Research article Special Issues

Perfusion-based co-culture model system for bone tissue engineering

  • Received: 07 May 2020 Accepted: 25 May 2020 Published: 29 May 2020
  • In this work, we report on a perfusion-based co-culture system that could be used for bone tissue engineering applications. The model system is created using a combination of Primary Human Umbilical Vein Endothelial Cells (HUVECs) and osteoblast-like Saos-2 cells encapsulated within a Gelatin Methacrylate (GelMA)-collagen hydrogel blend contained within 3D printed, perfusable constructs. The constructs contain dual channels, within a custom-built bioreactor, that were perfused with osteogenic media for up to two weeks in order to induce mineral deposition. Mineral deposition in constructs containing only HUVECs, only Saos-2 cells, or a combination thereof was quantified by microCT to determine if the combination of endothelial cells and bone-like cells increased mineral deposition. Histological and fluorescent staining was used to verify mineral deposition and cellular function both along and between the perfused channels. While there was not a quantifiable difference in the amount of mineral deposited in Saos-2 only versus Saos-2 plus HUVEC samples, the location of the deposited mineral differed dramatically between the groups and indicated that the addition of HUVECs within the GelMA matrix allowed Saos-2 cells, in diffusion limited regions of the construct, to deposit bone mineral. This work serves as a model on how to create perfusable bone tissue engineering constructs using a combination of 3D printing and cellular co-cultures.

    Citation: Stephen W. Sawyer, Kairui Zhang, Jason A. Horton, Pranav Soman. Perfusion-based co-culture model system for bone tissue engineering[J]. AIMS Bioengineering, 2020, 7(2): 91-105. doi: 10.3934/bioeng.2020009

    Related Papers:

  • In this work, we report on a perfusion-based co-culture system that could be used for bone tissue engineering applications. The model system is created using a combination of Primary Human Umbilical Vein Endothelial Cells (HUVECs) and osteoblast-like Saos-2 cells encapsulated within a Gelatin Methacrylate (GelMA)-collagen hydrogel blend contained within 3D printed, perfusable constructs. The constructs contain dual channels, within a custom-built bioreactor, that were perfused with osteogenic media for up to two weeks in order to induce mineral deposition. Mineral deposition in constructs containing only HUVECs, only Saos-2 cells, or a combination thereof was quantified by microCT to determine if the combination of endothelial cells and bone-like cells increased mineral deposition. Histological and fluorescent staining was used to verify mineral deposition and cellular function both along and between the perfused channels. While there was not a quantifiable difference in the amount of mineral deposited in Saos-2 only versus Saos-2 plus HUVEC samples, the location of the deposited mineral differed dramatically between the groups and indicated that the addition of HUVECs within the GelMA matrix allowed Saos-2 cells, in diffusion limited regions of the construct, to deposit bone mineral. This work serves as a model on how to create perfusable bone tissue engineering constructs using a combination of 3D printing and cellular co-cultures.



    加载中

    Acknowledgments



    This work was supported by (IGERT) DMR-DGE-1068780. This work was also partially supported by NIH R21GM129607 awarded to Pranav Soman. We would like to thank the Syracuse University Machine Shop, Lucas D. Albrecht, and Alex B. Filip for creating the polycarbonate bioreactors and printing the ABS cages. We would also like to thank Professor Megan E. Oest from SUNY Upstate Medical University for their aid in microCT analysis and histological sectioning, and Professor Zhen Ma for the use of his fluorescence microscope.

    Conflict of interest



    The authors declare no conflict of interest.

    Author contribution



    S.W.S., K.Z. J.A.H and P.S. wrote the manuscript; S.W.S. J.A.H and P.S. conceived and designed the experiments; S.W.S. and K.Z. performed cell studies and analyzed results.

    [1] Buckwalter JA, Glimcher MJ, Cooper RR (1995) Bone biology. Part I: Structure, blood supply, cells, matrix, and mineralization. J Bone Joint Surg 77: 1256-1275. doi: 10.2106/00004623-199508000-00019
    [2] McCarthy I (2006) The physiology of bone blood flow: a review. JB & JS 88: 4-9.
    [3] Laroche M (2002) Intraosseous circulation from physiology to disease. Joint Bone Spine 69: 262-269. doi: 10.1016/S1297-319X(02)00391-3
    [4] Mercado-Pagán ÁE, Stahl AM, Shanjani Y, et al. (2015) Vascularization in bone tissue engineering constructs. Ann Biomed Eng 43: 718-729. doi: 10.1007/s10439-015-1253-3
    [5] Rouwkema J, Westerweel PE, De Boer J, et al. (2009) The use of endothelial progenitor cells for prevascularized bone tissue engineering. Tissue Eng Part A 15: 2015-2027. doi: 10.1089/ten.tea.2008.0318
    [6] Krishnan L, Willett NJ, Guldberg RE (2014) Vascularization strategies for bone regeneration. Ann Biomed Eng 42: 432-444. doi: 10.1007/s10439-014-0969-9
    [7] Shanjani Y, Kang Y, Zarnescu L, et al. (2017) Endothelial pattern formation in hybrid constructs of additive manufactured porous rigid scaffolds and cell-laden hydrogels for orthopedic applications. J Mech Behav Biomed Mater 65: 356-372. doi: 10.1016/j.jmbbm.2016.08.037
    [8] Murphy WL, Peters MC, Kohn DH, et al. (2000) Sustained release of vascular endothelial growth factor from mineralized poly (lactide-co-glycolide) scaffolds for tissue engineering. Biomaterials 21: 2521-2527. doi: 10.1016/S0142-9612(00)00120-4
    [9] Lee KY, Peters MC, Anderson KW, et al. (2000) Controlled growth factor release from synthetic extracellular matrices. Nature 408: 998-1000. doi: 10.1038/35050141
    [10] Sheridan MH, Shea LD, Peters MC, et al. (2000) Bioabsorbable polymer scaffolds for tissue engineering capable of sustained growth factor delivery. J Controll Release 64: 91-102. doi: 10.1016/S0168-3659(99)00138-8
    [11] Peters MC, Polverini PJ, Mooney DJ (2002) Engineering vascular networks in porous polymer matrices. J Biomed Mater Res A 60: 668-678. doi: 10.1002/jbm.10134
    [12] Wang L, Fan H, Zhang ZY, et al. (2010) Osteogenesis and angiogenesis of tissue-engineered bone constructed by prevascularized β-tricalcium phosphate scaffold and mesenchymal stem cells. Biomaterials 31: 9452-9461. doi: 10.1016/j.biomaterials.2010.08.036
    [13] Yu H, VandeVord PJ, Gong W, et al. (2008) Promotion of osteogenesis in tissue-engineered bone by pre-seeding endothelial progenitor cells-derived endothelial cells. J Orthop Res 26: 1147-1152. doi: 10.1002/jor.20609
    [14] Villars F, Bordenave L, Bareille R, et al. (2000) Effect of human endothelial cells on human bone marrow stromal cell phenotype: role of VEGF? J Cell Biochem 79: 672-685. doi: 10.1002/1097-4644(20001215)79:4<672::AID-JCB150>3.0.CO;2-2
    [15] Santos MI, Reis RL (2010) Vascularization in bone tissue engineering: physiology, current strategies, major hurdles and future challenges. Macromol Biosci 10: 12-27. doi: 10.1002/mabi.200900107
    [16] Villars F, Guillotin B, Amedee T, et al. (2002) Effect of HUVEC on human osteoprogenitor cell differentiation needs heterotypic gap junction communication. Am J Physiol-Cell Ph 282: C775-C85. doi: 10.1152/ajpcell.00310.2001
    [17] Stegen S, van Gastel N, Carmeliet G (2015) Bringing new life to damaged bone: the importance of angiogenesis in bone repair and regeneration. Bone 70: 19-27. doi: 10.1016/j.bone.2014.09.017
    [18] Ghanaati S, Fuchs S, Webber MJ, et al. (2011) Rapid vascularization of starch–poly (caprolactone) in vivo by outgrowth endothelial cells in co-culture with primary osteoblasts. J Tissue Eng Regen M 5: e136-e143. doi: 10.1002/term.373
    [19] Guillotin B, Bareille R, Bourget C, et al. (2008) Interaction between human umbilical vein endothelial cells and human osteoprogenitors triggers pleiotropic effect that may support osteoblastic function. Bone 42: 1080-1091. doi: 10.1016/j.bone.2008.01.025
    [20] Unger RE, Sartoris A, Peters K, et al. (2007) Tissue-like self-assembly in cocultures of endothelial cells and osteoblasts and the formation of microcapillary-like structures on three-dimensional porous biomaterials. Biomaterials 28: 3965-3976. doi: 10.1016/j.biomaterials.2007.05.032
    [21] Ma JL, van den Beucken JJJP, Yang F, et al. (2011) Coculture of osteoblasts and endothelial cells: optimization of culture medium and cell ratio. Tissue Eng Part C 17: 349-357.
    [22] Black AF, Berthod F, L'Heureux N, et al. (1998) In vitro reconstruction of a human capillary-like network in a tissue-engineered skin equivalent. FASEB J 12: 1331-1340. doi: 10.1096/fasebj.12.13.1331
    [23] Chiu LLY, Montgomery M, Liang Y, et al. (2012) Perfusable branching microvessel bed for vascularization of engineered tissues. Proc Natl Acad Sci USA 109: E3414-E3423. doi: 10.1073/pnas.1210580109
    [24] Zheng Y, Chen J, Craven M, et al. (2012) In vitro microvessels for the study of angiogenesis and thrombosis. Proc Natl Acad Sci USA 109: 9342-9347. doi: 10.1073/pnas.1201240109
    [25] Chen YC, Lin RZ, Qi H, et al. (2012) Functional human vascular network generated in photocrosslinkable gelatin methacrylate hydrogels. Adv Funct Mater 22: 2027-2039. doi: 10.1002/adfm.201101662
    [26] Cuchiara MP, Gould DJ, McHale MK, et al. (2012) Integration of self-assembled microvascular networks with microfabricated PEG-based hydrogels. Adv Funct Mater 22: 4511-4518. doi: 10.1002/adfm.201200976
    [27] Chrobak KM, Potter DR, Tien J (2006) Formation of perfused, functional microvascular tubes in vitro. Microvasc Res 71: 185-196. doi: 10.1016/j.mvr.2006.02.005
    [28] Price GM, Wong KHK, Truslow JG, et al. (2010) Effect of mechanical factors on the function of engineered human blood microvessels in microfluidic collagen gels. Biomaterials 31: 6182-6189. doi: 10.1016/j.biomaterials.2010.04.041
    [29] Nichol JW, Koshy ST, Bae H, et al. (2010) Cell-laden microengineered gelatin methacrylate hydrogels. Biomaterials 31: 5536-5544. doi: 10.1016/j.biomaterials.2010.03.064
    [30] Park JH, Chung BG, Lee WG, et al. (2010) Microporous cell-laden hydrogels for engineered tissue constructs. Biotechnol Bioeng 106: 138-148.
    [31] Sadr N, Zhu M, Osaki T, et al. (2011) SAM-based cell transfer to photopatterned hydrogels for microengineering vascular-like structures. Biomaterials 32: 7479-7490. doi: 10.1016/j.biomaterials.2011.06.034
    [32] Therriault D, White SR, Lewis JA (2003) Chaotic mixing in three-dimensional microvascular networks fabricated by direct-write assembly. Nat Mater 2: 265-271. doi: 10.1038/nmat863
    [33] Golden AP, Tien J (2007) Fabrication of microfluidic hydrogels using molded gelatin as a sacrificial element. Lab Chip 7: 720-725. doi: 10.1039/b618409j
    [34] Miller JS, Stevens KR, Yang MT, et al. (2012) Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues. Nat Mater 11: 768-774. doi: 10.1038/nmat3357
    [35] Annabi N, Tamayol A, Uquillas JA, et al. (2014) 25th anniversary article: Rational design and applications of hydrogels in regenerative medicine. Adv Mater 26: 85-124. doi: 10.1002/adma.201303233
    [36] Bertassoni LE, Cardoso JC, Manoharan V, et al. (2014) Direct-write bioprinting of cell-laden methacrylated gelatin hydrogels. Biofabrication 6: 024105. doi: 10.1088/1758-5082/6/2/024105
    [37] Tan Y, Richards DJ, Trusk TC, et al. (2014) 3D printing facilitated scaffold-free tissue unit fabrication. Biofabrication 6: 024111. doi: 10.1088/1758-5082/6/2/024111
    [38] Wu W, DeConinck A, Lewis JA (2011) Omnidirectional printing of 3D microvascular networks. Adv Mater 23: H178-H183. doi: 10.1002/adma.201004625
    [39] Lee VK, Kim DY, Ngo H, et al. (2014) Creating perfused functional vascular channels using 3D bio-printing technology. Biomaterials 35: 8092-8102. doi: 10.1016/j.biomaterials.2014.05.083
    [40] Miller JS, Stevens KR, Yang MT, et al. (2012) Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues. Nat Mater 11: 768-774. doi: 10.1038/nmat3357
    [41] Kolesky DB, Truby RL, Gladman AS, et al. (2014) 3D bioprinting of vascularized, heterogeneous cell-laden tissue constructs. Adv Mater 26: 3124-3130. doi: 10.1002/adma.201305506
    [42] Skardal A, Zhang J, McCoard L, et al. (2010) Photocrosslinkable hyaluronan-gelatin hydrogels for two-step bioprinting. Tissue Eng Part A 16: 2675-2685. doi: 10.1089/ten.tea.2009.0798
    [43] Gao Q, He Y, Fu J-z, et al. (2015) Coaxial nozzle-assisted 3D bioprinting with built-in microchannels for nutrients delivery. Biomaterials 61: 203-215. doi: 10.1016/j.biomaterials.2015.05.031
    [44] Yang L, Shridhar SV, Gerwitz M, et al. (2016) An in vitro vascular chip using 3D printing-enabled hydrogel casting. Biofabrication 8: 035015. doi: 10.1088/1758-5090/8/3/035015
    [45] Tocchio A, Tamplenizza M, Martello F, et al. (2015) Versatile fabrication of vascularizable scaffolds for large tissue engineering in bioreactor. Biomaterials 45: 124-131. doi: 10.1016/j.biomaterials.2014.12.031
    [46] Sawyer SW, Shridhar SV, Zhang K, et al. (2018) Perfusion directed 3D mineral formation within cell-laden hydrogels. Biofabrication 10: 035013. doi: 10.1088/1758-5090/aacb42
    [47] Sladkova M, De Peppo GM (2014) Bioreactor systems for human bone tissue engineering. Processes 2: 494-525. doi: 10.3390/pr2020494
    [48] Rice JJ, Martino MM, De Laporte L, et al. (2013) Engineering the regenerative microenvironment with biomaterials. Adv Healthc Mater 2: 57-71. doi: 10.1002/adhm.201200197
    [49] Cartmell SH, Porter BD, García AJ, et al. (2003) Effects of medium perfusion rate on cell-seeded three-dimensional bone constructs in vitro. Tissue Eng 9: 1197-1203. doi: 10.1089/10763270360728107
    [50] Albrecht LD, Sawyer SW, Soman P (2016) Developing 3D scaffolds in the field of tissue engineering to treat complex bone defects. 3D Print Addit Manuf 3: 106-112. doi: 10.1089/3dp.2016.0006
    [51] Hutmacher DW (2001) Scaffold design and fabrication technologies for engineering tissues—state of the art and future perspectives. J Biomater Sci Poly Ed 12: 107-124. doi: 10.1163/156856201744489
    [52] Burg KJL, Porter S, Kellam JF (2000) Biomaterial developments for bone tissue engineering. Biomaterials 21: 2347-2359. doi: 10.1016/S0142-9612(00)00102-2
    [53] Reichert JC, Hutmacher DW (2011) Bone tissue engineering. Tissue Engineering Heidelberg: Springer, 431-456. doi: 10.1007/978-3-642-02824-3_21
    [54] Bose S, Vahabzadeh S, Bandyopadhyay A (2013) Bone tissue engineering using 3D printing. Mater Today 16: 496-504. doi: 10.1016/j.mattod.2013.11.017
    [55] Stevens MM (2008) Biomaterials for bone tissue engineering. Mater Today 11: 18-25. doi: 10.1016/S1369-7021(08)70086-5
    [56] Baranski JD, Chaturvedi RR, Stevens KR, et al. (2013) Geometric control of vascular networks to enhance engineered tissue integration and function. Proc Natl Acad Sci USA 110: 7586-7591. doi: 10.1073/pnas.1217796110
    [57] Lee VK, Lanzi AM, Ngo H, et al. (2014) Generation of multi-scale vascular network system within 3D hydrogel using 3D bio-printing technology. Cell Mol Bioeng 7: 460-472. doi: 10.1007/s12195-014-0340-0
    [58] Sawyer S, Oest M, Margulies B, et al. (2016) Behavior of encapsulated saos-2 cells within gelatin methacrylate hydrogels. J Tissue Sci Eng 7: 1000173. doi: 10.4172/2157-7552.1000173
    [59] Sawyer SW, Dong P, Venn S, et al. (2017) Conductive gelatin methacrylate-poly (aniline) hydrogel for cell encapsulation. Biomed Phys Eng Express 4: 015005. doi: 10.1088/2057-1976/aa91f9
    [60] Mikos AG, Sarakinos G, Lyman MD, et al. (1993) Prevascularization of porous biodegradable polymers. Biotechnol Bioeng 42: 716-723. doi: 10.1002/bit.260420606
  • bioeng-07-02-009-s001.pdf
  • Reader Comments
  • © 2020 the Author(s), licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0)
通讯作者: 陈斌, bchen63@163.com
  • 1. 

    沈阳化工大学材料科学与工程学院 沈阳 110142

  1. 本站搜索
  2. 百度学术搜索
  3. 万方数据库搜索
  4. CNKI搜索

Metrics

Article views(3883) PDF downloads(275) Cited by(5)

Article outline

Figures and Tables

Figures(5)

/

DownLoad:  Full-Size Img  PowerPoint
Return
Return

Catalog