First steps towards tissue engineering of small‐diameter blood vessels: Preparation of flat scaffolds of collagen and elastin by means of freeze drying

Abstract

Porous scaffolds composed of collagen or collagen and elastin were prepared by freeze drying at temperatures between -18 and -196 degrees C. All scaffolds had a porosity of 90-98% and a homogeneous distribution of pores. Freeze drying at -18 degrees C afforded collagen and collagen/elastin matrices with average pore sizes of 340 and 130 mum, respectively. After 20 successive cycles up to 10% of strain, collagen/elastin dense films had a total degree of strain recovery of 70% +/- 5%, which was higher than that of collagen films (42% +/- 6%). Crosslinking of collagen/elastin matrices either in water or ethanol/water (40% v/v) was carried out using a carbodiimide (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, EDC) in combination with a succinimide (N-hydroxysuccinimide, NHS) in the presence or absence of a diamine (J230) or by reaction with butanediol diglycidylether (BDGE), followed by EDC/NHS. Crosslinking with EDC/NHS or EDC/NHS/J230 resulted in matrices with increased stiffness as compared to noncrosslinked matrices, whereas sequential crosslinking with the diglycidylether and EDC/NHS yielded very brittle scaffolds. Ethanol/water was the preferred solvent in the crosslinking process because of its ability to preserve the open porous structure during crosslinking. Smooth muscle cells were seeded on the (crosslinked) scaffolds and could be expanded during 14 days of culturing.

Macrophage-mediated degradation of crosslinked collagen scaffolds

“…N-Hydroxy-succinimide (NHS) was added in an EDC:NHS ratio of 3:1. Excess EDC was rinsed from the matrix using 0.1 M Na 2 HPO 4 (pH 9.1) twice for 1 h, followed by washing with distilled water four times, each time for 30 min [32,33]. Samples were frozen again, then freezedried as described.…”

ECM-based triple layered scaffolds for vascular tissue engineering

“…ECM harvested from the small intestine, skin, liver, pancreas, and urinary bladder has been successfully used to promote the regeneration of tissues and organs in both animal models and human (14)(15)(16)(17)(18). Several types of mono-or multilayer vascular grafts containing collagen and elastin have been obtained using different techniques (9,(19)(20)(21)(22)(23). Nevertheless, only few studies have dealt with three-layered small-diameter blood vessel substitutes (24)(25)(26)(27).…”

Quantitative architectural description of tissue engineering scaffolds

“…This tends to give slightly lower values of porosity compared with density measurements, due to the limit of resolution of image data. 41,42 A line intercept method, in which the number of pore/solid interfaces crossing a line of known length is counted, is often used to measure pore size. 6,43 It must be noted that this provides a measure only of the pore cross-sections seen in a two-dimensional (2D) image.…”