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Matrix composition regulates three-dimensional network formation by endothelial cells and mesenchymal stem cells in collagen/fibrin materials

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Abstract

Co-cultures of endothelial cells (EC) and mesenchymal stem cells (MSC) in three-dimensional (3D) protein hydrogels can be used to recapitulate aspects of vasculogenesis in vitro. MSC provide paracrine signals that stimulate EC to form vessel-like structures, which mature as the MSC transition to the role of mural cells. In this study, vessel-like network formation was studied using 3D collagen/fibrin (COL/FIB) matrices seeded with embedded EC and MSC and cultured for 7 days. The EC:MSC ratio was varied from 5:1, 3:2, 1:1, 2:3 and 1:5. The matrix composition was varied at COL/FIB compositions of 100/0 (pure COL), 60/40, 50/50, 40/60 and 0/100 (pure FIB). Vasculogenesis was markedly decreased in the highest EC:MSC ratio, relative to the other cell ratios. Network formation increased with increasing fibrin content in composite materials, although the 40/60 COL/FIB and pure fibrin materials exhibited the same degree of vasculogenesis. EC and MSC were co-localized in vessel-like structures after 7 days and total cell number increased by approximately 70%. Mechanical property measurements showed an inverse correlation between matrix stiffness and network formation. The effect of matrix stiffness was further investigated using gels made with varying total protein content and by crosslinking the matrix using the dialdehyde glyoxal. This systematic series of studies demonstrates that matrix composition regulates vasculogenesis in 3D protein hydrogels, and further suggests that this effect may be caused by matrix mechanical properties. These findings have relevance to the study of neovessel formation and the development of strategies to promote vascularization in transplanted tissues.

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References

  1. Jain RK (2003) Molecular regulation of vessel maturation. Nat Med 9(6):685–693

    Article  PubMed  CAS  Google Scholar 

  2. Langer R, Vacanti JP (1993) Tissue engineering. Science 260(5110):920–926

    Article  PubMed  CAS  Google Scholar 

  3. Mikos AG, Herring SW, Ochareon P, Elisseeff J, Lu HH, Kandel R, Schoen FJ, Toner M, Mooney D, Atala A, Van Dyke ME, Kaplan DL, Vunjak-Novakovic G (2006) Engineering complex tissues. Tissue Eng 12(12):3307–3339

    Article  PubMed  CAS  Google Scholar 

  4. Novosel EC, Kleinhans C, Kluger PJ (2011) Vascularization is the key challenge in tissue engineering. Adv Drug Deliv Rev 63(4–5):300–311

    Article  PubMed  CAS  Google Scholar 

  5. Santos MI, Reis RL (2010) Vascularization in bone tissue engineering: physiology, current strategies, major hurdles and future challenges. Macromol Biosci 10(1):12–27

    Article  PubMed  CAS  Google Scholar 

  6. Lovett M, Lee K, Edwards A, Kaplan DL (2009) Vascularization strategies for tissue engineering. Tissue Eng Part B 15(3):353–370

    Article  CAS  Google Scholar 

  7. Vailhe B, Vittet D, Feige JJ (2001) In vitro models of vasculogenesis and angiogenesis. Lab Invest 81(4):439–452

    Article  PubMed  CAS  Google Scholar 

  8. Davis GE, Stratman AN, Sacharidou A, Koh W (2011) Molecular basis for endothelial lumen formation and tubulogenesis during vasculogenesis and angiogenic sprouting. Int Rev Cell Mol Biol 288:101–165

    Article  PubMed  CAS  Google Scholar 

  9. Koh W, Stratman AN, Sacharidou A, Davis GE (2008) In vitro three dimensional collagen matrix models of endothelial lumen formation during vasculogenesis and angiogenesis. Methods Enzymol 443:83–101

    Article  PubMed  CAS  Google Scholar 

  10. Shepherd B, Jay S, Saltzman W, Tellides G, Pober J (2009) Human aortic smooth muscle cells promote arteriole formation by coengrafted endothelial cells. Tissue Eng Part A 15:165–173

    Article  PubMed  CAS  Google Scholar 

  11. Au P, Tam J, Fukumura D, Jain R (2008) Bone marrow derived mesenchymal stem cells facilitate engineering of long-lasting functional vasculature. Blood 111:4551–4558

    Article  PubMed  CAS  Google Scholar 

  12. Ghanaati S, Fuchs S, Webber MJ, Orth C, Barbeck M, Gomes ME, Reis RL, Kirkpatrick CJ (2011) Rapid vascularization of starch-poly(caprolactone) in vivo by outgrowth of endothelial cells in co-culture with primary osteoblasts. J Tissue Eng Regen Med 5(6):e136–e143

    Article  PubMed  CAS  Google Scholar 

  13. Merfeld-Clauss S, Gollahalli N, March K, Traktuev D (2010) Adipose tissue progenitor cells directly interact with endothelial cells to induce vascular network formation. Tissue Eng Part A 16:2953–2966

    Article  PubMed  CAS  Google Scholar 

  14. Stratman AN, Malotte KM, Mahan RD, Davis MJ, Davis GE (2009) Pericyte recruitment during vasculogenic tube assembly stimulates endothelial basement membrane matrix formation. Blood 114(24):5091–5101

    Article  PubMed  CAS  Google Scholar 

  15. Hellstrom M, Gerhardt H, Kalen M, Li X, Eriksson U, Wolburg H, Betsholtz C (2001) Lack of pericytes lead to endothelial hyperplasia and abnormal vascular morphogenesis. J Cell Biol 153(3):543–553

    Article  PubMed  CAS  Google Scholar 

  16. Baluk P, Hashizume H, McDonald DM (2005) Cellular abnormalities of blood vessels as targets in cancer. Curr Opin Genet Dev 15(1):102–111

    Article  PubMed  CAS  Google Scholar 

  17. Ghajar CM, Kachgal S, Kniazeva E, Mori H, Costes SV, George SC, Putnam AJ (2010) Mesenchymal cells stimulate capillary morphogenesis via distinct proteolytic mechanisms. Exp Cell Res 316(5):813–825

    Article  PubMed  CAS  Google Scholar 

  18. Sorrell JM, Baber MA, Caplan AI (2009) Influence of adult mesenchymal stem cells on in vitro vascular formation. Tissue Eng Part A 15(7):1751–1761

    Article  PubMed  CAS  Google Scholar 

  19. Duffy GP, Ahsan T, O’Brien T, Barry F, Nerem RM (2009) Bone marrow-derived mesenchymal stem cells promote angiogenic processes in a time- and dose-dependent manner in vitro. Tissue Eng Part A 15(9):2459–2470

    Article  PubMed  CAS  Google Scholar 

  20. Ghajar CM, Blevins KS, Hughes CCW, George SC, Putnam AJ (2006) Mesenchymal stem cells enhance angiogenesis in mechanically viable prevascularized tissues via early matrix metalloproteinase upregulation. Tissue Eng 12(10):2875–2888

    Article  PubMed  CAS  Google Scholar 

  21. Gruber R, Kandler B, Holzmann P, Vogele-Kadletz M, Losert U, Fischer MB, Watzek G (2005) Bone marrow stromal cells provide a local environment that favors migration and formation of tubular structures of endothelial cells. Tissue Eng 11(5):896–903

    Article  PubMed  CAS  Google Scholar 

  22. Pati S, Khakoo AY, Zhao J, Jimenez F, Gerber MH, Harting M, Redell JB, Grill R, Matsuo Y, Guha S, Cox CS, Retz MS, Holcomb JB, Dash PK (2011) Human mesenchymal stem cells inhibit vascular permeability by modulating vascular endothelial cadherin/β-catenin signaling. Stem Cells Dev 20(1):89–101

    Article  PubMed  CAS  Google Scholar 

  23. Caplan AI (2008) All MSCs are pericytes? Cell Stem Cell 3(3):229–230

    Article  PubMed  CAS  Google Scholar 

  24. Ma J, van den Beucken JJJP, Yang F, Both SK, Cui F-Z, Pan J, Jansen JA (2011) Coculture of osteoblasts and endothelial cells: optimization of culture medium and cell ratio. Tissue Eng Part C Methods 17(3):349–357

    Article  PubMed  CAS  Google Scholar 

  25. Melero-Martin JM, De Obaldia ME, Kang S-Y, Khan ZA, Yuan L, Oettgen P, Bischoff J (2008) Engineering robust and functional vascular networks in vivo with human adult and cord blood-derived progenitor cells. Circ Res 103(2):194–202

    Article  PubMed  CAS  Google Scholar 

  26. Rouwkema J, De Boer J, Van Blitterswijk CA (2006) Endothelial cells assemble into a 3-dimensional prevascular network in a bone tissue engineering construct. Tissue Eng 12(9):2685–2693

    Article  PubMed  CAS  Google Scholar 

  27. Dietrich F, Lelkes PI (2006) Fine-tuning of a three-dimensional microcarrier-based angiogenesis assay for the analysis of endothelial-mesenchymal cell co-cultures in fibrin and collagen gels. Angiogenesis 9(3):111–125

    Article  PubMed  CAS  Google Scholar 

  28. Martineau L, Doillon CJ (2007) Angiogenic response of endothelial cells seeded dispersed versus on beads in fibrin gels. Angiogenesis 10:269–277

    Article  PubMed  Google Scholar 

  29. Kroon ME, van Schie MLJ, van der Vecht B, van Hinsbergh VWM, Koolwijk P (2002) Collagen type I retards tube formation by human microvascular endothelial cells in a fibrin matrix. Angiogenesis 5:257–265

    Article  PubMed  CAS  Google Scholar 

  30. Rowe SL, Stegemann J (2006) Interpenetrating collagen–fibrin composite matrices with varying protein contents and ratios. Biomacromolecules 7:2942–2948

    Article  PubMed  CAS  Google Scholar 

  31. Rowe SL, Stegemann JP (2009) Microstructure and mechanics of collagen–fibrin matrices polymerized using ancrod snake venom enzyme. J Biomech Eng 131(6):061012

    Article  PubMed  Google Scholar 

  32. Critser PJ, Kreger ST, Voytik-Harbin SL, Yoder MC (2010) Collagen matrix physical properties modulate endothelial colony forming cell-derived vessels in vivo. Microvasc Res 80(1):23–30

    Article  PubMed  CAS  Google Scholar 

  33. Kniazeva E, Kachgal S, Putnam AJ (2011) Effects of extracellular matrix density and mesenchymal stem cells on neovascularization in vivo. Tissue Eng Part A 17(7–8):905–914

    Article  PubMed  CAS  Google Scholar 

  34. Allen P, Melero-Martin J, Bischoff J (2011) Type I collagen, fibrin and PuraMatrix matrices provide permissive environments for human endothelial and mesenchymal progenitor cells to form neovascular networks. J Tissue Eng Regen Med 5(4):e74–e86

    Article  PubMed  CAS  Google Scholar 

  35. Wang L, Stegemann JP (2011) Glyoxal crosslinking of cell-seeded chitosan/collagen hydrogels for bone regeneration. Acta Biomater 7(6):2410–2417

    Article  PubMed  CAS  Google Scholar 

  36. Kotlarchyk MA, Shreim SG, Alvarez-Elizondo MB, Estrada LC, Singh R, Valdevit L, Kniazeva E, Gratton E, Putnam AJ, Botvinick EL (2011) Concentration independent modulation of local micromechanics in a fibrin gel. PLoS ONE 6(5):e20201

    Article  PubMed  CAS  Google Scholar 

  37. Collen A, Koolwijk P, Kroon ME, Van Hinsbergh VWM (1998) Influence of fibrin structure on the formation and maintenance of capillary-like tubules by human microvascular endothelial cells. Angiogenesis 2:153–165

    PubMed  CAS  Google Scholar 

  38. Seidlits SK, Drinnan CT, Petersen RR, Shear JB, Suggs LJ, Schmidt CE (2011) Fibronectin-hyaluronic acid composite hydrogels for three-dimensional endothelial cell culture. Acta Biomater 7(6):2401–2409

    Article  PubMed  CAS  Google Scholar 

  39. Bala K, Ambwani K, Gohil NK (2011) Effect of different mitogens and serum concentration on HUVEC morphology and characteristics: Implication on use of higher passage cells. Tissue Cell 43(4):216–222

    Article  PubMed  CAS  Google Scholar 

  40. Rao RR, He J, Leach JK (2010) Biomineralized composite substrates increase gene expression with nonviral delivery. J Biomed Mater Res Part A 94(2):344–354

    Google Scholar 

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Acknowledgments

This work was supported in part by a National Science Foundation Graduate Research Fellowship (to RRR), National Heart, Lung and Blood Institute grant HL085339 (to AJP), as well as by the University of Michigan Cardiovascular Center Summer Research Fellowship (to AWP).

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Biomedical Engineering, University of Michigan, 1101 Beal Ave., Ann Arbor, MI, 48109, USA

    Rameshwar R. Rao, Alexis W. Peterson, Jacob Ceccarelli, Andrew J. Putnam & Jan P. Stegemann

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  1. Rameshwar R. Rao
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  2. Alexis W. Peterson
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  3. Jacob Ceccarelli
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  4. Andrew J. Putnam
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  5. Jan P. Stegemann
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Correspondence to Jan P. Stegemann.

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Rao, R.R., Peterson, A.W., Ceccarelli, J. et al. Matrix composition regulates three-dimensional network formation by endothelial cells and mesenchymal stem cells in collagen/fibrin materials. Angiogenesis 15, 253–264 (2012). https://doi.org/10.1007/s10456-012-9257-1

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  • DOI: https://doi.org/10.1007/s10456-012-9257-1

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