as a model for type 2 diabetes to understand how post-traumatic HO development may be affected by this disease process. Male leptin-deficient (ob/ob) or wild-type (C57BL/6 background) mice aged 6-8 weeks underwent 30% total body surface area burn injury with left hind limb Achilles tenotomy. Micro-CT (μCT) imaging showed significantly lower HO volumes in diabetic mice compared with wild-type controls (0.70 vs. 7.02 mm(3), P < 0.01) 9 weeks after trauma. Ob/ob mice showed evidence of HO resorption between weeks 5 and 9. Quantitative real time PCR (qRT-PCR) demonstrated high Vegfa levels in ob/ob mice, which was followed by disorganized vessel growth at 7 weeks. We noted diminished chondrogenic gene expression (SOX9) and diminished cartilage formation at 5 days and 3 weeks, respectively. Tartrate-resistant acid phosphatase stain showed increased osteoclast presence in normal native bone and pathologic ectopic bone in ob/ob mice. Our findings suggest that early diminished HO in ob/ob mice is related to diminished chondrogenic differentiation, while later bone resorption is related to osteoclast presence.

FIG. 1.

(A) Micro-CT imaging of representative ob/ob and wild-type hind limbs at 5, 7, and 9 weeks post-trauma. Areas of HO are indicated in gray. (B) Total quantified HO volume at 5, 7, and 9 weeks post-trauma in ob/ob and wild-type mice (* P < 0.05). (C) Alkaline phosphatase staining of adipose-derived mesenchymal stem cells (adMSCs) from burned ob/ob and wild-type mice at 7 days. (D) Alizarin red staining of adMSCs from burned ob/ob and wild-type mice at 14 days. HO, heterotopic ossification.

FIG. 2.

(A) Vessel reconstruction after MICROFIL injection at 7 weeks of ob/ob and wild-type hind limbs depicts more vessel growth with disorganization in ob/ob mice. Cross sections taken from the top third, middle third, and bottom third of both legs are depicted to the right. Gray arrowheads = MICROFIL dye in vessels. (B) Mean vessel volume standardized to hind limb mass. (C) Staining for CD31 (black arrowheads) shows comparable amount of staining in both ob/ob and wild-type hind limbs after 3 weeks. (D) Staining for Hif1α (black arrowheads) is comparable between ob/ob and wild-type mice after 5 days. (E) Higher Hif1a RNA transcript level in the diabetic hind limb compared with wild type after 5 days, standardized to internal Gapdh levels [normalized ratios 3.33 (SD 1.44) vs. 1.00 (SD 0.52)]. (F) VEGF staining is increased in the ob/ob hind limb 5 days postinjury. (G) Vegfa RNA transcript levels are higher in the ob/ob hind limb after 5 days, standardized to internal Gapdh levels [normalized ratios 2.38 (SD 0.83) vs. 1.00 (SD 0.59)]. *P < 0.05.

FIG. 3.

(A) Pentachrome staining of injured ob/ob and wild-type hind limbs at 3 weeks shows strong alcian blue presence in wild-type mice indicative of cartilage deposition (black arrowheads). (B) SOX9 staining confirms the presence of chondrocytes (black arrowheads) and chondrogenic precursors (gray arrowheads) to a higher degree than seen in ob/ob mice. (C) Sox9 gene expression is not significantly elevated early after trauma in wild-type mice compared with ob/ob mice.

FIG. 4.

(A) Aniline blue stain of wt/wt and ob/ob sections 3 months after injury with regions of HO indicated (gray arrowheads) (4× magnification). (B) Aniline blue stain of normal bone and HO 3 months after injury in wt/wt and ob/ob mice (10×). (C) Representative osteocalcin immunofluorescent staining in normal bone and HO in wt/wt and ob/ob mice 3 months after injury (20×). (D) Increased osteocalcin staining per high-powered field in normal bone and HO of wt/wt mice compared with ob/ob mice (n = 3 sections for wt/wt and ob/ob mice).

FIG. 5.

(A) Decreasing trend in HO volume of ob/ob mice over time compared with wild-type mice (gray arrowheads). (B) Increased osteoclast activity based on TRAP staining of ob/ob and wild-type HO regions.