The role of stem cells in skeletal health and disease

National Institute Of Dental & Craniofacial Research

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Abstract

During the last fiscal year, the following advances were made: Biological Activity Skeletal stem cells/bone marrow stromal cells (SSC/BMSCs) have been shown to form cartilage in vitro by using a pellet culture type of assay. In these pellets, one can see bona fide chondrocytes lying in lacunae, surrounded by extracellular matrix that stains purple with toluidine blue (metachromasia). However, when pre-differentiated immature or mature pellets are transplanted in vivo, they are converted to a bone/marrow organ either through a hypertrophic phase as in the growth plate, or by direct remodeling without a hypertrophic phase. This strategy has failed to achieve formation of stable, hyaline-like cartilage, resistant to hypertrophy in vivo. We hypothesized that in vitro pre-differentiation of BMSCs is not necessary when cells are combined with an adequate scaffold that supports the formation of stable cartilage in vivo. In our recent study, nave (undifferentiated) human BMSCs were attached to dehydrothermally crosslinked stable fibrin microbeads (FMBs) without and with other scaffolds and transplanted subcutaneously into immunocompromised mice. Optimal formation of abundant, hypertrophy-resistant, ectopic hyaline-like cartilage was achieved when nave BMSCs were attached to FMBs covalently coated with hyaluronic acid. The cartilage that was formed was of human origin and was stable for at least 28 weeks in vivo (Stem Cells Translational Medicine 2019;8:586-592). Diseases Fibrous Dysplasia of bone/McCune-Albright Syndrome (FD/MAS, OMIM#174800) is a crippling skeletal disease caused by gain-of-function mutations of Gs-alpha. Enhanced bone resorption is a recurrent histological feature of FD and a major cause of fragility of affected bones. Previous work suggests that increased bone resorption in FD is driven by RANKL and some studies have shown that the anti-RANKL monoclonal antibody, denosumab, reduces bone turnover and bone pain in FD patients. RANKL, a mediator of osteoclast formation, is highly expressed by FD-BMSCs in bone lesions. However, the role of RANKL and osteoprotegerin (OPG, a soluble antagonist of RANKL) in FD pathophysiology is not yet understood. Serum levels of RANKL, OPG, and inactive RANKL-OPG complexes in FD patients with known disease burden and in normal volunteers were measured. RANK, RANKL, and Ki67 (a marker of proliferation) immunohistochemistry were assessed in FD tissue. Cultured FD and normal BMSCs were stimulated with prostaglandin E2 (PGE2 ) and 1,25 vitamin D3 to increase RANKL expression, and media levels of RANKL and OPG were measured. Osteoclastogenic induction by FD or normal BMSCs was assessed in co-cultures with normal peripheral monocytes. FD patients showed a 16-fold increase in serum RANKL compared with normal. OPG was moderately increased (24%), although the RANKL/OPG ratio was 12-fold higher in FD patients than normal. These measurements were positively correlated with the skeletal burden score (SBS), a validated marker of overall FD burden. No differences in serum inactive RANKL-OPG complexes were observed. In FD tissue, RANKL+ and Ki67+ fibroblastic cells were observed near RANK+ osteoclasts. High levels of RANKL were released by FD BMSCs cultures, but were undetectable in normal cultures. FD BMSCs released less OPG than normal BMSCs. FD, but not normal BMSCs, induced osteoclastogenesis in monocyte co-cultures, which was prevented by denosumab addition. These data are consistent with the role of RANKL as a driver in FD-induced osteoclastogenesis (J Bone Miner Res 2019;34:290-294). While the the above study was suggestive of an influence of denosumab on FD lesions, the effect of RANKL inhibition on the histopathology of FD and its impact on the natural history of the disease remained to be assessed. Consequently, the EF1-Gs R201C mice, which develop an FD-like phenotype, were treated with an anti-mouse RANKL monoclonal antibody. The treatment induced marked radiographic and microscopic changes at affected skeletal sites in two-month-old mice. The skeletal segments involved became sclerotic due to the deposition of new, highly mineralized bone within developing FD lesions and showed a higher mechanical resistance compared with affected segments from untreated transgenic mice. Similar changes were also detected in older mice with a full-blown skeletal phenotype. The administration of anti-mouse RANKL antibody arrested the growth of established lesions and, in young mice, prevented the appearance of new ones. However, after drug withdrawal, the newly formed bone was remodeled into FD tissue and the disease progression resumed in young mice. Taken together, our results demonstrate that the anti-RANKL antibody significantly affected the bone pathology and natural history of FD in the mouse. Pending further work on the prevention and management of relapse after treatment discontinuation, the pre-clinical study suggests that RANKL inhibition may be an affective therapeutic option for FD patients (J Bone Miner Res 2019 Epub ahead of print). Tissue Engineering Ectopic bone formation in mice is the gold standard for evaluation of osteogenic constructs. By regular procedures, usually only 4 constructs can be accommodated per mouse, limiting screening power. Combinatorial cassettes (combi-cassettes) hold up to 19 small, uniform constructs from the time of surgery, through time in vivo, and subsequent evaluation. Two types of bone tissue engineering constructs were tested in the combi-cassettes: i) a cell-scaffold construct containing primary human bone marrow stromal cells with hydroxyapatite/tricalcium phosphate particles (hBMSCs + HA/TCP) and ii) a growth factor-scaffold construct containing bone morphogenetic protein 2 in a gelatin sponge (BMP2+GS). Measurements of bone formation by histology, bone formation by X-ray microcomputed tomography (CT) and gene expression by quantitative polymerase chain reaction (qPCR) showed that constructs in combi-cassettes were similar to those created by regular procedures. Combi-cassettes afford placement of multiple replicates of multiple formulations into the same animal, which enables, for the first time, rigorous statistical assessment of: 1) the variability for a given formulation within an animal (intra-animal variability), 2) differences between different tissue-engineered formulations within the same animal and 3) the variability for a given formulation in different animals (inter-animal variability). Combi-cassettes enable a more high-throughput, systematic approach to in vivo studies of tissue engineering constructs (Biomaterials 2018;186:31-43).