Construction And Modification Of PLLA Nano-fibrous Scaffolds And Their Nanomechanical Properties

Scaffolds used in tissue engineering play a pivotal role in regenerative medicine, whichaims to regenerate and replace lost/dysfunctional tissues or organs. Scaffolds provide three-dimensional templates and synthetic extracellular matrix (ECM) environments for tissueregeneration. To mimicking the architecture and excellent surface properties of natural ECMcomponent such as the nano-fibrous structure of collagen is an important area of tissueengineering scaffolds.In this research, Poly (L-lactic acid)(PLLA) nano-fibrous scaffolds were preparedthrough a thermally induced phase separation (TIPS) process. The structural evolution andnanoscale properties of PLLA nanofiber formation during the early quenching period in aTIPS process have been investigated. The morphology, phase transition, crystallizationbehavior, chemical structure, surface property, hydrophobicity, variation of chainconformation, adhesion force and elastic modulus were studied by using SEM, WAXD,ATR-FTIR, DSC, XPS, contact angle measurement, AFM and force spectroscopymeasurements. The introduction of citric acid and oxygen plasma treatment are used for thesurface modification of PLLA scaffolds and the influence on cell behavior has been studied.The TIPS process include four basic steps: PLLA dissolved, gelation, solvent exchangeand freeze dried in vacuum.The observation suggested a temperature dependent phasebehavior during the early quenching period. The initial phase separation resulted in anamorphous gel with the condensation of nanoparticles, followed by the nucleation of PLLAcrystals and formed a ribbon-like structures. After gelation for enough time, an nano-fibousnetwork started to form and the nanofibers tended to be more uniform with the increase of thegelation time. The α-form crystal appeared after quenching for1min, corresponding to a geltemperature around15°C. A limited disorder α′-form crystal prevailed at lower geltemperature and the content of α′-phase in the mixture was increasing gradually with thegelation time. Along with the increase in the degree of crystallinity, structural transformationof the polymer toward a more ordered and compact state proceeded with the extending of thegelation time. During the formation of PLLA nanofibers, the possible surface segregation ofthe methyl groups which may have certain effect on the increase of water contact angle andsurface hydrophobicity.The biodegradability and biological property of the PLLA scaffolds with differentgelation time were studied by hydrolytic degradation and cell culture experiments. Theevolution of architecture, crystallinity, chemical structure, surface property and the polymer chain packing mode, during the formation of PLLA nanofibers, has a direct functionalconsequence in the hydrophobicity, biodegradability as well as biological property. Thecrystallization process and the increasing hydrophobicity negatively influenced the hydrolyticdegradation. The PLLA nano-fibrous scaffolds with longer gelation time provided a suitablesurface for cell attachment and proliferation.AFM (force spectroscopy, nanoindentation, nanolithography) and TEM are used toinvestigate the formation mechanism and nanomechanical properties (adhesion force,elasticity) of PLLA nanofibers. The TIPS process was believed to occur through spinodalliquid-liquid phase separation into a polymer-rich phase and a polymer-poor phase. Grain-likecrystals were formed by consequential crystallization of the polymer-rich phase. Thesecrystals arranged regularly to form the thinner fibrils(70-100nm) and then assembled laterallyinto larger nanofibers. The structural evolution of PLLA chains conformation and the chainpacking has a direct functional consequence in the nanoscale mechanical property. With moreordered and compact structure, PLLA nanofiber have higher adhesion force and elasticmodulus. The AFM cantilever tip is then used to draw a scratch on individual PLLA nanofiber.The deformation was produced by both compressional stress and shear stress. The structurebecomes more compact and ordered which increases the adhesion force and elasticity.In order to improve the surface hydrophobicity and cell affinity of PLLA nano-fibrousscaffolds, the introduction of citric acid and oxygen plasma treatment are used for the surfacemodification of PLLA nano-fibrous scaffolds prepared via TIPS process. The evolution ofarchitecture, physical and chemical structure, surface properties after surface modificationhave been characterized. Cell culture, cell seeding, cell viability, adhesion, proliferation,osteogenesis differentiation and RT-PCR are used to characterized the cytocompatibility andcell biological properties of PLLA nano-fibrous scaffolds after surface modification. Aftersurface modification, the introduction of a strongly polar group (–COOH) on PLLA surfacedecreased the contact angle and improved its hydrophilicity to a most suitable range for cellculture (40-80°). These scaffolds have a better cell affinity which were more beneficial forcell adhesion and spreading well with many small processes interacting with PLLA nanofibers.The cell proliferation rate and cell activity also have significantly increases. Alkalinephosphatase and calcium nodules were staining with BCIP/NBT and Alizarin red. All thesescaffolds showed a positive result. However, the alkaline phosphatase activity of the cells onthe scaffolds after surface modification is much better. The expression of osteogenic markers(ALP、COL、OCN and Cbfa-1/Runx-2) were detected by RT-PCR and the scaffolds aftersurface modification have better effect on improving the expression of osteogenic gene of mBMSCs. In conclude, all these PLLA scaffolds were benefit for the osteogenicdifferentiation of mBMSCs and it was much better on the scaffolds after surface modification.