多肽水凝胶在软骨再生工程中的应用

doi:10.1007/s12204-022-2507-5

摘要/Abstract

摘要： 关节软骨缺损被认为与骨关节炎的发展有关。相关组织再生的研究在骨关节炎的治疗中具有重要意义。用于软骨再生的支架应具有良好的组织相容性，力学性能及无细胞毒性，并能促进种子细胞的增殖和分化。多肽水凝胶中多肽序列的不同组合使其具有优异的生物可降解性和准确模拟软骨细胞细胞外基质的独特特性，以维持软骨表型的稳定性及促进关节透明软骨的再生。因此，多肽水凝胶在软骨再生中的应用前景广阔。本文系统综述了多肽水凝胶在软骨再生工程中的研究进展。对这些材料的特点、局限性和前景进行了评价。

关键词: 多肽水凝胶，聚氨基酸，关节软骨，软骨修复，软骨再生工程

Abstract: Articular cartilage defects are considered to be associated with the development of osteoarthritis. Research on relevant tissue regeneration is important in the treatment of osteoarthritis. The scaffolds applied incartilage regeneration should have good histocompatibility and mechanical properties, as well as no cytotoxicity,and promote the proliferation and differentiation of seed cells. Different combinations of peptide sequences inpolypeptide hydrogels endow them with unique characteristics including excellent biodegradability and accuratesimulation of the extracellular matrix of chondrocytes to maintain the stability of the chondrogenic phenotypeand facilitate articular hyaline cartilage regeneration. Thus, the application of polypeptide hydrogels for cartilage regeneration has a bright future. In this study, the research progress of polypeptide hydrogels used incartilage-regeneration engineering is systematically reviewed. The characteristics, limitations, and prospects ofthese materials are evaluated.

Key words: polypeptide hydrogel, poly amino acid, articular cartilage, cartilage repair, cartilage-regeneration engineering

中图分类号:

R 318.01

胡颖涵1, 朱泽宇1, 滕林2, 何雨石3, 邹德荣1 , 陆家瑜1. 多肽水凝胶在软骨再生工程中的应用[J]. J Shanghai Jiaotong Univ Sci, 2023, 28(4): 468-.

HU Yinghan1 (胡颖涵)，ZHU Zegu1 (朱泽宇)， TENG Lin2 (滕林)， HE Yushi3 (何雨石)，ZOU Derong1 (邹德荣)，LU Jiayu1*(陆家瑜). Applications of Polypeptide Hydrogels in Cartilage-Regeneration Engineering[J]. J Shanghai Jiaotong Univ Sci, 2023, 28(4): 468-.

参考文献

[1] GLYN-JONES S, PALMER A J R, AGRICOLA R,et al. Osteoarthritis [J]. The Lancet, 2015, 386(9991):376-387.
[2] JIN X Z, JONES G, CICUTTINI F, et al. Effect ofvitamin D supplementation on tibial cartilage volumeand knee pain among patients with symptomatic kneeosteoarthritis: A randomized clinical trial [J]. JAMA,2016, 315(10): 1005-1013.
[3] ARMIENTO A R, STODDART M J, ALINI M, et al.Biomaterials for articular cartilage tissue engineering:Learning from biology [J]. Acta Biomaterialia, 2018,65: 1-20.
[4] LUO Y Y, SINKEVICIUTE D, HE Y, et al. The minor collagens in articular cartilage [J]. Protein & Cell,2017, 8(8): 560-572.
[5] GUILAK F, NIMS R J, DICKS A, et al. Osteoarthritis as a disease of the cartilage pericellular matrix [J].Matrix Biology, 2018, 71/72: 40-50.
[6] KüHN K, D’LIMA D D, HASHIMOTO S, et al. Celldeath in cartilage [J]. Osteoarthritis and Cartilage,2004, 12(1): 1-16.
[7] KWON H, BROWN W E, LEE C A, et al. Surgicaland tissue engineering strategies for articular cartilageand meniscus repair [J]. Nature Reviews Rheumatology, 2019, 15(9): 550-570.
[8] CUCCHIARINI M, MADRY H. Biomaterial-guideddelivery of gene vectors for targeted articular cartilagerepair [J]. Nature Reviews Rheumatology, 2019, 15(1):18-29.
[9] TEMENOFF J S, MIKOS A G. Review: Tissue engineering for regeneration of articular cartilage [J]. Biomaterials, 2000, 21(5): 431-440.
[10] ORYAN A, SAHVIEH S. Effectiveness of chitosan scaffold in skin, bone and cartilage healing [J]. International Journal of Biological Macromolecules, 2017,104: 1003-1011.
[11] GUO T, NOSHIN M, BAKER H B, et al. 3D printedbiofunctionalized scaffolds for microfracture repair ofcartilage defects [J]. Biomaterials, 2018, 185: 219-231.
[12] MAKRIS E A, GOMOLL A H, MALIZOS K N, etal. Repair and tissue engineering techniques for articular cartilage [J]. Nature Reviews Rheumatology, 2015,11(1): 21-34.
[13] GUO T, FERLIN K M, KAPLAN D S, et al.Engineering niches for cartilage tissue regeneration[M]//Biology and engineering of stem cell niches.Boston: Academic Press, 2017.
[14] NIE X L, CHUAH Y J, ZHU W Z, et al. Decellularizedtissue engineered hyaline cartilage graft for articularcartilage repair [J]. Biomaterials, 2020, 235: 119821.
[15] RINGE J, BURMESTER G R, SITTINGER M. Regenerative medicine in rheumatic disease: Progress intissue engineering [J]. Nature Reviews Rheumatology,2012, 8(8): 493-498.
[16] HUANG B J, HU J C, ATHANASIOU K A. Cell-basedtissue engineering strategies used in the clinical repairof articular cartilage [J]. Biomaterials, 2016, 98: 1-22.
[17] JIANG Y Z, TUAN R S. Origin and function of cartilage stem/progenitor cells in osteoarthritis [J]. NatureReviews Rheumatology, 2015, 11(4): 206-212.
[18] GRACEFFA V, VINATIER C, GUICHEUX J, et al.Chasing chimeras: The elusive stable chondrogenicphenotype [J]. Biomaterials, 2019, 192: 199-225.
[19] LEE H P, GU L, MOONEY D J, et al. Mechanical confinement regulates cartilage matrix formationby chondrocytes [J]. Nature Materials, 2017, 16(12):1243-1251.
[20] WANG Y, CHEN Y, XU Y, et al. Effects of the bonding intensity between hyaluronan and gelatin on chondrogenic phenotypic maintenance [J]. Journal of Materials Chemistry B, 2020, 8: 9062-9074.
[21] VáZQUEZ-GONZáLEZ M, WILLNER I. Stimuliresponsive biomolecule-based hydrogels and their applications [J]. Angewandte Chemie International Edition, 2020, 59(36): 15342-15377.
[22] GAO J, ZHAN J, YANG Z M. Enzyme-instructed selfassembly (EISA) and hydrogelation of peptides [J]. Advanced Materials, 2020, 32(3): 1805798.
[23] DING X, ZHAO H M, LI Y Z, et al. Synthetic peptide hydrogels as 3D scaffolds for tissue engineering [J].Advanced Drug Delivery Reviews, 2020, 160: 78-104.
[24] FRENCH K M, SOMASUNTHARAM I, DAVIS M E.Self-assembling peptide-based delivery of therapeuticsfor myocardial infarction [J]. Advanced Drug DeliveryReviews, 2016, 96: 40-53.
[25] REN K X, HE C L, XIAO C S, et al. Injectable glycopolypeptide hydrogels as biomimetic scaffolds forcartilage tissue engineering [J]. Biomaterials, 2015, 51:238-249.
[26] FU K, WU H G, SU Z Q. Self-assembling peptidebased hydrogels: Fabrication, properties, and applications [J]. Biotechnology Advances, 2021, 49: 107752.
[27] CALIARI S R, BURDICK J A. A practical guide tohydrogels for cell culture [J]. Nature Methods, 2016,13(5): 405-414.
[28] CAI L L, LIU S, GUO J W, et al. Polypeptide-basedself-healing hydrogels: Design and biomedical applications [J]. Acta Biomaterialia, 2020, 113: 84-100.
[29] SONG Z Y, HAN Z Y, LV S X, et al. Syntheticpolypeptides: From polymer design to supramolecular assembly and biomedical application [J]. ChemicalSociety Reviews, 2017, 46(21): 6570-6599.
[30] LU Z H, LIU S J, LE Y G, et al. An injectable collagengenipin-carbon dot hydrogel combined with photodynamic therapy to enhance chondrogenesis [J]. Biomaterials, 2019, 218: 119190.
[31] MREDHA M T I, KITAMURA N, NONOYAMA T,et al. Anisotropic tough double network hydrogel fromfish collagen and its spontaneous in vivo bonding tobone [J]. Biomaterials, 2017, 132: 85-95.
[32] SHI W L, SUN M Y, HU X Q, et al. Structurally andfunctionally optimized silk-fibroin–gelatin scaffold using 3D printing to repair cartilage injury in vitro and invivo [J]. Advanced Materials, 2017, 29(29): 1701089.
[33] AISENBREY E A, BRYANT S J. The role of chondroitin sulfate in regulating hypertrophy during MSCchondrogenesis in a cartilage mimetic hydrogel underdynamic loading [J]. Biomaterials, 2019, 190/191: 51-62.
[34] PARMAR P A, CHOW L W, ST-PIERRE J P, etal. Collagen-mimetic peptide-modifiable hydrogels forarticular cartilage regeneration [J]. Biomaterials, 2015,54: 213-225.
[35] CHEN Z Y, ZHANG Q, LI H M, et al. Elastinlike polypeptide modified silk fibroin porous scaffoldpromotes osteochondral repair [J]. Bioactive Materials, 2021, 6(3): 589-601.
[36] HONG H, SEO Y B, KIM D Y, et al. Digital lightprocessing 3D printed silk fibroin hydrogel for cartilage tissue engineering [J]. Biomaterials, 2020, 232:119679.
[37] QI C, LIU J, JIN Y, et al. Photo-crosslinkable, injectable sericin hydrogel as 3D biomimetic extracellular matrix for minimally invasive repairing cartilage[J]. Biomaterials, 2018, 163: 89-104.
[38] LIU H, CHENG Y L, CHEN J J, et al. Componenteffect of stem cell-loaded thermosensitive polypeptidehydrogels on cartilage repair [J]. Acta Biomaterialia,2018, 73: 103-111.
[39] LEE S S, CHOI G E, LEE H J, et al. Layered doublehydroxide and polypeptide thermogel nanocompositesystem for chondrogenic differentiation of stem cells[J]. ACS Applied Materials & Interfaces, 2017, 9(49):42668-42675.
[40] KIM S H, LEE H R, YU S J, et al. Hydrogel-laden paper scaffold system for origami-based tissue engineering [J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(50):15426-15431.
[41] LAM J, CLARK E C, FONG E L S, et al. Evaluation of cell-laden polyelectrolyte hydrogels incorporating poly(l-Lysine) for applications in cartilage tissueengineering [J]. Biomaterials, 2016, 83: 332-346.
[42] LIN C, CROWLEY S T, UCHIDA S, et al. Treatmentof intervertebral disk disease by the administration ofmRNA encoding a cartilage-anabolic transcription factor [J]. Molecular Therapy: Nucleic Acids, 2019, 16:162-171.
[43] LI R, XU J B, WONG D S H, et al. Self-assembled Ncadherin mimetic peptide hydrogels promote the chondrogenesis of mesenchymal stem cells through inhibition of canonical Wnt/β-catenin signaling [J]. Biomaterials, 2017, 145: 33-43.
[44] KIM S J, KIM J E, KIM S H, et al. Therapeutic effects of neuropeptide substance P coupled with selfassembled peptide nanofibers on the progression of osteoarthritis in a rat model [J]. Biomaterials, 2016, 74:119-130.
[45] LU J J, SHEN X Z, SUN X, et al. Increased recruitment of endogenous stem cells and chondrogenic differentiation by a composite scaffold containing bonemarrow homing peptide for cartilage regeneration [J].Theranostics, 2018, 8(18): 5039-5058.
[46] ALMEIDA H V, ESWARAMOORTHY R,CUNNIFFE G M, et al. Fibrin hydrogels functionalized with cartilage extracellular matrix andincorporating freshly isolated stromal cells as aninjectable for cartilage regeneration [J]. Acta Biomaterialia, 2016, 36: 55-62.
[47] USTUN YAYLACI S, SARDAN EKIZ M, ARSLANE, et al. Supramolecular GAG-like self-assembled glycopeptide nanofibers induce chondrogenesis and cartilage regeneration [J]. Biomacromolecules, 2016, 17(2):679-689.
[48] VEGA S L, KWON M Y, SONG K H, et al. Combinatorial hydrogels with biochemical gradients for screening 3D cellular microenvironments [J]. Nature Communications, 2018, 9: 614.
[49] ARMIENTO A R, ALINI M, STODDART M J. Articular fibrocartilage - Why does hyaline cartilage failto repair? [J]. Advanced Drug Delivery Reviews, 2019,146: 289-305.
[50] GU L S, SHAN T T, MA Y X, et al. Novel biomedical applications of crosslinked collagen [J]. Trends inBiotechnology, 2019, 37(5): 464-491.
[51] SORUSHANOVA A, DELGADO L M, WU Z N, et al.The collagen suprafamily: From biosynthesis to advanced biomaterial development [J]. Advanced Materials, 2019, 31(1): 1801651.
[52] KLOTZ B J, GAWLITTA D, ROSENBERG A JW P, et al. Gelatin-methacryloyl hydrogels: Towards biofabrication-based tissue repair [J]. Trends inBiotechnology, 2016, 34(5): 394-407.
[53] ALTUNBAS A, POCHAN D J. Peptide-based andpolypeptide-based hydrogels for drug delivery and tissue engineering [J]. Topics in Current Chemistry, 2012,310: 135-167.
[54] DALY A C, CRITCHLEY S E, RENCSOK E M, et al.A comparison of different bioinks for 3D bioprinting offibrocartilage and hyaline cartilage [J]. Biofabrication,2016, 8(4): 045002.
[55] HAN L, XU J L, LU X, et al. Biohybrid methacrylatedgelatin/polyacrylamide hydrogels for cartilage repair[J]. Journal of Materials Chemistry B, 2017, 5(4): 731-741.
[56] HAN L, WANG M H, LI P F, et al. Mussel-inspiredtissue-adhesive hydrogel based on the polydopamine–chondroitin sulfate complex for growth-factor-free cartilage regeneration [J]. ACS Applied Materials & Interfaces, 2018, 10(33): 28015-28026.
[57] GAN D L, XU T, XING W S, et al. Mussel-inspireddopamine oligomer intercalated tough and resilientgelatin methacryloyl (GelMA) hydrogels for cartilageregeneration [J]. Journal of Materials Chemistry B,2019, 7(10): 1716-1725.
[58] PARMAR P A, ST-PIERRE J P, CHOW L W, etal. Enhanced articular cartilage by human mesenchymal stem cells in enzymatically mediated transientlyRGDS-functionalized collagen-mimetic hydrogels [J].Acta Biomaterialia, 2017, 51: 75-88.
[59] PARMAR P A, SKAALURE S C, CHOW L W, etal. Temporally degradable collagen–mimetic hydrogelstuned to chondrogenesis of human mesenchymal stemcells [J]. Biomaterials, 2016, 99: 56-71.
[60] PENG Y Y, YOSHIZUMI A, DANON S J, et al. AStreptococcus pyogenes derived collagen-like proteinas a non-cytotoxic and non-immunogenic cross-linkablebiomaterial [J]. Biomaterials, 2010, 31(10): 2755-2761.
[61] GHOLIPOURMALEKABADI M, SAPRU S,SAMADIKUCHAKSARAEI A, et al. Silk fibroinfor skin injury repair: Where do things stand? [J].Advanced Drug Delivery Reviews, 2020, 153: 28-53.
[62] CHENG G, DAVOUDI Z, XING X, et al. Advancedsilk fibroin biomaterials for cartilage regeneration [J].ACS Biomaterials Science & Engineering, 2018, 4(8):2704-2715.
[63] DU S, ZHANG J, ZHOU W T, et al. Interactionsbetween fibroin and sericin proteins from Antheraeapernyi and Bombyx mori silk fibers [J]. Journal of Colloid and Interface Science, 2016, 478: 316-323.
[64] KIM S H, YEON Y K, LEE J M, et al. Precisely printable and biocompatible silk fibroin bioink for digitallight processing 3D printing [J]. Nature Communications, 2018, 9: 1620.
[65] BASU A, KUNDURU K R, KATZHENDLER J, et al.Poly(α-hydroxy acid)s and poly(α-hydroxy acid-co-α-amino acid)s derived from amino acid [J]. AdvancedDrug Delivery Reviews, 2016, 107: 82-96.
[66] GELAIN F, SILVA D, CAPRINI A, et al. BMHP1-derived self-assembling peptides: Hierarchically assembled structures with self-healing propensity and potential for tissue engineering applications [J]. ACS Nano,2011, 5(3): 1845-1859.
[67] ZAMUNER A, CAVO M, SCAGLIONE S, et al. Design of decorated self-assembling peptide hydrogels asarchitecture for mesenchymal stem cells [J]. Materials,2016, 9(9): 727.
[68] CAO F Y, YIN W N, FAN J X, et al. A novel functionof BMHP1 and cBMHP1 peptides to induce the osteogenic differentiation of mesenchymal stem cells [J].Biomaterials Science, 2015, 3(2): 345-351.
[69] BOGUNOVIC L, WETTERS N G, JAIN A, et al. Invitro analysis of micronized cartilage stability in theknee: Effect of fibrin level, defect size, and defect location [J]. Arthroscopy: the Journal of Arthroscopic &Related Surgery, 2019, 35(4): 1212-1218.
[70] PENG Z, SUN H, BUNPETCH V, et al. The regulation of cartilage extracellular matrix homeostasis injoint cartilage degeneration and regeneration [J]. Biomaterials, 2021, 268: 120555.
[71] KIM J S, KIM T H, KANG D L, et al. Chondrogenicdifferentiation of human ASCs by stiffness control in3D fibrin hydrogel [J]. Biochemical and Biophysical Research Communications, 2020, 522(1): 213-219.
[72] DE MELO B A G, JODAT Y A, MEHROTRA S, etal. 3D printed cartilage-like tissue constructs with spatially controlled mechanical properties [J]. AdvancedFunctional Materials, 2019, 29(51): 1906330.
[73] KARGARPOUR Z, NASIRZADE J, STRAUSS F J,et al. Platelet-rich fibrin suppresses in vitro osteoclastogenesis [J]. Journal of Periodontology, 2020, 91(3):413-421.
[74] WONG C C, OU K L, LIN Y H, et al. Platelet-richfibrin facilitates one-stage cartilage repair by promoting chondrocytes viability, migration, and matrix synthesis [J]. International Journal of Molecular Sciences,2020, 21(2): 577.
[75] MCDERMOTT I D. Patellar chondral defect treatment with a cell-free polyglycolic acid-hyaluronanbased implant and platelet-rich fibrin glue after previously failed microfracture [J]. SAGE Open MedicalCase Reports, 2019, 7: 2050313X18823470.
[76] BARBON S, STOCCO E, MACCHI V, et al. Plateletrich fibrin scaffolds for cartilage and tendon regenerative medicine: From bench to bedside [J]. InternationalJournal of Molecular Sciences, 2019, 20(7): 1701.
[77] TIWARI S, BAHADUR P. Modified hyaluronic acidbased materials for biomedical applications [J]. International Journal of Biological Macromolecules, 2019,121: 556-571.
[78] ACAR H, SRIVASTAVA S, CHUNG E J, et al. Selfassembling peptide-based building blocks in medical applications [J]. Advanced Drug Delivery Reviews,2017, 110/111: 65-79.
[79] OKESOLA B O, WU Y H, DERKUS B, et al.Supramolecular self-assembly to control structural andbiological properties of multicomponent hydrogels [J].Chemistry of Materials, 2019, 31(19): 7883-7897.
[80] WOLF K J, KUMAR S. Hyaluronic acid: Incorporating the bio into the material [J]. ACS BiomaterialsScience & Engineering, 2019, 5(8): 3753-3765.
[81] DOU X Q, FENG C L. Amino acids and peptidebased supramolecular hydrogels for three-dimensionalcell culture [J]. Advanced Materials, 2017, 29(16):1604062.
[82] LI S Y, WANG X, CAO B, et al. Effects of nanoscalespatial arrangement of arginine-glycine-aspartate peptides on dedifferentiation of chondrocytes [J]. NanoLetters, 2015, 15(11): 7755-7765.
[83] QIAO Y S, LIU X Z, ZHOU X C, et al. Gelatin templated polypeptide co-cross-linked hydrogel for boneregeneration [J]. Advanced Healthcare Materials, 2020,9(1): 1901239.
[84] THAMBI T, LI Y, LEE D S. Injectable hydrogels forsustained release of therapeutic agents [J]. Journal ofControlled Release, 2017, 267: 57-66.
[85] ZHENG H Y, YOSHITOMI T, YOSHIMOTO K.Analysis of chirality effects on stem cell fate usingthree-dimensional fibrous peptide hydrogels [J]. ACSApplied Bio Materials, 2018, 1(3): 538-543.
[86] UMAN S, DHAND A, BURDICK J A. Recent advances in shear-thinning and self-healing hydrogels forbiomedical applications [J]. Journal of Applied Polymer Science, 2020, 137(25): 48668.
[87] YADAV N, CHAUHAN M K, CHAUHAN V S. Shortto ultrashort peptide-based hydrogels as a platformfor biomedical applications [J]. Biomaterials Science,2020, 8(1): 84-100.
[88] O’BRIEN S, BRANNIGAN R P, IBANEZ R, etal. Biocompatible polypeptide-based interpenetratingnetwork (IPN) hydrogels with enhanced mechanicalproperties [J]. Journal of Materials Chemistry B, 2020,8(34): 7785-7791.
[89] OKESOLA B O, LAU H K, DERKUS B, et al.Covalent co-assembly between resilin-like polypeptideand peptide amphiphile into hydrogels with controllednanostructure and improved mechanical properties [J].Biomaterials Science, 2020, 8(3): 846-857.
[90] ANNABI N, RANA D, SANI E S, et al. Engineeringa sprayable and elastic hydrogel adhesive with antimicrobial properties for wound healing [J]. Biomaterials,2017, 139: 229-243.
[91] JIN H L, WAN C, ZOU Z W, et al. Tumor ablationand therapeutic immunity induction by an injectablepeptide hydrogel [J]. ACS Nano, 2018, 12(4): 3295-3310.
[92] GRIFFIN D R, ARCHANG M M, KUAN C H, et al.Activating an adaptive immune response from a hydrogel scaffold imparts regenerative wound healing [J].Nature Materials, 2021, 20(4): 560-569.
[93] FAROKHI M, MOTTAGHITALAB F, FATAHI Y, etal. Overview of silk fibroin use in wound dressings [J].Trends in Biotechnology, 2018, 36(9): 907-922.
[94] KOIVUSALO L, KAUPPILA M, SAMANTA S, etal. Tissue adhesive hyaluronic acid hydrogels for sutureless stem cell delivery and regeneration of cornealepithelium and stroma [J]. Biomaterials, 2019, 225:119516.
[95] ZHU D Q, WANG H Y, TRINH P, et al. Elastin-likeprotein-hyaluronic acid (ELP-HA) hydrogels with decoupled mechanical and biochemical cues for cartilageregeneration [J]. Biomaterials, 2017, 127: 132-140.
[96] ZHANG X Z, CAI D D, ZHOU F F, et al. Targeting downstream subcellular YAP activity as a function of matrix stiffness with Verteporfin-encapsulatedchitosan microsphere attenuates osteoarthritis [J]. Biomaterials, 2020, 232: 119724.
[97] DAVIDSON M D, BAN E, SCHOONEN A C M, etal. Mechanochemical adhesion and plasticity in multi-fiber hydrogel networks [J]. Advanced Materials, 2020,32(8): 1905719.
[98] YANG J R, LI Y Q, LIU Y B, et al. Influence of hydrogel network microstructures on mesenchymal stemcell chondrogenesis in vitro and in vivo [J]. Acta Biomaterialia, 2019, 91: 159-172.
[99] JEYAKUMAR V, NICULESCU-MORZSA E,BAUER C, et al. Redifferentiation of articularchondrocytes by hyperacute serum and platelet richplasma in collagen type I hydrogels [J]. InternationalJournal of Molecular Sciences, 2019, 20(2): 316.
[100] BRETSCHNEIDER H, STIEHLER M, HARTMANNA, et al. Characterization of primary chondrocytes harvested from hips with femoroacetabular impingement[J]. Osteoarthritis and Cartilage, 2016, 24(9): 1622-1628.
[101] NOVAK T, SEELBINDER B, TWITCHELL C M, etal. Mechanisms and microenvironment investigation ofcellularized high density gradient collagen matrices viadensification [J]. Advanced Functional Materials, 2016,26(16): 2617-2628.