(A-16) In situ bioprinting of nanoengineered granular hydrogel scaffolds assembled from thermostable gelatin methacryloyl microgels

Introduction:: Bioprinting is a rapidly evolving field that holds promise for creating patient-specific, functional tissues for regenerative medicine. Granular hydrogels, composed of hydrogel microparticles (microgels) as building blocks, offer various advantages over bulk (conventional) hydrogels, most notably the ability to form scaffolds in situ and on demand with micron-size, cell-scale interconnected void spaces. Conventionally, three-dimensional (3D) bioprinting of microgels has been performed via tightly packing of microgels, which compromises the interconnected pores. To overcome this challenge, we previously introduced the first class of nanoengineered granular bioinks (NGB)[1] which enabled 3D bioprinting of granular scaffolds with preserved porosity while enhancing printability and shape fidelity. In NGB, we used reversible self-assembly of colloidal nanoclay adsorbed onto the microgel surface to dynamically enhance microgel-microgel interactions. However, NGB required low temperature working environment and storage to maintain the structure of thermosensitive gelatin methacryloyl (GelMA) microgels, limiting its potential for in situ bioprinting. To address this limitation, we have developed NGB 2.0 from thermostable GelMA microgel building blocks that enable in situ bioprinting at the physiological temperature.

Materials and Methods:: GelMA was synthesized according to our established protocol.[2] Briefly, 20 g of gelatin was dissolved in 200 mL of Dulbecco's phosphate-buffered saline (DPBS), followed by the dropwise addition of 16, 2.5, or 0.5 mL of methacrylic anhydride to the solution at 50 °C for the synthesis of GelMA with low, medium, or high degrees of methacryloyl substitution (DoS), respectively. The reaction was stopped after 2 h by adding an excessive amount of DPBS (400 mL). The solution was dialyzed against ultra-pure water for 10 days, lyophilized, and stored. Droplet fabrication was performed using a high-throughput step-emulsification microfluidic device.[3] GelMA (10% w/v in DPBS, 0.1% w/v photoinitiator) was used as the aqueous phase, and Novec 7500 engineered fluid (with 2% v/v surfactant) was used as the oil phase. GelMA droplets were stored at 4 °C to yield physically crosslinked microgels. Then, a Schiff-base reaction was conducted to fabricate thermostable microgels using a glutaraldehyde solution (0.15% v/v in DPBS) for 1 h at temperature < 4 °C while stirring at 400 rpm. The resulting thermostable microgels were washed five times with DPBS, packed via centrifugation at 3000 ×g, and exposed to light to form granular hydrogel scaffold (GHS). The thermostable microgels formed from high, medium, and low DoS GelMA were heated at 37 °C for 1 h and imaged to test their stability. The cytotoxicity of GHS made from thermostable microgels was measured using the Live/Dead and PrestoBlue in vitro assays.

Results, Conclusions, and Discussions::
Results and discussion:




The thermoresponsive nature of physically crosslinked GelMA microgels prevented the formation of scaffolds at body temperature,[4] thus limiting their application for in situ bioprinting in the surgical incisions or inside the body. To overcome this limitation, GelMA biopolymer was crosslinked via a Schiff-base reaction to produce thermostable microgels. GelMA microgels were fabricated using three different DoS. High DoS resulted in thermally unstable microgels because of insufficient primary amine groups for Schiff-base reaction (Figure 1A). Medium and low DoS resulted in thermostable microgels that were used for in situ fabrication of GHS via free radical photopolymerization (Figure 1B). Oscillatory rheology showed that the storage modulus of GelMA GHS made from thermostable microgels (TS GelMA GHS) was not significantly different from the GelMA GHS made from the physically crosslinked microgels (GelMA GHS), but both were significantly lower than the bulk GelMA scaffolds (Figures 1C-D). Furthermore, biocompatibility assessment of TS GelMA GHS using murine fibroblast cells showed consistently high cell viability and metabolic activity rates over a period of 7 days (Figures 1E-F). This study highlights the potential of using thermostable GelMA microgels for in situ bioprinting of GHS, which may have significant clinical implications in tissue engineering and regenerative medicine.




Conclusions:




In conclusion, the development of NGB 2.0 using thermostable GelMA microgels enables in situ scaffold formation at physiological temperature, advancing granular hydrogel bioprinting. The study demonstrates the successful fabrication of thermostable microgels and the formation of granular hydrogel scaffolds and bioinks at physiological temperature. We envision that NGB 2.0 will overcome the limitations of in situ 3D bioprinting beyond the traditional biofabrication window.[5]




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[1] Z. Ataie, S. Kheirabadi, J. W. Zhang, A. Kedzierski, C. Petrosky, R. Jiang, C. Vollberg, A. Sheikhi, Small 2022, 18, 2202390.




[2] Z. Ataie, A. Jaberi, S. Kheirabadi, A. Risbud, A. Sheikhi, J. Vis. Exp. 2022, 190, e64829.




[3] J. M. de Rutte, J. Koh, D. Di Carlo, Adv. Funct. Mater. 2019, 29, 1.




[4] A. Sheikhi, J. de Rutte, R. Haghniaz, O. Akouissi, A. Sohrabi, D. Di Carlo, A. Khademhosseini, Biomaterials 2019, 192, 560.




[5] W. Sun, B. Starly, A. C. Daly, J. A. Burdick, J. Groll, G. Skeldon, W. Shu, Y. Sakai, M. Shinohara, M. Nishikawa, J. Jang, D. W. Cho, M. Nie, S. Takeuchi, S. Ostrovidov, A. Khademhosseini, R. D. Kamm, V. Mironov, L. Moroni, I. T. Ozbolat, Biofabrication 2020, 12, 22002.