Collagen is widely used in medical field because of its excellent biocompatibility and bioactivity. To date, collagen for biomedical use is always derived from bovine or swine. The purpose of this study was to evaluate collagen-based biomaterials from non-mammalian donors for bone repair. Thus, tilapia skin collagen-hydroxyapatite (T-col/HAp) scaffolds were fabricated in three different proportions and then cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-N-hydroxysuccinimide (EDC-NHS). The scaffolds were evaluated for their microstructure, chemical and physical properties, mechanical strength and degradability. Then the in vitro responses of bone mesenchymal stem cells (BMSCs) to the scaffolds were investigated in terms of cellular proliferation, differentiation, and mineralization. At last, the scaffolds were implanted into rat skull critical defections to investigate the potential of osteogenic activities. As a result, the pore sizes and the porosities of the scaffolds were approximately 106.67–196.67 μm and 81.5%–66.7%. Pure collagen group showed a mechanical strength of 0.065 MPa, and the mechanical strength was significantly enhanced almost 17 times and 32 times in collagen/HAp ratio 1:4 and 1:9 groups. In vitro studies revealed the most prominent and healthy growth of BMSCs in collagen/HAp ratio 1:4 group. All the scaffolds showed certain osteogenic activities and those loaded with small amount of hydroxyapatite showed the strongest bioactivities. The micro-CT showed that the critical bone defect was almost filled with generated bone 6 months after implantation in collagen/HAp ratio 1:4 group. The biomechanics tests further confirmed the highest generated bone strength was in the collagen/HAp ratio 1:4 group. This study indicated aquatic collagen might be a potential alternative for type I collagen from mammals in bone tissue engineering. The combination of collagen and inorganic materials was also important and appropriate inorganic component loading can achieve both osteogenic quality and osteogenic efficiency to a certain extent.
RamshawJAShahNKBrodskyB. Gly-X-Y tripeptide frequencies in collagen: a context for host-guest triple-helical peptides. J Struct Biol1998; 122(1–2): 86–91.
2.
GhodbaneSADunnMG. Physical and mechanical properties of cross-linked type I collagen scaffolds derived from bovine, porcine, and ovine tendons. J Biomed Mater Res A2016; 104(11): 2685–2692.
3.
LiXXuJNicolescuCT, et al.Generation, endothelialization, and microsurgical suture anastomosis of strong 1-mm-diameter collagen tubes. Tissue Eng A2017; 23(7–8): 335–344.
4.
MatsumotoRUemuraTXuZ, et al.Rapid oriented fibril formation of fish scale collagen facilitates early osteoblastic differentiation of human mesenchymal stem cells. J Biomed Mater Res A2015; 103(8): 2531–2539.
5.
ChoiDJChoiSMKangHY, et al.Bioactive fish collagen/polycaprolactone composite nanofibrous scaffolds fabricated by electrospinning for 3D cell culture. J Biotechnol2015; 205: 47–58.
6.
YamamotoKIgawaKSugimotoK, et al.Biological safety of fish (tilapia) collagen. Biomed Res Int2014; 2014: 630757.
7.
LimYSOkYJHwangSY, et al.Marine collagen as a promising biomaterial for biomedical applications. Mar Drugs2019; 17(8): 467.
8.
HoyerBBernhardtAHeinemannS, et al.Biomimetically mineralized salmon collagen scaffolds for application in bone tissue engineering. Biomacromolecules2012; 13(4): 1059–1066.
9.
EastoeJE. The amino acid composition of fish collagen and gelatin. Biochem J1957; 65(2): 363–368.
10.
CoppolaDOlivieroMVitaleGA, et al.Marine collagen from alternative and sustainable sources: extraction, processing and applications. Mar Drugs2020; 18(4): 214–236.
11.
FitzsimmonsKMMartinez-GarciaRGonzalez‐AlanisP. Why tilapia is becoming the most important food fish on the planet. In: Proceedings 9th International Symposium Tilapia Aquaculture, 22 April 2011; 2011. pp. 1–8.
12.
GautieriAVesentiniSRedaelliA, et al.Hierarchical structure and nanomechanics of collagen microfibrils from the atomistic scale up. Nano Lett2011; 11(2): 757–766.
13.
LiaoSNgiamMChanCK, et al.Fabrication of nano-hydroxyapatite/collagen/osteonectin composites for bone graft applications. Biomed Mater2009; 4(2): 025019.
14.
BenayahuDPomeraniecLShemeshS, et al.Biocompatibility of a marine collagen-based scaffold in vitro and in vivo. Mar Drugs2020; 18(8): 420–430.
15.
Weiss-BilkaHEMeagherMJGargacJA, et al.Mineral deposition and vascular invasion of hydroxyapatite reinforced collagen scaffolds seeded with human adipose-derived stem cells. Biomater Res2019; 23: 15.
16.
YunokiSSugiuraHIkomaT, et al.Effects of increased collagen-matrix density on the mechanical properties and in vivo absorbability of hydroxyapatite-collagen composites as artificial bone materials. Biomed Mater2011; 6(1): 015012.
17.
LakraRKiranMSSaiKP. Fabrication of homobifunctional crosslinker stabilized collagen for biomedical application. Biomed Mater2015; 10(6): 065015.
18.
AdamiakKSionkowskaA. Current methods of collagen cross-linking: review. Int J Biol Macromol2020; 161: 550–560.
19.
NairMJohalRKHamaiaSW, et al.Tunable bioactivity and mechanics of collagen-based tissue engineering constructs: a comparison of EDC-NHS, genipin and TG2 crosslinkers. Biomaterials2020; 254: 120109.
20.
Skopinska-WisniewskaJTuszynskaMOlewnik-KruszkowskaE. Comparative study of gelatin hydrogels modified by various cross-linking agents. Materials (Basel)2021; 14(2): 396–410.
21.
DavidenkoNSchusterCFBaxDV, et al.Control of crosslinking for tailoring collagen-based scaffolds stability and mechanics. Acta Biomater2015; 25: 131–142.
22.
YangCR. Enhanced physicochemical properties of collagen by using EDC/NHS-crosslinking. B Mater Sci2012; 35(5): 913–918.
23.
ShangYLYaoSYQiaoXR, et al.Evaluations of marine collagen peptides from tilapia skin on experimental oral ulcer model of mice. Mater Today Commun2021; 26: 101893.
24.
LaemmliUK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature1970; 227(5259): 680–685.
25.
KaczmarekBSionkowskaAKozlowskaJ, et al.New composite materials prepared by calcium phosphate precipitation in chitosan/collagen/hyaluronic acid sponge cross-linked by EDC/NHS. Int J Biol Macromol2018; 107(Pt A): 247–253.
26.
SalehiMNaseri-NosarMEbrahimi-BaroughS, et al.Polyurethane/gelatin nanofibrils neural guidance conduit containing platelet-rich plasma and melatonin for transplantation of schwann cells. Cell Mol Neurobiol2018; 38(3): 703–713.
27.
VedhanayagamMAnandasadagopanSNairBU, et al.Polymethyl methacrylate (PMMA) grafted collagen scaffold reinforced by PdO-TiO2 nanocomposites. Mater Sci Eng C Mater Biol Appl2020; 108: 110378.
28.
NegishiJFunamotoSHashimotoY, et al.PLA-collagen composite scaffold fabrication by vacuum pressure impregnation. Tissue Eng C Methods2019; 25(12): 742–747.
29.
SelvakumarGLonchinS. Fabrication and characterization of collagen-oxidized pullulan scaffold for biomedical applications. Int J Biol Macromol2020; 164: 1592–1599.
30.
SongWKLiuDSunLL, et al.Physicochemical and biocompatibility properties of type I collagen from the skin of nile tilapia (oreochromis niloticus) for biomedical applications. Mar Drugs2019; 17(3): 137–150.
31.
JafariHListaASiekapenMM, et al.Fish collagen: extraction, characterization, and applications for biomaterials engineering. Polymers (Basel)2020; 12(10): 2230–2265.
32.
MatmarohKBenjakulSProdpranT, et al.Characteristics of acid soluble collagen and pepsin soluble collagen from scale of spotted golden goatfish (Parupeneus heptacanthus). Food Chem2011; 129(3): 1179–1186.
33.
BoudkoSFrankSKammererRA, et al.Nucleation and propagation of the collagen triple helix in single-chain and trimerized peptides: transition from third to first order kinetics. J Mol Biol2002; 317(3): 459–470.
34.
SongZLiuHChenL, et al.Characterization and comparison of collagen extracted from the skin of the Nile tilapia by fermentation and chemical pretreatment. Food Chem2021; 340: 128139.
35.
Grabska-ZielinskaSSionkowskaACarvalhoA, et al.Biomaterials with potential use in bone tissue regeneration-collagen/chitosan/silk fibroin scaffolds cross-linked by EDC/NHS. Materials (Basel)2021; 14(5): 1105.
36.
ShepherdDVShepherdJHGhoseS, et al.The process of EDC-NHS cross-linking of reconstituted collagen fibres increases collagen fibrillar order and alignment. APL Mater2015; 3(1): 014902.
37.
KarageorgiouVKaplanD. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials2005; 26(27): 5474–5491.
38.
BoseSRoyMBandyopadhyayA. Recent advances in bone tissue engineering scaffolds. Trends Biotechnol2012; 30(10): 546–554.
39.
GoodarziHHashemi-NajafabadiSBaheiraeiN, et al.Preparation and characterization of nanocomposite scaffolds (collagen/beta-TCP/SrO) for bone tissue engineering. Tissue Eng Regen Med2019; 16(3): 237–251.
40.
ParkSALeeSHKimWD. Fabrication of porous polycaprolactone/hydroxyapatite (PCL/HA) blend scaffolds using a 3D plotting system for bone tissue engineering. Bioproc Biosyst Eng2011; 34(4): 505–513.
41.
DiaoJOuYangJDengT, et al.3D-plotted beta-tricalcium phosphate scaffolds with smaller pore sizes improve in vivo bone regeneration and biomechanical properties in a critical-sized calvarial defect rat model. Adv Healthc Mater2018; 7(17): e1800441.
42.
MurphyCMHaughMGO’BrienFJ. The effect of mean pore size on cell attachment, proliferation and migration in collagen-glycosaminoglycan scaffolds for bone tissue engineering. Biomaterials2010; 31(3): 461–466.
GupteMJSwansonWBHuJ, et al.Pore size directs bone marrow stromal cell fate and tissue regeneration in nanofibrous macroporous scaffolds by mediating vascularization. Acta Biomater2018; 82: 1–11.
45.
RojaeeRFathiMRaeissiK. Controlling the degradation rate of AZ91 magnesium alloy via sol-gel derived nanostructured hydroxyapatite coating. Mater Sci Eng C Mater Biol Appl2013; 33(7): 3817–3825.
46.
DiomedeFMarconiGDFonticoliL, et al.Functional relationship between osteogenesis and angiogenesis in tissue regeneration. Int J Mol Sci2020; 21(9): 3242–3255.
47.
BohnerMMironRJ. A proposed mechanism for material-induced heterotopic ossification. Mater Today2019; 22: 132–141.
48.
HosseiniSNaderi-ManeshHValiH, et al.Contribution of osteocalcin-mimetic peptide enhances osteogenic activity and extracellular matrix mineralization of human osteoblast-like cells. Colloids Surf B Biointerfaces2019; 173: 662–671.
49.
QiaoXYangJShangY, et al.Magnesium-doped nanostructured titanium surface modulates macrophage-mediated inflammatory response for ameliorative osseointegration. Int J Nanomed2020; 15: 7185–7198.
50.
LiuCSunJ. Potential application of hydrolyzed fish collagen for inducing the multidirectional differentiation of rat bone marrow mesenchymal stem cells. Biomacromolecules2014; 15(1): 436–443.
51.
HongZMelloAYoshidaT, et al.Osteoblast proliferation on hydroxyapatite coated substrates prepared by right angle magnetron sputtering. J Biomed Mater Res A2010; 93(3): 878–885.
52.
NaitoHYoshimuraMMizunoT, et al.The advantages of three-dimensional culture in a collagen hydrogel for stem cell differentiation. J Biomed Mater Res A2013; 101(10): 2838–2845.
53.
MoonSOkYHwangS, et al.A marine collagen-based biomimetic hydrogel recapitulates cancer stem cell niche and enhances progression and chemoresistance in human ovarian cancer. Mar Drugs2020; 18(10): 498.
54.
MorelliTZhangSMonaghanE, et al.Three-dimensional volumetric changes after socket augmentation with deproteinized bovine bone and collagen matrix. Int J Oral Maxillofac Implants2020; 35(3): 566–575.
55.
RodriguesCVSerricellaPLinharesAB, et al.Characterization of a bovine collagen-hydroxyapatite composite scaffold for bone tissue engineering. Biomaterials2003; 24(27): 4987–4997.
56.
RodriguesSCSalgadoCLSahuA, et al.Preparation and characterization of collagen-nanohydroxyapatite biocomposite scaffolds by cryogelation method for bone tissue engineering applications. J Biomed Mater Res A2013; 101(4): 1080–1094.
57.
AddadSExpositoJYFayeC, et al.Isolation, characterization and biological evaluation of jellyfish collagen for use in biomedical applications. Mar Drugs2011; 9(6): 967–983.
58.
NagaiNMoriKSatohY, et al.In vitro growth and differentiated activities of human periodontal ligament fibroblasts cultured on salmon collagen gel. J Biomed Mater Res A2007; 82(2): 395–402.
59.
HadzikJKubasiewicz-RossPKunert-KeilC, et al.A silver carp skin derived collagen in bone defect treatment-A histological study in a rat model. Ann Anat2016; 208: 123–128.
60.
TangJSaitoT. Biocompatibility of novel type I collagen purified from tilapia fish scale: an in vitro comparative study. Biomed Res Int2015; 2015: 139476.
61.
XuXSuiBLiuX, et al.Superior low-immunogenicity of tilapia type I collagen based on unique secondary structure with single calcium binding motif over terrestrial mammals by inhibiting activation of DC intracellular Ca(2+)-mediated STIM1-Orai1/NF-small ka, CyrillicB pathway. Mater Sci Eng C Mater Biol Appl2021; 131: 112503.
62.
SugiuraHYunokiSKondoE, et al.In vivo biological responses and bioresorption of tilapia scale collagen as a potential biomaterial. J Biomater Sci Polym Ed2009; 20(10): 1353–1368.
63.
LohmannPWilluweitANeffeAT, et al.Bone regeneration induced by a 3D architectured hydrogel in a rat critical-size calvarial defect. Biomaterials2017; 113: 158–169.
64.
DelmasPDREGarneroPSeibelMJ, et al.The use of biochemical markers of bone turnover in osteoporosis. Osteoporos Int2000; 11: S2–S17.
65.
AardenEMWassenaarAMAlblasMJ, et al.Immunocytochemical demonstration of extracellular matrix proteins in isolated osteocytes. Histochem Cel Biol1996; 106(5): 495–501.
66.
DucyPDesboisCBoyceB, et al.Increased bone formation in osteocalcin-deficient mice. Nature1996; 382(6590): 448–452.
67.
NakashimaM. Induction of dentin formation on canine amputated pulp by recombinant human bone morphogenetic proteins (BMP)-2 and -4. J Dent Res1994; 73(9): 1515–1522.
68.
SivarajKKAdamsRH. Blood vessel formation and function in bone. Development2016; 143(15): 2706–2715.
69.
CoultasLChawengsaksophakKRossantJ. Endothelial cells and VEGF in vascular development. Nature2005; 438(7070): 937–945.
70.
GerberHPVuTHRyanAM, et al.VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med1999; 5(6): 623–628.
71.
LiuYBerendsenADJiaS, et al.Intracellular VEGF regulates the balance between osteoblast and adipocyte differentiation. J Clin Invest2012; 122(9): 3101–3113.
72.
HingKA. Bone repair in the twenty-first century: biology, chemistry or engineering?Philos Trans A Math Phys Eng Sci2004; 362(1825): 2821–2850.
73.
SuchanekWYoshimuraM. Processing and properties of hydroxyapatite-based biomaterials for use as hard tissue replacement implants. J Mater Res1998; 13(1): 94–117.
74.
JonesLThomsenJSMosekildeL, et al.Biomechanical evaluation of rat skull defects, 1, 3, and 6 months after implantation with osteopromotive substances. J Craniomaxillofac Surg2007; 35(8): 350–357.
75.
DengSHuYZhouJ, et al.TLR4 mediates alveolar bone resorption in experimental peri-implantitis through regulation of CD45(+) cell infiltration, RANKL/OPG ratio, and inflammatory cytokine production. J Periodontol2020; 91(5): 671–682.
76.
van EijdenTM. Biomechanics of the mandible. Crit Rev Oral Biol Med2000; 11(1): 123–136.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
