Modulatory Effects Of Mesenchymal Stem Cells On Osteoclast Formation And Function

Background and ObjectiveOsteoclasts are specialized cells derived from the hematopoietic stem cells (HSCs) and most of them adhere to bone surface, then secrete acid and lytic enzymes to degrade bone. In steady state conditions, only very low levels of osteoclasts are generated and take part in regulation of bone remodeling with osteoblasts in a concerted manner. Moreover, investigators have recently elucidated that osteoclasts also play a role in regulation of host defense and repair, inflammatory diseases and cancer bone metastasis. Osteoclastogenesis is highly dependent on the cellular microenvironment, which provides essential cytokines as well as important cell surface molecules.Mesenchymal stem cells (MSCs) are first identified in bone marrow and can differentiate into numerous kinds of cells under certain conditions. In addition, MSCs and their progenies are important supporting cells in bone marrow microenvironment and they supply numerous cytokines and cell surface signals in modulating the survival, proliferation, and differentiation of most hematopietic cells.Though both MSCs and osteoclasts reside in bone marrow microenvironment, however, seldom study so far was processed in the association of these two kinds of cells. In our study, the modulatory effect of MSCs on monocyte-derived osteoclast formation and function as well as the shift of the effect after MSCs were stimulated by TNF-αwas investigated in virtue of a reliable and efficient mouse MSC culture method. The roles of MSCs in osteoclastogenesis under inflammatory conditions were further explored in vivo and in vitro through the LPS injected mouse model and the rheumatoid arthritis synovial fluid (RASF). Furthermore, the effect on osteoclast formation of human RASF derived MSCs were further explored.Methods1. Isolated and cultured MSCs from mouse compact bone through our published method. To assess the tri-lineage differentiation capacity of these cells, osteogenic, adipogenic and chondrogenic inductions were performed in vitro. Moreover, the immunophenotypic features of the cells were analyzed by flow cytometry.2. Mouse CD11b+ monocytes were separated by MACSs and cultured onto MSC layers in the presence or absence of M-CSF and RANKL. The modulation effect of MSCs on the differentiation, maturation, survival and bone resorption of osteoclasts were explored.3. Pretreated by TNF-αor cultured in the presence of this cytokine, graded cell numbers of MSCs and CD11b+ monocytes were cultured with or without addition of different doses of M-CSF and RANKL. The bone resorption capacity of osteoclasts generated with the TNF-αstimulated MSCs were also explored by using elephant tusk slices. In some experiments, MSCs and monocytes were separated by a transwell to analyze whether MSCs and TNF-αstimulated MSCs regulate osteoclast formation through soluble factors. Based on the transwell assay data, the mRNA and protein levels of the OPG which was significant in suppressing osteoclast formation were analyzed by RT-PCR, real-time PCR and ELISA respectively. The mRNA level of some osteoclastogenesis associated cytokines including RANKL, M-CSF and interleukin-6(IL-6) were also analyzed by RT-PCR and real-time PCR.4. To search the mechanism closely related to TNF-αinduced OPG production in MSCs, the activation of MAPK pathway by TNF-αwere evaluated by western blot. Furthermore, the effect of the specific MAPK pathway inhibitors on OPG mRNA level was assayed by real-time PCR. In addition, the roles of the pathway inhibitors in osteoclast formation were explored.5. To explore the potential effect of TNF-αon MSC biological characteristics, immunophenotyping characteristics, multipotential differentiation capacities were validated as usual. The immunosuppressive properties of MSCs were analyzed by a Carboxyfluoresce indiacetate succinimidyl ester (CFSE) labeled CD3+ T lymphocytes proliferation assay.6. TNF-αlevels in the RASF samples from 20 patients were evaluated by ELISA. Then MSCs were pretreated with RASF samples and cultured with monocytes to generate osteoclasts. The relationship between TNF-αconcentration and osteoclast number was further explored.7. To investigate the modulatory effect on osteoclastogenesis under inflammatory condition in vivo, MSCs were given to LPS-injected mouse via intravenous injection. The femur bones of mice were sectioned and the osteoclast formation was identified by in situ TRAP staining.8. To test their regulatory effect on osteoclastogenesis, RASF-MSCs were co-cultured with CD14+ monocytes and osteoclasts were identified with in situ TRAP staining.Results:1. The average number of MSCs from one mouse usually reaches 107 in 14 days with a protocol for isolation and culture MSCs from mouse compact bone. The multilineage differentiation assays show that the mouse compact bone derived MSCs can readily differentiate into osteoblasts, adipocytes and chondrocytes in the improved induction medium. FACS analysis data show that these cells harbor a typical immunophenotypic profile of MSCs.2. MSCs can independently supported monocytes derived osteoclast formation and this effect is enhanced by M-CSF and RANKL. This stimulatory effect is MSC dose- and co-culture time-dependent. Moreover, ratio of TRAP+ MNCs/total TRAP+ cells is raised from 19% to 39% in the presence of MSCs. Furthermore, with the addition of MSCs, the number of survived TRAP+ MNCs is almost 2 times more than that in the absence of MSCs.3. Stimulated with TNF-α, MSCs turn to inhibit osteoclast formation. The suppressive effect is TNF-αdose- and time-dependent. Moreover, TNF-αstimulated MSCs begin to inhibit osteoclast formation at a lower MSCs/monocytes ratio of 1:5. Furthermore, the inhibitory effect maintain at least 12 days. In addition, the inhibitory effect is more remarkable in co-culture experiment with direct supplementation of TNF-α.4. MSCs and TNF-αstimulated MSCs exhibit different effects on resorbing function of osteoclasts. In the presence of MSCs, osteoclasts produce larger resorption pits on slices of elephant tusk dentine while smaller or no resorption pits are observed when monocytes are co-cultured with TNF-αstimulated MSCs or with direct supplementation of TNF-αto co-culture system.5. The transwell assay data show that MSCs and TNF-αstimulated MSCs regulate osteoclast formation partially through soluble factors. The RT-PCR results reveal that MSCs steadily express the mRNA of numerous osteoclastogenesis modulatory cytokines including RANKL, M-CSF, IL-6, and OPG. The real-time PCR data show that TNF-αis capable of up-regulating OPG gene expression in MSCs in a time- and dose-dependent manner. Consistently, ELISA results show an elevation of OPG secretion in a TNF-αdose-dependent manner. In addition, TNF-αstimulated MSCs slightly down-regulate the mRNA level of RANKL, IL-6, and M-CSF.6. Western blot results show that TNF-αinduced the phosphorylation of p38/MAPK, ERK/MAPK, and JNK/SAPK in MSCs. Real-time PCR data reveal that OPG mRNA elevation is partially blocked by SB23580HCL and JNK inhibitor II pretreatment but not PD98059. Similarly, SB23580HCL and JNK inhibitor II pretreatment before TNF-αstimulus partially rescue the osteoclast formation.7. TNF-αprimed MSCs retain their morphological feature, immunophenotyping characteristics, and multipotential differentiation capacities. The CFSE labeled T lymphocytes proliferation experiment demonstrate that TNF-αtreated MSCs remarkably inhibited Con A stimulated CD3+ T lymphocytes proliferation.8. The RASF assays show that MSCs promotes osteoclast formation when TNF-αconcentration is relatively low in RASF samples, while the cells inhibit osteoclast generation after treated by the samples with high TNF-αconcentration.9. The TRAP staining of femoral metaphysis shows that the MSC infusion remarkably suppresses the osteoclastogenesis in LPS-injected mouse.10. RASF-MSCs possess typical MSC phenotypes but exert decreased multi-differentiation capacity as compared with human bone marrow mesenchymal stem cells. Interestingly, the enhanced osteoclastogenesis is observed when RASF-MSCs are co-cultured with monocytes.Conclusions:1. MSCs exhibit a promoting effect on the differentiation, maturation, survival and bone resorption of monocytes derived osteoclasts. Numerous cytokines and cell surface signals supplied by MSCs may contribute to the supportive effect.2. MSCs turn to inhibit osteoclastogenesis upon stimulation with TNF-αand this alteration is partially dependent on the activation of P38/MAPK and JNK/SAPK pathway and the following OPG elevation. Pretreating MSCs with RASFs yield similar effects on osteoclast formation. Moreover, a single injection of MSCs ameliorates bone lesions in a LPS-induced bone resorption mouse model. It is the first report that MSCs act as conditional osteoclastogenesis inhibitors in inflammatory microenvironment.3. Compared with human bone marrow derived MSCs, RASF-MSCs exhibit an enhanced promoting effect on osteoclast formation, which indicates that distinct tissues derived MSCs may differentially regulate osteoclast formation.