| Backgrounds:Tumors, injuries and congenital malformation are the main causes of bone defects in maxillofacial area. And this problem has bothered clinicians for decades. For many years, researchers kept seeking for the therapeutic approaches for bone defects restoration. And bone tissue engineering is the one that draws the most attention. In traditional bone tissue engineering, bone marrow stroma cells (BMSCs) are the most widely used stem cells. BMSCs have the perfect ability to promote bone regeneration and bone mineralization. But there are still considerable issues for extensive clinical applications, such as the limited cell storage, the invasion in harvest and the severe complications. Hence for recent years, researches have started to test some other multipotent stromal cells from different sources for bone tissue engineering including:adipose-derived stem cells (ADSCs), myoblasts, chondrocytes, fibroblasts, umbilical cord blood cells, skin derived precursor cells, mesothelial cells and ovarian follicular granulose cells. Among all these cells, ADSC may be the most promising one for the reasons listed below:ADSCs have a generous storage in the subcutaneous fat tissue; they are much easier to isolate with minimum trauma; they grow fast in vitro with minimum culture condition requirements and they can differentiate into osteoblaste-like cells within a short time period of culture in proper osteogenic induction medium. Another key element for bone tissue engineering is the scaffold. So far, tricalcium phosphate (TCP) is the most popular biomaterial for bone tissue engineering. Actually there are two subtypes of TCP:a-TCP and β-TCP. Compared with the traditional a-TCP, P-TCP has a better biocompatibility. It could be degraded at approximately the same speed of bone regeneration without any debris left. The last component of bone tissue engineering is cytokines. 1α,25-Dihydroxyvitamin D3 (calcitrol) and its analogs have been used for osteoporosis treatment for decades due to their capability of increaseing the bone mineral density (BMD). However for recent years, it has been reported that 1,25(OH)2D3 and its analogs manipulate bone formation and bone resorption through direct interference of osteoblasts and osteoclasts activity via vitamin D receptor (VDR) which was detected in all three kinds of bone cells including osteoblast, osteocyte and osteoclast. So in this research we expected to establish a new tissue engineering therapeutic approach for bone defects restoration by combining ADSC, β-TCP and calcitrol or its analogs.Purpose:The purpose of this research was to evaluate the effect of bone defect restoration by the combination of ADSC, β-TCP and calcitrol through a series of in vivo and in vitro studies. Further speculation of the mechanisms of the manipulation of osteoblasts and osteoclasts function by calcitrol and its analog eldecalcitol was carried out. And finally the different effects of calcitrol and eldecalcitol in bone regeneration were compared in vivo.Materials and methods:In the first stage of the experiment, we isolated ADSCs from subcutaneous fat tissue of the rats, and submitted the ADSCs to osteoblastic differentiation induction through in vitro culture system. Alizarin red staining was introduced to evaluate the efficiency of osteogenic induction. Well differentiated ADSCs were then co-cultured with β-TCP for 3 days, and the complex was implanted into bone defects in the femora of rats. Calcitrol was administered intraperitoneally every other day postoperation. At day 7, day 14, and day 28 after the restoration, femora were harvested for histomorphology analysis. During the second stage of in vitro experiment, osteoblastic cell line MC3T3-E1 cells were induced with osteoblastic differentiation medium. Calcitrol and eldecalcitol were then added into the culture medium. The expressions of runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), and receptor activator of nuclear factor kappa-B ligand (RANKL) were evaluated through western blot analysis. And for the third stage, the femora defect animal model was used again, but with neither the ADSCs nor the β-TCP in order to evaluate of the differences between calcitrol and eldecalcitol in bone defects restoration with minimum variants. After the treatment of bone defects with calcitrol (CAL gtoup) and eldecalcitol (ELD group) for 28 days and 56 days, femora were harvested for histomorphology analysis.Results:The first stage experiment indicated that the combination of ADSC, P-TCP and calcitrol improved bone formation dramatically accompanied by the higher expression level of alkaline phosphatase (ALP) and Runx2. On the other hand, calcitrol suppressed osteoclastic bone resorption in the early stage of restoration of bone defects by reducing osteoclast number and suppressing the expression of cathepsin K (CK). The second stage experiment showed that both calcitrol and eldecalcitol promoted the expression of Runx2 and OPG, and suppressed the expression of RANKL of the MC3T3-E1 cells. However, calcitrol exhibited a stronger manipulation of MC3T3-E1 compared with eldecalcitol. In the last stage we found that, after 28 days of the treatment of the bone defects with calcitrol (CAL group), slightly more new bone volume was found than eldecalcitol treatment (ELD group). But no significant difference in new bone volume was noticed at day 56 between these two groups. However, CAL group showed smaller non-mineralized collagen area in the bone matrix than ELD group at both day 28 and 56. Through immunohistochemistry examination of CK, we noticed that since day 28, calcitrol showed a strong suppression of CK positive osteoclasts, but this effect did not come up in ELD group until day 56.Conclusion:1. After osteogenic induction, ADSCs promoted bone formation by upregulating the expression of ALP and Runx2 in vivo.2. Systemic administration of active vitamin D compound (AVDC) decreased the number of osteoclasts and suppressed the expression of CK.3. Calcitrol and eldecalcitol promoted the expression of Runx2 and OPG, and suppressed the expression of RANKL through direct interaction with osteoblasts.4. Compared with eldecalcitol, calcitrol promoted bone formation and mineralization of the defects relatively faster, and suppressed osteoclasts function from an earlier time point. Taken together, we speculated that the combination of ADSC, β-TCP and calcitrol might be a promising option for bone defects restoration. |
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