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Muscle fascia and periosteum have been used clinically to guide prefabrication of vascularized bone flaps for reconstruction of complex three-dimensional tissues. Although it seems that both locations have the capacity to generate vascularized bone, there have been no studies that directly compare different implantation sites. The authors performed a rigorous, quantitative, histomorphometric comparison of bone prefabrication in a large-animal model comparing graft implanted against muscle fascia and periosteum.Twenty skeletally mature domestic sheep were implanted with rectangular chambers containing equal weights of morcellized bone graft. Two chambers were implanted into each sheep, one with the open face apposed to the cambium layer of the rib periosteum and the other with the open face apposed to the fascia of the latissimus dorsi muscle. Animals were euthanized at 3, 6, 9, 12, and 24 weeks and the chambers were harvested. Tissue inside the chambers was analyzed for shape conformation to chamber geometry, gross tissue volume, and bone histomorphology.There were no differences in volume or shape of tissue formed in the chambers. However, chambers in contact with fascia consisted almost entirely of fibrovascular tissue, with progressive resorption of the morcellized bone graft and little evidence of new bone. Chambers in contact with periosteum showed active endochondral, direct, and appositional bone formation over time, with increasing calcified tissue area and new bone formation.Both periosteum and muscle fascia were able to vascularize bone grafts, but bone formation was higher in the periosteum. The periosteum appears to be a more suitable foundation from which to promote flap prefabrication.