The periosteum is a specialized connective tissue comprising of two layers and covering the outer surface of bone tissue. The outer fibrous layer contains mainly fibroblasts and elastic fibers. The inner osteogenic layer contains osteogenic progenitor cells of mesenchymal origin, including osteoblasts and preosteoblasts.

The periosteum displays various modalities of bone formation upon surgical stimulation. Radden and Fullmer, 1969 B. Radden and H. Fullmer, Morphological and histochemical studies of bone repair in the rat, Arch Oral Biol 14 (1969), pp. 1243–1252. Abstract | View Record in Scopus | Cited By in Scopus (4)Radden and Fullmer (1969) reported that mesenchymal cells in the periosteum show chondrogenic differentiation in a rat bone fracture model. Ueno et al. (2001) described how grafted periosteum taken from the tibia induce endochondral ossification in the muscle. Kagawa (2002) demonstrated that calvarial periosteum immediately differentiates into osteoblastic cells during the process of callus distraction in the rat. Sasano et al. (1999) noted that physical contact between periosteum and bone matrix induces osteoblastic differentiation of periosteal cells in a calvarial experimental model. In the embryonic stage, limbs were formed by endochondral ossification, whereas calvaria was formed by intramembraneous ossification. During development, the periosteum determined the skeletal form in bone remodeling.

Although the periosteum shows various bone forming patterns depending on the affected skeletal region and the healing conditions of the fracture, there have been no reports yet on the pattern of bone formation of released periosteum after surgical stimulation.

Differentiation and proliferation of osteogenic and chondrogenic mesenchymal cells are regulated by molecular signals. Sox9 is one of those molecular signals, and is a transcription factor belonging to the SRY family of high-mobility box proteins. In mice, Sox9 expression is present during the development of limbs by endochondral ossification in fibroblasts, prechondroblasts and chondrocytes, but absent in hypertrophic chondrocytes. Sox9 is also needed for chondrogenic mesenchymal cell condensation after chondrogenic differentiation and proliferation. Using mouse chimeras of normal and Sox9-deficient embryonic stem cells, the expression of Sox9 has been shown to be essential for chondrogenic differentiation and expression of type-2 collagen. These findings indicate that Sox9 plays a crucial role in the chondrogenic differentiation stage of periosteal endochondral ossification. However, the role of Sox9 in bone formation after periosteum release has not yet been elucidated.

Differences in periosteal bone formation on tibia and calvaria have not been revealed hitherto. Therefore, we have used the released periosteum model instead of the fracture model, because with the released periosteum model we can concentrate on the supply of osteo/chondrogenic cells from periosteum, whereas with fracture bone models, we would have to examine the supply of osteo/chondrogenic cells from bone marrow and endosteum. We used Sox9 as the pheno-type marker of prechondroblastic fibroblasts, and type-2 collagen as a marker of chondrocytes and hypertrophic chondrocytes. In the present study, we examined the immunohistochemical expression of Sox9 and type-2 collagen during the process of new bone formation after the surgical release of the periosteum from both tibia and calvaria.

An experimental group of 56 adult male Sprague–Dawley rats (300–400 g body weight) was obtained from Clea (Japan), was housed at 25 °C and fed a standard animal diet (Oriental, Osaka, Japan) and water ad libitum for 1 week prior to surgery.

The rats were randomly assigned to one of two test groups. One group was composed of 28 rats. All surgical procedures were performed under anesthesia with the use of sodium pentobarbital (10 mg/kg body weight). In group 1, surgery was performed on the tibia. A straight skin incision of approx. 20 mm long was made aseptically along the anterior border of the tibia. An incision was made on the periosteum and the flap was gently released. The periosteum and skin flaps were repositioned on the bone surface and sutured. In group 2, the same procedure was performed on calvaria. An elliptical skin incision was made along the temporal line. An incision was made on the periosteum and the periosteal flap was released. The skull bone was exposed through the periosteal flap. The flaps were repositioned to cover the bone and sutured on the calvaria.

Tibial and calvarial tissues were removed and fixed in 10% neutrally-buffered formalin, and then decalcified in 10% ethylene diaminetetraacetic acid (EDTA) for 14 days. The tissues were subsequently dehydrated using a graded ethanol series and embedded in paraffin. Six-?m-thick sections were cut and stained with hematoxylin and eosin.

The sections were immersed in a solution of 0.3% hydrogen peroxide for 15 min in order to inhibit endogenous peroxidase before blocking with 10% bovine serum in phosphate-buffered saline (PBS) for 30 min at room temperature. To reveal Sox9 and type-2 collagen expression, sections were then exposed to rabbit polyclonal anti-Sox9 antibody (Chemicom, Temecula, CA, USA) and rabbit polyclonal anti-type-2 collagen antibody (LSL, Japan), respectively, diluted 1:100 in PBS containing 3% bovine serum albumin (BSA) at 4 °C overnight. After incubation, sections were exposed to horseradish peroxidase (HRP)-labeled rat anti-rabbit IgG diluted 1:100 in PBS containing 3% BSA for 60 min at room temperature. After washing in PBS, sections were incubated for 5 min at room temperature in a medium containing 0.05% 3,3?-diaminobenzidine tetrahydrochloride, 0.01% hydrogen peroxide and 0.05 M Tris–HCl (pH 7.6), for visualization of immunoreactivity. The sections were counterstained with hematoxylin. As negative controls, sections were incubated in a solution of 3% BSA in PBS-lacking primary antibody.

After decalcification, specimens were post-fixed in 1% osmium tetroxide buffered with 0.1 M cacodylate (pH 7.4) for 60 min, dehydrated using a graded acetone series and embedded in Epon 812. Ultrathin sections were stained using uranyl acetate and lead citrate for fine structural observation using a Hitachi-800 electron microscope at an accelerating voltage of 100 kV.

Periosteum covered all bone surfaces as connective tissue consisting of a fibrous and an osteogenic layer both on tibia and calvaria, the fibrous layer consisted of fibroblastic cells separated by large bundles of collagen and elastic tissue fibers. The osteogenic layer was made up of osteoblasts and preosteoblasts. The fibrous layer of calvaria was thinner than that of tibia. Periosteal cells on tibia resembled cells on calvaria in histological appearance.

After surgery, the released periosteum comprised of the fibrous layer and part of the osteogenic layer. There were no osteogenic cells on the bone surface at the periosteum release site. At 3 days, the periosteum had changed into fibrous tissue. Many erythrocytes and inflammatory cells were presented. At 7 days, periosteum had undergone changes at the release site, displaying loose connective tissue. New bone formation by endochondral ossification was observed in this inflammatory tissue. These periosteal cells of the fibrous layer were markedly more abundant and had differentiated into chondroblasts and chondrocytes. Fibroblasts derived from periosteal cells displayed differentiation and proliferation. These chondrocytes and chondroblasts had formed a cartilage mass. At 14 days after surgery, periosteal bone formation appeared as a combination of intramembraneous and endochondral ossification. Chondrogenic cells derived from the periosteum had formed a cartilage mass. Hypertrophic and mature chondrocytes formed new external callus cartilage, which in turn was replaced by newly formed trabeculae of cancellous bone undergoing endochondral ossification. At 28 days after surgery, all newly formed cartilage had been changed into trabecular bone. The new trabecular bone was covered with a thick layer of fibrous tissue and consisted of immature bone.