Complications that arise from impaired fracture healing have considerable socioeconomic implications. Current research in the field of bone tissue engineering predominantly aims to mimic the mature bone tissue microenvironment. This approach, however, may produce implants that are intrinsically unresponsive to the cues present during the initiation of fracture repair. As such, this study describes the development of decellularized xenogeneic hyaline cartilage matrix in an attempt to mimic the initial reparative phase of fracture repair. Three approaches based on vacuum-assisted osmotic shock (Vac-OS), Triton X-100 (Vac-STx), and sodium dodecyl sulfate (Vac-SDS) were investigated. The Vac-OS methodology reduced DNA content below 50 ng/mg of tissue, while retaining 85% of the sulfate glycosaminoglycan content, and as such was selected as the optimal methodology for decellularization. The resultant Vac-OS scaffolds (decellularized extracellular matrix [dcECM]) were also devoid of the immunogenic alpha-Gal epitope. Furthermore, minimal disruption to the structural integrity of the dcECM was demonstrated using differential scanning calorimetry and fluorescence lifetime imaging microscopy. The biological integrity of the dcECM was confirmed by its ability to drive the chondrogenic commitment and differentiation of human chondrocytes and periosteum-derived cells, respectively. Furthermore, histological examination of dcECM constructs implanted in immunocompetent mice revealed a predominantly M2 macrophage-driven regenerative response both at 2 and 8 weeks postimplantation. These findings contrasted with the implanted native costal cartilage that elicited a predominantly M1 macrophage-mediated inflammatory response. This study highlights the capacity of dcECM from the Vac-OS methodology to direct the key biological processes of endochondral ossification, thus potentially recapitulating the callus phase of fracture repair.
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References
1.
GerstenfeldL.C., CullinaneD.M., BarnesG.L., GravesD.T., and EinhornT.A.Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J Cell Biochem, 88, 873, 2003.
MarsellR., and EinhornT.A.The biology of fracture healing. Injury, 42, 551, 2011.
4.
BiasibettiA., AlojD., Di GregorioG., MasseA., and SalomoneC.Mechanical and biological treatment of long bone non-unions. Injury, 36Suppl 4, S45, 2005.
5.
Gómez-BarrenaE., RossetP., LozanoD., StanoviciJ., ErmthallerC., and GerbhardF.Bone fracture healing: cell therapy in delayed unions and nonunions. Bone, 70, 93, 2015.
6.
SenM.K., and MiclauT.Autologous iliac crest bone graft: should it still be the gold standard for treating nonunions?. Injury, 38, 2, 2007.
7.
KinneyR.C., ZiranB.H., HirshornK., SchlattererD., and GaneyT.Demineralized bone matrix for fracture healing: fact or fiction? J Orthop Trauma, 24Suppl 1, S52, 2010.
8.
GruskinE., DollB.A., FutrellF.W., SchmitzJ.P., and HollingerJ.O.Demineralized bone matrix in bone repair: history and use. Adv Drug Deliv Rev, 64, 1063, 2012.
9.
ThompsonE.M., MatsikoA., FarrellE., KellyD.J., and O'BrienF.J.Recapitulating endochondral ossification: a promising route to in vivo bone regeneration. J Tissue Eng Regen Med, 9, 889, 2015.
10.
DaiJ., and RabieA.B.M.VEGF: an essential mediator of both angiogenesis and endochondral ossification. J Dent Res, 86, 937, 2007.
11.
FarrellE., van der JagtO.P., KoevoetW., et al.Chondrogenic priming of human bone marrow stromal cells: a better route to bone repair?. Tissue Eng Part C Methods, 15, 285, 2009.
12.
MackieE.J., AhmedY.A., TatarczuchL., ChenK.-S., and MiramsM.Endochondral ossification: how cartilage is converted into bone in the developing skeleton. Int J Biochem Cell Biol, 40, 46, 2008.
13.
GawlittaD., FarrellE., MaldaJ., CreemersL.B., AlblasJ., andDhertW.J.A.Modulating endochondral ossification of multipotent stromal cells for bone regeneration. Tissue Eng Part B Rev, 16, 385, 2010.
14.
BahneyC.S., HuD.P., TaylorA.J., et al.Stem cell-derived endochondral cartilage stimulates bone healing by tissue transformation. J Bone Miner Res, 29, 1269, 2014.
15.
HaradaN., WatanabeY., SatoK., et al.Matsushita, Bone regeneration in a massive rat femur defect through endochondral ossification achieved with chondrogenically differentiated MSCs in a degradable scaffold. Biomaterials, 35, 7800, 2014.
16.
MuragliaA., CorsiA., RiminucciM., et al.Formation of a chondro-osseous rudiment in micromass cultures of human bone-marrow stromal cells. J Cell Sci, 116, 2949, 2003.
17.
LutolfM.P., and HubbellJ.A.Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat Biotechnol, 23, 47, 2005.
18.
BadylakS.F.The extracellular matrix as a biologic scaffold material. Biomaterials, 28, 3587, 2007.
19.
BendersK.E.M., van WeerenP.R., BadylakS.F., SarisD.B.F., DhertW.J.A., and MaldaJ.Extracellular matrix scaffolds for cartilage and bone regeneration. Trends Biotechnol, 31, 169, 2013.
20.
ChengC.W., SolorioL.D., and AlsbergE.Decellularized tissue and cell-derived extracellular matrices as scaffolds for orthopaedic tissue engineering. Biotechnol Adv, 32, 462, 2014.
21.
VasW.J., ShahM., Al HosniR., OwenH.C., and RobertsS.J.Biomimetic strategies for fracture repair: engineering the cell microenvironment for directed tissue formation. J Tissue Eng, 8, 204173141770479, 2017.
22.
CrapoP.M., GilbertT.W., and BadylakS.F.An overview of tissue and whole organ decellularization processes. Biomaterials, 32, 3233, 2011.
23.
GonfiottiA., JausM.O., BaraleD., et al.The first tissue-engineered airway transplantation: 5-year follow-up results. Lancet, 383, 238, 2014.
24.
VisserJ., GawlittaD., BendersK.E.M., et al.Endochondral bone formation in gelatin methacrylamide hydrogel with embedded cartilage-derived matrix particles. Biomaterials, 37C, 174, 2014.
25.
GawlittaD., BendersK.E.M., VisserJ., et al.Decellularized cartilage-derived matrix as substrate for endochondral bone regeneration. Tissue Eng Part A, 21, 694, 2015.
26.
Sophia FoxA.J., BediA., and RodeoS.A.The basic science of articular cartilage: structure, composition, and function. Sports Health, 1, 461, 2009.
27.
CunniffeG.M., VinardellT., MurphyJ.M., et al.Porous decellularized tissue engineered hypertrophic cartilage as a scaffold for large bone defect healing. Acta Biomater, 23, 82, 2015.
28.
BourgineP.E., ScottiC., PigeotS., TchangL.A., TodorovA., and MartinI.Osteoinductivity of engineered cartilaginous templates devitalized by inducible apoptosis. Proc Natl Acad Sci U S A, 111, 17426, 2014.
29.
BardsleyK., KwarciakA., FreemanC., BrookI., HattonP., and CrawfordA.Repair of bone defects in vivo using tissue engineered hypertrophic cartilage grafts produced from nasal chondrocytes. Biomaterials, 112, 313, 2017.
30.
BahramiS., PlateU., DreierR., DuChesneA., WillitalG.H., and BrucknerP.Endochondral ossification of costal cartilage is arrested after chondrocytes have reached hypertrophic stage of late differentiation. Matrix Biol, 19, 707, 2001.
31.
OkihanaH., and ShimomuraY.Osteogenic activity of growth cartilage examined by implanting decalcified or devitalized ribs and costal cartilage zone, and living growth cartilage cells. Bone, 13, 387, 1992.
32.
GrecoK.V, FrancisL., SomasundaramM., et al.Characterisation of porcine dermis scaffolds decellularised using a novel non-enzymatic method for biomedical applications. J Biomater Appl, 30, 239, 2015.
33.
LangeP., GrecoK., PartingtonL., et al.Pilot study of a novel vacuum-assisted method for decellularization of tracheae for clinical tissue engineering applications. J Tissue Eng Regen Med, 11, 800, 2017.
34.
RobertsS.J., ChenY., MoesenM., SchrootenJ., and LuytenF.P.Enhancement of osteogenic gene expression for the differentiation of human periosteal derived cells. Stem Cell Res, 7, 137, 2011.
35.
MendesL.F., TamW.L., ChaiY.C., GerisL., LuytenF.P., and RobertsS.J.Combinatorial analysis of growth factors reveals the contribution of bone morphogenetic proteins to chondrogenic differentiation of human periosteal cells. Tissue Eng Part C Methods, 22, 473, 2016.
36.
BolanderJ., ChaiY.C., GerisL., et al.Wnt and Ca2+/PKC pathway activation predicts the bone forming capacity of periosteal cells in combination with calcium phosphates. Biomaterials, 86, 106, 2016.
37.
RobertsS.J., GerisL., KerckhofsG., DesmetE., SchrootenJ., and LuytenF.P.The combined bone forming capacity of human periosteal derived cells and calcium phosphates. Biomaterials, 32, 4393, 2011.
38.
AhrensP.B., SolurshM., and ReiterR.S.Stage-related capacity for limb chondrogenesis in cell culture. Dev Biol, 60, 69, 1977.
39.
LivakK.J., and SchmittgenT.D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods, 25, 402, 2001.
40.
GaliliU.The alpha-gal epitope and the anti-Gal antibody in xenotransplantation and in cancer immunotherapy. Immunol Cell Biol, 83, 674, 2005.
41.
WilliamsR.M., ZipfelW.R., and WebbW.W.Interpreting second-harmonic generation images of collagen I fibrils. Biophys J, 88, 1377, 2005.
SuhlingK., HirvonenL.M., LevittJ.A., et al.Fluorescence lifetime imaging (FLIM): basic concepts and some recent developments. Med Photonics, 27, 3, 2015.
44.
GilbertT.W., SellaroT.L., and BadylakS.F.Decellularization of tissues and organs. Biomaterials, 273, 675, 2006.
45.
SchwarzS., KoerberL., ElsaesserA.F., et al.Decellularized cartilage matrix as a novel biomatrix for cartilage tissue-engineering applications. Tissue Eng Part A, 18, 120720085805006, 2012.
46.
ButlerC.R., HyndsR.E., CrowleyC., et al.Vacuum-assisted decellularization: an accelerated protocol to generate tissue-engineered human tracheal scaffolds. Biomaterials, 124, 95, 2017.
47.
SutherlandA.J., BeckE.C., DennisS.C., et al.Decellularized cartilage may be a chondroinductive material for osteochondral tissue engineering. PLoS One, 10, e0121966, 2015.
48.
BendersK.E.M., BootW., CokelaereS.M., et al.Multipotent stromal cells outperform chondrocytes on cartilage-derived matrix scaffolds. Cartilage, 5, 221, 2014.
49.
RiderC.C.Heparin/heparan sulphate binding in the TGF-beta cytokine superfamily. Biochem Soc Trans, 34, 458, 2006.
50.
TurnbullJ., PowellA., and GuimondS.Heparan sulfate: decoding a dynamic multifunctional cell regulator. Trends Cell Biol, 11, 75, 2001.
51.
FrenchM.M., GomesR.R., TimplR., et al.Chondrogenic activity of the heparan sulfate proteoglycan perlecan maps to the N-terminal domain I. J Bone Miner Res, 17, 48, 2002.
52.
IngavleG.C., FreiA.W., GehrkeS.H., and DetamoreM.S.Incorporation of aggrecan in interpenetrating network hydrogels to improve cellular performance for cartilage tissue engineering. Tissue Eng Part A, 19, 1349, 2013.
53.
MohanN., GuptaV., SridharanB., SutherlandA., and DetamoreM.S.The potential of encapsulating “raw materials” in 3D osteochondral gradient scaffolds. Biotechnol Bioeng, 111, 829, 2014.
54.
JochmannK., BachvarovaV., and VortkampA.Reprint of: heparan sulfate as a regulator of endochondral ossification and osteochondroma development. Matrix Biol, 35, 239, 2014.
55.
LinY., LinH., LiuZ., WangK., and YanY.Improvement of a sample preparation method assisted by sodium deoxycholate for mass-spectrometry-based shotgun membrane proteomics. J Sep Sci, 37, 3321, 2014.
56.
ElderB.D., KimD.H., and AthanasiouK.A.Developing an articular cartilage decellularization process toward facet joint cartilage replacement. Neurosurgery, 66, 722, 2010.
57.
UtomoL., PleumeekersM.M., NimeskernL., et al.Preparation and characterization of a decellularized cartilage scaffold for ear cartilage reconstruction. Biomed Mater, 10, 15010, 2015.
58.
NagataS., HanayamaR., and KawaneK.Autoimmunity and the clearance of dead cells. Cell, 140, 619, 2010.
59.
WongM.L., and GriffithsL.G.Immunogenicity in xenogeneic scaffold generation: antigen removal vs. decellularization. Acta Biomater, 10, 1806, 2014.
60.
BadylakS.F., and GilbertT.W.Immune response to biologic scaffold materials. Semin Immunol, 20, 109, 2008.
61.
KonakciK.Z., BohleB., BlumerR., et al.Alpha-Gal on bioprostheses: xenograft immune response in cardiac surgery. Eur J Clin Invest, 35, 17, 2005.
62.
MangoldA., SzerafinT., HoetzeneckerK., et al.Alpha-Gal specific IgG immune response after implantation of bioprostheses. Thorac Cardiovasc Surg, 57, 191, 2009.
63.
ParkC.S., ParkS.-S., ChoiS.Y., YoonS.H., KimW.-H., and KimY.J.Anti alpha-Gal immune response following porcine bioprosthesis implantation in children. J Heart Valve Dis, 19, 124, 2010.
64.
GattazzoF., UrciuoloA., and BonaldoP.Extracellular matrix: a dynamic microenvironment for stem cell niche. Biochim Biophys Acta, 1840, 2506, 2014.
65.
HortonW.A., and MachadoM.M.Extracellular matrix alterations during endochondral ossification in humans. J Orthop Res, 6, 793, 1988.
66.
OrtegaN., BehonickD.J., and WerbZ.Matrix remodeling during endochondral ossification. Trends Cell Biol, 14, 86, 2004.
67.
HyllestedJ.L., VejeK., and OstergaardK.Histochemical studies of the extracellular matrix of human articular cartilage—a review. Osteoarthritis Cartilage, 10, 333, 2002.
68.
SchmitzN., LavertyS., KrausV.B., and AignerT.Basic methods in histopathology of joint tissues. Osteoarthritis Cartilage, 18, S113, 2010.
69.
ThanP.Differential scanning calorimetric examination of the osteoarthritic hyaline cartilage in rabbits. Thermochim Acta, 404, 149, 2003.
70.
TalbotC.B., LeverM.J., BenningerR.K.P., et al.Fluorescence lifetime imaging of articular cartilage. Int J Exp Pathol, 85, A31, 2008.
71.
AkkirajuH., and NoheA.Role of chondrocytes in cartilage formation, progression of osteoarthritis and cartilage regeneration. J Dev Biol, 3, 177, 2015.
72.
CaronM.M.J., EmansP.J., CoolsenM.M.E., et al.Redifferentiation of dedifferentiated human articular chondrocytes: comparison of 2D and 3D cultures. Osteoarthritis Cartilage, 20, 1170, 2012.
73.
TewS.R., PothacharoenP., KatopodiT., and HardinghamT.E.SOX9 transduction increases chondroitin sulfate synthesis in cultured human articular chondrocytes without altering glycosyltransferase and sulfotransferase transcription. Biochem J, 414, 231, 2008.
74.
ChenJ., WangY., ChenC., et al.Exogenous heparan sulfate enhances the TGF-β3-induced chondrogenesis in human mesenchymal stem cells by activating TGF-β/Smad signaling. Stem Cells Int, 2016, 1520136, 2016.
75.
KimS.-H., TurnbullJ., and GuimondS.Extracellular matrix and cell signalling: the dynamic cooperation of integrin, proteoglycan and growth factor receptor. J Endocrinol, 209, 139, 2011.
76.
Van der KraanP.M., BumaP., Van KuppeveltT., and Van Den BergW.B.Interaction of chondrocytes, extracellular matrix and growth factors: relevance for articular cartilage tissue engineering. Osteoarthritis Cartilage, 10, 631, 2002.
77.
OkaK., OkaS., SasakiT., et al.The role of TGF-beta signaling in regulating chondrogenesis and osteogenesis during mandibular development. Dev Biol, 303, 391, 2007.
78.
MurphyM.K., HueyD.J., HuJ.C., and AthanasiouK.A.TGF-β1, GDF-5, and BMP-2 stimulation induces chondrogenesis in expanded human articular chondrocytes and marrow-derived stromal cells. Stem Cells, 33, 762, 2015.
79.
EllmanM.B., AnH.S., MuddasaniP., and ImH.-J.Biological impact of the fibroblast growth factor family on articular cartilage and intervertebral disc homeostasis. Gene, 420, 82, 2008.
80.
ColnotC.Skeletal cell fate decisions within periosteum and bone marrow during bone regeneration. J Bone Miner Res, 24, 274, 2009.
81.
ZhangX., XieC., LinA.S.P., et al.Periosteal progenitor cell fate in segmental cortical bone graft transplantations: implications for functional tissue engineering. J Bone Miner Res, 20, 2124, 2005.
82.
RobertsS.J., van GastelN., CarmelietG., and LuytenF.P.Uncovering the periosteum for skeletal regeneration: the stem cell that lies beneath. Bone, 70, 10, 2015.
83.
LinZ., FatehA., SalemD.M., and IntiniG.Periosteum: biology and applications in craniofacial bone regeneration. J Dent Res, 93, 109, 2014.
84.
ZhengQ., ZhouG., MorelloR., ChenY., Garcia-RojasX., and LeeB.Type X collagen gene regulation by Runx2 contributes directly to its hypertrophic chondrocyte-specific expression in vivo. J Cell Biol, 162, 833, 2003.
85.
LiF., LuY., DingM., et al.Runx2 contributes to murine Col10a1 gene regulation through direct interaction with its cis-enhancer. J Bone Miner Res, 26, 2899, 2011.
86.
BrownB.N., ValentinJ.E., Stewart-AkersA.M., McCabeG.P., and BadylakS.F.Macrophage phenotype and remodeling outcomes in response to biologic scaffolds with and without a cellular component. Biomaterials, 30, 1482, 2009.
87.
OlsenB.R., ReginatoA.M., and WangW.Bone development. Annu Rev Cell Dev Biol, 16, 191, 2000.
88.
KlingerP., Surmann-SchmittC., BremM., et al.Chondromodulin 1 stabilizes the chondrocyte phenotype and inhibits endochondral ossification of porcine cartilage repair tissue. Arthritis Rheum, 63, 2721, 2011.
89.
ScottiC., TonnarelliB., PapadimitropoulosA., et al.Recapitulation of endochondral bone formation using human adult mesenchymal stem cells as a paradigm for developmental engineering. Proc Natl Acad Sci U S A, 107, 7251, 2010.
90.
OsingaR., Di MaggioN., TodorovA., et al.Generation of a bone organ by human adipose-derived stromal cells through endochondral ossification. Stem Cells Transl Med, 5, 1090, 2016.
91.
ScottiC., PiccininiE., TakizawaH., et al.Engineering of a functional bone organ through endochondral ossification. Proc Natl Acad Sci U S A, 110, 3997, 2013.
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