The Effect Of Diabetic Osteoblast On Differentiation Of Human Umbilical Cord Mesenchymal Stem Cell

Objective:The objective of this experimental study is to evaluate the osteogenic potential ofdiabetic osteoblast and insulin on human umbilical cord mesenchymal stem cells（HUCMSCs）in co-culture and suppose the possibility of HUCMSCs treating delayeddiabetic bone healing.Methods:Part1: Isolation and culture human osteoblasts from diabetes and non-diabetesHuman osteoblasts were isolated and cultured by the collagenase digestion method.They were identified by calcium and cobalt staining of alkaline phosphatase(ALP),immunohistochemical staining of type Ⅰ collagen, alizarin red staining for calciumnodules.Part2: Osteogenic potential of diabetic osteoblast on HUCMSCs in co-cultureTwo types of cells were indirectly co-cultured by transwell culture plates.Experiments were divided into three groups.There was no cells on the upper chamber ofgroup A; On the upper chamber of group B placed the non-diabetic osteoblasts; On theupper chamber of group C placed the diabetic osteoblasts; HUMSCs were placed on theinferior chamber of the three groups respectively. The growth and proliferation ofHUMSCs were observed by CCK-8assay in the first week. The expression of type Ⅰcollagen was tested by immunofluorescence staining on the10th day. ALP activity ofHUMSCs has been observed by cell alkaline phosphatase activity assay on the7th/10th/14th day. The relative expression level of osteocalcin mRNA in HUMSCs weredetected by RT-PCR menthod on21th day.Part3: The effect of insulin on osteogenic differentiation of HUMSCsIt was divided into insulin treated and non-insulin-treated group.Proliferation of HUMSCs was compared by CCK-8assay. Alkaline phosphatase activity colorimetricassay and RT-PCR method were performed to evaluated ALP activity and the relativeexpression levels of type I collagen protein mRNA and osteocalcin mRNA in HUMSCs.Result:1. The cultured cells were expressed ALP, type Ⅰ collagen, and calcium nodulescorresponding the characteristics of osteoblasts. The quantity of alkaline phosphatase,typeⅠ collagen and calcium nodules in diabetic osteoblasts expression was less.2. The proliferation of HUCMSCs of group A was slower than group B and group C,The quality of cell in group B more than group C, the difference was statistical significance(P <0.05).3. HUCMSCs were cultured after10days, The immunofluorescence staining of typeⅠ collagen of it was negative in group A. group B and group C were positive, thedifference between group B and C was not statistically significant (P>0.05).4. The alkaline phosphatase activity of group A has always been in the low expressionstate. The ALP activity increased in group B and C compared with group A. They reachedto peak at10th days and decreased at14days. The trendency of expression of ALP activityof group B and C was similar, However, the group B expressed higher ALP activityrelative to group C. The difference was statistical significance (P <0.05).5. The expressions of osteocalcin mRNA were detected by RT-PCR21th days afterco-culture in group B and C,there was no osteocalcin mRNA in group A. The expressionnumber of C group is less than group B.The difference was statistically significant (P <0.05).6. The HUCMSCs proliferated more quickly after insulin using. The HUCMSCs ofinsulin treated group expressed higher ALP activity, type I collagen protein mRNA andosteocalcin mRNA compared with non-insulin-treated group(P <0.05).Conclusion:This data leads us to conclude that co-culture of HUCMSCs with diabetic osteoblastspromotes osteogenesis and stabilizes the osteogenic potential of HUCMSCs,althoughdiabetic osteoblasts are impaired. Not only insulin could contribute to repair impairedosteoblast, but also promote proliferation and osteogenic differentiation of theHUCMSCs.The present results suggest that transplantation of HUCMSCs and insulin maybecome a useful strategy for cell-based bone regeneration in diabitic bone healing.