The role of gelatin and collagen in cell viability and osteogenic potential of an injectable chitosan-based scaffold containing LL37 peptide

Mahboubeh Rezazadeh , Vajihe Akbari, Azam Sadeghi, Naser Tavakoli

Abstract


Background and purpose: LL37 peptide is a human antimicrobial peptide with potential application in bone tissue engineering through the stimulation of cell proliferation and osteogenesis. The current study aimed to fabricate chitosan/gelatin/glycerophosphate (CTS/G/GP) and chitosan/collagen/glycerophosphate (CTS/C/GP) thermosensitive hydrogels loaded with LL37 and compared their ability to support cell growth, proliferation, and osteogenesis.

Experimental approach: The hydrogel systems were prepared by the physical mixture of chitosan, gelatin, collagen, and GP at the concentrations of 2.5, 1, 1, and 10% w/v, respectively. LL37 was added at a fixed concentration of 1 µg/mL of the hydrogels. The viscosity, friability, release properties, and biological experiments were evaluated based on standard procedures.

Findings/Results: The viscosity of CTS/C/GP increased to 7000 cP at 35 °C in 100-120 s, while for CTS/G/GP, the viscosity and gelation time were recorded as 14000 cP and 30 s, respectively. The friability percent for CTS/G/GP after 72 h was reported as 28%, which was significantly lower than that of 38% for CTS/C/GP. LL37 was released during 8 h from both scaffold systems, and it did not demonstrate any significant differences between the hydrogel systems. Cell viability and alkaline phosphatase activity revealed that the incorporation of LL37 in the hydrogels could accelerate cell proliferation compared to empty scaffolds, and it was higher in gelatin-containing scaffolds.

Conclusion and implications: LL37 was successfully loaded into both hydrogel systems and demonstrated the ability to accelerate cell proliferation and differentiation compared to the empty scaffold.

 

 


Keywords


Thermosensitive hydrogel; LL37 peptide; Bone tissue repair.

Full Text:

PDF

References


Hao Zh, Chen R, Chai Ch, Wang Y, Chen T, Li H, et al. Antimicrobial peptides for bone tissue engineering:diversity, effects and applications. Front Bioeng Biotechnol. 2022;10:1-20.DOI: 10.3389/fbioe.2022.1030162.

Kittaka M, Shiba H, Kajiya M, Fugita T, Iwata T, Rathvisal Kh, et al. The antimicrobial peptide LL37 promotes bone regeneration in a rat calvarial bone defect. Peptides. 2013;46:136-142.DOI: 10.1016/j.peptides.2013.06.001.

Visser R, Rico-Llanos G, Pulkkinen H, Becerra J. Peptides for bone tissue engineering. J Control Release. 2016;244:122-135.DOI: 10.1016/j.jconrel.2016.10.024.

He Y, Caiyun M, Shen X, Yuan Z, Liu J, Chen W, et al. Peptide LL-37 coating on micro-structured titanium implants to facilitate bone formation in vivo via mesenchymal stem cell recruitment. Acta Biomater. 2018;80:412-424.DOI: 10.1016/j.actbio.2018.09.036.

Megha KB, Syama S, Sangeetha V, Vandana V, Oyane A, Mohanan P. Development of a 3D multifunctional collagen scaffold impregnated with peptide LL-37 for vascularised bone tissue regeneration. Int J Pharm. 2024;652:123797.DOI: 10.1016/j.ijpharm.2024.123797.

Roseti L, Parisi V, Petretta M, Cavallo C, Desando G, Bartolotti I. Scaffolds for bone tissue engineering: state of the art and new perspectives. Mater Sci Eng C Marter Biol Appl. 2017;78:1246-1262.DOI: 10.1016/j.msec.2017.05.017.

Liu M, Zeng X, Ma C, Yi H, Ali Z, Mou X, et al. Injectable hydrogels for cartilage and bone tissue engineering. Bone Res. 2017;5:17014,1-20.DOI:10.1038/boneres.2017.14.

Kim H, Sun Shim W, Kim S, Lee K, Kang E, Kim J, et al. Injectable in situ-forming pH/Thermo-sensitive hydrogel for bone tissue engineering. Tissue Eng Part A. 2009;15(4):923-933.DOI: 10.1089/ten.tea.2007.0407.

Lopez-Gutierrez J, Ramos-Payan R, Romero-Quintana JG, Ayala-Ham A, Castro-Salazar Y, Castillo-Ureta H. Evaluation of biocompatibility and angiogenic potential of extracellular matrix hydrogel biofunctionalized with the LL-37 peptide. Biomed Mater Eng. 2023;34(6):25-29.DOI: 10.3233/BME-230022.

Levengood SL, Zhang M. Chitosan-based scaffolds for bone tissue engineering J Mater Chem B. 2014;2(21):3161-3184.DOI: 10.1039/C4TB00027G.

Costa-Pinto AR, Reis RL, Neves NM. Scaffolds based bone tissue engineering: the role of chitosan. Tissue Eng Part B Rev. 2011;17(5):331-347.DOI: 10.1089/ten.teb.2010.0704.

Bharathi R, Garesh S, Harini G, Vatsala K, Anushika R, Aravind S, et al. Chitosan-based scaffolds as drug delivery systems in bone tissue engineering. Int J Biol Macromol. 2022;222(Pt A):132-153.DOI: 10.1016/j.ijbiomac.2022.09.058.

Sun B, Ma W, Su F, Wang Y, Liu J, Wang D, et al. The osteogenic differentiation of dog bone marrow mesenchymal stem cells in a thermo-sensitive injectable chitosan/collagen/b-glycerophosphate hydrogel: in vitro and in vivo. J Mater Sci Mater Med. 2011;22(9):2111-2118.DOI: 10.1007/s10856-011-4386-4.

Yang M, He S, Su Z, Yang Z, Liang X, and Wu Y. Thermosensitive injectable chitosan/collagen/β-glycerophosphate composite hydrogels for enhancing wound healing by encapsulating mesenchymal stem cell spheroids. ACS Omega. 2020;5(23):21015-21023.DOI: 10.1021/acsomega.0c02580.

Dang Q, Liu K, Zhang Z, Liu C, Liu X, Xin Y, et al. Fabrication and evaluation of thermosensitive chitosan/collagen/-glycerophosphate hydrogels for tissue regeneration. Carbohydr Polym. 2017;167:145-157.DOI: 10.1016/j.carbpol.2017.03.053.

Huang Y, Lin Y, Chen C, Hsieh Y, Liaw C, Huang S, et al. Thermosensitive chitosan-gelatin-glycerol phosphate hydrogels as collagenase carrier for tendon-bone healing in a rabbit model. Polymers. 2020;12(2):436,1-15.DOI: 10.3390/polym12020436.

Kavya KC, Jayakumar R, Nair S, Chennazhi K. Fabrication and characterization of chitosan/gelatin/nSiO2 composite scaffold for bone tissue engineering. Int J Biol Macromol. 2013;59:255-263.DOI: 10.1016/j.ijbiomac.2013.04.023.

Kuttappan S, Mathew D, Nair M. Biomimetic composite scaffolds containing bioceramics and collagen/gelatin for bone tissue engineering- a mini review. Int J Biol Macromol. 2016;93(Pt B):1390-1401.DOI: 10.1016/j.ijbiomac.2016.06.043.

Li X, Huang X, Li L, Wu J, Yi W, Lai Y, et al. LL-37-coupled porous composite scaffold for the treatment of infected segmental bone defect. Pharmaceutics. 2023;15(1):88,1-12.DOI: 10.3390/pharmaceutics15010088.

Ye P, Yang Y, Qu Y, Yang W, Tan J, Zhang Ch, et al. LL-37 and bisphosphonate co-delivery 3D-scaffold with antimicrobial and antiresorptive activities for bone regeneration. Int J Biol Macromol. 2024;277(Pt 1):134091,1-15.DOI: 10.1016/j.ijbiomac.2024.134091.

Akbari V, Rezazadeh M, Ebrahimi Z. Comparison the effects of chitosan and hyaluronic acid-based thermally sensitive hydrogels containing rosuvastatin on human osteoblast-like MG-63 cells. Res Pharm Sci. 2020;15(1):97-106.DOI: 10.4103/1735-5362.278719.

Kim J, Kim I, Hyung Chod T, Lee K, Jung Hwang S, Taec G, et al. Bone regeneration using hyaluronic acid-based hydrogel with bone morphogenic protein-2 and human mesenchymal stem cells. Biomaterials. 2007;28(10):1830-1837.DOI: 10.1016/j.biomaterials.2006.11.050.

Yamaguchi M, Shinbo T, Kanamori T, Wang P, Niwa M, K Hiroyoshi, et al. Surface modification of poly (l-lactic acid) affects initial cell attachment, cell morphology, and cell growth. J Artif Organs. 2004;7(4):187-193.DOI: 10.1007/s10047-004-0267-7.

Dominijanni AJ, Devarasetty M, Forsythe SD, Votanopoulos KI, Soker S. Cell viability assays in three-dimensional hydrogels: a comparative study of accuracy. Tissue Eng Part C Methods. 2021;27(7):401-410.DOI: 10.1089/ten.tec.2021.0060.

Rezazadeh M, Parandeh M, Akbari V, Ebrahimi Z, Taheri A. Incorporation of rosuvastatin-loaded chitosan/chondroitin sulfate nanoparticles into a thermosensitive hydrogel for bone tissue engineering: preparation, characterization, and cellular behavior. Pharm Dev Technol. 2019;24(3):357-367.DOI: 10.1080/10837450.2018.1484765.

Santoro M, Tatara A, Antonios G. Mikos. Gelatin carriers for drug and cell delivery in tissue engineering. J Control Release. 2014;190:210-218.DOI: 10.1016/j.jconrel.2014.04.014.

Li L, Peng Y, Yuan Q, Sun J, Zhuang A, Bi X. Cathelicidin LL37 promotes osteogenic differentiation in vitro and bone regeneration in vivo. Front Bioeng Biotechnol. 2021;9:125,1-11.DOI: 10.3389/fbioe.2021.638494.

Anders E, Dahl S, Svensson D, Nilsson BO. LL-37-induced human osteoblast cytotoxicity and permeability occurs independently of cellular LL-37 uptake through clathrin-mediated endocytosis. Biochem Biophys Res Commun. 2018;501(1):280-285.DOI: 10.1016/j.bbrc.2018.04.235.

Shen X, Al-Baadani M, He H, Cai L, Wu Z, Yao L, et al. Antibacterial and osteogenesis performances of LL37-loaded titania nanopores in vitro and in vivo. Int J Nanomedicine. 2019;14:3043-3054.DOI: 10.2147/IJN.S198583.


Refbacks

  • There are currently no refbacks.


Creative Commons LicenseThis work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.