Fabrication And Property Evaluation Of Emulsion Electrospun Poly (Lactic-acid-co-ε-caprolactone) Tissue Engineering Scaffolds

Electrospinning is one of the most convenient technologies for nanofiber preparation. It is based on the principle of electrostatic force to form ultrafine nanoscale that have high surface to volume ratio, and may be tailored to formporous fibrous scaffolds. Owing to their wide scope of material ingredients and structural Versatility, electrospun nanofibers have several biomedical applications in the field of tissue engineering. Essentially, nanofibers can mimic natural extracellular matrix (ECM) and provide necessary micro-environmentsfor cell adhesion, proliferation and differentiation. Nanofiber scaffolds have also shown excellent scope in drug delivery. However, the conventional electrospinning method has several limitations to a successful drug delivery. For instance, most of the electrospinning methods fabricate drug-loaded nanofibers by blending drugs and spinning solutions. This often leads to two main challenging situations:(1) non-uniform drug solution due to mismatch of solvent polarities leading to uncontrolled drug release, and,(2) inactivation or aggregation of drug due to blending with organic solvents. Application of inactivated drugs may not only fail of a positive therapeutic effect, but also may trigger serious foreign body immune response. Emulsion electrospinning technique features use of an emulsifying agent that prevents drug inactivation by overcoming polarity differences of the solvents.The work presented here uses emulsion electrospinning to fabricate tissue engineering scaffolds. This work is divided in the following three parts:(3) Establishment of emulsion electrospinning systemModel drug bovine serum albumin (BSA) was dissolved in an aqueous solution, and organic polymer poly(lactic-acid-co-ε-caprolactone)(PLCL) was dissolved in organic chloroform solution. The two phase solutions were mixed and stirred into uniform emulsion for electrospinning process. Nanofibers with fluorescently tagged ingredients were observed by confocal laser scanning microscopy (CLSM), that confirmed the resultant core-shell structure. The hydrophilicity was evaluated by sessile drop water contact angle test. The contact angles of PLCL and PLCL_BSA scaffolds were135.10°and89.30°respectively, indicating significant improvement inHydrophilicity as a result of emulsion electrospinning. Hydrophilicity was shown beneficial for theattachment and behavior of mesenchymal stem cells (MSCs). The in vitro release study of BSA showed that only47.71%of BSA was released for up to28days, the abovementioned burst release phenomenon was alleviated effectively, and a long term release effect was obtained. Cell proliferation analysis showed that the proliferation on PLCL_BSA scaffolds was41.8%、85.0%and49.7%higher than that on PLCL scaffold at7,14and21days respectively indicating biocompatibility of emulsion electrospun scaffolds with MSCs. Thus, we show that emulsion electrospun scaffolds hold a higher potential to support cell attachment and proliferation than conventional electrospun scaffolds.(2) Fabrication and characterization of single factor (growth factor) encapsulated emulsion electrospun nanofibrous scaffoldsFor cardiovascular applications, Vascular Endothelial Growth Factor (VEGF) was used in the core of nanofiber. The core-shell nanofibers were fabricated by emulsion electrospinning and its core-shell structure, hydrophilicity, tensile property and biological properties were characterized. The biocompatibility of VEGF-encapsulated nanofibrous scaffolds were demonstrated by cell proliferation MTS assay. The VEGF release assay as studied using (1) dextran as VEGF protective agent, and (2) BSA as VEGF protective agent. While both agents promoted VEGF release but alleviated the burst release. It was observed that protective agents dextran and BSA had significant difference in their effect tin the initial24hours’ release with a release amount was1.0%and9.6%respectively. However, this difference of release amount was not observed for the following640hours recording11.6%and11.7%respectively for dextran and BSA. Cell proliferation using MTS assay indicated that VEGF-encapsulated scaffolds significantly improved the proliferation compared to the PLCL scaffolds. The cell proliferation on PLCL-VEGF-BSA was32.3%and 49.9%higher than that on PLCL scaffolds at10and20days respectively.The cell proliferation on PLCL-VEGF-DEX was14.6%and39.8%higher than that on PLCL scaffolds at10and20days respectively. These results indicated only the release but also functional activity of encapsulatedVEFG.Natural polymers are known to have superior biocompatibility with cells. Thus, improve the biocompatibility of nanofibers and enhance the protection VEGF in the core, natural polymer gelatin was used in the core solution along with VEGF. We analyzed the cardiogenic differentiationpotential of mesenchymal stem cells (MSCs) on these scaffolds. The cardiogenic differentiation of MSCs was triggered by5-azacytidine (5-AZA) treatment. Results show that the cell proliferation on PLCL/GV was35.5%,61.1%and73.4%higher than that on PLCL scaffolds at10,15and20days respectively. According to cell proliferation results,there was no significant difference between the cell proliferations before and after5-AZA treatment. The morphology of cells on fibrous scaffolds were evaluated by5-Chloromethylfluorescein Diacetate (CMFDA) staining. Results depicted increase in cellular size that appeared to be polygonal shapein process of contacting neighboring cells. Number and morphologies of cells growing on PLCL/GV scaffolds differed significantly from PLCL scaffolds. The cells on PLCL/GV scaffolds showed more cardiogenic phenotype such as larger size, polygonal shape and ability to form intercellular network. The undifferentiated MSCs have long shape, and they often grow in parallelpatterns in vitro, similar to fibroblasts. The immunocytochemistry resultsindicated that the cells on PLCL/GV scaffolds expressed more of alpha-actinnin and cardiac-specific myosin heavy chain (MHC) than PLCL indicating potential of PLCL/GV as an MSC differentiation platform.(3) Fabrication and Characterization of dual factor encapsulated emulsion electrospun nanofibersThe emulsion electrospun nanofibers of PLCL with hydroxyapatite (HA)encapsulated in the shell and while, laminnin, was encapsulated in the core (PLCL/HA/Lam) were fabricated using the unique core-shell emulsion electrospun procedure. Human osteoblasts were cultured on the scaffolds for21days in osteogenic medium. MTS assay shows that the cell proliferation on electrospun PLCL/HA/Lam, PLCL/HA and PLCL/Lam scaffolds was better than that on TCP at7days. There onwards, the cell proliferation on PLCL/HA/Lam was23.3%、12.0%and10.4%higher than those on TCP, PLCL/HA and PLCL/Lam scaffolds respectively.The alkaline phosphatase activity (ALP) of cells was significantly higher on PLCL/HA/Lam than PLCL/HA and PLCL/Lam scaffolds at days14and days21(p<0.05),which may be caused by the synergistic function of HA and laminin. Energy dispersive x-ray (EDX) data show the calcium content of TCP, PLCL/HA, PLCL/Lam and PLCL/HA/Lam scaffolds was0%,0.05%,0.09%and0.51%respectively, which was consistent with the results of alizarin red staining (ARS).The results of cell proliferation, bone protein expression, ALP, alizarin red s staining and mineralizationstrongly support that dual factor encapsulated nanofibers may significantly improve the proliferation, maturation and biological of osteoblasts making them potential for use in bone tissue engineering.