L. Buttafoco

Preparation and evaluation of molecularly-defined collagen-elastin-glycosaminoglycan scaffolds for tissue engineering.

Extracellular matrix components are valuable building blocks for the preparation of biomaterials involved in tissue engineering, especially if their biological, chemical and physical characteristics can be controlled. In this study, isolated type I collagen fibrils, elastin fibres and chondroitin sulphate (CS) were used for the preparation of molecularly-defined collagen-elastin-glycosaminoglycan scaffolds. A total of 12 different scaffolds were prepared with four different ratios of collagen and elastin (1:9, 1:1, 9:1 and 1:0), with and without chemical crosslinking, and with and without CS. Collagen was essential to fabricate coherent, porous scaffolds. Electron microscopy showed that collagen and elastin physically interacted with each other and that elastin fibres were enveloped by collagen. By carbodiimide-crosslinking, amine groups were coupled to carboxylic groups and CS could be incorporated. More CS could be bound to collagen scaffolds (10%) than to collagen-elastin scaffolds (2.4-8.5% depending on the ratio). The attachment of CS increased the water-binding capacity to up to 65%. Scaffolds with a higher collagen content had a higher tensile strength whereas addition of elastin increased elasticity. Scaffolds were cytocompatible as was established using human myoblast and fibroblast culture systems. It is concluded that molecularly-defined composite scaffolds can be composed from individual, purified, extracellular matrix components. Data are important in the design and application of tailor-made biomaterials for tissue engineering.

Tissue engineering of blood vessels: characterization of smooth-muscle cells for culturing on collagen-and-elastin-based scaffolds.

Tissue engineering offers the opportunity to develop vascular scaffolds that mimic the morphology of natural arteries. We have developed a porous three‐dimensional scaffold consisting of fibres of collagen and elastin interspersed together. Scaffolds were obtained by freeze‐drying a suspension of insoluble type I collagen and insoluble elastin. In order to improve the stability of the obtained matrices, they were cross‐linked by two different methods. A water‐soluble carbodi‐imide, alone or in combination with a diamine, was used for this purpose: zero‐ or non‐zero‐length cross‐links were obtained. The occurrence of cross‐linking was verified by monitoring the thermal behaviour and the free‐amino‐group contents of the scaffolds before and after cross‐linking. Smooth‐muscle cells (SMCs) were cultured for different periods of time and their ability to grow and proliferate was investigated. SMCs were isolated from human umbilical and saphenous veins, and the purity of the cultures obtained was verified by staining with a specific monoclonal antibody (mAb). Cultured cells were also identified by mAbs against muscle actin and vimentin. After 14 days, a confluent layer of SMCs was obtained on non‐cross‐linked scaffolds. As for the cross‐linked samples, no differences in cell attachment and proliferation were observed between scaffolds cross‐linked using the two different methods. Cells cultured on the scaffolds were identified with an anti‐(α‐smooth‐muscle actin) mAb. The orientation of SMCs resembled that of the fibres of collagen and elastin. In this way, it may be possible to develop tubular porous scaffolds resembling the morphological characteristics of native blood vessels.

The use of an oriented, collagen-based matrix for nerve regeneration in the central nervous system

Since surface properties of hydroxyapatite (HA) play an important role in its performance, surface modification of HA has gained much attention from researchers. Silane coupling agents have been the focus of the research. In this study, an effective surface modification method was developed using hexamethylene diisocyanate as a coupling agent. Polyethylene glycol (Mw=1500) was successfully coupled to the surface of nano-size apatite particles (nano-apatite). Various methods were used to characterize the surface-modified nano-apatite. Infra-red spectra confirmed the existence of a layer of polymer with both urethane and ether linkage on the surface of nano-apatite. The amount of grafted polymer as determined by total organic carbon analysis (TOC) and thermal gravimetric analysis (TGA) was about 20% in weight. Solid 1H MAS NMR spectra indicated that the amount of hydroxyl groups of nano-apatite was decreased by 7.7% after surface grafting reaction. It is concluded that the surface hydroxyl groups of nano-apatite have the reactivity towards isocyanate groups.