All animals experienced good postoperative recoveries: their surgical wounds healed well, and their microbiological tests showed no infection, because the implants were sterilized process by irradiated for sterilization. The cell samples from marrow aspirates (20–35 mL collected for each animal), for AOC 10 + and 14 + (animals with graft cellularized, ten and fourteen weeks before surgery), were obtained from the first pass, and for AOC 6 + and 18 + (animals with graft cellularized, six and eighteen weeks before surgery), were obtained from the second pass. In all cultures, regardless of the time of expansion, the cells had fusiform morphology characteristics of myofibroblastic cells. A similar process of culture isolation of bone marrow-derived mesenchymal stem cells was reviewed later and showed osteogenic and chondrogenic differentiation [8], which was different from the experimental cells.
The radiographic evolution of the grafts consolidation in the AOC 6+, 10 +, 14 +, and 18 + animals and the AOC 6 -, 10 -, 14 - and 18 - animals are illustrated in Figure 1. The parameters of the radiographic score of the two animals euthanized at 0, 4, 6, 10, 14 and 18 weeks are illustrated in Figure 2.
In both sheep, the grafts were aligned with the distal and proximal fragments (Figure 1A and D). After 4 weeks, the radiotransparent lines were less clear in the AOC 6 + animals because they were partially filled by new bone formation (Figure 1B). Bone formation in this region of the graft was significantly higher in the AOC 6- animals (Figure 1C) than in the animals treated with noncellularized grafts (Figure 1F).
Two months after surgery, in the AOC 10 + animals (Figure 1C), the graft was completely covered by new bone formation on the entire lateral surface. The osteotomy line with the host bone was practically imperceptible in most joints of the graft, indicating radiographic bone healing. The perforations of the cortical graft were partially filled by new bone formation, leaving few unconcluded holes. In the same period, for the AOC 10 - sheep (Figure 1G), the holes in the cortical graft and bone formation around the graft remained patent, although osteotomy was not significant and no callus formed a bone bridge between the graft and the host bone. The radiotransparent line remained visible in both outbreaks of osteotomy.
Two months after surgery, the AOC 14 + sheep (Figure 1C) showed large bone formations involving the lateral and medial osteotomy of the graft. Although the osteotomy lines were still evident, there was a formation of bony bridges joining the fragments in the proximal and distal focus. In the AOC 14 - sheep (Figure 1G), the bone formation was small and limited to the lateral focus of the proximal graft, but the osteotomy line was difficult to see due to the new bone formation.
In the AOC 18 + sheep, after three months (Figure 1D) the graft was completely consolidated and remodeled: it was no longer possible to identify the focus of the osteotomy. In the AOC 18 - sheep (Figure 1I), the osteotomy focus was consolidated proximally and distally and remained fully visible, indicating delayed consolidation. At 18 weeks, the cellularized grafts (Figure 1E) showed an advanced process of remodeling, with the thickening of the cortical region and partial reduction of the thickness of the callus formed at the foci of the osteotomy. For sheep in the control group (Figure 1J), although the graft was consolidated at both foci of the osteotomy, it was still possible to visualize the radiotransparent line in distal focus.
At the first analysis point (6 weeks), perforations were clearly identified in the cortical grafts, and no difference was observed between the two animals with regards to the filling of the holes by new bone formation. However, as in the radiographic evaluation, the formation of the callus and the periosteal reaction were more evident in the animals with cellularized grafts (Figure 2A and B, AOC 6 + and 6 -). At 10 and 14 weeks, the animals treated with cellularized grafts (AOC 10 + and 14 +) had holes that were less patent than the control animals (AOC 10 - and AOC 14 -) due to increased bone formation and, consequently, greater obliteration of the perforations (Figure 2A, B, C and D).
After 18 weeks, the perforations of the cortical bone of the recipient cellularized grafts (AOC 18+) were completely filled by new bone formation and were no longer displaying tomographic sections (Figure 3A to 3G following complete healing of the bone and comparative disappearance of holes dependent of therapy and 3H as gross cross section of new formed bone structure, AOC 18 + and 18 -).
Histopathological evaluation was performed on the entire length of the graft, including the interface with the host bone. To standardize the results and thus facilitate the comparative analysis of the bone repair process in the presence and absence of AOC, the histological findings were correlated between the animals euthanized at each of the four observation points.
After 6 weeks, in the AOC 6 + animal, the holes of the compact bone graft were filled and consisted largely of bone formation and structured thick beams interconnected with marked osteoblastic activity. In the peripheral portion of the hole, the neoformation of immature bone tissue predominated and was related with high osteogenic cell activity. In the compact bone graft, there were frequent outbreaks of osteoclastic resorption of both the Haversian and cortico-endosteal surface. Haversian resorption occurred in an organized manner, and the presence of cutting-cones was characteristic of bone remodeling. The main components of the medullary filling at this vantage point were tissues and beams of neoformed bone in similar proportions. The peri-trabecular tissue in the general aspect was loose and highly vascularized, morphologically similar to the bone marrow stroma (Figure 4A+ to D+).
For the AOC 6 - animal, interfaces with the host bone graft were filled by dense connective tissue and bone formation inside the hole penetrated into the medullary cavity. However, the main difference observed by the comparison of the AOC 6 - animal with the AOC 6 + animal was the composition of the tissue filling the medullary space (Figure 4A- to D-).
After 10 weeks of observation, the animals treated with cellularized grafts (AOC 10 +) had bone continuity in the region of the distal osteotomy. The compact bone graft was in the process of remodeling, and morphological structures of osteons were often identified. Although much of the graft that filled the medullary cavity consisted of bone formation, this was not uniform, and the beams filling the cavity were not structured because the interconnected distal edge of the graft and the host bone were joined by trabecular bone (Figure 5A+ to D+).
In the AOC 10 + animal, most of the tissue present in the marrow cavity of the graft was replaced by new bone. In the animal treated with a noncellularized graft (AOC 10 - ), extensive areas of connective tissue and fbro-vascular tissue was observed and just a little or no osteogenic activity, differently from results found for AOC 10+ (Figure 5A- to D-).
In the AOC 14 + animal, both interfaces of the graft were filled by newly formed bone, which established the union between the grafted bone and native bone. In the AOC 14 - animal, the medullary filling was not consistent and differed depending on the proximity to the edges of the host bone. In the regions immediately adjacent to the interfaces of the graft, the marrow space was filled mostly by dense connective tissue containing empty cavities and newly formed vessels (Figure 6A+ to D+ and A- to D-).
After 18 weeks, in the animals treated with cellularized grafts (AOC 18 +), the regions of the graft interfaces were no longer histologically identifiable. The compact bone graft in this region comprised morphological structures with deposits of osteons, concentric lamellar bone osteocytes and round vascular structures. With the completion of the process of consolidation and remodeling of the callus, the endosteal and periosteal surfaces showed no more cellular activity, lying covered with thin ledge osteoid unrelated osteoblasts that featured inactive or latent state bone surfaces (Figure 7A+ to D+).
The comparison between the two animals euthanized after 18 weeks showed significant differences in the pattern and temporal evolution of the bone healing process at the graft site. Unlike what was observed in AOC 18 + animals, where the remodeling of the bone callus had been completed, the AOC 18 – animal (Figure 7A- to D-) exhibited a periosteal reaction covering a large part of the graft, including the region of the osteotomies. This finding confirmed that the process of graft consolidation was still in progress.
Inside the graft tissue, there was no uniformity in the filling composition. In the central portion, the tissue was represented by beams of bone formed from the cortico-endosteal surface and the intramedullary tissue and by fibrovascular tissue unattached to osteogenesis. The filling in the less central tissue alternated between beams with thick bone cell activity foci, a good standard of connectivity and related connective tissue stroma with features of bone marrow fat and areas represented exclusively by intensely vascularized tissue, unrelated to training of bone.