Role of gender in burn-induced heterotopic ossification and mesenchymal cell osteogenic differentiation

Abstract

Background: Heterotopic ossification most commonly occurs after burn injury, joint arthroplasty, and trauma. Male gender has been identified as a risk factor for the development of heterotopic ossification. It remains unclear why adult male patients are more predisposed to this pathologic condition than adult female patients. In this study, the authors use their validated tenotomy/burn model to explore differences in heterotopic ossification between male and female mice.

Methods: The authors used their Achilles tenotomy and burn model to evaluate the osteogenic potential of mesenchymal stem cells of male and female injured and noninjured mice. Groups consisted of injured male (n = 3), injured female (n = 3), noninjured male (n = 3), and noninjured female (n = 3) mice. The osteogenic potential of cells harvested from each group was assessed through RNA and protein levels and quantified using micro-computed tomographic scan. Histomorphometry was used to verify micro-computed tomographic findings, and immunohistochemistry was used to assess osteogenic signaling at the site of heterotopic ossification.

Results: Mesenchymal stem cells of male mice demonstrated greater osteogenic gene and protein expression than those of female mice (p < 0.05). Male mice in the burn group formed 35 percent more bone than female mice in the burn group. This bone formation correlated with increased pSmad and insulin-like growth factor 1 signaling at the heterotopic ossification site in male mice.

Conclusions: The authors demonstrate that male mice form quantitatively more bone compared with female mice using their burn/tenotomy model. These findings can be explained at least in part by differences in bone morphogenetic protein and insulin-like growth factor 1 signaling.

Figure 1. Assessment and quantification of HO at 5, 7, 9 and 15 weeks post-injury

A. Micro-CT images of ectopic bone formation in burned and unburned mice. Green indicates heterotopic bone. White indicates the normal bone. B. Qualitative analysis of HO volume by week. Threshold set at 1250 HU. Values represent mean amount of HO formed ± standard deviation. Error bars represent standard deviation.

Figure 2. Histological assessment of inflammation and soft tissue swelling one week post-burn+tenotomy injury

Tenotomy site at one week post-burn. Arrows indicate inflammation around cut end of the Achilles tendon. Sample stained via H&E. All images at 10× (right) and 20× (left) magnification.

Figure 3. Histological assessment of cartilage formation, soft tissue mineralization, and early evidence of ectopic bone formation at three weeks post-burn+tenotomy injury

Arrows indicate sites of cartilage formation. Slides stained with pentachrome. All images at 4× and 10× magnification.

Figure 4. Alkaline phosphatase (ALP) and Alizarin Red staining in MSC derived from injured and sham-treated male and female mice

A. Qualitative and quantitative analysis of alkaline phosphatase activity; representative images collected from each experimental subset. B. Qualitative and quantitiative analysis of alizarin red staining demonstrating in vitro mineralization potential; representative images collected from each experimental subset. Quantification of alizarin red staining in each experimental subset. Asterisk indicates significance p<0.05. Error bars indicate standard deviation (SD).

Figure 5. pSmad 1/5/8 and IGF-1 protein expression in MSCs derived from injured and sham-treated male and female mice

A. Quantification and representative immunoblots of pSmad 1/5/8 normalized via densitometry. Normalization via alpha-tubulin. Asterisk indicates significance p<0.05. B. Quantification and representative immunoblots of IGF-1 normalized via densitometry. Normalization via alpha-tubulin. Error bars indicate SD. Male mice after burn injury respond with greater activation of the BMP cascade as manifested by higher levels of pSmad and IGF-1 compared to females after burn injury.

Figure 6. IHC staining for pSmad or IGF-1 in the healing soft tissue of male and female mice at 3 weeks post-burn + tenotomy injury

A. IHC staining for pSmad. Samples stained via IHC for pSmad. B. IHC staining for IGF-1. Samples stained via IHC for IGF-1. Representative images of early heterotopic ossification at the tenotomy site 3 weeks post-burn injury in male (left) and female (right) mice. All images at 10× magnification. Arrows indicate positivity for pSmad or IGF-1.