Aminolysis Modification And Cytocompatibility Of Ester-containing Polymeric Biomaterials

The purpose of this study is to fabricate the cytocompatible surface or interface on polymeric materials via developing novel modification methods. Firstly, amino groups were covalently introduced onto polycaprolactone (PCL) surface by the aminolyzing reaction between 1,6-hexanediamine and the ester groups of PCL. The occurrence of the aminolysis and the introduction of free NH₂ groups were verified qualitatively by fluorescence spectroscopy where rhodamine B isothiocyanate was employed to label NH₂ groups, and quantitatively by absorbance spectroscopy where ninhydrin was used to react with NH₂ to generate a blue product. Due to the presence of deep pores on PCL membrane, the aminolysis reaction could penetrate as deep as 30μm to yield NH2 density as high as 2x10^-7 mol/cm² .By using the NH₂ groups as active sites, biocompatible macromolecules such as gelatin, chitosan or collagen was further immobilized on the aminolyzed PCL membrane via a cross-linking agent, glutaraldehyde. X-ray photoelectron spectroscopy (XPS) and surface wettability measurements confirmed the coupling of the biomacromolecules. The endothelial cell culture proved that the cytocompatibility of the aminolyzed PCL was improved slightly regardless of the NH2 amount on the surface. After immobilization of the biomacromolecules, however, the cell attachment and proliferation ratios were improved obviously, and the cells showed a similar morphology to those on tissue culture polystyrene. Measurement of von Willebrand factor (vWF) secreted by these endothelial cells (ECs) verified the endothelial function.Other biomaterials containing ester groups like polylactide and polyurethane can also be modified via aminolysis between amino groups from diamine and ester groups from polymer chain to improve their cytocompatibility of human endothelial cells. The improvement of EC compatibility is influenced somewhat by the substrate bulk properties because amino groups introduced by aminolyzing reaction cannot cover the entire substratum. Among the three polymers we studied, the EC compatibility of aminolyzed poly(L-lactic acid) is the best. After further immobilization of biomolecules like gelatin or collagen on the aminolyzed substrata, the cytocompatibility is improved greatly regardless of the bulk attributes. The measurements of vWF and 6-keto-PGF_1α, both which are synthesized and secreted by human blood vessel endothelial cells, verified the endothelium function.The introduction of the amino groups may neutralize the acid generated during the polyester scaffold degradation and reduce the inflammation around the implanting scaffold. Moreover, it also provides the possibility to modify polymer surface in a simpler manner, for example, layer-by-layer (LBL) assembly of charged species, because these aminolyzed polyesters can be used as polycationic substrata. This technique is more practical and important for scaffolds with irregular shape and inner structure, where traditional methods are generally unavailable. To verify the feasibility of this technology, a simple polyanion, poly(styrene sulfonate, sodium salt) (PSS), and biocompatible chitosan were chosen to deposit onto aminolyzed poly(L-lactic acid) (PLLA) membrane surface in a layer-by-layer assembly manner. The layer-by-layer deposition process of PSS and chitosan was monitored by UV-Vis absorbance spectroscopy, energy transfer by fluorescence spectroscopy after the polyelectrolytes were labeled by fluorescein (FITC) and rhodamine, respectively, and advancing contact angle measurements. The layer thickness increased with the increase of the deposited layers. The existed chitosan obviously improved the cytocompatibility of PLLA to human endothelial cells. The cell attachment, activity, proliferation and endothelial function (vWF secretion) on the PLLA membranes assembled with 3 or 5 bilayers of PSS/chitosan (with chitosan as the outermost layer) were better than those with 1 bilayer of PSS/chitosan or the control PLLA. These cells also showed morphology of an elongated shape with abundant cytoplasm and a confluent cell layer was reached after cultured for 4d.In order to avoid the bad influence of substratum or inner molecules on the material cytocompatibility, more biocompatible polyelectrolytes like chondroitin sulfate and collagen type I were chosen to deposit onto the aminolyzed PLLA membrane. Human endothelial cells cultured in vitro demonstrated that only one bilayer of chondroitin sulfate and collagen was enough to improve the cytocompatibility of PLLA.Biodegradable PCL was also modified through the combination of photo-oxidation and methacrylic acid (MAA) grafting copolymerization initiated under UV light. The covalent immobilization of gelatin on PMAA grafted PCL surface was consequently performed by using condensing agent, l-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride. The occurrence of grafting copolymerization of PMAA and further immobilization of gelatin was confirmed by ATR-FTIR and X-ray photoelectron spectroscopy (XPS) characterizations. The existence of the carboxyl groups grafted on PCL surface was verified quantitatively by absorbance spectroscopy where rhodamine 6G was employed to react with carboxyl groups to generate an absorbance at 512nm. The endothelial cell culture proved that the cytocompatibility of modified PCL membrane is related to the surface hydrophilicity. Onlymoderate hydrophilicity can support and accelerate the growth of human endothelial cells (ECs). For this system, the COOH density on PCL surface within 3^<sup>6><10"7 mol/cm2 is more favorable for the ECs growth.Using PLLA film as substratum, we modified the PLLA surface via aminolysis and further collagen immobilization, aminolysis and layer-by-layer self-assembly with chondroitin sulfate and collagen type I, and PMAA grafting and collagen immobilization using ED AC as condensing agent, respectively. The human endothelial cells cultured in vitro showed that the cell proliferation, activity and vWF and 6-keto-PGFia (the stable expression of prostacyclin) of all modified PLLA surface increased at a different extent, comparing with the control PLLA. Moreover, the cytocompatibility of collagen- immobilization PLLA is better than that of other modified PLLA. However, we did not find the big difference between these surface modification methods at the aspect of PLLA cytocompatibility to human endothelial cells.Increased cell spreading and growth to form a viable endothelial layer on luminal surface can improve the healing and integrating of the vascular graft. Using the method of aminolysis and further gelatin immobilization, PU vascular scaffold was modified to enhance cell-material interaction. SEM and confocal laser scanning microscopy (CLSM) observations have found that the gelatin-immobilized PU vascular scaffold has formed a monolayer of endothelial intima on the luminal surface after HUVECs were cultured for 6d. Therefore, the aminolysis and the following biomacromolecule immobilization is a promising way to accelerate the endothelium regeneration, which is crucial for blood vessel tissue engineering.The objective of tissue engineering is to regenerate natural tissues with some shape via the composite of biodegradable scaffold and living cells. Therefore, layer-by-layer self-assembly technique based on aminolysis, which is not restricted to substratum and can be used for coating arbitrarily-shaped objects, was used to modify PLLA porous scaffold to improve the chondrocytes compatibility. PLLA porous scaffold with good interconnectivity was fabricated using the technique of thermally induced phase separation (TIPS). Polyelectrolytes of chondroitin sulfate and collagen type I were chosen to deposit alternately on the aminolyzed PLLA scaffold. From the culture of chondrocytes digested from New Zealand rabbit ear with 3⁴ weeks old in vitro, the modified PLLA scaffold showed better cell spreading and more evenly distributed than the control scaffold. After 12d culture, the chondrocytes permeated the entire PLLA porous scaffold, and grew very well under confocal laser scanning microscopy (CLSM) observation. Therefore, a novel modification technique, i.e. layer-by-layer self-assembly, on materials with complicated shape has been developed. Itis believed to attract more and more attention in tissue engineering due to its simplicity and versatility.