טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentRachmiel Adi
SubjectCharacterization of Membranous Bone Regeneration
Following Osteotomy and Gradual Distraction.
Effect of TGF-Beta on the Osteogenesis
Process
DepartmentDepartment of Medicine
Supervisor Assistant Professor Dina Lewinson


Abstract

Distraction osteogenesis is a method of generating new bone by gradual distraction. In the first stage of the study a maxillary osteotomy was performed in 5 sheep. An external distraction device was used for maxillary lengthening of 40mm at a rate of 1mm/day twice a day for 21 days. New bone was evident clinically, radiographically and histologically. At the end of lengthening slender bone trabeculae rimmed by osteoblasts were observed. TRAP histochemistry demonstrated the bone remodeling. The trabeculae thickened later, with a mixture of lamellar and woven bone and 1 year later resembled the non distracted bone.

In the second stage, after the same procedure, in 5 sheep, on the fourth postoperative day before lengthening, 2mg rhTGF-b were injected to one side of the distracted maxilla. The purpose of the study was: 1. To follow the early events in bone formation and revascularization during maxillary distraction. 2. To study the influence of rhTGF-b on the bone regeneration process. The methods used for analysis were histological, histochemical, histomorphometrical, immunohistochemical, and ultrastructural.

A fibrin clot is formed during the 5 days of latency, and after 5 days of distraction was replaced by mesenchymal cells with high proliferation rate and capillaries. After 10 days of distraction the regenerated tissue can be divided into three zones: (1) a central zone - occupied by polygonal mesenchyme-like cells and spindle-shaped cells (mesenchymal area or proliferative area). (2) A paracentral zone - an intense apoptotic process leads to decreased number of cells embedded in wavy collagen fibers (fibroblastic area or collagenous area). In the transition from the central to the paracentral zone, condensations of mesenchymal cells and onion-like configurations embedded in basement membrane can be seen. (3) Most distally and proximally in continuation with old bone, delicate new woven bone trabeculae which became aligned with osteoblasts. Their tips recruit preosteoblasts from the distracted tissue and these further differentiate into osteoblasts which contribute to the trabecular growth. The trabeculae get gradually mineralized. Immunohistochemical staining by specific antibodies to markers of endothelial cells, Tie-2 and FVIIIrAg, demonstrated positive staining also of the mesenchymal condensations, the inner cells of the onion-like configurations, the preosteoblasts and the osteoblasts. On the other hand, the skeletal marker PTHrP stained most mesenchymal, endothelial cells and osteogenic cells. At the area that was injected with TGF-b1 we observed an earlier trabecular formation with increased recruitment of preosteoblasts at the edges. Morphometric analysis indicated that the surface area of the tissue occupied by bone trabeculae was larger in the TGF-b-injected side than in the control side.

Conclusions:
1. The distraction force creates a pool of undifferentiated mesenchymal cells with osteogenic potential and capillary formation. 2. The common antigenicity of two specific endothelial markers: Tie-2, FVIIIrAg and a skeletal marker PTHrP indicates that vascular cells may contribute to osteogenic cells. 3. The onion-like configurations might be unique for membranous bone regeneration. 4. A single dose of TGF-b at the end of latency period is sufficient to accelerate the bone formation process. This fact makes possible clinical application for improving bone quality and for shortening the time of device-bearing by the patients.