本文關(guān)鍵詞：納米羥基磷灰石—殼聚糖支架復(fù)合外周血間充質(zhì)干細胞修復(fù)大鼠脛骨缺損的實驗研究　出處：《南方醫(yī)科大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 外周血間充質(zhì)干細胞 納米羥基磷灰石 殼聚糖 支架 骨缺損

【摘要】：研究背景:先天性畸形、復(fù)雜性創(chuàng)傷、腫瘤切除以及骨病等都可能造成臨界性骨缺損,骨組織無法完成自我修復(fù)而往往需要大量的骨質(zhì)進行重建。傳統(tǒng)治療臨界骨缺損有自體骨移植、異體骨移植以及人工合成材料等方法。但是供體來源缺乏、免疫排斥、容易傳播傳染性疾病以及耐用程度有限等均不同程度地限制各自的臨床應(yīng)用。骨組織工程有望克服上述缺陷并成功修復(fù)骨缺損而成為研究的熱點,其往往將間充質(zhì)干細胞移植至有三維結(jié)構(gòu)的支架材料當中,通過成骨誘導(dǎo)而促進新骨形成。間充質(zhì)干細胞(MSCs)在體外能夠自我增殖,有較強多向分化潛能并且容易向成骨細胞分化,無免疫排斥而成為骨組織工程較理想的種子細胞。骨髓間充質(zhì)干細胞(BMMSCs)有較強的增殖及成骨分化能力,成為公認的修復(fù)骨缺損的細胞來源。但是其存在取材過程創(chuàng)傷較大、數(shù)量、增殖和分化能力隨著患者年齡增大而出現(xiàn)明顯下降等缺陷。相對而言,外周血間充質(zhì)干細胞(PBMSCs)是通過極其微創(chuàng)和相對簡單的方式來獲得的,經(jīng)濟成本較低,因而有可能成為替代BMMSCs的較為理想的種子細胞。目前普遍的觀點認為對于可以應(yīng)用至骨組織工程修復(fù)的材料而言,單一材料無法滿足其需求,復(fù)合材料才是更好的選擇。納米羥基磷灰石-殼聚糖(nHA-CS)為三維多孔結(jié)構(gòu)的復(fù)合支架材料,具有良好的生物相容性、可降解性及骨傳導(dǎo)性,能夠滿足構(gòu)建組織工程化骨的要求。本實驗擬將搭載PBMSCs的nHA-CS復(fù)合支架材料移植至大鼠脛骨5mm離斷性骨缺損,觀察其修復(fù)效果。研究目的觀察納米羥基磷灰石-殼聚糖(nHA-CS)支架材料復(fù)合外周血間充質(zhì)干細胞(PBMSCs)修復(fù)大鼠臨界性骨缺損的效果。研究方法1.采用密度梯度離心及貼壁生長法分離培養(yǎng)大鼠PBMSCs并行體外擴增,檢測其表面標志物以及成骨分化潛能。2.共沉淀法制備nHA-CS支架材料,掃描電鏡(SEM)觀察其立體結(jié)構(gòu),測定其孔隙率及體外降解率。3.選擇SD大鼠脛骨5mm離斷性骨缺損作為實驗?zāi)Ｐ?42只大鼠隨機分為3組:A組曠置不處理;B組僅填充nHA-CS;C組移植PBMSCs-nHA-CS。在術(shù)后8周及12周分別處死動物,通過X線、Micro-CT以及組織學檢測結(jié)果判斷骨缺損修復(fù)情況。4.采用SPSS 20.0統(tǒng)計軟件對所有的數(shù)據(jù)進行分析。采用均值±標準差(X±SD)表示數(shù)據(jù)結(jié)果。采用單因素方差分析(One-wayANOVA)進行多組資料比較,采用最小顯著差異法(Least significant difference,LSD)進行組間比較,若方差不齊則采用Dunnett'S T3法;P值0.05認為差異有統(tǒng)計學意義。研究結(jié)果1.分離培養(yǎng)的PBMSCs呈長梭形形態(tài),能在體外擴增,流式細胞術(shù)檢測PBMSCs表面標志物符合MSCs表達。PBMSCs在體外經(jīng)成骨誘導(dǎo)可向成骨細胞分化,ALP表達陽性,鈣結(jié)節(jié)形成。2.制備的nHA-CS支架具有三維多孔網(wǎng)絡(luò)狀結(jié)構(gòu),孔徑介于100-200μm,孔隙率高且彼此相通。3.X線、Micro-CT以及組織學檢測結(jié)果顯示PBMSCs-nHA-CS組修復(fù)臨界骨缺損的效果明顯優(yōu)于nHA-CS組或空白組,且Lane-Sandhu X評分、BMD、BV以及成骨面積等定量檢測均提示PBMSCs-nHA-CS組高于nHA-CS組或空白組,結(jié)果具有顯著性差異(P0.05)。結(jié)論本實驗證明PBMSCs可以成為骨組織工程良好的細胞來源,有良好的應(yīng)用前景。PBMSCs-nHA-CS能夠成功修復(fù)大鼠脛骨5mm離斷性缺損,其修復(fù)效果優(yōu)于nHA-CS或空白組。
[Abstract]:Background: congenital malformations, complexity of trauma, tumor excision and bone disease are likely critical bone defect caused by bone tissue, unable to complete the self repair and often require a large amount of bone reconstruction. The traditional treatment of critical bone defect with autogenous bone graft, allograft and artificial synthetic materials and other methods. But the lack of donor organs, immune rejection, easy to spread of infectious diseases and durability limited to varying degrees of restrictions respectively. Clinical application of bone tissue engineering is expected to overcome the defect and repair bone defect successfully has become a research hotspot, which tend to mesenchymal stem cells transplanted to the scaffold material with three-dimensional structure, and promote new bone induction by bone formation. Mesenchymal stem cells (MSCs) in vitro can have strong self proliferation, multilineage differentiation potential and easy osteoblastic differentiation, without immune rejection And become the ideal seed cells for bone tissue engineering. Bone marrow mesenchymal stem cells (BMMSCs) and osteogenic differentiation capacity strong proliferation and repair of bone defects become recognized source of cells. But there were large number of trauma, proliferation and differentiation ability of patients with increased age and decreased significantly. Defect. In contrast, peripheral blood mesenchymal stem cells (PBMSCs) are obtained through extremely minimally invasive and relatively simple way, low cost, and may become the ideal seed cells to replace BMMSCs. The general idea that can be used for bone tissue engineering to repair materials, a single material can not meet the requirements, the composite is a better choice. Nano hydroxyapatite chitosan (nHA-CS) composite scaffolds for three-dimensional porous structure, has good biological compatibility, can be Degradation and bone conduction, can meet the requirements of the construction of tissue engineered bone. The experiment will be equipped with PBMSCs of 5mm nHA-CS composite scaffold transplanted to rat severed bone defect, to observe the repair effect. Objective to study nano hydroxyapatite chitosan (nHA-CS) composite scaffold of peripheral blood mesenchymal stem cells (PBMSCs) critical bone defect repair in rats. Methods 1. by density gradient centrifugation and adherent cells were isolated and cultured rat PBMSCs parallel in vitro, the detection of surface markers and osteogenic differentiation potential of.2. co precipitation method to prepare nHA-CS scaffolds, scanning electron microscope (SEM) to observe the stereo the structure, determination of the porosity and degradation rate in vitro of.3. SD 5mm transection of rat tibial bone defect as experimental model, 42 rats were randomly divided into 3 groups: group A no exclusion treatment; group B filled nHA-CS group C PBMSCs transplantation; -nHA-CS. after 8 weeks and 12 weeks were sacrificed by animal, X-ray, Micro-CT and histological examination results to determine the repair of bone defect by.4. on all of the data were analyzed by SPSS 20 statistical software. The mean standard deviation (X + SD). The results indicates that the data with single factor analysis of variance (One-wayANOVA) of group data comparison, using the least significant difference method (Least significant difference, LSD) were compared between the two groups, if homogeneity of variance using Dunnett'S T3 method; P value of 0.05 is considered statistically significant. Results: 1. PBMSCs isolated from the long fusiform shape, can be amplified in vitro and flow cytometry PBMSCs the surface markers with MSCs expression of.PBMSCs in vitro osteogenic induction of osteogenic differentiation, the expression of ALP, the formation of calcium nodules nHA-CS scaffolds prepared by.2. with three-dimensional porous network structure, the aperture is 100-2 00 m, high porosity and shared.3. X-ray, Micro-CT and histological examination showed that the PBMSCs-nHA-CS group to repair critical bone defect is better than that of nHA-CS group and blank group, and the Lane-Sandhu X score, BMD, BV and bone area quantitative detection showed PBMSCs-nHA-CS group than in nHA-CS group or control group, the results were significant the difference (P0.05). Conclusion PBMSCs can be a good source of cells for bone tissue engineering, has good application prospects of.PBMSCs-nHA-CS can successfully repair tibia of 5mm rats isolated defects, the repair effect is better than nHA-CS or the control group.

【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R687

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