Evidence for Reperfusion Injury in Cortical Bone as a Function of Crush Injury Ischemia Duration: A Rabbit Bone Chamber Study A. S. HSIEH, 1 H. WINET, 1,2 J. Y. BAO, 2 H. GLAS, 3 and H. PLENK, JR. 3 1 USC Department of Biomedical Engineering, University of Southern California, Los Angeles, CA, USA 2 Orthopedic Hospital and University of Southern California Department of Orthopedics, Los Angeles, CA, USA 3 Bone and Biomaterials Research, Institute for Histology and Embryology, University of Vienna, Vienna, Austria A model for critical limb ischemia was produced by occlud- ing femoral vessels in 24 rabbits with a pneumatic cuff for 0, 2, 4, or 6 hours. Immediate sequelae and subsequent creeping substitution of cortical bone were observed in vivo using an implanted tibial window, the optical bone chamber implant (with intravital microscopy), and then by light and fluores- cence microscopy of fluorochrome-labeled and surface- stained ground sections of retrieved implants. Six rabbits were used as controls (0 h) for each ischemia treatment, and the animals were monitored for 5 weeks postocclusion. A subpopulation of 13 implants was retrieved after euthaniza- tion and then histologically assessed for bone necrosis and remodeling. The hypothesis tested was that reperfusion in- jury during the 24 h after occluder release (reperfusion phase), and vessel perfusion/caliber, angiogenesis, and net bone resorption during the 5 subsequent weeks (creeping substitution phase), would exhibit ischemia duration-depen- dent effects. All animals could bear weight on the affected limb to ambulate by 1 week posttreatment. Two-way analysis of variance (ANOVA) comparison of the resulting data con- firmed a significant difference between control and ischemia- treated rabbits for: (1) vessel perfusion/reperfusion; (2) ves- sel caliber; and (3) net bone resorption. Vascular responses to 4 vs. 6 h of ischemia were not significantly different, but net bone resorption was strictly ischemia duration-dependent. The conclusion that reperfusion injury was the mechanism spreading ischemia to more vessels was supported by a decrease in reperfusion and caliber of vessels, and an in- crease in vascular permeability and leukocyte adherence during the reperfusion phase. It is postulated that reperfu- sion injury produces a secondary ischemia that amplifies the occlusion-created primary ischemia and, in the present work, may have been succeeded by progressive episodes of isch- emia, similar to the infarction pattern of ischemic hearts. (Bone 28:94 –103; 2001) © 2001 by Elsevier Science Inc. All rights reserved. Key Words: Ischemia; Reperfusion injury; Osteonecrosis; An- giogenesis; Bone resorption; Creeping substitution; Bone cham- ber; Intravital microscopy; Histomorphology. Introduction Ischemia has been identified as an etiological agent in a number of orthopedic clinical presentations, including: (1) failure of replants and vascularized transplants 14 ; (2) so-called avascular/ ischemic osteonecrosis (ION) of the femoral head and other bone regions 10 ; (3) functional deficits following tourniquet applica- tion 4,12 ; (4) compartment syndrome 11,17 ; and (5) crush injury. 19 With the exception of ION, skeletal muscle has been the primary focus for investigating the pathophysiology of these diseases because of its systemic sequelae, particularly myoglobinuria. An understanding of the effects of ischemia on cortical bone is critical not only for development of agents to reverse them, but for detecting stages of recovery that may be amenable to treat- ment. For example, dead bone is resorbed and replaced by new bone by a process called “creeping substitution.” If resorption occurs much faster than formation, a deficit in bone mass may develop, which weakens it mechanically. An agent that slows osteoclast activity or accelerates osteogenesis, delivered at a specific timepoint following an ischemic insult, may be war- ranted. In any case, limb ischemia is not a single-tissue concern and the effect of any treatment must be compatible with the recovery needs of the integrated structure. This study examines the effect of ischemia on one limb tissue, cortical bone. The tool used by this laboratory to study in vivo responses of tibial cortical bone is the optical bone chamber implant (BCI), which is an in situ window containing a compartment into which vessels and then bone grow and can be observed through an intravital microscope (IVM). Bone growth into the compartment differs from surrounding cortical bone in that it is a dense mixture of both woven and lamellar bone. 6 The BCI is related to the human “skin tube chamber” 15 and rabbit ear chamber 24 used to study ischemia. A histologic section of an in situ chamber is presented in Figure 1. Results from a preliminary BCI study indicate that reperfusion (return of blood flow to vessels) fol- lowing 10 h of primary ischemia (experimentally imposed oc- clusion) from common iliac artery compression was accompa- nied by a secondary ischemia (recurrence of blood flow deficit following a period of apparent flow recovery). The extent of secondary ischemia, coupled with abnormal vessel leakage and increased adherence of leukocytes to endothelium has suggested Address for correspondence and reprints: Dr. H. Winet, Orthopedic Hospital/UCLA, 2400 Flower Street, Los Angeles, CA 90007. E-mail: hwinet@laoh.ucla.edu Bone Vol. 28, No. 1 January 2001:94 –103 94 © 2001 by Elsevier Science Inc. 8756-3282/01/$20.00 All rights reserved. PII S8756-3282(00)00415-4