Preparation of Three-Dimensional Vascularized MSC Cell Sheet Constructs for Tissue Regeneration

Engineering three-dimensional (3D) vascularized constructs remains a challenge due to the inability to form rich microvessel networks. In this study we engineered a prevascularized 3D cell sheet construct for tissue regeneration using human bone marrow-derived mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells as cell sources. hMSCs were cultured to form a thick cell sheet, and human umbilical vein endothelial cells (HUVECs) were then seeded on the hMSCs sheet to form networks. The single prevascularized HUVEC/hMSC cell sheet was folded to form a 3D construct by a modified cell sheet engineering technique. In vitro results indicated that the hMSCs cell sheet promoted the HUVECs cell migration to form networks in horizontal and vertical directions. In vivo results showed that many blood vessels grew into the 3D HUVEC/hMSC cell sheet constructs after implanted in the subcutaneous pocket of immunodeficient mice. The density of blood vessels in the prevascularized constructs was higher than that in the nonprevascularized constructs. Immunohistochemistry staining further showed that in vitro preformed human capillaries in the prevascularized constructs anastomosed with the host vasculature to form functional blood vessels. These results suggest the promising potential of this 3D prevascularized construct using hMSCs cell sheet as a platform for wide applications in engineering vascularized tissues.

[1]  T. Okano,et al.  Network formation through active migration of human vascular endothelial cells in a multilayered skeletal myoblast sheet. , 2013, Biomaterials.

[2]  G. Davis,et al.  Biosynthesis, Remodeling, and Functions During Vascular Morphogenesis and Neovessel Stabilization , 2005 .

[3]  Masayuki Yamato,et al.  Endothelial Cell Coculture Within Tissue-Engineered Cardiomyocyte Sheets Enhances Neovascularization and Improves Cardiac Function of Ischemic Hearts , 2008, Circulation.

[4]  Tadashi Sasagawa,et al.  Design of prevascularized three-dimensional cell-dense tissues using a cell sheet stacking manipulation technology. , 2010, Biomaterials.

[5]  J. Hubbell,et al.  Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering , 2005, Nature Biotechnology.

[6]  C James Kirkpatrick,et al.  The rapid anastomosis between prevascularized networks on silk fibroin scaffolds generated in vitro with cocultures of human microvascular endothelial and osteoblast cells and the host vasculature. , 2010, Biomaterials.

[7]  T. Okano,et al.  Reproducible subcutaneous transplantation of cell sheets into recipient mice , 2011, Nature Protocols.

[8]  M. Pittenger,et al.  Multilineage potential of adult human mesenchymal stem cells. , 1999, Science.

[9]  Andrés J. García,et al.  Engineering more than a cell: vascularization strategies in tissue engineering. , 2010, Current opinion in biotechnology.

[10]  Domenico Ribatti,et al.  Endothelialization approaches for viable engineered tissues , 2012, Angiogenesis.

[11]  Teruo Okano,et al.  Pulsatile Myocardial Tubes Fabricated With Cell Sheet Engineering , 2006, Circulation.

[12]  K. Alitalo,et al.  Molecular regulation of angiogenesis and lymphangiogenesis , 2007, Nature Reviews Molecular Cell Biology.

[13]  P. Ma,et al.  Nanofibrous Scaffolds for Dental and Craniofacial Applications , 2012, Journal of dental research.

[14]  T. Okano,et al.  Bioengineering of a functional sheet of islet cells for the treatment of diabetes mellitus. , 2009, Biomaterials.

[15]  D. Kohane,et al.  Engineering vascularized skeletal muscle tissue , 2005, Nature Biotechnology.

[16]  T. Okano,et al.  Treatment of oesophageal ulcerations using endoscopic transplantation of tissue-engineered autologous oral mucosal epithelial cell sheets in a canine model , 2006, Gut.

[17]  A. Perets,et al.  Enhancing the vascularization of three-dimensional porous alginate scaffolds by incorporating controlled release basic fibroblast growth factor microspheres. , 2003, Journal of biomedical materials research. Part A.

[18]  S. Levenberg,et al.  Vascularization--the conduit to viable engineered tissues. , 2009, Tissue engineering. Part B, Reviews.

[19]  W. Khan,et al.  A systematic review on preclinical and clinical studies on the use of scaffolds for bone repair in skeletal defects. , 2013, Current stem cell research & therapy.

[20]  P. Menasché,et al.  Cell delivery: intramyocardial injections or epicardial deposition? A head-to-head comparison. , 2009, The Annals of thoracic surgery.

[21]  Minna Kellomäki,et al.  A review of rapid prototyping techniques for tissue engineering purposes , 2008, Annals of medicine.

[22]  Paolo De Coppi,et al.  Regenerative medicine as applied to solid organ transplantation: current status and future challenges , 2011, Transplant international : official journal of the European Society for Organ Transplantation.

[23]  L. Romer,et al.  Endothelial cell adhesion, signaling, and morphogenesis in fibroblast-derived matrix. , 2009, Matrix biology : journal of the International Society for Matrix Biology.

[24]  Rakesh K Jain,et al.  Molecular regulation of vessel maturation , 2003, Nature Medicine.

[25]  Mitsuo Umezu,et al.  Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro , 2012, Nature Protocols.

[26]  C. Lattermann,et al.  Osteochondral allografts: state of the art. , 2009, Clinics in sports medicine.

[27]  Detlev Drenckhahn,et al.  Pericyte involvement in capillary sprouting during angiogenesis in situ , 1992, Cell and Tissue Research.

[28]  Martin Fussenegger,et al.  Scaffold-free cell delivery for use in regenerative medicine. , 2010, Advanced drug delivery reviews.

[29]  R. Soetikno,et al.  Treatment of oesophageal ulcerations using endoscopic transplantation of tissue-engineered autologous oral mucosal epithelial cell sheets in a canine model , 2006, Gut.

[30]  A. Fernando,et al.  Towards understanding the mode of action of the multifaceted cell adhesion receptor CD146. , 2009, Biochimica et biophysica acta.

[31]  Esther Novosel,et al.  Vascularization is the key challenge in tissue engineering. , 2011, Advanced drug delivery reviews.

[32]  Masayuki Yamato,et al.  Periodontal regeneration with multi-layered periodontal ligament-derived cell sheets in a canine model. , 2009, Biomaterials.

[33]  A. Khademhosseini,et al.  Osteogenic and angiogenic potentials of monocultured and co-cultured human-bone-marrow-derived mesenchymal stem cells and human-umbilical-vein endothelial cells on three-dimensional porous beta-tricalcium phosphate scaffold. , 2013, Acta biomaterialia.

[34]  Marcin Maruszewski,et al.  Effectiveness of haemodialysis access with an autologous tissue-engineered vascular graft: a multicentre cohort study , 2009, The Lancet.

[35]  Arnold I Caplan,et al.  All MSCs are pericytes? , 2008, Cell stem cell.

[36]  K. Hirschi,et al.  Pericytes in the microvasculature. , 1996, Cardiovascular research.

[37]  Masayuki Yamato,et al.  Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface , 2004, Transplantation.