3D-printed GelMA/SA/CMCS hydrogel scaffolds containing Cynomorium songaricum polysaccharide for critical bone defect repair

Abstract

Critical bone defect repair remains a major challenge in orthopedics. Cynomorium songaricum polysaccharide (CSP), derived from the traditional medicinal plant Cynomorium songaricum Rupr. in China, demonstrates excellent anti-inflammatory and osteogenic properties. Given these promising biological activities, we developed a novel therapeutic approach using a hydrogel composite scaffold incorporating CSP (GAC-C) for treating critical-sized bone defects. The composite scaffold was fabricated by embedding CSP into a methacrylated gelatin (GelMA)/sodium alginate (SA)/carboxymethyl chitosan (CMCS) blend via three-dimensional (3D) printing technology. The structural, mechanical, and biological properties of GAC-C were characterized, and osteogenic performance was evaluated both in vitro with rat bone marrow stromal cells (rBMSCs) and in vivo using a critical-sized bone defect model. Results indicated that the GAC-C scaffold demonstrated excellent biocompatibility, promoted osteogenic differentiation of rBMSCs, and enhanced bone integration and repair. Thus, the GAC-C scaffold has the potential for effectively repairing criticalsized bone defects.

Keywords

3D-printed hydrogel / Critical bone defect / Cynomorium songaricum polysaccharide / Osteogenic / differentiation

Funding

This work was financially supported by the Key Research and Development Program - Social Development Field(25YFFA064) the Natural Science Foundation Exploration Project of Zhejiang Province (LY23H060012), and the Zhejiang Provincial Medical and Health Science and Technology Plan (2024KY986).

Conflict of interest

The authors declared no competing interests.

References

[1] Shineh G, Patel K, Mobaraki M, Tayebi L. Functional approaches in promoting vascularization and angiogenesis in bone critical-sized defects via delivery of cells, growth factors, drugs, and particles. J Funct Biomater. 2023; 14(2):99.doi: 10.3390/jfb14020099

[2] Zhang L, Yang G, Johnson BN, Jia X. Three-dimensional (3D) printed scaffold and material selection for bone repair. Acta Biomater. 2019; 84:16-33.doi: 10.1016/j.actbio.2018.11.039

[3] Huang Y, Zhang L, Ji Y, et al. A non-invasive smart scaffold for bone repair and monitoring. Bioact Mater. 2023; 19:499-510.doi: 10.1016/j.bioactmat.2022.04.034

[4] Wang C, Huang W, Zhou Y, et al. 3D printing of bone tissue engineering scaffolds. Bioact Mater. 2020; 5(1):82-91.doi: 10.1016/j.bioactmat.2020.01.004

[5] Yuan X, Zhu W, Yang Z, et al. Recent advances in 3D printing of smart scaffolds for bone tissue engineering and regeneration. Adv Mater. 2024; 36(34):e2403641.doi: 10.1002/adma.202403641

[6] Fu JN, Wang X, Yang M, et al. Scaffold-based tissue engineering strategies for osteochondral repair. Front Bioeng Biotechnol. 2021;9:812383.doi: 10.3389/fbioe.2021.812383

[7] Koushik TM, Miller CM, Antunes E. Bone tissue engineering scaffolds: function of multi-material hierarchically structured scaffolds. Adv Healthc Mater. 2023; 12(9):e2202766.doi: 10.1002/adhm.202202766

[8] Klotz BJ, Gawlitta D, Rosenberg A, Malda J, Melchels FPW. Gelatin-methacryloyl hydrogels: towards biofabricationbased tissue repair. Trends Biotechnol. 2016; 34(5):394-407.doi: 10.1016/j.tibtech.2016.01.002

[9] Zhou B, Jiang X, Zhou X, et al. GelMA-based bioactive hydrogel scaffolds with multiple bone defect repair functions: therapeutic strategies and recent advances. Biomater Res. 2023; 27(1):86.doi: 10.1186/s40824-023-00422-6

[10] Zhang X, Zhang H, Zhang Y, et al. 3D printed reduced graphene oxide-GelMA hybrid hydrogel scaffolds for potential neuralized bone regeneration. J Mater Chem B. 2023; 11(6): 1288-1301.doi: 10.1039/d2tb01979e

[11] Qi J, Wu H, Liu G. Novel strategies for spatiotemporal and controlled BMP-2 delivery in bone tissue engineering. Cell Transplant. 2024;33:9636897241276733.doi: 10.1177/09636897241276733

[12] Wang T, Yi W, Zhang Y, et al. Sodium alginate hydrogel containing platelet-rich plasma for wound healing. Colloids Surf B Biointerfaces. 2023;222:113096.doi: 10.1016/j.colsurfb.2022.113096

[13] Chen Q, Yang ZR, Du S, Chen S, Zhang L, Zhu J. Polyphenolsodium alginate supramolecular injectable hydrogel with antibacterial and anti-inflammatory capabilities for infected wound healing. Int J Biol Macromol. 2024; 257(Pt 1):128636.doi: 10.1016/j.ijbiomac.2023.128636

[14] Hu X, Zhang Z, Wu H, et al. Progress in the application of 3D-printed sodium alginate-based hydrogel scaffolds in bone tissue repair. Biomater Adv. 2023;152:213501.doi: 10.1016/j.bioadv.2023.213501

[15] Wang Y, Zhou X, Jiang J, et al. Carboxymethyl chitosan-enhanced multi-level microstructured composite hydrogel scaffolds for bone defect repair. Carbohydr Polym. 2025;348(Pt B):122847.doi: 10.1016/j.carbpol.2024.122847

[16] Shi Z, Yang Q, Pang Y, et al. The osteogenesis and the biologic mechanism of thermo-responsive injectable hydrogel containing carboxymethyl chitosan/sodium alginate nanoparticles towards promoting osteal wound healing. Int J Biol Macromol. 2023; 224:533-543.doi: 10.1016/j.ijbiomac.2022.10.142

[17] Paek K, Woo S, Song SJ, et al. A well plate-based GelMA photo-crosslinking system with tunable hydrogel mechanical properties to regulate the PTH-mediated osteogenic fate. Biofabrication. 2024; 16(2):025022.doi: 10.1088/1758-5090/ad2a7e

[18] Liying Q, Yining Y, Yongjian S, et al. Incorporation of carboxymethyl chitosan (CMCS) for the modulation of physio-chemical characteristics and cell proliferation environment of the composite hydrogel microspheres. Biomed Mater. 2024; 19(6):065003.doi: 10.1088/1748-605X/ad7565

[19] Naahidi S, Jafari M, Logan M, et al. Biocompatibility of hydrogel-based scaffolds for tissue engineering applications. Biotechnol Adv. 2017; 35(5):530-544.doi: 10.1016/j.biotechadv.2017.05.006

[20] Jin J, Yang Y, Yang J, et al. Macrophage metabolic reprogramming-based diabetic infected bone defect/bone reconstruction though multi-function silk hydrogel with exosome release. Int J Biol Macromol. 2024; 278(Pt 4):134830.doi: 10.1016/j.ijbiomac.2024.134830

[21] Amrita , Arora A, Sharma P, Katti DS. , Pullulan-based composite scaffolds for bone tissue engineering: improved osteoconductivity by pore wall mineralization. Carbohydr Polym. 2015; 123:180-189.doi: 10.1016/j.carbpol.2015.01.038

[22] Xue C, Chen L, Wang N, et al. Stimuli-responsive hydrogels for bone tissue engineering. Biomater Transl. 2024; 5(3):257-273.doi: 10.12336/biomatertransl.2024.03.004

[23] Mpuhwe NA, Kim GN, Koh YH. 2)-incorporated hydrogel scaffolds with antimicrobial efficacy. Materials (Basel). 2025; 18(5):1125.doi: 10.3390/ma18051125

[24] Ji S, Zhao Y, Zhai X, et al. A dual-crosslinked hydrogel based on gelatin methacryloyl and sulfhydrylated chitosan for promoting wound healing. Int J Mol Sci. 2023; 24(3):2447.doi: 10.3390/ijms24032447

[25] Meng HC, Wang S, Li Y, Kuang YY, Ma CM. Chemical constituents and pharmacologic actions of Cynomorium plants. Chin J Nat Med. 2013; 11(4):321-329.doi: 10.1016/S1875-5364(13)60049-7

[26] Wang J, Zhang J, Zhao B, Wu Y, Wang C, Wang Y. Structural features and hypoglycaemic effects of Cynomorium songaricum polysaccharides on STZ-induced rats. Food Chem. 2010; 120(2):443-451.doi: 10.1016/j.foodchem.2009.10.034

[27] You WL, Xu ZL. Curculigoside promotes osteogenic differentiation of ADSCs to prevent ovariectomized-induced osteoporosis. J Orthop Surg Res. 2021; 16(1):279.doi: 10.1186/s13018-021-02389-3

[28] Wang C, Liu C, Liang C, et al. Role of berberine thermosensitive hydrogel in periodontitis via PI3K/AKT pathway in vitro. Int J Mol Sci. 2023; 24(7):6364.doi: 10.3390/ijms25105104

[29] Meng J, Zhang W, Wang C, et al. Catalpol suppresses osteoclastogenesis and attenuates osteoclast-derived bone resorption by modulating PTEN activity. Biochem Pharmacol. 2020;171:113715.doi: 10.1016/j.bcp.2019.113715

[30] Zhang J, Chen X, Han L, et al.Research progress in traditional applications, phytochemistry, pharmacology, and safety evaluation of Cynomorium songaricum. Molecules. 2024; 29(5):941.doi: 10.3390/molecules29050941

[31] Negut I, Bita B, Groza A. Polymeric coatings and antimicrobial peptides as efficient systems for treating implantable medical devices associated-infections. Polymers. 2022; 14(8):1611.doi: 10.3390/polym14081611

[32] Lai Y, Li Y, Cao H, et al. Osteogenic magnesium incorporated into PLGA/TCP porous scaffold by 3D printing for repairing challenging bone defect. Biomaterials. 2019; 197: 207-219.doi: 10.1016/j.biomaterials.2019.01.013

[33] Lai Y, Cao H, Wang X, et al. Porous composite scaffold incorporating osteogenic phytomolecule icariin for promoting skeletal regeneration in challenging osteonecrotic bone in rabbits. Biomaterials. 2018; 153:1-13.doi: 10.1016/j.biomaterials.2017.10.025

[34] Fang J, Zhao X, Li S, et al. Protective mechanism of artemisinin on rat bone marrow-derived mesenchymal stem cells against apoptosis induced by hydrogen peroxide via activation of c-Raf-Erk1/2-p90(rsk)-CREB pathway. Stem Cell Res Ther. 2019; 10(1):312.doi: 10.1186/s13287-019-1419-2

[35] Cui Z, Zhou L, Huang J, et al. Dual-model biomanufacturing of porous biomimetic scaffolds with concentrated growth factors and embedded endothelial vascular channels for bone defect regeneration. Chem Eng J. 2024;483:148933.doi: 10.1016/j.cej.2024.148933

[36] Eufinger H, Rasche C, Lehmbrock J, et al. Performance of functionally graded implants of polylactides and calcium phosphate/calcium carbonate in an ovine model for computer assisted craniectomy and cranioplasty. Biomaterials. 2007; 28(3):475-485.doi: 10.1016/j.biomaterials.2006.08.055

[37] Mao Q, Wang Y, Li Y, et al. Fabrication of liver microtissue with liver decellularized extracellular matrix (dECM) bioink by digital light processing (DLP) bioprinting. Mater Sci Eng: C. 2020; 109:110625.doi: https://doi.org/10.1016/j.msec.2020.110625

[38] Qiao Y, Liu X, Zhou X, et al. Gelatin templated polypeptide co-cross-linked hydrogel for bone regeneration. Adv Healthc Mater. 2020; 9(1):e1901239.doi: 10.1002/adhm.201901239

[39] Jin J, Wang D, Qian H, et al. Precision pore structure optimization of additive manufacturing porous tantalum scaffolds for bone regeneration: a proof-of-concept study. Biomaterials. 2025;313:122756.doi: 10.1016/j.biomaterials.2024.122756

[40] Ma D, Wang J, Zheng M, et al. Degradation behavior of ZE21C magnesium alloy suture anchors and their effect on ligament-bone junction repair. Bioact Mater. 2023; 26:128-141.doi: 10.1016/j.bioactmat.2023.02.021

[41] Zhou K, Yu P, Shi X, et al. Hierarchically porous hydroxyapatite hybrid scaffold incorporated with reduced graphene oxide for rapid bone ingrowth and repair. ACS Nano. 2019; 13(8):9595-9606.doi: 10.1021/acsnano.9b04723

[42] Zhang X, Li Y, Ma Z, He D, Li H. Modulating degradation of sodium alginate/bioglass hydrogel for improving tissue infiltration and promoting wound healing. Bioact Mater. 2021; 6(11):3692-3704.doi: 10.1016/j.bioactmat.2021.03.038

[43] Trujillo S, Gonzalez-Garcia C, Rico P, et al. Engineered 3D hydrogels with full-length fibronectin that sequester and present growth factors. Biomaterials. 2020;252:120104.doi: 10.1016/j.biomaterials.2020.120104

[44] Jiang Q, Zhou L, Yang Y, et al. Injectable NGF-loaded double crosslinked collagen/hyaluronic acid hydrogels for irregular bone defect repair via neuro-guided osteogenic process. Chem Eng J. 2024;497:154627.doi: 10.1016/j.cej.2024.154627

[45] Huang W, Liu C, Liu F, Liu Z, Lai G, Yi J. Hinokiflavone induces apoptosis and inhibits migration of breast cancer cells via EMT signalling pathway. Cell Biochem Funct. 2020; 38(3):249-256.doi: 10.1002/cbf.3443

[46] Zhou Y, Liu X, She H, Wang R, Bai F, Xiang B. A silk fibroin/ chitosan/nanohydroxyapatite biomimetic bone scaffold combined with autologous concentrated growth factor promotes the proliferation and osteogenic differentiation of BMSCs and repair of critical bone defects. Regen Ther. 2022; 21:307-321.doi: 10.1016/j.reth.2022.08.006

[47] Hu H, Wang D, Li L, Yin H, He G, Zhang Y. Role of microRNA-335 carried by bone marrow mesenchymal stem cells-derived extracellular vesicles in bone fracture recovery. Cell Death Dis. 2021; 12(2):156.doi: 10.1038/s41419-021-03430-3

[48] Zhong X, Xiu LL, Wei GH, et al. Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation. Acta Pharmacol Sin. 2011; 32(5):591-600.doi: 10.1038/aps.2011.15

[49] Wang J, Sun Y, Liu Y, et al. Effects of platelet-rich fibrin on osteogenic differentiation of Schneiderian membrane derived mesenchymal stem cells and bone formation in maxillary sinus. Cell Commun Signal. 2022; 20(1):88.doi: 10.1186/s12964-022-00844-0

[50] Ma X, Liu J, Yang L, Zhang B, Dong Y, Zhao Q. Cynomorium songaricum prevents bone resorption in ovariectomized rats through RANKL/RANK/TRAF6 mediated suppression of PI3K/AKT and NF-κB pathways. Life Sci. 2018; 209:140-148.doi: https://doi.org/10.1016/j.lfs.2018.08.008

[51] Fili S, Karalaki M, Schaller B. Therapeutic implications of osteoprotegerin. Cancer Cell Int. 2009;9:26.doi: 10.1186/1475-2867-9-26

[52] Yan K, Wu C, Ye Y, et al. A 20 inhibits osteoclastogenesis via TRAF6-dependent autophagy in human periodontal ligament cells under hypoxia. Cell Prolif. 2020; 53(3): e12778.doi: 10.1111/cpr.12778

[53] Spicer PP, Kretlow JD, Young S, Jansen JA, Kasper FK, Mikos AG. Evaluation of bone regeneration using the rat critical size calvarial defect. Nat Protoc. 2012; 7(10):1918-1929.doi: 10.1038/nprot.2012.113

[54] Luo T, Tan B, Zhu L, Wang Y, Liao J. A review on the design of hydrogels with different stiffness and their effects on tissue repair. Front Bioeng Biotechnol. 2022;10:817391.doi: 10.3389/fbioe.2022.817391

[55] Xu FF, Zhu H, Li XM, et al. Intercellular adhesion molecule-1 inhibits osteogenic differentiation of mesenchymal stem cells and impairs bio-scaffold-mediated bone regeneration in vivo. Tissue Eng Part A. 2014; 20(19-20):2768-2782.doi: 10.1089/ten.TEA.2014.0007

[56] Gargalionis AN, Adamopoulos C, Vottis CT, Papavassiliou AG, Basdra EK. Runx2 and polycystins in bone mechanotransduction: challenges for therapeutic opportunities. Int J Mol Sci. 2024; 25(10):5291.doi: 10.3390/ijms25105291

[57] Blair HC, Larrouture QC, Li Y, et al. Osteoblast differentiation and bone matrix formation in vivo and in vitro. Tissue Eng Part B Rev. 2017; 23(3):268-280.doi: 10.1089/ten.TEB.2016.0454