Experimental Plastic Surgery, Clinic for Plastic and Hand Surgery, Klinikum Rechts der Isar, Technische Universität München, D-81675 Munich, Germany.
J Control Release. 2012 Aug 10;161(3):852-60. doi: 10.1016/j.jconrel.2012.04.048. Epub 2012 May 22.
While chronically ischaemic tissues are continuously exposed to hypoxia, the primary angiogenic stimulus, they fail to appropriately respond to it, as hypoxia-regulated angiogenic factor production gradually undergoes down-regulation, thus hindering adaptive angiogenesis. We have previously reported on two strategies for delivering on demand hypoxia-induced signalling (HIS) in vivo, namely, implanting living or non-viable hypoxic cell-matrix depots that actively produce factors or act as carriers of factors trapped within the matrix during in vitro pre-conditioning, respectively. This study aims to improve this approach through the development of a novel, injectable system for delivering cell-free matrix HIS-carriers. 3D spiral collagen constructs, comprising an inner cellular and outer acellular compartment, were cultured under hypoxia (5% O₂). Cell-produced angiogenic factors (e.g. VEGF, FGF, PLGF, IL-8) were trapped within the nano-porous matrix of the acellular compartment as they radially diffused through it. The acellular matrix was mechanically fragmented into micro-fractions and added into a low temperature (5 °C) thermo-responsive type I collagen solution, which underwent a collagen concentration-dependent solution-to-gel phase transition at 37 °C. Levels of VEGF and IL-8, delivered from matrix fractions into media by diffusion through collagen sol-gel, were up-regulated by day 4 of hypoxic culture, peaked at day 8, and gradually declined towards the baseline by day 20, while FGF levels were stable over this period. Factors captured within matrix fractions were bioactive after 3 months freeze storage, as shown by their ability to induce tubule formation in an in vitro angiogenesis assay. This system provides a minimally invasive, and repeatable, method for localised delivery of time-specific, cell-free HIS factor mixtures, as a tool for physiological induction of spatio-temporally controlled angiogenesis.
虽然慢性缺血组织持续暴露于缺氧环境中，这是主要的血管生成刺激因素，但它们无法对此做出适当反应，因为缺氧调节的血管生成因子的产生逐渐受到下调，从而阻碍了适应性血管生成。我们之前报道了两种在体内按需传递缺氧诱导信号（HIS）的策略，即分别植入活的或非存活的缺氧细胞-基质储存库，这些储存库在体外预处理过程中主动产生因子或作为基质内被困因子的载体，或者植入活的或非存活的缺氧细胞-基质储存库，这些储存库在体外预处理过程中主动产生因子或作为基质内被困因子的载体。本研究旨在通过开发一种新的、可注射的细胞外基质 HIS 载体传递系统来改进这种方法。3D 螺旋胶原构建体由内细胞和外细胞外区室组成，在缺氧（5% O₂）条件下培养。细胞产生的血管生成因子（如 VEGF、FGF、PLGF、IL-8）在它们通过细胞外区室的径向扩散过程中被困在纳米多孔基质中。细胞外基质被机械地破碎成微片段，并添加到低温（5°C）热响应型 I 型胶原溶液中，该溶液在 37°C 下经历胶原浓度依赖性的溶胶-凝胶相转变。通过胶原溶胶-凝胶扩散从基质片段递送到培养基中的 VEGF 和 IL-8 水平在缺氧培养的第 4 天升高，在第 8 天达到峰值，然后在第 20 天逐渐降至基线，而 FGF 水平在此期间保持稳定。在 3 个月的冷冻储存后，从基质片段中捕获的因子仍然具有生物活性，这表现为它们能够在体外血管生成实验中诱导管腔形成。该系统提供了一种微创且可重复的局部递送电镜特异性、细胞外 HIS 因子混合物的方法，可作为生理诱导时空可控血管生成的工具。
