Allergy Research Group, School of Molecular Medical Sciences, Queen's Medical Centre, The University of Nottingham, Nottingham NG7 2UH, UK.
Biomed Mater. 2012 Aug;7(4):045011. doi: 10.1088/1748-6041/7/4/045011. Epub 2012 Jun 1.
The amniotic membrane (AM) is considered as a natural cell culture substrate and has occasionally been exploited in regenerative medicine especially for ocular surface reconstruction and dermal wound healing applications. However, its use is limited by its relatively weak mechanical strength, difficulty during manual handling and susceptibility to proteolytic degradation in vivo. Therefore, in this study we aimed to enhance the mechanical and biological characteristics of the AM by enzymatically cross-linking it using tissue transglutaminase (TG)-a calcium-dependent enzyme capable of forming stable ε(γ-glutamyl)lysine cross-linkages. Using a biological catalyst such as TG does not only prevent denaturation during sample preparation but also minimizes the potential of residual chemical cross-linking agents compared to alternative methodologies. Human AM, sourced from elective caesarean sectioning, were treated with TG, bovine serum albumin and/or a no-treatment control. Samples were then compared in terms of their physical and (scanning electron microscopy (SEM), transparency, mechanical strength, susceptibility to proteolytic degradation) biological characteristics (in vitro cell culture, activation of dendritic cells (DC)) and their in vivo biocompatibility/angiogenic capacity (chick chorioallantoic membrane assay). TG-treated AM exhibited enhanced mechanical strength and greater resistance to proteolytic/collagenase degradation compared to the control(s). SEM imaging of the TG-treated membrane summarized a significantly closer association and greater interconnectivity of individual collagen fibres yet it had no effect on the overall transparency of the AM. In vitro cell culture demonstrated no detrimental effect of TG-treatment on the AM in terms of cell attachment, spreading, proliferation and differentiation. Moreover, an 'immune response' was not elicited based on extended in vitro culture with human-monocyte-derived DC. Interestingly, the TG-treated AM still allowed angiogenesis to occur and in some instances, demonstrated an enhancement compared to the control (n = 5). We hereby demonstrate that treating the AM with the cross-linking enzyme, TG, results in a novel biomaterial with enhanced mechanical and biological characteristics. Above all, this modified membrane demonstrates greater strength, maintains in vitro cell growth, retains optical transparency and allows angiogenesis to occur without inducing an immune response. Altogether, this study demonstrates the feasibility of TG as an alternate cross-linking treatment for the production of novel biomaterials and suggests that TG-treated AM may now be more commonly exploited as a therapeutic dressing for ocular or wound applications.
羊膜（AM）被认为是一种天然的细胞培养底物，并且偶尔在再生医学中被利用，特别是用于眼表面重建和皮肤伤口愈合应用。然而，其使用受到其相对较弱的机械强度、手动处理的难度以及在体内易受蛋白水解降解的限制。因此，在这项研究中，我们旨在通过使用组织转谷氨酰胺酶（TG）对 AM 进行酶交联来增强其机械和生物学特性，TG 是一种能够形成稳定的ε（γ-谷氨酰基）赖氨酸交联的钙依赖性酶。与替代方法相比，使用生物催化剂（如 TG）不仅可以防止样品制备过程中的变性，而且还可以最大程度地减少残留化学交联剂的潜在风险。从选择性剖腹产中获得的人 AM 用 TG、牛血清白蛋白和/或无处理对照进行处理。然后根据其物理和（扫描电子显微镜（SEM）、透明度、机械强度、对蛋白水解降解的敏感性）生物学特性（体外细胞培养、树突状细胞（DC）的激活）以及体内生物相容性/血管生成能力（鸡胚绒毛尿囊膜测定）对样品进行比较。与对照相比，TG 处理的 AM 表现出增强的机械强度和更大的抗蛋白水解/胶原酶降解能力。TG 处理膜的 SEM 成像总结了单个胶原纤维之间更紧密的关联和更大的连通性，但对 AM 的整体透明度没有影响。体外细胞培养表明，TG 处理对 AM 在细胞附着、铺展、增殖和分化方面没有不利影响。此外，基于与人类单核细胞衍生的 DC 的延长体外培养，没有引发“免疫反应”。有趣的是，TG 处理的 AM 仍允许血管生成发生，并且在某些情况下，与对照相比，显示出增强（n = 5）。我们在此证明，用交联酶 TG 处理 AM 会产生一种具有增强的机械和生物学特性的新型生物材料。最重要的是，这种改性膜具有更高的强度，保持体外细胞生长，保持光学透明度并允许血管生成发生，而不会引起免疫反应。总之，这项研究证明了 TG 作为生产新型生物材料的替代交联处理的可行性，并表明 TG 处理的 AM 现在可能更常用于眼或伤口应用的治疗敷料。
