生物衍生骨支架材料的组织相容性研究

目的 了解不同处理方法制得的3种生物衍生骨支架材料的组织相容性。方法 将物理化学方法处理制得的复合型完全脱蛋白骨（composite fully deproteinized bone,CFDB)、部分脱蛋白骨（partially deproteinized bone,PDPB)、部分脱钙骨（partially decalcified bone,PDCB)3种材料各10块分别植入兔肌肉内及骨膜下，进行一般观察、血清抗体检测、局部细胞免疫评估、常规HE染色，观测3种材料对机体的毒性作用、免疫效应及骨膜成骨的影响。结果 3种材料均无毒性作用，且能引导周围组织长入材料内；3种材料对骨膜成骨无不良影响，且能促进骨膜形成的软骨或类骨组织钙化形成新骨，并有一定的骨结合能力；3种材料引起机体免疫反应强弱为：PDCB＞PDPB＞CFDB。结论 CFDB、PDPB、PDCB3种天然生物衍生骨材料皆有良好的生物相容性。     

异种肌腱基质填充材料的组织相容性

目的 通过动物实验及组织学观察,研究评价所研制的异种肌腱基质填充材料的组织相容性.方法 健康杂种犬15只,体质量10～13 kg,雌雄不限.于脊柱两侧皮下分别置入异种肌腱基质填充材料(实验组)和硅橡胶材料(对照组),术后14、30、60 d分别取材,进行大体观察和组织学观察.结果 两组动物均在术后研究期间生存良好,置入部位均未见组织坏死、积液及化脓感染.术后14 d,实验组和对照组均见纤维组织增生包绕,炎性反应轻到中度;术后30 d,两组炎性反应轻微;术后60 d,实验组未见炎性反应,置入材料完整,未见肉芽肿.对照组置入硅胶材料周围有薄层纤维组织增生,炎性反应不明显.术后相同时间点,实验组与对照组组织学评分比较,差异无统计学意义(P＞0.05).结论 异种肌腱基质填充材料具有良好的组织相容性.     

仿生复合人工骨材料的组织工程细胞相容性研究

目的　探讨组织工程化人工骨的仿生构建模式及具有不同细胞相容性支架材料的选择。方法　分别采用人工合成材料生物活性玻璃陶瓷 (BGC)与双相羟基磷灰石 (HA/β TCP)为支架材料 ,与聚 DL 乳酸 (PDLLA)复合后 (重量比分别为 65∶1及 50∶1 )再复合I型胶原及重组合人类骨形态发生蛋白 (rhBMP 2 ) ,与兔骨膜成骨细胞 (密度 1× 1 0 6 /ml)复合培养 ,进行一般与超微形态学观察 ,测定生长曲线及成骨细胞碱性磷酸酶 (ALP)、骨钙素和I型胶原合成量的测定 ,比较细胞粘附能力、增殖活力及成骨活性。结果　生物活性玻璃陶瓷、双相羟基磷灰石分别与成骨细胞复合后的粘附能力、增殖活力 (1d组 :0 .380± 0 .0 32 ,0 .2 50± 0 .0 1 9;7d组 :0 .950± 0 .0 60 ,0 .650±0 .0 4 0 )、成骨活性 (3H 脯氨酸掺入活性 :6 .2 2 8± 1 .785 ,3 .81 8± 0 .858;骨钙素含量 :0 .70 9± 0 .1 1 5 ,0 .386± 0 .0 93 ;碱性磷酸酶 :0 .1 2 3± 0 .0 32 ,0 .0 83± 0 .0 1 8) ,前者均优越于后者。结论　该种仿生组织工程化人工骨的构建模式能较满意模拟天然骨优势 ,生物活性玻璃陶瓷、双相羟基磷灰石两种材料的共混体 ,有可能成为骨组织工程较理想的支架材料     

纳米羟基磷灰石-40%二氧化锆生物陶瓷材料组织相容性评价

[目的]评估纳米羟基磷灰石-二氧化锆生物陶瓷材料组织相容性.[方法]根据ISO10993-1标准,采用细胞毒性试验、急性毒性试验、溶血试验和体内植入(90 d)试验对纳米羟基磷灰石-二氧化锆生物陶瓷材料组织相容性进行评估.[结果]纳米羟基磷灰石-二氧化锆生物陶瓷材料组织相容性的细胞毒性评分小于I级,细胞生长无明显抑制现象,无急性毒性反应,无溶血反应,体内植入符合植入材料生物学评价要求.[结论]纳米羟基磷灰石-二氧化锆生物陶瓷材料具有良好的组织相容性,作为骨组织工程中生物支架材料具有广阔临床应用前景.     

修复神经缺损的组织工程支架材料的研制及其生物相容性研究

目的 研制一种可用于临床神经损伤修复的人工桥接物．并对其进行微观空间结构观察及生物相容性的研究。方法期利用生物相容性较好且可降解的I型胶原和明胶经混合溶解及冷淋后形成具有单一纵向微管的神经桥接物。将该材料植入BALB／C小鼠股部肌袋中，分为实验组及对照组在材料植入的第7、14、21、35天等不同时间段进行组织学观察及血液生化学的检测。结果 术后各组小鼠如常．伤口I期愈合、组织学观察及血液学的检测提示该桥接物具有较好的生物相容性及可吸收性。结论 该材料可用作神经损伤后修复的基础及临床研究的组织工程桥接物。     

衍生肌腱支架材料的细胞相容性研究

目的 探讨衍生肌腱支架材料（tendon derivation biomaterials，TDBM）与肌腱细胞的细胞相容性及肌腱细胞在此三维支架上培养的生物学行为，为肌腱组织工程新型支架材料的应用提供依据。方法将肌腱细胞与TDBM体外复合培养，设置单纯肌腱细胞培养为对照组，进行形态学观察，并检测细胞增殖、细胞周期、细胞DNA倍体水平及肌腱细胞凋亡率。通过^3H-脯氨酸（^3H-Proline）掺入试验了解材料对肌腱细胞胶原合成的影响。结果 肌腱细胞在TDBM上呈梭形生长，TDBM组细胞第2天进入对数增长期，倍增时间为3d，而单纯肌腱细胞培养对照组倍增时间为3．75d。TDBM组与单纯肌腱细胞培养对照组比较，^3H-Proline掺入值差异无统计学意义（P〉0．05），说明细胞功能未受影响。与支架材料复合培养的肌腱细胞DNA指数为0．96，增殖指数较对照组高10．1％，提示肌腱细胞在三维胶原支架上生长速度快，增殖能力强。结论TDBM具有良好的细胞相容性，可作为肌腱细胞的有效载体应用于组织工程化肌腱的构建。     

可注射性纳米组织工程骨的生物相容性

目的 以可注射性纳米材料与共培养的成骨细胞和血管内皮细胞复合,构建可注射性组织工程骨,并观察其体外实验的生物相容性.方法 将共培养的成骨细胞和血管内皮细胞与可注射性纳米材料体外复合培养,噻唑蓝(MTT)比色法测定细胞活性,检测碱性磷酸酶(ALP)活性,并行激光共聚焦显微镜及扫描电镜观察,进行形态学和功能测定.结果 细胞复合材料后保持正常的分裂增殖速度,细胞的ALP活性与单纯细胞培养的对照组差异无统计学意义(P>0.05).激光共聚焦显微镜及扫描电镜观察可见,细胞能在可注射性纳米材料上良好地增殖、生长,细胞的活性未受到材料的影响.结论 可注射性纳米材料具有良好的细胞相容性,可作为骨组织工程可注射性载体材料.     

血管组织工程中支架材料的应用与进展

血管组织支架是血管组织工程研究的重点内容。制备支架的材料包括天然材料和人工合成材料。天然材料具有良好的细胞和组织相容性,人工合成的聚合物支架在结构形状、机械强度及规模化生产方面均具有很大的优势,但二者均有很多问题要克服。近年来,该领域有了很大发展,出现了杂交支架、纳米材料支架等新型支架。就血管支架材料的研究进展进行综述。     

蚕丝及胶原海绵与颌下腺细胞相容性的实验研究

目的　探讨SD鼠颌下腺细胞在蚕丝和胶原海绵支架上生物相容性 ,为颌下腺组织工程中支架选择提供依据。方法　体外培养SD鼠颌下腺细胞 ,传至第 2代 ,将浓度为 5 0× 10 6/ml细胞分别接种在蚕丝和胶原海绵支架上。于接种后 1、3、5、7d取材 ,进行细胞计数、倒置相差显微镜及扫描电镜观察等。评价颌下腺细胞在蚕丝和胶原海绵支架上生长情况。结果　颌下腺细胞在蚕丝与胶原海绵支架上均能黏附增殖及分化 ,在胶原海绵上生长较佳。结论　蚕丝和胶原海绵均可为组织工程化颌下腺体外构筑的支架材料 ,但胶原海绵更优于蚕丝。     

Improved retention of endothelial cells seeded on polyurethane small-diameter vascular grafts modified by a recombinant RGD-containing protein

Sponge-type small-diameter vascular grafts were fabricated from a medical-grade polyurethane, Pellethane 2363-80A, by utilization of a salt casting technique. The grafts were compliance matched with a storage modulus of 0.53 +/- 0.08 MPa. The luminal surface of grafts was modified with a thin layer ( approximately 40 micro m) of gelatin crosslinked by epoxide. Then a special Arg-Gly-Asp (RGD)-containing recombinant protein, named CBD-RGD (cellulose binding domain RGD-containing protein), was coated onto the gelatin layer. The platelet adhesion and activation on such a gelatin/CBD-RGD modified surface was significantly reduced. Human umbilical vein endothelial cells were seeded more efficiently onto the modified grafts. There was also a substantial reduction in the subsequent loss of cells from the graft surface following perfusion in vitro. The cell number retained on the modified graft was enhanced by three times after 1 h of perfusion, and by eight times after 3 h of perfusion (retention rate approximately 63%). The retention after 3 h of perfusion could be further increased to nearly 100% if the lined endothelium on gelatin/CBD-RGD modified graft was cultured for another week before perfusion. The modified surface was also shown to help canine external jugular vein endothelial cells to maintain the round cell morphology in vitro.     

Cytotoxicity and endothelial cell adhesion of lyophilized and irradiated bovine pericardium modified with silk fibroin and chitosan

Grafts of biological tissues have been used since the 1960s as an alternative to the mechanical heart prostheses. Nowadays, the most consolidated treatment to bovine pericardial (BP) bioprostheses is the crosslinking with glutaraldehyde (GA), although GA may induce calcification in vivo. In previous work, our group demonstrated that electron beam irradiation applied to lyophilized BP in the absence of oxygen promoted crosslinks among collagen fibers of BP tissue. In this work, the incorporation of silk fibroin (SF) and chitosan (CHIT) in the BP not treated with GA was studied. The samples were irradiated and then analyzed for their cytotoxicity and the ability of adhesion and growth of endothelial cells. Initially, all samples showed cytotoxicity. However, after a few washing cycles, the cytotoxicity due to acetic acid and ethanol residues was removed from the biomaterial making it suitable for the biofunctional test. The samples modified with SF/CHIT and electron beam irradiated favored the adhesion and growth of endothelial cells throughout the tissue.     

两种非吸收性肌腱缝线的对比研究

目的 对两种常用非吸收性肌腱缝线的生物力学特性及组织相容性进行对比研究,为临床肌腱修复术的缝线选择提供实验依据.方法 取3-0 TICRON聚酯缝线和3-0真丝缝线各100根,分为两组.A组:3-0 TICRON聚酯缝线;B组:3-0真丝缝线.将2组缝线各80根分别随机埋入40只SD大鼠腹部皮下,术后1、2、3、4周各组取材,对缝线周围组织进行形态学观察及HE染色,观察缝线周围炎性反应程度及缝线与周围组织粘连程度;对缝线进行生物力学测定,测定缝线初始张力强度及术后4个时间点的张力强度.结果 A组术后1、2周时缝线周围炎性反应明显,3、4周时炎性反应逐渐减轻至消退.B组术后各个时间段缝线周围炎性反应均较A组严重,并且炎性反应消退慢.3-0 TICRON聚酯缝线的初始张力强度为(19.02±0.63)N、机体内4周维持张力强度为(17.80±0.64)N,大于3-0真丝缝线的初始张力强度(15.40± 0.44)N和机体内4周维持张力强度(9.79±0.54)N,并且3-0 TICRON聚酯缝线张力强度的衰减速度小于3-0真丝缝线.结论 3-0TICRON聚酯缝线组织相容性好,局部炎性反应轻,抗张力强度维持时间长,衰减速度慢,修复肌腱断裂比3-0真丝缝线理想.     

Early results of infrainguinal arterial reconstruction with a modified biological conduit

We implanted 112 glutaraldehyde-fixed bovine carotid artery grafts (BioPolyMeric [BPM]) for infrainguinal reconstruction in 107 legs of 98 patients. Indications for surgery were disabling claudication in 28%, rest pain in 33% and tissue loss in 39%. In 32%, BPM bypass followed failed ipsilateral reconstruction. Autologous vein was either absent or inadequate in 60% of cases. BPM was used preferentially over vein in above-knee bypasses. The distal anastomosis was to the above-knee popliteal artery in 40%, to the below-knee popliteal artery in 35%, and to the tibial arteries in 25%. Follow-up was available from one to 25 months, with a mean of nine months. Wound complications developed after 9% of operations, including seven (6%) graft infections. Both patent grafts that became infected were salvaged. Four patients (4%) died within 60 days of surgery due to cardiac complications. Life-table primary and secondary patencies of all grafts were 64% and 65% at one year, and 48% and 62% at two years, respectively. The only factor significantly affecting graft patency was the location of the distal anastomosis (p<.01). Primary patencies at one and two years to the above-knee popliteal artery were 90% and 80%, to the below-knee popliteal artery were 56% and 37%, and to the infrapopliteal arteries were 34% and 26%. Bypass to 16% of extremities resulted in amputation, including 5% that were amputated with patent grafts. No limb loss occurred as a result of operation for claudication. In conclusion, BPM grafts provide early results comparable to saphenous vein above the knee. Future randomization with synthetic grafts is necessary to define the role of BPM grafts below the knee.Presented at the Tenth Annual Meeting of the Southern California Vascular Surgical Society, September 27–29, 1991, Marina Del Rey, California.     

丝素膜代人工硬脑膜的组织相容性研究

目的　探讨丝素膜为人硬脑膜修补材料的可能性。方法　取新西兰兔额顶部正中切口 ,作双侧颞顶部开颅术 ,在手术显微镜下 ,剪除兔硬脑膜和蛛网膜 1.0cm× 0 .8cm ,硬脑膜缺损处分别用牛心包和不同的丝素膜膜修补。结果　术后无伤口感染、脑膜炎及抽搐现象 ,行为及进食无异常变化。早期 ,局部炎症反应轻微。晚期 ,呈轻度慢性炎性反应 ,致密丝素膜与脑皮层之间可见薄层假膜形成。脑皮质形态正常。结论　对制作工艺加以改进 ,丝素膜有望成为一种新型的脑膜替代材料。     

Porous silk scaffolds can be used for tissue engineering annulus fibrosus

There is no optimal treatment for symptomatic degenerative disc disease which affects millions of people worldwide. One novel approach would be to form a patch or tissue replacement to repair the annulus fibrosus (AF) through which the NP herniates. As the optimal scaffold for this has not been defined the purpose of this study was to determine if porous silk scaffolds would support AF cell attachment and extracellular matrix accumulation and whether chemically decorating the scaffold with RGD peptide, which has been shown to enhance attachment for other cell types, would further improve AF cell attachment and tissue formation. Annulus fibrosus cells were isolated from bovine caudal discs and seeded into porous silk scaffolds. The percent cell attachment was quantified and the cell morphology and distribution within the scaffold was evaluated using scanning electron microscopy. The cell-seeded scaffolds were grown for up to 8 weeks and evaluated for gene expression, histological appearance and matrix accumulation. AF cells attach to porous silk scaffolds, proliferate and synthesize and accumulate extracellular matrix as demonstrated biochemically and histologically. Coupling the silk scaffold with RGD-peptides did not enhance cell attachment nor tissue formation but did affect cell morphology. As well, the cells had higher levels of type II collagen and aggrecan gene expression when compared to cells grown on the non-modified scaffold, a feature more in keeping with cells of the inner annulus. Porous silk is an appropriate scaffold on which to grow AF cells. Coupling RGD peptide to the scaffold appears to influence AF cell phenotype suggesting that it may be possible to select an appropriate scaffold that favours inner annulus versus outer annulus differentiation which will be important for tissue engineering an intervertebral disc.     

羟基磷灰石多孔层修饰镍钛合金的组织相容性评价

目的 评价羟基磷灰石多孔层修饰镍钛合会的组织相容性.方法 选取健康成年雄兔24只.随机分为实验组(C组)和对照组(R组).选取左后肢股骨下1/3外侧为植入区,C组植入羟基磷灰石多孔层修饰镍钛合金.R组植入镍钛合金.分别于术后1、2、4、8周处死动物.观察各期骨组织局部反应、骨缺损修复情况.检测肝脏镍离子含量.并用免疫组化染色法测定TGF-β1表达阳性细胞的百分比.结果 R组各期肝脏镍离子含量测定均明显高于C组,且差异有统计学意义(P<(0.05);C组术后1、2、4周TGF-β1表达阳性细胞的百分比明显高于R组,且差异有统计学意义(P<0.05).结论 羟基磷灰石涂层能显著抑制镍钛合金中镍离子的释放;羟基磷灰石涂层能刺激骨组织中TGF-β1的表达.促进骨缺损的修复;羟基磷灰石多孔层修饰镍钛合金具有很好的组织相容性.     

丝素蛋白降低聚丙烯补片表面粘连的研究

目的 观察聚丙烯补片经丝素蛋白涂覆后,其表面粘连程度的变化,并探讨其作用机制.方法 将40只Sprague-Dawle大鼠随机分为两组,每组各20只.按组于腹腔内分别植入丝素蛋白涂覆聚丙烯材料(丝素面朝向腹腔,聚丙烯面朝向腹壁)和聚丙烯补片,于术后3、7、14、90 d分批处死大鼠,比较植入补片表面粘连程度及粘连面积,并行病理及扫描电镜检查.结果 丝素蛋白涂覆聚丙烯材料组,术后3、7、14、90d检查基本无腹腔内粘连形成,补片表面粘连面积占总面积的(6.04±4.78)%,粘连程度为(0.8±0.4);单纯聚丙烯组全部大鼠术后并发腹腔内粘连面积占总面积的(86.61±12.25)%,粘连程度为(3.4±0.6),有5只(5/20,25.0%)大鼠分别于术后2～9d因肠管与聚丙烯粘连引起肠梗阻或肠瘘死亡,两组的术后腹腔内粘连面积和粘连程度差异均存在统计学意义(P均<0.05).结论 丝素材料能促进腹膜间皮细胞长入,于聚丙烯补片表面腹膜化,形成新的间皮细胞层,可有效降低聚丙烯补片与腹腔内器官发生粘连的程度及范围.     

Evaluation of stretched electrospun silk fibroin matrices seeded with urothelial cells for urethra reconstruction

Background

We investigated the feasibility of urethral reconstruction using stretched electrospun silk fibroin matrices.

Materials and methods

A novel electrospun silk fibroin matrix was prepared. The structure of the material was assessed by scanning electron microscopy and a porosity test. Canine urothelial cells were isolated, expanded, and seeded onto the material for 1 wk to obtain a tissue-engineered graft. The tissue-engineered graft was assessed using hematoxylin and eosin staining and scanning electron microscopy. A dorsal urethral mucosal defect was created in nine female beagle dogs. In the experimental group, tissue-engineered mucosa was used to repair urethra mucosa defects in six dogs. No substitute was used in the three dogs of the control group. Retrograde urethrography was performed at 1, 2, and 6 mo after grafting. The urethral grafts were analyzed grossly and histologically.

Results

Scanning electron microscope and a porosity test revealed that the material had a three-dimensional porous structure. Urothelial cells grew on the material and showed good biocompatibility with the stretched silk fibroin matrices. Canines implanted with tissue-engineered mucosa voided without difficulty. Retrograde urethrography revealed no signs of stricture. Histologic staining showed gradual epithelial cell development and stratified epithelial layers at 1, 2, and 6 mo. The canines in the control group showed difficulty in voiding. Retrograde urethrography showed urethra stricture. Histologic staining showed that no or only one layer of epithelial cells developed. A severe inflammatory reaction was also observed in the control group.

Conclusions

Stretched electrospun silk fibroin matrices have good biocompatibility with urothelial cells, which could prove to be a potential material for use in urethra reconstruction.