纳米羟基磷灰石/壳聚糖/半水硫酸钙为可注射骨组织工程支架材料的可行性

背景：前期实验采用仿生学原理制备了可注射性纳米羟基磷灰石/壳聚糖/半水硫酸钙复合材料,但其与骨髓间充质干细胞的生物相容性还不十分清楚。目的：探讨纳米羟基磷灰石/壳聚糖/半水硫酸钙作为注射型骨组织工程支架材料的可行性。方法：将第3代兔骨髓间充质干细胞与可注射纳米羟基磷灰石/壳聚糖/半水硫酸钙支架复合培养,作为实验组;以单纯接种培养的骨髓间充质干细胞为对照组,倒置显微镜下观察细胞生长情况,MTT法检测细胞增殖,扫描电镜观察细胞在材料表面生长与增殖。将纳米羟基磷灰石/壳聚糖/半水硫酸钙支架埋植在家兔背部肌袋内,埋植后2,4,6,8周进行病理学观察。结果与结论：实验组细胞生长、增殖良好,与对照组无明显差异。支架埋植后2周,材料周围有中等量中性粒细胞、淋巴细胞和巨细胞浸润,可见小血管与纤维母细胞增生,材料已被炎性细胞分割、围绕散碎;埋植后4周,可见少量淋巴细胞、纤维母细胞聚集,炎症反应进一步消退,肌纤维排列、形态正常;埋植后6周,材料周围炎症反应轻微,组织水肿不明显;埋植后8周,炎症反应基本消退,材料基本降解完成,肌纤维形态基本正常。表明纳米羟基磷灰石/壳聚糖/半水硫酸钙复合物具有良好的细胞相容性和生物降解性,可作为注射型支架材料。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

壳聚糖水凝胶与星形胶质细胞体外生物相容性评价的实验研究

研究壳聚糖水凝胶材料与星形胶质细胞的体外生物相容性,初步探讨壳聚糖水凝胶作为神经组织工程支架材料的可行性.利用氯化壳聚糖、β-甘油磷酸钠和羟乙基纤雏素制备壳聚糖水凝胶,MTT法评价其细胞毒性;体外培养鉴定新生Wistar大鼠脑皮层星形胶质细胞;壳聚糖水凝胶与星形胶质细胞体外共培养,观察星形胶质细胞在材料上的生长;MTT...     

壳聚糖水凝胶与星形胶质细胞体外生物相容性评价的实验研究

研究壳聚糖水凝胶材料与星形胶质细胞的体外生物相容性,初步探讨壳聚糖水凝胶作为神经组织工程支架材料的可行性。利用氯化壳聚糖、β-甘油磷酸钠和羟乙基纤维素制备壳聚糖水凝胶,MTT法评价其细胞毒性;体外培养鉴定新生Wistar大鼠脑皮层星形胶质细胞;壳聚糖水凝胶与星形胶质细胞体外共培养,观察星形胶质细胞在材料上的生长;MTT法检测接种后1、35、、7d的细胞增殖度。体外成功制备壳聚糖水凝胶,该材料无细胞毒性;体外分离、培养得到状态良好的星形胶质细胞;体外星形胶质细胞在材料上培养呈星形,生长良好,有分支状突起形成;MTT结果表明,材料-细胞共培养组中的细胞增殖度明显高于单纯细胞组（P〈0.001）。壳聚糖水凝胶与星形胶质细胞在体外具有良好的生物相容性,有望作为神经组织工程支架材料。     

京尼平交联脱细胞纤维环基质/壳聚糖水凝胶与兔纤维环源干细胞的生物相容性

BACKGROUND:To improve the mechanical properties and uncontrollability of degradation of decellularized matrix, we manufactured genipin cross-linked decellularized annulus fibrosus matrix/chitosan hydrogels as annulus fibrosus tissue-engineered scaffold.     

可注射水凝胶在心肌梗死治疗中的研究进展

急性心肌梗死后由于大量心肌细胞坏死、纤维瘢痕形成等原因造成心室重构和心功能下降,严重影响心梗患者的远期预后.天然材料或人工合成的水凝胶是一种新兴的生物替代材料,具有良好的亲水性和组织相容性,可模拟心肌细胞微环境的生化和机械特性,目前已广泛地应用于心肌梗死的治疗研究中.水凝胶可单独应用,也可作为细胞/药物运载工具和平台.研究表明:将水凝胶-干细胞-药物复合物进行心肌注射后,可以增加室壁厚度,促进新生血管形成,提高移植干细胞的存活率,并实现生物活性药物的控释,在一定程度上抑制梗死后心室重构,促进心功能的恢复.就目前可注射水凝胶在治疗心肌梗死方面的相关研究进展作一综述.     

可注射温敏淫羊藿苷壳聚糖水凝胶促进家兔桡骨骨折的愈合

背景：淫羊藿可显著促进体外培养成骨细胞的增殖、分化和成熟,刺激骨形成。 目的：观察可注射温敏淫羊藿苷壳聚糖水凝胶修复家兔桡骨骨折愈合的效果。 方法：麻醉下将36只兔左右桡骨截断,随机分为实验组、对照组和空白组,前两组分别在骨折端周围注入可注射温敏携淫羊藿苷壳聚糖水凝胶、壳聚糖水凝胶,空白组骨折区未注入任何材料。 结果与结论：3组骨折均于术后6周时愈合。X射线片显示,术后2,4, 6周,实验组骨折修复区单位面积平均灰度值及成骨细胞计数均明显高于对照组、空白组(P < 0.05),且实验组成骨细胞增生活跃。说明可注射温敏携淫羊藿苷壳聚糖水凝胶具有促成骨活性,可促进骨折愈合。     

壳聚糖的生物相容性*

背景：壳聚糖是惟一一种被广泛应用于生物医学工程领域的碱性、带有正电荷的天然多糖,其生物相容性是决定这些应用价值的关键。 目的：综述了壳聚糖的生物相容性,包括组织相容性、血液相容性和力学相容性。 方法：由第一作者检索1990/2011 PubMed数据库、中国知网数据库及万方数据库有关壳聚糖及其衍生物在生物医学上的应用和生物相容性等方面的文献。 结果与结论：壳聚糖作为可生物降解高分子材料具有良好的组织相容性及与人体组织相匹配所需要的力学相容性,被逐渐应用于人工皮肤、手术缝合线、眼科修复、人工骨骼、牙齿修复、肿瘤治疗等方面。但壳聚糖的促凝血作用使其血液相容性很差,目前很多研究关注于寻找解决这一问题的方法,改善其血液相容性,扩展其在生物医学工程上的应用领域,使其更加安全有效地与人体心血管系统直接接触。 关键词：壳聚糖;组织相容性;血液相容性;力学相容性;生物材料 doi:10.3969/j.issn.1673-8225.2012.12.034     

含中药三七的壳聚糖/明胶水凝胶复合止血材料的制备和性能评价

目的制备负载中药三七的壳聚糖/明胶水凝胶复合止血材料(PN/CMC/GMs),并对其性能进行评价。方法利用冷冻干燥法制备PN/CMC/GMs,利用扫描电镜观察其形态,流变仪观察其流变学性能,溶胀测试其吸水膨胀率,细胞毒性实验检测其生物相容性,并利用SD大鼠肝脏出血模型检测其快速止血效果。结果制备了PN/CMC/GMs,呈网格状结构,具有一定的孔隙率。随着三七粉含量的增加,PN/CMC/GMs的模量也相应的增加,机械强度增加。PN/CMC/GMs具有较好的吸水膨胀功能,可形成压迫止血和浓缩血液实现快速止血,且具有良好的生物相容性。止血实验表明,PN/CMC/GMs对大鼠肝脏损伤的止血时间和止血效果均优于空白对照组。结论PN/CMC/GMs具有良好的止血效果和生物相容性,具有进一步研究的价值和临床应用前景。     

载银水凝胶或载抗生素壳聚糖水凝胶在抑菌性能和细胞毒性方面的研究

目的研究壳聚糖水凝胶,壳聚糖载银水凝胶和壳聚糖载抗生素水凝胶短期的抑菌功效和细胞毒性。方法通过添加交联剂后制备壳聚糖水凝胶,并有效装载银离子或硫酸庆大霉素。进行抑菌实验和累计释放实验了解壳聚糖基水凝胶的抗菌性能和药物控释性。通过使用材料的浸提液检测这三种水凝胶的细胞毒性。结果抑菌实验结果表明壳聚糖水凝胶,壳聚糖载银水凝胶和壳聚糖载抗生素水凝胶均能有效抑制金黄色葡萄球菌的增殖。且壳聚糖载抗生素水凝胶具有最佳的抑菌性能且极大地抑制了生物膜的形成。体外药物释放显示抗生素在7天内的累计释放多于60%;而银离子的释放低于10%。细胞毒性实验表明这三个凝胶材料无明显细胞毒性。结论壳聚糖基水凝胶具有良好的短期抑菌效果,可降解,且无明显细胞毒性,在骨科应用方面有着巨大的前景。     

温敏性壳聚糖水凝胶对大鼠成骨细胞的毒性

背景：温敏性壳聚糖与多种细胞相容性良好,是组织工程中不可多得的优良载体,但其对成骨细胞毒性研究相对缺乏。 目的：验证温敏性壳聚糖水凝胶对成骨细胞的毒性。 方法：成骨细胞在温敏性壳聚糖水凝胶中进行培养,显微镜下观察细胞形态及扩增情况,同时,SD大鼠成骨细胞在不同浓度的温敏性壳聚糖水凝胶浸提液中体外培养24,48,72,96 h,MTT法测定细胞相对增殖率,判断细胞毒性的级别。 结果与结论：SD大鼠成骨细胞在温敏性壳聚糖水凝胶中培养24 h内镜下观察呈圆形,48 h后开始伸出触角并扩增;温敏性壳聚糖水凝胶浸提液中培养的各组细胞在不同时间点相对增殖率在92%~112%之间,各浓度的温敏性壳聚糖水凝胶材料浸提液的细胞毒性均为0级或1级,完全符合生物材料的安全评价标准。     

Retention and biodistribution of microspheres injected into ischemic myocardium

Intramyocardial injection of therapeutic agents may enhance heart repair after infarction. Incomplete retention of intramyocardial injections has been reported, but modes of loss are undefined. We determined the fate of neutron-activated microspheres injected into acutely ischemic rat myocardium using saline, Pluronic F127, or Matrigel as vehicle. Twenty minutes after injection in saline, 63% +/- 12% of 10-mum microspheres was retained in the heart. Similar retention was observed after 6 days. Injection site leakage accounted for 14% +/- 5% of the microspheres, whereas exit via coronary veins resulted in 11.2% +/- 9.5% collecting in the lungs. Microspheres distribution to other organs was minimal. Retention of 40-mum microspheres was similar to that observed with the 10-mum microspheres. Pluronic F127 and Matrigel reduced immediate leakage to 4% +/- 1% and 2% +/- 1%, respectively. Surprisingly, microsphere retention in the heart was not improved at 20 min using either gelling vehicle, suggesting that leakage occurs over a prolonged period. Thus, most injected particles are retained in the ischemic rat heart following direct injection, but significant fractions are lost from the injection site and through coronary veins. Gelling agents reduced short-term leakage, but failed to enhance longer-term retention. Hydrogels with stiffer mechanical properties might enhance retention and reduce variability.     

Covalently crosslinked chitosan hydrogel: properties of in vitro degradation and chondrocyte encapsulation

In vitro degradation and chondrocyte-encapsulation of chitosan hydrogel made of crosslinkable and water-soluble chitosan derivative (CML) at neutral pH and body temperature were studied with respect to weight loss, cytoviability, DNA content and cell morphology. In vitro degradation of the chitosan hydrogels was sensitive to their crosslinking degree and existence of lysozyme in the solution. Chitosan hydrogel (Gel-I5) fabricated from 1% CML and 5mM ammonium persulfate (APS)/N,N,N',N'-tetramethylethylenediamine (TMEDA) displayed no degradation in phosphate buffered saline (PBS) after 18d, but degraded completely at 8d in 1mg/ml lysozyme/PBS. The chitosan hydrogel fabricated from 10mM APS/TMEDA was non-degradable even in lysozyme/PBS solution after 18d. The hydrogel loaded with chondrocytes in cell culture medium, however, was susceptible to degradation during the in vitro culture. In vitro culture of the encapsulated chondrocytes in the chitosan hydrogel demonstrated that the cells retained round shaped morphology and could survive through a 12d-culture period, although the DNA assay detected an overall reduction of the cell number. These features provide a great opportunity to use the chitosan hydrogel as an injectable scaffold in tissue engineering and orthopaedics.     

Synthesis,characterization and therapeutic efficacy of a biodegradable,thermoresponsive hydrogel designed for application in chronic infarcted myocardium

Injection of a bulking material into the ventricular wall has been proposed as a therapy to prevent progressive adverse remodeling due to high wall stresses that develop after myocardial infarction. Our objective was to design, synthesize and characterize a biodegradable, thermoresponsive hydrogel for this application based on copolymerization of N-isopropylacrylamide (NIPAAm), acrylic acid (AAc) and hydroxyethyl methacrylate-poly(trimethylene carbonate) (HEMAPTMC). By evaluating a range of monomer ratios, poly(NIPAAm-co-AAc-co-HEMAPTMC) at a feed ratio of 86/4/10 was shown to be ideal since it formed a hydrogel at 37 °C, and gradually became soluble over a 5 month period in vitro through hydrolytic cleavage of the PTMC residues. HEMAPTMC, copolymer and degradation product chemical structures were verified by NMR. No degradation product cytotoxicity was observed in vitro. In a rat chronic infarction model, the infarcted left ventricular (LV) wall was injected with the hydrogel or phosphate buffered saline (PBS). In the PBS group, LV cavity area increased and contractility decreased at 8 wk (p < 0.05 versus pre-injection), while in the hydrogel group both parameters were preserved during this period. Tissue ingrowth was observed in the hydrogel injected area and a thicker LV wall and higher capillary density were found for the hydrogel versus PBS group. Smooth muscle cells with contractile phenotype were also identified in the hydrogel injected LV wall. The designed poly(NIPAAm-co-AAc-co-HEMAPTMC) hydrogel of this report may thus offer an attractive biomaterial-centered treatment option for ischemic cardiomyopathy.     

Delivery of basic fibroblast growth factor with a pH-responsive, injectable hydrogel to improve angiogenesis in infarcted myocardium

A pH- and temperature-responsive, injectable hydrogel has been designed to take advantage of the acidic microenvironment of ischemic myocardium. This system can improve therapeutic angiogenesis methods by providing spatio-temporal control of angiogenic growth factor delivery. The pH- and temperature-responsive random copolymer, poly(N-isopropylacrylamide-co-propylacrylic acid-co-butyl acrylate) (p[NIPAAm-co-PAA-co-BA]), was synthesized by reversible addition fragmentation chain transfer polymerization. This polymer was a liquid at pH 7.4 and 37?°C but formed a physical gel at pH 6.8 and 37?°C. Retention of biotinylated basic fibroblast growth factor (bFGF) between 0 and 7 days after injection into infarcted rat myocardium was 10-fold higher with hydrogel delivery versus saline. Following 28 days of treatment in vivo, capillary and arteriolar densities were increased 30-40% by polymer?+?bFGF treatment versus saline?+?bFGF or polymer-only controls. Treatment with polymer?+?bFGF for 28 days resulted in a 2-fold improvement in relative blood flow to the infarct region versus day 0, whereas saline?+?bFGF or polymer-only had no effect. Fractional shortening determined by echocardiography was significantly higher following treatment with polymer?+?bFGF (30?±?1.4%) versus saline (25?±?1.2%) and polymer alone (25?±?1.8%). By responding to local changes in pH- and temperature in an animal model of ischemia, this hydrogel system provided sustained, local delivery of bFGF, improved angiogenesis, and achieved therapeutic effects in regional blood flow and cardiac function.     

Antibody targeting of stem cells to infarcted myocardium

Hematopoietic stem cell (HSC) therapy for myocardial repair is limited by the number of stem cells that migrate to, engraft in, and proliferate at sites of injured myocardium. To alleviate this limitation, we studied whether a strategy using a bispecific antibody (BiAb) could target human stem cells specifically to injured myocardium and preserve myocardial function. Using a xenogeneic rat model whereby ischemic injury was induced by transient ligation of the left anterior descending artery (LAD), we determined the ability of a bispecific antibody to target human CD34+ cells to specific antigens expressed in ischemic injured myocardium. A bispecific antibody comprising an anti-CD45 antibody recognizing the common leukocyte antigen found on HSCs and an antibody recognizing myosin light chain, an organ-specific injury antigen expressed by infarcted myocardium, was prepared by chemical conjugation. CD34+ cells armed and unarmed with this BiAb were injected intravenously in rats 2 days postmyocardial injury. Immunohistochemistry studies showed that the armed CD34+ cells specifically localized to the infarcted region of the heart, colocalized with troponin T-stained cells, and colocalization with vascular structures. Compared to unarmed CD34+ cells, the bispecific antibody improved delivery of the stem cells to injured myocardium, and such targeted delivery was correlated with improved myocardial function 5 weeks after infarction (p < .01). Bispecific antibody targeting offers a unique means to improve the delivery of stem cells to facilitate organ repair and a tool to study stem cell biology.     

Changes in the microvasculature of ischemic and infarcted myocardium.

The fine structure of the small vessels in experimental myocardial infarcts of 10- to 360-minute duration was studied in 32 dogs and compared with the appearance of the small vessels in the corresponding normal myocardium. Following 10 to 60 minutes of coronary artery occlusion, increasing numbers of endothelial cells showed a marked swelling which was consistent with the presence of an intracellular edema and frequently resulted in various degrees of obstruction of the vessel lumen. After 120 minutes of ischemia, not all endothelial cells were obviously swollen, but all showed signs of degeneration, including changes in organelles similar to those in surrounding muscle cells. A progressive intensification of degenerative changes, particularly with respect to the continuity of the endothelial lining and the integrity of the membrane systems of individual endothelial cells, was observed at 3, 4, 5, and 6 hours after coronary artery ligation, and cell debris could be seen in the lumina of most small vessels.     

A hydrogel material for plastic and reconstructive applications injected into the subcutaneous space of a sheep

Soft tissue reconstruction using tissue-engineered constructs requires the development of materials that are biocompatible and support cell adhesion and growth. The objective of this study was to evaluate the use of macroporous hydrogel fragments that were formed using either unmodified alginate or alginate covalently linked with the fibronectin cell adhesion peptide RGD (alginate-RGD). These materials were injected into the subcutaneous space of adult, domesticated female sheep and harvested for histological comparisons at 1 and 3 months. In addition, the alginate-RGD porous fragments were seeded with autologous sheep preadipocytes isolated from the omentum, and these cell-based constructs were also implanted. The results from this study indicate that both the alginate and alginate-RGD subcutaneous implants supported tissue and vascular ingrowth. Furthermore, at all time points of the experiment, a minimal inflammatory response and capsule formation surrounding the implant were observed. The implanted materials also maintained their sizes over the 3-month study period. In addition, the alginate-RGD fragments supported the adhesion and proliferation of sheep preadipocytes, and adipose tissue was present within the transplant site of these cellular constructs, which was not present within the biomaterial control sites.     

Self-assembling peptide nanofibers and skeletal myoblast transplantation in infarcted myocardium

Cell transplantation is currently limited by poor graft retention and survival in the postinfarction scar. Because this issue could potentially be addressed by embedding cells in bioinjectable scaffolds and boosting cell survival pathways, we induced a myocardial infarction in 72 rats to assess the effects of different self-assembling peptides with or without platelet-derived growth factor (PDGF-BB) on survival of transplanted skeletal myoblasts. Two weeks after coronary artery ligation, rats were randomized to receive in-scar injections of culture medium (controls, n = 11), self-assembling peptide (RAD16-I) nanofibers (NF, n = 9), skeletal myoblasts (n = 12), or skeletal myoblasts in combination with NF (n = 8). In separate experiments with different self-assembling peptides (RAD16-II), rats received in-scar injections of culture medium (controls, n = 6), skeletal myoblasts (n = 10), PDGF-loaded peptides (n = 7), or skeletal myoblasts (5 x 10(6)) in combination with PDGF-loaded peptides (n = 9). After 1 month, left ventricular function, as assessed by echocardiography, was not improved in either of the experimental groups compared with controls. This correlated with the failure of RAD16-I peptides or PDGF-loaded RAD16-II peptides to improve myoblast survival despite a greater angiogenesis. In vitro experiments confirmed that the number of myoblasts decreased over time when seeded on nanofiber gels. These data suggest that the optimal use of biomaterial scaffolds for survival of transplanted cells will require specific tailoring of the biomaterial to the cell type.