Bioprinting can be defined as 3D patterning of living cells and other biologics by filling and assembling them using a computer-aided layer-by-layer deposition approach to fabricate living tissue and organ analogs for tissue engineering. The presence of cells within the ink to use a 'bio-ink' presents the potential to print 3D structures that can be implanted or printed into damaged/diseased bone tissue to promote highly controlled cell-based regeneration and remineralization of bone. In this study, it was shown for the first time that chitosan solution and its composite with nanostructured bone-like hydroxyapatite (HA) can be mixed with cells and printed successfully. MC3T3-E1 pre-osteoblast cell laden chitosan and chitosan-HA hydrogels, which were printed with the use of an extruder-based bioprinter, were characterized by comparing these hydrogels to alginate and alginate-HA hydrogels. Rheological analysis showed that all groups had viscoelastic properties. It was also shown that under simulated physiological conditions, chitosan and chitosan-HA hydrogels were stable. Also, the viscosity values of the bio-solutions were in an applicable range to be used in 3D bio-printers. Cell viability and proliferation analyses documented that after printing with bio-solutions, cells continued to be viable in all groups. It was observed that cells printed within chitosan-HA composite hydrogel had peak expression levels for early and late stages osteogenic markers. It was concluded that cells within chitosan and chitosan-HA hydrogels had mineralized and differentiated osteogenically after 21 days of culture. It was also discovered that chitosan is superior to alginate, which is the most widely used solution preferred in bioprinting systems, in terms of cell proliferation and differentiation. Thus, applicability and printability of chitosan as a bio-printing solution were clearly demonstrated. Furthermore, it was proven that the presence of bone-like nanostructured HA in alginate and chitosan hydrogels improved cell viability, proliferation and osteogenic differentiation.

Methacrylated gelatin (Gel-MA) is a commonly used biomaterial in bioprinting applications. The Gel-MA synthesis procedure is inadequate and needs to be improved, particularly from the point of optimization and efficacy. We report a significantly faster (by 5 min) and effective method to controllably synthesize Gel-MA using microwave energy (Mw at 1000 W power) with ≥90% degree of methacrylation (DM) even with the use of a very low concentration of methacrylic anhydride (MA). Rheological and mechanical analyses indicated that Gel-MA synthesized by Mw-assisted methacrylation enabled the formation of hydrogels that are more elastic and stronger and have a lower degradation rate (∼27% at 35 days) than Gel-MA synthesized by the conventional method. The viscosity values of the Gel-MA bioink were in the range applicable for use in 3D bioprinters. Additionally, Mw-assisted methacrylated Gel-MA hydrogels that have mechanically superior properties significantly enhanced the viability, attachment, proliferation, alkaline phosphatase (ALP) activity, mineral deposition, and mRNA expression levels of osteogenic genes of MC3T3-E1 preosteoblastic cells.

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Nowadays, scientists focus on the development of tissue-specific and personalized bio-ink that can be used in 3D bioprinting technologies. Platelet-rich plasma (PRP) is a person-specific source that is used as a therapeutic adjunct for the treatment of cartilage damage because it offers a cocktail of growth factors that are necessary for wound healing and tissue regeneration. However, PRP treatments in the clinic are not satisfactory and require upgrading, especially the point of maintaining bioactivity. In this study, we presented PRP as a photo-activated and photo-crosslinkable bio-ink in terms of tissue-specific structures for the first time. We achieved long-term and constant rate growth factor release and bioactivity protection of PRP with satisfactory mechanical characteristics. Photo-crosslinked PRP hydrogel was enabled by the addition of microwave-induced methacrylated gelatin (Gel-MA), which is connected to platelets in PRP via integrin receptors in its structure and chemically cross-linked upon UV irradiation (300–500 nm). Photo-activation of PRP was realized by a polychromatic light source in the near-infrared region (PAC, 600–1200 nm). Our results showed that Gel-MA/PRP hydrogels with the desired mechanical properties (low degradation rate and high mechanical strength) released growth factors at a constant rate for the long-term by the periodic PAC application. In vitro cell culture studies (viability, proliferation, morphology, histology, immunochemistry, biochemistry, gene expression analyses) proved that proliferation and differentiation of the ATDC5 cells increased in the periodically light-applied Gel-MA/PRP hydrogel without any external chemical agents.

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