Three-dimensional printed scaffolds with sodium alginate/chitosan/mineralized collagen for promoting osteogenic differentiation_Journal of Biomedical Engineering

Authors：

YANG Bo ¹ ,  LIAN Xiaojie ^1,2 , FENG Haonan ¹ , QIN Tingwei ¹ , LYU Song ¹ , LIU Zehua ¹ , FU Tong ¹

1. Department of Biomedical Engineering, College of artificial intelligence, Taiyuan University of Technology, Jinzhong, Shanxi 030600, P. R. China;
2. Shanxi Provincial Key Laboratory of Functional Proteins, Taiyuan University of Technology, Jinzhong, Shanxi 030600, P. R. China;

Corresponding?author：

LIAN Xiaojie, Email: yuhalian@126.com

Keywords：

Three-dimensional printing; Sodium alginate/chitosan scaffold; Mineralized collagen; Osteogenic differentiation

DOI：

10.7507/1001-5515.202501043

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

The three-dimensional (3D) printed bone tissue repair guide scaffold is considered a promising method for treating bone defect repair. In this experiment, chitosan (CS), sodium alginate (SA), and mineralized collagen (MC) were combined and 3D printed to form scaffolds. The experimental results showed that the printability of the scaffold was improved with the increase of chitosan concentration. Infrared spectroscopy analysis confirmed that the scaffold formed a cross-linked network through electrostatic interaction between chitosan and sodium alginate under acidic conditions, and X-ray diffraction results showed the presence of characteristic peaks of hydroxyapatite, indicating the incorporation of mineralized collagen into the scaffold system. In the in vitro collagen release experiments, a weakly alkaline environment was found to accelerate the release rate of collagen, and the release amount increased significantly with a lower concentration of chitosan. Cell experiments showed that scaffolds loaded with mineralized collagen could significantly promote cell proliferation activity and alkaline phosphatase expression. The subcutaneous implantation experiment further verified the biocompatibility of the material, and the implantation of printed scaffolds did not cause significant inflammatory reactions. Histological analysis showed no abnormal pathological changes in the surrounding tissues. Therefore, incorporating mineralized collagen into sodium alginate/chitosan scaffolds is believed to be a new tissue engineering and regeneration strategy for achieving enhanced osteogenic differentiation through the slow release of collagen.

1.	Niu X, Fan R, Tian F, et al. Calcium concentration dependent collagen mineralization. Mater Sci Eng C, 2017, 73: 137-143.
2.	Vázquez Sanz C, Victoria Rodríguez I, Forriol F, et al. Variation in juvenile long bone properties as a function of age: mechanical and compositional characterization. Materials, 2023, 16(4): 1637.
3.	Petruska J A, Hodge A J. A subunit model for the tropocollagen macromolecule. PNAS, 1964, 51(5): 871-876.
4.	Xu Z, Zhao W, Wang Z, et al. Structure analysis of collagen fibril at atomic-level resolution and its implications for intra-fibrillar transport in bone biomineralization. PCCP, 2018, 20(3): 1513-1523.
5.	Orgel J P R O, Irving T C, Miller A, et al. Microfibrillar structure of type I collagen in situ. PNAS, 2006, 103(24): 9001-9005.
6.	Niu X, Feng Q, Wang M, et al. Porous nano-HA/collagen/PLLA scaffold containing chitosan microspheres for controlled delivery of synthetic peptide derived from BMP-2. J Controlled Release, 2009, 134(2): 111-117.
7.	Chatzipanagis K, Iafisco M, Roncal-Herrero T, et al. Crystallization of citrate-stabilized amorphous calcium phosphate to nanocrystalline apatite: a surface-mediated transformation. CrystEngComm, 2016, 18(18): 3170-3173.
8.	Liu D, Nie W, Li D, et al. 3D printed PCL/SrHA scaffold for enhanced bone regeneration. Chem Eng J, 2019, 362: 269-279.
9.	Bidarra S J, Barrias C C, Granja P L. Injectable alginate hydrogels for cell delivery in tissue engineering. Acta Biomater, 2014, 10(4): 1646-1662.
10.	Axpe E, Oyen M L. Applications of alginate-based bioinks in 3D bioprinting. Int J Mol Sci, 2016, 17(12): 1976.
11.	Wu Q, Maire M, Lerouge S, et al. 3D printing of microstructured and stretchable chitosan hydrogel for guided cell growth. Biosystems, 2017, 1(6): 1700058.
12.	Bergonzi C, Di Natale A, Zimetti F, et al. Study of 3D-printed chitosan scaffold features after different post-printing gelation processes. Sci Rep, 2019, 9(1): 362.
13.	Shukla S K, Mishra A K, Arotiba O A, et al. Chitosan-based nanomaterials: A state-of-the-art review. Int J Biol Macromol, 2013, 59: 46-58.
14.	Chen L, Wu C, Chen S, et al. Biomimetic mineralizable collagen hydrogels for dynamic bone matrix formation to promote osteogenesis. J Mater Chem B, 2020, 8(15): 3064-3075.
15.	Qiu Z Y, Cui Y, Tao C S, et al. Mineralized collagen: rationale, current status, and clinical applications. Materials, 2015, 8(8): 4733-4750.
16.	Jayabalan M, Shalumon K T, Mitha M K, et al. Effect of hydroxyapatite on the biodegradation and biomechanical stability of polyester nanocomposites for orthopaedic applications. Acta Biomater, 2010, 6(3): 763-775.
17.	Tuzlakoglu K, Santos M I, Neves N, et al. Design of nano-and microfiber combined scaffolds by electrospinning of collagen onto starch-based fiber meshes: a man-made equivalent of natural extracellular matrix. Tissue Eng Part A, 2011, 17(3-4): 463-473.
18.	Kuttappan S, Mathew D, Nair M B. Biomimetic composite scaffolds containing bioceramics and collagen/gelatin for bone tissue engineering-A mini review. Int J Biol Macromol, 2016, 93: 1390-1401.
19.	Oryan A, Alidadi S, Moshiri A, et al. Bone regenerative medicine: classic options, novel strategies, and future directions. J Orthop Surg Res, 2014, 9: 1-27.
20.	S?ther H V, Holme H K, Maurstad G, et al. Polyelectrolyte complex formation using alginate and chitosan. Carbohydr Polym, 2008, 74(4): 813-821.
21.	Ouyang L, Yao R, Zhao Y, et al. Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells. Biofabrication, 2016, 8(3): 035020.
22.	Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem., 1976, 72(1-2): 248-254.
23.	Adhikari J, Perwez M S, Das A, et al. Development of hydroxyapatite reinforced alginate–chitosan based printable biomaterial-ink. Nano-Struct Nano-Objects, 2021, 25: 100630.
24.	Eriksen E F. Cellular mechanisms of bone remodeling. Rev Endocr Metab Disord, 2010, 11: 219-227.
25.	González-Vázquez A, Planell J A, Engel E. Extracellular calcium and CaSR drive osteoinduction in mesenchymal stromal cells. Acta Biomater, 2014, 10(6): 2824-2833.
26.	Gu X, Masters K S. Role of the MAPK/ERK pathway in valvular interstitial cell calcification. Am J Physiol Heart Circ Physiol, 2009, 296(6): H1748-H1757.
27.	Reyes C D, García A J. α2β1 integrin‐specific collagen‐mimetic surfaces supporting osteoblastic differentiation. J Biomed Mater Res A, 2004, 69(4): 591-600.
28.	Fassett John T, Tobolt Diane, Nelsen Christopher J, et al. The role of collagen structure in mitogen stimulation of ERK, cyclin D1 expression, and G1-S progression in rat hepatocytes. J Biol Chem, 2003, 278(34): 31691-31700.
29.	Rajshankar D, Wang Y, McCulloch C A. Osteogenesis requires FAK‐dependent collagen synthesis by fibroblasts and osteoblasts. FASEB J, 2017, 31(3): 937-953.
30.	Kim K I, Jeong D S, Yoon T J, et al. Inhibition of collagen production by ICG-001, a small molecule inhibitor for Wnt/β-catenin signaling, in skin fibroblasts. J Dermatol Sci, 2017, 86(1): 76-78.
31.	Hiraishi N, Gondo T, Shimada Y, et al. Effect of borate, fluoride and strontium ions on biomimetic nucleation of calcium phosphate studied using solid-state nuclear magnetic resonance and X-ray diffraction. Dent Mater, 2024, 40(2): 210-218.
32.	Pandolfi F, Altamura S, Frosali S, et al. Key role of DAMP in inflammation, cancer, and tissue repair. Clin Ther, 2016, 38(5): 1017-1028.
33.	Shao J, Weng L, Li J, et al. Regulation of macrophage polarization by mineralized collagen coating to accelerate the osteogenic differentiation of mesenchymal stem cells. ACS Biomater Sci Eng, 2022, 8(2): 610-619.

1. Niu X, Fan R, Tian F, et al. Calcium concentration dependent collagen mineralization. Mater Sci Eng C, 2017, 73: 137-143.
2. Vázquez Sanz C, Victoria Rodríguez I, Forriol F, et al. Variation in juvenile long bone properties as a function of age: mechanical and compositional characterization. Materials, 2023, 16(4): 1637.
3. Petruska J A, Hodge A J. A subunit model for the tropocollagen macromolecule. PNAS, 1964, 51(5): 871-876.
4. Xu Z, Zhao W, Wang Z, et al. Structure analysis of collagen fibril at atomic-level resolution and its implications for intra-fibrillar transport in bone biomineralization. PCCP, 2018, 20(3): 1513-1523.
5. Orgel J P R O, Irving T C, Miller A, et al. Microfibrillar structure of type I collagen in situ. PNAS, 2006, 103(24): 9001-9005.
6. Niu X, Feng Q, Wang M, et al. Porous nano-HA/collagen/PLLA scaffold containing chitosan microspheres for controlled delivery of synthetic peptide derived from BMP-2. J Controlled Release, 2009, 134(2): 111-117.
7. Chatzipanagis K, Iafisco M, Roncal-Herrero T, et al. Crystallization of citrate-stabilized amorphous calcium phosphate to nanocrystalline apatite: a surface-mediated transformation. CrystEngComm, 2016, 18(18): 3170-3173.
8. Liu D, Nie W, Li D, et al. 3D printed PCL/SrHA scaffold for enhanced bone regeneration. Chem Eng J, 2019, 362: 269-279.
9. Bidarra S J, Barrias C C, Granja P L. Injectable alginate hydrogels for cell delivery in tissue engineering. Acta Biomater, 2014, 10(4): 1646-1662.
10. Axpe E, Oyen M L. Applications of alginate-based bioinks in 3D bioprinting. Int J Mol Sci, 2016, 17(12): 1976.
11. Wu Q, Maire M, Lerouge S, et al. 3D printing of microstructured and stretchable chitosan hydrogel for guided cell growth. Biosystems, 2017, 1(6): 1700058.
12. Bergonzi C, Di Natale A, Zimetti F, et al. Study of 3D-printed chitosan scaffold features after different post-printing gelation processes. Sci Rep, 2019, 9(1): 362.
13. Shukla S K, Mishra A K, Arotiba O A, et al. Chitosan-based nanomaterials: A state-of-the-art review. Int J Biol Macromol, 2013, 59: 46-58.
14. Chen L, Wu C, Chen S, et al. Biomimetic mineralizable collagen hydrogels for dynamic bone matrix formation to promote osteogenesis. J Mater Chem B, 2020, 8(15): 3064-3075.
15. Qiu Z Y, Cui Y, Tao C S, et al. Mineralized collagen: rationale, current status, and clinical applications. Materials, 2015, 8(8): 4733-4750.
16. Jayabalan M, Shalumon K T, Mitha M K, et al. Effect of hydroxyapatite on the biodegradation and biomechanical stability of polyester nanocomposites for orthopaedic applications. Acta Biomater, 2010, 6(3): 763-775.
17. Tuzlakoglu K, Santos M I, Neves N, et al. Design of nano-and microfiber combined scaffolds by electrospinning of collagen onto starch-based fiber meshes: a man-made equivalent of natural extracellular matrix. Tissue Eng Part A, 2011, 17(3-4): 463-473.
18. Kuttappan S, Mathew D, Nair M B. Biomimetic composite scaffolds containing bioceramics and collagen/gelatin for bone tissue engineering-A mini review. Int J Biol Macromol, 2016, 93: 1390-1401.
19. Oryan A, Alidadi S, Moshiri A, et al. Bone regenerative medicine: classic options, novel strategies, and future directions. J Orthop Surg Res, 2014, 9: 1-27.
20. S?ther H V, Holme H K, Maurstad G, et al. Polyelectrolyte complex formation using alginate and chitosan. Carbohydr Polym, 2008, 74(4): 813-821.
21. Ouyang L, Yao R, Zhao Y, et al. Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells. Biofabrication, 2016, 8(3): 035020.
22. Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem., 1976, 72(1-2): 248-254.
23. Adhikari J, Perwez M S, Das A, et al. Development of hydroxyapatite reinforced alginate–chitosan based printable biomaterial-ink. Nano-Struct Nano-Objects, 2021, 25: 100630.
24. Eriksen E F. Cellular mechanisms of bone remodeling. Rev Endocr Metab Disord, 2010, 11: 219-227.
25. González-Vázquez A, Planell J A, Engel E. Extracellular calcium and CaSR drive osteoinduction in mesenchymal stromal cells. Acta Biomater, 2014, 10(6): 2824-2833.
26. Gu X, Masters K S. Role of the MAPK/ERK pathway in valvular interstitial cell calcification. Am J Physiol Heart Circ Physiol, 2009, 296(6): H1748-H1757.
27. Reyes C D, García A J. α2β1 integrin‐specific collagen‐mimetic surfaces supporting osteoblastic differentiation. J Biomed Mater Res A, 2004, 69(4): 591-600.
28. Fassett John T, Tobolt Diane, Nelsen Christopher J, et al. The role of collagen structure in mitogen stimulation of ERK, cyclin D1 expression, and G1-S progression in rat hepatocytes. J Biol Chem, 2003, 278(34): 31691-31700.
29. Rajshankar D, Wang Y, McCulloch C A. Osteogenesis requires FAK‐dependent collagen synthesis by fibroblasts and osteoblasts. FASEB J, 2017, 31(3): 937-953.
30. Kim K I, Jeong D S, Yoon T J, et al. Inhibition of collagen production by ICG-001, a small molecule inhibitor for Wnt/β-catenin signaling, in skin fibroblasts. J Dermatol Sci, 2017, 86(1): 76-78.
31. Hiraishi N, Gondo T, Shimada Y, et al. Effect of borate, fluoride and strontium ions on biomimetic nucleation of calcium phosphate studied using solid-state nuclear magnetic resonance and X-ray diffraction. Dent Mater, 2024, 40(2): 210-218.
32. Pandolfi F, Altamura S, Frosali S, et al. Key role of DAMP in inflammation, cancer, and tissue repair. Clin Ther, 2016, 38(5): 1017-1028.
33. Shao J, Weng L, Li J, et al. Regulation of macrophage polarization by mineralized collagen coating to accelerate the osteogenic differentiation of mesenchymal stem cells. ACS Biomater Sci Eng, 2022, 8(2): 610-619.

Journal of Biomedical Engineering

Latest ArticlesThree-dimensional printed scaffolds with sodium alginate/chitosan/mineralized collagen for promoting osteogenic differentiation

Abstract Full text Figures/Tables Video References Cited by

Format

Content