Modulation of keratocyte phenotype by collagen fibril nanoarchitecture in membranes for corneal repair

Type I collagen membranes with tailored fibril nanoarchitectures were fabricated through a vitrification processing, which mimicked, to a degree, the collagen maturation process of corneal stromal extracellular matrix in vivo. Vitrification was performed at a controlled temperature of either 5 °C or 39 °C at a constant relative humidity of 40% for various time periods from 0.5 wk up to 8 wk. During vitrification, the vitrified collagen membranes (collagen vitrigels, CVs) exhibited a rapid growth in fibrillar density through the evaporation of water and an increase in fibrillar stiffness due to the formation of new and/or more-stable interactions. On the other hand, the collagen fibrils in CVs maintained their D-periodicity and showed no significant difference in fibrillar diameter, indicating preservation of the native states of the collagen fibrils during vitrification. Keratocyte phenotype was maintained on CVs to varying degrees that were strongly influenced by the collagen fibril nanoarchitectures. Specifically, the vitrification time of CVs mainly governed the keratocyte morphology, showing significant increases in the cell protrusion number, protrusion length, and cell size along with CV vitrification time. The CV vitrification temperature affected the regulation of keratocyte fibroblasts' gene expressions, including keratocan and aldehyde dehydrogenase (ALDH), demonstrating a unique way to control the expression of specific genes in vitro.     

Effects of insulin-like growth factor 2 and its receptor expressions on corneal repair

Limbal stem cell (LSC) on the basal layer of cornea plays an important role in the epithelial repair after corneal injury as it can proliferate, differentiate and migrate into injury sites under the direction of cytokines. This study explored the signaling pathway and cellular mechanism between corneal epithelial cells LSC, on a mouse model with mechanic corneal injury. Ipsilateral corneal mechanic injury model was prepared on mice using the contralateral eye as the control. Tissues from both central and peripheral regions of cornea were collected, cultured and quantified for expression of various cytokines including epidermal growth factor (EGF), fibroblast growth factor-β (FGF-β), heparin-like growth factor (HGF), keratinocyte growth factor (KGF), transforming growth factor-β1 (TGF-β1), IGF-1 and IGF-2. The effects of these factors on the differentiation of LSC and fibroblasts were also studied. Most of those cytokines had elevated gene expressions after the corneal injury. Among those IGF-2 had significantly increased expression, along with the high expression of IGF-2 receptor in corneal peripheral cells. IGF-2 also induced the differentiation of LSC into keratin-12-positive cells. Further studies showed the prominent expression of α-actin in injured tissues, suggesting the potential transformation of fibroblasts into myofibroblasts. Both IGF-2 and its receptor had elevated expressions after corneal injury. They may facilitate the transformation of LSC into epithelial cells, in addition to the role in transformation from fibroblasts to myofibroblasts.     

骨修复用胶原复合支架材料研究进展

胶原蛋白作为骨组织工程支架材料具有许多优点,文章综述了骨修复用胶原复合支架材料的研究现状,仿天然骨成分、结构和特性胶原复合支架材料是骨组织工程研究发展的趋势。     

组织损伤修复中的生物力学问题

生物力学对损伤组织修复的影响及调控研究方兴未艾。组织修复与再生医学的发展为生物力学的研究提供了新平台,而生物力学则成为推动组织修复与再生医学发展的重要因素。对《医用生物力学》2016年第5期发表的"生物力学与组织修复"专栏论文进行简要的分析、述评,同时对该领域的研究进行回顾和展望。     

Collagen-PVA aligned nanofiber on collagen sponge as bi-layered scaffold for surface cartilage repair

Researchers have made bi-layered scaffolds but mostly for osteochondral repairs. The anatomic structure of human cartilage has different zones and that each has varying matrix morphology and mechanical properties is often overlooked. Two bi-layered collagen-based composites were made to replicate the superficial and transitional zones of an articular cartilage. Aligned and random collagen-PVA nanofibers were electrospun onto a freeze-dried collagen sponge to make the aligned and random composites, respectively. The morphology, swelling ratio, degradation and tensile properties of the two composites were examined. Primary porcine chondrocytes were cultured on the composites for three weeks and their proliferation and secretion of glycosaminoglycan (GAG) and type II collagen were measured. The influences of the cell culture on the tensile properties of the composites were studied. The nanofiber layer remained adhered to the sponge after three weeks of cell culture. Both composites lost 30–35% of their total weight in a saline buffer after three weeks. The tensile strength and Young’s modulus of both composites increased after three weeks of chondrocyte culture (p < 0.05). The aligned composite with extracellular matrix deposition had a Young’s modulus (0.35 MPa) similar to that of articular cartilage reported in literature (0.36–0.8 MPa). The chondrocytes on both aligned and random composites proliferated and secreted similar amounts of GAG and type II collagen. They were seen embedded in lacunae after three weeks. The aligned composite may be more suitable for articular cartilage repair because of the higher tensile strength from the aligned nanofibers on the surface that can better resist wear.     

3D打印胶原/壳聚糖支架改善大鼠脊髓损伤后神经功能恢复

文题释义：壳聚糖：为一种天然多糖,是虾蟹等低等动物外壳的重要成分,具有一定的机械强度,并且具有良好的生物相容性和抗菌性,在生物工程领域具有较好的应用前景。 3D生物打印：是组织工程中最重要的技术之一。目前常用的三维生物打印方法包括喷墨打印、挤压生物打印和激光生物打印,选择好合适的材料后,在计算机指导下根据所选择的生物材料和细胞类型逐层准确地打印出所设计的结构。 背景：3D打印技术可以根据需求制备出满足脊髓植入形状、大小和表面形态要求的生物支架。 目的：观察3D打印胶原/壳聚糖支架对脊髓损伤大鼠神经功能恢复的影响。 方法：将胶原和壳聚糖按2∶1的质量比混合,采用冷冻干燥法制备普通胶原/壳聚糖支架,采用3D打印机制备3D打印胶原/壳聚糖支架,分别测量两种支架的孔隙率和弹性模量,电镜观察支架形态。将神经干细胞分别与3D打印胶原/壳聚糖支架、普通胶原/壳聚糖支架共培养,进行扫描电镜观察与CCK-8检测。将40只雌性SD大鼠(由中国人民解放军医学科学院军事科学院提供)随机分成4组：假手术组、脊髓损伤组、普通胶    原/壳聚糖支架组和3D打印胶原/壳聚糖支架组,后3组制作脊髓全横断损伤模型,普通胶原/壳聚糖支架组和3D打印胶原/壳聚糖支架组损伤处填充对应的支架材料,术后相应时间点进行后肢功能BBB评分、斜坡实验、神经电生理检测与磁共振平扫。实验方案经天津市神经创伤重点实验室伦理委员会批准。 结果与结论：①扫描电镜显示,3D打印胶原/壳聚糖支架具有互连的多孔结构,普通胶原/壳聚糖支架内部结构紊乱;②神经干细胞在3D打印胶原/壳聚糖支架表面生长良好,完全伸展,且3D打印胶原/壳聚糖支架表面神经干细胞的活性显著高于普通胶原/壳聚糖支架组(P < 0.05);③3D打印胶原/壳聚糖支架的孔隙率与弹性模量均高于普通胶原/壳聚糖支架组(P < 0.05);④3D打印胶原/壳聚糖支架组术后3-8周的BBB评分高于脊髓损伤组、普通胶原/壳聚糖支架组(P < 0.05),术后4,6,8周的斜坡实验角度大于脊髓损伤组、普通胶原/壳聚糖支架组(P < 0.05);⑤3D打印胶原/壳聚糖支架组术后8周的运动诱发电位振幅、体感诱发电位振幅大于脊髓损伤组与普通胶原/壳聚糖支架组(P < 0.05),运动诱发电位潜伏期、体感诱发电位潜伏期短于脊髓损伤组与普通胶原/壳聚糖支架组(P < 0.05);⑥磁共振平扫显示与脊髓损伤组及普通胶原/壳聚糖支架组比较,3D打印胶原/壳聚糖支架组损伤处具有较好的连续性与较多的神经纤维束通过;⑦结果表明,3D打印胶原/壳聚糖支架可促进脊髓损伤大鼠神经功能的修复。 ORCID: 0000-0001-5771-8222(史新宇) 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     

应用胶原-壳聚糖桥接管引导大鼠坐骨神经再生

目的:观察胶原-壳聚糖桥接管促进大鼠坐骨神经损伤后再生与修复的作用。方法:用壳聚糖和胶原蛋白按1∶3的比例采用冷冻干燥法制成胶原-壳聚糖复合导管;将20只雄性Wistar大鼠随机分为2组,胶原-壳聚糖导管组和硅胶管组,切断坐骨神经,建立10mm的缺损动物模型;分别用胶原-壳聚糖导管和硅胶管进行桥接。于术后不同时间对2组动物进行大体观察,并于术后14周进行电生理、组织学及逆行示踪检测,比较2组大鼠坐骨神经的再生和功能恢复情况。结果:术后14周2组动物坐骨神经再生和功能恢复情况的各项检测指标显示,胶原-壳聚糖桥接组明显优于硅胶管桥接组。结论:胶原-壳聚糖导管可用来桥接损伤神经,在周围神经缺损修复方面具有良好的应用前景。     

聚乳酸/矿化胶原复合人工骨修复材料在兔股骨髁松质骨缺损中的应用研究*

目的 研究聚乳酸/矿化胶原人工骨修复材料（NBG）在兔股骨髁松质骨缺损中的疗效,以及该材料与自体骨、骨髓混合使用的效果,考察材料的安全性及有效性。方法 在兔双侧股骨髁造成松质骨缺损,将60只雄性大白兔进行不同骨材料植入。试验分为5组：空白（Blank）组、人工骨（NBG）组、人工骨混骨髓（NBG+ABM）组、人工骨混自体骨（NBG+AGB）组、自体骨（AGB）组,通过影像学观察、组织学评价、生物力学分析等方法考察5 组材料的骨修复性能。结果 观察新生骨和周围骨质结合情况,拍摄X线片并进行灰度值分析以及进行CT三维重建分析,不同时间点的组织切片观察,均表明NBG组、NBG+ABM组、NBG+AGB组、AGB组骨修复效果良好,生物力学测试也表明各实验组相对于空白组可以承载较高的压力载荷。AGB组、NBG+AGB组、NBG+ABM组、NBG组修复效果依次降低,但均接近完全修复。结论 聚乳酸/矿化胶原基人工骨修复材料有良好的生物相容性及骨诱导性,是良好的骨植入材料。单独使用即可以获得良好的骨修复效果,也可以和自体骨或骨髓混合使用,修复效果更加明显。     

超声引导注射富血小板血浆修复坐骨神经挤压伤

文题释义：富血小板血浆：是通过离心自体全血而得到的含高浓度血小板的血浆。已证实富血小板血浆中含有多种生长因子,如血小板源性生长因子、转化生长因子β、类胰岛素生长因子1、血管内皮生长因子、神经生长因子、脑源神经营养因子、肝细胞生长因子等。这些生长因子对组织的修复和再生起着重要作用。富血小板血浆由自体血液取样制备,因取材方便、制备较简单,且具有较好的促进组织再生等优点,已被广泛用于骨科领域,如：肌肉、肌腱、韧带、软骨损伤,创面修复,骨折、骨性关节炎、脊柱融合等,并逐年被应用于神经再生的研究。 背景：富血小板血浆富含影响肌腱、韧带、肌肉和骨愈合的生长因子,在此基础上,研究人员逐渐认识到富血小板血浆激活后释放的分子可调节周围神经早期炎症、激活许旺细胞、促进巨噬细胞极化及阻止胶原纤维的增生,成为神经功能恢复的关键驱动力。 目的：探讨超声引导富血小板血浆注射修复坐骨神经挤压伤的价值。 方法：将28只新西兰大白兔(北京隆安动物繁殖中心提供)随机分为4组,每组7只：正常组暴露右侧坐骨神经后直接缝合;对照组建立右侧坐骨神经挤压损伤模型;单频次注射组建立右侧坐骨神经挤压损伤模型,术后24 h于超声引导下在损伤神经周围注射自体富血小板血浆;多频次注射组建立右侧坐骨神经挤压损伤模型,术后24 h于超声引导下在损伤神经周围注射自体富血小板血浆,此后第3,5周各注射1次。术后12周,进行再生神经的组织学、形态学评价及失神经支配肌肉的湿质量恢复与组织学检测。实验经解放军总医院实验动物管理委员会批准(2015-x10-02)。 结果与结论：①与对照组比较,单频次与多频次注射组再生轴突NF-200、S100染色累积吸光度值明显升高(P均< 0.05);多频次注射组二者累积吸光度值高于单频次注射组(P均< 0.05),但仍低于正常组(P均< 0.05);②与对照组比较,单频次与多频次注射组有髓神经纤维密度、有髓神经纤维直径及髓鞘厚度明显增加(P均< 0.05);多频次注射组3指标多于单频次注射组(P均< 0.05),但仍低于正常组(P均< 0.05);③与对照组比较,单频次与多频次注射组肌肉湿质量、肌纤维横截面积增加(P均< 0.05);多频次注射组两指标多于单频次注射组(P均< 0.05),但仍少于正常组(P均< 0.05);④结果表明,超声引导自体富血小板血浆多频次注射治疗坐骨神经挤压伤具有良好的效果。 ORCID: 0000-0002-1974-3221(朱亚琼) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Microvascular injury and repair in acute human bacterial pyelonephritis

Summary Acute inflammatory cell-capillary endothelial cell interactions, related to injury and repair, were investigated light and electron microscopically in acute human bacterial pyelonephritis. In inflammatory infiltrate-adjacent microvessels, the small capillaries were completely occluded by leukocyte plugs and the large capillaries were densely filled with acute inflammatory cells adhering to the endothelium. Severe damage to small and large capillaries was observed around endothelium adherent, degranulated neutrophil granulocytes containing phagocytosed bacteria. There were spaces in the endothelium, degradation of the vascular basement membrane, of the perivascular interstitial matrix and of collagen fibrils, with fibrin deposition and vessel wall fragmentation. In the small capillaries relatively distant from the interstitial infiltrates, emigration of leukocytes was frequently seen. Around the escaping cells the endothelial lining displayed occasional discontinuities, allowing leakage of vascular fluid into the interstitial space. Some small capillaries not related to the infiltrate were occluded by fibrin thrombi with apparent damage to the endothelial cells and disruption of the capillary wall. Various reparative changes were noticed in association with this change including capillary neovascularization. The findings confirm the existence of polymorphonuclear leukocyte-mediated injury of capillaries during the development of inflammatory responses in acute pyelonephritis.Some parts of this paper were presented at the XVIth Congress of the IAP, Vienna, Austria, August 31–September 5, 1986A Fellow of the Deutscher Akademischer Austauschdienst, Bonn, FRG     

Macrophage involvement in the kidney repair phase after ischaemia/reperfusion injury

Macrophage infiltration is a common feature of the early phase of renal ischaemia/reperfusion injury. Indeed, it is generally regarded as the cause of tissue injury in this phase, although it is also clear that it can lead to tissue repair in other phases. In order to ascertain whether macrophages are directly involved in the repair/late phase, which follows the pro-inflammatory and injury process of renal ischaemia/reperfusion, we used two different approaches based on macrophage depletion. Firstly, we produced renal ischaemia in mice that were previously treated with clodronate liposome. Secondly, during reperfusion we re-injected RAW 264.7 to macrophage-depleted mice 24 h prior to sacrifice. The results showed that regeneration, as evaluated by stathmin and PCNA markers, was macrophage-dependent: it was blocked when macrophage depletion was provoked and recovered with macrophage re-injection. The cytokine profile revealed the influence of the inflammatory environment on kidney repair: pro-inflammatory cytokines (MCP-1, MIP-1alpha) increased during the early stages of reperfusion, coinciding with low regeneration, and the anti-inflammatory cytokine IL-10 increased during the longer periods of reperfusion when regeneration was more evident. We conclude that macrophages induce renal regeneration after ischaemia/reperfusion, depending on the inflammatory milieu.     

Dexamethasone transiently attenuates up-regulation of endostatin/collagen XVIII following traumatic brain injury

Endostatin/collagen XVIII is a specific inhibitor of endothelial proliferation and migration in vitro. It has also been shown to have anti-angiogenic activity and tumor growth inhibitory activity in vivo and in vitro. Here we studied expression of endostatin/collagen XVIII in a rat traumatic brain injury (TBI) model, focusing on the early phase. A significant up-regulation of endostatin/collagen XVIII in TBI began as early as 24 h post-TBI. Double-staining experiment revealed that the major resource of endostatin/collagen XVIII(+) cells in our TBI rat model was a subpopulation of reactivated microglia/macrophages. Our data further showed that dexamethasone attenuated up-regulation of endostatin/collagen XVIII expression at days 1 and 2, but not at day 4, post-TBI, indicating that dexamethasone might possess an early and transient influence to the angiogenesis following TBI.     

Injectable co-gels of collagen and decellularized vascular matrix improve MSC-based therapy for acute kidney injury

Transplantation of mesenchymal stem cells (MSCs) is promising for treatment of acute kidney injury (AKI), but their therapeutic effects are often limited under normal conditions. In this study, we prepared the co-gels of decellularized vascular matrix and collagen, and investigated whether the co-gels increase the therapeutic potentials of MSCs on AKI. In vitro studies indicated that the co-gels enhanced the paracrine effects of MSCs, and significantly reduced the apoptosis of MSCs under oxidative environments. When the co-gels were co-transplanted with MSCs into the kidney of model rats with ischemia-reperfusion (I/R)-induced AKI, the survival and paracrine effects of MSCs were enhanced in the injured kidney. More importantly, the co-gels increased the therapeutic effects of MSCs for AKI, as indicated by cell apoptosis, tissue damage, vascularization and renal function. Therefore, the co-gels of decellularized vascular matrix and collagen improved the therapeutic effects of MSCs, and might be promising for AKI treatment.     

Demineralized dentin matrix composite collagen material for bone tissue regeneration

Demineralized dentin matrix (DDM) had been successfully used in clinics as bone repair biomaterial for many years. However, particle morphology of DDM limited it further applications. In this study, DDM and collagen were prepared to DDM composite collagen material. The surface morphology of the material was studied by scanning electron microscope (SEM). MC3T3-E1 cells responses in vitro and tissue responses in vivo by implantation of DDM composite collagen material in bone defect of rabbits were also investigated. SEM analysis showed that DDM composite collagen material evenly distributed and formed a porous scaffold. Cell culture and animal models results indicated that DDM composite collagen material was biocompatible and could support cell proliferation and differentiation. Histological evaluation showed that DDM composite collagen material exhibited good biocompatibility, biodegradability and osteoconductivity with host bone in vivo. The results suggested that DDM composite collagen material might have a significant clinical advantage and potential to be applied in bone and orthopedic surgery.     

过氧化物酶6作用下神经干细胞对面神经损伤的修复研究

目的探索过氧化物酶6(Prdx6)作用下神经干细胞(NSC)对面神经离断性损伤的修复效果。方法选择新生6 h内SD大鼠6只,雌雄不限,体质量100 g左右。选择清洁级雄性成年SD大鼠30只,体质量(220±50)g,鼠龄6~8周。从新生6 h内SD大鼠取脑内分离NSC,采用悬浮的方法体外培养NSC,并进行NSC特异性标记蛋白Nestin的免疫荧光染色鉴定。将清洁级雄性成年SD大鼠分为A组(假手术组)、B组(溶媒组)、C组(Prdx6组)、D组(NSC组)、E组(NSC联合Prdx6组),每组6只。除A组外,其余各组采用手术离断方法对大鼠左侧面神经颊支离断形成5 mm缺损,制作SD大鼠面神经缺损模型,使用大鼠同种自体脱细胞动脉导管作为移植支架进行NSC移植治疗或其他处理。分别在第2、4、6、8周对各组大鼠进行面神经麻痹评分;术后8周利用尼氏染色观察各组大鼠面神经核团内的神经元情况;术后8周利用透射电子显微镜观察再生神经的恢复情况。结果取第3代神经球,用激光共聚焦电子显微镜检测到NSC特异性标记蛋白Nestin阳性表现。获得无细胞成分的脱细胞动脉导管。除A组外,术后第2、4、6、8周E组大鼠面神经恢复情况最佳,D组次之。术后8周,除A组(29.83个±2.64个)外,E组神经元数量(27.83个±1.47个)最多,其次为D组(24.17个±0.98个),差异都有统计学意义(P<0.05)。各组大鼠面神经再生后通过透射电子显微镜观察,A组神经髓鞘的厚度与成熟度最好,神经外膜完整,束膜清晰,轴突横切面近圆形;其次为E组,细胞器丰富,结构清晰,形态规则,但未达到正常,且E组面神经恢复效果优于D组。结论Prdx6可以促进NSC对面神经损伤的修复效果。     

大鼠应激性胃粘膜损伤及修复中肥大细胞的异质性表现

目的：观察大鼠胃壁两型肥大细胞在应激性胃粘膜损伤及修复中的形态变化。方法：运用光镜和电镜技术观察正常、应激、修复多个不同时相组大鼠腺胃部的肥大细胞。结果：粘膜损伤及修复过程中粘膜肥大细胞(MMC)和结缔组织肥大细胞(CTMC)的数量无显著改变。在胃粘膜应激损伤过程中,MMC和CTMC脱颗粒率均明显增高,其中MMC的反应更为灵敏。在胃粘膜修复过程中MMC的脱颗粒率仍明显增高。MMC与嗜酸性粒细胞、神经纤维等关系密切。结论：MMC和CTMC可能均参与了应激性胃粘膜损伤及修复过程,MMC的反应较CTMC更为灵敏,提示两型肥大细胞在功能上呈异质性表现,在此过程中MMC的作用可能更为重要。     

局部注射复方倍他米松联合类人胶原蛋白瘢痕修复硅凝胶治疗增生性瘢痕的疗效评价

目的 评价局部注射复方倍他米松注射液联合类人胶原蛋白瘢痕修复硅凝胶治疗增生性瘢痕的疗效.方法 将2018年12月至2020年10月沈阳积水潭医院收治的70例增生性瘢痕患者按随机数字表法分为实验组和对照组,每组35例.实验组采取局部注射复方倍他米松注射液+外涂类人胶原蛋白瘢痕修复凝胶治疗,将复方倍他米松注射液(≤40 m...     

The linear-ordered collagen scaffold-BDNF complex significantly promotes functional recovery after completely transected spinal cord injury in canine

Spinal cord injury (SCI) is still a worldwide clinical challenge for which there is no viable therapeutic method. We focused on developing combinatorial methods targeting the complex pathological process of SCI. In this study, we implanted linear-ordered collagen scaffold (LOCS) fibers with collagen binding brain-derived neurotrophic factor (BDNF) by tagging a collagen-binding domain (CBD) (LOCS + CBD-BDNF) in completely transected canine SCI with multisystem rehabilitation to validate its potential therapeutic effect through a long-term (38 weeks) observation. We found that LOCS + CBD-BDNF implants strikingly promoted locomotion and functional sensory recovery, with some dogs standing unassisted and transiently moving. Further histological analysis showed that administration of LOCS + CBD-BDNF reduced lesion volume, decreased collagen deposits, promoted axon regeneration and improved myelination, leading to functional recovery. Collectively, LOCS + CBD-BDNF showed striking therapeutic effect on completely transected canine SCI model and it is the first time to report such breakthrough in the war with SCI. Undoubtedly, it is a potentially promising therapeutic method for SCI paralysis or other movement disorders caused by neurological diseases in the future.     

胶原/羟基磷灰石复合材料的制备及用于骨缺损修复的研究现状

通过模拟天然骨的结构,制备胶原/羟基磷灰石复合材料,与天然骨具有相似的组成、结构和性能,并具有良好的生物活性和生物降解性。本文就胶原/羟基磷灰石复合材料的制备方法、仿生形成机制、表征手段及骨缺损修复的应用等进行综述,并展望其未来发展方向。