Comparative 2-year outcomes of conventional and accelerated corneal collagen crosslinking in progressive keratoconus

AIM:To compare the safety and efficacy of conventional versus accelerated(9 mW/cm^2)corneal collagen crosslinking(CXL)in progressive keratoconus at the 2-year follow-up.METHODS:In this prospective study,consecutive progressive keratoconus patients were randomized to receive either conventional CXL(CCXL)or accelerated CXL(ACXL;using hydroxypropyl methylcellulose-assisted riboflavin imbibition for 10 min at 9 mW/cm^2).Visual,refractive,keratometric,topographic,and aberrometric outcomes and stromal demarcation line depth(DLD)measurements were compared at the end of a 2-year follow-up.RESULTS:Thirty-two eyes from 32 patients in the CCXL and 27 eyes from 27 patients in the ACXL groups completed 2-year follow-up.At 2y post-CXL,both uncorrected and corrected visual acuities improved significantly in both groups.The improvements in keratometric readings,flattening rate(flattening of the maximum keratometry more than 1 D),3 topographic indices,and vertical coma were significantly better in the CCXL group compared to the ACXL group(P<0.05).The DLD as measured by anterior segment optical coherence tomography or in vivo confocal microscopy was better detectable and significantly deeper in the CCXL group compared to the ACXL group.The deeper DLD was found to be significantly correlated with improvements in the mean keratometry measurements.Progression was noted in 11.1%of eyes in the ACXL group,whereas progression was not observed in any patient eye in the CCXL group.CONCLUSION:In this prospective randomized study,ACXL is less effective in halting the progression of keratoconus at a 2-year follow-up compared to CCXL.

Physiochemical and Biological Properties of Modified Collagen Sponge from Porcine Skin

The aim of the present study was to compare one-step method to EDC/NHS crosslinking （EDC/NHS group） and one-step simultaneous method to EDC/NHS crosslinking and heparin immobilization （EDC/NHS- Heparin group） in improving physiochemical and biological properties of native collagen sponge （Control group）. Modified collagen sponge overcome the disadvantages of native collagen sponge. IR spectra suggest the change of the functional groups. DSC data indicate that the stability of caloric transformation in EDC/NHS group is slightly higher than that of EDC/NHS-Heparin group. The crosslinking degree, stability against enzymes, stability in morphologically and biomechanical properties of EDC/NHS-Heparin group are higher than those of EDC/NHS group, whereas, the water-binding capacity in EDC/NHS-Heparin group is lower than that of EDC/NHS group. HUVECs in EDC/NHS-Heparin group scaffold proliferate fast, migrate well and distribute uniformly. One-step simultaneous method gains the better effects in above aspects, heparinized collagen matrices increase in angiogenic potential and suit for defect repairing and tissue engineering.

Protective effects of riboflavin-UVA-mediated posterior sclera collagen cross-linking in a guinea pig model of form-deprived myopia

AIM:To evaluate the effect of posterior sclera collagen cross-linking induced by riboflavin-ultraviolet A(UVA)on form-deprived myopia in guinea pigs.METHODES:Twenty-five pigmented guinea pigs of 3-week-old were randomly assigned into 4 groups that included normal control(NOR,n=7),form-deprived(FDM,n=7),normal with riboflavin-UVA cross-linking(NOR+CL,n=5)and form-deprived with cross-linking(FDM+CL,n=6).The NOR+CL group and the FDM+CL group received the riboflavin-UVA induced cross-linking at day 0.FDM was induced by monocularly deprived with facemask in the right eyes.The refraction,axial length and corneal curvature were measured by retinoscopy,A-scan and keratometer respectively in scheduled time points(day 0 and 1,2,3,4 wk after form-deprivation).At the end of 4 weeks’experiment,stress-strain tests of sclera were measured and morphological changes of sclera and retina were examined.RESULTS:After 4 wk,the interocular difference of refractive error were-0.11±0.67,-2.93±0.56,1.10±0.58,and-1.63±0.41 D in the NOR,FDM,NOR+CL,and FDM+CL groups respectively.Mixed-effect linear model revealed significant effect of FDM(P<0.01)and CL(P<0.001).Also,after 4 wk,the interocular difference of axial length were 0.01±0.04,0.29±0.07,-0.13±0.06,and 0.11±0.05 mm in the NOR,FDM,NOR+CL,and FDM+CL group.Mixedeffect linear model revealed significant effect of FDM(P<0.001)and CL(P<0.01).As for corneal curvature,significant interocular difference have not found between any of the two groups.At the end of this experiment,the ultimate stress and elastic modulus were found significantly increased in both CL groups.But no difference was found in the groups without cross-linked.There was no abnormality observed in the retina and RPE cells of the treated eyes.CONCLUSION:The posterior sclera collagen crosslinking induced by riboflavin-UVA can slow down the progress of myopia and increase the sclera biomechanical strength in the guinea pig model of form-deprived myopia.

Design of Medical Devices Using Spongy Sheet Composed of Hyaluronic Acid and Collagen

This research aims to obtain useful information for development of medical devices such as wound dressing and tissue anti-adhesive product, using a spongy sheet composed of hyaluronic acid (HA) and collagen (Col). The spongy sheets were manufactured by freeze vacuum drying of HA and Col aqueous solution, followed by UV irradiation to introduce intermolecular crosslinks between Col molecules. These spongy sheets are referred to as Sponge-A (ratio of HA/Col = 5/1) and Sponge-B (ratio of HA/Col = 5/5). Both surfaces of Sponge-A and Sponge-B treated with UV irradiation for 15 minutes are referred to as Sponge-A-15 and Sponge-B-15, respectively. The weight change of spongy sheet was determined by immersing a peace of spongy sheet in water at 37°C. The weight of sponge-A-15 collected 1/2, 1, 3, 7 days after immersion in water was 63.5%, 62.1%, 56.6%, 54.4% of the original weight, respectively. The weight of Sponge-B-15 was 78.3%, 76.7%, 79.1%, 71.9% of the original weight, respectively. The weight change of spongy sheet was determined by immersing a peace of spongy sheet in water containing collagenase at 37°C. The weight of Sponge-A-15 collected 6, 8, 10, 12 hours after immersion in water containing collagenase (0.0005 w/v%) was 65.7%, 59.8%, 57.9%, 55.2% of the original weight, respectively. The weight of Sponge-B-15 was 63.5%, 52.1%, 42.0%, 43.2% of the original weight, respectively. This spongy sheet is considered to have the unique structure, where HA molecules are entrapped in an intermolecular cross-linked network structure of Col molecules. When immersed in water containing collagenase, the weight loss of spongy sheet is accelerated by easy extraction of HA molecules from the enzymatic degraded Col network structure. The performance of wound dressing and tissue anti-adhesive product is considered to depend on appropriate ratio of HA and Col, and also on appropriate rate of intermolecular crosslinks between Col molecules. These findings obtained in this study provide useful information for product development such as wound dressing and tissue anti-adhesive product.

Visual rehabilitation in low-moderate keratoconus:intracorneal ring segment implantation followed by same-day topography-guided photorefractive keratectomy and collagen cross linking

AIMTo present the results of same-day topography-guided photorefractive keratectomy (TG-PRK) and corneal collagen crosslinking (CXL) after previous intrastromal corneal ring segment (ISCR) implantation for keratoconus.

Study on Different Modification Methods of Collagen for Tissue Engineering

Because of the excellent biocompatibility and its specific amino sequences,collagen is an ideal biomedical material for tissue engineering applications. But collagen is usually lack of mechanical strength to form a rigid 3-D matrix and lack of ability to resist collagenase. In order to be a tissue engineering scaffold,collagen must strengthen its structures by modifying with chemical crosslinkers. Chemical crosslinkers used for modifying collagen fibers include glutaraldehyde(GA),epoxy compounds(PC) and carbodiimides (EDC). The aim of this study is to choose the best chemical crosslinker from the three reagents. In terms of the resistance to collagenase degradation,chemical cross-linking with PC provided the best protection; in terms of the mechanical characterization,chemical cross-linking with GA provided the best;and in terms of the biocompatibility,chemical cross-linking with EDC provided the best.There is not a reagent which has all merits for collagen crosslinking,so we should select the crosslinking reagent as the demands of use ask.

不同交联方法构建的胶原基支架及其性能表征

分别采用1-乙基-(3-二甲氨基丙基)碳酰二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)交联法、甲基丙烯酸酐(MA)修饰光聚合交联法和苯基-2,4,6-三甲基苯甲酰基磷酸锂(LAP)光交联法制备了3种胶原基支架材料,并对其微观结构、理化性能和生物学性能进行了评价。结果表明:这3种交联法制备的胶原基支架材料的孔洞结构具有显著差异,其孔径范围均为50~250μm,符合组织修复材料对孔径的需求。此外,通过MA复合光交联所得的胶原基支架材料(Col-MA),其抗压强度可达2.17 MPa,满足组织工程理想支架材料对力学性能的基本要求。生物相容性实验结果表明,这3种支架材料均能够为细胞生长提供合适的空间以及供应足够的营养物质。

胶原蛋白交联方法及其研究进展

胶原蛋白具有优异的生物相容性、高效的生物降解性、低免疫原性以及胶原宿主和组织之间的协调性,但其机械性能和抗酶解速率都较低,通过对胶原蛋白进行交联,以提高其结构的稳定性,分析了物理交联、化学交联和生物交联制备的胶原蛋白材料的优缺点,并展望了其未来的发展方向。

胶原-壳聚糖微球的制备与载药性能研究

本文采用胶原与壳聚糖作为原材料,利用壳聚糖的pH响应性,制备了一种无添加交联剂的复合微球,并通过天狼星红染色法和羟脯氨酸分析法,测定复合微球中胶原的含量。此外,对微球的形貌、粒径、热稳定性以及持水性能进行了系统性分析。通过将姜黄素作为模拟药物,对微球的载药性能进行了探索。

单宁酸-纳米协同改性胶原纤维多孔材料的制备及其油水分离性能

以胶原纤维为代表的生物质基多孔材料因其具有可再生、储量丰富等特点且保留胶原纤维独特的多尺度孔隙结构以及具有优异的分离能力、可重复使用和可生物降解等优势,近年在含油废水处理等领域备受关注。针对胶原纤维多孔材料存在着油水分离效率低等问题,本文利用单宁酸与金属离子(铝和锆)掺杂锂藻土纳米片的协同交联体系来改性胶原纤维,制备了一种具有油水分离性能的胶原纤维基多孔材料。结果表明:该协同交联体系可提高胶原纤维多孔材料的自支撑性、成型性和多孔性,同时未改变胶原的三股螺旋构象并可起到稳定胶原微结构的作用;协同交联改性后胶原纤维多孔材料的收缩温度提升至90℃以上,接触角提升至98°以上,油水分离效率提升至68%以上,表明其热稳定性、疏水性以及油水分离性能均得以改善。

Targeted protein delivery:carbodiimide crosslinking influences protein release from microparticles incorporated within collagen scaffolds

Tissue engineering response may be tailored via controlled,sustained release of active agents from protein-loaded degradable microparticles incorporated directly within three-dimensional(3D)ice-templated collagen scaffolds.However,the effects of covalent crosslinking during scaffold preparation on the availability and release of protein from the incorporated microparticles have not been explored.Here,we load 3D ice-templated collagen scaffolds with controlled additions of poly-(DL-lactide-co-glycolide)microparticles.We probe the effects of subsequent N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride crosslinking on protein release,using microparticles with different internal protein distributions.Fluorescein isothiocyanate labelled bovine serum albumin is used as a model protein drug.The scaffolds display a homogeneous microparticle distribution,and a reduction in pore size and percolation diameter with increased microparticle addition,although these values did not fall below those reported as necessary for cell invasion.The protein distribution within the microparticles,near the surface or more deeply located within the microparticles,was important in determining the release profile and effect of crosslinking,as the surface was affected by the carbodiimide crosslinking reaction applied to the scaffold.Crosslinking of microparticles with a high proportion of protein at the surface caused both a reduction and delay in protein release.Protein located within the bulk of the microparticles,was protected from the crosslinking reaction and no delay in the overall release profile was seen.

跨上皮快速角膜胶原交联术治疗进展期圆锥角膜患者的效果

目的探讨跨上皮快速角膜胶原交联术(CXL)治疗进展期圆锥角膜患者的效果。方法选取2017年10月至2021年3月于哈尔滨爱尔眼科医院诊治的60例(65眼)进展期圆锥角膜患者,均行跨上皮快速CXL,比较患者治疗前后裸眼视力(UCVA)、最佳矫正视力(BCVA)、屈光状态、角膜透明及眼压。结果术后3、6个月,患者UCVA均高于术前及术后1、12个月,且术后12个月高于术后1个月,差异有统计学意义(P<0.05)。术后3、6个月,患者BCVA高于术前及术后1、12个月,且术后6个月高于术后3个月,术后12个月高于术后1个月,差异有统计学意义(P<0.05)。术前及术后不同时间点的球镜度比较差异无统计学意义;术后1个月柱镜度低于术前,术后12个月柱镜度高于术后1、3、6个月,差异有统计学意义(P<0.05)。术前及术后1、3、6、12个月,患者眼压比较差异无统计学意义。随访期间,所有患者未出现角膜感染,晶状体保持透明。结论跨上皮快速CXL治疗进展期圆锥角膜效果显著,可有效改善患者视力水平,具有较高应用价值。

胶原交联的改性研究进展(二)

胶原蛋白作为一种在各类结缔组织中广泛存在的重要结构蛋白,其独特的三螺旋结构和形成交联网络的能力,为其提供了显著的机械强度和耐久性。通过不同的交联方法,特别是化学交联,可以显著改善胶原蛋白的物理和化学性质,从而拓宽其在医学、食品工业、环保和化妆品等领域的应用范围。各种交联剂,包括戊二醛、EDC/NHS体系、酚酸、京尼平以及纳米粒子等,均显示出增强胶原蛋白力学性能、热稳定性和抗酶降解能力的潜力。这些性能的提升,为胶原蛋白基材料的生物医学应用,如血管移植、软骨修复、器官再生、角膜修复以及在食品包装和保湿化妆品中的应用,提供了重要支持。

创伤性骨折患者术后血清骨代谢指标与骨愈合延迟的相关性

目的探讨创伤性骨折患者术后血清骨代谢指标与骨愈合延迟的相关性。方法回顾性分析本院2022年1月至2023年12月收治的54例创伤性骨折延迟愈合患者为研究组,同期选择54例创伤性骨折正常愈合患者为对照组。对比两组患者手术前后血清骨代谢指标[1型胶原氨基端延长肽(P1NP)、骨钙素(OST)、β-胶联降解产物(β-CTx)]水平,用受试者工作特征(ROC)曲线分析血清P1NP、OST及β-CTx对创伤性骨折患者术后骨愈合延迟的预测价值。结果研究组与对照组术后4周及8周血清P1NP、OST及β-CTx水平均分别高于同组术前及术后1周(P<0.05),对照组术前、术后1周、术后4周及术后8周的血清P1NP、OST及β-CTx水平均分别高于同时间的研究组(P<0.05)。ROC曲线分析显示血清P1NP、OST、β-CTx水平及联合检测对创伤性骨折术后骨愈合延迟预测的曲线下面积分别为0.734、0.729、0.705、0.865。结论骨代谢状态可能对创伤性骨折患者术后骨愈合情况有一定影响,临床可综合测定患者血清骨代谢指标,预测创伤性骨折患者术后骨愈合延迟风险。

Protein expression changes in cornea after collagen crosslinking

Riboflavin/UV-mediated corneal collagen cross-linking can increase the mechanical strength of the cornea and prevent or delay corneal expansion and keratoconus progression.We performed quantitative analysis of protein iTRAQ in rabbit eye white matter after cross-linking to explore the changes of protein expression in cornea at different times after cross-linking and to understand the process of corneal stroma remodeling after cross-linking.The screening conditions are fold Change
1.2 and P-value<0.05,we identified 713 and 38 differentially expressed proteins in cornea at 1 week and 1 month after cross-linking.There were 16 differentially expressed proteins at two time points after corneal cross-linking.By annotating the functions of these proteins,we identified some proteins that affect the mechanical properties of the cornea,and these proteins are involved in cell growth,oxidative stress response,and signal transduction in the cornea.It has a guiding role in studying the corneal stroma remodeling process after collagen crosslinking.

交联方法对草鱼皮胶原蛋白海绵性能的影响

为探讨不同交联方法对草鱼皮胶原蛋白海绵材料性能的影响,实验以草鱼鱼皮为原料,提取、纯化胶原蛋白并制备胶原海绵材料。在此基础上,分别用紫外交联、热交联、戊二醛交联以及EDC/NHS交联方法处理胶原海绵,通过测定材料的交联度、热变性温度、拉伸强度和体外抗酶降解性能,比较了不同方法的交联效果。结果发现,提取所得的草鱼皮胶原蛋白为典型的Ⅰ型胶原;经不同方法交联处理后,海绵材料交联度依次为戊二醛(72.0%)>EDC/NHS(32.5%)>热交联(29.9%)>紫外交联(15.6%);与对照胶原材料相比,戊二醛处理后,胶原材料的热变性峰值温度(67.4℃)、最大拉伸强度(125.6 kPa)和体外抗酶降解性能均有显著提升(P<0.05);EDC/NHS处理后,胶原材料的热变性焓显著提升(6.86 J/g),同时材料的拉伸强度(98.6 kPa)和体外抗酶降解性能也得到适度增加(P<0.05)。紫外交联和热交联对草鱼皮胶原材料性能的改善作用比较有限,并可能导致胶原分子的部分变性。红外光谱的分析结果表明,戊二醛处理可导致草鱼皮胶原三螺旋分子内产生新的共价键交联从而使材料性能改善,而EDC/NHS处理主要导致胶原分子间产生新的氢键交联并可提高胶原材料的稳定性。研究表明,戊二醛和EDC/NHS交联能有效提高胶原海绵材料的性能,而热交联和紫外交联对材料性能的改善作用非常有限。

交联温度对京尼平交联胶原/壳聚糖组织工程支架的影响

[目的]通过研究材料的孔径、交联度、溶胀率、降解率、细胞毒性及组织相容性的变化,探讨交联温度对京尼平交联胶原/壳聚糖支架的影响。[方法]采用冷冻干燥法制备胶原/壳聚糖复合多孔支架,分别于4℃、20℃、36℃条件下,在0.5%京尼平水溶液中交联24h。以未交联的胶原/壳聚糖复合多孔支架作为对照,评价所得支架的孔径、交联度、溶胀率、降解率、细胞毒性及组织相容性特点。[结果]随交联温度升高,支架的交联度明显增大,溶胀率和降解率逐渐减小。4℃组支架交联度47.88%±6.4%,溶胀率为721%±46%,4周后降解3.95%±6.4%;20℃组支架交联度67.69%±3.6%,溶胀率为662%±72%,4周降解0.91%±5.9%;36℃组支架交联度70.32%±5.7%,溶胀率为635%±27%,4周降解0.66%±7.3%,三组上述观察指标均优于未交联组(P/0.01)。[结论]京尼平交联可以显著降低胶原/壳聚糖支架的降解率和溶胀率,且对支架结构和生物相容性无明显影响。交联温度增加可以提高支架的交联度和抗降解能力,较快获得具有良好生物相容性和降解率的组织工程支架。

静电纺胶原/丝素复合微纳米纤维的制备及细胞相容性研究

采用静电纺丝技术制备胶原/丝素复合微纳米纤维,对其理化性能进行表征并观察其细胞相容性。以六氟异丙醇(HFIP)为溶剂,将胶原和丝素以100∶0、70∶30、50∶50、30∶70、0∶100的质量比共混进行电纺。制备的五种材料经戊二醛蒸汽交联12 h。采用扫描电镜、红外光谱、X射线衍射、热重分析和拉伸力学性能测试等方法对其理化性能进行表征。材料种植成纤维细胞后,通过扫描电镜和噻唑兰(MTT)比色法观察其细胞相容性。结果显示制备的纤维平均直径在550～1 100 nm之间,随着丝素含量的增加纤维平均直径增加。交联后纤维的β化程度、结晶度和热稳定性均有一定提高,且随着丝素含量的增加提高越明显;交联后材料的力学性能优于交联前;当丝素含量为70%时,纤维膜的平均断裂强度为(8.70±1.05)MPa,高于其它配比的纤维膜。细胞在材料表面生长状态良好;丝素含量为70%组的细胞粘附和增殖高于其它组,与细胞培养板相比无显著性差异,表明其细胞相容性良好,可望成为一种新型的组织工程支架材料。

不同交联方法对离体猪巩膜交联效果的比较

背景研究表明化学交联剂可加强巩膜胶原的强度，但交联效果受不同交联方法的影响。目的研究不同交联方法对猪巩膜化学交联效果的影响。方法离体4h以内的新鲜猪眼球70只分为5个组，分别用全眼球交联法和巩膜条带交联法在质量分数1％京尼平溶液、体积分数1％戊二醛溶液或PBS中37℃水浴40rain。交联后每组取10个10ram^4ITlm的颞侧巩膜条带于Instron5848型微力测试仪下进行拉伸实验，测定巩膜组织的弹性模量和拉伸应力；每组其他的4个样本于水浴箱内测试巩膜组织的最大热收缩温度。结果机械力学实验结果表明，1％京尼平溶液处理后，全眼球交联法巩膜条带的弹性模量值为（8．98±1．81）MPa，而巩膜条带交联后的值是（10．85±1．83）MPa，前者约为后者的82．8％，两种交联方法的巩膜弹性模量值间的差异有统计学意义（￡=3．375，P=0．003）；1％戊二醛溶液处理后，全眼球交联法巩膜条带的弹性模量值是（12．78±2．91）MPa，巩膜条带交联后的值是（18．25±5．16）MPa，前者约为后者的70．0％，两种交联方法间的差异有统计学意义（t=4．007，P=0．001）。在5％、10％、15％和20％的应变条件下，全眼球交联法巩膜组织的拉伸应力分别为巩膜条带交联法的54．9％～90．1％，均明显弱于巩膜条带交联法，差异均有统计学意义（P〈O．05）。热力学实验表明，1％京尼平处理后全眼球交联法巩膜的热收缩温度为（68．8±O．9）℃，巩膜条带交联法为（74．8-＋1．3）℃，两种交联方法间的差异有统计学意义（f=11．129，P=0．000）。1％戊二醛处理后全眼球交联法巩膜的热收缩温度为（73．3±O．9）℃，巩膜条带交联法为（79．3±1．3）℃，两种交联方法间的差异有统计学意义（t=11．112，P=0．000）。结论不同交联方法对猪巩膜化学交联后的力学效果存在影响，巩膜条带交联后巩膜的交联强度略好于全眼球交联。

环氧化合物和碳化二亚胺交联pADM的性能比较

采用丙三醇缩水甘油醚(GPE)和碳化二亚胺(EDC)对脱细胞真皮基质(pADM)分别进行交联改性,通过对交联后材料EC(GPE)-pADM、EDC-pADM的交联效率、XPS分析、KM鼠经口急毒、浸提液细胞毒性的表征和比较,探寻改性方法与材料性能之间的基本关系。研究结果表明,EDC比EC具有更高的交联效率;EC-pADM的亲水性能比EDC-pADM更好;EDC-pADM浸提液对KM鼠表现出一定经口毒性,而EC-pADM的毒性相对较低;两者浸出液细胞毒性检测均合格。综合而言,EC-pADM的性能优于EDC-pADM。