Collagenous scaffolds supplemented with hyaluronic acid and chondroitin sulfate used for wound fibroblast and embryonic nerve cell culture

Abstract

Background. Tissue engineering is a strategy aimed at improving the regeneration of injured tissues.Objectives. The aim of the present study was to determine whether a tri-copolymer composed of crosslinked collagen, chondroitin sulfate and hyaluronic acid (Col + CS + HA) provides a better environment for fibroblast and embryonic nerve cell culture than a collagenous scaffold (Col).Material and methods. The porosity of each of the matrices was characterized with a scanning electron microscope. Fibroblasts were isolated from rat wound granulation tissue (polypropylene net implanted subcutaneously). Embryonic nerve cells were obtained from the brains of rat embryos. The cells were applied to scaffolds and then stained with bisbenzimide to calculate cell entrapment within the material. The metabolic activity of the cells cultured within the scaffolds was tested using the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.Results. The Col scaffolds had a homogenously porous structure with a pore diameter of 50 μm for 70% of pores. The pore diameter in the tri-copolymer (Col + HA + CS) ranged from 24 to 160 μm (95% of total pore volume). Four times more cells (fibroblasts and embryonic nerve cells) were trapped within the superficial part of the collagenous scaffold than that of the tri-copolymer. On the third day of culture the metabolic activity of the fibroblasts within the 2 tested scaffolds was significantly higher than in the control conditions (cell culture on a laminin-coated surface). Also, the embryonic nerve cells demonstrated increased metabolic activity in Col + CS + HA scaffolds than the Col scaffolds.Conclusions. Both fibroblasts and embryonic nerve cells could be seeded within the 2 tested scaffolds. Both the scaffolds provide good conditions for fibroblast culture. However, the Col + CS + HA tri-copolymer is preferable for embryonic nerve cell engineering.

Intra‑cerebral implantation of a variety of collagenous scaffolds with nervous embryonic cells

“…Flow cytometry studies have found that the embryonic nerve cells seeded within the scaffolds contained cells differentiating into neurons (MAP2-positive cells) (7,8); these are able to replace the damaged nervous tissue at the site of trauma, and are hence, crucial for tissue reconstruction. Scaffolds populated with embryonic nerve cells are also believed to be a source of cytokines that may accelerate the regeneration process.…”

“…Embryonic rats were euthanized by decapitation before brain isolation. The embryos at day 17 of gestation were isolated from the uterus of the pregnant female rat in sterile cold PBS (7,8). Once removed from the embryos, the brains were rinsed with PBS, and the meninges and blood vessels were excised.…”

“…For cell cultures, the medium composed of MACS NeuroBrew-21, gentamycin (25 µg/ml) and fungizone (2.5 µg/ml) was applied. The cells were packaged on the scaffold at a density of 9×10 3 cells/sample in 96 well plates (7). After three days of cultivation, the scaffolds were implanted into the brains.…”

“…The isolated cells were identified in the earlier studies (7,8). The embryonic nerve cells were nestin negative but they were morphologically differentiated (7,8).…”

Natural-Based Biomaterials for Peripheral Nerve Injury Repair

“…Collagen is a very good vehicle for drug and cell delivery ( Dalamagkas et al, 2016 ) and it is possible to modify its physical features along different parts of the same conduit ( Aigner et al, 2020 ). The different binding domains on it give the possibility to create topographical cues that guide the axon regrowth ( Stang et al, 2005a , b ) facilitating cell adhesion, survival and migration ( Alberti et al, 2014 ; Sulong et al, 2014 ; Drobnik et al, 2017a , b ). Many researchers have shown that collagen has biological properties superior to other materials available in the market for nerve scaffold fabrication and it is also effective over a nerve gap distance of at least 15 mm ( Archibald et al, 1991 ).…”