An Study On Human Alveolar Bone Marrow Mesenchymal Stem Cells Osteogenic Differentiation Of Type 2 Diabetes Mellitus With Periodontitis

Objective: There is a close association between diabetes mellitus and periodontitis. Diabetes increases the prevalence, extent and severity of the periodontitis, but the mechanisms between diabetes mellitus and periodontitis remains poorly understood. Our previous experiments had demonstrated that differentiation capacity of human periodontal ligament stem cells(PDLSCs) isolated from type 2 diabetes mellitus patients with chronic periodontitis had decreased obviously, while it had not been reported about the osteogenic differentiation of bone marrow mesenchymal stem cells obtained from alveolar cavity(BMMSCs) of type 2 diabetes mellitus patients with chronic periodontitis. The aim of our research was to investigate the osteogenic differences of the BMMSCs obtained from alveolar bone of healthy volunteers(H-BMMSCs), chronic periodontitis patients with(D-BMMSCs) and without type 2 diabetes mellitus human(P-BMMSCs), exploring if the change of BMMSCs osteogenesis ability might play an important role in the periodontitis progress of type 2 diabetes mellitus patients.Methods : In this study, we extracted the bone pieces from alveolar cavity of healthy volunteers, periodontitis patients and periodontitis with type 2 diabetes mellitus, H-BMMSCs, P-BMMSCs and D-BMMSCs were cultured for primary passage stem cells by tissue piece anchorage primary culture method and isolated using limited dilution technique in vitro; The surface molecules of these BMMSCs were identificated by flow cytometry(FCM); The third passage of H-BMMSCs, P-BMMSCs and D-BMMSCs were chosen to culture with osteogenic induction solution. After 7-day osteogenic induction, ALP staining and ALP activation was to be tested. At the same time, the gene expression of osteogenic(ALP, Col-I, BSP, Runx-2) was assayed by real time RT-PCR; The calcified nodules were observed and compared by exerting Alizarin red staining after 21-day osteogenic induction.Results:1. BMMSCs were cultured for primary passage stem cells by tissue piece anchorage primary culture method in vitro. 5 samples from healthy volunteers, 4 samples(periodontitis) and 3 samples(T2DM with periodontitis) were successfully cultured from alveolar bone tissue. BMMSCs were cultured about 3-5 days in vitro, it could be seen there were a few cells around the alveolar bone pieces, after about 3 weeks, 80% confluence could be reached and most of them appeared the spindle shape, but a few of them were polygon. The morphology of the three groups of BMMSCs isolated by limited dilution technique were similar.2. Molecular identification of cell surface was detected by flow cytometry: thepositive rates of CD105, CD44, CD90, CD73: H-BMMSCs: 98.14%、99.98%、99.42%、99.61%; P-BMMSCs: 97.90%, 99.91%, 99.95%, 99.93%; D-BMMSCs: 93.58%, 99.98%,98.27%, 99.63%; and the percentage positivity for other surface molecular such as CD34,CD19, CD45, CD11 b, HLA-DR: 0.5%(H-BMMSCs), 0.07%(P-BMMSCs), 0.5%(D-BMMSCs).3. Comparison of osteogenic differentiation: after 7-day osteogenic induction, ALP staining showed H-BMMSCs was the deepest group, and P-BMMSCs group appeared deeper colour than D-BMMSCs group; ALP/AKP test kit was used to test the level of ALP and the differences among these three group showed statistical significance(P<0.05). After 21-day osteogenic induction, three groups appeared some calcified nodules, and alizarin red staining showed that the D-BMMSCs group had less calcified nodules than the other groups.4. RT-PCR was performed to explore the expression of osteogenic related gene of H-BMMSCs, P-BMMSCs, D-BMMSCs, the results showed that after 7-day osteogenic induction, the m RNA expression of ALP, Col-I, BSP, Runx-2 in the D-BMMSCs were obviously lower than those of H-BMMSCs and P-BMMSCs(P<0.05).Conclusion: Our study isolated BMMSCs from alveolar cavity of periodontitis patientswith type 2 diabetes mellitus and it was turned out to have similar stem cells characteristics with P-BMMSCs and H-BMMSCs by clone formation assay and phenotypic molecules detection. While they all appeared the spindle shape, it was obvious that osteogenic capacity of D-BMMSCs was weaker than H-BMMSCs and P-BMMSCs which indicated that type 2 diabetes mellitus as a systemic factors might weaken the alveolar bone regeneration to promote the progress of periodontitis. These findings could provide the theoretic and experimental basis for mechanisms between type 2 diabetes mellitus and periodontitis.