Yannas I V, Burke J F, Gordon P L, Huang C, Rubenstein R H
J Biomed Mater Res. 1980 Mar;14(2):107-32. doi: 10.1002/jbm.820140203.
Detailed methodology is described for the reproducible preparation of collagen--glycosaminoglycan (GAG) membranes with known chemical composition. These membranes have been used to cover satisfactorily large experimental full-thickness skin wounds in guinea pigs over the past few years. Such membranes have effectively protected these wounds from infection and fluid loss for over 25 days without rejection and without requiring change or other invasive manipulation. When appropriately designed for the purpose, the membranes have also strongly retarded wound contraction and have become replaced by newly synthesized, stable connective tissue. In our work, purified, fully native collagen from two mammalian sources is precipitated from acid dispersion by addition of chondroitin 6-sulfate. The relative amount of GAG in the coprecipitate varies with the amount of GAG added and with the pH. Since coprecipitated GAG is generally eluted from collagen fibers by physiological fluids, control of the chemical composition of membranes is arrived at by crosslinking the collagen--GAG ionic complex with glutaraldehyde, or, alternately, by use of high-temperature vacuum dehydration. Appropriate use of the crosslinking treatment allows separate study of changes in membrane composition due to elution of GAG by extracellular fluid in animal studies from changes in composition due to enzymatic degradation of the grafted or implanted membrane in these studies. Exhaustive in vitro elution studies extending up to 20 days showed that these crosslinking treatments insolubilize in an apparently permanent manner a fraction of the ionically complexed GAG, although it could not be directly confirmed that glutaraldehyde treatment covalently crosslinks GAG to collagen. By contrast, the available evidence suggests strongly that high-temperature vacuum dehydration leads to formation of chemical bonds between collagen and GAG. Procedures are described for control of insolubilized and "free" GAG in these membranes as well as for control of the molecular weight between crosslinks (Mc). The insolubilized GAG can be controlled in the range 0.5--10 wt. % while "free" GAG can be independently controlled up to at least 25 wt. %; Mc can be controlled in the range 2500--40,000. Studies by infrared spectroscopy have shown that treatment of collagen--GAG membranes by glutaraldehyde or under high-temperature vacuum does not alter the configuration of the collagen triple helix in the membranes. Neither do these treatments modify the native banding pattern of collagen as viewed by electron microscopy. Collagen--GAG membranes appear to be useful as chemically well-characterized, solid macromolecular probes of biomaterial--tissue interactions.
本文描述了一种可重复制备具有已知化学成分的胶原蛋白 - 糖胺聚糖（GAG）膜的详细方法。在过去几年中，这些膜已被用于令人满意地覆盖豚鼠的大面积实验性全层皮肤伤口。此类膜已有效地保护这些伤口在超过25天的时间里免受感染和液体流失，且无排斥反应，也无需更换或进行其他侵入性操作。当为此目的进行适当设计时，这些膜还能强烈抑制伤口收缩，并被新合成的稳定结缔组织所替代。在我们的工作中，通过添加硫酸软骨素6 - 硫酸盐，从两种哺乳动物来源的纯化、完全天然的胶原蛋白的酸性分散液中沉淀出胶原蛋白。共沉淀物中GAG的相对含量随添加的GAG量和pH值而变化。由于共沉淀的GAG通常会被生理流体从胶原纤维上洗脱下来，因此通过用戊二醛交联胶原蛋白 - GAG离子复合物，或者交替使用高温真空脱水来控制膜的化学成分。适当使用交联处理可以在动物研究中区分由于细胞外液洗脱GAG导致的膜成分变化与这些研究中由于移植或植入膜的酶降解导致的成分变化。长达20天的详尽体外洗脱研究表明，这些交联处理以一种明显永久的方式使一部分离子复合的GAG不溶解，尽管无法直接证实戊二醛处理使GAG与胶原蛋白共价交联。相比之下，现有证据强烈表明高温真空脱水导致胶原蛋白和GAG之间形成化学键。文中描述了控制这些膜中不溶性和“游离”GAG以及控制交联点之间分子量（Mc）的程序。不溶性GAG可以控制在0.5 - 10 wt.%的范围内，而“游离”GAG可以独立控制至至少25 wt.%；Mc可以控制在2500 - 40,000的范围内。红外光谱研究表明，用戊二醛或在高温真空下处理胶原蛋白 - GAG膜不会改变膜中胶原三螺旋的构型。电子显微镜观察也表明这些处理不会改变胶原蛋白的天然条纹图案。胶原蛋白 - GAG膜似乎可用作生物材料 - 组织相互作用的化学性质明确的固体大分子探针。
