Through our experimental study we found that the decrease in bone quality typically following limb transplant depends in part on recipient factors. As measured by micro-CT, all parameters of bone quality were significantly better in limbs transplanted between juvenile rats than between limbs transplanted from juvenile rats to adult rats, indicating that age related alterations in the hormonal milieu in the adult rats resulted in inferior bone quality in the transplanted limbs. To our knowledge, measuring bone quality using 3-D micro-CT, and comparing bone growth simultaneously, was performed for the first time in this study, which makes this study unique.
Kline et al.  noted that osteoporosis occurred following limb transplantation between animals of different ages; however, that study primarily dealt with the growth characteristics of growth plates following limb transplantation, without establishing the relationship between the degree of osteoporosis and the age of the recipient animal. They attributed the post transplantation osteoporosis to factors relating to both the non-weight-bearing, and lack of external stress conditions following limb transplantation. However, their hypothesis may be incorrect, because in a previous hindlimb transplanted rat model there were no significant differences in functional improvement between rats that received physiotherapy and rats that did not, except for foot drop . However, they did not evaluate bone quality in this study.
From the results of our experimental study we found that host dependent age related hormonal factors play a vital role in the etiopathogenesis of post transplantation osteoporosis. Gourian et al.  showed that age related changes in bone mineral content (BMC) are due to the mineralization process itself and not imbalance in the remodeling process. Tissue age can vary within the same bone specimen due to reabsorption of bone by osteoclasts and formation by osteoblasts. Juvenile bone placed in an adult hormonal environment (heterochronografts) suffers much greater bone loss than juvenile bone placed in a juvenile hormonal environment (isochronografts). Of course, other factors such as circulation, neuronal control, bodily responses to stress and transplantation also play roles in maintaining the bone quality of transplanted allogenic bone from the donor.
The results of the above experiment suggest that even after transplanting the limbs of juvenile animals into adult animals (heterochronografts), the growth plates in the transplanted limbs retained their properties of longitudinal growth and continued to grow at the same rate in the new adult environment as they would have in the juvenile environment. The increase in length of the heterochronograft limbs was not significantly different from the increase in length of the isochronograft limbs. In addition, the increase in length of the isochronograft limbs was not significantly different from the increase in length of the non-operated contralateral hind limbs. Our results are similar to those of Kline et al. , who reported that a mature hormonal environment does not inhibit the longitudinal growth of immature growth plates. Kline et al. observed maintenance of growth in juvenile limbs transplanted into adult rats. They also studied growth plate morphology in transplanted limbs, and observed that all transplanted limbs demonstrate maintenance of growth plate morphology and columnar organization . However, they did not assess the bone quality among these groups.
The increase in length of the heterochronograft limbs was, however, significantly greater than the increase in length of the non-operated contralateral hind limbs of the adult rats. In other words, after the age of 3 weeks, the internal environment of the host ceases to have a decisive role in the determination of the growth characteristics of the growth plate, and the increase in length of the growth plate is primarily determined by local transplantable factors that are expressed prior to transplantation by interactions between the inducing factors and inherited genomes. We can explain the increase in length of the adult non-operated limbs by the fact that the growth pattern in rats differs from that in humans, in that the growth plates in rats remain open later into adult life, though the growth rate at 10 weeks of age is a fraction of the rate at 3 weeks of age [21, 22]. The temporal analysis of rat growth plate shows cessation of growth with age, despite the presence of a physis . For this reason, we should be cautious regarding blind extrapolation of these results to humans, and rather, emphasize that these findings need to be confirmed in a clinical setting. Chiu et al. , in a similar study involving limb transplantation between animals of different ages, observed that the transplanted bone achieved only 70% of the normal growth in length. This finding was corroborated by Drzewiecki et al.  who also found that after limb transplantation the transplanted bone could not achieve normal growth potential, but noted that the maximum growth (91% of normal) was observed in heterochronografts. However, in both of these studies the nerves of the transplanted limbs were not sutured, and the animals were non-weight bearing, so the failure to achieve full growth could be attributed to the effects of denervation and lack of external stress. However, in our experiment blood vessels and nerves were sutured with microvascular anastomosis, minimizing the ischemic time, and therefore we could allow weight bearing in the subject group.
Stevens et al.  studied the growth of epiphyseal plate allografts after microvascular transplantation in rabbits of different ages, and found that the growth rate depended on the age of the donor epiphyseal plate and was independent of the age of the recipient. Glickman et al.  studied epiphyseal growth after microvascular transplantation to sites of different growth potential, and reaffirmed that growth potential of an epiphyseal plate transplant is a function of the donor, irrespective of the recipient site to which it is transplanted. Our report would further support their findings that the growth of epiphysis is an inherent property of the donor, while the quality of bone depends upon the internal environment of recipient's body.
There are reports in which microvascular epiphyseal transplants have been used to reconstruct the extremities of children whose epiphyseal plates were damaged or surgically removed as a result of disease or trauma. Vilkki  performed microvascular transplantations of the metatarsophalangeal joint with whole metatarsal bone in the treatment of radial club hand in nine children. At the average follow-up of six years they found that the deformity of the wrist had been reduced and growth of the ulna had been maintained due to intact functions of transplanted metatarsophalangeal joints. Innocenti et al [14–17] described the treatment of loss of the distal part of the radius, including the physis and epiphysis in skeletally immature patients, by performing vascularized proximal fibula transfers based on the anterior tibial artery. This included the physis, and a variable length of the diaphysis, and found a consistent and predictable longitudinal growth of the transferred fibula. On the basis of these findings, Innocenti et al. proposed that vascularized epiphyseal transfer is the only possible procedure that can solve the dual problem of replacement of osseous defect and restoration of longitudinal growth in the case of loss or damage to epiphyseal plates. Our report also supports the clinical implications of transferring intact joints with epiphyseal growth potentials on various congenital disorders, like epiphyseal dyplasia, tibial hemimelia, and psuedarthorosis of tibia (type IV), in which transplanted limbs could continue growth due to the intact inherent properties of epiphyseal growth plates from the donor, while bone quality would match the donor bone quality.
The major limitation of this study is that we did not use any control groups in our study, such as the comparison of bone and epiphyseal growth properties in a sham transplantation group. However, if we had used a control group with sham transplantation, all transplanted limbs would have necrosed due to lack of vacularity and lack of use. Another weak point is that we did not perform dynamic bone histometric parameters (after tetracycline labeling) of the healthy donor and recipient as well as of the transplanted limbs. We should have performed micro CT in the contralateral limbs of both groups to assess differences in limb bone quality. So far there are no clinical reports of allogenic transfers of growth plates. Additionally, the numbers of animals in recipient adult and juvenile groups were not equal and small, mainly due to limitations of funds. However, this is the first study in which micro CT was used to assess transplanted limb bone quality. Additionally we found consistent results in each experiment. Therefore we suggest further research on this issue, taking into consideration all of these weak points, to confirm our results. However, this study will provide useful information should such a procedure become feasible in the future.