Experimental Study On The Use Of Autos Adipose-derived Stem Cells Compounded With Heterogeneous Cancellous Bone Scaffold In The Repair Of Rabbit Skull Defect

Bone defect caused by trauma, infection, tumour extirpation and so on remains a difficult problem in orthopedics clinic. Ever since a long time ago, people have been trying to use variant bone, heterogeneous bone, artificial materials to solve this problem, but the result is not so satisfactory. Autologous bone transplantation remains the gold standard of bone defect therapy.Along with the development of tissue engineering technology in the past few years, trying to solve the problem above through bone tissue engineering becomes a hot pot. Bone tissue engineering includes seed cells, scaffold material and biological active factors. The problem of seed cell is the chief point and essential element of bone tissue engineering, and also is the prerequisite for further development of bone tissue engineering. How to obtain the seed cell of sufficient quantity and favorable function has been a bottleneck in this field. In our study, we use adipose-derived stem cells(ADSCs) as seed cells. We culure ADSCs in vitro, induce them along osteogenic lineages, compound them with self-made heterogeneous cancellous bone scaffold, and use them in the reparation of rabbit bone defect,to investigate the application prospect of ADSCs be used as seed cell in bone tissue engineering for the reparation of bone defect.Objective:We extract adipose-derived stem cells(ADSCs)from rabbit adipose tissue, induce them differentiate along osteogenic lineage, seed the osteogenic-induced ADSCs and non-induced ADSCs respectively onto self-made bull cancellous bone scaffold, to study the cyto-compatiblity and tissue-compatiblity of the self-made bull cancellous bone scaffold. If the cyto-compatiblity and tissue-compatiblity are both well , we will transplant the cell-scaffold compounds to the rabbit skull defect site, to investigate the application prospect of the tissue-engineered artificial bone which is got from ADSCs and bull cancellous bone scaffold for the reparation of bone defect.Methods1 The extraction, cultivation, osteogenic-induction of ADSCs: We obtain adipose tissue from posterior neck of the rabbit, get ADSCs through comminution, digestion of the adipose tissue, culture ADSCs and apply them with appropriate circumstance for propagation, induce the ADSCs differentiate along osteogenic lineage. The alkaline phosphatase contains in the cells is measured quantitatively, and alizarin red staining is conducted, to assure the effect of osteogenic-induction.2 The preparation of the heterogeneous cancellous bone scaffold and the study of it's biocompatibility: The cancellous bone of bull femur head got from the market is made into cancellous bone scaffold. We seed the osteogenic-induced and non-induced ADSCs respectively onto the cancellous bone scaffold, calculate the adhere rate of this two different kinds of cell, culture the cell-scaffold compounds in vitro, observe the cells living on the scaffold through scanning electron microscope, to study the cyto-compatibility of the scaffold. Then we embed the cell-scaffold and scaffold merely respectively on rabbit back subcutaneously, and harvest them 10 days later, make HE staining, to study the tissue-compatibility of the scaffold.3 Only if the biocompatibility of the self-made heterogeneous cancellous bone scaffold is well, we transport the scaffold, scaffold+ ADSCs, scaffold+osteoinduced ADSCs respectively to rabbit skull defect site, and harvest the skull 12 weeks after the operation, take X-ray photography, Micro-CT scanning, and HE staining, analyze the quantitative difference of the skull defect reparation among those different groups. To investigate the prospect of ADSCs be used as seed cell, and also self-made heterogeneous cancellous bone scaffold be used as scaffold in bone tissue engineering.Result1 There is no significant difference between the appearance of the ADSCs and osteoinduced ADSCs under microscope. The content of ALP notably increases when ADSCs are osteoinduced, the difference has statistical significance; Alizarin red staining is positive when ADSCs are osteoinduced for 2 weeks ,and is negative in non-induced group. These tell us that the ADSCs differentiate along osteogenic lineage after been osteoinduced.2 Both the ADSCs antero-osteoinduced and posterior- osteoinduced can live well on the self-made heterogeneous cancellous bone scaffold, and the difference of the adhere rates in 24 hours of the two kinds of cell is not statistical significant(P>0.05).HE staining shows that the cells can stay alive 10 days after the cell-scaffold compounds been implanted subcutaneously. The biocompatibility of the self-made heterogeneous cancellous bone scaffold is well.3 Through X-ray photography, we quantitatively measure the reparation of the skull defect. We know that the difference between the ADSCs-scaffold group, the osteoinduced ADSCs-scaffold group , the merely scaffold group and the vacant control group is not statistical significant (P>0.05)but those who has the biggest reparation area is in the osteoinduced ADSCs-scaffold group. Through the analysis of Micro-CT scanning, we know that the amount of bone formation of the osteoinduced ADSCs-scaffold group is the most, and the difference is statistical significant(P<0.05). Through HE staining we can see the scaffold being absorbed, and we also see that there is new bone formation, bone formation is especially significant in the osteoinduced ADSCs-scaffold group.ConclusionAs the biocompatibility of the self-made heterogeneous cancellous bone scaffold is very good, it can be used as scaffold in bone tissue engineering. ADSCs can be osteogenic induced in vitro, as been compounded with self-made heterogeneous cancellous bone scaffold, osteoinduced ADSCs can repair bone defect well. ADSCs can be used as seed cell in bone tissue engineering.