In vitro evaluation of adipose-derived stromal vascular fraction combined with osteochondral integrated scaffold

doi:10.3969/j.issn.2095-4344.3230

Abstract

Abstract: BACKGROUND: A large number of animal and clinical experiments have confirmed that local transplantation of adipose-derived stromal vascular fraction can promote articular cartilage repair.
OBJECTIVE: To explore the feasibility of constructing tissue-engineered cartilage with acellular dermal matrix/biomineralized collagen integrated bone cartilage scaffold combined with autologous adipose-derived stromal vascular fraction.
METHODS: The groin fat pads of both sides of the female rabbit were taken out under sterile conditions, and the stromal vascular fraction was obtained by enzyme digestion. Flow cytometry was used to detect the surface specific protein and observe the ability of adipogenic, osteogenic and chondrogenic differentiation. Adipose-derived stromal vascular fraction (experimental group), adipose-derived mesenchymal stem cells containing transforming growth factor-β3 (cartilage induction group) and adipose-derived mesenchymal stem cells containing 10% fetal bovine serum (control group) were co-cultured on acellular dermal matrix/biomineralized collagen scaffold. CCK8 assay was used to detect cell proliferation. The number of cells was observed under fluorescence microscope. Real time-quantitative PCR detection was used to detect cartilage related gene expression. The content of glycosaminoglycan in extracellular matrix was determined by dimethylmethylene blue colorimetry.
RESULTS AND CONCLUSION: (1) The results of flow cytometry showed that the adipose-derived stromal vascular fraction had high expression of CD44, CD105 and CD90, and low expression of CD14, CD19 and CD45. Adipose-derived stromal vascular fraction had the ability to differentiate into adipocytes, osteoblasts and chondrocytes. (2) CCK8 assay showed that with the extension of culture time, the number of three kinds of cells on the acellular dermal matrix/biomineralized collagen scaffold increased, and the number of cells in the experimental group was higher than that in the other two groups (P < 0.05). Under the fluorescence microscope, the number of cells in the experimental group was more than that in the other two groups at 7 days after culture. (3) Real time-quantitative PCR results showed that at 21 days after culture, the mRNA expression levels of Sox9, proteoglycan and type II collagen in the experimental group were higher than those in the other two groups (P < 0.05). (4) Dimethylmethylene blue colorimetry results demonstrated that at 7, 14, and 21 days, the content of glycosaminoglycan in the experimental group was higher than that in the other two groups (P < 0.05). (5) The results showed that adipose-derived stromal vascular fraction combined with acellular dermal matrix / biomineralized collagen scaffold could effectively support cartilage formation in vitro.

Key words: bone, material, adipose derived stromal vascular fraction, acellular dermal matrix, biomineralized collagen, integrated scaffold, cartilage defect, osteochondral

CLC Number:

Chen Lei, Zheng Rui, Jie Yongsheng, Qi Hui, Sun Lei, Shu Xiong. In vitro evaluation of adipose-derived stromal vascular fraction combined with osteochondral integrated scaffold[J]. Chinese Journal of Tissue Engineering Research, 2021, 25(22): 3487-3492.

Figures/Tables 6

References

[1] HUEY DJ, HU JC, ATHANASIOU KA. Unlike bone, cartilage regeneration remains elusive. Science. 2012;338(6109):917-927.
[2] GUGJOO MB, SHARMA GT, AITHAL HP, et al. Cartilage tissue engineering: Role of mesenchymal stem cells along with growth factors & scaffolds. Indian J Med Res. 2016;144(3):339-347.
[3] ALBRIGHT JC, DAOUD AK. Microfracture and Microfracture Plus. Clin Sports Med. 2017;36(3):501-507.
[4] KRAEUTLER MJ, HOUCK DA, MCQUEEN MB, et al. A Cost-Effectiveness Analysis of Surgical Treatment Modalities for Chondral Lesions of the Knee: Microfracture, Osteochondral Autograft Transplantation, and Autologous Chondrocyte Implantation. Orthop J Sports Med. 2017; 5(5):1-8.
[5] GOMOLL AH, MADRY H, KNUTSEN G, et al. The subchondral bone in articular cartilage repair: current problems in the surgical management. Knee Surg Sports Traumatol Arthrosc. 2010;18(4):434-447.
[6] ASIK M, CIFTCI F, SEN C, et al. The microfracture technique for the treatment of full-thickness articular cartilage lesions of the knee: midterm results. Arthroscopy. 2008;24(11):1214-1220.
[7] ARMIENTO AR, STODDART MJ, ALINI M, et al. Biomaterials for articular cartilage tissue engineering: Learning from biology. Acta Biomater. 2018;65:1-20.
[8] PATEL JM, WISE BC, BONNEVIE ED, et al. A Systematic Review and Guide to Mechanical Testing for Articular Cartilage Tissue Engineering. Tissue Eng Part C Methods. 2019;25(10):593-608.
[9] WALTER SG, OSSENDORFF R, SCHILDBERG FA, et al. Articular cartilage regeneration and tissue engineering models: a systematic review. Arch Orthop Trauma Surg. 2019;139(3):305-316.
[10] KWON H, BROWN WE, LEE CA, et al. Surgical and tissue engineering strategies for articular cartilage and meniscus repair. Nat Rev Rheumatol. 2019;15(9):550-570.
[11] MEHRANFAR S, ABDI RAD I, MOSTAFAV E, et al. The use of stromal vascular fraction (SVF), platelet-rich plasma (PRP) and stem cells in the treatment of osteoarthritis: an overview of clinical trials. Artif Cells Nanomed Biotechnol. 2019;47(1):882-890.
[12] BANSAL H, COMELLA K, LEON J, et al. Intra-articular injection in the knee of adipose derived stromal cells (stromal vascular fraction) and platelet rich plasma for osteoarthritis. J Transl Med. 2017; 15(1):141.
[13] 郑蕊,杰永生,陈磊,等.脱细胞真皮基质/生物矿化胶原一体化骨软骨支架的制备及其生物相容性的研究[J].中国医药生物技术, 2019,14(2):121-126.
[14] 舒雄,杰永生,郑蕊,等.激活的富血小板血浆促进Pellet 培养的脂肪间充质干细胞成软骨样分化[J].中国医药生物技术,2018, 13(4):328-333.
[15] ZHOU W, LIN J, ZHAO K, et al. Single-Cell Profiles and Clinically Useful Properties of Human Mesenchymal Stem Cells of Adipose and Bone Marrow Origin. Am J Sports Med. 2019;47(7):1722-1733.
[16] ZHAO Z, FAN C, CHEN F, et al. Progress in Articular Cartilage Tissue Engineering: A Review on Therapeutic Cells and Macromolecular Scaffolds. Macromol Biosci. 2020;20(2):e1900278.
[17] BAKHSHAYESH A, BABAIE S, NASRABADI HT, et al. An overview of various treatment strategies, especially tissue engineering for damaged articular cartilage. Artif Cells Nanomed Biotechnol. 2020;48(1):1089-1104.
[18] CIPOLLARO L, CIARDULLI MC, PORTA GD, et al. Biomechanical issues of tissue-engineered constructs for articular cartilage regeneration: in vitro and in vivo approaches. Br Med Bull. 2019;132(1):53-80.
[19] LEMOINE M, CASEY SM, O’BYRNE JM, et al. The development of natural polymer scaffold-based therapeutics for osteochondral repair. Biochem Soc Trans. 2020;48(4):1433-1445.
[20] Dan X, Wei L, Li JJ, et al. Engineering 3D functional tissue constructs using self-assembling cell-laden microniches. Acta Biomater. 2020; 114:170-182.
[21] BAPTISTA LS. Adipose stromal/stem cells in regenerative medicine: Potentials and limitations. World J Stem Cells. 2020;12(1):1-7.
[22] WEN YT, DAI NT, HSU SH. Biodegradable water-based polyurethane scaffolds with a sequential release function for cell-free cartilage tissue engineering. Acta Biomater. 2019;88:301-313.
[23] CARVALHO MS, SILVA JC, UDANGAWA RN, et al. Co-culture cell-derived extracellular matrix loaded electrospun microfibrous scaffolds for bone tissue engineering. Mater Sci Eng C Mater Biol Appl. 2019;99:479-490.
[24] TAKAHASHI H, OKANO T. Thermally-triggered fabrication of cell sheets for tissue engineering and regenerative medicine. Adv Drug Deliv Rev. 2019;138:276-292.
[25] NEO PY, TEH TK, TAY AS, et al. Stem cell-derived cell-sheets for connective tissue engineering. Connect Tissue Res. 2016;57(6): 428-442.
[26] CHOI JS, CHAE DS, RYU HA, et al. Transplantation of human adipose tissue derived-SVF enhance liver function through high anti-inflammatory property. Biochim Biophys Acta Mol Cell Biol Lipids. 2019;1864(12):158526.
[27] KILINC MO, SANTIDRIAN A, MINEV I, et al. The ratio of ADSCs to HSC-progenitors in adipose tissue derived SVF may provide the key to predict the outcome of stem-cell therapy. Clin Transl Med. 2018;7(1):5.
[28] SOLODEEV I, ORGIL M, COHEN M, et al. Cryopreservation of Stromal Vascular Fraction Cells Reduces Their Counts but Not Their Stem Cell Potency. Plast Reconstr Surg Glob Open. 201;7(7):e2321.
[29] MORANDI EM, PLONER C, WOLFRAM D, et al. Risk factors and complications after body-contouring surgery and the amount of stromal vascular fraction cells found in subcutaneous tissue. Int Wound J. 2019;16(6):1545-1552.
[30] ZANATA F, BOWLES A, FRAZIER T, et al. Effect of Cryopreservation on Human Adipose Tissue and Isolated Stromal Vascular Fraction Cells: In Vitro and In Vivo Analyses. Plast Reconstr Surg. 2018;141(2): 232e-243e.
[31] MEHRANFAR S, RAD IA, MOSTAFAV E, et al. The use of stromal vascular fraction (SVF), platelet-rich plasma (PRP) and stem cells in the treatment of osteoarthritis: an overview of clinical trials. Artif Cells Nanomed Biotechnol. 2019;47(1):882-890.
[32] YOKOTA N, HATTORI M, OHTSURU T, et al. Comparative Clinical Outcomes After Intra-articular Injection With Adipose-Derived Cultured Stem Cells or Noncultured Stromal Vascular Fraction for the Treatment of Knee Osteoarthritis. Am J Sports Med. 2019;47(11):2577-2583.
[33] FRANKLIN SP, STOKER AM, BOZYNSKI CC, et al. Comparison of Platelet-Rich Plasma, Stromal Vascular Fraction (SVF), or SVF with an Injectable PLGA Nanofiber Scaffold for the Treatment of Osteochondral Injury in Dogs. J Knee Surg. 2018;31(7):686-697.
[34] DONGEN JA, HARMSEN MC, STEVENS HP, et al. Isolation of Stromal Vascular Fraction by Fractionation of Adipose Tissue. Methods Mol Biol. 2019;1993:91-103.
[35] YAO Y, DONG Z, LIAO Y, et al. Adipose Extracellular Matrix/Stromal Vascular Fraction Gel: A Novel Adipose Tissue-Derived Injectable for Stem Cell Therapy. Plast Reconstr Surg. 2017;139(4):867-879.
[36] SCHNEIDER MC, CHU S, RANDOLPH MA, et al. An in vitro and in vivo comparison of cartilage growth in chondrocyte-laden matrix metalloproteinase-sensitive poly(ethylene glycol) hydrogels with localized transforming growth factor β3. Acta Biomater. 2019;93: 97-110.
[37] JIA ZF, WANG SJ, LIANG YJ, et al. Combination of kartogenin and transforming growth factor-β3 supports synovial fluid-derived mesenchymal stem cell-based cartilage regeneration. Am J Transl Res. 2019;11(4):2056-2069.
[38] QU D, ZHU JP, CHILDS HR, et al. Nanofiber-based transforming growth factor-β3 release induces fibrochondrogenic differentiation of stem cells. Acta Biomater. 2019;93:111-122.
[39] CHEN YR, ZHOU ZX, ZHANG JY, et al. Low-Molecular-Weight Heparin-Functionalized Chitosan-Chondroitin Sulfate Hydrogels for Controlled Release of TGF-β3 and in vitro Neocartilage Formation. Front Chem. 2019;7:745.
[40] NAKAGAWA Y, FORTIER LA, MAO JJ, et al. Long-term Evaluation of Meniscal Tissue Formation in 3-dimensional-Printed Scaffolds With Sequential Release of Connective Tissue Growth Factor and TGF-β3 in an Ovine Model. Am J Sports Med. 2019;47(11):2596-2607.