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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Biomaterials. Author manuscript; available in PMC 2014 July 1.
Published in final edited form as:
PMCID: PMC3702175
NIHMSID: NIHMS474487

Engineered cell-laden human protein-based elastomer

Abstract

Elastic tissue equivalence is a vital requirement of synthetic materials proposed for many resilient, soft tissue engineering applications. Here we present a bioelastomer made from tropoelastin, the human protein that naturally facilitates elasticity and cell interactions in all elastic tissues. We combined this protein’s innate versatility with fast non-toxic fabrication techniques to make highly extensible, cell compatible hydrogels. These hydrogels can be produced in less than a minute through photocrosslinking of methacrylated tropoelastin (MeTro) in an aqueous solution. The fabricated MeTro gels exhibit high extensibility (up to 400%) and superior mechanical properties that outperform other photocrosslinkable hydrogels. MeTro gels were used to encapsulate cells within a flexible 3D environment and to manufacture highly elastic 2D films for cell attachment, growth, and proliferation. In addition, the physical properties of this fabricated bioelastomer such as elasticity, stiffness, and pore characteristics were tuned through manipulation of the methacrylation degree and protein concentration. This photocrosslinkable, functional tissue mimetic gel benefits from the innate biological properties of a human elastic protein and opens new opportunities in tissue engineering.

Keywords: Elastin, photocrosslinking, cell-laden hydrogel, tropoelastin, elastomer

1. Introduction

The golden standard for elastic tissue engineering is a moldable, substantially elastic, biologically compatible, human-based protein polymer that provides for the encapsulation and surface growth of multiple cell types [13]. Available elastomers include polysiloxanes [4, 5], polyurethanes [6, 7], polyhydroxyalkanoates [8, 9], poly(diol citrates) [10], poly(glycerolsebacate) [11, 12], and hybrid alginate/polyacrylamide [13]. These artificial chemical elastomers have some limitations. For example, they may require high temperatures and cytotoxic agents in their synthesis making them incompatible with viable cell encapsulation. They do not possess the innate ability to bind cells and require artificial derivatization to address this deficiency. The general release of toxic components during breakdown in vivo stand in the way of their broad approval as implantable materials in humans [1114].

We used a biomimetic approach to make a new class of engineered elastic materials by drawing on elastin’s ubiquitous, functional persistence in human elastic tissues. Elastin is required to maintain structural integrity, confer elasticity and signal cells to coordinate their activities. For example, blood vessels, skin, heart, lung, bladder and elastic cartilage all contain elastin. Tropoelastin is the natural monomeric precursor of elastin. We took the perspective that materials based on recombinant tropoelastin would yield elastic tissue equivalents if the protein could be photocrosslinked in less than a minute.

Most elastin is formed in the womb with the slow cross-linking of tropoelastin following its lysine-specific oxidation by lysyl oxidase. Various crosslinking approaches have been used to produce 3D elastin-based hydrogels from recombinant human tropoelastin [15, 16], α-elastin [1719], and engineered elastin-like polypeptides (ELPs) [2022]. The resulting hydrogels generally show biocompatibility and support cell growth both in vitro and in vivo, however, low mechanical properties [23, 24] and a non-homogenous cell penetration into the 3D structures of these hydrogels [15] are issues. Furthermore the use of cytotoxic conditions including chemical crosslinkers [15, 25], organic solvents [18, 26], prolonged UV exposure [27] and high pressure [17, 19] in the fabrication of these hydrogels prevents viable cell encapsulation during hydrogel formation.

To address these challenges, we use a synthetic path to generate highly elastic 3D cell-laden tropoelastin-based hydrogels by brief, light-initiated crosslinking of methacrylated tropoelastin (MeTro). The use of recombinant human tropoelastin containing 35 lysine residues per molecule is expected to promote cell-adhesive properties of the engineered construct. We hypothesized that our approach may enable us to control the physical properties of fabricated elastic hydrogels, such as pore characteristics, swelling behavior, and mechanical properties by changing the methacrylation degree and polymer concentration. In vitro studies were also conducted to assess the suitability of the MeTro gel for both 3D elastic environments for cell encapsulation and 2D films for surface growth of multiple cell types.

2. Materials and Methods

2.1. Tropoelastin synthesis

Recombinant human tropoelastin isoform SHELΔ26A (Synthetic Human Elastin without domain 26A) corresponding to amino acid residues 27–724 of GenBank entry AAC98394 was purified from Escherichia coli on a multi-gram scale using fermentation, selective alcohol solubilization and reverse phase HPLC methodologies as previously described [28, 29]. SHELΔ26A represents the 60 kDa mature form of the most frequently observed secreted splice form of the protein following removal of the signal peptide.

2.2. MeTro synthesis and characterization

Tropoelastin was methacrylated by the addition of methacrylate anhydride (MA, Sigma) to a 10% (w/v) tropoelastin solution in phosphate buffered saline (PBS; Invitrogen) and reacted for 12 h at 4 °C. The solution was then diluted and dialyzed (Slide-A-Lyzer MINI, 3.5K MWCO) against distilled water at 4 °C for 48 h and lyophilized to yield MeTro. Various concentrations of MA (e.g. 8, 15, 20% (v/v)) were used to influence the degree of methacrylation. 1H NMR analysis was used to calculate the methacrylation degree of MeTro. The effect of methacrylation on the coacervation behavior and secondary structure of tropoelastin was investigated using UV spectrophotometry and circular dichroism (CD) analysis, respectively.

2.2.1. 1H NMR

1H NMR spectra of tropoelastin and MeTro solutions in D2O (1% (w/v)) were obtained on a Varian Inova-500 NMR spectrometer and used to calculate the extent of methacrylation. NMR was performed at 4 °C to prevent coacervation of Me Tro solutions during analysis. The degree of methacrylation was calculated using the peak values between 4.9 and 6 ppm from methacrylate groups and the peak at 2.6 ppm from lysine residues in tropoelastin and defined as the number of methacrylated lysines divided by the total number of lysines.

2.2.2. UV Spectrophotometry

The coacervation behavior of 1% (w/v) tropoelastin and MeTro solutions in PBS were assayed by monitoring turbidity through light scattering (300 nm) using a spectrophotometer (Shimadzu UV-1601). The turbidity of each solution was assayed for 10 min at temperatures ranging from 4 °C to 45 °C. The temperature was con trolled by connecting the cuvette holder to a recirculating water bath. To investigate the effect of methacrylation degree on coacervation behavior, MeTro macromers with varying methacrylation degrees were used. Following each measurement, the solutions were cooled to 4 °C for 10 min. At each temperature, the maximum variation in turbidity was recorded and expressed as a percentage of maximum turbidity for each MeTro solution to generate a series of coacervation curves.

2.2.3. Circular dichroism

Tropoelastin and MeTro samples were dissolved in water at 0.015% (w/v) and monitored using 0.1 cm quartz cuvettes in a Jasco J-815 CD spectrometer. Five spectral accumulations were obtained for each sample. Spectra were measured from 184 to 240 nm at 20 °C. Data were expressed in terms of the mean residue ellipticity (deg cm2 dmol−1). Secondary structure analysis of the CD spectra was performed with CONTIN, SELCON3 and CDSSTR programs through the CDPro software package using reference set SP43 [30].

2.3. Hydrogel fabrication and characterization

MeTro macromers with different degrees of methacrylation were used to make photocrosslinked MeTro hydrogels. To prepare the hydrogels, various concentrations of MeTro solutions (e.g. 5, 10, 15% (w/v)) were prepared in PBS containing 0.5% (w/v) 2-hydroxy-1-(4-(hydroxyethoxy) phenyl)-2-methyl-1-propanone (Irgacure 2959, CIBA Chemicals) at 4 °C. Photocrosslinked MeTro gel was formed by pipetting 10 µl of the solution between two glass coverslips that were separated by a 150 µm spacer and exposing to 6.9 mW/cm2 UV light (360–480 nm) for 35 sec. MeTro solutions with varying methacrylation degrees were used to examine the effects of the extent of methacrylation on hydrogel characteristics including pore architecture, swelling ratio, and tensile and compressive properties. GelMA hydrogels were prepared by using 10% (w/v) GelMA with 80% methacrylation degree as previously described [31] and used as controls for mechanical properties characterization. Poly (ethylene glycol) dimethacrylate (PEGDMA, MW 1000 Da) hydrogels were also formed [32] and used as controls for the in vitro studies.

2.3.1. Swelling ratio

The swelling behavior of MeTro hydrogels was evaluated at 37 °C in PBS using previously reported procedures [17, 25]. To prepare MeTro hydrogels for swelling ratio measurements, 40 µL MeTro solution was first injected into a custom-made polydimethylsiloxane (PDMS) mold (diameter: 5 mm and depth: 1 mm). A glass coverslip was placed on the mold before exposing to UV light for 180 sec. Samples were then collected from the mold and detached from the slide after soaking in PBS for 5 min. To calculate swelling ratios, the gels were lyophilized and their dry weights were recorded. The samples were then incubated in PBS for 24 h after which the excess liquid was removed from the swollen gels and the samples were reweighed. The swelling ratio was then calculated as the ratio of mass increase to the mass of dry samples. The effect of methacrylation degree and MeTro concentrations on the swelling ratio was obtained. At least 5 samples were tested for each condition.

2.3.2. Scanning electron microscopy (SEM)

SEM analysis was used to determine the pore characteristics of the fabricated MeTro hydrogels and also to examine cell growth on the surfaces of hydrogels. SEM images of MeTro gels were obtained by using a FEI/Philips XL30 FEG SEM (15 KV). Lyophilized samples were mounted on aluminum stubs and gold coated prior to SEM analysis. The effects of methacrylation degree and polymer concentrations on the pore characteristics of MeTro hydrogels were examined. The average pore sizes of fabricated MeTro gels were calculated using Image J software. For pore size measurement, at least 100 pores were measured based on 3 images from each of 5 samples per condition.

2.3.3. Mechanical characterization

Compressive and tensile properties of MeTro gels were assessed using a mechanical tester (Instron Model 5542) with a 10 N load cell. For compression tests, MeTro gels were prepared as described previously for swelling ratio measurements. Hydrogels were swelled for 4 h in PBS prior to mechanical testing. A digital caliper was used to measure the thickness (1 ± 0.2 mm) and diameter (5 ± 0.3 mm) of each hydrogel. Cyclic uniaxial compression tests in an unconfined state were then performed using a cross speed of 30 µm/s and a 60% strain level according to previously described procedures [18, 25]. Hydrogels were cyclically preconditioned for 7 cycles after which they were subjected to another loading and unloading and the compression (mm) and load (N) were recorded at the 8th cycle using Bluehill software. The tangent slope of the linear region of the stress-strain curve for the 8th cycle was reported as the compressive modulus. The energy loss for the 8th cycle was also calculated based on the area between the loading and unloading curves. The effects of methacrylation degree and MeTro concentrations on the compressive properties of MeTro gels were determined by using various concentrations of MeTro solutions (5, 10, 5% (w/v)) with varying methacrylation degrees (31%, 44%, and 48%). At least 5 samples were tested for each condition.

For tensile tests, samples that were 20 ± 0.3 mm in length, 4 ± 0.3 mm in width, and 1 ± 0.1 mm in thickness were prepared following the procedure described previously for hydrogel fabrication. The gels were swelled for 4 h in PBS and then mounted onto the mechanical tester. The tensile grips were covered with fine sand paper to eliminate slippage. The test was performed at 1 mm/min strain rate until failure. The tensile properties of samples including elastic modulus (the tangent slope of the stress-strain curve), ultimate stress (stress at failure), and maximum strain or extensibility (strain level at failure) were reported. The effect of methacrylation degree and MeTro concentration on tensile properties of hydrogels were investigated. In addition, the extensibilities of native pulmonary artery and aorta of sheep were obtained to compare with the strain failure of the fabricated MeTro gel. At least 5 gels were tested per condition.

2.4. In vitro studies

2.4.1. Cell culture

Immortalized green fluorescent protein (GFP)-expressing human umbilical vein endothelial cells (HUVEC; ATCC) and NIH 3T3 fibroblasts were used in this study. HUVECs were cultured in a 5% CO2 humidified incubator at 37 °C in endothelial basal medium (EBM-2; Lonza) supplemented with endothelial growth BulletKit (Lonza) and 100 units/ml penicillin-streptomycin (Gibco, USA). 3T3 fibroblast cells were maintained in Dulbecco's modified eagle medium (DMEM; Invitrogen) modified with 10% fetal bovine serum (FBS) and 100 units/ml penicillin-streptomycin. Cells were passaged approximately 2 times per week and media was changed every 2 days.

2.4.2. Surface seeding (2D culture)

For cell adhesion and proliferation studies, MeTro gels were made following exposure to 6.9 mW/cm2 UV light (360–480 nm) for 35 s on 3-(trimethoxysilyl)propyl methacrylate (TMSPMA, Sigma) treated glass. The slides containing MeTro were seeded with cells (2×105 cells/scaffold) and incubated for 7 days. Media were changed every second day.

2.4.3. Cell viability

Cell viability was determined by using a Live/Dead assay Kit (Invitrogen, USA) according to the manufacturer’s instructions. To perform cell viability assay, the cells were stained with 0.5 µl/ml calcein AM and 2 µl/ml ethidium homodimer-1 (EthD-1) in PBS. Two hundred µl calcein AM/(EthD-1) solution was added to each well of a 24 well plate containing hydrogels. The well plate was then incubated at 37 °C for 20 min after which the live (green) and dead (red) cells were observed by using an inverted fluorescence microscope (Nikon TE 2000-U, Nikon instruments Inc., USA). The number of live and dead cells was counted by ImageJ software using at least 4 images from different areas of 3 gels for each condition. Cell viability was then calculated based on the number of live cells divided by the total cell number.

2.4.4. Cell adhesion, proliferation and spreading

Rhodamine-phalloidin (Alexa-Fluor 594; Invitrogen) and 4',6-diamidino-2-phenylindole (DAPI; Sigma) staining was used to investigate cellular attachment, proliferation, and spreading on the surfaces of fabricated hydrogels. For DAPI staining, the cell-seeded scaffolds were first fixed in 4% (v/v) paraformaldehyde (Electron Sciences) solution in PBS for 30 min. The samples were then incubated in a 0.1% (v/v) DAPI solution in PBS for 10 min at 37 °C to stain the cell nuclei. The stained samples were then washed twice with PBS before visualizing with an inverted fluorescence microscope. ImageJ software was used to count the DAPI stained nuclei and assess the proliferation of the cells on the surfaces of gels at various culture times. To stain F-actin filaments of the cell, cell-seeded gels were fixed in 4% (v/v) paraformaldehyde for 30 min. The cells were then permeabilized in a 0.1% (w/v) Triton X-100 solution in PBS for 20 min and blocked in 1% (w/v) bovine serum albumin (BSA) for 1 h. The samples were then incubated in a solution of 1:40 ratio of Alexa Fluor-594 phalloidin in 0.1% BSA for 45 min at room temperature to stain the actin cytoskeleton.

2.4.5. Cell encapsulation (3D culture)

Cells were trypsinized and resuspended in MeTro solution containing 0.5% (w/v) photoinitiator at a concentration of 5×106 cells/ml. Cell-laden MeTro gels were made following exposure to 6.9 mW/cm2 UV light (360–480 nm) for 35 s on TMSPMA treated glass. The gels were then washed 3 times with PBS and incubated for 7 days in 3T3 medium in a culture incubator at 37°C.

2.5. Statistical analysis

Data were compared using one-way ANOVA followed by Bonferroni’s post-hoc test (GraphPad Prism 5.02) software. Error bars represent the mean ± standard deviation (SD) of measurements (*p<0.05, **p<0.01, and ***p<0.001).

3. Results and discussion

In this study, we synthesized MeTro gels with tunable physical properties, which can be used in both 3D elastic environments for cell encapsulation and 2D films for cellular attachment and growth. These hydrogels can be made in a variety of shapes and sizes, and present several advantages over other elastin-derived hydrogels and synthetic elastomers. First, MeTro gels are formed rapidly within 35 seconds, which is shorter than the typical time (~ 24 h) required to chemically crosslink elastin-based hydrogels [15, 17]. Second, the photocrosslinking of MeTro gels in aqueous solution eliminates the use of toxic crosslinkers [15, 25] and organic solvents [18] during the hydrogel synthesis. Third, the potential to directly encapsulate cells rapidly within the MeTro gels allows for a pronounced cell distribution throughout the matrix, overcoming problems associated with minimal cell penetration into 3D matrices post-casting due to common issues such as low porosity, pore connectivity and small pore sizes [15, 17]. Fourth, unlike other synthetic elastomers such as polyurethane [33], the use of biodegradable MeTro gels do not cause cytotoxicity through in vivo degradation, as the gels are formed from a human protein. Fifth, tropoelastin-based materials are more stable [15] compared to some biodegradable elastomers such as PGS [34], thus providing mechanical support until new tissues form. Sixth, the presence of the full secreted tropoelastin amino acid sequence including the cell-interactive C-terminus imparts cell-interactive functions to the MeTro gels by providing endogenous integrin αVβ3 cell binding sites [35].

We first investigated the effect of methacrylation on the coacervation behavior and secondary structure of tropoelastin. Then, we used MeTro prepolymers with varying extents of methacrylation to form elastic hydrogels with tunable physical properties. The effects of methacrylation degree and polymer concentrations on the pore characteristics, swelling ratios, and mechanical properties (both tensile and compressive behaviors) of fabricated hydrogels were studied. In addition, we demonstrated functional performance for two cell types (HUVECs, and 3T3 fibroblasts) in supporting their growth on and embedded within MeTro hydrogels.

3.1. Synthesis of MeTro

MeTro was synthesized using recombinant human tropoelastin [36] and methacrylic anhydride. The degree of methacrylate functionalization was controlled by specific methacrylic anhydride concentrations and was quantified by 1H NMR analysis (Fig. 1A). MeTro polymers with methacrylation degrees of 31%, 44%, and 48% were produced with 8% (v/v), 15% (v/v), and 20% (v/v) methacrylic anhydride, respectively. The resultant MeTro solutions were lyophilized prior to use. To confirm the integrity of MeTro, we assessed the effect of methacrylation on secondary structure and functional protein performance by coacervation. Coacervation of tropoelastin is an inverse temperature transition process of massive protein assembly that plays a critical role in elastin formation. This temperature transition is mainly due to the intermolecular hydrophobic association of tropoelastin molecules in an aqueous environment and so is sensitive to protein concentration, pH, salt, and impurities [37]. On this basis, acrylate modification at lysine residues increased the protein’s hydrophobicity and gave a lower coacervation temperature (Fig. 1B). CD spectra of tropoelastin and MeTro each displayed an intense negative band at 200 nm, consistent with substantial secondary disorder, and a shoulder centered at 222 nm, demonstrating the presence of α-helical structure each of which is normal for tropoelastin [36, 38] (Fig. 1C). Quantification of secondary structure revealed that methacrylation of tropoelastin did not significantly alter secondary structure (Fig. 1d). The secondary structure calculated for all samples were approximately 12% α-helix, 18% β-sheet, 13% β-turn, 9% polyproline type-II turn and 48% unresolved, which are similar to those reported for bovine tropoelastin [39] and elastin [40].

Figure 1
Effect of methacrylation on tropoelastin

3.2. Fabrication and physical properties of MeTro hydrogels

Highly elastic and porous 3D MeTro hydrogels were prepared by UV irradiation (6.9 mW/cm2) of MeTro prepolymer solutions for 35 sec. Average pore size, swelling ratio, and mechanical performance were tailored by varying both the extent of methacrylation and MeTro concentration.

We found that the methacrylation degree significantly influenced both pore characteristics and swelling behavior of fabricated hydrogels. The average pore size of 10% (w/v) MeTro gels decreased from 51 ± 9 µm to 23.4 ± 5.8 µm and 11.7 ± 3.3 µm as the methacrylation degree was increased from 31% to 44% and 48%, respectively (Fig. 2Ai–iv) (P<0.01). In addition, swelling ratios decreased from 4.1 ± 0.3 to 2.3 ± 0.3 and 1.2 ± 0.2 g liquid / g protein as the degree of methacrylation was increased from 31% to 44% and 48%, respectively (Fig. 2Av). On this basis, higher degrees of methacrylation are likely to enhance the crosslinking density, and lead to reduced pore sizes and swelling ratio of the resulting MeTro gels.

Figure 2
Pore characteristics and equilibrium swelling properties of MeTro gels

In contrast we found that protein concentration had no effects on the swelling ratios and pore architectures of the hydrogels as shown in Fig. 2B. All MeTro gels made using various concentrations of MeTro (5, 10, 15% (w/v)) at 31% methacrylation contained large pores (44 µm – 63.5 µm) on their surfaces and within gel cross-sections (Fig. 2Bi–vii), conducive to nutrient and oxygen diffusion as well as removal of metabolic waste in 3D constructs.

The mechanical characteristics of MeTro hydrogels were analyzed by tensile and unconfined cyclic compression tests. Our fabricated material exhibited superior mechanical properties compared to other photocrosslinkable gels, including substantial extensibility up to 400% before rupture (Fig. 3A), reversible deformation with low energy loss, and high resilience upon stretching. The fabricated hydrogels showed soft tissue-like elastic moduli between 2.8 ± 0.6 kPa and 14.8 ± 1.9 kPa with an ultimate tensile strength ranging from 12.5 ± 2.2 kPa to 39.3 ± 2.5 kPa that was tuned by both the extent of methacrylation and MeTro concentration (Fig. 3B–E, Table 1). In addition, the extensibility of the fabricated MeTro gels was significantly decreased by increasing the methacrylation degree and polymer concentration. For example, the strain at break for a 10% (w/v) MeTro gel decreased 2.5-fold from 332 ± 48 to 135 ± 11 when the methacrylation degree was enhanced from 31% to 48%. This can be due to the enhanced degree of crosslinking in MeTro gels with higher methacrylation, which resulted in an increase in material stiffness and consequently decrease in elasticity. MeTro gels exhibit higher elastic moduli and extensibility compared to other photocrosslinkable hydrogels such as 10% (w/v) GelMA gel at 80% methacrylation [31] (modulus: 3.3 ± 0.7 kPa, extensibility: ~ 100%).

Figure 3
Tensile properties of MeTro hydrogels
Table 1
Mechanical characterization of photocrosslinked MeTro hydrogels

The extensibility of native pulmonary artery was 204 ± 9% and 158 ± 25% in the circumferential and radial directions, respectively. In addition, in longitudinal direction, the strain level of failure for aorta was 266 ± 55 % and in circumferential direction was 282 ± 48 %. The extensibility of fabricated MeTro gel ranging from 135–400% depending on the methacrylation degree and polymer concentration, is in the maximum strain range of native pulmonary artery and aorta. The unique elastic properties of this photocrosslinkable MeTro gel point to its potential as a suitable cell-interactive protein-based matrix for the engineering of elastic tissues such as blood vessel, skin, lung, and cardiac tissue because of the important role played by elastin in mechanical and cell-interactive functions in these tissues.

The compressive behavior of fabricated hydrogels was evaluated by applying cyclic compression on the hydrogels (Fig 4). Gels deformed reversibly following 8 cycles of loading and unloading at 60% strain. The hydrogels displayed energy losses of ~24.1–30.5% depending on MeTro concentration, which is comparable to the energy loss of purified native elastin at 23 ± 10% [41]. The compressive modulus increased from 8.8 ± 0.4 to 14.8 ± 1.1 and 159.7 ± 18.8 kPa when the MeTro concentration was increased from 5 to 10 and 15% (w/v). The high compressive modulus of 15% (w/v) MeTro gel is likely due to the increased viscosity of this higher protein concentration prepolymer solution which gives rise to an enhanced crosslinking density within the fabricated hydrogels after exposure to UV. In contrast, the low concentration of the 5% (w/v) MeTro prepolymer solution limited the UV crosslinking of the solution, which resulted in the formation of hydrogels with lower compressive modulus.

Figure 4
Unconfined compressive behavior of MeTro gels

It is likely that the compression data were dominated by the packing density of tropoelastin molecules. In contrast, extensional behavior is a complex combination of crosslinking density and extensibility of the constituent tropoelastin components. On this basis the compressive modulus showed a marked dependence on concentration as evidenced by sharp increase between 10% (w/v) and 15% (w/v) gels.

In addition, cyclic loading and unloading had no effect on the stiffness of the gels and all of the hydrogels fully recovered immediately after each unloading without any permanent deformation. Most other hydrogels exhibit high hysteresis and permanent deformation after unloading. For example, in a recent study highly stretchable hybrid alginate/polyacrylamide gels weaken by their second loading but somewhat recover if that loading is delayed by a day [13]. The elasticity and recoverable energy dissipation in our material is dictated by water interactions with repeated hydrophobic regions in tropoelastin while hydrophilic domains are responsible for crosslinking. On stretching, exposed hydrophobic domains organize structured water and lead an entropic drive to mechanical collapse; in this way the hydrophobic regions contract upon unloading, leading to recovery. MeTro gels have higher compressive moduli over elastin-based gels that use a variety of crosslinks and also other photocrosslikable hydrogels [1, 31, 32, 42, 43], making it a unique, stretchable human protein-based gel that can be considered for the repair of elastic tissues.

3.3. Cell interactions with MeTro hydrogels

MeTro gels with 10% (w/v) protein concentration and 31% methacrylation degree were used to examine the cell interactive capabilities (e.g. cell viability, adhesion, spreading and proliferation) of the resulting hydrogels. We used HUVECs and 3T3 fibroblasts as model attachment cells. Cells were cultured for a week on the surfaces of MeTro hydrogels. Cellular viability, adhesion, spreading, and proliferation on the hydrogels were examined by using calcein-AM/ethidium homodimer Live/Dead assay, SEM analysis, and F-actin/DAPI staining, respectively. As shown in Fig. 5M, cell viability was >92% for MeTro gels seeded with fibroblasts (Fig. 5A–C) and HUVECs (Fig. 5D–F) over a 7-day culture. In addition, fibroblasts (Fig 5G) and endothelial cells (Fig. 5H) adhered to and formed monolayers on the surfaces of MeTro hydrogels, however, cell did not grow on the control PEG hydrogel (Fig 5I and 5L). These cells had spread as indicated by the staining of cell F-actin filaments and nuclei with rhodamine-phalloidin/DAPI (Fig. 5I–J). The capacity of MeTro gel surfaces to support cell proliferation was evidenced by the increasing number of DAPI stained nuclei per given hydrogel area during culture (Fig. 5N). These results demonstrated the capacity for fabricated MeTro gels to act as cell-interactive substrates with applications for the formation of 2D films or coating agent. The excellent cell-interactive properties of MeTro gels is likely due to integrin-based cell-binding sites within the tropoelastin molecules [35].

Figure 5
2D studies in vitro on MeTro hydrogels

The viability of 3T3 fibroblasts encapsulated within the 3D structures of highly elastic MeTro gels was investigated over a 7-day culture. As shown in Fig 6A–D, when 3T3 fibroblasts were encapsulated within the 3D structure of 10 and 15% (w/v) MeTro hydrogels with 31% methacrylation degree, they remained viable for at least a week in culture (Fig. 6E). This demonstrated the compatibility of this range of MeTro gels with 3D cell encapsulation. To our best knowledge, this is the first highly elastic human protein-based hydrogel that is able to embed viable cells within its 3D structures.

Figure 6
3D in vitro studies on MeTro hydrogels

We have previously fabricated elastin-based hydrogels through chemical crosslinking of tropoelastin and soluble elastin using various types of crosslinking agents such as hexamethylene diisocyanate [18], glutaraldehyde [25], and bis(sulfosuccinimidyl) suberate [15]. In all these formulations, cells were seeded on the surfaces of the materials after hydrogel formation, as the use of toxic crosslinkers did not allow for cell embedding prior to hydrogel formation. Limited cellular penetration and growth within 3D structures of these gels [15] were the main limitations for tissue engineering applications. Electrospinning techniques [26] and dense gas carbon dioxide (CO2) [17, 18] were used to enhance the pore sizes and consequently facilitate cell penetration and infiltration into the 3D structures of these elastin-based biomaterials. However, non-homogenous cellular distribution within these elastic gels was an issue. The MeTro gel developed in this study overcomes these limitations and introduces a new class of bioelastomer with tunable elasticity and the capacity for cell encapsulation in a 3D elastic environment.

Due to these unique properties, MeTro hydrogels can serve as suitable biomaterials for engineering diverse elastic tissues where elasticity plays an important role in their functions. MeTro confers the additional advantages of viable cell encapsulation within 3D hydrogels while allowing for functional, cell surface colonization. This is the first report of a cytocompatible, elastic tissue equivalent with excellent mechanical properties and cell-interactive behavior.

4. Conclusion

In this study, we demonstrated the construction of a cell-laden human protein-based hydrogel, MeTro. The extraordinary properties of MeTro gels include high extensibility, recoverability upon loading and unloading, and ability for fast polymerization and easy method of synthesis, suitability for both 3D cell encapsulation and 2D cell seeding. These features point out that MeTro constructs are a unique class of materials for various tissue engineering application that rely on elasticity while allowing for cell encapsulation and surface growth. In many applications including the engineering of elastic tissues, the use of hydrogels is often severely limited by their low mechanical properties and stretchability. Compromised mechanical properties of hydrogels for cell encapsulation can lead to unintended cell release and death, while low toughness limits durability. Consequently, hydrogels of superior stiffness, extensibility, and recoverability are likely to improve performance in these applications.

Acknowledgments

N.A. acknowledges the support from the National Health and Medical Research Council. A.K. acknowledges funding from the National Science Foundation CAREER Award (DMR 0847287), the office of Naval Research Young National Investigator Award, the National Institutes of Health (HL092836, DE019024, EB012597, AR057837, DE021468, HL099073, EB008392), and the Presidential Early Career Award for Scientists and Engineers (PECASE). A.S.W. acknowledges funding from the Australian Research Council, Australian Defense Health Foundation and National Health & Medical Research Council. A.S.W. is the Scientific Founder of Elastagen Pty Ltd.

Footnotes

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The authors declare no conflict of interest in this work.

Author contributions

N.A. and S.M.M. designed the experiments and wrote the paper. N.A. and S.M.M performed experiments and analyzed the data. P.Z. performed experiments. G.C.U. revised the manuscript. A.S.W. and A.K. provided conceptual input, designed experiments, and revised the paper.

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