Angiogenesis and growth of isografted bone: quantitative in vivo assay in nude mice.
BACKGROUND
Understanding the regulation of vascularization and formation 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 facilitates the in vivo assessment of angiogenesis and growth of bone.


METHODS
Fresh, cryopreserved (frozen in the presence or absence of 10% dimethyl sulfoxide (DMSO)) or boiled neonatal femora were transplanted into dorsal skin fold chambers in adult mice of the identical strain, and angiogenesis and growth were monitored over 16 days. Computerized analysis of brightfield and epifluorescence images was employed to characterize the process of angiogenesis. Bone formation was quantified in vivo by the use of oxytetracycline.


RESULTS
Reperfusion of pre-existing blood vessels of the graft was observed only in fresh transplanted femora, whereas femora of all experimental groups elicited angiogenic response from the host tissue. The rank order of the angiogenic 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 microns/day) and in cartilage diameter (41 microns/day).


CONCLUSIONS
Our study demonstrates that (a) angiogenesis and growth of transplanted femora can be chronically assessed using in vivo microscopy; (b) the introduction of oxytetracycline for in vivo fluorescence microscopy allows the differential quantification of bone and cartilage growth; and (c) cryoprotection using DMSO enhances restoration of angiogenic potency after freezing. We consider this assay an excellent experimental model to study in vivo effects of agents or procedures that potentially modulate angiogenesis and growth of bone.
Leunig
M
M
Steele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston.
Yuan
F
F
Berk
D A
DA
Gerweck
L E
LE
Jain
R K
RK
D000818:Animals; D001846:Bone Development; D016025:Bone Transplantation; D001842:Bone and Bones / Q000098:blood supply* / Q000187:drug effects*; D015925:Cryopreservation; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005269:Femur / Q000098:blood supply / Q000254:growth & development / Q000637:transplantation; D006358:Hot Temperature; D008297:Male; D008722:Methods; D051379:Mice; D008819:Mice, Nude; D009389:Neovascularization, Pathologic / Q000503:physiopathology*; D014185:Transplantation, Isogeneic
D004121:Dimethyl Sulfoxide