GelMA Hydrogel Reinforced with 3D Printed PEGT/PBT Scaffolds for Supporting Epigenetically-Activated Human Bone Marrow Stromal Cells for Bone Repair
Abstract
:1. Introduction
2. Materials and Methods
2.1. Macromer Preparation
2.2. GelMA Hydrogel Synthesis
2.3. Three-Dimensional Printing of PEGT/PBT Scaffolds
2.4. Cell Culture and MI192 Treatment
2.5. Compressive Testing of GelMA–PEGT/PBT Constructs
2.6. Proliferation and Viability Assay
2.6.1. Proliferation Assay
2.6.2. Live/Dead Staining
2.7. Osteogenic Induction
2.8. Alkaline Phosphatase-Specific Activity (ALPSA) Assay
2.9. Histological Analysis
2.10. Immunohistochemical Analysis
2.11. Statistical Analysis
3. Results
3.1. GelMA Hydrogels Support the Proliferation and Viability of MI192-Pre-Treated hBMSCs
3.2. MI192 Enhances Osteogenic Differentiation of hBMSCs within GelMA Hydrogels
3.3. MI192 Pre-Treatment Promotes the Extracellular Matrix Mineralisation of hBMSCs within GelMA Hydrogels
3.4. MI192 Induced the Mineralisation of hBMSCs in PEGT/PBT-Reinforced GelMA Hydrogels
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Dimitriou, R.; Jones, E.; McGonagle, D.; Giannoudis, P.V. Bone regeneration: Current concepts and future directions. BMC Med. 2011, 9, 66. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Baroli, B. From natural bone grafts to tissue engineering therapeutics: Brainstorming on pharmaceutical formulative requirements and challenges. J. Pharm. Sci. 2009, 98, 1317–1375. [Google Scholar] [CrossRef] [PubMed]
- Calori, G.M.; Mazza, E.; Colombo, M.; Ripamonti, C. The use of bone-graft substitutes in large bone defects: Any specific needs? Injury 2011, 42, S56–S63. [Google Scholar] [CrossRef] [PubMed]
- Shegarfi, H.; Reikeras, O. Review article: Bone transplantation and immune response. J. Orthop. Surg. (Hong Kong) 2009, 17, 206–211. [Google Scholar] [CrossRef]
- Amini, A.R.; Laurencin, C.T.; Nukavarapu, S.P. Bone tissue engineering: Recent advances and challenges. Crit. Rev. Biomed. Eng. 2012, 40, 363–408. [Google Scholar] [CrossRef] [Green Version]
- Howard, D.; Buttery, L.D.; Shakesheff, K.M.; Roberts, S.J. Tissue engineering: Strategies, stem cells and scaffolds. J. Anat. 2008, 213, 66–72. [Google Scholar] [CrossRef]
- Amariglio, N.; Hirshberg, A.; Scheithauer, B.W.; Cohen, Y.; Loewenthal, R.; Trakhtenbrot, L.; Paz, N.; Koren-Michowitz, M.; Waldman, D.; Leider-Trejo, L.; et al. Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient. PLoS Med. 2009, 6, 221–231. [Google Scholar] [CrossRef]
- Herberts, C.A.; Kwa, M.S.G.; Hermsen, H.P.H. Risk factors in the development of stem cell therapy. J. Transl. Med. 2011, 9, 29. [Google Scholar] [CrossRef] [Green Version]
- Moschidou, D.; Mukherjee, S.; Blundell, M.P.; Drews, K.; Jones, G.N.; Abdulrazzak, H.; Nowakowska, B.; Phoolchund, A.; Lay, K.; Ramasamy, T.S.; et al. Valproic acid confers functional pluripotency to human amniotic fluid stem cells in a transgene-free approach. Mol. Ther. 2012, 20, 1953–1967. [Google Scholar] [CrossRef] [Green Version]
- Lunyak, V.V.; Rosenfeld, M.G. Epigenetic regulation of stem cell fate. Hum. Mol. Genet. 2008, 17, R28–R36. [Google Scholar] [CrossRef]
- Huynh, N.C.; Everts, V.; Ampornaramveth, R.S. Histone deacetylases and their roles in mineralized tissue regeneration. Bone Rep. 2017, 7, 33–40. [Google Scholar] [CrossRef] [PubMed]
- Duncan, H.F.; Smith, A.J.; Fleming, G.J.P.; Cooper, P.R. Hdaci: Cellular effects, opportunities for restorative dentistry. J. Dent. Res. 2011, 90, 1377–1388. [Google Scholar] [CrossRef] [PubMed]
- Paino, F.; La Noce, M.; Tirino, V.; Naddeo, P.; Desiderio, V.; Pirozzi, G.; De Rosa, A.; Laino, L.; Altucci, L.; Papaccio, G. Histone deacetylase inhibition with valproic acid downregulates osteocalcin gene expression in human dental pulp stem cells and osteoblasts: Evidence for hdac2 involvement. Stem Cells 2014, 32, 279–289. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Xu, S.; De Veirman, K.; Evans, H.; Santini, G.C.; Vande Broek, I.; Leleu, X.; De Becker, A.; Van Camp, B.; Croucher, P.; Vanderkerken, K.; et al. Effect of the hdac inhibitor vorinostat on the osteogenic differentiation of mesenchymal stem cells in vitro and bone formation in vivo. Acta Pharmacol. Sin. 2013, 34, 699–709. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Man, K.; Brunet, M.Y.; Fernandez-Rhodes, M.; Williams, S.; Heaney, L.M.; Gethings, L.A.; Federici, A.; Davies, O.G.; Hoey, D.; Cox, S.C. Epigenetic reprogramming enhances the therapeutic efficacy of osteoblast-derived extracellular vesicles to promote human bone marrow stem cell osteogenic differentiation. J. Extracell. Vesicles 2021, 10, e12118. [Google Scholar] [CrossRef]
- Lawlor, L. The Effect of Hdac Inhibitor mi192 on Stem Cell Behaviour-The Potential of Utilising mi192 for Bone Tissue Engineering. Ph.D. Thesis, University of Leeds, Leeds, UK, 2016. [Google Scholar]
- Man, K.; Lawlor, L.; Jiang, L.-H.; Yang, X.B. The selective histone deacetylase inhibitor mi192 enhances the osteogenic differentiation efficacy of human dental pulp stromal cells. Int. J. Mol. Sci. 2021, 22, 5224. [Google Scholar] [CrossRef] [PubMed]
- Bai, X.; Gao, M.Z.; Syed, S.; Zhuang, J.; Xu, X.Y.; Zhang, X.Q. Bioactive hydrogels for bone regeneration. Bioact. Mater. 2018, 3, 401–417. [Google Scholar] [CrossRef]
- Yue, S.; He, H.; Li, B.; Hou, T. Hydrogel as a biomaterial for bone tissue engineering: A review. Nanomaterials 2020, 10, 1511. [Google Scholar] [CrossRef]
- Nichol, J.W.; Koshy, S.T.; Bae, H.; Hwang, C.M.; Yamanlar, S.; Khademhosseini, A. Cell-laden microengineered gelatin methacrylate hydrogels. Biomaterials 2010, 31, 5536–5544. [Google Scholar] [CrossRef] [Green Version]
- Van den Bulcke, A.I.; Bogdanov, B.; De Rooze, N.; Schacht, E.H.; Cornelissen, M.; Berghmans, H. Structural and rheological properties of methacrylamide modified gelatin hydrogels. Biomacromolecules 2000, 1, 31–38. [Google Scholar] [CrossRef]
- Loessner, D.; Meinert, C.; Kaemmerer, E.; Martine, L.C.; Yue, K.; Levett, P.A.; Klein, T.J.; Melchels, F.P.W.; Khademhosseini, A.; Hutmacher, D.W. Functionalization, preparation and use of cell-laden gelatin methacryloyl-based hydrogels as modular tissue culture platforms. Nat. Protoc. 2016, 11, 727–746. [Google Scholar] [CrossRef] [Green Version]
- Cidonio, G.; Alcala-Orozco, C.R.; Lim, K.S.; Glinka, M.; Mutreja, I.; Kim, Y.H.; Dawson, J.I.; Woodfield, T.B.F.; Oreffo, R.O.C. Osteogenic and angiogenic tissue formation in high fidelity nanocomposite laponite-gelatin bioinks. Biofabrication 2019, 11, 035027. [Google Scholar] [CrossRef]
- Zoratto, N.; Di Lisa, D.; de Rutte, J.; Sakib, M.N.; Silva, A.R.A.E.; Tamayol, A.; Di Carlo, D.; Khademhosseini, A.; Sheikhi, A. In situ forming microporous gelatin methacryloyl hydrogel scaffolds from thermostable microgels for tissue engineering. Bioeng. Transl. Med. 2020, 5, e10180. [Google Scholar] [CrossRef]
- Lim, K.S.; Schon, B.S.; Mekhileri, N.V.; Brown, G.C.J.; Chia, C.M.; Prabakar, S.; Hooper, G.J.; Woodfield, T.B.F. New visible-light photoinitiating system for improved print fidelity in gelatin-based bioinks. ACS Biomater. Sci. Eng. 2016, 2, 1752–1762. [Google Scholar] [CrossRef] [PubMed]
- Kessler, L.; Gehrke, S.; Winnefeld, M.; Huber, B.; Hoch, E.; Walter, T.; Wyrwa, R.; Schnabelrauch, M.; Schmidt, M.; Kuckelhaus, M.; et al. Methacrylated gelatin/hyaluronan-based hydrogels for soft tissue engineering. J. Tissue Eng. 2017, 8, 2041731417744157. [Google Scholar] [CrossRef] [PubMed]
- Schuurman, W.; Levett, P.A.; Pot, M.W.; van Weeren, P.R.; Dhert, W.J.A.; Hutmacher, D.W.; Melchels, F.P.W.; Klein, T.J.; Malda, J. Gelatin-methacrylamide hydrogels as potential biomaterials for fabrication of tissue-engineered cartilage constructs. Macromol. Biosci. 2013, 13, 551–561. [Google Scholar] [CrossRef]
- Brown, G.C.J.; Lim, K.S.; Farrugia, B.L.; Hooper, G.J.; Woodfield, T.B.F. Covalent incorporation of heparin improves chondrogenesis in photocurable gelatin-methacryloyl hydrogels. Macromol. Biosci. 2017, 17, 1700158. [Google Scholar] [CrossRef] [PubMed]
- Malda, J.; Visser, J.; Melchels, F.P.; Jungst, T.; Hennink, W.E.; Dhert, W.J.A.; Groll, J.; Hutmacher, D.W. 25th anniversary article: Engineering hydrogels for biofabrication. Adv. Mater. 2013, 25, 5011–5028. [Google Scholar] [CrossRef] [PubMed]
- Celikkin, N.; Mastrogiacomo, S.; Jaroszewicz, J.; Walboomers, X.F.; Swieszkowski, W. Gelatin methacrylate scaffold for bone tissue engineering: The influence of polymer concentration. J. Biomed. Mater. Res. A 2018, 106, 201–209. [Google Scholar] [CrossRef] [PubMed]
- Schon, B.S.; Schrobback, K.; van der Ven, M.; Stroebel, S.; Hooper, G.J.; Woodfield, T.B.F. Validation of a high-throughput microtissue fabrication process for3Dassembly of tissue engineered cartilage constructs. Cell Tissue Res. 2012, 347, 629–642. [Google Scholar] [CrossRef]
- Mekhileri, N.V.; Lim, K.S.; Brown, G.C.J.; Mutreja, I.; Schon, B.S.; Hooper, G.J.; Woodfield, T.B.F. Automated3Dbioassembly of micro-tissues for biofabrication of hybrid tissue engineered constructs. Biofabrication 2018, 10, 024103. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Man, K.; Mekhileri, N.; Lim, K.; Jiang, L.H.; Woodfield, T.; Yang, X.B. Mi192 induced epigenetic reprogramming enhances the therapeutic efficacy of human bone marrows stromal cells for bone regeneration. Bone 2021, 153, 116138. [Google Scholar] [CrossRef] [PubMed]
- Man, K.; Barroso, I.A.; Brunet, M.Y.; Federici, A.S.; Peacock, B.; Hoey, D.A.; Cox, S.C. Controlled release of epigenetically-enhanced extracellular vesicles from a gelma/nanoclay composite hydrogel to promote bone repair. Int. J. Mol. Sci. 2022, 23, 832. [Google Scholar] [CrossRef] [PubMed]
- Yang, X.B.; Webb, D.; Blaker, J.; Boccaccini, A.R.; Maquet, V.; Cooper, C.; Oreffo, R.O. Evaluation of human bone marrow stromal cell growth on biodegradable polymer/bioglass composites. Biochem. Biophys. Res. Commun. 2006, 342, 1098–1107. [Google Scholar] [CrossRef]
- Maroni, P.; Brini, A.T.; Arrigoni, E.; de Girolamo, L.; Niada, S.; Matteucci, E.; Bendinelli, P.; Desiderio, M.A. Chemical and genetic blockade of hdacs enhances osteogenic differentiation of human adipose tissue-derived stem cells by oppositely affecting osteogenic and adipogenic transcription factors. Biochem. Biophys. Res. Commun. 2012, 428, 271–277. [Google Scholar] [CrossRef]
- Hu, X.Q.; Zhang, X.; Dai, L.H.; Zhu, J.X.; Jia, Z.Q.; Wang, W.P.; Zhou, C.Y.; Ao, Y.F. Histone deacetylase inhibitor trichostatin a promotes the osteogenic differentiation of rat adipose-derived stem cells by altering the epigenetic modifications on runx2 promoter in a bmp signaling-dependent manner. Stem Cells Dev. 2013, 22, 248–255. [Google Scholar] [CrossRef]
- Lai, J.Y.; Li, Y.T. Functional assessment of cross-linked porous gelatin hydrogels for bioengineered cell sheet carriers. Biomacromolecules 2010, 11, 1387–1397. [Google Scholar] [CrossRef]
- Barroso, I.A.; Man, K.; Robinson, T.E.; Cox, S.C.; Ghag, A.K. Photocurable gelma adhesives for corneal perforations. Bioengineering 2022, 9, 53. [Google Scholar] [CrossRef]
- Bertlein, S.; Brown, G.; Lim, K.S.; Jungst, T.; Boeck, T.; Blunk, T.; Tessmar, J.; Hooper, G.J.; Woodfield, T.B.F.; Groll, J. Thiol-ene clickable gelatin: A platform bioink for multiple3Dbiofabrication technologies. Adv. Mater. 2017, 29, 1703404. [Google Scholar] [CrossRef]
- Noshadi, I.; Hong, S.; Sullivan, K.E.; Sani, E.S.; Portillo-Lara, R.; Tamayol, A.; Shin, S.R.; Gao, A.E.; Stoppel, W.L.; Black, L.D.; et al. In vitro and in vivo analysis of visible light crosslinkable gelatin methacryloyl (gelma) hydrogels. Biomater. Sci. 2017, 5, 2093–2105. [Google Scholar] [CrossRef]
- Fang, X.X.; Xie, J.; Zhong, L.X.; Li, J.R.; Rong, D.M.; Li, X.S.; Ouyang, J. Biomimetic gelatin methacrylamide hydrogel scaffolds for bone tissue engineering. J. Mater. Chem. B 2016, 4, 1070–1080. [Google Scholar] [CrossRef] [PubMed]
- Lin, X.; Patil, S.; Gao, Y.G.; Qian, A.R. The bone extracellular matrix in bone formation and regeneration. Front. Pharmacol. 2020, 11, 757. [Google Scholar] [CrossRef] [PubMed]
- Carvalho, M.S.; Cabral, J.M.S.; da Silva, C.L.; Vashishth, D. Bone matrix non-collagenous proteins in tissue engineering: Creating new bone by mimicking the extracellular matrix. Polymers 2021, 13, 1095. [Google Scholar] [CrossRef] [PubMed]
- Boissinot, M.; Inman, M.; Hempshall, A.; James, S.R.; Gill, J.H.; Selby, P.; Bowen, D.T.; Grigg, R.; Cockerill, P.N. Induction of differentiation and apoptosis in leukaemic cell lines by the novel benzamide family histone deacetylase 2 and 3 inhibitor mi-192. Leuk. Res. 2012, 36, 1304–1310. [Google Scholar] [CrossRef]
- Tozzi, G.; De Mori, A.; Oliveira, A.; Roldo, M. Composite hydrogels for bone regeneration. Materials 2016, 9, 267. [Google Scholar] [CrossRef] [Green Version]
- Xavier, J.R.; Thakur, T.; Desai, P.; Jaiswal, M.K.; Sears, N.; Cosgriff-Hernandez, E.; Kaunas, R.; Gaharwar, A.K. Bioactive nanoengineered hydrogels for bone tissue engineering: A growth-factor-free approach. Acs Nano 2015, 9, 3109–3118. [Google Scholar] [CrossRef]
- Paul, A.; Manoharan, V.; Krafft, D.; Assmann, A.; Uquillas, J.A.; Shin, S.R.; Hasan, A.; Hussain, M.A.; Memic, A.; Gaharwar, A.K.; et al. Nanoengineered biomimetic hydrogels for guiding human stem cell osteogenesis in three dimensional microenvironments. J. Mater. Chem. B 2016, 4, 3544–3554. [Google Scholar] [CrossRef] [Green Version]
- Liu, B.; Li, J.Q.; Lei, X.; Miao, S.; Zhang, S.S.; Cheng, P.Z.; Song, Y.; Wu, H.; Gao, Y.; Bi, L.; et al. Cell-loaded injectable gelatin/alginate/laponite (r) nanocomposite hydrogel promotes bone healing in a critical-size rat calvarial defect model. Rsc. Adv. 2020, 10, 25652–25661. [Google Scholar] [CrossRef]
- Sakkers, R.J.; de Wijn, J.R.; Dalmeyer, R.A.; van Blitterswijk, C.A.; Brand, R. Evaluation of copolymers of polyethylene oxide and polybutylene terephthalate (polyactive): Mechanical behaviour. J. Mater. Sci. Mater. Med. 1998, 9, 375–379. [Google Scholar] [CrossRef]
- Deschamps, A.A.; Claase, M.B.; Sleijster, W.J.; de Bruijn, J.D.; Grijpma, D.W.; Feijen, J. Design of segmented poly(ether ester) materials and structures for the tissue engineering of bone. J. Control. Release 2002, 78, 175–186. [Google Scholar] [CrossRef]
- Galarraga, J.H.; Locke, R.C.; Witherel, C.E.; Stoeckl, B.D.; Castilho, M.; Mauck, R.L.; Malda, J.; Levato, R.; Burdick, J.A. Fabrication of msc-laden composites of hyaluronic acid hydrogels reinforced with mew scaffolds for cartilage repair. Biofabrication 2021, 14, 014106. [Google Scholar] [CrossRef] [PubMed]
- Sun, M.; Chi, G.; Xu, J.; Tan, Y.; Xu, J.; Lv, S.; Xu, Z.; Xia, Y.; Li, L.; Li, Y. Extracellular matrix stiffness controls osteogenic differentiation of mesenchymal stem cells mediated by integrin alpha5. Stem Cell Res. Ther. 2018, 9, 52. [Google Scholar] [CrossRef] [PubMed]
- Ghasemi-Mobarakeh, L.; Prabhakaran, M.P.; Tian, L.; Shamirzaei-Jeshvaghani, E.; Dehghani, L.; Ramakrishna, S. Structural properties of scaffolds: Crucial parameters towards stem cells differentiation. World J. Stem Cells 2015, 7, 728–744. [Google Scholar] [CrossRef] [PubMed]
- Li, Y.; Chu, J.S.; Kurpinski, K.; Li, X.; Bautista, D.M.; Yang, L.; Sung, K.L.P.; Li, S. Biophysical regulation of histone acetylation in mesenchymal stem cells. Biophys. J. 2011, 100, 1902–1909. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lv, L.; Tang, Y.; Zhang, P.; Liu, Y.; Bai, X.; Zhou, Y. Biomaterial cues regulate epigenetic state and cell functions-a systematic review. Tissue Eng. Part B Rev. 2018, 24, 112–132. [Google Scholar] [CrossRef]
- Man, K.; Brunet, M.Y.; Louth, S.; Robinson, T.E.; Fernandez-Rhodes, M.; Williams, S.; Federici, A.S.; Davies, O.G.; Hoey, D.A.; Cox, S.C. Development of a bone-mimetic3Dprinted ti6al4v scaffold to enhance osteoblast-derived extracellular vesicles’ therapeutic efficacy for bone regeneration. Front. Bioeng. Biotechnol. 2021, 9, 757220. [Google Scholar] [CrossRef] [PubMed]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Man, K.; Alcala, C.; Mekhileri, N.V.; Lim, K.S.; Jiang, L.-H.; Woodfield, T.B.F.; Yang, X.B. GelMA Hydrogel Reinforced with 3D Printed PEGT/PBT Scaffolds for Supporting Epigenetically-Activated Human Bone Marrow Stromal Cells for Bone Repair. J. Funct. Biomater. 2022, 13, 41. https://doi.org/10.3390/jfb13020041
Man K, Alcala C, Mekhileri NV, Lim KS, Jiang L-H, Woodfield TBF, Yang XB. GelMA Hydrogel Reinforced with 3D Printed PEGT/PBT Scaffolds for Supporting Epigenetically-Activated Human Bone Marrow Stromal Cells for Bone Repair. Journal of Functional Biomaterials. 2022; 13(2):41. https://doi.org/10.3390/jfb13020041
Chicago/Turabian StyleMan, Kenny, Cesar Alcala, Naveen V. Mekhileri, Khoon S. Lim, Lin-Hua Jiang, Tim B. F. Woodfield, and Xuebin B. Yang. 2022. "GelMA Hydrogel Reinforced with 3D Printed PEGT/PBT Scaffolds for Supporting Epigenetically-Activated Human Bone Marrow Stromal Cells for Bone Repair" Journal of Functional Biomaterials 13, no. 2: 41. https://doi.org/10.3390/jfb13020041