3D printed hollow pipe double-crosslinked hydrogel tissue engineering scaffold

doi:10.12307/2025.424

Abstract

Abstract: BACKGROUND: When cultivating cells with high demand for structure and oxygen, it is necessary to construct a three-dimensional biological scaffold with hollow pipe structure to make sure the cells get enough nutrients and oxygen. In recent years, hydrogel tissue engineering scaffolds with hollow pipe structure have been paid more and more attention.
OBJECTIVE: The biological scaffold material based on sodium alginate was combined with coaxial printing technology to prepare a tissue engineering scaffold with a hollow pipe structure, and the cells were inoculated by perfusion to verify its biological properties.
METHODS: The sodium alginate-acrylamide mixed printing solution was prepared, and the parameters such as the printing speed of the inner and outer layers in the coaxial printing process, sodium alginate concentration, and calcium chloride concentration in the receiving dish were controlled to realize the printing of the tissue engineering scaffold with a hollow pipe — sodium alginate-polyacrylamide double-crosslinked hydrogel. The microstructure and elastic modulus of the scaffold were characterized. Mouse fibroblasts were injected into hollow pipes of tissue engineering scaffolds. Cell compatibility was observed by living/dead cell staining.
RESULTS AND CONCLUSION: (1) By exploring the printing parameters in the printing process, when the inner printing speed was constant, the outer diameter of the hollow pipe increased with the increase of the flow rate of the outer printing solution, and the inner diameter increased slightly. When the flow rate of the outer layer printing solution was constant, and the flow rate of the inner layer solution was increased, the outer diameter of the hollow pipe was basically unchanged, and the inner diameter was significantly improved. (2) Experimental results showed that the concentration of sodium alginate was 2.5%. Excessive concentration was not conducive to the fusion of multi-layer structure layers, and the mechanical properties of hydrogels prepared at too low concentration were insufficient. (3) The elastic modulus of the double-crosslinked hydrogel was higher, generally higher than 200 kPa, and increased with the increase of the concentration of calcium chloride, and reached the maximum value of 375 kPa when the concentration of calcium chloride in the inner layer was 2% and the concentration of calcium chloride in the receiving dish was 0.3%. (4) The staining of live and dead cells after the tissue engineering scaffold perfusion cells in vitro showed that the cells were distributed along the axis of the hollow pipe and had a higher survival rate, but the cell concentration was lower than that during perfusion. (5) The results show that the sodium alginate-polyacrylamide double-crosslinked hydrogel has strong mechanical properties while retaining good biocompatibility, and can be used in the construction of tissue engineering scaffolds with hollow pipes, and the method of “first preparing the scaffold, then inoculating the cells” also avoids the traditional “cells and printing solution are mixed and then prepared” method to limit the scaffold material and processing method.

Key words: coaxial printing, tissue engineering, double-crosslinked hydrogel, sodium alginate, acrylamide, ion crosslinking, scaffold

CLC Number:

Wang Renzhi, Chen Yuanfen, Li Jinwei. 3D printed hollow pipe double-crosslinked hydrogel tissue engineering scaffold[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(16): 3432-3439.

Figures/Tables 6

2.2 多层同轴打印工艺参数优化 3D海藻酸钠-聚丙烯酰胺双交联水凝胶立体结构由单根中空管道按“蛇形线”排布，并且相邻两层呈90°交错叠加而成。为了稳定可靠地获得由中空丝线构成的3D海藻酸盐水凝胶立体结构，需要对玻璃皿内的低浓度氯化钙溶液进行测试。针对不同浓度海藻酸钠-丙烯酰胺混合打印溶液，分别在含有不同浓度氯化钙溶液的玻璃皿内进行打印实验，判断打印所获得的多层结构是否有层与层之间的连接，实验结果如图4A示。实验结果显示，2%与2.5%海藻酸钠混合打印溶液在不影响中空管道成形的前提下能实现层与层之间的丝线融合，并且所需氯化钙溶度选择范围较广。3D海藻酸盐-聚丙烯酰胺双交联水凝胶的立体结构如图4B，立体结构由单条中空丝线构建而成，相邻两层交错排布，通过海藻酸盐离子交联特性形成凝胶连接，形成一个整体。将在2.5%海藻酸钠-丙烯酰胺混合溶液、2%氯化钙溶液、外层打印溶液流速为0.2 mL/min、内层打印溶液流速为0.5 mL/min下所制备的立体水凝胶结构放入超纯水中，使用显微镜对其结构进行观察，如图4C所示，相邻两层呈现交错排列。为了获得更好的立体结构，需要对打印平台的移动速度进行测试，以匹配打印过程中的海藻酸钠溶液流速。该项测试使用的海藻酸钠浓度为2.5%、内层氯化钙浓度为2%、玻璃皿内氯化钙浓度为0.3%。在不同的海藻酸钠溶液流速下逐步提升平台移动速度，使打印针头按照设定的路线移动，通过观察丝线的成形效果确定平台移动速度的适用范围。如图4D所示，当平台移动速度小于海藻酸钠溶液流速数值的8倍时，丝线无法按照设定的路线成形，丝线的形态扭曲、变形；当平台移动速度为海藻酸钠溶液流速数值的8-18倍时，丝线能够按照设定的路线成形，丝线形态笔直，结构良好；当平台移动速度大于海藻酸钠溶液流速数值的18倍时，丝线无法按照设定的路线成形，由于打印平台移动速度较快，丝线发生拖拽，影响立体支架的成形。由以上实验获得打印参数可选择范围，确定打印实验参数：2%及2.5%海藻酸钠-丙烯酰胺混合溶液，玻璃皿内0.1%-0.3%氯化钙溶液，海藻酸钠-丙烯酰胺混合溶液打印速率0.2-0.5 mL/min，内层氯化钙溶液打印速率0.5-2 mL/min，XY移动平台速度2-7 mm/s。 "

References

[1] KHADEMHOSSEINI A, LANGER R. A decade of progress in tissue engineering. Nat Protoc. 2016;11(10):1775-1781.
[2] ROUWKEMA J, RIVRON NC, VAN BLITTERSWIJK CA. Vascularization in tissue engineering. Trends Biotechnol. 2008;26(8):434-441.
[3] NTEGE EH, SUNAMI H, SHIMIZU Y. Advances in regenerative therapy: A review of the literature and future directions. Regen Ther. 2020;14:136-153.
[4] KOLESKY DB, HOMAN KA, SKYLAR-SCOTT MA, et al. Three-dimensional bioprinting of thick vascularized tissues. Proc Natl Acad Sci U S A. 2016; 113(12):3179-3184.
[5] ZHU W, QU X, ZHU J, et al. Direct 3D bioprinting of prevascularized tissue constructs with complex microarchitecture. Biomaterials. 2017;124: 106-115.
[6] PRIYA SG, JUNGVID H, KUMAR A. Skin tissue engineering for tissue repair and regeneration. Tissue Eng Part B Rev. 2008;14(1):105-118.
[7] RATCLIFFE A. Tissue engineering of vascular grafts. Matrix Biol. 2000; 19(4):353-357.
[8] MOLLICA PA, BOOTH-CREECH EN, REID JA, et al. 3D bioprinted mammary organoids and tumoroids in human mammary derived ECM hydrogels. Acta Biomater. 2019;95:201-213.
[9] XUE L, GREISLER HP. Biomaterials in the development and future of vascular grafts. J Vasc Surg. 2003;37(2):472-480.
[10] DATTA P, AYAN B, OZBOLAT IT. Bioprinting for vascular and vascularized tissue biofabrication. Acta Biomater. 2017;51:1-20.
[11] JAIN RK, AU P, TAM J, et al. Engineering vascularized tissue. Nat Biotechnol. 2005;23(7):821-823.
[12] BOLAND T, XU T, DAMON B, et al. Application of inkjet printing to tissue engineering. Biotechnol J. 2006;1(9):910-917.
[13] BOLAND T, MIRONOV V, GUTOWSKA A, et al. Cell and organ printing 2: fusion of cell aggregates in three-dimensional gels. Anat Rec A Discov Mol Cell Evol Biol. 2003;272(2):497-502.
[14] Gu BK, Choi DJ, Park SJ, et al. 3D Bioprinting Technologies for Tissue Engineering Applications. Adv Exp Med Biol. 2018;1078:15-28.
[15] DUAN B, HOCKADAY LA, KANG KH, et al. 3D bioprinting of heterogeneous aortic valve conduits with alginate/gelatin hydrogels. J Biomed Mater Res A. 2013;101(5):1255-1264.
[16] Albrecht DR, Underhill GH, Wassermann TB, et al. Probing the role of multicellular organization in three-dimensional microenvironments. Nat Methods. 2006;3(5):369-375.
[17] KHALIL S, SUN W. Bioprinting endothelial cells with alginate for 3D tissue constructs. J Biomech Eng. 2009;131(11):111002.
[18] ATTALLA R, PUERSTEN E, JAIN N, et al. 3D bioprinting of heterogeneous bi- and tri-layered hollow channels within gel scaffolds using scalable multi-axial microfluidic extrusion nozzle. Biofabrication. 2018;11(1):015012.
[19] SHAO L, GAO Q, XIE C, et al. Directly coaxial 3D bioprinting of large-scale vascularized tissue constructs. Biofabrication. 2020;12(3):035014.
[20] DOLATI F, YU Y, ZHANG Y, et al. In vitro evaluation of carbon-nanotube-reinforced bioprintable vascular conduits. Nanotechnology. 2014;25(14):145101.
[21] HECHT H, SREBNIK S. Structural Characterization of Sodium Alginate and Calcium Alginate. Biomacromolecules. 2016;17(6):2160-2167.
[22] SMIDSRØD O, SKJÅK-BRAEK G. Alginate as immobilization matrix for cells. Trends Biotechnol. 1990;8(3):71-78.
[23] LI L, FANG Y, VREEKER R, et al. Reexamining the egg-box model in calcium-alginate gels with X-ray diffraction. Biomacromolecules. 2007;8(2):464-468.
[24] BOATENG JS, MATTHEWS KH, STEVENS HN, et al. Wound healing dressings and drug delivery systems: a review. J Pharm Sci. 2008; 97(8):2892-2923.
[25] FANG Y, AL-ASSAF S, PHILLIPS GO, et al. Multiple steps and critical behaviors of the binding of calcium to alginate. J Phys Chem B. 2007; 111(10):2456-2462.
[26] ROSIAK J, BUROZAK K, PKALA W, et al. Polyacrylamide hydrogels as sustained release drug delivery dressing materials. Radiat Phys Chem. 1983;22(3-5):907-915.
[27] FERNANDEZ ED, LOPEZ-CABARCOS LE, MIJANGOS CA, et al. Viscoelastic and swelling properties of glucose oxidase loaded polyacrylamide hydrogels and the evaluation of their properties as glucose sensors. Polymer. 2005;46(7): 2211-2217.
[28] ZHANG Y, YU Y, CHEN H, et al. Characterization of printable cellular micro-fluidic channels for tissue engineering. Biofabrication. 2013;5(2):025004.
[29] SUN JY, ZHAO X, ILLEPERUMA WR, et al. Highly stretchable and tough hydrogels. Nature. 2012;489(7414):133-136.
[30] YANG X, LU Z, WU H, et al. Collagen-alginate as bioink for three-dimensional (3D) cell printing based cartilage tissue engineering. Mater Sci Eng C Mater Biol Appl. 2018;83:195-201.