Gene expression of TGF-beta, TGF-beta receptor, and extracellular matrix proteins during membranous bone healing in rats

Poorly healing mandibular fractures and osteotomies can be troublesome comp lications of craniomaxillofacial trauma and reconstructive surgery. Gene th erapy may of fer ways of enhancing bone formation by altering the expressio n of desired growth factors and extracellular matrix molecules. The elucida tion of suitable candidate genes for therapeutic intervention necessitates investigation of the endogenously expressed patterns of growth;th factors d uring normal (i.e., successful) fracture repair. Transforming growth factor pi (TGF-beta I), its receptor (T beta-RII), and the extracellular matrix proteins osteocalcin and type I collagen are thoug ht to be important in long-bone (endochondral) formation, fracture healing, and osteoblast proliferation. However, the spatial and temporal expression patterns of these molecules during membranous bone repair remain unknown. In this study, 94 adult rats underwent mandibular osteotomy with rigid exte rnal fixation. In addition, four identically treated rats that underwent sh am operation (i.e., no osteotomy) were used as controls. Four experimental animals were then killed at each time point (3, 5, 7, 9, 23, and 97 dais af ter the procedure) to examine gene expression of TGF-beta 1 and T beta-RII, osteocalcin, and type I collagen. Northern blot analysis was wed to compar e gene expression of these molecules in experimental animals with that in c ontrol animals (i.e., nonosteotomized; n = 4). In addition, TGF-beta I and T beta-RII proteins were immunolocalized in an additional group of nine ani mals killed on postoperative days 3, 7, and 37. The results of Northern blot analysis demonstrated a moderate increase (1.7 times) in TGF-beta 1 expression 7 days postoperatively; TGF-beta 1 express ion returned there after to near baseline levels. T beta-RII mRNA expressio n was downregulated shortly after osteotomy but then increased, reaching a peak of 1.8 times the baseline level on postoperative day 9. Osteocalcin mR NA expression was dramatically downregulated shortly after osteotomy and re mained low during the early phases of fracture repair. Osteocalcin expressi on trended slowly up upward as healing continued, reaching peak expression by day 37 (1.7 times the control level). In contrast, collagen type I alpha I mRNA expression was acutely down-regulated shortly after osteotomy, peak ed on postoperative days 5, and then decreased at later time points. Histologic samples from animals killed 3 days after osteotomy demonstrated TGF-beta 1 protein localized to inflammatory: cells and extracellular matri x within the fracture gap, periosteum, and peripheral soft tissues. On post operative day 7, TGF-beta I staining was predominantly localized to the ost eotomized bone edges, periosteum, surrounding soft tissues, and residual in flammatory: cells. By postoperative day 37, complete bony healing was obser ved, and TGF-beta I staining was localized to the newly formed bone matric. and areas of remodeling. On postoperative clq 3, T beta-RII immunostaining localized to inflammatory cells within the Fracture gap, periosteal cells, and surrounding soft tiss ues. By day 7, T beta-RII staining localized to osteoblasts of the fracture gap but was most intense within osteoblasts and mesenchymal cells of the o steotomized bone edges. On postoperative day 37, T beta-RII protein was see n in osteocytes, osteoblasts, and the newly formed periosteum in the remode ling bone. These observations agree with those of previous in vivo studies of endochon dral bone formation, growth, and healing. In addition, these results implic ate TGF-beta I biological activity in the regulation of osteoblast migratio n, differentiation, and proliferation during mandibular fracture repair. Fu rthermore, comparison of these data with gene expression during mandibular distraction osteogenesis may provide useful insights into the treatment of poorly healing fractures because distraction osteogenesis has been shown to be effective in the management of these difficult clinical, cases.