Department of Biomedical Engineering and Center for Materials Innovation, Washington University, St Louis, MO 63130, USA.
Biomaterials. 2010 Nov;31(33):8642-50. doi: 10.1016/j.biomaterials.2010.07.085. Epub 2010 Aug 16.
A critical element in the formation of scaffolds for tissue engineering is the introduction of concentration gradients of bioactive molecules. We explored the use of poly(ethylene glycol) (PEG) microspheres fabricated via a thermally induced phase separation to facilitate the creation of gradients in scaffolds. PEG microspheres were produced with different densities (buoyancies) and centrifuged to develop microsphere gradients. We previously found that the time to gelation following phase separation controlled the size of microspheres in the de-swollen state, while crosslink density affected swelling following buffer exchange into PBS. The principle factors used here to control microsphere densities were the temperature at which the PEG solutions were reacted following phase separation in aqueous sodium sulfate solutions and the length of the incubation period above the 'cloud point'. Using different temperatures and incubation times, microspheres were formed that self-assembled into gradients upon centrifugation. The gradients were produced with sharp interfaces or gradual transitions, with up to 5 tiers of different microsphere types. For proof-of-concept, concentration gradients of covalently immobilized proteins were also assembled. PEG microspheres containing heparin were also fabricated. PEG-heparin microspheres were incubated with fluorescently labeled protamine and used to form gradient scaffolds. The ability to form gradients in microspheres may prove to be useful to achieve better control over the kinetics of protein release from scaffolds or to generate gradients of immobilized growth factors.
在组织工程支架的形成中，一个关键因素是引入生物活性分子的浓度梯度。我们探索了使用聚乙二醇（PEG）微球通过热诱导相分离来促进支架中梯度的形成。PEG 微球具有不同的密度（浮力），并通过离心来开发微球梯度。我们之前发现，相分离后凝胶化的时间控制着去溶胀状态下微球的大小，而交联密度影响缓冲液交换到 PBS 后的溶胀。这里用于控制微球密度的主要因素是 PEG 溶液在硫酸钠盐溶液中相分离后反应的温度，以及高于“浊点”的孵育期的长度。使用不同的温度和孵育时间，微球可以在离心时自组装成梯度。梯度可以形成锐利的界面或逐渐过渡，最多可以有 5 层不同类型的微球。为了验证概念，还组装了共价固定化蛋白质的浓度梯度。还制备了含有肝素的 PEG 微球。用荧光标记的鱼精蛋白孵育含有肝素的 PEG 微球，并用于形成梯度支架。在微球中形成梯度的能力可能有助于更好地控制支架中蛋白质释放的动力学，或产生固定化生长因子的梯度。
