Injuries to the marrow cavity result in rapid endosteal bone formation followed by bone remodeling and regeneration of the marrow. It is not known whether this process is affected by age, although the quality of marrow is markedly different in young and old animals. Whereas young animals have red marrow and comparatively high levels of mesenchymal stem cells, old animals have yellow marrow characterized by increased levels of fat cells and they have fewer mesenchymal stem cells. To test if marrow restoration differs as a function of age, we used the rat tibial bone marrow ablation model, which has been used to examine calcification during osteogenesis, effects of metal implants on osteointegration and remodeling of bone graft substitutes during marrow cavity restoration. These previous studies were conducted in 3-month old immunocompetent rats but analysis of many biomaterials requires the use of immune deficient animals;​ however, it is not known whether this will affect the healing process. Accordingly, we assessed bone marrow healing in nude rats aged 1 month, 3-months and 10-months using micro-CT and histomorphometry, and compared the results to our previous work using Sabra strain rats. Thus, we determined if restoration of bone marrow is age dependent;​ if differences in healing can be detected by micro-CT;​ if the quality of marrow differs in young and old rats;​ and if the time course of healing in 3- month immunocompromised animals is comparable to that seen in normal rats of the same age. After determining that there was a difference in the kinetics of the healing of aged rats we tested a new biomaterial which is suppose to induce the healing in aged rats. Porcine Bone Marrow Matrix (PBMM), is a material produced by decellularizing porcine bone marrow matrix. Boston Scientific provided it to us in a sterile liquid suspension. We hypothesized that PBMM would provide a growth factor enriched scaffold that would enhance repopulation of the marrow cavity with multipotent stem cells.

Methods:​ Marrow was ablated in the left tibia of seven rats (rNu/rNu) per time point. At 0, 7, 14, 21, 28, 35 and 42 days post-surgery, the treated tibia and the contralateral tibia were harvested, fixed in 70% ethanol for 24 h and post-fixed in buffered formalin. Both tibias were scanned using microCT and trabecular BV/TV calculated. Mid-sagittal sections of decalcified paraffin embedded bones were stained with haematoxylin and eosin. BV/TV was calculated using ImagePro. Left tibias from untreated animals were used as controls for histology. Using this software we were also able to determine fat cell number per marrow cavity. Pilot study using PBMM was also conducted in aged rats and was part of the aged group study. From this pilot study we chose one time point to test the PBMM as an enhancer of bone marrow restoration. For this study we used five different groups testing the PBMM in a dose dependent study. As mention above, left tibia was treated while the right served as a control and was not treated.

Results:​ Micro-CT analysis for the 1-month animals showed high increase in bone formation and on day 21 the marrow was restored. High increase in bone formation was also noticed in 3-month animals on days 7 and 14, however their bone formation was significantly lower compared to 1-month old animals. By day 21 remodeling had reduced the area of trabecular bone by 50%. 10-month animals had less trabecular bone at days 7 and 14, the levels were sustained through 21 days. Histomorphometry indicated that bone formation peaked at day 7 in 1-month old rats with remodeling underway by day 14, as noted previously for Sabra strain rats. For 3-month old rats bone formation peaked at day 7 as well, but restoration occurred only on day 21. However, in 10-month rats, peak bone formation occurred on day 14, with remodeling on day 28. In the pilot study of PBMM we got promising results for enhanced bone marrow restoration as we saw a significant decrease in trabecular bone when compare to day 42 control (No PBMM). However, when we proceed to the next study did not see this effect again. PBMM had an effect on the marrow cavity but it was not significantly different than with our control group.

Discussion:​ The significance of this study is in the development of a model that enables us to view bone marrow restoration in aged rats. Using this model we can examine various materials such as implants and bone grafts to learn more about bone recovery processes in general and also to test materials that might induce healing in older populations, in particular. As science advances so does human life expectancy. The aged population around the world is growing;​ in order to guaranty quality of life one will need to fully understand bone healing in elderly populations. Conclusions:​ Endosteal bone formation and remodeling in 3-month nude rats is comparable to 3-month immunocompetent rats. Aged animals produce less primary bone that younger animals and remodeling is initiated later. Differences in micro-CT and histomorphometric analyses may reflect a reduction in calcification of the osteoid in the 10-month old animals. PBMM des not appear to enhance marrow restoration in aged rats.