The Skeletal Biology Section has continued to study post-natal skeletal stem cells (SSCs, also known as """"""""mesenchymal"""""""" stem cells). SSCs are a subset of cells found in the bone marrow stromal cell (BMSC) population, capable of forming bone, cartilage, the stroma that supports blood formation and marrow fat cells. Based on the remarkable ability of SSCs to regenerate a complete bone/marrow organ, a major goal has been to further develop our techniques for bone regeneration in a number of pre-clinical studies in preparation for clinical trials in human patients. However, differences between SSCs and BMSCs derived from bones that develop from different anatomical origins; e.g., of the axial/appendicular skeleton that develop from mesoderm and of the craniofacial skeleton that develop from neuroectoderm, are not well known. In consideration of the use of BMSCs for human craniofacial reconstruction, initial studies addressing this issue were completed. ? ? Canine Cranial Reconstruction Using Autologous Bone Marrow Stromal Cells:? ? Initiation of clinical studies using autologous BMSC transplantation requires the demonstration of effective bone formation in sizable transplants in a large animal model as well as noninvasive techniques for evaluating transplant success. To address these issues, we obtained bone marrow from the femora of six dogs and expanded BMSCs in tissue culture. Autologous BMSC-hydroxyapatite/tricalcium phosphate (HA/TCP) transplants were introduced into critical-sized calvarial defects (ones that would never heal on their own) and contralateral control skull defects received HA/TCP vehicle alone. At intervals ranging from 2 to 20 months, transplants were biopsied or harvested for histological and mechanical analysis. Noninvasive studies, including quantitative computed tomography scans and ultrasound were simultaneously obtained. In all animals, BMSC-containing transplants formed significantly more bone than their control counterparts. BMSC-associated bone possessed mechanical properties similar to the adjacent pre-existing bone, confirmed by both ultrasound and ex vivo analysis. Evaluation by quantitative computed tomography confirmed that the extent of bone formation demonstrated by histology could be discerned through noninvasive means. These results show that autologous cultured BMSC transplantation is a feasible therapy in clinical-sized bone defects and that such transplants can be assessed non-invasively, suggesting that this technique has potential for use in patients with certain types of bone defects.? In Vivo Bone Formation By Human Bone Marrow Stromal Cells - Reconstruction of the Mouse Calvarium and Mandible:? ? Previously, we showed that mouse BMSCs, combined with a collagen carrier, can close critical-sized mouse cranial defects, but that this new bone had a poor union with the adjacent bone. When human BMSCs are transplanted into immunocompromised mice (to avoid graft versus host rejection) for the purpose of engineering new bone, best results were achieved if the cells are combined with hydroxyapatite/tricalcium phosphate particles (HA/TCP), rather than with collagen sponges. We demonstrated that transplantation of cultured human BMSCs in conjunction with HA/TCP particles can be used to successfully close mouse craniofacial bone defects, and that removal of the periosteum from the cranial bone significantly enhanced union with the transplant. Transplants were followed for up to 96 weeks and were found to change in morphology but not bone content after 8 weeks; this constituted the first description of the use of human BMSCs placed long-term to heal bone defects. New bone formation continued to form in the oldest transplants, confirmed by tetracycline labeling, due to normal bone turnover. Additionally, the mechanic properties (as determined by measurement of the elastic modulus) of this engineered bone resembled that of the normal mouse calvarium, and our use of atomic force microscopy (AFM)-based nano-indentation offered us the first opportunity to compare these small transplants against equally minute pieces of mouse bone. Similar transplants were also placed onto the mandible to determine if our technique could also be used for alveolar ridge augmentation. These transplants also formed bone and bone unions, but to a lesser degree than what were seen in the calvarium. Further development will be needed in order to improve the use of BMCS/HA/TCP constructs for this type of application. However, our results provide insights into the long-term behavior of newly engineered orthotopic bone from human cells, and further refine our ability to regenerate bone with good integration at the margins of the defect. ? ? Differences Between Bone Marrow Stromal Cells From Different Anatomical Sites: ? ? Autologous grafts from axial and appendicular bones are commonly used to repair orofacial bone defects, but often result in unfavorable outcomes. And as, noted above, BMSCs derived from the appendicular skeleton were less effective in regenerating alveolar bone than cranial bone. These observations, along with the fact that many bone abnormalities are limited to craniofacial bones, suggests that there are significant differences in bone metabolism in orofacial, axial and appendicular bones. It is plausible that these differences are dictated by site-specificity of embryological progenitor cells and osteogenic properties of resident multipotent human bone marrow stromal cells (BMSCs). This study investigated skeletal site-specific phenotypic and functional differences between orofacial (maxilla and mandible) and axial (iliac crest) BMSCs in vitro and in vivo. Primary cultures of maxilla, mandible and iliac crest BMSCs were established with and without osteogenic inducers. Site-specific characterization included colony forming efficiency, cell proliferation, life span before senescence, relative presence of surface markers, adipogenesis, osteogenesis, and transplantation into immunocompromised mice to compare bone regenerative capacity. Compared with iliac crest BMSCs, orofacial BMSCs (OF-BMSCs) proliferated more rapidly with delayed senescence, expressed higher levels of alkaline phosphatase and demonstrated more calcium accumulation in vitro. Cells isolated from the three skeletal sites were variably positive for STRO 1, a marker of BMSCs. In vitro, iliac BMSCs (I-BMSCs) were more responsive to osteogenic and adipogenic induction. In vivo, OF-BMSCs formed more bone, while I-BMSCs formed more trabecular bone that included hematopoietic tissue. These data demonstrate that BMSCs from the same donor differ in vitro and in vivo in a skeletal site-specific fashion and identified orofacial marrow stromal cells as unique cell populations. Further understanding of site-specific properties of BMSCs and their impact on site-specific bone diseases and regeneration are needed.? ? Human ES Cells? ? We have now successfully established the techniques to grow the Presidentially approved hES cell line, HSF6, in an undifferentiated state. The cells are now being analyzed for chromosomal abnormalities before we begin our studies. These cells will be used to incorporate mutations in genes of interest (in particular, GNAS, the gene that is mutated in Fibrous Dysplasia of Bone and the McCune-Albright Syndrome) by molecular recombineering techniques to generate a human based model system for the study of disease.
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