HHS Public Access Author manuscript Author Manuscript
Acta Biomater. Author manuscript; available in PMC 2016 November 01. Published in final edited form as: Acta Biomater. 2015 November ; 27: 151–166. doi:10.1016/j.actbio.2015.09.002.
Bioengineering vascularized tissue constructs using an injectable cell-laden enzymatically crosslinked collagen hydrogel derived from dermal extracellular matrix Kuan-Chih Kuoa, Ruei-Zeng Linb, Han-Wen Tiena, Pei-Yun Wua, Yen-Cheng Lic, Juan M. Melero-Martinb, and Ying-Chieh Chena,*
Author Manuscript
a
Department of Applied Science, National Hsinchu University of Education, Hsinchu 30014, Taiwan, ROC
b
Department of Cardiac Surgery, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA
c
Material and Chemical Research Laboratories, Industrial Technology Research Institute, Hsinchu 31040, Taiwan, ROC
Abstract
Author Manuscript
Tissue engineering promises to restore or replace diseased or damaged tissue by creating functional and transplantable artificial tissues. The development of artificial tissues with large dimensions that exceed the diffusion limitation will require nutrients and oxygen to be delivered via perfusion instead of diffusion alone over a short time period. One approach to perfusion is to vascularize engineered tissues, creating a de novo three-dimensional (3D) microvascular network within the tissue construct. This significantly shortens the time of in vivo anastomosis, perfusion and graft integration with the host. In this study, we aimed to develop injectable allogeneic collagen-phenolic hydroxyl (collagen-Ph) hydrogels that are capable of controlling a wide range of physicochemical properties, including stiffness, water absorption and degradability. We tested whether collagen-Ph hydrogels could support the formation of vascularized engineered tissue graft by human blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSC) in vivo. First, we studied the growth of adherent ECFCs and MSCs on or in the hydrogels. To examine the potential formation of functional vascular networks in vivo, a liquid pre-polymer solution of collagen-Ph containing human ECFCs and MSCs, horseradish peroxidase and hydrogen peroxide was injected into the subcutaneous space or abdominal muscle defect of an immunodeficient mouse before gelation, to form a 3D cell-laden polymerized construct. These results showed that extensive human ECFC-lined vascular networks can be generated within 7 days, the engineered vascular density inside collagen-Ph hydrogel constructs can be manipulated through refinable mechanical properties and proteolytic degradability, and these networks can form functional anastomoses with the existing vasculature to further support the survival of host muscle tissues. Finally, optimized conditions of the cell-
Author Manuscript *
Corresponding author at: Department of Applied Science, National Hsinchu University of Education, No. 521, Nanda Rd., Hsinchu City 30014, Taiwan, ROC. [email protected] (Y.-C. Chen).. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2015.09. 002.
Kuo et al.
Page 2
Author Manuscript
laden collagen-Ph hydrogel resulted in not only improving the long-term differentiation of transplanted MSCs into mineralized osteoblasts, but the collagen-Ph hydrogel also improved an increased of adipocytes within the vascularized bioengineered tissue in a mouse after 1 month of implantation.
Keywords Collagen hydrogels; Vascularization; Tissue engineering
1. Introduction
Author Manuscript Author Manuscript Author Manuscript
Creating large functional and transplantable tissues for organ replacement have failed in the past because of the limited diffusion distance of oxygen and nutrients to a few hundred micrometers. This can lead to necrosis at the central region of the large-sized tissueengineered or highly vascularized tissues [1–3]. Host blood vessels usually take days or weeks to invade the centre of engineered tissue, so during this process an insufficient vascular supply leads the tissue to nutrient depletion and ischemia, which can compromise cell viability and function [2,4,5]. To overcome this problem, nutrients and oxygen need to be delivered via perfusion instead of diffusion alone [3]. Aside from current strategies promoting angiogenesis from the host, an alternative concept is to pre-vascularize a tissue that creates a vascular network within the tissue construct prior to implantation [6]. Vascularization generally refers to the formation of an in vitro well-connected microvessel network within an implantable tissue construct through vasculogenesis. Following implantation, this vasculature can rapidly anastomose with the host and enhances tissue survival and function [1,3]. Furthermore, microfabrication techniques have been applied as a means to control space and direct the growth of vascular networks in vitro [7–10]; however, these pre-engineered networks are too simple to grow and reform in response to specific physiological demands from the organs they are supporting in the host [1,11]. Therefore, we have demonstrated the formation of vascular networks, de novo, from encapsulating endothelial colony-forming cells (ECFCs) and mesenchymal stem cells (MSC) in the liquid matrix prior to gelation, and injected or implanted subcutaneously in immune-deficient mice to form a 3D cell-laden vascularized construct within one week [12–14]. ECFCs circulating in peripheral blood participate in the formation of new blood vasculature and have been a promising source in producing non-invasive large quantities of autologous endothelial cells for clinical use [13,15]. Together with suitable support from scaffolds, MSCs can function as pericytes to promote vessel formation and maturation through secretion of specific proangiogenic cytokines [14,16]. Meanwhile, transplanted ECFCs provided critical angiocrine factors needed to preserve MSC as viable and further support ultimately long-term differentiation of transplanted MSCs to osteoblasts to form vascularized engineered bone tissue constructs by inducing specific stimulants of BMP-2 [12]. A critical requirement for engineering a tissue is the use of a suitable scaffold to mimic structural and functional properties of the natural extracellular matrix (ECM) that includes providing appropriate binding sites for cell–material interactions, mechanical properties to maintain cell function prior to host remodeling without negatively impacting the
Acta Biomater. Author manuscript; available in PMC 2016 November 01.
Kuo et al.
Page 3
Author Manuscript Author Manuscript
development of the capillary network, and biodegradation that matches the deposition rate of new extracellular matrix protein by the host [6]. Over the last few years, a variety of naturalbased hydrogels [17] have been shown to be compatible with endothelial cell-mediated vascular morphogenesis, including natural materials (type I collagen gel [18–22], fibrin gels [19,23], Matrigel [12,24]), semi-synthetic materials, i.e. modified natural material (photocrosslinkable methacrylated gelatin [10,14,16] and enzymatic-crosslinkable tyraminemodified gelatin hydrogels [25,26]). However, there still remains a significant discrepancy in how physicochemical properties of scaffolds, such as mechanical properties, density of cell-adhesive ligand (RGD) or degradable sites (MMP) of scaffolding affect the angiogenic potential of endothelial cells (EC) to form functional blood vessels in vivo. RGD-mediated and integrin-dependent bindings on native collagen nanofibrins support the development of sufficient matrix-transduced tensional forces necessary for EC sprouting and tube formation [27]. The stiffness of self-assembled collagen matrices affects cell spreading, migration, growth and matrix remodeling, all of which are critical components of vascular formation [20,28]. Varying collagen concentration of polymerized 3D collagen gels that alter matrix stiffness and fibril density has been shown to alter EC lumen size and tube length [18– 20,28,29], but in this case the RGD is dependent on collagen concentrations. These issues raised the difficulties of discerning which matrix properties induce EC responses. The aforementioned results provide initial evidence that specific collagen matrix physical properties are important parameters in determining the ability of matrices to guide vessel formation. However, most studies [19,26,28,29] have not been followed up with investigations addressing whether physicochemical properties of matrices impact upon vessel formation and if they further influence the success of creating vascularized tissue construct in vivo, which would lead to more clinically relevant cell-based therapies.
Author Manuscript Author Manuscript
So far, there have only been a few successful thick vascularized engineered tissue constructs, because the lack of proper scaffolding material that cannot only support vascularization in a short time period, but also directs transplanted MSCs differentiation into a specific lineage, remains a major challenge [4,5,11]. Although collagen offers an ideal injectable gel system in vascular tissue engineering, the extensive contraction, poor stiffness, rapid degradation and temperature instability of collagen gels limit their practical applications in regenerating and engineering living tissues, such as adipose or bone tissue grafts [22,30]. Thus, the development of collagen-based scaffolds that can maintain the mechanical integrity of the tissue, support the development and growth of complex microvessel networks, while simultaneously improving the mature differentiation of stem cells to meet the functional requirements of specific tissue, is more desirable. In response to these limitations, we aimed to develop chemical functionalization of natural-derived ECM proteins, i.e. injectable collagen-Ph hydrogels, which are capable of having controlled physicochemical properties over a wide range. To test the biocompatibility and potential for applications in tissue engineering, the ECFCs and MSCs were seeded on or inside collagenPh hydrogels to study the cell adhesion, proliferation and function in vitro. Pre-clinical studies with immunodeficient mice were carried out and demonstrated that functional human vascular networks can be generated in situ by means of post-injectable enzymatic polymerization that enables tuning of the final vascular density inside the collagen-Ph hydrogel constructs. We hypothesized that this cell-laden collagen-Ph construct would
Acta Biomater. Author manuscript; available in PMC 2016 November 01.
Kuo et al.
Page 4
Author Manuscript
rapidly anastomose with host vasculature and improve vascularization and survival of the host abdominal muscle defect with the formation of ECFC-lined vascular networks throughout and in-between skeletal muscle fibers in the host. Finally, feasibility studies toward an ultimate goal of bioengineered 3D vascularized tissue grafts were developed and characterized. Our studies importantly suggest the capacity of ECM-based cell delivery hydrogel systems to incorporate physicochemical cues to modulate vessel formation and further evaluate the feasibility to engineer vascularized transplantable artificial tissues in vivo.
2. Materials and methods 2.1. Extraction of murine collagen-Ph hydrogels from epidermis
Author Manuscript Author Manuscript
Before collagen extraction, murine epidermal tissue was cut into small pieces (90%) throughout a wide range of HRP (0–1250 unit/ml) (Fig. 4a) and H2O2 (0–78 μM) (Fig. 4b); cell viability was only negatively affected beyond 156 μM of H2O2 exposure directly into medium. However, addition of HRP and H2O2 would react and combine immediately with collagen-Ph conjugates to form hydrogels, with residual and unreactive H2O2 decreasing, and which did not affect the viability capacity of ECFCs and MSCs in 2 days of culture. In further studies, we then selected a working range of H2O2 (19–78 μM) used to crosslink hydrogels and discussed the effects of collagen-Ph hydrogels on ECFC and MSCs.
Author Manuscript
3.4. Cell behavior of ECFCs and MSCs grown on murine collagen-Ph hydrogels We studied whether varying amounts of H2O2 and collagen-Ph could be used as parameters to modulate cell behavior grown on collagen-Ph hydrogels. To investigate the effect of collagen-Ph hydrogels on cell attachment and proliferation, in vitro studies were carried out using ECFCs and MSCs as models. Cell proliferation was assessed by the live/dead assay (Invitrogen), as shown in Figs. 5, S5 and S6. We sought to understand whether increasing the degree of polymerization of collagen-Ph hydrogels (Fig. 3) could compromise cellular behavior by altering concentrations of murine collagen-Ph and H2O2. First, concentrations Acta Biomater. Author manuscript; available in PMC 2016 November 01.
Kuo et al.
Page 13
Author Manuscript Author Manuscript Author Manuscript
of collagen-Ph in the range of 0.5–1% did not affect the capacity of ECFCs and MSCs to attach (>82%, Fig. 5a), proliferate (>123% for ECFCs and >155% for MSCs, Fig. 5c) and survive (>91%, Fig. 5b and d) compared with cells grown on rat tail collagen (BD) gels. Secondly, various amounts of H2O2 (19–78 μM) did not affect the capacity of ECFCs and MSCs to attach (>80% for ECFCs and >84% for MSCs, Fig. 5a) and survive (>92% for ECFCs and >93% for MSCs, Fig. 5b) compared with cells grown on rat tail collagen (BD) gels. However, the degree of cross-linking through adjustment of H2O2 concentration modulated cell proliferation and spreading on collagen-Ph hydrogels (Fig. 5c). For longer culture of 2 days, with increasing H2O2 from 19 to 78 μM, the MSC proliferation rate was maintained at 155.4 ± 7.7% and 157.0 ± 11.3% at H2O2 levels of 19 and 39 μM, respectively, and then decreased to 99.8 ± 6.6% at H2O2 of 78 μM (Fig. 5c). Additionally, we explored the spreading area of ECFC on collagen-Ph hydrogels at different cross-linked degrees at concentrations of H2O2 (19 μM: 1948.9 ± 737.6 μM2; 39 μM: 2088.2 ± 834.5 μM2; 78 μM: 1891.1 ± 904.9 μM2) were larger than ECFCs spread out on rat tail collagen gels (950.7 ± 354.3 μM2). However, MSCs retain almost the same spread size grown on collagen-Ph hydrogels (19 μM: 1623.2 ± 702.7 μM2; 39 μm: 1828.1 ± 675.1 μm2; 78 μm: 1908.6 ± 736.0 μm2) and rat tail collagen gels (1495.4 ± 600.3 μm2) (Fig. 5e). There was no statistically significant increase in cell spread and/or proliferation observed when the amounts of collagen-Ph conjugates and H2O2 concentration increased from 0.5% to 1% (w/v) at 19 μM of H2O2 and 19 to 39 μm at 1% (w/v) of collagen-Ph conjugates, respectively. However, a further increase of H2O2 concentrations to 78 μM at 1% (w/v) of collagen-Ph conjugates resulted in a significant decline of proliferation rate on ECFC and MSCs, which could be attributed to its high stiffness (G′ > 900 Pa). The results indicate that the amounts of H2O2 used in cross-linking collagen-Ph hydrogels significantly altered the cell proliferation behavior and largely improved the ECFCs and MSCs ability to spread. As reported previously, cell proliferation was often directly correlated to the stiffness of the substrate where cells resided and were grown in 2D culture [43–45]. 3.5. Monoculture of ECFCs or MSCs and co-culture of ECFCs and MSCs inside murine collagen-Ph hydrogels
Author Manuscript
Previous studies have shown that cross-linking degrees do compromise the cell viability and ability to spread, especially on primary stem cells [14,16,19,46,47]. To assess the effect of the degree of cross-linking of murine collagen-Ph hydrogels on 3D cell behavior, the 3D cell culture system was established and investigated in vitro. ECFCs and MSCs were encapsulated into collagen-Ph hydrogels independently and co-cultured in endothelial growth medium 2 supplemented with FBS (2%, Hyclone), SingleQuots containing human epidermal growth factor, human recombinant fibroblast growth factor-b, vascular endothelial growth factor, insulin-like growth factor, ascorbic acid, heparin, gentamicin/ amphotericin-B (Lonza), and 1x PS (Invitrogen). Cells were harvested into each hydrogel after 1 and 2 days of culture, and viability of encapsulated cells was evaluated using the live/ dead kit to accurately distinguish the percentage of viable cells within each hydrogel (Fig. 6). For monoculture, the cell viability was above 92% for ECFCs and 97% for MSCs with increasing concentrations of collagen-Ph hydrogels from 0.5% to 1% at 19 μM of H2O2 and 78 unit/μl of HRP. Cell viability was above 89% for ECFCs and 97% for MSCs throughout various amounts of H2O2 (19–78 μm) at 78 unit/μl of HRP after 2 days in culture (Fig. 6a Acta Biomater. Author manuscript; available in PMC 2016 November 01.
Kuo et al.
Page 14
Author Manuscript Author Manuscript Author Manuscript
and d). Specifically, we found that increasing the concentration of H2O2 from 19 to 78 μm progressively diminished the capacity of both ECFCs and MSCs to spread inside collagenPh hydrogels (Fig. 6b and e) as well as the ability to migrate through collagen-Ph hydrogels (Fig. 6c and f). Qualitative images of intact hydrogels showed that the ECFCs spread well and underwent self-assembly to form interconnections, indicating an ability to form capillary network structures inside collagen-Ph hydrogels. Moreover, these resulting structures rapidly regressed after 2 days of culture and the same phenomena were observed in rat tail collagen gels and other published literature [3,41,48,49] (Figs. 6b and S7). On the other hand, MSCs were able to spread in all of the collagen-Ph hydrogels used, although the interconnected cellular network that formed was less extensive as the cross-linking degree increased (Figs. 6e and S8). These results were anticipated because higher concentrations of H2O2 increase the degree of polymerization on collagen-Ph hydrogels; in fact, provision of exogenous collagenase partially recovered the migratory capacity of the cells at higher H2O2 concentrations of 156 μm (data not shown), suggesting that cell spread and motility were likely inhibited as a result of too much collagen-Ph hydrogel cross-linking, resulting in stiffer and lower biodegradation. Next, we evaluated the ability of ECFCs and MSCs to proliferate, spread and organise when co-cultured (2:3 ECFC/MSC ratio) inside murine collagen-Ph hydrogels with different cross-linking degrees (Fig. 6g). Similar to monoculture, we found that both viability of ECFCs and MSCs slightly decreased with increased concentrations of H2O2 at any given time point (19 μM: 92.0 ± 4.2%; 39 μm: 90.4 ± 2.9%; 78 μm: 88.4 ± 4.5%; 156 μM), which was slightly lower than cells inside rat tail collagen gels (97.1 ± 1.0%) after 2 days of culture (Fig. 6g). After 6 days of co-culture, cell viability inside murine collagen-Ph hydrogels with various H2O2 increased to above 93% (19 μM: 93.1 ± 3.4%; 39 μM: 95.5 ± 1.9%; 78 μm: 93.9 ± 3.2%; 156 μM) but was still a little lower than cells inside rat tail collagen gels (97.8 ± 3.3%). In summary, the crosslinking degree modulated cell spreading and motility in collagen-Ph hydrogels compared with various concentrations of collagen-Ph conjugates. Moreover, we observed important differences between the monocultures and the co-cultures. The overall survival of ECFCs was increased by the presence of MSCs, although their survival was still compromized by higher cross-linking degrees. 3.6. Anastomosis of engineered and host vasculatures
Author Manuscript
We therefore compared the extent of vascular network formation in murine collagen-Ph constructs to rat tail collagen type-I gels that commonly used in this field of research. Rat tail collagen type-I cell-laden constructs were formed by adding human ECFCs and MSCs to a solution of collagen type-I (3 mg/ml), then 250 μl (2 × 106 cells; 2:3 ECFC: MSC ratio) was injected subcutaneously into nude mice before gelation. As shown in Fig. 7, as expected from our previous results [7,18,30,32], rat tail type-I collagen were suitable for ECFC/MSCmediated vascular network formation (131 ± 24 lumens/mm2). For the murine collagen-Ph cell-laden constructs, 250 μl of Ca/Mg-free DPBS containing 0.4–1% (w/v) collagen-Ph conjugates, 78 units/μl of HRP, 19–78 μM of H2O2, and human ECFCs and MSCs (2 × 106 cells; 2:3 ECFC: MSC ratio) was injected subcutaneously, then, after 7 days in vivo, the constructs were recovered and evaluated. H&E staining revealed that vascular network formation was affected by collagen-Ph concentration, as the number of lumens inside the construct increased with decreasing collagen-Ph concentrations (Fig. 7a–d). Quantitative
Acta Biomater. Author manuscript; available in PMC 2016 November 01.
Kuo et al.
Page 15
Author Manuscript Author Manuscript
evaluation of the explants revealed a significant increase in the total number of perfused blood vessels with a decrease in collagen-Ph concentrations from 1% (4 ± 5 lumens/mm2) to 0.5% (81 ± 40 lumens/mm2), followed by a decrease at lower collagen-Ph concentrations of 0.4% (47 ± 19 lumens/mm2) (Fig. 7c). The same trend of an increase in the percentage of the newly formed mature human microvessels, identified by staining for hCD31+/ aSMA+ (Fig. 7e), followed by a decrease with increasing collagen-Ph concentration (Fig. 7b and d), was seen within the collagen-Ph hydrogel. Moreover, we examined whether amounts of H2O2 (19, 39 and 78 μM) used to crosslink the 1% collagen-Ph gel modulated functional perfused vessel formation in vivo. H&E staining revealed few perfused blood vessels (
Acta Biomater. Author manuscript; available in PMC 2016 November 01. Published in final edited form as: Acta Biomater. 2015 November ; 27: 151–166. doi:10.1016/j.actbio.2015.09.002.
Bioengineering vascularized tissue constructs using an injectable cell-laden enzymatically crosslinked collagen hydrogel derived from dermal extracellular matrix Kuan-Chih Kuoa, Ruei-Zeng Linb, Han-Wen Tiena, Pei-Yun Wua, Yen-Cheng Lic, Juan M. Melero-Martinb, and Ying-Chieh Chena,*
Author Manuscript
a
Department of Applied Science, National Hsinchu University of Education, Hsinchu 30014, Taiwan, ROC
b
Department of Cardiac Surgery, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA
c
Material and Chemical Research Laboratories, Industrial Technology Research Institute, Hsinchu 31040, Taiwan, ROC
Abstract
Author Manuscript
Tissue engineering promises to restore or replace diseased or damaged tissue by creating functional and transplantable artificial tissues. The development of artificial tissues with large dimensions that exceed the diffusion limitation will require nutrients and oxygen to be delivered via perfusion instead of diffusion alone over a short time period. One approach to perfusion is to vascularize engineered tissues, creating a de novo three-dimensional (3D) microvascular network within the tissue construct. This significantly shortens the time of in vivo anastomosis, perfusion and graft integration with the host. In this study, we aimed to develop injectable allogeneic collagen-phenolic hydroxyl (collagen-Ph) hydrogels that are capable of controlling a wide range of physicochemical properties, including stiffness, water absorption and degradability. We tested whether collagen-Ph hydrogels could support the formation of vascularized engineered tissue graft by human blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSC) in vivo. First, we studied the growth of adherent ECFCs and MSCs on or in the hydrogels. To examine the potential formation of functional vascular networks in vivo, a liquid pre-polymer solution of collagen-Ph containing human ECFCs and MSCs, horseradish peroxidase and hydrogen peroxide was injected into the subcutaneous space or abdominal muscle defect of an immunodeficient mouse before gelation, to form a 3D cell-laden polymerized construct. These results showed that extensive human ECFC-lined vascular networks can be generated within 7 days, the engineered vascular density inside collagen-Ph hydrogel constructs can be manipulated through refinable mechanical properties and proteolytic degradability, and these networks can form functional anastomoses with the existing vasculature to further support the survival of host muscle tissues. Finally, optimized conditions of the cell-
Author Manuscript *
Corresponding author at: Department of Applied Science, National Hsinchu University of Education, No. 521, Nanda Rd., Hsinchu City 30014, Taiwan, ROC. [email protected] (Y.-C. Chen).. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2015.09. 002.
Kuo et al.
Page 2
Author Manuscript
laden collagen-Ph hydrogel resulted in not only improving the long-term differentiation of transplanted MSCs into mineralized osteoblasts, but the collagen-Ph hydrogel also improved an increased of adipocytes within the vascularized bioengineered tissue in a mouse after 1 month of implantation.
Keywords Collagen hydrogels; Vascularization; Tissue engineering
1. Introduction
Author Manuscript Author Manuscript Author Manuscript
Creating large functional and transplantable tissues for organ replacement have failed in the past because of the limited diffusion distance of oxygen and nutrients to a few hundred micrometers. This can lead to necrosis at the central region of the large-sized tissueengineered or highly vascularized tissues [1–3]. Host blood vessels usually take days or weeks to invade the centre of engineered tissue, so during this process an insufficient vascular supply leads the tissue to nutrient depletion and ischemia, which can compromise cell viability and function [2,4,5]. To overcome this problem, nutrients and oxygen need to be delivered via perfusion instead of diffusion alone [3]. Aside from current strategies promoting angiogenesis from the host, an alternative concept is to pre-vascularize a tissue that creates a vascular network within the tissue construct prior to implantation [6]. Vascularization generally refers to the formation of an in vitro well-connected microvessel network within an implantable tissue construct through vasculogenesis. Following implantation, this vasculature can rapidly anastomose with the host and enhances tissue survival and function [1,3]. Furthermore, microfabrication techniques have been applied as a means to control space and direct the growth of vascular networks in vitro [7–10]; however, these pre-engineered networks are too simple to grow and reform in response to specific physiological demands from the organs they are supporting in the host [1,11]. Therefore, we have demonstrated the formation of vascular networks, de novo, from encapsulating endothelial colony-forming cells (ECFCs) and mesenchymal stem cells (MSC) in the liquid matrix prior to gelation, and injected or implanted subcutaneously in immune-deficient mice to form a 3D cell-laden vascularized construct within one week [12–14]. ECFCs circulating in peripheral blood participate in the formation of new blood vasculature and have been a promising source in producing non-invasive large quantities of autologous endothelial cells for clinical use [13,15]. Together with suitable support from scaffolds, MSCs can function as pericytes to promote vessel formation and maturation through secretion of specific proangiogenic cytokines [14,16]. Meanwhile, transplanted ECFCs provided critical angiocrine factors needed to preserve MSC as viable and further support ultimately long-term differentiation of transplanted MSCs to osteoblasts to form vascularized engineered bone tissue constructs by inducing specific stimulants of BMP-2 [12]. A critical requirement for engineering a tissue is the use of a suitable scaffold to mimic structural and functional properties of the natural extracellular matrix (ECM) that includes providing appropriate binding sites for cell–material interactions, mechanical properties to maintain cell function prior to host remodeling without negatively impacting the
Acta Biomater. Author manuscript; available in PMC 2016 November 01.
Kuo et al.
Page 3
Author Manuscript Author Manuscript
development of the capillary network, and biodegradation that matches the deposition rate of new extracellular matrix protein by the host [6]. Over the last few years, a variety of naturalbased hydrogels [17] have been shown to be compatible with endothelial cell-mediated vascular morphogenesis, including natural materials (type I collagen gel [18–22], fibrin gels [19,23], Matrigel [12,24]), semi-synthetic materials, i.e. modified natural material (photocrosslinkable methacrylated gelatin [10,14,16] and enzymatic-crosslinkable tyraminemodified gelatin hydrogels [25,26]). However, there still remains a significant discrepancy in how physicochemical properties of scaffolds, such as mechanical properties, density of cell-adhesive ligand (RGD) or degradable sites (MMP) of scaffolding affect the angiogenic potential of endothelial cells (EC) to form functional blood vessels in vivo. RGD-mediated and integrin-dependent bindings on native collagen nanofibrins support the development of sufficient matrix-transduced tensional forces necessary for EC sprouting and tube formation [27]. The stiffness of self-assembled collagen matrices affects cell spreading, migration, growth and matrix remodeling, all of which are critical components of vascular formation [20,28]. Varying collagen concentration of polymerized 3D collagen gels that alter matrix stiffness and fibril density has been shown to alter EC lumen size and tube length [18– 20,28,29], but in this case the RGD is dependent on collagen concentrations. These issues raised the difficulties of discerning which matrix properties induce EC responses. The aforementioned results provide initial evidence that specific collagen matrix physical properties are important parameters in determining the ability of matrices to guide vessel formation. However, most studies [19,26,28,29] have not been followed up with investigations addressing whether physicochemical properties of matrices impact upon vessel formation and if they further influence the success of creating vascularized tissue construct in vivo, which would lead to more clinically relevant cell-based therapies.
Author Manuscript Author Manuscript
So far, there have only been a few successful thick vascularized engineered tissue constructs, because the lack of proper scaffolding material that cannot only support vascularization in a short time period, but also directs transplanted MSCs differentiation into a specific lineage, remains a major challenge [4,5,11]. Although collagen offers an ideal injectable gel system in vascular tissue engineering, the extensive contraction, poor stiffness, rapid degradation and temperature instability of collagen gels limit their practical applications in regenerating and engineering living tissues, such as adipose or bone tissue grafts [22,30]. Thus, the development of collagen-based scaffolds that can maintain the mechanical integrity of the tissue, support the development and growth of complex microvessel networks, while simultaneously improving the mature differentiation of stem cells to meet the functional requirements of specific tissue, is more desirable. In response to these limitations, we aimed to develop chemical functionalization of natural-derived ECM proteins, i.e. injectable collagen-Ph hydrogels, which are capable of having controlled physicochemical properties over a wide range. To test the biocompatibility and potential for applications in tissue engineering, the ECFCs and MSCs were seeded on or inside collagenPh hydrogels to study the cell adhesion, proliferation and function in vitro. Pre-clinical studies with immunodeficient mice were carried out and demonstrated that functional human vascular networks can be generated in situ by means of post-injectable enzymatic polymerization that enables tuning of the final vascular density inside the collagen-Ph hydrogel constructs. We hypothesized that this cell-laden collagen-Ph construct would
Acta Biomater. Author manuscript; available in PMC 2016 November 01.
Kuo et al.
Page 4
Author Manuscript
rapidly anastomose with host vasculature and improve vascularization and survival of the host abdominal muscle defect with the formation of ECFC-lined vascular networks throughout and in-between skeletal muscle fibers in the host. Finally, feasibility studies toward an ultimate goal of bioengineered 3D vascularized tissue grafts were developed and characterized. Our studies importantly suggest the capacity of ECM-based cell delivery hydrogel systems to incorporate physicochemical cues to modulate vessel formation and further evaluate the feasibility to engineer vascularized transplantable artificial tissues in vivo.
2. Materials and methods 2.1. Extraction of murine collagen-Ph hydrogels from epidermis
Author Manuscript Author Manuscript
Before collagen extraction, murine epidermal tissue was cut into small pieces (90%) throughout a wide range of HRP (0–1250 unit/ml) (Fig. 4a) and H2O2 (0–78 μM) (Fig. 4b); cell viability was only negatively affected beyond 156 μM of H2O2 exposure directly into medium. However, addition of HRP and H2O2 would react and combine immediately with collagen-Ph conjugates to form hydrogels, with residual and unreactive H2O2 decreasing, and which did not affect the viability capacity of ECFCs and MSCs in 2 days of culture. In further studies, we then selected a working range of H2O2 (19–78 μM) used to crosslink hydrogels and discussed the effects of collagen-Ph hydrogels on ECFC and MSCs.
Author Manuscript
3.4. Cell behavior of ECFCs and MSCs grown on murine collagen-Ph hydrogels We studied whether varying amounts of H2O2 and collagen-Ph could be used as parameters to modulate cell behavior grown on collagen-Ph hydrogels. To investigate the effect of collagen-Ph hydrogels on cell attachment and proliferation, in vitro studies were carried out using ECFCs and MSCs as models. Cell proliferation was assessed by the live/dead assay (Invitrogen), as shown in Figs. 5, S5 and S6. We sought to understand whether increasing the degree of polymerization of collagen-Ph hydrogels (Fig. 3) could compromise cellular behavior by altering concentrations of murine collagen-Ph and H2O2. First, concentrations Acta Biomater. Author manuscript; available in PMC 2016 November 01.
Kuo et al.
Page 13
Author Manuscript Author Manuscript Author Manuscript
of collagen-Ph in the range of 0.5–1% did not affect the capacity of ECFCs and MSCs to attach (>82%, Fig. 5a), proliferate (>123% for ECFCs and >155% for MSCs, Fig. 5c) and survive (>91%, Fig. 5b and d) compared with cells grown on rat tail collagen (BD) gels. Secondly, various amounts of H2O2 (19–78 μM) did not affect the capacity of ECFCs and MSCs to attach (>80% for ECFCs and >84% for MSCs, Fig. 5a) and survive (>92% for ECFCs and >93% for MSCs, Fig. 5b) compared with cells grown on rat tail collagen (BD) gels. However, the degree of cross-linking through adjustment of H2O2 concentration modulated cell proliferation and spreading on collagen-Ph hydrogels (Fig. 5c). For longer culture of 2 days, with increasing H2O2 from 19 to 78 μM, the MSC proliferation rate was maintained at 155.4 ± 7.7% and 157.0 ± 11.3% at H2O2 levels of 19 and 39 μM, respectively, and then decreased to 99.8 ± 6.6% at H2O2 of 78 μM (Fig. 5c). Additionally, we explored the spreading area of ECFC on collagen-Ph hydrogels at different cross-linked degrees at concentrations of H2O2 (19 μM: 1948.9 ± 737.6 μM2; 39 μM: 2088.2 ± 834.5 μM2; 78 μM: 1891.1 ± 904.9 μM2) were larger than ECFCs spread out on rat tail collagen gels (950.7 ± 354.3 μM2). However, MSCs retain almost the same spread size grown on collagen-Ph hydrogels (19 μM: 1623.2 ± 702.7 μM2; 39 μm: 1828.1 ± 675.1 μm2; 78 μm: 1908.6 ± 736.0 μm2) and rat tail collagen gels (1495.4 ± 600.3 μm2) (Fig. 5e). There was no statistically significant increase in cell spread and/or proliferation observed when the amounts of collagen-Ph conjugates and H2O2 concentration increased from 0.5% to 1% (w/v) at 19 μM of H2O2 and 19 to 39 μm at 1% (w/v) of collagen-Ph conjugates, respectively. However, a further increase of H2O2 concentrations to 78 μM at 1% (w/v) of collagen-Ph conjugates resulted in a significant decline of proliferation rate on ECFC and MSCs, which could be attributed to its high stiffness (G′ > 900 Pa). The results indicate that the amounts of H2O2 used in cross-linking collagen-Ph hydrogels significantly altered the cell proliferation behavior and largely improved the ECFCs and MSCs ability to spread. As reported previously, cell proliferation was often directly correlated to the stiffness of the substrate where cells resided and were grown in 2D culture [43–45]. 3.5. Monoculture of ECFCs or MSCs and co-culture of ECFCs and MSCs inside murine collagen-Ph hydrogels
Author Manuscript
Previous studies have shown that cross-linking degrees do compromise the cell viability and ability to spread, especially on primary stem cells [14,16,19,46,47]. To assess the effect of the degree of cross-linking of murine collagen-Ph hydrogels on 3D cell behavior, the 3D cell culture system was established and investigated in vitro. ECFCs and MSCs were encapsulated into collagen-Ph hydrogels independently and co-cultured in endothelial growth medium 2 supplemented with FBS (2%, Hyclone), SingleQuots containing human epidermal growth factor, human recombinant fibroblast growth factor-b, vascular endothelial growth factor, insulin-like growth factor, ascorbic acid, heparin, gentamicin/ amphotericin-B (Lonza), and 1x PS (Invitrogen). Cells were harvested into each hydrogel after 1 and 2 days of culture, and viability of encapsulated cells was evaluated using the live/ dead kit to accurately distinguish the percentage of viable cells within each hydrogel (Fig. 6). For monoculture, the cell viability was above 92% for ECFCs and 97% for MSCs with increasing concentrations of collagen-Ph hydrogels from 0.5% to 1% at 19 μM of H2O2 and 78 unit/μl of HRP. Cell viability was above 89% for ECFCs and 97% for MSCs throughout various amounts of H2O2 (19–78 μm) at 78 unit/μl of HRP after 2 days in culture (Fig. 6a Acta Biomater. Author manuscript; available in PMC 2016 November 01.
Kuo et al.
Page 14
Author Manuscript Author Manuscript Author Manuscript
and d). Specifically, we found that increasing the concentration of H2O2 from 19 to 78 μm progressively diminished the capacity of both ECFCs and MSCs to spread inside collagenPh hydrogels (Fig. 6b and e) as well as the ability to migrate through collagen-Ph hydrogels (Fig. 6c and f). Qualitative images of intact hydrogels showed that the ECFCs spread well and underwent self-assembly to form interconnections, indicating an ability to form capillary network structures inside collagen-Ph hydrogels. Moreover, these resulting structures rapidly regressed after 2 days of culture and the same phenomena were observed in rat tail collagen gels and other published literature [3,41,48,49] (Figs. 6b and S7). On the other hand, MSCs were able to spread in all of the collagen-Ph hydrogels used, although the interconnected cellular network that formed was less extensive as the cross-linking degree increased (Figs. 6e and S8). These results were anticipated because higher concentrations of H2O2 increase the degree of polymerization on collagen-Ph hydrogels; in fact, provision of exogenous collagenase partially recovered the migratory capacity of the cells at higher H2O2 concentrations of 156 μm (data not shown), suggesting that cell spread and motility were likely inhibited as a result of too much collagen-Ph hydrogel cross-linking, resulting in stiffer and lower biodegradation. Next, we evaluated the ability of ECFCs and MSCs to proliferate, spread and organise when co-cultured (2:3 ECFC/MSC ratio) inside murine collagen-Ph hydrogels with different cross-linking degrees (Fig. 6g). Similar to monoculture, we found that both viability of ECFCs and MSCs slightly decreased with increased concentrations of H2O2 at any given time point (19 μM: 92.0 ± 4.2%; 39 μm: 90.4 ± 2.9%; 78 μm: 88.4 ± 4.5%; 156 μM), which was slightly lower than cells inside rat tail collagen gels (97.1 ± 1.0%) after 2 days of culture (Fig. 6g). After 6 days of co-culture, cell viability inside murine collagen-Ph hydrogels with various H2O2 increased to above 93% (19 μM: 93.1 ± 3.4%; 39 μM: 95.5 ± 1.9%; 78 μm: 93.9 ± 3.2%; 156 μM) but was still a little lower than cells inside rat tail collagen gels (97.8 ± 3.3%). In summary, the crosslinking degree modulated cell spreading and motility in collagen-Ph hydrogels compared with various concentrations of collagen-Ph conjugates. Moreover, we observed important differences between the monocultures and the co-cultures. The overall survival of ECFCs was increased by the presence of MSCs, although their survival was still compromized by higher cross-linking degrees. 3.6. Anastomosis of engineered and host vasculatures
Author Manuscript
We therefore compared the extent of vascular network formation in murine collagen-Ph constructs to rat tail collagen type-I gels that commonly used in this field of research. Rat tail collagen type-I cell-laden constructs were formed by adding human ECFCs and MSCs to a solution of collagen type-I (3 mg/ml), then 250 μl (2 × 106 cells; 2:3 ECFC: MSC ratio) was injected subcutaneously into nude mice before gelation. As shown in Fig. 7, as expected from our previous results [7,18,30,32], rat tail type-I collagen were suitable for ECFC/MSCmediated vascular network formation (131 ± 24 lumens/mm2). For the murine collagen-Ph cell-laden constructs, 250 μl of Ca/Mg-free DPBS containing 0.4–1% (w/v) collagen-Ph conjugates, 78 units/μl of HRP, 19–78 μM of H2O2, and human ECFCs and MSCs (2 × 106 cells; 2:3 ECFC: MSC ratio) was injected subcutaneously, then, after 7 days in vivo, the constructs were recovered and evaluated. H&E staining revealed that vascular network formation was affected by collagen-Ph concentration, as the number of lumens inside the construct increased with decreasing collagen-Ph concentrations (Fig. 7a–d). Quantitative
Acta Biomater. Author manuscript; available in PMC 2016 November 01.
Kuo et al.
Page 15
Author Manuscript Author Manuscript
evaluation of the explants revealed a significant increase in the total number of perfused blood vessels with a decrease in collagen-Ph concentrations from 1% (4 ± 5 lumens/mm2) to 0.5% (81 ± 40 lumens/mm2), followed by a decrease at lower collagen-Ph concentrations of 0.4% (47 ± 19 lumens/mm2) (Fig. 7c). The same trend of an increase in the percentage of the newly formed mature human microvessels, identified by staining for hCD31+/ aSMA+ (Fig. 7e), followed by a decrease with increasing collagen-Ph concentration (Fig. 7b and d), was seen within the collagen-Ph hydrogel. Moreover, we examined whether amounts of H2O2 (19, 39 and 78 μM) used to crosslink the 1% collagen-Ph gel modulated functional perfused vessel formation in vivo. H&E staining revealed few perfused blood vessels (
