Abstract An in vivo system of membranous bone for- mation during distraction has been investigated in order to follow cells that express vascular markers with the ob- jective of understanding the neovascularization process. Concomitantly, sustained proliferation of preskeletal cells was achieved through the application of mechanical force. New capillaries and leading edges that arose by angiogenesis from the periosteal and mucosal surfaces and invaded the central zone of the regenerating distrac- tion tissue temporally preceded the growth of delicate woven bone trabeculae from both edges of the cut bone. Concentrically arranged ‘onion-like’ configurations were abundant in paracentral zones and in association with mesenchymal condensations, suggesting their de novo formation in situ. Vascular specific markers, the angio- poietin receptor Tie-2 and factor VIII-related antigen (FVIIIrAg), were localized immunohistochemically in order to follow cells of vascular origin. Endothelial cells of the new capillaries, centrally located cells of the concentric configurations, pericytes, and most of the adjacent polygonal mesenchymal cells stained positively with specific antibodies to both antigens. Moreover, pre- osteoblasts and osteoblasts that lie adjacent to or already embedded in the osteiod of the newly formed trabeculae were also FVIIIrAg and Tie-2 immunopositive. As the source of the bone-forming cells in regenerating tissue during distraction is not yet fully understood, this observation might support the possibility of their vascular origin. Keywords Distraction osteogenesis · Tie-2 · Factor VIII-related antigen · Osteoblasts · PTHrP Introduction Distraction osteogenesis of facial membranous bones pro- vides an extraordinary new in vivo system of bone forma- tion. In this system, bone is generated by stretching a cal- lus that develops following osteotomy of midfacial bones. In earlier studies of our group we have described the early and late events in the development of the regenerating bone and have already noticed the intensive neovascular- ization process that accompanies the osteogenic one (Rachmiel et al. 1998). We observed three different modes of revascularization of the tissue that regenerated during the applied distraction force: venous sinusoids sprouted from the edges of the cut bone parallel to the growing new bone trabeculae, new capillaries sprouted by angiogenesis from the periosteal and mucosal surfaces into the centrally located area of the distraction tissue which is mainly pop- ulated by mesenchyme-like cells, and lumen-devoid cel- lular complexes encapsulated in basement membrane, that we termed ‘onion-like’ configurations, were ob- served mainly in paracentral zones that were populated with fibroblast-like cells secreting a collagen-rich matrix (Rachmiel et al., manuscript submitted for publication). In the latter study we suggested that microvessels were formed in situ by a novel mode of vascularization as sprouts from the ‘onion-like’ configurations that make contact with capillaries that arose by angiogenesis. In the present study we bring supportive evidence for the vascu- lar nature of these special cellular complexes and new evidence underscoring an old hypothesis of a possible vascular origin for osteoprogenitors. In the regenerating tissue we looked for the protein expression of the recep- tor for angiopoietin-1, theTie-2 receptor tyrosine kinase, and for factor VIII-related antigen (FVIIIrAg). Both were used as accepted markers for vascular cells (Han et al. 1992; McComb et al. 1982; Mukai et al. 1980; Partanen and Dumont 1999; Sato et al. 1995; Suri et al. 1996). A. Rachmiel ( ✉ ) 103, Yefe-Nof Street, Haifa 34454, Israel e-mail: adi_rach@netvision.net.il Tel.: +972-4-8421443, Fax: +972-4-8428126 D. Lewinson · G. Maor · N. Rozen · S. Stahl · A. Rachmiel Department of Anatomy and Cell Biology, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel I. Rabinovich · A. Rachmiel Department of Oral and Maxillofacial Surgery, Rambam Medical Center, Haifa, Israel Histochem Cell Biol (2001) 116:381–388 DOI 10.1007/s004180100331 ORIGINAL PAPER Dina Lewinson · Gila Maor · Nimrod Rozen Iaron Rabinovich · Shay Stahl · Adi Rachmiel Expression of vascular antigens by bone cells during bone regeneration in a membranous bone distraction system Accepted: 27 August 2001 / Published online: 18 October 2001 © Springer-Verlag 2001