Bone tissue engineering strategy involves the 3D scaffolds and appropriate cell types promoting the replacement of the damaged area. In this work, we aimed to develop a fast and reliable clinically relevant protocol for engineering viable bone grafts, using cryopreserved adipose tissue-derived mesenchymal stromal cells (MSCs) and composite 3D collagen-nano-hydroxyapatite (nanoHA) scaffolds. Xeno- and DMSO-free cryopreserved MSCs were perfusion-seeded into the biomimetic collagen/nanoHA scaffolds manufactured by cryotropic gelation and their osteoregenerative potential was assessed in vitro and in vivo. Cryopreserved MSCs retained the ability to homogenously repopulate the whole volume of the scaffolds during 7 days of post-thaw culture. Moreover, the scaffold provided a suitable microenvironment for induced osteogenic differentiation of cells, confirmed by alkaline phosphatase activity and mineralization. Implantation of collagen-nanoHA cryogels with cryopreserved MSCs accelerated woven bone tissue formation, maturation of bone trabeculae, and vascularization of femur defects in immunosuppressed rats compared to cell-free collagen-nanoHA scaffolds. The established combination of xeno-free cell culture and cryopreservation techniques together with an appropriate scaffold design and cell repopulation approach accelerated the generation of viable bone grafts.

Biochemistry department 5 N Karazin Kharkiv National University Kharkiv Ukraine

Biochemistry department Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine Kharkiv Ukraine

Laboratory for Cryochemistry of BioPolymers A N Nesmeyanov Institute of Organoelement Compounds Russian Academy of Sciences Moscow Russian Federation

Laboratory of Connective Tissue Morphology Department of transplantology and experimental modeling with an experimental biological clinic Department of Joint Pathology Sytenko Institute of Spine and Joint Pathology National Academy of Medical Sciences of Ukraine Kharkiv Ukraine

Neuroregeneration department Institute of Experimental Medicine of the Czech Academy of Sciences Prague Czech Republic

See more in PubMed

Caddeo S, Boffito M, Sartori S. Tissue engineering approaches in the design of healthy and pathological in vitro tissue models. Front Bioeng Biotechnol. 2017;5(Aug):40. https://doi.org/10.3389/fbioe.2017.00040

Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res. 2007;100(9):1249-1260. https://doi.org/10.1161/01.RES.0000265074.83288.09

Rogulska O, Petrenko Y, Petrenko A. DMSO-free cryopreservation of adipose-derived mesenchymal stromal cells: expansion medium affects post-thaw survival. Cytotechnology. 2017;69(2):265-276. https://doi.org/10.1007/s10616-016-0055-2

Weng L, Beauchesne PR. Dimethyl sulfoxide-free cryopreservation for cell therapy: a review. Cryobiology. 2020;94:9-17. https://doi.org/10.1016/j.cryobiol.2020.03.012

Awan M, Buriak I, Fleck R, et al. Dimethyl sulfoxide: a central player since the dawn of cryobiology, is efficacy balanced by toxicity? Regen Med. 2020;15(3):1463-1491. https://doi.org/10.2217/rme-2019-0145

Wang W, Yeung KWK. Bone grafts and biomaterials substitutes for bone defect repair: a review. Bioact Mater. 2017;2(4):224-247. https://doi.org/10.1016/j.bioactmat.2017.05.007

Sayin E, Rashid RH, Rodríguez-Cabello JC, Elsheikh A, Baran ET, Hasirci V. Human adipose derived stem cells are superior to human osteoblasts (HOB) in bone tissue engineering on a collagen-fibroin-ELR blend. Bioact Mater. 2017;2(2):71-81. https://doi.org/10.1016/j.bioactmat.2017.04.001

Mizuno M, Fujisawa R, Kuboki Y. Type I collagen-induced osteoblastic differentiation of bone-marrow cells mediated by collagen-?2?1 integrin interaction. J Cell Physiol. 2000;184(2):207-213. https://doi.org/10.1002/1097-4652(200008)184:2<207::AID-JCP8>3.0.CO;2-U

Chattopadhyay S, Raines RT. Review collagen-based biomaterials for wound healing. Biopolymers. 2014;101(8):821-833. https://doi.org/10.1002/bip.22486

Uota M, Arakawa H, Kitamura N, Yoshimura T, Tanaka J, Kijima T. Synthesis of high surface area hydroxyapatite nanoparticles by mixed surfactant-mediated approach. Langmuir. 2005;21(10):4724-4728. https://doi.org/10.1021/la050029m

Hayrapetyan A, Bongio M, Leeuwenburgh SCG, Jansen JA, van den Beucken JJJP. Effect of nano-HA/collagen composite hydrogels on Osteogenic behavior of mesenchymal stromal cells. Stem Cell Rev Reports. 2016;12(3):352-364. https://doi.org/10.1007/s12015-016-9644-x

Lickorish D, Ramshaw JAM, Werkmeister JA, Glattauer V, Howlett CR. Collagen-hydroxyapatite composite prepared by biomimetic process. J Biomed Mater Res. 2004;68A(1):19-27. https://doi.org/10.1002/jbm.a.20031

Salgado CL, Grenho L, Fernandes MH, Colaço BJ, Monteiro FJ. Biodegradation, biocompatibility, and osteoconduction evaluation of collagen-nanohydroxyapatite cryogels for bone tissue regeneration. J Biomed Mater Res Part A. 2016;104(1):57-70. https://doi.org/10.1002/jbm.a.35540

Liu Z, Yin X, Ye Q, et al. Periodontal regeneration with stem cells-seeded collagen-hydroxyapatite scaffold. J Biomater Appl. 2016;31(1):121-131. https://doi.org/10.1177/0885328216637978

Chocholata P, Kulda V, Babuska V. Fabrication of scaffolds for bone-tissue regeneration. Materials (Basel). 2019;12(4):568. https://doi.org/10.3390/ma12040568

Eltom A, Zhong G, Muhammad A. Scaffold techniques and designs in tissue engineering functions and purposes: a review. Adv Mater Sci Eng. 2019;2019:1-13. https://doi.org/10.1155/2019/3429527

Lozinsky VI. Cryostructuring of polymeric systems. 55. Retrospective view on the more than 40 years of studies performed in the A.N.Nesmeyanov institute of organoelement compounds with respect of the cryostructuring processes in polymeric systems. Gels. 2020;6(3):1-59. https://doi.org/10.3390/gels6030029

Lozinsky VI. Polymeric cryogels as a new family of macroporous and supermacroporous materials for biotechnological purposes. Russ Chem Bull. 2008;57(5):1015-1032. https://doi.org/10.1007/s11172-008-0131-7

Hixon KR, Lu T, Sell SA. A comprehensive review of cryogels and their roles in tissue engineering applications. Acta Biomater. 2017;62:29-41. https://doi.org/10.1016/j.actbio.2017.08.033

Razavi M, Qiao Y, Thakor AS. Three-dimensional cryogels for biomedical applications. J Biomed Mater Res Part A. 2019;107(12):2736-2755. https://doi.org/10.1002/jbm.a.36777

Petrenko YA, Ivanov RV, Petrenko AY, Lozinsky VI. Coupling of gelatin to inner surfaces of pore walls in spongy alginate-based scaffolds facilitates the adhesion, growth and differentiation of human bone marrow mesenchymal stromal cells. J Mater Sci Mater Med. 2011;22(6):1529-1540. https://doi.org/10.1007/s10856-011-4323-6

Katsen-Globa A, Meiser I, Petrenko YA, et al. Towards ready-to-use 3-D scaffolds for regenerative medicine: adhesion-based cryopreservation of human mesenchymal stem cells attached and spread within alginate-gelatin cryogel scaffolds. J Mater Sci Mater Med. 2014;25(3):857-871. https://doi.org/10.1007/s10856-013-5108-x

Lozinsky VI, Kulakova VK, Ivanov RV, Petrenko AY, Rogulska OY, Petrenko YA. Cryostructuring of polymer systems. 47. Preparation of wide porous gelatin-based cryostructurates in sterilizing organic media and assessment of the suitability of thus formed matrices as spongy scaffolds for 3D cell culturing. e-Polymers. 2018;18(2):175-186. https://doi.org/10.1515/epoly-2017-0151

Raina DB, Isaksson H, Teotia AK, Lidgren L, Tägil M, Kumar A. Biocomposite macroporous cryogels as potential carrier scaffolds for bone active agents augmenting bone regeneration. J Control Release. 2016;235:365-378. https://doi.org/10.1016/j.jconrel.2016.05.061

Rodrigues SC, Salgado CL, Sahu A, Garcia MP, Fernandes MH, Monteiro FJ. Preparation and characterization of collagen-nanohydroxyapatite biocomposite scaffolds by cryogelation method for bone tissue engineering applications. J Biomed Mater Res Part A. 2013;101A(4):1080-1094. https://doi.org/10.1002/jbm.a.34394

Petrenko YA, Ivanov RV, Lozinsky VI, Petrenko AY. Comparison of the methods for seeding human bone marrow mesenchymal stem cells to macroporous alginate cryogel carriers. Bull Exp Biol Med. 2011;150(4):543-546. https://doi.org/10.1007/s10517-011-1185-3

Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells Raff M, ed. Mol Biol Cell. 2002;13(12):4279-4295. https://doi.org/10.1091/mbc.e02-02-0105

Blande IS, Bassaneze V, Lavini-Ramos C, et al. Transplantation and cellular engineering: adipose tissue mesenchymal stem cell expansion in animal serum-free medium supplemented with autologous human platelet lysate. Transfusion. 2009;49(12):2680-2685. https://doi.org/10.1111/j.1537-2995.2009.02346.x

Dankberg F, Persidsky MD. A test of granulocyte membrane integrity and phagocytic function. Cryobiology. 1976;13(4):430-432. https://doi.org/10.1016/0011-2240(76)90098-5

Rampersad SN. Multiple applications of Alamar blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors. 2012;12(9):12347-12360. https://doi.org/10.3390/s120912347

Morris SM, Kepka-Lenhart D, McGill RL, Curthoys NP, Adler S. Specific disruption of renal function and gene transcription by cyclosporin A. J Biol Chem 1992;267(19):13768-13771. https://pubmed.ncbi.nlm.nih.gov/1618871/. Accessed February 6, 2021.

Rittié L. Method for picrosirius red-polarization detection of collagen fibers in tissue sections. Methods in Molecular Biology. Vol 1627. New York: Humana Press; 2017:395-407. https://doi.org/10.1007/978-1-4939-7113-8_26

Podorozhko EA, Kurskaya EA, Kulakova VK, Lozinsky VI. Cryotropic structuring of aqueous dispersions of fibrous collagen: influence of the initial pH values. Food Hydrocolloids, 2000;14(2):111-120. https://doi.org/10.1016/s0268-005x(99)00054-5.

Becherucci V, Piccini L, Casamassima S, et al. Human platelet lysate in mesenchymal stromal cell expansion according to a GMP grade protocol: a cell factory experience. Stem Cell Res Ther. 2018;9(1):124. https://doi.org/10.1186/s13287-018-0863-8

Petrenko YA, Rogulska OY, Mutsenko V V, Petrenko AY. A sugar pretreatment as a new approach to the Me2SO- and xeno-free cryopreservation of human mesenchymal stromal cells. Cryo Letters 2014;35(3):239-246. https://pubmed.ncbi.nlm.nih.gov/24997842/. Accessed February 6, 2021.

Rogulska O, Tykhvynska O, Revenko O, et al. Novel cryopreservation approach providing off-the-shelf availability of human multipotent Mesenchymal stromal cells for clinical applications. Stem Cells Int. 2019;2019:1-11. https://doi.org/10.1155/2019/4150690

Trivedi S, Srivastava K, Saluja TS, et al. Hydroxyapatite-collagen augments osteogenic differentiation of dental pulp stem cells. Odontology. 2020;108(2):251-259. https://doi.org/10.1007/s10266-019-00464-0

Bou Assaf R, Fayyad-Kazan M, Al-Nemer F, et al. Evaluation of the osteogenic potential of different scaffolds embedded with human stem cells originated from Schneiderian membrane: an in vitro study. Biomed Res Int. 2019;2019:1-10. https://doi.org/10.1155/2019/2868673

Bhuiyan DB, Middleton JC, Tannenbaum R, Wick TM. Bone regeneration from human mesenchymal stem cells on porous hydroxyapatite-PLGA-collagen bioactive polymer scaffolds. Biomed Mater Eng. 2017;28(6):671-685. https://doi.org/10.3233/BME-171703

Yang X, Li Y, He W, Huang Q, Zhang R, Feng Q. Hydroxyapatite/collagen coating on PLGA electrospun fibers for osteogenic differentiation of bone marrow mesenchymal stem cells. J Biomed Mater Res Part A. 2018;106(11):2863-2870. https://doi.org/10.1002/jbm.a.36475

Wu S, Xiao Z, Song J, Li M, Li W. Evaluation of BMP-2 enhances the osteoblast differentiation of human amnion mesenchymal stem cells seeded on nano-hydroxyapatite/collagen/poly(l-Lactide). Int J Mol Sci. 2018;19(8):2171. https://doi.org/10.3390/ijms19082171

Gigante A, Manzotti S, Bevilacqua C, Orciani M, Di Primio R, Mattioli-Belmonte M. Adult mesenchymal stem cells for bone and cartilage engineering: effect of scaffold materials. Eur J Histochem. 2009;52(3):169-174. https://doi.org/10.4081/1208

Calabrese G, Giuffrida R, Fabbi C, et al. Collagen-hydroxyapatite scaffolds induce human adipose derived stem cells Osteogenic differentiation in vitro. PLoS ONE. 2016;11(3):e0151181. https://doi.org/10.1371/journal.pone.0151181