Short Communication
Manipulating Mesenchymal Stem Cells for Vascular Tissue Engineering
Feng Zhao*
Department of Biomedical Engineering, Michigan Technological University, USA

Human mesenchymal stem cells (hMSCs) have generated great attention in tissue engineering applications due to their extensive proliferative capacity in vitro, multilineage differentiation potential, and immune response modulation ability in vivo [1- 9]. hMSCs also secrete a variety of “trophic factors”, including growth factors, cytokines, and adhesion molecules, that can alter the tissue microenvironment and thereby rejuvenate or repair diseased tissues and cells [2,7,10,11]. Therefore, the hMSCinduced in vivo repair of dysfunctional tissues can be a result of either differentiation or secretion of trophic factors, which alter the milieu so as to regenerate the dysfunctional tissue or cells.
The hMSCs have been proven to be an excellent starting material when combined with biodegradable scaffolds, either in their undifferentiated or differentiated states, for the regeneration of damaged vascular tissues. hMSCs are “trophic” and highly regenerative [7,9]. They can adapt to the microenvironment in vivo, and be directed by the growth factors released by platelets and vascular cells after vessel injury (PDGF, TGF-β1, and bFGF) toward a vessel reparative function [7,8]. Most notably, the in vivo experiments demonstrated that aligned hMSC constructs facilitate endothelial cell (EC) and smooth muscle cell (SMC) recruitment and organization in addition to providing excellent long-term graft patency, implying that local cues within injured vessels in vivo may direct hMSCs toward a vessel repairing function [1]. The unique properties of hMSCs offer us opportunities to use this single cell type to engineer an off-the-shelf tissue engineered blood vessel (TEBV) that can be readily used as allografts by any patient without time concerns.
Cell sheet engineering enables nondestructive cell harvest from thermosensitive polymer-coated surface by controlling the conformational change of the polymer coating [12,13]. The thermosensitive polymers change their hydrophobicity to hydrophilicity when the environmental temperature decreased below the lower critical solution temperature (LCST) of the polymers. This technique avoids the use of proteolytic enzyme to digest the ECM structure and intracellular junction, thus conserves the cell sheet completeness to the maximum extent [13]. Hydroxybutyl chitosan (HBC) is a thermosensitive polymer derived from the biopolymer chitosan, a polysaccharide with similar structure to glycosaminoglycans (GAG) [14,15]. When blended with collagen, the obtained polymer complex coating favors the hMSC attachment, proliferation and phenotypic expression, in addition to the easy removal of hMSC cell sheets from the coated substrate surfaces upon exposure to a temperature lower than the LCST of HBC. In our previous study, we have successfully used the HBC to coat the nanogratings for the ease of harvesting aligned hMSC cell sheets. In the next step, we will use the prealigned hMSC cell sheets to produce a TEBV with well-defined 3D cellular organization similar to that of the SMC organization in natural vessels.
The optimal functionality of a tissue depends on its appropriate histological organization. In natural blood vessels, the SMCs and reinforcing extracellular matrix (ECM) fibers form an elastomotor helix inclined to the vessel centerline [16-18]. The angle between the elastomotor helix winding and the longitudinal axis of the vessel is 30-50o in large arteries, and gradually increases as the vessel diameter decreases [17,18]. The alignment of cells also plays an important role in providing tissues with stronger mechanical properties. Inspired by the structure of natural blood vessels, we are trying to mimic the 3D spiral and interwoven organization of SMCs in real blood vessels with hMSCs. Towards this goal, we firstly fabricate hMSCs into cell sheets that have a high degree of alignment and confluency. We then engineer them into a scaffold-free tissue engineered vascular graft, with one cell sheet layer inclined 30−50o to the centerline, and the second layer perpendicularly to the first. We anticipate that the endurance of TEBVs to pressure and stretch stress of blood flow will be significantly improved by the 3D cellular organization that highly mimics the orientation of SMCs.
To mimic the 3D spiral and interwoven organization of SMCs in real blood vessels, it is crucial to fabricate an hMSC cell sheet that has a high degree of alignment and confluency. A grated substrate can effectively orient cells [19,20]. Other than the width, the depth of the grating is also an important parameter. At the microscale, deep gratings appear to produce a non-uniform cell sheet [21]. The portion of the cell layer grown on the ridges tends to be thinner, rendering the cell sheet more prone to tearing during handling and processing. Furthermore, deep grooves would likely lead to an increase in the time required for an intact sheet to form [21]. We have previously studied hMSC alignment on nanogratings, and established that nanopatterns with a grating depth of 250 nm exert a more pronounced effect than micropatterns in aligning cells [22,23]. However, although the nanogratings align hMSCs, the cells have a great tendency to grow into an uneven patchy layer [24]. A desirable cell sheet should comprise cells forming tight junctions with each other and secrete plenty of ECM proteins to hold the cell sheet together [25-27]. A non-uniform or patchy structure could make the cell sheet vulnerable to tearing during handling, in addition to compromising the quality of the engineered tissue. Another complication of culturing hMSCs on nanopatterns is the differentiation driven by nanotopographical cues. Nanostructures stimulate hMSCs to differentiate along the neuronal, myogenic, and osteogenic lineages in a proliferative, non-differentiation medium, while decreasing their proliferation [23,28,29].
Low O2 is a native physiological condition of the hMSC niche, which effectively supports hMSC survival, maintains their primitive status, increases the ECM secretion, and considerably improves the uniformity of cell layers [24,30-32]. We have previously demonstrated that hMSCs grown under 2% O2 conditions secret abundant ECM proteins [24,31,32], facilitating the cells to grow into even layers with highly aligned morphology on nanogratings [24]. Moreover, the cells maintained elevated self-renewal ability and preserved higher multi-lineage differentiation ability of the hMSCs than their counterpart cultured under conventional 20% O2 [24,32]. Thus, for engineering vascular grafts using hMSCs, it is necessary to maintain their undifferentiated status of hMSCs during the process of fabricating hMSC vascular grafts. Our previous studies have shown that physiologically low O2 conditions favor the in vitro expansion of hMSCs, prevent their differentiation, stimulate the secretion of ECM proteins, and considerably improve the uniformity of cell layers [24,31,32]. It is also an important environmental parameter that regulates the developmental process and metabolic behavior of blood vessels [33,34]. Therefore, we are incorporating physiologically low O2 conditions in the fabrication process, ensuring a high quality hMSC cell sheet and a subsequent mechanically strong TEBV while maintaining the “trophic” and regenerative properties of hMSCs.
Once wrapped around the temporary mandrel, the 3D tubular cellular assemblies need to be further matured to fuse all the cell layers together. Static culture impairs diffusion of nutrients and O2 [35] to 3D tissue constructs. In addition, blood vessels reside in a dynamic environment in vivo. During the cardiac cycle, the arteries are exposed to significant mechanical strains and variable O2 levels. The shear stress in the human arteries is in the range of 15∼30 dyn/cm2 [36], whereas the O2 level is about 12% (90 mmHg) on average [34,37]. Dynamic culture systems have been widely utilized [38-41] to mimic physical effects of blood flow and pressure in engineering vascular grafts [42-45]. To provide sufficient nutrients and also mature the TEBVs in the natural environment of coronary arteries, it is necessary to develop a bioreactor system that can replicate the physiologically relevant O2 and flow conditions of coronary arteries in one single unit. Therefore, we are currently developing a novel bioreactor system that has the capacity to stabilize the TEBVs in a hydrodynamic microenvironment by providing controllable O2 tension and physiological pulsatile force to the 3D tubular cellular constructs.

  1. Hashi CK, Zhu Y, Yang GY, Young WL, Hsiao BS, Wang K, et al. Antithrombogenic property of bone marrow mesenchymal stem cells in nanofibrous vascular grafts. Proc Natl Acad Sci U S A. 2007; 104: 11915-11920.
  2. Wagner J, Kean T, Young R, Dennis JE, Caplan AI. Optimizing mesenchymal stem cell-based therapeutics. Curr Opin Biotechnol. 2009; 20: 531-536.
  3. Németh K, Leelahavanichkul A, Yuen PS, Mayer B, Parmelee A, Doi K,et al. Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat Med. 2009; 15: 42-49.
  4. Groh ME, Maitra B, Szekely E, Koç ON. Human mesenchymal stem cells require monocyte-mediated activation to suppress alloreactive T cells. Exp Hematol. 2005; 33: 928-934.
  5. Ren GW, et al. The interaction between mesenchymal stem cells and the immune system. Cell Research. 2008; 18.
  6. Ren G, Zhang L, Zhao X, Xu G, Zhang Y, Roberts AI, et al. Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell. 2008; 2: 141-150.
  7. Caplan AI, Correa D. The MSC: an injury drugstore. Cell Stem Cell. 2011; 9: 11-15.
  8. Salem HK, Thiemermann C. Mesenchymal stromal cells: current understanding and clinical status. Stem Cells. 2010; 28: 585-596.
  9. Petrie Aronin CE, Tuan RS. Therapeutic potential of the immunomodulatory activities of adult mesenchymal stem cells. Birth Defects Res C Embryo Today. 2010; 90: 67-74.
  10. Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. J Cell Biochem. 2006; 98: 1076-1084.
  11. Haynesworth SE, Baber MA, Caplan AI. Cytokine expression by human marrow-derived mesenchymal progenitor cells in vitro: effects of dexamethasone and IL-1 alpha. J Cell Physiol. 1996; 166: 585-592.
  12. Kumashiro Y, Yamato M, Okano T. Cell attachment-detachment control on temperature-responsive thin surfaces for novel tissue engineering. Ann Biomed Eng. 2010; 38: 1977-1988.
  13. Elloumi-Hannachi I, Yamato M, Okano T. Cell sheet engineering: a unique nanotechnology for scaffold-free tissue reconstruction with clinical applications in regenerative medicine. J Intern Med. 2010; 267: 54-70.
  14. Dang JM, Sun DD, Shin-Ya Y, Sieber AN, Kostuik JP, Leong KW. Temperature-responsive hydroxybutyl chitosan for the culture of mesenchymal stem cells and intervertebral disk cells. Biomaterials. 2006; 27: 406-418.
  15. Dang JM, Leong KW. Myogenic Induction of Aligned Mesenchymal Stem Cell Sheets by Culture on Thermally Responsive Electrospun Nanofibers. Adv Mater. 2007; 19: 2775-2779.
  16. Kupriianov VV. [Spiral arrangement of muscular elements in the walls of blood vessels and their role in hemodynamics]. Arkh Anat Gistol Embriol. 1983; 85: 46-54.
  17. Holzapfel GA, Gasser TC, Ogden RW. A new constitutive framework for arterial wall mechanics and a comparative study of material models. Journal of Elasticity. 2000; 61: 1-48.
  18. Medvedev AE, Samsonov VI, Fomin VM. Rational structure of blood vessels. Journal of Applied Mechanics and Technical Physics. 2006; 47:324-329.
  19. Vernon RB, Gooden MD, Lara SL, Wight TN. Microgrooved fibrillar collagen membranes as scaffolds for cell support and alignment. Biomaterials. 2005; 26: 3131-3140.
  20. Sarkar S, Dadhania M, Rourke P, Desai TA, Wong JY. Vascular tissue engineering: microtextured scaffold templates to control organization of vascular smooth muscle cells and extracellular matrix. Acta Biomater. 2005; 1: 93-100.
  21. Isenberg BC, Tsuda Y, Williams C, Shimizu T, Yamato M, Okano T, et al. A thermoresponsive, microtextured substrate for cell sheet engineering with defined structural organization. Biomaterials. 2008;29: 2565-2572.
  22. Yim EK, Reano RM, Pang SW, Yee AF, Chen CS, Leong KW. Nanopatterninduced changes in morphology and motility of smooth muscle cells. Biomaterials. 2005; 26: 5405-5413.
  23. Yim EK, Pang SW, Leong KW. Synthetic nanostructures inducing differentiation of human mesenchymal stem cells into neuronal lineage. Exp Cell Res. 2007; 313: 1820-1829.
  24. Zhao F, Veldhuis JJ, Duan Y, Yang Y, Christoforou N, Ma T, et al. Low oxygen tension and synthetic nanogratings improve the uniformity and stemness of human mesenchymal stem cell layer. Mol Ther. 2010;18: 1010-1018.
  25. Chan BP, Leong KW. Scaffolding in tissue engineering: general approaches and tissue-specific considerations. Eur Spine J. 2008; 17 Suppl 4: 467-479.
  26. Yang J, Yamato M, Shimizu T, Sekine H, Ohashi K, Kanzaki M, et al. Reconstruction of functional tissues with cell sheet engineering. Biomaterials. 2007; 28: 5033-5043.
  27. Auger Fa, Remy-Zolghadri M, Grenier G, et al. The Self-Assembly Approach for Organ Reconstruction by Tissue Engineering. J Regen Med. 2000; 1: 75-86.
  28. Oh S, Brammer KS, Li YS, Teng D, Engler AJ, Chien S, et al. Stem cell fate dictated solely by altered nanotube dimension. Proc Natl Acad Sci U S A. 2009; 106: 2130-2135.
  29. Dang JM, Leong KW. Myogenic Induction of Aligned Mesenchymal Stem Cell Sheets by Culture on Thermally Responsive Electrospun Nanofibers. Adv Mater. 2007; 19: 2775-2779.
  30. Mylotte LA, Duffy AM, Murphy M, O'Brien T, Samali A, Barry F, et al. Metabolic flexibility permits mesenchymal stem cell survival in an ischemic environment. Stem Cells. 2008; 26: 1325-1336.
  31. Grayson WL, Zhao F, Izadpanah R, Bunnell B, Ma T. Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs. J Cell Physiol. 2006; 207: 331-339.
  32. Grayson WL, Zhao F, Bunnell B, Ma T. Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells. Biochem Biophys Res Commun. 2007; 358: 948-953.
  33. Poiani GJ, Tozzi CA, Yohn SE, Pierce RA, Belsky SA, Berg RA, et al.Collagen and elastin metabolism in hypertensive pulmonary arteries of rats. Circ Res. 1990; 66: 968-978.
  34. Csete M. Oxygen in the cultivation of stem cells, in Stem Cell Biology: Development and Plasticity. J. Ourednik, et al., Editors. 2005, New York Acad Sciences: New York. 1-8.
  35. Martin I, Wendt D, Heberer M. The role of bioreactors in tissue engineering. Trends Biotechnol. 2004; 22: 80-86.
  36. Morawietz H, Talanow R, Szibor M, Rueckschloss U, Schubert A, Bartling B, et al. Regulation of the endothelin system by shear stress in human endothelial cells. J Physiol. 2000; 525 Pt 3: 761-770.
  37. Vogiatzis I, Georgiadou O, Koskolou M, Athanasopoulos D, Kostikas K, Golemati S, et al. Effects of hypoxia on diaphragmatic fatigue in highly trained athletes. J Physiol. 2007; 581: 299-308.
  38. Opitz F, Schenke-Layland K, Richter W, Martin DP, Degenkolbe I,Wahlers T, et al. Tissue engineering of ovine aortic blood vessel substitutes using applied shear stress and enzymatically derived vascular smooth muscle cells. Ann Biomed Eng. 2004; 32: 212-22.
  39. Seliktar D, Black RA, Vito RP, Nerem RM. Dynamic mechanical conditioning of collagen-gel blood vessel constructs induces remodeling in vitro. Ann Biomed Eng. 2000; 28: 351-362.
  40. Webb AR, Macrie BD, Ray AS, Russo JE, Siegel AM, Glucksberg MR, et al. In vitro characterization of a compliant biodegradable scaffold with a novel bioreactor system. Ann Biomed Eng. 2007; 35: 1357-1367.
  41. Hahn MS, McHale MK, Wang E, Schmedlen RH, West JL. Physiologic pulsatile flow bioreactor conditioning of poly(ethylene glycol)-based tissue engineered vascular grafts. Ann Biomed Eng. 2007; 35: 190- 200.
  42. McCulloch AD, Harris AB, Sarraf CE, Eastwood M. New multi-cue bioreactor for tissue engineering of tubular cardiovascular samples under physiological conditions. Tissue Eng. 2004; 10: 565-573.
  43. Mironov V, Kasyanov V, McAllister K, Oliver S, Sistino J, Markwald R. Perfusion bioreactor for vascular tissue engineering with capacities for longitudinal stretch. J Craniofac Surg. 2003; 14: 340-347.
  44. Nasseri BA, Pomerantseva I, Kaazempur-Mofrad MR, Sutherland FW, Perry T, Ochoa E, et al. Dynamic rotational seeding and cell culture system for vascular tube formation. Tissue Eng. 2003; 9: 291-299.
  45. Sodian R, Lemke T, Fritsche C, Hoerstrup SP, Fu P, Potapov EV, et al. Tissue-engineering bioreactors: a new combined cell-seeding and perfusion system for vascular tissue engineering. Tissue Eng. 2002; 8:863-70.

Cite this article: Zhao F (2013) Manipulating Mesenchymal Stem Cells for Vascular Tissue Engineering. JSM Biotechnol Bioeng 1(2): 1012.
Right Table
Content:   Home  |  Aims & Scope  |  Early Online  |  Current Issue  | 
Journal Info:   Editorial Board  |  Article Processing Charges  |  FAQs
Contact Us
2952 Market Street, Suite 140
San Diego, California 92102, USA
Tel: 1-619-373-8030
Fax: 1-619-793-4845
Toll free number: 1-800-762-9856
Copyright © 2013 JSciMed Central. All rights reserved.