富血小板血浆及带血管筋膜在组织工程骨血管化中的作用组织影像学观察

背景：富血小板血浆中的多种生长因子及带血管筋膜是否具有促进骨再生及血管化作用尚在争论之中。目的：拟从组织影像学角度评价富血小扳血浆及带血管筋膜对骨髓基质干细胞／脱钙骨基质复合的组织工程骨血管化作用效果。设计、时间及地点：单一样本观察及动物同体对照实验，于200410／200711在青岛大学医学院附属医院中心实验室完成。材料：1-12月龄健康杂种犬12只，体质量20～25妇，雌雄各半。方法：④采用密度梯度离心法从犬骨髓中分离骨髓基质干细胞，体外贴壁培养扩增、成骨诱导培养。取杂种犬的股骨制备脱钙骨基质，并与骨髓基质干细胞复合。②将每只犬的背部分为4个区，A，B区，植入加富血小板血浆的骨髓基质干细胞，脱钙骨基质复合体，C，D区，仅植入骨髓基质干细胞／脱钙骨基质复合体。A，C区植入物用背阔肌带血管肌筋膜包囊；B，D区植入物用背部不带血管的浅筋膜包裹。分别在4，8，12周各取4只，雌雄各半，麻醉后处死取标本。主要观察指标：④采用倒置相差显微镜观察骨髓基质干细胞形态，四甲基偶氮唑盐法测定细胞生长曲线。②行改良钙钴法碱性磷酸酶染色，VonKossa法、茜素红法，钙结节染色观察诱导成骨细胞形态特征。③应用倒置相差显微镜、扫描电镜观察复合体的结构。④行大体标本、X射线数字影像及组织学观察评价。结果：①诱导成骨分化的骨髓基质干细胞分泌的碱性磷酸酶及钙结节染色均呈阳性。②骨髓基质干细胞和成骨诱导细胞的生长曲线呈典型的S形。⑨同种异体脱钙骨基质与骨髓基质干细胞复合培养后，细胞上架率高，生长状态好，增殖迅速，能分泌大量细胞外基质。④各时间点A，B，C区在成骨的骨量、骨光学密度、血管化程度等方面均明显优于D组（P〈0．05）。结论：经组织影像学观察证实，富血小板血浆和带血管肌筋膜均能促进组织工程骨的成骨和血管化，且两者具有协同作用。

Platelet-rich plasma and latissimus dorsi fascia with blood vessels in the vascularization of tissue-engineered bone: A histological & radiological observation

BACKGROUND: It is still disputed whether several growth factors of platelet-rich plasma and the fascia with blood vessels can promote bone regeneration and vascularization. OBJECTIVE: To evaluate the effects of platclet-rich plasma and the fascia with blood vessels on the vascularization of tissue-engineered bone composited by marrow stromal stem cells (MSCs) and decalcified bone matrix (DBM). DESIGN, TIME AND SETTING: The present single-sample observation, self-control animal experiment was performed at the Central Laboratory Center, Affiliated Hospital of Qingdao University Medical College between October 2004 and November 2007. MATERIALS: Twelve healthy hybrid dogs, aged 11-12 months, weighing 20-25 kg, equal number of males and females, were included for this study. METHODS: ①MSCs were isolated from dog bone marrow by density gradient centrifugation, followed by in vitro adherent culture and ontogenesis culture.Dog femur was taken for preparation of DBM and composited with MSCs.②The back of each dog was divided into 4 regions (A,B,C,and D).In the regions A and B, DBM/MSCs/platelet-rich plasma composites were transplanted. In the regions C and D, DBM/MSCs composites were transplanted. The implants for the regions A and C were wrapped with latissimus dorsi fascia with blood vessels. The implants for the regions B and D were wrapped with blood vessels-free superficial fascia from back. At weeks 4,8,and 12,4 dogs comprising 2 males and 2 females, were sacrificed following anesthesia for specimen harvesting. MAIN OUTCOME MEASURES:①MSC morphology was observed utilizing a phase contrast microscope, and cell growth curves were portrayed by methyl thiazolyl tetrazolium colorimetric assay.②Morphology of osteoblasts induced was observed by modified alkaline phospharase (ALP) staining (Ca-Co method), Von Kossa method, alizarin red method and calcium node staining.③Composite structure was observed through the use of inverted phase contrast microscope and scanning electron microscope.④Gross, radiological, and histological observations. RESULTS: ①Both induced osteoblasts and MSCs exhibited ALP and calcium node stainings positive.②The growth curves of induced osteoblasts and MSCs presented with typical "S" shape.③Following composite culture of allogenic DBM and MSCs, MSCs grew well, rapidly proliferated and could secrete a great amount of extraccllular matrix.④At each time point, osteogenic bone mass, bone absorbance, and vascularization were better in regions A,B,and C than in region D (P＜0.05). CONCLUSION: Histological and radiological observations have confirmed that both platelet-rich plasma and the fascia with blood vessels can synergistically promote the formation and vascularization of tissue-engineered bone.