Neovascularization of engineered tissues for clinical translation: Where we are, where we should be?

Abstract

One of the key challenges in engineering three-dimensional tissue constructs is the development of a mature microvascular network capable of supplying sufficient oxygen and nutrients to the tissue. Recent angiogenic therapeutic strategies have focused on vascularization of the constructed tissue, and its integration in vitro; these strategies typically combine regenerative cells, growth factors (GFs) with custom-designed biomaterials. However, the field needs to progress in the clinical translation of tissue engineering strategies. The article first presents a detailed description of the steps in neovascularization and the roles of extracellular matrix elements such as GFs in angiogenesis. It then delves into decellularization, cell, and GF-based strategies employed thus far for therapeutic angiogenesis, with a particularly detailed examination of different methods by which GFs are delivered in biomaterial scaffolds. Finally, interdisciplinary approaches involving advancement in biomaterials science and current state of technological development in fabrication techniques are critically evaluated, and a list of remaining challenges is presented that need to be solved for successful translation to the clinics.

FIG. 1.

Schematic representation of tissue engineering strategies for therapeutic angiogenesis and interdisciplinary approaches to facilitate angiogenesis. Decellularization of native organs has been used to obtain immuno-compatible tissues as a scaffold where a suitable ECM matrix is present. Neovascularization can be induced by using pre-vascularization, in vivo cell delivery, and co-culturing strategies or delivering angiogenic growth factors. Different techniques such as electrospinning, spatial micropatterning, 3D printing have been used to facilitate angiogenesis at the desired site.

FIG. 2.

Morphogenetic stages with major angiogenic regulatory growth factors and extracellular matrix molecules involved in neovascularization. Endothelial progenitors are differentiated from mesodermal cells as first step of vasculogenesis (a). EPCs differentiates into ECs (b). Organization of primitive vascular network are achieved by vacuole and lumen formation, branching and sprouting (c). Vessel is matured and stabilized by recruitment of SMC (d). Vessel destabilization occurs owing to SMC detachment (e). Nascent vessel sprouting occurs with the migration of endothelial cells up a growth factor gradient in response to biochemical growth factors (f). Vessel regression is observed during tissue repair and regeneration (g). Reprinted by permission from M. P. Lutolf and J. A. Hubbell, Nat. Biotechnol. 23(1), 47–55 (2005). Copyright 2005 Springer Nature Customer Service Center GmbH, Springer Nature.

FIG. 3.

Schematic illustration of three different processes involved in graft nutrition (a). In vitro revascularization (b). Endothelialized capillary-like tubes are formed in a skin construct. Human (red) and mouse ECs (green) were co-localized (arrows) or branched (arrowheads). Reprinted with permission from Tremblay et al., Am. J. Transplant. 5, 1002 (2005). Copyright 2005 John Wiley & Sons, Inc.

FIG. 4.

Schematic representation for vascularized tissue formation by sandwich method (a) human umbilical vein endothelial cells (HUVECs) sandwiched between myoblast sheets with the help of a gelatin-coated plunger, cultured up to 3 days, and stained with UEA-I (red) and anti-desmin antibody (green) for HUVECs and myoblasts, respectively (b) and (c). Observation of neovascularization with anti-human CD31 antibody (green) staining in five-layered myoblast sheet constructs with (d) and without HUVECs (e). Notations f, c, and m represent the fibrin gel, cell sheet construct, and muscle tissue, respectively. Reprinted with permission from Sasagawa et al., Biomaterials 31, 1646 (2010). Copyright 2010 Elsevier.

FIG. 5.

Human endothelial progenitor cells improved limb salvage. (a) Reprinted with permission from Kalka et al., Proc. Natl. Acad. Sci. U. S. A. 97, 3422 (2000). Copyright 2000 National Academy of Sciences, USA. The co-culture of HUVECs and MSCs led to high tubulogenesis (c) and (d) compared to HUVEC only culture (B). Reprinted by permission from Zhang et al., J. Huazhong Univ. Sci. Technol., Med. Sci. 32, 173–180 (2012). Copyright 2012 Springer Nature Customer Service Center GmbH, Springer Nature.

FIG. 6.

Isolation and encapsulation of islets limit mass transfer. Compared to the native pancreas (a), diffusion dramatically reduced for the majority of cells in islets (especially in the core of the cell mass) as a result of loss of blood perfusion following isolation from the acinar tissue (b). Furthermore, microencapsulation increases the distance of islet cells to the surrounding fluid or blood vessels (c). Dark green represents greater mass transport.

FIG. 7.

Angiogenic growth factor delivery strategies. (a) Physical encapsulation of growth factors which provides sustained and local GF's release at the target site with better retention of biological activity. (b) Natural or augmented materials affinity to angiogenic factor in which sustained and localized release are obtained by use of ionic complexation between oppositely charged groups on GFs and biomaterial scaffolds. Binding interaction through (c) affinity mediated by ECM proteins. (d) Affinity mediated by heparin. (e) Affinity mediated by immobilized heparin. (f) Prolonged signaling by high-affinity glycosaminoglycans (GAGs) prolongs the retention of GFs within the scaffold, increases their stability, and protects them from denaturation by heat, inactivation at acidic pH inactivation, and proteolytic degradation. (g) Direct conjugation provides more prolonged GF retention and release. (h) The cell-mediated release makes the system responsive to environmental stimuli (such as pH, temperature, proteolytic cleavage site, ions, light, drug, magnetic, and electric field) which provide temporal control over GF. (i) Multiple and sequential delivery provide better recapitulation of an in vivo microenvironment where more than one factor has involved the process.

FIG. 8.

Angioma formation and angiogenesis in rats treated with phVEGF (a). Reprinted with permission from Schawrz et al., J. Am. Coll. Cardiol. 35, 1323 (2000). Copyright 2000 Elsevier. Enhanced angiogenesis by VEGF-expressing Marrow MSCs (reddish-brown). An increased number of vessels were observed in groups treated with gene delivery (b) compared to the control group (c). Reprinted with permission from Yang et al., Cardiology 107, 17 (2007). Copyright 2007 Karger Publishers, Basel, Switzerland. Serial Single-photon emission computed tomography (SPECT) myocardial perfusion images showed the bolus delivery of rhVEGF restored circulation and promoted angiogenesis in ischemic tissues (D). Reprinted with permission from Henry et al., Am. Heart. J. 142, 872 (2001). Copyright 2001 Elsevier. VEGF encapsulated in PLA scaffolds present in CAM. Histological analyses showed increased vessel number (arrowheads) in the PLA-VEGF scaffold (f) compared to control (e). Reprinted with permission from Kanczler et al., Biochem. Biophys. Res. Commun. 352,” 135 (2007). Copyright 2007 Elsevier. Hematoxylin and eosin staining images of mice ear tissue, heparin-HA-VEGF hydrogel showed greater neovascularization with well-defined vascular borders (g) compared to HA-VEGF specimen (h) and control (I). Reprinted with permission from Pike et al., Biomaterials 27, 5242 (2006). Copyright 2006 Elsevier. The covalent conjugation of ephrinA1 and PDGF to PEGDA hydrogels showed greater neovascularization (k) compared to PDGF-BB alone conjugation (j). Reprinted with permission from Saik et al., Biomacromolecules 12(7), 2715–2722 (2011). Copyright 2011 American Chemical Society. Vascularization analysis through fluorescent images perfused with lectin (green) to label vasculature after 14 days of implantation in mice in non-degradable microgel with VEGF (l), degradable microgel without VEGF (m), degradable microgel with a bolus injection of VEGF (n) and degradable microgel with VEGF (o). Reprinted with permission from Foster et al., Biomaterials 113,170 (2017). Copyright 2017 Elsevier. Fluorescence live (green)/dead (red) image of H5V cells on collagen scaffolds showed greater vessel formation (yellow arrows) in co-immobilized growth factor groups compared to single growth factor groups and control (p). Reprinted with permission from Chiu et al., Biomaterials 31, 226 (2010). Copyright 2010 Elsevier.

FIG. 9.

Experimental design for in vitro and in vivo studies (a), where human skeletal muscle-derived cells and human umbilical vein endothelial cells were seeded in parenchymal spaces and in microvessels respectively while for in vivo studies, full-thickness punches of scaffolds with/without cells were implanted in nude rats. Immunostaining with anti-rat CD31 for a native blood vessel (b), Subcutaneous (SC) scaffold without (c) or with cells (d), and IP scaffold without (e) and with cells (f). Scale bar: 25 μm. Reprinted with permission from Ye et al., Biomaterials 34, 10007 (2013). Copyright 2013 Elsevier.

FIG. 10.

Schematics of microfabrication design (a), in step 1, to fabricate an external perfusion housing, PDMS is replica molded. In step 2, the interior of the housing is coated with a photoinitiator. In step 3, the housing is injected with photopolymerizable PEG precursors, and to fabricate hydrogel microchannels within the external PDMS housing, mask-directed photolithography is used. In step 4, the PDMS–PEG multilayer device is conformally sealed to coverglass and perfused with media and buffer. The last schematic shows the spatial relationship of the perfused media (red) and buffer (blue) microchannels to PEG hydrogel (cyan) regions imaged for analysis. Immunohistochemistry (IHC) of microvascular network formation at 96 h (B), where HUVECs (green), 10T1/2 cells (red), and nuclei (blue) Reprinted with permission from Cuchiara et al., Adv. Funct. Mater. 22, 4511 (2012). Copyright 2012 John Wiley & Sons, Inc.

FIG. 11.

Schematics of microvascular network conditions for optimization of self-assembly of microvasculature (a), where (i) iPSC-ECs only, (ii) iPSC-ECs + PCs, (iii) iPSC-ECs + PCs + ACs. Confocal images of microvascular networks after 7 days (b), where (i) iPSC-ECs only (CD31, green), (ii) co-culture with PCs (F-actin, red), (iii) tri-culture with ACs [Glial fibrillary acidic protein (GFAP), magenta]. Scale bars indicate 100 μm. Reproduced with permission Campisi et al., Biomaterials 180, 117 (2018). Copyright 2018 Authors, licensed under a Creative Commons Attribution (CC BY) license.

FIG. 12.

Explants analyses after two weeks of implantation. Schematics for multi cells containing 3D bioprinted cardiac tissue constructs with three different geometries. (a) In vivo grafting of bulk and 3D bioprinted hydrogels after 15 days of implantation (first column from left), immunofluorescence staining to visualize CMs orientation (second column), and vessels (third column) while polar graphs are quantifying CMs orientation (fourth column). Arrows indicate vascularization, dash lines in column explant shows implantation site, dash lines in column CMs shows the organization of vasculature. TNN1, DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride), vWF, and Lamin A/C were used to stain CM, nucleus, vasculature, and capillaries originated from human endothelial cells, respectively (b). Reproduced with permission from Maiullari et al., Sci. Rep. 8, 13532 (2018). Copyright 2018 Authors, licensed under a Creative Commons Attribution (CC BY) license.