The fundamental electrical properties of bone have been attributed to the organic collagen and the inorganic mineral component; however, contributions of individual components within bone tissue toward the measured electrical properties are not known. In our study, we investigated the electrical properties of cell-mediated mineral deposition process and compared our results with cell-free mineralization.
Materials and Methods:
Saos-2 cells encapsulated within gelatin methacrylate (GelMA) hydrogels were chemically stimulated in osteogenic medium for a period of 4 weeks. The morphology, composition, and mechanical properties of the mineralized constructs were characterized using bright-field imaging, scanning electron microscopy (SEM) energy-dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy (FITR), nuclear magnetic resonance spectroscopy (NMR), micro-CT, immunostaining, and mechanical compression tests. In parallel, a custom-made device was used to measure the electrical impedance of mineralized constructs. All results were compared with cell-free GelMA hydrogels mineralized through the simulated body fluid approach.
Results:
Results demonstrate a decrease in the electrical impedance of deposited mineral in both cell-mineralized and cell-free mineralized samples.
Conclusions:
This study establishes a model system to investigate in vivo and in vitro mineralization processes.
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References
1.
Florencio-SilvaR, SassoGRdS, Sasso-CerriE, et al.Biology of bone tissue: Structure, function, and factors that influence bone cells. Biomed Res Int, 2015; 2015:421746.
2.
ManolagasSC, JilkaRL. Bone marrow, cytokines, and bone remodeling—Emerging insights into the pathophysiology of osteoporosis. N Engl J Med, 1995; 332:305–311.
3.
WittkowskeC, ReillyGC, LacroixD, et al.In vitro bone cell models: Impact of fluid shear stress on bone formation. Front Bioeng Biotechnol, 2016; 4:87.
4.
BoonrungsimanS, GentlemanE, CarzanigaR, et al.The role of intracellular calcium phosphate in osteoblast-mediated bone apatite formation. Proc Natl Acad Sci U S A, 2012; 109:14170–14175.
5.
MarshallD, JohnellO, WedelH.Meta-analysis of how well measures of bone mineral density predict occurrence of osteoporotic fractures. BMJ, 1996; 312:1254–1259.
6.
Hoang-KimA, GelsominiL, LucianiD, et al.Fracture healing and drug therapies in osteoporosis. Clin Cases Miner Bone Metab, 2009; 6:136.
7.
Dolatshahi-PirouzA, NikkhahM, GaharwarAK, et al.A combinatorial cell-laden gel microarray for inducing osteogenic differentiation of human mesenchymal stem cells. Sci Rep, 2014; 4:3896.
8.
ManY, WangP, GuoY, et al.Angiogenic and osteogenic potential of platelet-rich plasma and adipose-derived stem cell laden alginate microspheres. Biomaterials, 2012; 33:8802–8811.
9.
SawyerS, OestM, MarguliesB, et al.Behavior of encapsulated Saos-2 cells within gelatin methacrylate hydrogels. J Tissue Sci Eng, 2016; 7:2.
10.
BurdickJA, AnsethKS. Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering. Biomaterials, 2002; 23:4315–4323.
11.
ChatterjeeK, Lin-GibsonS, WallaceWE, et al.The effect of 3D hydrogel scaffold modulus on osteoblast differentiation and mineralization revealed by combinatorial screening. Biomaterials, 2010; 31:5051–5062.
12.
SawyerSW, ShridharSV, ZhangK, et al.Perfusion directed 3D mineral formation within cell-laden hydrogels. Biofabrication, 2018; 10:035013.
13.
FedorovichNE, AlblasJ, de WijnJR, et al.Hydrogels as extracellular matrices for skeletal tissue engineering: State-of-the-art and novel application in organ printing. Tissue Eng, 2007; 13:1905–1925.
14.
ThieleJ, MaY, BruekersSMC, et al.25th anniversary article: Designer hydrogels for cell cultures: A materials selection guide. Adv Mater, 2014; 26:125–148.
15.
QuarlesLD, YohayDA, LeverLW, et al.Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: An in vitro model of osteoblast development. J Bone Miner Res, 1992; 7:683–692.
16.
BenoitDSW, SchwartzMP, DurneyAR, et al.Small functional groups for controlled differentiation of hydrogel-encapsulated human mesenchymal stem cells. Nat Mater, 2008; 7:816.
17.
ButteryLDK, BourneS, XynosJD, et al.Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells. Tissue Eng, 2001; 7:89–99.
18.
BielbyRC, BoccacciniAR, PolakJM, et al.In vitro differentiation and in vivo mineralization of osteogenic cells derived from human embryonic stem cells. Tissue Eng, 2004; 10:1518–1525.
19.
KangH, ShihY-RV, HwangY, et al.Mineralized gelatin methacrylate-based matrices induce osteogenic differentiation of human induced pluripotent stem cells. Acta Biomater, 2014; 10:4961–4970.
20.
XavierJR, ThakurT, DesaiP, et al.Bioactive nanoengineered hydrogels for bone tissue engineering: A growth-factor-free approach. ACS Nano, 2015; 9:3109–3118.
21.
Carles-CarnerM, SalehLS, BryantSJ. The effects of hydroxyapatite nanoparticles embedded in a MMP-sensitive photoclickable PEG hydrogel on encapsulated MC3T3-E1 pre-osteoblasts. Biomed Mater, 2018; 13:045009.
22.
KohataK, ItohS, HoriuchiN, et al.The role of the collaborative functions of the composite structure of organic and inorganic constituents and their influence on the electrical properties of human bone. Biomed Mater Eng, 2016; 27:305–314.
23.
Hronik-TupajM, KaplanDL. A review of the responses of two-and three-dimensional engineered tissues to electric fields. Tissue Eng Part B Rev, 2012; 18:167–180.
24.
FukadaE, YasudaI.On the piezoelectric effect of bone. J Phys Soc J, 1957; 12:1158–1162.
25.
ShuY, BaumannMJ, CaseED, et al.Surface microcracks signal osteoblasts to regulate alignment and bone formation. Mater Sci Eng C, 2014; 44:191–200.
26.
ClarkeB.Normal bone anatomy and physiology. Clin J Am Soc Nephrol, 2008; 3:S131–S139.
27.
BassettCAL, PawlukRJ, PillaAA. Acceleration of fracture repair by electromagnetic fields. A surgically noninvasive method. Ann N Y Acad Sci, 1974; 238:242–262.
28.
ItoH, ShiraiY.The efficacy of ununited tibial fracture treatment using pulsing electromagnetic fields. J Nippon Med Sch, 2001; 68:149–153.
29.
PickeringS, ScammellB.Electromagnetic fields for bone healing. Int J Low Extrem Wounds, 2002; 1:152–160.
30.
SierpowskaJ, LammiM, HakulinenM, et al.Effect of human trabecular bone composition on its electrical properties. Med Eng Phys, 2007; 29:845–852.
31.
McKibbinB.The biology of fracture healing in long bones. J Bone Joint Surg Br, 1978; 60:150–162.
32.
ChenYX, YangS, YanJ, et al.A novel suspended hydrogel membrane platform for cell culture. J Nanotechnol Eng Med, 2015; 6:021002.
33.
OyaneA, KimHM, FuruyaT, et al.Preparation and assessment of revised simulated body fluids. J Biomed Mater Res A, 2003; 65:188–195.
34.
KikuchiM, IkomaT, ItohS, et al.Biomimetic synthesis of bone-like nanocomposites using the self-organization mechanism of hydroxyapatite and collagen. Compos Sci Technol, 2004; 64:819–825.
35.
SawyerSW, DongP, VennS, et al.Conductive gelatin methacrylate-poly (aniline) hydrogel for cell encapsulation. Biomed Phys Eng Express, 2017; 4:015005.
36.
WuY, ChenYX, YanJ, et al.Fabrication of conductive gelatin methacrylate–polyaniline hydrogels. Acta Biomater, 2016; 33:122–130.
37.
ZhouL, TanG, TanY, et al.Biomimetic mineralization of anionic gelatin hydrogels: Effect of degree of methacrylation. RSC Adv, 2014; 4:21997–22008.
38.
TanG, ZhouL, NingC, et al.Biomimetically-mineralized composite coatings on titanium functionalized with gelatin methacrylate hydrogels. Appl Surf Sci, 2013; 279:293–299.
39.
ChangMC, TanakaJ.FT-IR study for hydroxyapatite/collagen nanocomposite cross-linked by glutaraldehyde. Biomaterials, 2002; 23:4811–4818.
40.
TasAC.Calcium metal to synthesize amorphous or cryptocrystalline calcium phosphates. Mater Sci Eng C, 2012; 32:1097–1106.
41.
CaiMM, SmithER, HoltSG. The role of fetuin-A in mineral trafficking and deposition. Bonekey Rep, 2015; 4:672.
42.
SannigrahiP, IngallE. Polyphosphates as a source of enhanced P fluxes in marine sediments overlain by anoxic waters: Evidence from 31 P NMR. Geochem Trans, 2005; 6:52.
43.
LiuY, WuG, de GrootK. Biomimetic coatings for bone tissue engineering of critical-sized defects. J R Soc Interface, 2010; 7:S631.
44.
Vasquez-SanchoF, AbdollahiA, DamjanovicD, et al.Flexoelectricity in bones. Adv Mater, 2018; 30:1705316.
45.
HuW-W, HsuY-T, ChengY-C, et al.Electrical stimulation to promote osteogenesis using conductive polypyrrole films. Mater Sci Eng C, 2014; 37:28–36.
46.
ThrivikramanG, LeePS, HessR, et al.Interplay of substrate conductivity, cellular microenvironment, and pulsatile electrical stimulation toward osteogenesis of human mesenchymal stem cells in vitro. ACS Appl Mater Interfaces, 2015; 7:23015–23028.
47.
HabrakenWJEM, TaoJ, BrylkaLJ, et al.Ion-association complexes unite classical and non-classical theories for the biomimetic nucleation of calcium phosphate. Nat Commun, 2013; 4:1507.
48.
VeisA, DorveeJR. Biomineralization mechanisms: A new paradigm for crystal nucleation in organic matrices. Calcif Tissue Int, 2013; 93:307–315.
49.
LiuQ, HuangS, MatinlinnaJP, et al.Insight into biological apatite: Physiochemical properties and preparation approaches. Biomed Res Int, 2013; 2013.
50.
MarelliB, GhezziCE, BarraletJE, et al.Collagen gel fibrillar density dictates the extent of mineralization in vitro. Soft Matter, 2011; 7:9898–9907.
51.
HerbstE.Electric stimulation of bone growth and repair: A review of different stimulation methods. Burny F, Herbst E, Hinsenkamp M In: Electric Stimulation of Bone Growth and Repair. Berlin, Germany: Springer, 1978; 1–13.
52.
BalmerTW, VesztergomS, BroekmannP, et al.Characterization of the electrical conductivity of bone and its correlation to osseous structure. Sci Rep, 2018; 8:8601.
53.
WangY, AzaïsT, RobinM, et al.The predominant role of collagen in the nucleation, growth, structure and orientation of bone apatite. Nat Mater, 2012; 11:724.
54.
BigiA, BoaniniE, PanzavoltaS, et al.Bonelike apatite growth on hydroxyapatite–gelatin sponges from simulated body fluid. J Biomed Mater Res, 2002; 59:709–715.
55.
ZhangL-J, FengX-S, LiuH-G, et al.Hydroxyapatite/collagen composite materials formation in simulated body fluid environment. Mater Lett, 2004; 58:719–722.
56.
RobinM, AlmeidaC, AzaïsT, et al.Involvement of 3D osteoblast migration and bone apatite during in vitro early osteocytogenesis. Bone, 2016; 88:146–156.
57.
UchihashiK, AokiS, MatsunobuA, et al.Osteoblast migration into type I collagen gel and differentiation to osteocyte-like cells within a self-produced mineralized matrix: A novel system for analyzing differentiation from osteoblast to osteocyte. Bone, 2013; 52:102–110.
58.
KimS-S, ParkMS, GwakS-J, et al.Accelerated bonelike apatite growth on porous polymer/ceramic composite scaffolds in vitro. Tissue Eng, 2006; 12:2997–3006.
59.
LiJ, LiaoH, SjöströmM.Characterization of calcium phosphates precipitated from simulated body fluid of different buffering capacities. Biomaterials, 1997; 18:743–747.
60.
HabibovicP, BarrereF, Van BlitterswijkCA, et al.Biomimetic hydroxyapatite coating on metal implants. J Am Ceram Soc, 2002; 85:517–522.
61.
SaY, YangF, de WijnJR, et al.Physicochemical properties and mineralization assessment of porous polymethylmethacrylate cement loaded with hydroxyapatite in simulated body fluid. Mater Sci Eng C, 2016; 61:190–198.
62.
MiH-Y, JingX, SalickMR, et al.Morphology, mechanical properties, and mineralization of rigid thermoplastic polyurethane/hydroxyapatite scaffolds for bone tissue applications: Effects of fabrication approaches and hydroxyapatite size. J Mater Sci, 2014; 49:2324–2337.
63.
ZadpoorAA.Relationship between in vitro apatite-forming ability measured using simulated body fluid and in vivo bioactivity of biomaterials. Mater Sci Eng C, 2014; 35:134–143.
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