Objective To compare the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) and anterior cruciate ligament derived mesenchymal stem cells (ACL-MSCs) from ratsin vitro. Methods Ten male SPF-level BN rats, weighing 200-220 g, were selected to obtain anterior cruciate ligaments and bone marrows, and ACL-MSCs and BMSCs were isolated for passage culture respectively under sterile condition. The cell morphology was observed, and the cells at passage 3 were used to detect the surface markers of CD34, CD45, CD90, and CD29 by flow cytometry, the ability of cell proliferation by cell counting kit 8 (CCK-8), and colony formation ability by clone forming test. The mRNA levels of differentiation related genes [alkaline phosphatas (ALP), bone gamma-carboxyglutamate protein, runt related transcription factor 2, bone morphogenetic protein 2 (BMP-2), secreted phosphoprotein 1 (Spp1), collagen type II α1 (Col2α1), Aggrecan (Acan), Sox9, peroxisome proliferator activated receptor γ2 (PPARγ2), and CCAAT-enhancer-binding protein-α] were also determined by real-time fluorescent quantitative PCR. Results BMSCs and ACL-MSCs had similar morphology, adherent cells displaying long fusiform. The immunoprofile of ACL-MSCs and BMSCs at passage 3 was positive for CD29 and CD90 and was negative for CD45 and CD34. The absorbance (A) value of ACL-MSCs (1.11±0.08) was significantly higher than that of BMSCs (0.78±0.05) (t=3.599,P=0.023); the number of colonies of ACL-MSCs [(53.00±5.51)/hole] was significantly more than that of BMSCs [(30.67±4.84)/hole] (t=3.045,P=0.038). The results of toluidine blue staining, alizarin red staining, and oil red O staining were positive in BMSCs and ACL-MSCs at 21 days after osteogenic, chondrogenic, and adipogenic induction. The mRNA expressions of BMP-2, Spp1, Col2α1, Acan, Sox9, and PPARγ2 in ACL-MSCs were significantly higher than those in BMSCs (P<0.01). Conclusion The proliferation potential of ACL-MSCs is greater than that of BMSCs, and the former is apt to differentiate into chondrocytes. ACL-MSCs are promising cells to promote tendon-bone healing.
Objective To investigate the effect of bone morphogenetic protein 2 (BMP-2) and dexamethason (DXM) on proliferation and differentiation of human dental pulp cellsin vitro. Methods Primary human dental pulp cells were cultured in vitro by tissue culture method. The 3rd generation cells were used to identify cell phenotype for vimentin and cytokeratin by immunocytochemistry staining. The 3-5 generations of human dental pulp cells were randomly divided into 4 groups: 100 ng/mL BMP-2 (group A), 1×10–8 mol/L DXM (group B), and both 100 ng/mL BMP-2 and 1×10–8 mol/L DXM (group C) were added; neither BMP-2 nor DXM was added in group D as control group. The cell growth curve was drawn at 1, 3, 5, and 7 days after culture. The expressions of osteo/dentanogenic genes including alkaline phosphatase (ALP), dentin sialophoshoprotein (DSPP), and dentin matrix protein 1 (DMP-1) were detected by RT-PCR analysis at 5 and 7 days after culture, the ratio between the positive staining area and the total area by ALP staining at 14 days, and absorbance (A) value at 562 nm by alizarin red staining at 21 days after culture. Results Human dental pulp cells were successfully isolated and cultured, which were long fusiform and showed a positive reaction for vimentin and a negative reaction for cytokeratin. The growth curve indicated that cells increased with the extending of incubation time, reached a peak at 5 days, then reduced at 7 days to the level at 3 days. At 5 days after culture, the cells were significantly more in groups A, B, and C than group D (P<0.05), in group C than group A (P<0.05), and in group A than group B (P<0.05). RT-PCR analysis showed that the mRNA expressions of ALP, DSPP, and DMP-1 at 5 days were significantly higher in groups A, B, and C than group D (P<0.05), and in group C than groups A and B (P<0.05), but no significant difference was found between groups A and B (P>0.05); the mRNA expression of DSPP in groups A, B, and C was significantly higher than that in group D (P<0.05), but there was no significant difference in mRNA expressions between other groups at 7 days (P>0.05). At 14 days, positive staining in varying degrees was observed in each group, especially in group C; the ratio between the positive staining area and the total area was significantly higher in group C than groups A, B, and D (P<0.05), and in groups A and B than group D (P<0.05), but there was no significant difference between groups A and B (P>0.05). At 21 days, there were a variety of mineralized nodules in groups A, B, and C in nonuniformly scattered or clustered distribution, but no mineralized nodules were observed in group D. TheA values of mineralized nodules showed significant difference between groups (P<0.05). Conclusion BMP-2 may be more effective in promoting proliferation of human dental pulp cells than DXM. Combined application of BMP-2 and DXM can remarkably promote the proliferation and differentiation of human dental pulp cells.