Regulatory Mechanisms Of Mesenchymal Stem Cells Transplantation On Systemic Osteoporosis In Collagen Induced Arthritis Mice

Background:The secondary osteoporosis is the principal reason of bone structural deterioration in patients with rheumatoid arthritis(RA).Studies on the mechanism involved in osteoporosis among RA patients and collagen induced arthritis(CIA)models were focused on the over-activated osteoclast(OC)activity previously.Bone marrow mesenchymal stem cells(BMMSCs)have potential to differentiate into osteoblast(OB).Abnormal osteoblastic differentiation ability were observed in RA patients and CIA models.In our clinical studies,we found that umbilical cord-mesenchymal stem cells(UC-MSCs)transplantation was a novel and effective therapeutic option for refractory RA,and we also observed an increased bone mineral density(BMD)in patients with osteoporosis after UC-MSCs transplantation,however,the mechanism remains unclear.Here,we hypothesized that allogeneic MSCs transplantation could promote the differentiation of BMMSCs into OB and/or inhibit bone marrow monocytes(BMMs)from differentiating into OC in CIA models with osteoporosis.The present study will provide novel evidence for osteoprosis therapy by allogeneic MSCs in patients with RA.Objective:To investigate the effects of UC-MSCs transplantation on joint damage and systemic osteoporosis in CIA mice and to explore the mechanism by which UC-MSCs modulate the osteogenic differentiation of BMMSCs and OC differentiation of BMMs in CIA mice.Methods:DBA/1 male mice aged 6-8 weeks were induced to CIA models by injecting type II Collagen(CII).CIA models were successfully set up on day 28 post initial immunization.Then the CIA mice were devided into four groups,including CIA control(untreated)group,UC-MSCs transplantation goup,anti-tumour necrosis factor alpha(anti-TNF?)treatment group and zoledronic acid(ZA)treatment group.The CIA mice were treated for 8 weeks.We evaluated the arthritis score of CIA mice every 3 days.Micro computed tomography(micro-CT)were used to analyze the bone morphology parameters at the end point.The pathological manifestation of knee joints and femurs were evaluated by hematoxylin-eosin(H&E)staining.MSCs were isolated from bone marrow of CIA and DBA/1 mice,and cultured in the osteogenic mediun.Osteogenic differentiation was determined by alkaline phosphatase(ALP)staining and ALP activity on day 7.The expression of the osteogenic markers ALP,Osterix and type I collagen(COL-I)were analyzed by real time polymerase chain reaction(RT-PCR)and western blot(WB)on day 7 and day 14 respectively.Meanwhile,BMMSCs from CIA mice were cultured in the osteogenic medium with bone morphogenetic protein 2(BMP-2)and/or TNFa,then we determined the osteogenic differentiation ability.Moreover,monocytes were isolated from bone marrow of CIA mice and DBA/1 mice,then induced to OC in the presence of macrophage colony-stimulating factor(M-CSF)and receptor activator of nuclear factor-KB ligand(RANKL).OC differentiation was determined by tartrate-resistant acid phosphatase(TRAP)staining and mRNA levels of nuclear factor of activated T cells(NFAT)2 and osteoprotegerin(OPG).Results:The arthritis score was significantly reduced in UC-MSCs transplantation and anti-TNFa treated CIA group compared with control mice(P<0.001).There was no significant difference of arthritis score between ZA treated CIA group compared with control mice.Micro-CT showed that the BMD(P<0.01),trabecular bone volume/total volume(BV/TV)(P<0.001)and trabecular number(Tb.N)(P<0.01)of the femur were significantly decreased in CIA mice,compared with that in DBA/1 mice.H&E staining also showed a reduced Tb.N in CIA mice.Whereas these parameters were partially improved in UC-MSCs treated CIA mice compared with control mice.Impaired osteogenic differentiation functions were shown by decreased ALP activity(P<0.001),reduced gene and protein levels of osteogenic marker genes(P<0.05)in CIA mice compared with DBA/1 mice.UC-MSCs treatment significantly upregulated the impaired osteogenic differentiation ability in CIA mice(P<0.05).Moreover,addition of BMP-2 in the osteogenic medium enhanced the osteogenic differentiation ability in CIA mice(P<0.05).TRAP staining and OC counting showed that OC number was increased in CIA mice compared to that in DBA/1 mice(P<0.001).RT-PCR showed that the mRNA levels of NFAT2 were elevated in CIA mice compared to that in DBA/1 mice(P<0.01),however the OPG mRNA levels were decreased in CIA mice(P<0.05).UC-MSCs treatment downregulated the enhanced OC differentiation in CIA mice(P<0.05).Conclusion:The present study demonstrated that CIA mice developed osteoporosis after attacked for 8 weeks.UC-MSCs transplantation not only improved the joint damage significantly but also played a positive role in osteoporosis in CIA mice.The osteogenic differentiation ability of BMMSCs in CIA mice was impaired.The OC differentiation function of BMMs in CIA mice was enhanced.UC-MSCs transplantation could upregulate the osteogenic differentiation and downregulate the OC differentiation in CIA mice.Thus,we provide strong evidence that MSCs ameliorate inflammation-induced systemic bone loss in CIA mice by upregulating osteogenic differentiation and reducing OC differentiation.