Silk fibroin (SF) has garnered significant attention as a natural polymer for fabricating porous scaffolds in various engineering applications. However, the limited osteoinductive property of SF has hindered its efficacy in bone repair applications. In this study, we constructed an SF-based injectable porous microcarrier that is doped with laponite (LAP), containing magnesium ions (Mg2+). The influence of freezing temperatures and concentrations of SF and LAP on the structural parameters of SF-LAP microcarriers was investigated. The SF-LAP microcarrier exhibited a porosity of 76.7 ± 1.2% and a controlled pore size of 24.6 ± 4.0 μm. At the 6 weeks of in vitro degradation test, a mild alkaline level in culture medium containing SF-LAP microcarriers was detected. The release of Mg2+ from the SF-LAP microcarrier was maintained at a concentration within the range of 1.2–2.3 mM during the 6 weeks. The seeded human adipose-derived stem cells in the SF-LAP microcarrier demonstrated a significant enhancement in osteogenic differentiation compared with cells seeded in the pure SF microcarrier, as evidenced by quantitative alkaline phosphatase activity and the expression of osteogenic marker genes. These findings underscore the potential of the SF-LAP microcarrier as an ideal cell carrier in the treatment of bone defects.
Impact Statement
Mg alloys and Mg-incorporated biodegradable implants have been widely applied in the repair of bone defects. This work aims at fabricating the SF microcarrier-LAP, which exhibited good biocompatibility and pro-osteogenic properties while avoiding the detrimental effect on osteogenesis caused by the accumulated high concentration of Mg2+ generated with the degradation of magnesium-containing biomaterials. This SF-LAP microcarrier shows potential application as a cell-delivering vehicle in bone tissue engineering.
Get full access to this article
View all access options for this article.
References
1.
KimHD, AmirthalingamS, KimSL, et al.Biomimetic materials and fabrication approaches for bone tissue engineering. Adv Healthc Mater, 2017; 6(23):1700612; doi: 10.1002/adhm.201700612
2.
BunpetchV, ZhangZY, ZhangX, et al.Strategies for MSC expansion and MSC-based microtissue for bone regeneration. Biomaterials, 2019; 196:67–79; doi: 10.1016/j.biomaterials.2017.11.023
3.
HandralHK, WyrobnikTA, LamAT. Emerging trends in biodegradable microcarriers for therapeutic applications. Polymers (Basel), 2023; 15(6):1487; doi: 10.3390/polym15061487
4.
ZhouZ, WuW, FangJ, et al.Polymer-based porous microcarriers as cell delivery systems for applications in bone and cartilage tissue engineering. Int Mater Rev, 2021; 66(2):77–113; doi: 10.1080/09506608.2020.1724705
Perucca OrfeiC, TalòG, ViganòM, et al.Silk/Fibroin microcarriers for mesenchymal stem cell delivery: Optimization of cell seeding by the design of experiment. Pharmaceutics, 2018; 10(4):200; doi: 10.3390/pharmaceutics10040200
7.
FangJ, WangD, HuF, et al.Strontium mineralized silk fibroin porous microcarriers with enhanced osteogenesis as injectable bone tissue engineering vehicles. Mater Sci Eng C Mater Biol Appl, 2021; 128:112354; doi: 10.1016/j.msec.2021.112354
8.
LuetchfordKA, ChaudhuriJB, De BankPA. Silk fibroin/gelatin microcarriers as scaffolds for bone tissue engineering. Mater Sci Eng C Mater Biol Appl, 2020; 106:110116; doi: 10.1016/j.msec.2019.110116
9.
VeigaA, CastroF, RochaF, et al.Silk-based microcarriers: Current developments and future perspectives. IET Nanobiotechnol, 2020; 14(8):645–653; doi: 10.1049/iet-nbt.2020.0058
10.
WangQ, ZhangY, LiB, et al.Controlled dual delivery of low doses of BMP-2 and VEGF in a silk fibroin-nanohydroxyapatite scaffold for vascularized bone regeneration. J Mater Chem B, 2017; 5(33):6963–6972; doi: 10.1039/c7tb00949f
11.
BessaPC, BalmayorER, AzevedoHS, et al.Silk fibroin microparticles as carriers for delivery of human recombinant BMPs. Physical characterization and drug release. J Tissue Eng Regen Med, 2010; 4(5):349–355; doi: 10.1002/term.245
12.
Ho-Shui-LingA, BolanderJ, RustomLE, et al.Bone regeneration strategies: Engineered scaffolds, bioactive molecules and stem cells current stage and future perspectives. Biomaterials, 2018; 180:143–162; doi: 10.1016/j.biomaterials.2018.07.017
13.
SarisNE, MervaalaE, KarppanenH, et al.Magnesium. An update on physiological, clinical and analytical aspects. Clin Chim Acta, 2000; 294(1–2):1–26; doi: 10.1016/s0009-8981(99)00258-2
14.
KianiF, WenC, LiY. Prospects and strategies for magnesium alloys as biodegradable implants from crystalline to bulk metallic glasses and composites-A review. Acta Biomater, 2020; 103:1–23; doi: 10.1016/j.actbio.2019.12.023
15.
WalkerJ, ShadanbazS, WoodfieldTB, et al.Magnesium biomaterials for orthopedic application: A review from a biological perspective. J Biomed Mater Res B Appl Biomater, 2014; 102(6):1316–1331; doi: 10.1002/jbm.b.33113
16.
LaiY, LiY, CaoH, 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
17.
ZhouB, LinW, LongY, et al.Notch signaling pathway: Architecture, disease, and therapeutics. Signal Transduct Target Ther, 2022; 7(1):95; doi: 10.1038/s41392-022-00934-y
18.
NourisaJ, Zeller-PlumhoffB, HelmholzH, et al.Magnesium ions regulate mesenchymal stem cells population and osteogenic differentiation: A fuzzy agent-based modeling approach. Comput Struct Biotechnol J, 2021; 19:4110–4122; doi: 10.1016/j.csbj.2021.07.005
19.
Díaz-TocadosJM, HerenciaC, Martínez-MorenoJM, et al.Magnesium chloride promotes osteogenesis through notch signaling activation and expansion of mesenchymal stem cells. Sci Rep, 2017; 7(1):7839; doi: 10.1038/s41598-017-08379-y
20.
Retegi-CarriónS, Ferrandez-MonteroA, EguiluzA, et al.The effect of Ca(2+) and Mg(2+) ions loaded at degradable PLA membranes on the proliferation and osteoinduction of MSCs. Polymers (Basel), 2022; 14(12):2422; doi: 10.3390/polym14122422
21.
CaiZ, LiuX, HuM, et al.In situ enzymatic reaction generates magnesium-based mineralized microspheres with superior bioactivity for enhanced bone regeneration. Adv Healthc Mater, 2023; 12(24):e2300727; doi: 10.1002/adhm.202300727
22.
YuW, ZhaoH, DingZ, et al.In vitro and in vivo evaluation of MgF(2) coated AZ31 magnesium alloy porous scaffolds for bone regeneration. Colloids Surf B Biointerfaces, 2017; 149:330–340; doi: 10.1016/j.colsurfb.2016.10.037
23.
MiaoS, ZhouJ, LiuB, et al.A 3D bioprinted nano-laponite hydrogel construct promotes osteogenesis by activating PI3K/AKT signaling pathway. Mater Today Bio, 2022; 16:100342; doi: 10.1016/j.mtbio.2022.100342
24.
HuangZ, GuH, YinX, et al.Bone regeneration using injectable poly (γ-benzyl-L-glutamate) microspheres loaded with adipose-derived stem cells in a mouse femoral non-union model. Am J Transl Res, 2019; 11(5):2641–2656.
25.
GaoL, HuangZ, YanS, et al.Sr-HA-graft-Poly(γ-benzyl-l-glutamate) nanocomposite microcarriers: Controllable Sr (2+) release for accelerating osteogenenisis and bony nonunion repair. Biomacromolecules, 2017; 18(11):3742–3752; doi: 10.1021/acs.biomac.7b01101
26.
LiuG, ZhangY, LiuB, et al.Bone regeneration in a canine cranial model using allogeneic adipose derived stem cells and coral scaffold. Biomaterials, 2013; 34(11):2655–2664; doi: 10.1016/j.biomaterials.2013.01.004
27.
RockwoodDN, PredaRC, YücelT, et al.Materials fabrication from Bombyx mori silk fibroin. Nat Protoc, 2011; 6(10):1612–1631; doi: 10.1038/nprot.2011.379
28.
HsiehWC, ChangCP, LinSM. Morphology and characterization of 3D micro-porous structured chitosan scaffolds for tissue engineering. Colloids Surf B Biointerfaces, 2007; 57(2):250–255; doi: 10.1016/j.colsurfb.2007.02.004
29.
NgYC, BerryJM, ButlerM. Optimization of physical parameters for cell attachment and growth on macroporous microcarriers. Biotechnol Bioeng, 1996; 50(6):627–635; doi: 10.1002/(sici)1097-0290(19960620)50:6<627::Aid-bit3>3.0.Co;2-m
30.
TsaiAC, JeskeR, ChenX, et al.Influence of microenvironment on mesenchymal stem cell therapeutic potency: From planar culture to microcarriers. Front Bioeng Biotechnol, 2020; 8:640; doi: 10.3389/fbioe.2020.00640
31.
MandalBB, KunduSC. Cell proliferation and migration in silk fibroin 3D scaffolds. Biomaterials, 2009; 30(15):2956–2965; doi: 10.1016/j.biomaterials.2009.02.006
32.
LeemYH, LeeKS, KimJH, et al.Magnesium ions facilitate integrin alpha 2- and alpha 3-mediated proliferation and enhance alkaline phosphatase expression and activity in hBMSCs. J Tissue Eng Regen Med, 2016; 10(10): e527–e536; doi: 10.1002/term.1861
33.
YoshizawaS, BrownA, BarchowskyA, et al.Magnesium ion stimulation of bone marrow stromal cells enhances osteogenic activity, simulating the effect of magnesium alloy degradation. Acta Biomater, 2014; 10(6):2834–2842; doi: 10.1016/j.actbio.2014.02.002
34.
QinH, WengJ, ZhouB, et al.Magnesium ions promote in vitro rat bone marrow stromal cell angiogenesis through notch signaling. Biol Trace Elem Res, 2023; 201(6):2823–2842; doi: 10.1007/s12011-022-03364-7
35.
MonfouletLE, BecquartP, MarchatD, et al.The pH in the microenvironment of human mesenchymal stem cells is a critical factor for optimal osteogenesis in tissue-engineered constructs. Tissue Eng Part A, 2014; 20(13-14):1827–1840; doi: 10.1089/ten.TEA.2013.0500
