富含血小板血浆双相接种法构建组织工程骨

目的：传统的细胞接种法接种效率低,影响组织工程骨的成骨质量。本研究预探讨富含血小板血浆双相接种法是否可以提高细胞接种效率、优化组织工程骨的构建。方法：分别用富含血小板血浆、乏血小板血浆重悬BMSCs,灌注于三维多孔PLGA支架中,凝血酶促凝,体外培养。通过扫描电镜观察支架表面的细胞分布;通过检测支架内DNA含量、ALP含量、成骨基因表达情况分析细胞的接种效率、增殖和分化情况,并与传统的接种方法进行比较。结果：富含血小板血浆和乏血小板血浆双相接种法均能提高细胞与三维支架的结合效率;富含血小板血浆还可以促进支架内细胞的增殖,同时不影响细胞的分化。结论：双相接种法可以提高细胞的接种效率;与普通双相接种法相比,富含血小板血浆双相接种法可以进一步优化组织工程骨的构建。     

Fresh bone marrow introduction into porous scaffolds using a simple low‐pressure loading method for effective osteogenesis in a rabbit model

Recent advances in tissue engineering techniques have allowed porous biomaterials to be combined with osteogenic cells for effective bone regeneration. We developed a simple low‐pressure cell‐loading method using only syringes and stopcocks, and examined the effect of this method on osteogenesis when applied to the combination of highly porous β‐tricalcium phosphate (β‐TCP) and fresh autologous bone marrow. Both block and granule β‐TCP scaffolds were used to prepare implants in three different ways: without bone marrow as a control, with bone marrow that was allowed to penetrate spontaneously under atmospheric pressure (AP group), and with bone marrow that was seeded under low pressure (ULP group). These implants were transplanted into rabbit intramuscular sites, and the samples were examined biologically and histologically. The penetration efficiency of the block implants after marrow introduction was significantly higher in the ULP group than in the AP group. In the transplanted block samples, alkaline phosphatase activity was significantly higher in the ULP group at 2 weeks after implantation, and significantly more newly formed bone was observed in the ULP group at both 5 and 10 weeks compared with the AP group. Similar results were observed even in the experiment using β‐TCP granules, which are smaller than the blocks and frequently used clinically. Because of its convenience and safety, this low‐pressure method might be a novel, effective treatment to promote osteogenesis with bone marrow in clinical bone reconstruction surgeries. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1–7, 2009     

玉米醇溶蛋白仿生组织工程支架材料体内外诱导成骨的实验研究

目的 检测玉米醇溶蛋白(zein)仿生三维多孔支架材料的生物相容性、骨诱导性和生物可降解性. 方法 将人骨髓基质干细胞(BMSCs)载人海藻酸钠或zein制成复合物.通过扫描电镜观察、噻唑蓝分析法以及碱性磷酸酶染色与定量检测,分别比较zein和海藻酸钠对BMSCs体外黏附、增殖和分化的影响.取24只8周龄裸鼠,随机分为两组,制成肌袋植入异位成骨模型.对照组单纯植入zein,实验组植入zein与兔BMSCs复合物.术后2、4、8和12周取材进行影像学和组织学观察. 结果 与海藻酸钠相比,zein在任何时间段均能够更好地促进兔BMSCs的体外增殖和分化,差异有统计学意义(P<0.05).同时,与单纯植入zein相比,BMSCs能够增强zein体内诱导成骨的作用. 结论 zein具有良好的生物相容性、骨诱导性和生物可降解性.     

Orbital seeding of mesenchymal stromal cells increases osteogenic differentiation and bone-like tissue formation

In bone tissue engineering (TE), an efficient seeding and homogenous distribution of cells is needed to avoid cell loss and damage as well as to facilitate tissue development. Dynamic seeding methods seem to be superior to the static ones because they tend to result in a more homogeneous cell distribution by using kinetic forces. However, most dynamic seeding techniques are elaborate or require special equipment and its influence on the final bone tissue-engineered construct is not clear. In this study, we applied a simple, dynamic seeding method using an orbital shaker to seed human bone marrow–derived mesenchymal stromal cells (hBMSCs) on silk fibroin scaffolds. Significantly higher cell numbers with a more homogenous cell distribution, increased osteogenic differentiation, and mineral deposition were observed using the dynamic approach both for 4 and 6 hours as compared to the static seeding method. The positive influence of dynamic seeding could be attributed to both cell density and distribution but also nutrient supply during seeding and shear stresses (0.0-3.0 mPa) as determined by computational simulations. The influence of relevant mechanical stimuli during seeding should be investigated in the future, especially regarding the importance of mechanical cues for bone TE applications. Our results highlight the importance of adequate choice of seeding method and its impact on developing tissue-engineered constructs. The application of this simple seeding technique is not only recommended for bone TE but can also be used for seeding similar porous scaffolds with hBMSCs in other TE fields.     

Promotion of osteogenesis in tissue‐engineered bone by pre‐seeding endothelial progenitor cells‐derived endothelial cells

In addition to a biocompatible scaffold and an osteogenic cell population, tissue‐engineered bone requires an appropriate vascular bed to overcome the obstacle of nutrient and oxygen transport in the 3D structure. We hypothesized that the addition of endothelial cells (ECs) may improve osteogenesis and prevent necrosis of engineered bone via effective neovascularization. Osteoblasts and ECs were differentiated from bone marrow of BALB/c mice, and their phenotypes were confirmed prior to implantation. Cylindrical porous polycaprolactone (PCL)‐hydroxyapatite (HA) scaffolds were synthesized. ECs were seeded on scaffolds followed by seeding of osteoblasts in the EC‐OB group. In the OB group, scaffolds were only seeded with osteoblasts. The cell‐free scaffolds were denoted as control group. A 0.4‐cm‐long segmental femur defect was established and replaced with the grafts. The grafts were evaluated histologically at 6 weeks postimplantation. In comparison with the OB group, the EC‐OB group resulted in a widely distributed capillary network, osteoid generated by osteoblasts and absent ischemic necroses. Pre‐seeding scaffold with ECs effectively promoted neovascularization in grafts, prevented the ischemic necrosis, and improved osteogenesis. The integration of bone marrow‐derived ECs and osteoblasts in porous scaffold is a useful strategy to achieve engineered bone. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1147–1152, 2008     

胎儿骨髓间充质干细胞复合PLGA支架的形态学观察

目的 探讨胎儿骨髓间充质干细胞(fetal bone marrow mesenehymal stem cells，fBMSCs)与低热高压法制作的聚丙交酯-乙交酯(PLGA)支架材料复合培养。构建组织工程化骨的可行性。方法 预制PLGA支架材料，取5月胎儿肱骨和股骨骨髓，用单核细胞分离液分离fBMSCs，含双抗的低糖DMEM原代和传代培养，倒置显微镜下观察细胞生物学特性，用F1TC标记的抗CDl05、CD44和用PE标记的抗CD34、CD29对第4代细胞进行流式细胞鉴定，并与PLGA支架材料复合培养l周后行扫描电镜观察。结果原代及传代培养的fBMSCs增殖较迅速，第4代CD105阳性细胞占84．08％，CD29阳性细胞占88．77％，CD44阳性细胞占89．53％。复合PLGA支架材料细胞生长状态良好。结论 骨髓间充质干细胞(bone mesenchymal stem cells，BMSCs)是骨组织工程适宜的种子细胞，与低热高压制作的PLGA支架材料复合可体外构建组织工程骨。     

骨形态发生蛋白-2活性多肽修饰的重组胶原矿化骨对骨髓基质干细胞生物学行为的影响

目的 探讨骨形态发生蛋白-2(BMP-2)活件多肽修饰的重组胶原矿化骨复合材料对骨髓基质干细胞(BMSCs)增殖、黏附及分化等生物学行为的影响. 方法制备重组胶原矿化骨支架材料,将BMP-2活性多肽通过交联剂共价结合到材料上,扫描电镜观察支架材料表面微观形貌;取第3代BMSCs接种到材料上,以未结合多肽的重组胶原矿化骨作为对照,采用MTT法检测BMSCs在材料表面的增殖;沉淀法检测BMSCs在材料表面的黏附率;扣描电镜观察比较BMSCs在材料表面的生长形态;通过检测细胞中的碱性磷酸酶活性及钙含量,观察BMSCs在材料表面的分化情况. 结果扫描电镜结果显示:支架材料旱多孔状;X射线光电子能谱法证实BMP-2活性多肽成功共价结合到材料表面;BMP-2活性多肽修饰的重组胶原矿化骨复合材料表面BMSCs的黏附和向成骨细胞方向分化能力均高于对照组,差异有统计学意义(P＜0.05),而BMSCs的增殖能力与对照组相比,差异无统计学意义(P＞0.05). 结论BMP-2活性多肽可以显著改善重组胶原矿化骨复合材料的细胞相容性和生物活性,经BMP-2活性多肽修饰的重组胶原矿化骨复合材料是一种理想的骨组织工程支架材料.     

Improved endothelial cell seeding with cultured cells and fibronectin-coated grafts

A possible approach to the low seeding efficiency of endothelial cells into prosthetic grafts is to increase the number of cells to be seeded in cell culture and improve seeding efficiency by graft precoating with fibronectin. The effect of cell culture on cell adhesion is unknown, however, and fibronectin also binds fibrin, which may increase the thrombogenicity of the graft luminal surface. To investigate these questions, freshly harvested canine jugular vein endothelial cells from six animals and similar cells harvested from six primary and eight secondary cell cultures were labeled with 111Indium and seeded into 5 cm, 4 mm PTFE grafts coated with fibronectin, using similar uncoated PTFE grafts as controls. Platelet accumulation and distribution on six similar coated and uncoated grafts placed in canine carotid, external jugular arterial venous shunts for 2 hr were also determined using autogenous 111Indium-labeled platelets. Significant differences between group means were determined using the paired Student's t test. Results reveal that seeding efficiency is significantly better in all groups of coated grafts compared to uncoated grafts (P less than 0.01). Cells derived from cell culture also had significantly higher seeding efficiencies than freshly harvested cells when seeded into coated grafts (P less than 0.05) and tended to have higher seeding efficiencies than harvested cells when seeded into uncoated grafts (P = 0.53). Fibronectin coating increased mean platelet accumulation on the entire graft luminal surface, but not to a statistically significant degree (P greater than 0.1). Whether this increased seeding efficiency will improve graft endothelialization remains to be investigated.     

Bone tissue engineering in a critical size defect compared to ectopic implantations in the goat.

Since the application of the autologous bone graft, the need for an alternative has been recognized. Tissue engineering (TE) of bone by combining bone marrow stromal cells (BMSCs) with a porous scaffold, is considered a promising technique. In this study we investigated the potential of tissue engineered bone to heal a critical sized defect in the goat. Orthotopic bone formation was compared to ectopic bone formation in comparable constructs. TE constructs were prepared from goat BMSCs and porous biphasic calcium phosphate ceramic scaffolds. These constructs and scaffolds without cells were implanted paired in critical sized iliac wing defects. Comparable samples were implanted intramuscularly. After 9 (n=7) and 12 (n=8) weeks implantation, the samples were analyzed histomorphometrically. After 9-weeks implantation in the iliac wing defect, significantly more bone apposition was found in the TE condition. After 12 weeks, the defects were almost completely filled with bone, but no significant advantage of TE was determined anymore. This contrasted with the intramuscular samples where TE implants showed significantly more bone at both time points. In conclusion, bone TE is feasible in critical sized defects. However, when appropriate osteoconductive/inductive materials are applied the effect of cell seeding may be temporary.     

Optimization of bone tissue engineering in goats: a peroperative seeding method using cryopreserved cells and localized bone formation in calcium phosphate scaffolds

BACKGROUND: Bone tissue engineering by combining cultured bone marrow stromal cells with a porous scaffold is a promising alternative for the autologous bone graft. Drawbacks of the technique include the delay necessary for cell culture and the complicated logistics. We investigated methods to bypass these drawbacks. Furthermore, we investigated the localization of bone formation inside the scaffold. METHODS: Bone marrow stromal cells from seven goats were culture expanded and cryopreserved. One week before surgery, some of the cells were thawed, cultured, and seeded on porous calcium phosphate scaffolds. The constructs were cultured for another week until implantation. The remaining cryopreserved cells were thawed just before implantation and peroperatively resuspended in plasma before combining with the scaffold. Scaffolds impregnated with fresh bone marrow, devitalized cultured constructs, and empty scaffolds served as controls. All samples were implanted in the back muscles of the goats for 9 weeks. RESULTS: Histologic examination showed minimal (<1%) bone in the empty and devitalized scaffolds, 4.2 +/- 5.1 bone area percent in the bone marrow samples, and significantly more bone in both the cultured and peroperatively seeded constructs (11.7 +/- 2.5 and 14.0 +/- 2.0%). The peripheral 350 microm of the implants contained significantly less bone. CONCLUSION: Peroperative preparation of osteogenic constructs with cryopreserved cells is feasible. These constructs yield substantially more bone than the scaffolds alone or scaffolds impregnated with fresh bone marrow. Bone deposition is much less on the scaffold periphery.     

Tissue engineering in cardiovascular surgery: MTT, a rapid and reliable quantitative method to assess the optimal human cell seeding on polymeric meshes.

OBJECTIVE: Currently used valve substitutes for valve replacement have certain disadvantages that limit their long-term benefits such as poor durability, risks of infection, thromboembolism or rejection. A tissue engineered autologous valve composed of living tissue is expected to overcome these shortcomings with natural existing biological mechanisms for growth, repair, remodeling and development. The aim of the study was to improve cell seeding methods for developing tissue-engineered valve tissue. METHODS: Human aortic myofibroblasts were seeded on polyglycolic acid (PGA) meshes. Cell attachment and growth of myofibroblasts on the PGA scaffolds with different seeding intervals were compared to determine an optimal seeding interval. In addition, scanning electron microscopy study of the seeded meshes was also performed to document tissue development. RESULTS: There was a direct correlation between cell numbers assessed by direct counting and MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertra-zoliu m bromide) assay. Both attach rate and cell growth seeded on meshes with long intervals (24 and 36 h) were significantly higher than those seeded with short intervals (2 and 12 h) (P<0.01), there was no significant difference between 24- and 36-h seeding interval. Scanning electron microscopy also documented more cell attachment with long seeding intervals resulting in a more solid tissue like structure. CONCLUSION: It is feasible to use human aortic myofibroblasts to develop a new functional tissue in vitro. Twenty-four hours is an optimal seeding interval for seeding human aortic myofibroblasts on PGA scaffolds and MTT test is a rapid and reliable quantitative method to assess the optimal human cell seeding on polymeric meshes.     

血管平滑肌细胞在几种基质膜片上生长情况及三种接种培养方法的比较

目的 筛选适合于组织工程血管构建的可降解基质材料及最佳的接种培养方式。方法 将血管平滑肌细胞接种于几种现有的可降解基质材料上，采用MTT法观察细胞在几种基质材料上的生长情况；比较静态、水平摇床、旋转接种三种接种培养方式。结果 细胞相容性比较好的材料有：PGA、胶原加黏多糖、脱钙骨等。三种接种方法的接种率为：静态1．5％。水平摇床7．2％，旋转接种培养53．5％。结论 PGA、胶原加黏多糖、脱钙骨三种材料适合于组织工程血管再造的应用。而三种接种培养方式以旋转接种培养的效率最高。     

双层血管细胞种植提高人工血管内皮细胞粘附性

目的　采用血管内皮细胞 (EC)和平滑肌细胞 (SMC)双层种植方法 ,提高人工血管腔面EC的保存率。　方法　聚四氟乙烯 (PTFE)人工血管经纤维连结蛋白预衬处理 ,管腔面先后种植SMC和EC ,将受试的人工血管接入脉冲式体外灌注装置灌注 1h ,计算比较灌注前后受试标本SMC和EC密度。　结果　灌注 1h后 ,单纯EC种植组 61%细胞脱落 ,单纯SMC种植组 3 6%细胞脱落 ,而双层细胞种植组仅有 2 7%EC脱落 ,双层细胞种植组细胞保存率明显高于单纯EC种植组 (P <0 0 1)。低流速预灌注未能改善EC的保存率。　结论　在EC和人工血管壁之间种植SMC可提高EC的保存率     

Construction of an autologous tissue-engineered venous conduit from bone marrow-derived vascular cells: optimization of cell harvest and seeding techniques

Background

Currently available vascular grafts for pediatric cardiovascular operations are limited by their inability to grow. Tissue-engineering techniques can be used to create vascular grafts with the potential for repair, remodeling, and growth. This study demonstrates the feasibility of constructing an autologous tissue-engineered venous conduit from bone marrow-derived vascular cells (BMVCs) in the ovine animal model.

Methods

Ovine mononuclear cells were isolated from the bone marrow, cultured in endothelial growth medium, and characterized with immunocytochemistry. Biodegradable tubular scaffolds were constructed from polyglycolic acid mesh coated with a copolymer of poly[ε-caprolactone-l-lactide]. Scaffolds were seeded at various cell concentrations and incubation times to optimize seeding conditions for the construction of an autologous venous conduit. Using optimized conditions, 6 tissue-engineered vascular grafts were implanted as inferior vena cava interposition grafts in juvenile lambs. Grafts were assessed for patency at days 1 to 30 postoperatively and explanted for histological and immunohistochemical analysis.

Results

A mixed cell population of BMVCs consisting of smooth muscle cells and endothelial cells was cultured from ovine sternal bone marrow. A seeding concentration of 2 × 10⁶ cells/cm² and 7 days of postseeding incubation were optimal for creating a confluent cellular layer on the polyglycolic acid/poly[ε-caprolactone-l-lactide]) scaffold. Grafts were explanted up to 4 weeks postoperatively. All grafts were patent without evidence of thrombosis. Histological evaluation of the explanted grafts demonstrated neo-endothelialization. Graft wall was composed of neo-tissue made up of residual polymer matrix, mesenchymal cells, and extracellular matrix without evidence of calcification.

Conclusions

Bone marrow-derived vascular cells, containing endothelial and smooth muscle cells, can be isolated and cultured from ovine sternal bone marrow and used as a cell source for vascular tissue engineering. Our optimized techniques for BMVC harvest and seeding onto biodegradable scaffolds can be used for studying autologous tissue-engineered vascular grafts in the ovine animal model.     

Endothelial cell seeding

Endothelial cell seeding is the transplantation of vascular endothelial cells to denuded vascular surfaces. Seeding theoretically reduces the probability of graft or vessel thrombosis and of neointimal fibrous hyperplasia. Thus far, clinical seeding trials disclosed modest improvements in patency and the development of hyperplastic anastomotic lesions in failed grafts. Seeding inefficiency theoretically contributes to anastomotic hyperplasia. The inefficiency is linked to two steps in the seeding process, namely harvesting and cell retention. Of these, cell retention on the seeded surface is the more critical. Priorities for future research should be set first on the retention of seeded endothelium in vitro and second on improved and standardized methods of cell harvesting.     

Endothelial cell seeding of small-caliber synthetic grafts in the baboon

Endothelial cell (EC) seeding has been proposed as a method to improve the performance of small-caliber synthetic vascular prostheses. Seeding experiments to date have all been carried out in the dog. This study investigates EC seeding of small-caliber Dacron carotid interposition grafts compared with contralateral control grafts in the baboon. Surface thrombogenicity was assessed at 24 hours, 2 weeks, and 4 weeks after implantation with indium 111-labeled autologous platelets. Morphologic and immunohistochemical techniques were used to assess the identity and homogeneity of the EC inoculum before seeding and to identify cell types on the harvested grafts. There was no significant difference in patency rates between seeded and control grafts at 5 weeks. Platelet accumulation on seeded grafts was significantly less (p less than 0.05) than on paired controls at 2 and 4 weeks after implantation. The luminal lining of seeded grafts had more cellular ingrowth, less adherent thrombus, and more surface cells with the morphologic and histochemical characteristics of EC than did the lining of controls. EC seeding reduces the platelet reactivity and accelerates EC coverage of small-caliber grafts in the baboon.     

Endothelial cell seeding of polytetrafluoroethylene vascular grafts in humans: a preliminary report

The importance of initial human trials with autologous endothelial seeding lies not only in the implementation of a promising idea but also in the fact that canine data are only partially applicable to humans. The surface area of jugular veins in humans is much smaller than in dogs and considerably longer grafts are needed. Moreover, the reproductive capacity of adult human endothelial cells under in vivo conditions, which probably determines the success of seeding more than the seeding density, is also uncertain. Therefore the efficiency of autologous endothelial seeding in humans was investigated in 18 patients undergoing distal femoropopliteal bypass surgery. The average surface area of the jugular veins was 4.9 +/- 1.7 cm2 with an average cell yield of 32.6 +/- 18.0 x 10(4). The mean number of seeded cells per square centimeter of graft surface was 3.1 x 10(3). In a follow-up extending for 14 weeks, plasma levels of platelet factor 4 and beta-thromboglobulin as well as the platelet function in the whole blood aggregometer showed significantly better results in the seeded group. Plasma thromboxane B2, uptake and survival of indium 111-labeled platelets, and Doppler ultrasound investigations also favored the seeded group, but the results were statistically insignificant. No difference at all was found for the platelet dense granule compounds, releasable adenosine triphosphate and platelet serotonin. Thus our findings did not indicate the development of a closed endothelialized surface after 14 weeks, which is a period three times as long as the one required for confluent endothelial cell coverage in dogs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Treatment of osteonecrosis of the femoral head with hBMP-2-gene-modified tissue-engineered bone in goats

The efficacy of beta-tricalcium phosphate (beta-TCP) loaded with bone morphogenetic protein-2 (BMP-2)-gene-modified bone-marrow mesenchymal stem cells (BMSCs) was evaluated for the repair of experimentally-induced osteonecrosis of the femoral head in goats. Bilateral early-stage osteonecrosis was induced in adult goats three weeks after ligation of the lateral and medial circumflex arteries and delivery of liquid nitrogen into the femoral head. After core decompression, porous beta-TCP loaded with BMP-2 gene- or beta-galactosidase (gal)-gene-transduced BMSCs was implanted into the left and right femoral heads, respectively. At 16 weeks after implantation, there was collapse of the femoral head in the untreated group but not in the BMP-2 or beta-gal groups. The femoral heads in the BMP-2 group had a normal density and surface, while those in the beta-gal group presented with a low density and an irregular surface. Histologically, new bone and fibrous tissue were formed in the macropores of the beta-TCP. Sixteen weeks after implantation, lamellar bone had formed in the BMP-2 group, but there were some empty cavities and residual fibrous tissue in the beta-gal group. The new bone volume in the BMP-2 group was significantly higher than that in the beta-gal group. The maximum compressive strength and Young's modulus of the repaired tissue in the BMP-2 group were similar to those of normal bone and significantly higher than those in the beta-gal group. Our findings indicate that porous beta-TCP loaded with BMP-2-gene-transduced BMSCs are capable of repairing early-stage, experimentally-induced osteonecrosis of the femoral head and of restoring its mechanical function.     

转化生长因子β1缓释载体诱导骨髓基质干细胞成软骨分化

目的探讨转化生长因子β1（TGF—β1）缓释对骨髓基质干细胞诱导成软骨分化的影响。方法乳化交联法制备TGF—β1壳聚糖缓释微球并将其与壳聚糖支架复合，将骨髓基质干细胞置于复合载体中立体培养，通过HE染色、Ⅱ型胶原免疫组化染色、扫描电镜观察复合载体对细胞的增殖、分化及功能的影响。结果骨髓基质干细胞于复合载体中培养，细胞形态类似软骨细胞，并可见细胞增殖及细胞外基质分泌，Ⅱ型胶原免疫组织化学染色示基质中有Ⅱ型胶原表达，其细胞增殖率及Ⅱ型胶原分泌功能均较对照组明显增强。结论复合载体能够控制TGF-β1的释放并保持其活性，可促进骨髓基质干细胞的增殖并诱导其向软骨细胞分化。     

绵羊BMSCs与光接枝改性前后聚羟基丁酸-羟基异戊酯共培养的对比研究

目的 评价光接枝改性对聚羟基丁酸-羟基异戊酯(copolymers of 3-hydroxybutyrate and 3-hy-droxyvalerate,PHBV)与绵羊BMSCs相容性的影响. 方法 取6月龄雄性绵羊1只,体重17 kg,髂后棘穿刺抽取骨髓,全骨髓贴壁培养法分离培养BMSCs.制备PHBV膜、光接枝PHBV膜、PHBV支架及光接枝PHBV支架.取第3代BMSCs以1 ×105/mL接种至培养板,分别与PHBV膜和光接枝PHBV膜复合培养,接种后1、2、6 h计算细胞在两种膜上的黏附率;另将BMSCs以1×104/孔接种于两种膜上,接种后6 h、3 d及1、3周取材,扫描电镜观察细胞在两种膜上的生长情况;另将BMSCs以2×105/孔接种于两种支架上,接种后3 d及1、3周取材,扫描电镜观察细胞在两种支架上的生长情况;再将BMSCs以2 × 104/孔接种于两种膜上,5 d后收集细胞,流式细胞仪分析细胞周期;再将BMSCs以1 ×107/孔接种于两种支架上,接种后4、8、12d测定细胞内蛋白质和DNA含量. 结果 接种后1 h,PHBV膜上的细胞黏附率为37.5%±5.3%,光接枝PHBV膜为52.7%±6.0%,二者比较差异有统计学意义(P＜0.05);接种后2、6 h,两种支架上的细胞黏附率比较差异无统计学意义(P＞0.05).扫描电镜观察光接枝PHBV膜以及光接枝PHBV支架上的细胞黏附较早,接种后1周两种材料上的细胞数较多,且细胞分泌物亦较多.流式细胞仪检测见两种膜上的BMSCs均为正常二倍体细胞,未见异倍体细胞,细胞周期和增殖指数比较差异均无统计学意义(P＞0.05).BMSCs接种至两种支架上4、8、12 d,细胞内DNA、蛋白质含量比较差异均无统计学意义(P＞0.05). 结论 光接枝表面改性增强了PHBV对绵羊BMSCs的早期吸附,改善了其生物相容性.