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1 Chinese Journal of Tissue Engineering Research August 19, 2016 Vol.20, No.34 Cytodext-3/ ( ). Cytodext-3 /[J] (34): DOI: /j.issn ORCID: () Cytodext-3 Cytodex t-3 Cytodext-3 / DNA Cytodex-3 Cytodext-3 / Cytodext-3 / :R318 :A : (2016) Cytodext-3 Cytodext-3 / Cytodext-3 DNA Cytodext-3 / (P < 0.05) 1 d 14 d DNA DNA (P < 0.05)Cytodext-3 / 5104 P.O. Box 10002, Shenyang

2 . Cytodext-3 / Yin He-yong, Master, PLA Institute of Orthopedics, General Hospital of Chinese PLA, Beijing , China; School of Medicine, Nankai University, Tianjin , China (Z )(863)(2012AA020502) Constructing injectable tissue-engineered cartilage using cytodex-3 microcarrier and alginate hydrogel Corresponding author: Peng Jiang, M.D., Associate researcher, Master s supervisor, PLA Institute of Orthopedics, General Hospital of Chinese PLA, Beijing , China; Yin He-yong 1, 2, Sun Zhen 1, Li Pan 3, Yu Xiao-ming 1, Xu Yi-chi 1, Sun Xun 1, 2, Xiao Bo 1, Wang Yu 1, Wang Ai-yuan 1, Guo Quan-yi 1, Xu Wen-jing 1, Lu Shi-bi 1, Peng Jiang 1 ( 1 PLA Institute of Orthopedics, General Hospital of Chinese PLA, Beijing , China; 2 School of Medicine, Nankai University, Tianjin , China; 3 Hebei Medical University, Shijiazhuang , Hebei Province, China) Abstract BACKGROUND: Alginate hydrogel and microcarrier both can be used as injectable scaffolds, but their shortcomings such as poor mechanical property and poor plasticity remain unresolved. OBJIECTIVE: To explore the feasibility of constructing an injectable tissue-engineered cartilage with cytodex-3 microcarrier/alginate hydrogel composite. METHODS: Injectable cytodex-3 microcarrier/alginate hydrogel composite scaffold and injectable alginate hydrogel scaffold were established, and the mechanical properties of the two scaffolds were detected. Chondrocytes-seeded cytodex-3 microcarrier was obtained after incubated in the bioreactor, and then composited with alginate hydrogel as experimental group; chondrocytes were co-cultured with alginate hydrogel as control group. Subsequently, cell viability and ability of DNA and glycosaminoglycan synthesis were detected. RESULTS AND CONCLUSION: The Young s modulus of the experimental group was significantly higher than that of the control group (P < 0.05). And in the control group, chondrocytes were in a round shape and evenly distributed in the alginate hydrogel; in the experimental group, chondrocytes adhered on the scaffold surface and evenly distributed in the scaffold. After 1 day of culture, both viable and numerous dead chondrocytes could be found in both two scaffolds; and after 14-day culture, there were no dead chondrocytes in both two scaffolds, abundant proliferating chondrocytes maintained a high cell viability, and the number of chondrocytes in the experimental group was significantly higer than that of the control group. What s more, the contents of DNA and glycosaminoglycans were in a rise with time in both two groups, which were significantly higher in the experimental group than the control group (P < 0.05). These results suggest that the cytodex-3 microcarrier/alginate hydrogel composite is a promising injectable scaffold in cartilage tissue engineering. Subject headings: Chondrocytes; Stents; Tissue Engineering Funding: the Beijing Science and Technology Project, No. Z ; the National High-tech Research and Development Program of China (863), No. 2012AA Cite this article: Yin HY, Sun Z, Li P, Yu XM, Xu YC, Sun X, Xiao B, Wang Y, Wang AY, Guo QY, Xu WJ, Lu SB, Peng J. Constructing injectable tissue-engineered cartilage using cytodex-3 microcarrier and alginate hydrogel. Zhongguo Zuzhi Gongcheng Yanjiu. 2016;20(34): Introduction [1] [2-3] [4-5] 250 µm [6-8] (phco 2 ) 40 [9] ISSN CN /R CODEN: ZLKHAH 5105

3 . Cytodext-3 / Cytodext-3 Cytodext-3 Cytodext- [10-11] / 1Materials and methods SCXK-() Cytodext-3 Sigma DMEM Gibco DNA Invitrogen Synthecon BX-51DP70 Olympus ElectroForce 3320 BOSS PBS3 1 cm 1 cm 1 cm0.15% 37 2 h 10%DMEM1 500 r/min 5 min10%dmed 37 5%CO 2 3 d 0.25% [12-13] P24 cm 4 cm Cytodext-3/ Cytodex-3 Cytodex-31 g100 ml PBS( Ca 2+ Mg 2+ )4 hpbs 5 minpbs50 mlpbs ( kpa)15 min 4 1 ml 2.4% 102 mmol/l CaCl 2 Cytodext-3/ 1 ml 2.4% 102 mmol/l CaCl 2 Cytodext P2 20 r/min1 min30 min24 h 50 r/min 37 5%CO % 102 mmol/l CaCl P2 1 ml 2.4% 102 mmol/l CaCl BOSS [14-16] d 5 mg/l FDA5 minpbs3 5 µmg/l PI5 minpbs [17] DNA d3 4(30 mmol/l 55 mmol/l0.15 mmol/l CaCl 2 )30 min 5106 P.O. Box 10002, Shenyang

4 . Cytodext-3 / 60 DNADNA [18] d3 19 [19] 1.6 SPSS 13.0 x _ ±s SNKP < Results 2.1 Cytodext-3/ Cytodext-3/ 1 1 Cytodext-3 / Figure 1 Gross observation of the cytodex-3 microcarrier/ alginate hydrogel composite scaffold 2.2 Cytodext-3/ [(24.22±2.32)(0.78±0.063) kpap < 0.05] d 2AD3 d 7 d 2BE14 d 2CF 2.4 DNA DNA DNA DNA (P < 0.05)3 2.5 (P < 0.05)4 3Discussion 4 cm 2 [20-22] 3 [3] [23] [24] Cytodext-3 / 1 / DNA ISSN CN /R CODEN: ZLKHAH 5107

5 . Cytodext-3 / A B C D E F DNA(µg) d 3 d 7 d 14 d 21 d 3 DNA Figure 3 DNA contents in the chondrocytes of the two groups at different time points of culture DNA (P < 0.05) (µg) 2 Figure 2 Dead/living chondrocytes stained in the two scaffolds at different time points of culture ABC 1714 d DE F 1714 d d 3 d 7 d 14 d 21 d 4 Figure 4 Glycosaminoglycan contents in the chondrocytes of the two groups at different time points of culture (P < 0.05) [25] [26] [14] Cytodext-3 Cytodext-3 / Cytodext-3 / CNKI 5108 P.O. Box 10002, Shenyang

6 . Cytodext-3 / 3 ( ) 4 References [1] Johnstone B,Alini M,Cucchiarini M,et al.tissue engineering for articular cartilage repair the state of the art. Eur Cells Mater.2013;25: [2] Matsunaga D,Akizuki S,Takizawa T,et al.repair of articular cartilage and clinical outcome after osteotomy with microfracture or abrasion arthroplasty for medial gonarthrosis.knee.2007;14(6): [3],. [J].,2013,17(41): [4] Eyrich D,Brandl F,Appel B,et al.long-term stable fibrin gels for cartilage engineering.biomaterials. 2007; 28(1): [5] Balakrishnan B,Jayakrishnan A.Self-cross-linking biopolymers as injectable in situ forming biodegradable scaffolds.biomaterials.2005;26: [6],,,. [J]., 2000,20(9): [7],,,. [J].,2007,11(14): [8],,,. [J]., 2015, 19(34): [9],,,. [J]., 2007,11(48): [10],,,. [J]., 2007, 15(10): ,803. [11],,. [J]., 2012,16(8): [12] Shao X,Goh JC,Hutmacher DW,et al.repair of large articular osteochondral defects using hybrid scaffolds and bone marrow-derived mesenchymal stem cells in a rabbit model. Tissue Eng. 2006;12: [13] Yang Q,Peng J,Guo Q,et al.a cartilage ECM-derived 3-D porous acellular matrix scaffold for in vivo cartilage tissue engineering with PKH26-labeled chondrogenic bone marrow-derived mesenchymal stem cells. Biomaterials.2008;29: [14] Hong Y,Gao C,Xie Y,et al.collagen-coated polylactide microspheres as chondrocyte microcarriers. Biomaterials.2005;26: [15] Bouwmeester PS,Kuijer R,Homminga GN,et al.a retrospective analysis of two independent prospective cartilagerepair studies: autogenous perichondrial grafting versus subchondraldrilling 10 years post-surgery. J Orthop Res.2002;20(2): [16] Harris JD,Siston RA,Pan X,et al. Flanigan, Autologous chondrocyte implantation: a systematic review.j Bone Joint Surg Am.2010;92(12): [17] Chung HJ,Go DH,Bae JW,et al.synthesis and characterization of Pluronic grafted chitosan copolymer as a novel injectable biomaterial.curr Appl Phys. 2005; 5: [18] Choi YS,Park S,Suh H.Adipose tissue engineering usingmesenchymal stem cells attached to injectable PLGA spheres.biomaterials.2005;26: [19] Matricali GA,Dereymaeker GPE,Luyten FP.Donor site morbidity after articular cartilage repair procedures : a review.acta Orthop Belg.2010;76: [20] Schrobback K, Klein TJ,Crawford R,et al.effects of oxygen and culture system on in vitro propagation and redifferentiation of osteoarthritic human articular chondrocytes.cell Tissue Res.2012;347: [21] Cummings LJ,Waters SK, Tissue growth in a rotating bioreactor. Part II: fluid flow and nutrient transport problems. Math Med Biol.2007;24: [22] Li YY,Cheng HW,Cheung KMet al.mesenchymal stem cell-collagen microspheres for articular cartilage repair: cell density and differentiation status.acta Biomater. 2014;10: [23] Malda J,van Blitterswijk CA,Grojec M,et al.expansion of bovine chondrocytes on microcarriers enhances redifferentiation.tissue Eng. 2003;9: [24] Schrobback K,Klein TJ,Schuetz M,et el.adult human articular chon-drocytes in a microcarrier-based culture system: expansion and redifferentiation.j Orthop Res 2011;29: [25] Mathieu M,Vigier S,Labour MN,et al.induction of mesenchymal stem cell differentiation and cartilage formation by cross-linker-free collagen microspheres. Eur Cell Mater.2014;28: [26] Hong Y,Gao C,Xie Y,et al.collagen-coated polylactide microspheres as chondrocyte microcarriers. Biomaterials.2005;26: ISSN CN /R CODEN: ZLKHAH 5109