METHODS IN LABORATORY INVESTIGATION - ANGIOGENESIS AND GROWTH OF ISOGRAFTED BONE - QUANTITATIVE IN-VIVO ASSAY IN NUDE-MICE

BACKGROUND: Understanding the regulation of vascularization and format ion of bone after skeletal trauma is essential for the development of methods to promote healing. The lack of information on the biology of bone healing led us to establish an experimental model that facilitate s the in vivo assessment of angiogenesis and growth of bone. EXPERIMEN TAL DESIGN: Fresh, cryopreserved (frozen in the presence or absence of 10% dimethyl sulfoxide (DMSO)) or boiled neonatal femora were transpl anted into dorsal skin fold chambers in adult mice of the identical st rain, and angiogenesis and growth were monitored over 16 days. Compute rized analysis of brightfield and epifluorescence images was employed to characterize the process of angiogenesis. Bone formation was quanti fied in vivo by the use of oxytetracycline. RESULTS: Reperfusion of pr e-existing blood vessels of the graft was observed only in fresh trans planted femora, whereas femora of all experimental groups elicited ang iogenic response from the host tissue. The rank order of the angiogeni c response was: fresh > cryopreservation with DMSO > cryopreservation without DMSO > boiled. Growth of femora was completely abolished after cryopreservation or boiling. Only fresh transplanted femora increased in length (95 mu m/day) and in cartilage diameter (41 mu m/day). CONC LUSIONS: Our study demonstrates that (a) angiogenesis and growth of tr ansplanted femora can be chronically assessed using in vivo microscopy ; (b) the introduction of oxytetracycline for in vivo fluorescence mic roscopy allows the differential quantification of bone and cartilage g rowth; and (c) cryoprotection using DMSO enhances restoration of angio genic potency after freezing. We consider this assay an excellent expe rimental model to study in vivo effects of agents or procedures that p otentially modulate angiogenesis and growth of bone.