Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, USA.
J Microencapsul. 2009 Sep;26(6):544-55. doi: 10.1080/02652040802500473.
Currently employed bone tissue engineered scaffolds often lack the potential for vascularization, which may be enhanced through the incorporation of and regulated release of angiogenic factors. For this reason, the objective here was to fabricate and characterize protein-loaded amino acid ester polyphosphazene (Pphos)-based scaffolds and evaluate the novel sintering method used for protein incorporation, a method which will ultimately allow for the incorporation of proangiogenic agents. To test the hypothesis, Pphos and their composite microspheres with nanocrystalline hydroxyapatite (Pphos-HAp) were fabricated via the emulsion solvent evaporation method. Next, bovine serum albumin (BSA)-containing microsphere matrices were created using a novel solvent-non-solvent approach for protein loading. The resulting protein (BSA) loaded circular porous microsphere based scaffolds were characterized for morphology, porosity, protein structure, protein distribution and subsequent protein release pattern. Scanning electron microscopy revealed porous microsphere scaffolds with a smooth surface and sufficient level of sintering, illustrated by fusion of adjacent microspheres. The porosity measured for the poly(ethyl phenylalanato:glycinato)phosphazene (PNPhGly) and poly(ethyl phenylalanato:glycinato)phosphazene-hydroxyapatite (PNPhGly-HAp) scaffolds were 23 +/- 0.11% and 18 +/- 4.02%, respectively, and within the range of trabecular bone. Circular dichroism confirmed an intact secondary protein structure for BSA following the solvent sintering method used for loading and confocal microscopy verified that FITC-BSA was successfully entrapped both between adjacent microspheres and within the surface of the microspheres while sintering. For both Pphos and their composite microsphere scaffolds, BSA was released at a steady rate over a 21 day time period, following a zero order release profile. HAp particles in the composite scaffolds served to improve the release profile pattern, underscoring the potential of HAp for growth factor delivery. Moreover, the results of this work suggest that the solvent-non-solvent technique for protein loading is an optimal one that will allow for future development of angiogenic factor-loaded Pphos matrices with the capacity to invoke neovascularization.
目前使用的骨组织工程支架往往缺乏血管生成的潜力，而通过加入和调节血管生成因子的释放可以增强这种潜力。出于这个原因，本研究的目的是制备和表征载蛋白的氨基酸酯聚膦腈（Pphos）基支架，并评估用于蛋白质掺入的新型烧结方法，这种方法最终将允许掺入促血管生成剂。为了验证假设，通过乳液溶剂蒸发法制备了 Pphos 及其与纳米晶羟基磷灰石（Pphos-HAp）的复合微球。接下来，使用新型溶剂-非溶剂方法制备了含有牛血清白蛋白（BSA）的微球基质用于蛋白质负载。所得的载蛋白（BSA）的圆形多孔微球支架进行形态学、孔隙率、蛋白结构、蛋白分布和随后的蛋白释放模式的特征分析。扫描电子显微镜显示多孔微球支架具有光滑的表面和足够的烧结水平，相邻微球的融合说明了这一点。聚（苯丙氨酸乙酯：甘氨酸乙酯）膦腈（PNPhGly）和聚（苯丙氨酸乙酯：甘氨酸乙酯）膦腈-羟基磷灰石（PNPhGly-HAp）支架的孔隙率分别为 23 +/- 0.11%和 18 +/- 4.02%，在小梁骨的范围内。圆二色性确认了 BSA 在用于负载的溶剂烧结方法之后具有完整的二级蛋白结构，共聚焦显微镜证实 FITC-BSA 成功地在相邻微球之间以及在微球的表面上被包埋，同时进行烧结。对于 Pphos 及其复合微球支架，BSA 在 21 天的时间内以零级释放曲线的形式以稳定的速率释放。复合支架中的 HAp 颗粒改善了释放曲线模式，突出了 HAp 用于生长因子传递的潜力。此外，这项工作的结果表明，用于蛋白质负载的溶剂-非溶剂技术是一种优化的技术，它将允许未来开发具有诱导新血管形成能力的载有血管生成因子的 Pphos 基质。
