Effects of Differentiation and Antisenescence from BMSCs to Hepatocy

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Effects of Differentiation and Antisenescence from BMSCs to Hepatocy-Like Cells of the PAAm-IGF-1/TNF‑α Biomaterial Runcai Yang,†,‡ Lifang Wu,†,‡ Jiehong Chen,†,‡ Wuya Chen,† Lin Zhang,† Li Zhang,† Rong You,† Liang Yin,† Chu-Hua Li,† and Yan-Qing Guan*,†,§ †

School of Life Science, South China Normal University, Guangzhou 510631, China MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China

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S Supporting Information *

ABSTRACT: Aiming at the cells’ differentiation phenomenon and senescence problem in liver tissue engineering, this work is designed to synthesize three different chargeable polymers (polypropylene acid (PAAc), polyethylene glycol (PEG), and polypropylene amine (PAAm)) coimmobilized by the insulin-like growth factor 1 (IGF-1) and tumor necrosis factor-α (TNF-α). We explore the hepatocyte differentiation effect and the antisenecence effect of PSt-PAAm-IGF-1/TNF-α biomaterial which was selected from the three different chargeable polymers in bone marrow mesenchymal stem cells (BMSCs). Our work will establish a model for studying the biochemical molecular regulation mechanism and signal transduction pathway of cell senescence in liver tissue engineering, which provide a molecular basis for developing biomaterials for liver tissue engineering. KEYWORDS: liver tissue engineering, coimmobilized IGF-1 plus TNF-α, BMSCs, antisenescence, hepatocyte differentiation, molecular mechanism

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the proliferation of liver cells.10,11 Moreover, aggressive researchers have reported a series of biomaterials with good mechanical performance and biocompatibility by means of various modification methods,12−14 which on the other hand push forward the frontiers of knowledge of the signal transduction between biomaterial and liver cell.15,16 However, so far little work on the lifetime issue of liver cells has been available, although the source and amplification of liver cells are addressed well. It is known that after cell planting, the lifetime issue becomes particularly critical for constructing tissue engineering due to a variety of cell deaths. A large area of cell death may result in serious calcification.17 The most direct reason is believed to be the low vitality and short lifetime of the seeding cells as well as the toxicity of the biomaterials degradation. In order to solve this problem, a promising strategy is to make use of the cell senescence characteristic in liver tissue engineering so that the cell−biological mechanisms can be explored comprehensively, noting that such a strategy seems to be of much less concern in the literature on liver tissue engineering.

INTRODUCTION The liver is one of the most important organs of our body, but a variety of liver diseases seriously affect people’s health.1 Liver transplantation is currently the best way to solve liver failure and liver cancer, and liver tissue engineering is a creative way to build a new liver which can be implanted.2 In fact, researchers have synthesized the natural liver in vivo and built the tissueengineered artificial liver system with certain functions and viability.3,4 However, due to the complexity of physiological function and structure of liver tissue, roadmaps toward a largescale application of liver tissue remain challenging. Given this state of the art, a comprehensive understanding of the function and molecular regulation of the liver cells is highly appealing in order to construct a functional and transplantable tissueengineered liver. Along this line, a large amount of research focusing on liver cells, cytokines, and scaffolds has been reported, including extensive investigations on adhesion, proliferation, differentiation, and apoptosis of various liver cells induced by biomaterials.5−7 Meanwhile, a proper choice of suitable growth factor remains to be one of the key challenges in liver tissue engineering. It is noted that tumor necrosis factor (TNF) as positive regulator of cytokine is involved in the angiogenesis process.2,8,9 Although insulin will not directly promote liver cell division, it can cooperate with other growth factors to enhance © XXXX American Chemical Society

Received: August 18, 2016 Accepted: September 26, 2016

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DOI: 10.1021/acsami.6b10377 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Schematic diagram showing (A) the synthesis of photoactive IGF-1 and TNF-α and (B) the preparation of biomaterials.

pathway provides an opportunity to control the cellular senescence. Similarly, the Rb was also found to be activated by another CDK inhibitor p16 by suppressing the cyclin D/ CDK4,6 complex, disclosing the similar final common pathways for irreversible growth arrest. On the other hand, partially in order to overcome the toxicity issue of biomaterials in tissue engineering, surface modification technology remains to be the common employed approach. The photochemical surface modification has been well demonstrated to be efficient and highly preferred,27−29 not only for functional activity consideration but also for its biological compatibility. In our earlier works,30−35 we developed various approaches of photoimmobilization to fix TNF-α/IFN-γ onto polystyrene, polyurethane, and other biomaterials. This technique was proved simple and efficient, and consequently, a series of investigations on the human gynecologic cancer HeLa, OVCAR-3, and/or MCF-7 cells death regulations were carried out. These approaches provide a feasible and efficient platform on which the underlying

It is noted that cellular senescence can be induced by DNA damage, oxidative stress, antineoplastic drugs, and other external stimulus, which might lead to the senescence-like growth arrests.18−20 Conventional research revealed two major cell signal transduction pathways regulation roadmaps: p53/ p21 pathway and/or p16 pathway, although many different stimulating factors were shown to be effective in inducing the cellular senescence.21,22 In detail, the tumor suppressor proteins, p5323,24 and Rb, 25,26 either concurrently or independently, regulating the onset of cellular senescence. The two types of proteins were frequently found to lose their genes in cancer cells, allowing the unlimited proliferation. In response to those stimuli which lead to the cellular senescence, phosphorylated and stabilized p53 is able to activate its target genes including the cyclin-dependent kinase inhibitor p21 gene. The protein product of the p53 pathway can then activate the Rb via inhibiting a cyclin-dependent kinase complex (cyclin E/ CDK2). It was also observed that the hypo-phosphorylated Rb can inhibit the transcription of E2F target genes which arrest the cells in the G1 phase of the cell cycle. This regulation B


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Figure 2. Measured zeta potential of AzPhPAAc, AzPhPEG, and AzPhPAAm (A). Measured Raman spectra of the modified PSt-AzPhPAAc-IGF-1/ TNF-α, PSt-AzPhPEG-IGF-1/TNF-α, and PSt-AzPhPAAm-IGF-1/TNF-α (B).

photoimmobilization method, so as to prepare three types of polymer-based functional biomaterials. A schematic drawing of the whole procedure is given in Figure 1. Subsequently, a series of microstructural and biological characterizations will be performed in order to reveal the impact of these functional biomaterials on the cellular senescence and their molecular regulation mechanisms on hepatic stellate cells (HSCs). In addition, the NH2/positively charged PAAm-IGF-1/TNF-α biomaterial, which has the best antisenescence effect, is proved to have a positive influence during the differentiation of BMSCs to hepatocytes and also BMSCs’ cellular senescence. It is known that the gene regulation aspect of cell senescence issue in liver tissue engineering will provide important experimental and theoretical basis for uncovering the cellular senescence signaling pathways of seed cells and designing promising biomaterials with novel molecular mechanisms.

mechanisms of the cellular senescence in the circumstance of tissue engineering with biomaterials can be investigated. For the cell for liver tissue engineering, mesenchymal stem cells (MSCs) are multipotent progenitor cells characterized by their ability to both self-renew and differentiate into tissues of mesodermal origin included osteoblasts, adipocytes, and chondrocytes myocytes.36 Animal bone marrow mesenchymal stem cells (BMSCs) are the most common source of MSC for clinical and research use. BMSCs are directly isolated from bone marrow aspirates based on their ability of adhering to plastic when plated in monolayer culture and thereafter replicate ex vivo to form a phenotypically homogeneous population of cells. The plasticity or transdifferentiation potential of MSCs is not limited to mesenchymal derivatives, since under appropriate cell culture conditions and stimulation by certain exogenous or endogenous bioactive factors, BMSCs have also been differentiated into endodermal (hepatocytes) and neuroectodermal (neurons) cells.37,38 In this paper, we start from three types of polymers with different functional groups and chargeable properties on the surface: polypropylene acid (PAAc), polyethylene glycol (PEG), and polyacrylamide (PAAm). The surfaces of these polymers will be coimmobilized with insulin-like growth factor 1 (IGF-1) plus tumor necrosis factor-α (TNF-α) using the

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EXPERIMENTAL SECTION

Preparation of Three Different Chargeable Biopolymers. The preparation of photoactive PAAc, PEG, PAAm, IGF-1, and TNFα was described previously,28,30 as shown in Figure 1. All treatments were carried out in the dark. Then the prepared AzPhIGF-1 and AzPhTNF-α at a dose of 10 ng/well were separately added into the 24 tissue culture plates which were immobilized with AzPhPAAc, C


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Figure 3. (A) X-ray photoelectron spectrometer (XPS or ESCA) for chemical analysis was used to analyze three chargeable polymers and coimmobilized IGF-1 plus TNF-α three chargeable polymers. (B) Surface morphology and microstructure were investigated by atomic force microscopy (AFM).

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AzPhPEG, and AzPhPAAm, respectively, with detailed procedure described in ref 30, followed by drying the plates with AzPhIGF-1 and AzPhTNF-α in air at 4 °C. These plates were irradiated with the UV light (15 W) for 20 min at a distance of 2 cm. Due to the highly active azido group, AzPhIGF-1 and AzPhTNF-α can be successfully coimmobilized onto the polystyrene culture plates, constituting the as-prepared biomaterials. These plates were then thoroughly washed with PBS(−) (pH 7.4) 30 times and then stored at 4 °C. Cell Culture. The hepatic stellate cells (HSCs) obtained from the Sun Yat-Sen University were subcultured on the plastic tissue culture dishes in RPMI1640 medium supplemented with 10% FCS, 100 μg/ mL penicillin, and 100 μg/mL streptomycin in a humidified 5% CO2 atm at 37 °C. Two sets of HSCs (1 × 105 cells/well) were seeded in the 24-well cell culture polystyrene plates and then treated by the free IGF-1 plus TNF-α and the coimmobilized IGF-1 plus TNF-α for given time periods (24 h). Then the SA-β-gal staining was performed as described.39 Bone marrow mesenchymal stem cells (BMSCs) obtained from the Sun Yat-Sen University were subcultured on the plastic tissue culture dishes in Dulbecco’s modified eagle medium (Gibco, USA) supplemented with 10% fetal bovine serum (Cellgro, USA), 100 μg/ mL penicillin, and 100 μg/mL streptomycin in a humidified 5% CO2 atmosphere at 37 °C. Statistical Analysis. Statistical analyses were conducted using SPSS17.0. One-way analysis of variance (ANOVA) was used to analyze differences between groups under different conditions. Results were analyzed by the Student’s t test. AP values less than 0.05 were considered statistically significant. For further details, refer to the Supporting Information.

RESULTS Characterization of the Biomaterials. The zeta potentials of three different chargeable biomaterials are showed in Figure 2A. The zeta potentials of both pure PAAc and AzPhPAAc were negative, about −15 and −10 mV, respectively. This suggested PAAc was negatively charged and the quantity of electric charge of AzPhPAAc was diminished by immobilization with 4-azidoaniline hydrochloride. On the contrary, the zeta potentials of both pure PAAm and AzPhPAAm were positive, about 20 and 10 mV, repectively, which means the PAAm carries a positive charge while the quantity of electric charge of AzPhPAAm was decreased. However, the zeta potentials of the PEG and AzPhPEG were about 0 mV, indicating they carried no charge. Additionally, the azidophenyl groups in the AzPhPAAc, AzPhPEG, AzPhPAAm, AzPhIGF-1, and AzPhTNF-α were characterized by Fourier transform infrared spectroscopy (Figure S1). To detect the chemical structure the surface-modified polymeric plates, we used Raman spectra and electron spectroscopy (ESCA) (shown in Figures 2B and 3A). The result of Raman spectra (Figure 2B) shows PSt-AzPhPAAcIGF-1/TNF-α exhibits the C−O stretched at 1137 cm−1 compared with PSt-AzPhPAAc. For the PSt-AzPhPEG-IGF-1/ TNF-α biomaterial, the amide bonds stretched at 3070 cm−1 while the PSt-AzPhPEG exhibits the C−N at 1081 cm−1. It suggests that the photoimmobilization breaks the C−H of the D


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Figure 4. Efficient influence of hepatocyte differentiation of BMSCs induced by NH2/positively charged polymer biomaterials. (A) PAS staining for glycogen deposits in BMSCs during the hepatocyte differentiation. Bar: 50 μm. (B) Immunocytochemical analysis of ALB protein expression in BMSCs during the hepatocyte differentiation. Bar: 50 μm. (C) RT-PCR and qPCR analysis of mRNA of AFP gene in BMSCs during the hepatocyte differentiation. (D) Measured urea products in BMSCs during hepatocyte differentiation. Significant increases at (*) p < 0.05, (**) 0.01 < p < 0.001, and (***) p < 0.001 in comparison with the free group are identified. Only one of three representative experiments is shown here. (E) Protein expression and quantitative analysis of NF-κB, IL-6, K-ras, and STAT3 expressions in BMSCs. Protein expression in 48 h was determined using BandScan software.

also checked. The result shows the highest grafting ratio, i.e., 100%, further demonstrating the complete drug loading and high grafting efficiency on the PSt-PAAc, PSt-PEG, and PStPAAm surfaces. Furthermore, we employed atomic force microscopy (AFM) to observe the morphology of the IGF-1/TNF-α layer (Figure 3), which shows the peak height, average rough (Ra), and surface coverage of the coimmobilized IGF-1 plus TNF-α. For PSt-AzPhPAAc-IGF-1/TNF-α, the peak height is 23.68 nm with Ra of 3.49 nm. For PSt-AzPhPEG-IGF-1/TNF-α, the peak height is 24.1 nm with Ra of 3.55 nm. For PSt-AzPhPAAmIGF-1/TNF-α, the peak height is 24.97 nm with Ra of 2.03 nm.

PSt-AzPhPEG and the IGF-1 or TNF-α bonding which are the sources of amide bonds. Similar to PSt-AzPhPEG-IGF-1/TNFα, the amide bond and C−N bond appear in the spectrum at 1081 cm−1. Compared with PSt-AzPhPAAm, in the spectrum of PSt-AzPhPAAm-IGF-1/TNF-α biomaterial, the peak of the N− H bond was moved to 1603 cm−1. The remarkable change in Raman spectra demonstrates the successful attachment of IGF1 and TNF-α onto the polymeric substrates. In Figure 3A, the ESCA survey scans analysis of chemical bonding shows that the C−N is enhanced up to 14.72%, 12.10%, and 16.20% from 11.45%, 5.95%, and 0%. The grafting ratios of AzPhTNF-α and AzPhIGF-1 for the three kinds of prepared biomaterials were E


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Figure 5. Effect of antisenescence of BMSCs induced by NH2/positively charged polymer biomaterials during hepatocyte differentiation (PStAzPhPAAm, PSt-AzPhPAAm-IGF-1, PSt-AzPhPAAm-TNF-α, and PSt-AzPhPAAm-IGF-1/TNF-α biomaterial). (A) SA-β-Gal staining of BMSCs cultured on the prepared biomaterials during hepatocyte differentiation. Bar: 50 μm. (B) DAPI staining of BMSCs during hepatocyte differentiation. Bar: 200 μm. (C) Cell viability analysis of BMSCs during hepatocyte differentiation. (D) ROS assay of BMSCs during hepatocyte differentiation. (E) RT-PCR and qPCR analysis of mRNA of SOD gene in BMSCs during the hepatocyte differentiation. Significant increases at (*) p < 0.05, (**) 0.01 < p < 0.001, (***) p < 0.001 in comparison with the free group are identified. Only one of three representative experiments is shown here.

The formation of different peak heights and the protein particles on the surface suggest the IGF-1 plus TNF-α is crosslinked with the PSt-AzPhPAAc, PSt-AzPhPEG, and PStAzPhPAAm. PAAm-IGF-1/TNF-α Biomaterial Inhibits Cell Senescence in HSCs. In order to clarify the cell senescence evolution on the prepared biomaterials, we first employed HSCs as our cell model. What we found is that NH2/positively charged PSt-AzPhPAAm-IGF-1/TNF-α has the highest inhib-

ition effect of cell senescence (Figures S2−S5). As a result, we chose this biomaterial for following a study on whether it is positively influenced by the differentiation of BMSCs to hepatocytes. Considering the signal cytokine (IGF-1 or TNF-α) immobilized on the PSt-AzPhPAAm, we did four tests of biomaterial groups including PSt-AzPhPAAm, IGF-1 immobilized PSt-AzPhPAAm (PSt-AzPhPAAm-IGF-1), TNF-α immoF


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differentiate into hepatocytes triggered by NH2/positively charged PSt-AzPhPAAm-IGF-1/TNF-α biomaterial. PAAm-IGF-1/TNF-α Biomaterial Plays a Role in Antisenescence of BMSCs Differentiated into Hepatocytes. To study the effect of antisenescence of BMSCs on our biomaterials, we employed SA-β-gal, DAPI staining. In Figure 5A, results indicated that PSt-AzPhPAAm-IGF-1/TNF-α inhibited senescence in BMSCs effectively. Blue stain of SAβ-gal can only be observed in PSt-AzPhPAAm biomaterial and PSt-AzPhPAAm-IGF-1 biomaterial after culturing for 7 days. Indeed, senescence was exhibited in PSt-AzPhPAAm-TNF-α group, since senescence-associated heterochromatin foci (SAHF) occurred in all PSt-AzPhPAAm, PSt-AzPhPAAmIGF-1, and PSt-AzPhPAAm-TNF-α biomaterials after day 7 (Figure 5B). Moreover, cell viability was assessed by the MTT method. All of the BMSCs’ cell viability was reduced significantly when incubation in differentiation medium at 7 days and then began to go up gradually. Relatively, BMSCs’ cell viability grown on PSt-AzPhPAAm-IGF-1/TNF-α biomaterial trended to be higher than the control’s (Figure 5C). Moreover, we examined the intracellular ROS level with DCF-DA tab analysis. We found that BMSCs elicited a rapid ROS generation at day 7 and day 14 during the differentiation and decrease at day 21 (Figure 5D). However, BMSCs cultured on PSt-AzPhPAAm-IGF-1/TNF-α biomaterial displayed the ability of relieve the products of ROS compared with those cultured on PSt-AzPhPAAm which had a statistical significance labeled by symbol *(p < 0.05). Furthermore, the expression of the SOD mRNA was corresponding to the result of ROS (Figure 5E). At the differentiated protocol of day 21, the SOD mRNA level of BMSCs cultured on PSt-AzPhPAAm-IGF-1/ TNF-α was significantly increased compared with the control group (labeled by symbol ***, p < 0.001).

bilized PSt-AzPhPAAm (PSt-AzPhPAAm-TNF-α), and PStAzPhPAAm-IGF-1/TNF-α for the following research. PAAm-IGF-1/TNF-α Biomaterial Promotes BMSCs Differentiate into Hepatocytes. To identify whether the BMSCs differentiated into functional hepatocytes, we detected the hepatocellular molecule such as albumin (ALB) and glycogen products (Figure 4A and 4B). The result of immunocytochemical staining showed that the differentiated BMSCs cultivated on the PSt-AzPhPAAm-IGF-1/TNF-α biomaterial expressed more ALB since day 14. Furthermore, the intense PAS staining associated with stored glycogen was observed in differentiated BMSCs culture on the coimmobilized biomaterials at day 7, which in the control group was observed at day 14. Apparently, PSt-AzPhPAAm-IGF-1/TNF-α biomaterial can induce BMSCs to differentiate into mature hepatocytes. Total RNA was isolated at days 1, 7, 14, and 21 after differentiation of the BMSCs into hepatic lineage, and the expressions of several hepatic genes were examined by reverse transcription-PCR (RT-PCR) and a real-time quantitative pcr detecting system (qPCR). Undifferentiated BMSCs did not express alpha fetoprotein (AFP) mRNA (Figure 4C). During hepatic differentiation culture, the expression of AFP mRNA was increased at day 7. The relative amount of AFP mRNA transcribed by BMSCs cultured on PSt-AzPhPAAm biomaterial was decreased at day 21 and kept high level transcription of AFP mRNA on PSt-AzPhPAAm-IGF-1/TNF-α biomaterial. To compare the level of AFP mRNA expression in differentiated BMSCs cultured on PSt-AzPhPAAm biomaterial and PStAzPhPAAm-IGF-1/TNF-α biomaterial, we quantified the AFP mRNA products relative to GAPDH products. The result of qPCR is in accordance to RT-PCR. On PSt-AzPhPAAm biomaterial, BMSCs transcribe AFP mRNA between day 7 and day 14 and return to a low level at day 21. On PSt-AzPhPAAmTNF-α biomaterial and PSt-AzPhPAAm-IGF-1/TNF-α biomaterial, the levels of AFP mRNA transcription in BMSCs are upregulated steadily about 8 times at day 21 compared with day 1. The results of urea assay showed undifferentiated BMSCs did not produce urea. After induction of differentiation protocol, urea can be examined (Figure 4D). That of BMSCs culture on PSt-AzPhPAAm biomaterial for 7 days was 39.22 μg/L, while that of BMSCs culture on PSt-AzPhPAAm-IGF-1/-TNF-α biomaterial was 71.61 μg/L, making a statistically significance (p < 0.05) between these groups. At day 21, the products of urea of BMSCs cultured on PSt-AzPhPAAm-IGF-1/TNF-α biomaterial have an much higher significance labeled by symbol *** (p < 0.001) compared to BMSCs culture on PStAzPhPAAm biomaterial. Next, the expression of the proteins associated with cell differentiation was detected by Western blotting (Figure 4E). At day 21, the expression of NF-κB, K-ras, IL-6, and STAT3 in PSt-AzPhPAAm-IGF-1/TNF-α was upregulated compared with other groups. Specifically, expression of IL-6 was increased ∼40% in the PSt-AzPhPAAm-IGF-1/TNF-α group, while there were no significant changes when only IGF-1 or TNF-α was modified on the materials. Alsp, STAT3 was increased by nearly 100% in this group at the same time. More distinctions of this upregulated trend occurred in the expression of K-ras and NFκB between different groups. They climbed ∼1.5 times in the PSt-AzPhPAAm-IGF-1/TNF-α group compared to the control group. These results indicate a promotion of BMSCs

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DISCUSSION Hepatic tissue engineering, an innovative way to construct an implantable liver, has the potential to alleviate the organ donor need. On one hand, many new polymers that respond to thermal changes and release imbedded or attached growth factors/other mediators are being developed. They have degradation characteristics and properties ideal for growth, viability, and attachment. On the other hand, for cell growth in the tissue-engineering field, many ethical considerations are recognized. The important issues include which cell source is safe for patients, which kind of cells should be used, whether they are embryonic stem cells or oval progenitors, and how the cells should be stored and cultured.40,41 Actually, liver tissue engineering is a multidisciplinary field including but not limited to polymer chemistry, polymer physics, developmental biology, and biomedical engineering.42 However, to date, the cellular biology mechanisms for hepatic tissue engineering are largely unknown. Among these mechanisms, the cellular senescence43,44 was described more than four decades ago when Hayflick and colleagues showed that normal cells have a limited ability to proliferate in culture. Soon after this discovery one of two important hypotheses stemmed from the fact that tissue regeneration and repair can deteriorate with age. The cellular senescence was proposed to recapitulate the aging or regenerative capacity loss of cells in vivo. In this context, the cellular senescence was considered deleterious because it contributes to decrements in tissue renewal and function.45 However, to the best of our knowledge, G


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ACS Applied Materials & Interfaces the cellular senescence may never be touched in the hepatic tissue engineering studies. Although further investigations to elucidate all the mechanisms clearly, including inhibition of the p53/p21 pathway and (or) p16 pathway which regulate the high expression of CDK4 induced by coimmobilized IGF-1 plus TNF-α, are needed,39,46−48 we may present a generic mechanistic model for the induced cell senescence, as shown in Figure S5. Our data presented here are very helpful for illustrating how the inhibition of cellular senescence was induced by different chargeable polymers with IGF-1 plus TNF-α synergism. Due to the fact that the cellular senescence inhibited by biomaterials is a key event in determining cell growth and survival,49−54 our studies may uncover some novel mechanisms, whereby used antisenescence biomaterials can enhance cell lifetime. These findings may complement the use of free IGF-1 plus TNF-α that specifically inhibits Ink4 family or/and Cip/Kip family in promoting cell growth and survival for liver tissue engineering. (For further details, refer to the Supporting Information.) In addition, by the antisenescence studies of three kinds of biomaterials, we considered that the PSt-AzPhPAAm-IGF-1/ TNF-α biomaterial is the best scaffold for the following research of BMSCs differentiation and antisenescence. As part of the interleukin-6 (IL-6) family, OSM was the key factor to mediate the mature hepatocyte differentiation of BMSCs which activated the signal transducer and activator of transcription (STAT3).55 It is reported that nuclear factor-κB (NF-κB) promotes products of IL-6 which play an important role in early liver regeneration by the Jak/STAT3 pathway.56,57 NF-κB is a dimeric transcription factor which is robustly activated by TNF-α and has pleiotropic effects in a wide variety of cellular programs. Although the role of TNF-α signaling in inflammation and apoptosis has been well described, TNF-α is also capable of stimulating hepatocyte proliferation.58,59 In our study, the immobilized TNF-α may combine with the receptor to activate the NF-κB signal pathway to promote the BMSCs to differentiate into hepatocytes. The deeper mechanism will be focused on in our further study. In addition to the cytokines, cell−cell contact may induce differentiation signals. The IGF-1 and TNF-α coimmobilized on the PSt-AzPhPAAm play a role in the antisenescence which stimulates the proliferation of BMSCs. During the long-term differentiation protocol, the effect of antisenescence of the PStAzPhPAAm-IGF-1/TNF-α may promote the differentiation of BMSCs via the influence of cell−cell communication. We present a generic mechanistic model for the regulation of differentiation and cell senescence in BMSCs by the prepared biomaterials as shown in Figure 6.

Figure 6. Schematic representation of the proposed mechanism on the regulation of differentiation and cell senescence in BMSCs by the prepared biomaterials.

mechanism and signal transduction pathway of cell senescence in liver tissue engineering.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b10377. Detailed experimental section; synthesis of AzPhPAAc, AzPhPEG, AzPhPAAm, AzPhIGF-1, and AzPhTNF-α; coimmobilized IGF-1 plus TNF-α enhances hsc cell survival; coimmobilized IGF-1 plus TNF-α biomaterials inhibit cell senescence in HSCs; coimmobilized IGF-1 plus TNF-α biomaterials inhibit cell cycle G1 arrest in HSCs; schematic representation of the proposed mechanism on the regulation of antisenescence in HSCs by the prepared biomaterials (PDF)

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CONCLUSION In summary, we demonstrated the preparation, physical and chemical characteristics, and molecular mechanism of antisenescence regulation to hepatic stellate cells induced by three different chargeable polymers coimmobilized with IGF-1 plus TNF-α. As a result, the NH2/positively charged PStAzPhPAAm-IGF-1/TNF-α biomaterial displayed the best effect of antisenescence in both HSCs and BMSCs. More specifically, it is not only the protection from cell senescence but also the promotion for the hepatocyte differentiation of BMSCs by this biomaterial that we discovered. Our work thus establishes a model for studying the biochemical molecular regulation

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AUTHOR INFORMATION

Corresponding Author

*Tel.: (+86-20)85211241. E-mail: [email protected]. Author Contributions

Y.-Q. G. conceived and designed the experiments. R.Y. (Runcai Yang), L.W., W.C., L.Z. (Lin Zhang), and L.Y. carried out the characterization experiments. R.Y. (Runcai Yang), L.W., J.C., L.Z. (Li Zhang), and R.Y. (Rong You) did the in vitro experiments of BMSCs. R.Y. (Runcai Yang), L.W., J.C., L.Z. (Lin Zhang), and C.-H.L. did the in vitro experiments of HSCs. H


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W.C. carried out the art works. L.W., W.C., and Y.-Q.G. cowrote the paper. All authors contributed to analyzing and reviewing the data in this manuscript. Author Contributions

‡ R.Y., L.W., and J. C.: These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

The authors acknowledge the financial support by the National Natural Science Foundation of China (31170919, 31370967), the Science and Technology Planning Project of Guangdong Province, China (No. 2015A020212033), the Guangdong Province Universities and Colleges Pearl River Scholar Fund Scheme (2014), China, and the Innovative Entrepreneurial Training for college students of South China Normal University (No.201610574025), China.

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