| 【Background】According to the incomplete statistics by national center for disease control, thedisease and injury in cartilage defect rate is about1.5%-3%, with the improvement ofsports consciousness, and transportation accident increases, the incidence is increasingyear by year. Although, at present there are many treatments to repair the articularcartilage defects, such as autogenous cartilage transplantation, allogeneic cartilagetransplantation,synthetic biological material and so on. But these are unsatisfactory causeof the inconvenience and difficult to operation, and costs too much. Tissue engineeringprovides a new choice to cartilage damage repair, convenient operate, safe and effective，economical and practical cartilage defect repair methods are widely research and exploration. Convenient, budget, safety and effective are the purpose in our researchproject. We try to use the undifferentiating adipose derived stem cells (ADSCs) andplatelet rich fibrin (PRF) as a cartilage repair materials to repair the rabbit’s ear cartilagefull thickness defect, and make groundwork for the further study in cartilage tissueengineering.【Objective】To explore the feasibility of repairing ear cartilage whole layer defect in the rabbit bycombined the undifferentiating autologous or allogeneic adipose derived stem cells(ADSCs) and platelet rich fibrin (PRF) which as a new kind of cartilage repair material,and detecting the immune rejection.【Methods】1. The ADSCs were cultured using the tissue culture method derives from therabbit’back adiposetissue, using the FCM, HE staining, adipogenic and osteogenicinduction to identify the stem cell characteristics. And a lot of ADSCs will culture usefor transplantation in vitro.2. The PRF are made by the rabbit’s whole blood, and composite with the ADSCs as anew kind of materials to repair the cartilage full-thickness defect.3. Set up a cartilage defect animal model. Make a full-thickness cartilage defect on therabbit auricle by trauma.3months later, check the cartilage defect repair situation bythe general observation and histological observation (HE and alcian blue staining).4. Group of experiments. We selected30rabbits,4ear cartilage whole layer defectswere operated in each rabbit, then the defects were randomly divided into6groups:group A, as blank control; group B, autologous ADSCs as the repair material; groupC, allogeneic ADSCs as the repair material; group D, self PRF were used for repaired;group E, the implant is autologous ADSCs and PRF compounds; group F, the implantis allogeneic ADSCs and PRF compounds. The rabbits were sacrificed at1,2,3months. The reconstructive cartilage was evaluated by gross observation and HEstaining, and use IPP6.0for half quantitative analysis. 5. Immunology monitoring. We taken the rabbit’s whole blood and plasma before andafter the experiment1w,2w,4w,3months, then detecting the CD4, CD8and IL2,IL-4. The date was analysed using SPSS16.0software.6. Statistical analysis. All the data using SPSS16.0software for statistical, and the dosedata record as the mean plus or minus the standard deviation form, multiple sampleswere compared using analysis of variance, two two compared using the SNK-qinspection, inspection level alpha is0.05on both sides.【Results】1. The stem cells which separation and cultivate from rabbit adipose tissue is appears asa long spindle, self-renewal, the bility of proliferation, and has the capacity todifferentiation into fat and ossification cells. And the FCM result showed that thestem cells expression of the ADSCs specificity surface antigen CD29, the positiveexpression rate was (91.2+2.9)%, and CD31negative expression, expression ratewas（2.9±1.5）%, its biological characteristics stability.2. The PRF is a kind of tough, elastic material, it is very suitable for transplant to use inthe tissue engineering.3. Successfully established experimental animal model. The gross observation, HEand alcian blue staining, all of these results were showed that cartilage defect areawithout new cartilage formation.4. After transplantation for3months, the HE and alcian blue staining results showedthat, group A was almost no new cartilage formation.In group B and group C, therewere small amount of new cartilage tissue on the edge of the defect area,but thedefect is still obviously, as similar as the blank control group; In grour D, the grossobservation see that a layer of membrane reform in the cartilage defect area, and thehistological result show that is new cartilage tissue; In the group E and group F, thedefect area covered by new cartilage tissue, and it’s surface is smooth, consistentwith the cartilage tissue around them. The IPP6.0statistical results showed that thecartilage defect repair rate was: group A（,1.68±0.17）%;group B,(15.9±0.87)%,group C,(15.4±0.91)%; group D,(32.0±2.76)%; group E,(87.59±3.21)%; group F,(85.77±4.88)%. The SPSS16.0statistical results showed that there is no statisticaldifference between the group B and group C (P>0.05), the same result is esistbetween the group E and group F (P>0.05).But there was statistical difference amongthe rest groups (P<0.01).5. The immune detection results show that, there were no statistical difference (P>0.05)between the experimental group and the blank control group before and after thecomposite implant. And the HE staining results also show that allograft ADSCsimplantation is no lymphocyte infiltration in the organization, all the result indicatesthat allogeneic ADSCs implant will not causing obvious immune rejection.【Conclusion】1. The stem cells which separation and cultivate from rabbit adipose tissue, has themultidirectional differentiation potential and self proliferation ability, can be used asa source of seed cell for tissue engineering.2. This experimental animal model is easy to operate and convenient to observe, so, it isa kind of ideal cartilage defect model.3. Autologous or allogeneic ADSCs composite PRF as a kind of material can effectivelyrepair rabbit’s ear full-thickness cartilage defects,and there is no difference betweenthe two groups.4. Allogeneic ADSCs transplantation will not cause a significant immune rejection. |
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