Investigating the effect of sterilisation methods on the physical properties and cytocompatibility of methyl cellulose used in combination with alginate for 3D-bioplotting of chondrocytes

Abstract

For both the incorporation of cells and future therapeutic applications the sterility of a biomaterial must be ensured. However, common sterilisation techniques are intense and often negatively impact on material physicochemical attributes, which can affect its suitability for tissue engineering and 3D printing. In the present study four sterilisation methods, autoclave, supercritical CO2 (scCO2) treatment, UV-and gamma () irradiation were evaluated regarding their impact on material properties and cellular responses. The investigations were performed on methyl cellulose (MC) as a component of an alginate/methyl cellulose (alg/MC) bioink, used for bioprinting embedded bovine primary chondrocytes (BPCs). In contrast to the autoclave, scCO2 and UV-treatments, the-irradiated MC resulted in a strong reduction in alg/MC viscosity and stability after extrusion which made this method unsuitable for precise bioprinting. Gel permeation chromatography analysis revealed a significant reduction in MC molecular mass only after-irradiation, which influenced MC chain mobility in the Ca 2+crosslinked alginate network as well as gel composition and microstructure. With regard to cell survival and proteoglycan matrix production, the results determined UV-irradiation and autoclaving as the best candidates for sterilisation. The scCO2-treatment of MC resulted in an unfavourable cell response indicating that this method needs careful optimisation prior to application for cell encapsulation. As proven by consistent FT-IR spectra, chemical alterations could be excluded as a cause for the differences seen between MC-treatments on alg/MC behaviour. This investigation provides knowledge for the development of a clinically appropriate 3D-printing-based fabrication process to produce bioengineered tissue for cartilage regeneration.

3D Bioprinting of Functional Islets of Langerhans in an Alginate/Methylcellulose Hydrogel Blend

“…The Alg/MC blend has previously been reported as a highly suitable paste for 3D bioprinting with favorable characteristics for cell survival and allows for the fabrication of macroporous cell‐laden constructs. Furthermore, we could also show in previous work that Alg/MC scaffolds additionally contain micropores, which we attribute in part to the sole presence and in part to the release of a large fraction of the MC from plotted scaffolds within the first few days . All scaffolds used in the present study were comprised of only four layers in height, but as proven by Schütz et al with this material a much greater height can be achieved without loss of shape accuracy, which is an important feature for upscaling.…”

“…For experiments performed in vitro only differences up to day 7 would be of consequence, since under cell culture conditions the ability of islets to react to glucose stimulation is reduced and finally lost over a maximum of 10 days in culture; however, lasting stability is an important factor for eventual transplantation. While it is well known that presence of chelating agents can lead to a loss of crosslinking ions from ionically crosslinked alginate gels, which results in a lesser resistance against mechanical pressure also in Alg/MC scaffolds, previous in vivo studies showed alginate capsules to be stable for years after transplantation . Since presence of MC and its release from the scaffolds do not lead to a disintegration of the scaffolds in vitro, a quicker degradation in vivo than is known from plain alginate is not to be expected.…”

“…In this study, we used an alginate‐based composite material for the 3D bioprinting of pancreatic islets isolated from rat, alginate/methylcellulose (Alg/MC) which has, in contrast to plain alginate, a high shape fidelity ideal for bioprinting. Out of the two biopolymer components in the Alg/MC hydrogel, only the alginate is crosslinked by divalent cations, whereas MC remains un‐crosslinked and is released from the scaffold over time . Furthermore, the addition of MC to the alginate did not impair the survival of mammalian cells; it has previously been shown that MC is neither cytotoxic nor allergenic, and cannot be taken up by human cells or digested into toxic derivatives in the human body .…”

Biodegradable Polymers as Drug Delivery Systems for Bone Regeneration

“…Cellulose is also a material with excellent biocompatibility [59], and the cellulose/hyaluronic acid complex has been clinically applied as a barrier material for preventing postoperative adhesion [60]. As a scaffold for regenerative medicine, research has been conducted on the use of methyl cellulose for cartilage regeneration [61]. Chakraborty et al [62] evaluated web-shaped cellulose nanofibers as a scaffold for bone regeneration and reported good results on MTT assay and scanning electron microscopy (SEM) when the MC3T3-E1 osteoblast cell line was cultured on the scaffold.…”

The Use of Electrospun Organic and Carbon Nanofibers in Bone Regeneration

“…Cellulose has excellent biocompatibility [55]. A composite of chitosan/cellulose was used as a gauze as a barrier for preventing postoperative adhesion after abdominal surgery [56], and methyl cellulose [57] was studied for its use as a scaffold in cartilage regeneration. Trivedi et al cultured human osteoblasts on chitosan/cellulose hydrogel beads and demonstrated their potential as a scaffold for bone regeneration [58].…”