Burn injury enhances bone formation in heterotopic ossification model

Abstract

Objective: To demonstrate the pro-osteogenic effect of burn injury on heterotopic bone formation using a novel burn ossicle in vivo model.

Background: Heterotopic ossification (HO), or the abnormal formation of bone in soft tissue, is a troubling sequela of burn and trauma injuries. The exact mechanism by which burn injury influences bone formation is unknown. The aim of this study was to develop a mouse model to study the effect of burn injury on heterotopic bone formation. We hypothesized that burn injury would enhance early vascularization and subsequent bone formation of subcutaneously implanted mesenchymal stem cells.

Methods: Mouse adipose-derived stem cells were harvested from C57/BL6 mice, transfected with a BMP-2 adenovirus, seeded on collagen scaffolds (ossicles), and implanted subcutaneously in the flank region of 8 adult mice. Burn and sham groups were created with exposure of 30% surface area on the dorsum to 60°C water or 30°C water for 18 seconds, respectively (n = 4/group). Heterotopic bone volume was analyzed in vivo by micro-computed tomography for 3 months. Histological analysis of vasculogenesis was performed with platelet endothelial cell adhesion molecule staining. Osteogenic histological analysis was performed by Safranin O, Picrosirius red, and aniline blue staining. Qualitative analysis of heterotopic bone composition was completed with ex vivo Raman spectroscopy.

Results: Subcutaneously implanted ossicles formed heterotopic bone. Ossicles from mice with burn injuries developed significantly more bone than sham control mice, analyzed by micro-computed tomography at 1, 2, and 3 months (P < 0.05), and had enhanced early and late endochondral ossification as demonstrated by Safranin O, Picrosirius red, and aniline blue staining. In addition, burn injury enhanced vascularization of the ossicles (P < 0.05). All ossicles demonstrated chemical composition characteristic of bone as demonstrated by Raman spectroscopy.

Conclusions: Burn injury increases the predilection to osteogenic differentiation of ectopically implanted ossicles. Early differences in vascularity correlated with later bone development. Understanding the role of burn injury on heterotopic bone formation is an important first step toward the development of treatment strategies aimed to prevent unwanted and detrimental heterotopic bone formation.

Figure 1

Burn injury increases angiogenesis in ossicles 7 days after implantation. A, PECAM quantification of ASC-seeded ossicles after burn injury or nonburn (sham) control (*, P < 0.05). B, Representative images at 20× of PECAM stain of ASC-seeded ossicles after burn injury or nonburn control (sham) 7 days after ossicle implantation. Circle indicates PECAM positive circular structure.

Figure 2

Burn injury enhances early osteogenic predilection of ASC-seeded ossicles. A, Early endochondral ossification seen in ASC-seeded ossicles. Left to Right, Images of the ASC ossicle with Safranin O staining at 4×, 10×, and 20× are shown 7 days after ossicle implantation. Black arrows point to regions of early cartilage formation with positive Safranin O staining. B, OCN immunohistochemical stain of ASC-seeded ossicles at 7 days shown at 20× and 40× magnification. Arrows indicate regions of OCN positive staining (cytoplasmic). Negative control slides were not incubated with primary OCN antibody, but otherwise similarly processed.

Figure 3

Burn injury enhances bone formation in ASC-seeded ossicles. A, 3-Dimensional reconstruction of representative ossicles from nonburn control (sham) and burn injury mice at 1, 2, and 3 months. B, Quantification of bone volume at 1, 2, and 3 months comparing burn with nonburn control (sham) (*, P < 0.05, n = 4/group).

Figure 4

Burn injury enhances early collagen deposition and late lamellar bone deposition in ASC-seeded ossicles. Picrosirius red stain of ASC-seeded ossicles in nonpolarized (left) and polarized light (right) at 7 days and 3 months in burn and non-burn control (sham) mice. Picrosirius red quantification using Adobe Photoshop showing analysis of percentage of Picrosirius red staining under polarized light (*, P < 0.05).

Figure 5

Burn injury increases osteoid formation by histology at 12 weeks. A, Representative slides of ASC seeded ossicles stained with aniline blue showing mature osteoid. B, Aniline blue quantification using Adobe Photoshop analysis of aniline blue staining (*, P < 0.05). C, OCN immunohistochemical stain of ASC-seeded ossicles at 12 weeks shown at 20× and 40× magnification. Arrows indicate regions of OCN positive staining (cytoplasmic). Negative control slides were not incubated with primary OCN antibody, but otherwise similarly processed.

Figure 6

Chemical composition of bone is not different between burn and nonburn control (sham) groups as demonstrated by Raman spectroscopy of sham and burn ASC seeded ossicles. Spectra are normalized to bone mineral band (960 cm⁻¹) and overlain to compare similarity of chemical composition signaling.