Characterisation and Expression of Osteogenic and Periodontal Markers of Bone Marrow Mesenchymal Stem Cells (BM-MSCs) from Diabetic Knee Joints

Abstract

Type 2 diabetes mellitus (T2DM) represents a significant health problem globally and is linked to a number of complications such as cardiovascular disease, bone fragility and periodontitis. Autologous bone marrow mesenchymal stem cells (BM-MSCs) are a promising therapeutic approach for bone and periodontal regeneration; however, the effect of T2DM on the expression of osteogenic and periodontal markers in BM-MSCs is not fully established. Furthermore, the effect of the presence of comorbidities such as diabetes and osteoarthritis on BM-MSCs is also yet to be investigated. In the present study, BM-MSCs were isolated from osteoarthritic knee joints of diabetic and nondiabetic donors. Both cell groups were compared for their clonogenicity, proliferation rates, MSC enumeration and expression of surface markers. Formation of calcified deposits and expression of osteogenic and periodontal markers were assessed after 1, 2 and 3 weeks of basal and osteogenic culture. Diabetic and nondiabetic BM-MSCs showed similar clonogenic and growth potentials along with comparable numbers of MSCs. However, diabetic BM-MSCs displayed lower expression of periostin (POSTN) and cementum protein 1 (CEMP-1) at Wk3 osteogenic and Wk1 basal cultures, respectively. BM-MSCs from T2DM patients might be suitable candidates for stem cell-based therapeutics. However, further investigations into these cells' behaviours in vitro and in vivo under inflammatory environments and hyperglycaemic conditions are still required.

Keywords: bone marrow mesenchymal stem cells; bone regeneration; diabetes.

Figure 1

CFUF-Fs and PDT assays of nondiabetic (ND) and diabetic (D) BM-MSCs. (A) Representative CFU-Fs of early-passage BM-MSCs isolated from an ND and (B) a D donor. (C) CFU-Fs% in ND and D donors. (D) Population doubling, (E) accumulative population doubling and (F) population doubling time in ND and D BM-MSCs. Data are presented as mean ± SD (n = 3) and analysed using unpaired t test.

Figure 2

Enumeration of MSC population in diabetic (D) BM-MSCs. (A) Gating to exclude dead cells and debris based on their forward and side scatter. (B) Gating to include living cells based on their negative uptake of fixable viability stain. (C) Gating to include CD73⁺CD90⁺ cells in quadrant Q1-UR. (D) Gating to include CD73⁺CD90⁺CD105⁺CD14⁻ cells in quadrant Q2-LR. (E) Gating to include CD73⁺CD90⁺CD105⁺CD14⁻CD19⁻CD34⁻ in quadrant Q3-LL. (F) Gating to include CD73⁺CD90⁺CD105⁺CD14⁻CD19⁻CD34⁻CD45⁻HLA-DR⁻ in quadrant Q4-LL. The same approach was employed with BM-MSCs from nondiabetic (ND) and D donors. (G) Expression of MSC markers in ND and D BM-MSCS. (H) Enumeration of MSC population in ND and D MSCs. Data are presented as mean ± SD (n = 3) and analysed using unpaired t test.

Figure 3

Comparing osteogenic differentiation of diabetic (D) and nondiabetic (ND) BM-MSCs: (a) Alkaline phosphatase stain of D and ND BM-MSCs under basal and osteogenic conditions after 1, 2 and 3 weeks of culture. (b) Alizarin Red (AR) stain of ND and D BM-MSCs under basal and osteogenic conditions after 1, 2 and 3 weeks of culture. (c) Quantification of AR stain of ND and D BM-MSCs. Data are presented as mean ± SD (n = 3), analysed using unpaired t test and showed no significant difference (* p < 0.05)).

Figure 4

Comparing the relative changes in gene expression of periodontal markers (top panel: COL1A1, POSTN and CEMP-1) and osteogenic markers (lower panel: ALPL, Runx2, OCN) in ND and D BM-MSCs cultured under basal or osteogenic media for 1, 2 and 3 weeks as well as baseline untreated cells (0 time point). Data are presented as mean value ± SEM (n = 3) normalised to housekeeping (HPRT1) and were statistically analysed using unpaired t test for unmatched groups, paired t test for matched groups and repeated measures ANOVA for comparing time points, * p < 0.05.

Figure 5

Comparing the relative changes in gene expression of bone homeostasis markers: RANKL, OPG, OPG/RANKL ratio) in ND and D BM-MSCs cultured under basal or osteogenic media for 1, 2 and 3 weeks as well as baseline untreated cells (0 time point). Data are presented as mean value ± SEM (n = 3) normalised to housekeeping (HPRT1) and were statistically analysed using unpaired t test for unmatched groups, paired t test for matched groups and repeated measures ANOVA for comparing time points. There was no significant difference between D and ND BMSCS, culture conditions or time points (NS > 0.05).