Bone Marrow-Derived Mesenchymal Stem Cells Encapsulated in

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BMSCs Encapsulated in Functionalized Gellan gum/ Collagen Hydrogel for Effective Vascularization Hong Chen, Yajie Zhang, Pi Ding, Tingting Zhang, Yue Zan, Tianyu Ni, Rong Lin, Min Liu, and Renjun Pei ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00361 • Publication Date (Web): 10 Oct 2018 Downloaded from http://pubs.acs.org on October 13, 2018

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ACS Applied Bio Materials

Grraphical Abstract

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BMSCs Encapsulated in Functionalized Gellan gum/Collagen Hydrogel for Effective Vascularization

Hong Chen†‡, Yajie Zhang†§, Pi Ding†, Tingting Zhang†‡, Yue Zan†‡, Tianyu Ni†, Rong Lin‡, Min Liu*⊥, Renjun Pei*†§

†

CAS Key Laboratory for Nano-Bio Interface, Division of Nanobiomedicine, Suzhou

Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou, 215123, China. ⊥

‡

Institute for Interdisciplinary Research, Jianghan University, Wuhan, 430056, China.

School of Pharmacy, Xi’an Jiaotong University, Xi’an, 710061, China.

§

School of Nano Technology and Nano Bionics, University of Science and

Technology of China, Hefei, 230026, China.


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Abstract Gellan gum hydrogel holds great potential in tissue engineering, but the high phase transition temperature greatly inhibits the applications in biomedical field. In this study, gellan gum was modified with methacrylic anhydride, and then the phase transition temperature was reduced. The functionalized gellan gum together with type I

collagen

was

gelled

by

ion/photo

dual-crosslinking

for

fabricating

BMSCs-encapsulating hydrogel for vascularization. After optimizing the ratio between gellan gum and collagen, the hydrogel with proper pore size and mechanical properties were prepared. The WST assay demonstrated that the hydrogel could offer excellent microenvironment for cell survival and proliferation. Finally, RT-qPCR suggests that the hydrogel could promote BMSCs to differentiate into endothelial cells.

Together,

this

work

provides

a

general

strategy

for

fabricating

BMSCs-encapsulating hydrogel in one step, which has the potential for 3D-printing live cell scaffold for study of vasculogenic differentiation.

Keywords: gellan gum/collagen hydrogel, photo-crosslinking, BMSCs-encapsulating hydrogel, vascularization



INTRODUCTION Stem cell regenerative medicine is a field which aims to fabricate tissue-like constructs in vitro or in vivo through biomaterials-based scaffolds and living stem cells. This method will change the traditional treatments for diseases and make significant innovation in medical field.1-3 However, these tissue engineering technologies were succeeded in some small or thin tissues or organs over the past several decades, such as skin, cartilage,4-5 but failed to fabricate large or thick tissues or organs such as liver, heart and lung. One of the most important reasons is the deficiency of the nutrition and waste exchanging in the scaffold interior, thus causing cell death.6-10 Therefore, the introduction of vascularization into biomaterial scaffolds is an effective way to solve this challenge. Typical two-dimensional (2D) cell culture systems are not able to fully mimic the in vivo microenvironment, while three-dimensional (3D) scaffolds could mimic the in vivo environment that interacts with cells directly and maintains a dynamic regulatory for tissue morphogenesis. Especially for the regenerative medicine, an ideal tissue engineering scaffold is the most important factor in tissue engineering.11 The formation of new blood vessels is mediated by interactions among endothelial cells (ECs), growth factors (such as VEGF) and scaffold. Therefore, the delivery of growth factors and the property of scaffold have great impact on neovascularization.12-13 Hydrogel, a 3D network, can absorb and keep plenty of water, which is widely used for cell encapsulating and loading in tissue engineering.14-18 Different from the conventional cell-laden 3D scaffolds which seed cells into the already prepared


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scaffolds, current strategies for tissue engineering focus on fabricating 3D tissue constructs through encapsulating living BMSCs into appropriate biomaterials in one step, which has better control over cellular distribution.19 Therefore, the materials for constructing scaffold are the crucial factor since the cells are suspended in the hydrogel precursor solution before gelation. Gellan gum, an anionic linear microbial polysaccharide, possesses well biocompatibility and biodegradability which is widely used in foods and industries. Currently, the gellan gum based hydrogels attracted more and more attentions in drug delivery and tissue engineering due to the good temperature sensitivity and divalent ion response. However, the high phase transition temperature greatly inhibits its application for constructing cell-encapsulating scaffolds.20-29 Previous work demonstrated that grafting with vinyl-group could decrease the phase transition temperature.25 Moreover, collagen, a major component of extracellular matrix, is widely

studied

in

biomedical

field

since

collagen

contains

RGD

(Arginine-Glycine-Aspartic) sequences in its structure which can increase the adhesion and proliferation of stem cells.30-33 Therefore, by combining the advantages of gellan gum and collagen, we can construct a hydrogel which not only possesses certain mechanical properties, but also offers a suitable microenvironment for cell adhesion and proliferation. In this study, we constructed a BMSCs-encapsulating hydrogel based on vinyl-group modified gellan gum and collagen through a double crosslinking method, which not only made gellan gum and collagen soluble in water at room temperature,



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but also solidified the construct which adapted the requirement for vascular differentiation. The pore size and mechanical properties were well performed, and the results suggested that this hydrogel system could be used as vascular differentiation. Furthermore, the real-time quantitative PCR (RT-qPCR) analyses of vasculogenic differentiation markers, including PECAM-1, KDR, TEK, vWF were well characterized at various time points, especially the different cultural environment (3D culture and 2D culture) was investigated in details. Together, this double crosslinking hydrogel system holds great potential in 3D bioprinting and could be used as scaffold for vasculogenic differentiation.

EXPERIMENTAL SECTION Materials and instruments Gellan

gum

and

methacrylic

anhydride

were

purchased

from

Sigma.

2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I 2959) was purchased from BASF. Fetal bovine serum (FBS), Dulbecco’s modified eagle medium (low glucose) and TrypLETM express enzyme were obtained from Gibco (Life Technologies, USA). Live/Dead Viability/Cytotoxicity kit and PicoGreen dsDNA Assay kit were purchased from Invitrogen. PrimeScriptTM RT Reagent Kit and SYBR® Premix Ex TaqTM were purchased from Takara. BMSCs were isolated according to the previous literatures and passage 4-6 was used in all the experiments.34Rat type I collagen was obtained in our lab. All the other regents were obtained from domestic suppliers and used as received.


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1

H NMR spectra were recorded on a Varian NMR spectrometer at 400 MHz.

WST assay was measured on BiotekCytation 3. The morphology of the hydrogel was observed under scan electron microscope (SEM, Quanta FEG 250). The cellular images were observed by confocal laser scanning microscopy (CLSM, Nikon A1). The storage modulus (G') and loss modulus (G″) of hydrogels were measured using a Haake rotational rheometer (RS6000). RT-PCR was performed on Mastercycler® nexus (Eppendorf).

Synthesis of functionalized gellan gum and collagen Methacrylated gellan gum (MeGG) was synthesized according to the previous literature.35 Briefly, gellan gum (GG, 1g) was dissolved in 100 mL of deionized water at 90 oC until a clear solution was obtained. Then, the GG solution was cooled to 50 o

C, and methacrylic anhydride (3 mL) was added dropwise into the above solution.

Meanwhile, the pH was maintained at 8.0 with NaOH (5.0 mol/L). The reaction was continued for 6 h, and then the modified MeGG solution was purified by dialysis (Mw = 14 kDa), followed by lyophilization and stored at -20 oC. Collagen (300 mg) was stirred in 1% acetic acid solution (60 mL) at room temperature until a clear solution was obtained. Thereafter, the pH of the above solution was adjusted to 7.4, followed by adding tween-20 (545 µL), triethylamine (1.5 mL). Finally, glycidyl methacrylate (1 mL) was added dropwise into the above solution. After stirring overnight, the mixture was precipitated in excess ethanol, and the final product was obtained by centrifugation and lyophilization.36



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Preparation of MeGG/Col and MeGG hydrogels MeGG was dissolved at 2% and 3% (w/v) in PBS under stirring at 70 oC, followed by adding I 2959 (0.5% w/v). Then, the solution was cooled to room temperature, and the functionalized collagen was added. The mixture was sonicated for several minutes until a clear solution was obtained, and the final concentration of collagen was determined to be 1 or 2 mg/mL. Afterward, the obtained solution (100 µL) was injected into the transwell, and then immersed into the CaCl2 (0.1 M) for 1 min. Thereafter, the pre-solidified hydrogel (diameter: 8 mm, thickness: 2 mm) was further reinforced under UV light (365 nm) for another 3 min to obtain MeGG/Col hydrogel. MeGG hydrogel was obtained at the same procedure but without adding collagen, which was used as a control.

Characterization of hydrogels Firstly, the morphology of freeze-dried hydrogel was imaged through SEM after gold sputtering. Afterward, the swelling kinetic of the as-prepared hydrogels were investigated. Briefly, MeGG/Col hydrogel was immersed in 2 mL of PBS at 37 oC under mild shaking. Then, the hydrogel (n=3) was taken out from PBS and the hydrogel surface was quickly blotted by filter paper at different time points. Thereafter, the wet weight of each hydrogel was measured (wt) and compared to the initial wet weight (w0). The swelling ratio (Sr) was defined according to Eq. (1). Sr (%) = (wt - w0)/w0×100


(1)

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After that, the mechanical property of MeGG-Col hydrogel was measured by rheometer with 0.5 N constant pressure. MeGG/Col hydrogel (5 mm thick, 16 mm diameter) was used in this experiment. The storage modules G′ and loss modules G′′ were measured by a Haake rotational rheometer at 0.1% strain frequency sweep (0.1-10 Hz). Finally, in vitro degradation of MeGG/Col hydrogel was also investigated. Weighed hydrogels (w1, n=3) were hydrolytically degraded in 2 mL of PBS at 37 oC for one month, and PBS was replaced every 3 days. The samples were taken out to lyophilize at different time points, and the weight was defined as w2. The remaining ratio (Rk) was defined according to Eq. (2). Rk (%) = w2 / w1 ×100

(2)

BMSCs culture and proliferation within hydrogel WST assay was employed to evaluate the potential proliferation of the hydrogels. Briefly, rat BMSCs were isolated from the bone marrow of 4w-5w SD rats, and cultured

in

low-glucose

DMEM

supplemented

with

12%

FBS,

1%

penicillin/streptomycin. After preparing the hydrogel precursor solution described above, a cell suspension of BMSCs (107 cells/mL) was added and mixed together, followed by solidifying in CaCl2 and UV light. Thereafter, the proliferation of BMSCs-encapsulating hydrogels was performed with WST assay according to the manufacturer’s instruction. In addition, the live/dead status of BMSCs within hydrogel was evaluated through Live/Dead Viability/Cytotoxicity kit at different time



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points, and the images were directly observed under confocal laser scanning microscopy.

BMSCs vasculogenic differentiation The BMSCs-encapsulating hydrogels (100 µL) were prepared as described above and cultured in vesculogeic medium supplemented with 50 ng/mL vascular endothelial growth factor (VEGF), 10 ng/mL basic fibroblast growth factor (bFGF) for 7, 14, 21 and 28 days, and the hydrogel cultured in complete DMEM was set as control. The medium was changed every 3 days. At each time point, the hydrogels were taken out and subjected to RT-qPCR. After taking out from the culture medium, the hydrogels were rinsed with PBS three times. After that, the mRNA expression levels of vasculogenic-related genes were assessed at 7, 14, 21 and 28 days by RT-PCR, including platelet/endothelial cell adhesion molecule 1 (PECAM-1), kinase insert domain protein receptor (KDR), endothelial-specific receptor tyrosine kinase (TEK) and von willebrand factor (vWF). The relative expressions for the target genes were then normalized to that of the calibrator reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers for RT-qPCR are listed in Table 1. Table 1 RT-qPCR primer sequences used in this study. Genes

Prime sequence

Product Length (bp)

Forward primer: 5'-TTCAATGGCACAGTCAAGGC-3' 101

GAPDH Reverse primer: 5'-TCACCCCATTTGATGTTAGCG-3'


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Forward primer: 5'-TAGCGGGATGAAATCTTTGG-3' KDR

207 Reverse primer: 5'-TTGGTGAGGATGACCGTGTA-3' Forward primer: 5'-CCGTGCTGCTGAACAACTTA-3' 201

TEK Reverse primer: 5'-AATAGCCGTCCACGATTGTC-3' Forward primer: 5'-GCTCCAGCAAGTTGAAGACC-3' vWF

163 Reverse primer: 5'-GCAAGTCACTGTGTGGCACT-3'

PECAM

Forward primer: 5'-CGAAATCTAGGCCTCAGCAC-3' 227

-1

Reverse primer: 5'-CTTTTTGTCCACGGTCACCT-3'

Briefly, total cellular RNA extraction from BMSCs-encapsulating hydrogels cultured in non-vasculogenic and vasculogenic media were performed via TRIzol Plus RNA purification kit according to manufacturer’s instruction at each time point, and BMSCs in 6-well plate were used as control. The purity of the RNA was assessed using A260/280 nm. Thereafter, 500 ng of RNA was reverse transcribed into cDNA using PrimeScriptTM RT Reagent Kit. RT-PCR was performed on Mastercycler® nexus using SYBR Green Ⅱ PCR Kit. Passage 4 BMSCs at 0 day was set as the calibrator control and the target gene expression was normalized by non-regulated reference gene expression (GAPDH). PCR cycling parameters: an initial denaturation of 10 min at 95oC followed by 50 cycles of 30 s at 95oC, 30 s at 56oC, and 60 s at 72oC. Data collection was enabled at 72oC in each cycle.

Statistical analysis All the experiment results were reported as mean ± standard deviation for in vitro studies. The statistical data analysis was conducted using Origin Pro 8.5 program



and p < 0.05 was considered statistically significant.

RESULTS AND DISCUSSION Synthesis of MeGG/Col hydrogel In recent years, gellan gum hydrogel holds great potential in tissue engineering, which was usually prepared via physical crosslinking induced by temperature or divalent cations. When the temperature decreases, the gellan molecules undergo a rapid random-coil to double-helix phase transition to form the hydrogel, or when some cations, especially divalent cations are present, the hydrogel can also be formed.26, 37-38 However, the phase transition temperature of gellan gum was too high, which is not suitable for cell survival. Meanwhile, the divalent cations such as Ca2+ or Mg2+ could induce crosslinking of gellan gum, but the crosslinking mediated by ions was not stable due to ion exchange. In this work, we aimed to fabricate cell-encapsulating hydrogel with rapid gelation rate and mild crosslinking condition. As shown in Scheme 1, we took a two-step crosslinking method in this work. First of all, the gellan gum and collagen were modified with vinyl groups, and the gelation point of gellan gum was optimized through changing the grafting ratio of methacrylic anhydride. After that, the hydrogel was treated with low concentration of Ca2+ solution to form the pre-hydrogel rapidly. Finally, the pre-hydrogel was re-solidified under UV light to prevent degradation of ion exchange.


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Scheme 1 Preparation of BMSCs-encapsulating MeGG/Col hydrogel

1

H NMR spectroscopy was carried out to demonstrate whether methacrylic

anhydride or glycidyl methacrylate was successfully grafted onto gellan gum or collagen. As shown in Figure 1. The characterized peaks between δ5.50 ppm and 6.50 ppm suggest that vinyl-group was successfully grafted onto both gellan gun and collagen. The grafting ratio of methacrylic anhydride onto gellan gum is 60%, and the degree of methacrylation for MeGG was calculated by the ratio of average intensity of the methyl proton peaks of the methacrylate groups over the average intensity of the methyl groups of the rhamnose. Meanwhile, we also found that the grafting ratio of methacrylic anhydride onto gellan gum directly influenced the properties of MeGG. For example, when the grafting ratio was as high as 100%, the properties of temperature sensitivity and Ca2+ response diminished or even disappeared. However, when the grafting ratio was lower to 50%, the temperature sensitivity was similar to gellan gum which could form hydrogel rapidly at about 50 oC and thus affect cell



encapsulation. The possible reason was that the steric effects arising from the introduction of methacrylic group may slow down the helix-helix aggregation process and thus reduce the gelation temperature39. Therefore, the grafting ratio of methacrylic anhydride onto gellan gum in this work was defined as about 60%.

Figure 1 A. Synthesis route of MeGG and Col-GMA. B. 1H NMR spectroscopies of MeGG and Col-GMA

Characterization of MeGG/Col hydrogel Scaffold plays a key role in tissue engineering, not only directly supporting the cell adhesion and proliferation, but also affecting cell migration, differentiation. Therefore, the related physical and chemical properties were well performed. Firstly of all, the internal structure of the hydrogels was investigated through SEM. As shown in Figure


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2, we fabricated two kinds of hydrogels for each component, and the w/v ratio was 2% and 3%, respectively. For the GG hydrogel group which was prepared in 0.1 M Ca2+ bath, no porous structure was observed at each ratio (Figure 2A). After modifying with methacrylic anhydride, the MeGG hydrogel was prepared through 0.1M Ca2+ bath (1 min) and UV light (365 nm, 5 min), porous structure was observed (Figure 2A), but the average pore size was about 30 µm, which is too small to rapidly exchange of nutrition and waste. Afterward, the functionalized collagen was introduced to change the pore size. As shown in Figure 2B, when the final concentration of collagen was 1 mg/mL, the pore size was about 50-100 µm. While the final concentration of collagen was 2 mg/mL, the pore size was obviously expanded to about 100-300 µm which maybe offer a more suitable microenvironment for cell adhesion and proliferation. By considering the viscosity, the hydrogel consisting of MeGG (2%) and Col-GMA (2 mg/mL) was chosen for further experiment.



Figure 2 The SEM images of GG and MeGG hydrogels (A), MeGG/Col hydrogels (B).

After that, the degradation rate of MeGG/Col hydrogels was measured in PBS at 37 oC for a month. As shown in Figure 3A, with the time went on, the loss mass was gradually decreased and lost about 25% weight in a month. The differentiation of stem cell usually needs a relative long time, maybe a month or even longer. Hence, the degradation with a proper rate is much suitable for stem cell differentiation.


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Furthermore, the swelling kinetics of MeGG/Col hydrogel in vitro was assessed by immersing the hydrogels in PBS for 24h. As shown in Figure 3B, all the hydrogels was rapidly shrank after immersing into PBS. After immersing into PBS for 1 h, the hydrogel presented a significant deswelling of nearly 13% and reached equilibrium for about 5 h, for about 26%. The presence of ions could increase the double helix formation and the establishment of junction zone, thus leading to the formation of more crosslinked networks.38 Finally, the mechanical property was measured by a Haake rotational rheometer at 0.1% strain-frequency sweep (0.1-10 Hz). For the MeGG/Col hydrogel, the storage modules G' is about 1 KPa. Combined these results obtained above, the MeGG/Col hydrogel could be used for study of vascular differentiation.



Figure 3. A. In vitro hydrolytic degradation of MeGG/Col hydrogel in PBS (pH 7.4) at 37 oC. B. Swelling kinetics of MeGG/Col hydrogel in PBS at 37 oC. C. The storage modulus (G′) and loss modulus (G″) of MeGG/Col hydrogel.

Proliferation of BMSCs encapsulating in hydrogels


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Whether hydrogel offers a suitable microenvironment for cell survival and proliferation is of critical importance. Therefore, the cell viability was prior to be investigated. WST assay was carried out to evaluate the cell survival and proliferation within hydrogels. As shown in Figure 4A, the optical density was about 1.0 in the first three day, but, the optical density rapidly increased to 1.78 at 7th day. Furthermore, live/dead staining was carried out to investigate the live/dead status of BMSCs encapsulated in hydrogels (Figure 4B). The live cells were stained as green fluorescence dots while the dead cells were stained as red fluorescence dots. We could observe many green fluorescence dots at the first three days and a few red fluorescence dots. With time went on, the number of green fluorescence dots significantly increased in comparison with that of the first three days, and very few red fluorescence dots could be observed. This result is quite agreement with the WST assay, and the 3D view of the live/dead status further demonstrated it (Figure 4C). All these results suggest that the short time of ultraviolet irradiation and low concentration of Ca2+ bath had very small impact on cell survival.40 Therefore, the MeGG/Col hydrogel could offer a suitable microenvironment for cell adhesion and proliferation. The excellent biocompatibility is mainly assigned to the addition of collagen in the hydrogel system. Previous work demonstrated that collagen contained specific cell binding sites, particularly the RGD amino acid sequence. The interaction between RGD and integrin receptors on the BMSC surface will stabilize the cytoskeleton onto the surface of scaffold and avoid mechanical detaching and further promote the BMSCs adhesion and proliferation within hydrogels.41-42



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Figure 4. The proliferation (A) and the live/dead status, **p

Bone Marrow-Derived Mesenchymal Stem Cells Encapsulated in

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