稻壳作为缓释碳源及载体的改性研究

稻壳可作为废水处理的外加碳源, 通过适当改性处理可提高其应用性能。为探索稻壳的改性条件, 以不同浓度的NaOH、Ca(OH)2、NaClO为改性试剂对稻壳进行改性处理, 并研究了改性后稻壳的表面结构、芽孢杆菌吸附量、静态释碳量、可生化性以及成分含量变化。结果表明: 6% NaOH、0.9% Ca(OH)2和3% NaClO处理对稻壳表面糙化、芽孢杆菌吸附性和静态释碳能力有良好的提升效果。在此三组中, 6% NaOH处理后稻壳可生化效果最佳, CD600增长率为其他处理组的4倍; 纤维素含量增加了16.03%, 灰分含量显著降低, 仅剩4.9%; 且结构改性效果最为明显, 适用于稻壳改性优化。     

PHBHHx膜表面亲水改性及其与神经干细胞生物相容性

对羟基丁酸-羟基己酸共聚酯(PHBHHx)膜进行表面改性,研究神经干细胞(NSCs)在改性后的PHBHHx膜表面的贴附、增殖及分化情况,为开发新型脑组织工程支架材料奠定基础。采用溶剂挥发法制备PHBHHx膜,扫描电镜观察其表面性状;分别通过脂肪酶处理,NaOH处理的方法对PHBHHx膜进行表面改性,测量接触角以检测膜表面亲水性。分离培养孕14.5 d大鼠胚胎大脑皮质NSCs,接种在表面改性后的PHBHHx膜表面进行体外培养,扫描电镜观察膜表面细胞形态,MTT法检测细胞活力,免疫细胞化学染色观察NSCs存活和分化情况。结果显示,与未处理的PHBHHx膜相比,脂肪酶、NaOH处理能够显著提高PHBHHx膜表面亲水性,增加NSCs在PHBHHx膜表面贴附数量;NSCs在改性后的PHBHHx膜表面能够良好地存活并分化为神经元和胶质细胞。结果提示PHBHHx膜表面碱处理通过提高材料表面亲水性和粗糙程度,增加其与NSCs的生物相容性,改性后的PHBHHx材料是一种非常有潜力的新型脑组织工程支架材料,有望在NSCs移植修复脑损伤中发挥作用。     

纳米结构Ti/TiN涂层对NiTi合金生物相容性的影响

目的：评价纳米结构Ti/TiN涂层对镍钛形状记忆合金（含镍50.6at%）生物相容性的影响,为生物医用纳米结构Ti/TiN涂层表面改性的NiTi合金材料生物安全性提供依据。方法：不影响基体的形状记忆性或超弹性效应的前提下,采用真空过滤电弧离子镀技术,在NiTi合金表面沉积一层纳米结构Ti/TiN涂层,分别对表面改性前和改性后的NiTi合金样品进行体外细胞毒性实验、溶血实验和血小板粘附实验,探索纳米结构Ti/TiN涂层对NiTi合金生物相容性的影响。结果：表面具有纳米结构Ti/TiN涂层的NiTi合金无细胞毒性,H9C2（2-1）细胞相容性优于涂层前,细胞形态典型,粘附数量明显大于涂层前。纳米结构Ti/TiN涂层有改善NiTi合金的血液相容性作用,其溶血率从2.1%降至涂层改性后的1.2%,同时,血小板黏附量和聚集程度小于处理前的NiTi合金。结论：纳米Ti/TiN涂层能够显著改善NiTi合金的生物相容性。     

医用外科拉链细胞毒性试验研究

将医用外科拉链的 16 4 0浸出液与小鼠成纤维细胞L92 9悬液 (1x10 4/ml)共培养 ,观察其对L92 9细胞生长抑制的影响 ,结果表明 ,该外科拉链对L92 9细胞形态、生长和增殖没有影响 ,无细胞毒性作用 ,具有良好的生物相容性 ,符合生物材料应用要求     

MTT法评价医用骨科材料的细胞毒性

目的:评价三种常用医用骨科材料的细胞毒性。方法:通过制备表面阳极氧化钛合金(Ti-6Al-4V)材料、聚醚醚酮(PEEK)材料和β-磷酸三钙(β-TCP)材料的浸提液与L929细胞接触,进行MTT试验。结果:所有样品浸提液的细胞相对增殖率(RGR)均≥80%,细胞毒性反应分级为0至1级。结论:这三类材料的0.2g/ml浸提液均显示无明显的细胞毒性。     

微弧氧化后锆铜铝银非晶合金的细胞相容性研究

目的:探讨微弧氧化(micro-arc oxidation,MAO)后的Zr46(Cu4.5/5.5Ag1/5.5)46Al8(at%)(本文简称Zr-Cu-Al-Ag非晶合金)的细胞相容性。方法:按照国家标准制备300 V、350 V和400 V电压MAO处理的Zr-Cu-Al-Ag非晶合金、铸态Zr-Cu-Al-Ag非晶合金以及TI6Al4V合金试件的浸提液用于培养L929细胞,阴性组的L929细胞用含10%小牛血清的DMEM溶液培养,阳性组的L929细胞用含64 g/L苯酚和10%小牛血清的DMEM溶液培养,通过四唑盐(MTT)比色法分析试件的细胞相容性。结果:MAO处理的Zr-Cu-Al-Ag非晶合金细胞毒性评级为0,其浸提液中的L929细胞状态良好,细胞增殖曲线呈上升趋势,三个MAO组的吸光度值高于铸态Zr-Cu-Al-Ag非晶合金组、TI6Al4V合金组和阳性对照组(P0.05),但与阴性对照组无明显差别(P0.05)。结论:MAO提高了Zr-Cu-Al-Ag非晶合金表面的细胞相容性。     

下颌下腺脱细胞基质材料的生物相容性及毒理学研究

为探讨下颌下腺脱细胞基质支架材料的生物相容性,应用3%TritonX-100对SD大鼠的下颌下腺组织进行脱细胞处理,制备脱细胞基质支架材料,将该材料的浸提液注入小鼠体内进行全身急性毒性试验,观察小鼠全身反应.将该材料植入Wistar鼠肌内进行体内植入试验,不同时间观察支架材料与组织反应.用传代培养的第2代下颌下腺细胞与支架材料体外复合培养,第7 d时进行MTT检测,观察支架材料对细胞增殖的影响.全身急性毒性试验结果显示,实验组与对照组无显著性差别(P＞0.05),体内植入试验2、4、8 W时光镜下表现与对照组基本相似,MTT检测结果,细胞相对增长率为91.66%,支架材料的毒性为0级.结果可见,经3%TritonX-100脱细胞处理后所制备的下颌下腺生物衍生支架材料具有良好的生物相容性,对机体无毒害作用.     

腹膜间皮细胞与聚羟基乙酸的细胞相容性研究

目的：研究腹膜间皮细胞（Peritoneal mesothelial cell,PMC）与可吸收生物材料聚羟基乙酸（Polyglycolicacids,PGA）的相容性,为组织工程化尿道的构建奠定基础。方法：采用酶消化法,从SD大鼠腹腔中分离出PMC,常规传代,取第2代细胞与PGA混合培养,形成细胞-生物材料复合物。每两天换液1次,观察细胞形态和黏附生长状况。体外培养1周后,行扫描电镜观察和RT-PCR检测。结果：复合物体外培养1周后,PMC能成功黏附在PGA支架上,并分泌大量细胞外基质。RT-PCR提示CK-AE1/AE3和Vimentin均呈阳性表达,PMC表型未发生改变。结论：PMC与PGA有良好的细胞相容性,PGA可作为组织工程化腹膜样组织的支架材料。     

g-HA/PLA复合材料的细胞反应和血液相容性研究

将表面改性后的羟基磷灰石颗粒同聚乳酸复合得到新型复合材料可望用于骨替代领域, 本文旨在研究此种复合材料的血液相容性和细胞反应. 将L-乳酸低聚物接枝到羟基磷灰石表面, 得到接枝羟基磷灰石颗粒. 之后, 将g-HA颗粒同PLA进行共混获得g-HA/PLA复合材料. 先前研究表明, 由于提高了聚合物基体和HA颗粒之间的界面黏附力, 这些材料的拉伸性能得到了明显提高. 为进一步考察这些材料在骨修复及其他整形外科方面的潜在应用, 进行了一系列体内和体外实验来测试其细胞反应及血液相容性. 体外实验表明, g-HA/PLA复合材料有利于L-929细胞的生长. 复合材料的溶血率低于纯PLA. 皮下植入实验表明, g-HA/PLA复合材料的软组织反应比较合适. 以上结果提示, g-HA/PLA复合材料是一种安全的材料, 有望用于组织工程研究.     

用流式细胞术评价一种新型人工肝材料的生物相容性

目的通过用流式细胞术（FCM）观察丙稀酰胺接枝改性聚丙烯膜 （PP-g-AAm）的生物相容性,来评价FCM在检测医用生物材料的生物相容性中的作用.方法：用材料浸提液培养L929细胞24 h后用FCM检测细胞增殖周期; 用改性前、后的膜材料分别与新提取的PRP（富含血小板血浆）和PBMC（末梢血单个核细胞）孵育培养后,用FCM分别检测血小板和PBMC的激活标志物CD62P、CD63和CD69.结果：PP-g-AAm 组的PI与阴性组及空白对照组比较差异无统计学意义（P=0.063,P=0.053）,而与阳性对照组比较差异有统计学意义（P=0.002）.PP-g-AAm膜组CD62P、CD63及CD69的表达率明显少于对照组（P=0.042, P=0.004,P=0.013）.结论：PP-g-AAm无细胞毒性并具有良好的生物相容性,FCM在生物材料的生物相容性评价中有着广泛的应用价值.     

雪旺氏细胞与同种异体骨支架的体外共培养研究

目的:探讨雪旺细胞(Schwann’s cells,SCs)在同种异体骨支架上的生物相容性,体外构建组织工程骨神经化模型。方法:利用新鲜人体骨骼制备同种异体骨支架材料,检测其物理性能;采用优化方法提取新生SD大鼠坐骨、臂丛神经培养SCs,实验分为三维培养实验组(SCs+同种异体骨)、二维培养对照组(SCs+胶原玻片),S-100抗体免疫荧光染色鉴定SCs纯度;细胞计数法检测两组细胞增殖特点;细胞接种后第3、7天取样,扫描电镜观察。结果:同种异体骨支架具有良好的三维孔隙结构,适宜细胞贴附生长;S-100免疫荧光染色证实SCs纯度95%;扫描电镜检测显示两组SCs均可正常粘附增殖,细胞间排布规律相似,培养早期实验组SCs胞体更加细长,伪足更加明显,随着培养时间的延长表现出较强的迁移能力;细胞增殖检测:两组SCs生长曲线特征基本一致,支架材料对SCs无毒性作用。结论:同种异体骨支架SCs具有良好的生物相容性,其三维立体多孔结构有利于SCs的粘附与迁移,初步构建了体外组织工程骨神经化模型。     

成年猴雪旺细胞的在体增殖和体外迁移的研究

为了探讨成年猴雪旺氏细胞的在体增殖和体外迁移的能力,我们对用神经结扎术结扎的A组6只3-13岁雄性恒河猴的腓肠神经进行植块培养,部分细胞培养在聚酯纤维上,2-4周后作抗S-100抗体免疫组化染色和电镜观察;B组2只未做结扎的新生猴腓肠神经培养作为对照.结果显示A组雪旺氏细胞平均在培养的第5天从神经段中迁出,年幼者早于成年猴;细胞在纤维上以螺旋状向前迁移;雪旺氏细胞抗S-100蛋白抗体染色阳性;电镜显示,雪旺氏细胞包卷纤维,但是,未见髓鞘形成.B组神经段培养2周仍无雪旺氏细胞迁出.研究表明,结扎神经使其发生瓦勒氏变性,经植块培养、纯化,能够获得可用于移植的成年猴的雪旺氏细胞.     

微重力条件下心肌细胞在聚羟基乙酸纤维支架上的三维固定化培养

以 1d龄Wistar乳鼠的心室肌组织为心肌细胞的来源 ,采用胰蛋白酶消化及细胞差速贴壁分离心肌细胞 ,以未经修饰和经鼠尾胶原溶液浸泡修饰的聚羟基乙酸 (polyglycolicacid ,PGA)纤维支架作为心肌细胞体外三维 (3D)固定化培养的支架 ,比较心肌细胞在静置培养体系及微重力培养体系下的生长、形态和收缩状况。心肌细胞在未经处理的PGA纤维支架 3D固定化培养时 ,心肌细胞在其上的分布不均匀 ,大部分心肌相互连接形成球状聚集体 ;PGA纤维支架经鼠尾胶原溶液浸泡处理后 ,心肌细胞在其上的分布较为均匀 ,细胞形态多呈梭形或不规则状 ,心肌细胞自律性搏动的幅度加大。在模拟微重力培养条件下 ,心肌细胞在经鼠尾胶原溶液浸泡修饰的PGA纤维支架上的分布更为均匀 ,心肌细胞形成具有自律性同步收缩特性、面积约为 15mm2 的类组织样 3D结构     

皮肤细胞复合改性聚乳酸构建组织工程皮肤

目的:探讨以改性聚乳酸为细胞外基质网架构建组织工程皮肤的可行性。方法:采用盐溶法制备机械性能得到部分改进的聚乳酸多孔泡沫网架,向改进的聚乳酸网架接种真皮成纤维细胞和表皮角质形成细胞,以普通聚乳酸支架作为对照,构建组织工程皮肤。体外培养一周,对网架进行形态学观察。主要观察指标:①一般形态观察②组织学观察。结果:复层组织工程皮肤在结构上与正常皮肤相似,具有真皮、表皮双层结构。改性聚乳酸网架上有双层细胞生长,生长的细胞与网架接触,并且在其表面形成较为明显而连续的细胞层。随着培养时间的延长,发生了一系列变化:表皮部分细胞层数逐渐增多,真皮部分细胞也逐渐增多,并向表皮层深入,位于表皮与网架之间。结论:双醛淀粉作为良好的增柔剂在改善聚乳酸网架的机械性能的同时,也具有良好的细胞相容性,不影响细胞的生长增殖和代谢,可以进一步用作组织工程皮肤的支架材料。     

Aging Schwann cells in vitro

Schwann cells (SCs) can support the regeneration of lesioned fiber tracts of the peripheral and central nervous system and have been transplanted alone or in combination with synthetic nerve guides. For neuronal tissue engineering purposes, the cells must be isolated from small biopsies and expanded in vitro. In this study we analyze the impact of cell expansion on 9 different cell parameters, comparing short- and long-term cultured rat SCs, which we refer to as 'young' and 'old' or 'aged' cells, respectively. In comparison to young SCs, old SCs doubled the axonal outgrowth from dorsal root ganglion explants and displayed only one-third as much adhesion to the gray and white matter of spinal cord cryosections. In a 3-dimensional extracellular matrix the two cell populations showed very different cellular responses with regard to cell morphology and cell-cell adhesion. Cell proliferation of old SCs was independent of serum components and was not hampered by contact inhibition. In addition, population doubling times were reduced by a factor of almost three compared to those of young SCs. Despite considerable karyotype changes, with an average of 68.7 chromosomes versus 42 in native rat cells, old SCs did not show any increase in telomerase activity and loss of anchorage dependence--characteristics that are typical of tumor cells. The data also provide biological insights into which cell characteristics (proliferation and adhesion, for example) are functionally clustered and either change or remain constant with aging in vitro. Though the data indicate a lack of tumorigenic transformation coupled with increased neurite outgrowth-promoting activity after extensive SC expansion in vitro, thus suggesting better regeneration qualities, we strongly recommend that in vitro aged rat SCs (>11 passages) should not be employed for tissue engineering.     

Effects of Bone Marrow Stromal Cell-conditioned Medium on Primary Cultures of Peripheral Nerve Tissues and Cells

Implantation of bone marrow stromal cells (MSCs) produces an improved functional outcome of peripheral nerve repair. In this study, rat dorsal root ganglion (DRG) explants, rat DRG neurons, and rat Schwann cells (SCs) were treated with monkey MSC-conditioned medium, respectively, and then subjected to MTT assay, Bromodeoxyuridine/Hoechst 33342 double staining, flow cytometry, immunohistochemistry, real-time quantitative PCR, and Western blot analysis, respectively. The results showed that MSC-conditioned medium enhanced axon growth and neurogenesis in cultured DRG explants, augmented cell survival of and expression of NF and GAP-43 by cultured DRG neurons, promoted cell survival and proliferation of cultured SCs, and increased the expression of NGF, BDNF, and bFGF in cultured SCs. We also found that mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) 1/2 pathway was involved in the enhanced cell proliferation of SCs evoked by MSC-conditioned medium. The data of this study might help the understanding of MSCs-based treatment for peripheral nerve repair.     

Correlation between scaffold in vivo biocompatibility and in vitro cell compatibility using mesenchymal and mononuclear cell cultures

The aim of this study was to compare the cell compatibility of silk and polyglycolic acid (PGA) scaffolds cultured in vitro with mesenchymal stem cells (MSCs) and peripheral blood mononuclear cells (PBMCs) to their biocompatibility in vivo following implantation. Scaffolds were knitted with silk or PGA thread and the average efficiency of cell attachment was 35 ± 4% and 17 ± 2% in the PGA and silk scaffold groups. Thus, the initial attachment of the MSC cells to the PGA scaffold was superior to the initial attachment of the cells on the silk scaffold. After 21 days in culture, the average cell density on the silk scaffold was \text5\text.8 ±\text0\text.5 ×\text10^\text5 {\text{5}}{\text{.8}} \pm {\text{0}}{\text{.5}} \times {\text{10}}^{\text{5}} cells, and the average cell density of the PGA scaffolds was \text6\text.34 ±\text0\text.5 ×\text10^\text5 {\text{6}}{\text{.34}} \pm {\text{0}}{\text{.5}} \times {\text{10}}^{\text{5}} cells. In addition, there was no cell cytoxicity observed with either scaffold. However, the immune response of in vitro cultured PBMCs was significantly higher with the PGA scaffold than with the silk scaffold. The proliferation of the PBMCs cultured on the PGA scaffold was two times greater than that of those cultured on the silk scaffold after 3 days of culture. In addition, the secretion of IL-1 by the PBMCs cultured on the PGA scaffold was superior to that of the PBMCs cultured on the silk scaffold. The secretion of IL-1β and IFN-γ was increased by about 50% when the PBMCs were cultured with the PGA scaffold. Silk and PGA scaffolds were also implanted subcutaneous in rats. Histological evaluation of the scaffold explants revealed the presence of monocytes and macrophages in PGA scaffold. The inflammatory tissue reaction was more conspicuous on the PGA scaffold than on the silk scaffold. These results suggest that the results of in vitro PBMC cultures were more closely related to the in vivo results of implantation than the results of in vitro MSC cultures.     

Soluble forms of NCAM and F3 neuronal cell adhesion molecules promote Schwann cell migration: identification of protein tyrosine phosphatases zeta/beta as the putative F3 receptors on Schwann cells

Neural cell adhesion molecule (NCAM) and F3 are both axonal adhesion molecules which display homophilic (NCAM) or heterophilic (NCAM, F3) binding activities and participate in bidirectional exchange of information between neurones and glial cells. Engineered Fc chimeric molecules are fusion proteins that contain the extracellular part of NCAM or F3 and the Fc region of human IgG1. Here, we investigated the effect of NCAM-Fc and F3-Fc chimeras on Schwann cell (SC) migration. Binding sites were identified at the surface of cultured SCs by chimera coated fluorospheres. The functional effect of NCAM-Fc and F3-Fc binding was studied in two different SC migration models. In the first, migration is monitored at specific time intervals inside a 1-mm gap produced in a monolayer culture of SCs. In the second, SCs from a dorsal root ganglion explant migrate on a sciatic nerve cryosection. In both systems addition of the chimeras significantly increased the extent of SC migration and this effect could be prevented by the corresponding anti-NCAM or anti-F3 blocking antibodies. Furthermore, antiproteoglycan-type protein tyrosine phosphatase zeta/beta (RPTPzeta/beta) antibodies identified the presence of RPTPzeta/beta on SCs and prevented the enhancing effect of soluble F3 on SC motility by 95%. The F3-Fc coated Sepharose beads precipitated RPTPzeta/beta from SC lysates. Altogether these data point to RPTPzeta/beta is the putative F3 receptor on SCs. These results identify F3 and NCAM receptors on SC as potential mediators of signalling occurring between axons and glial cells during peripheral nerve development and regeneration.     

Polyhydroxyalkanoates: Current applications in the medical field

Polyhydroxyalkanoates (PHAs) are a class of biopolyesters that are synthesized intracellularly by microorganisms, mainly by different genera of eubacteria. These biopolymers have diverse physical and chemical properties that also classify them as biodegradable in nature and make them compatible to living systems. In the last two decades or so, PHAs have emerged as potential useful materials in the medical field for different applications owing to their unique properties. The lower acidity and bioactivity of PHAs confer them with minimal risk compared to other biopolymers such as poly-lactic acid (PLA) and poly-glycolic acid (PGA). Therefore, the versatility of PHAs in terms of their non-toxic degradation products, biocompatibility, desired surface modifications, wide range of physical and chemical properties, cellular growth support, and attachment without carcinogenic effects have enabled their use as in vivo implants such as sutures, adhesion barriers, and valves to guide tissue repair and in regeneration devices such as cardiovascular patches, articular cartilage repair scaffolds, bone graft substitutes, and nerve guides. Here, we briefly describe some of the most recent innovative research involving the use of PHAs in medical applications. Microbial production of PHAs also provides the opportunity to develop PHAs with more unique monomer compositions economically through metabolic engineering approaches. At present, it is generally established that the PHA monomer composition and surface modifications influence cell responses.PHA synthesis by bacteria does not require the use of a catalyst (used in the synthesis of other polymers), which further promotes the biocompatibility of PHA-derived polymers.