A Biomimetic Poly(vinyl alcohol)âCarrageenan Composite Scaffold

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A biomimetic poly (vinyl alcohol)-carrageenan composite scaffold with oriented microarchitecture Yabin Zhang, Lei Ye, Jing Cui, Boguang Yang, Hong Sun, Junjie Li, and Fanglian Yao ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00535 • Publication Date (Web): 22 Mar 2016 Downloaded from http://pubs.acs.org on March 27, 2016

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ACS Biomaterials Science & Engineering

A biomimetic poly (vinyl alcohol)-carrageenan composite scaffold with oriented microarchitecture Yabin Zhang †, Lei Ye †, Jing Cui ‡, Boguang Yang †, Hong Sun*, ‡, Junjie Li*, § and Fanglian Yao*, †,⊥

†

School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.

‡

Department of Basic Medical Sciences, North China University of Science and Technology,

Tangshan 063000, China. ⊥

Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin University,

Tianjin 300072, China §

Department of Advanced Interdisciplinary Studies, Institute of Basic Medical Sciences and

Tissue Engineering Research Center, Academy of Military Medical Science, Beijing 100850, China.

*Corresponding author at: 1.School of Chemical Engineering and Technology, Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin University, Tianjin 300072, China. Tel.: +86-22-27402893; Fax: +86-22-27403389 E-mail address: [email protected] (Fanglian Yao)

2. Department of Basic Medical Sciences, North China University of Science and Technology, Tangshan 063000, China. Tel.: +86-315-3725740; Fax: +86-315-3726552 E-mail address: [email protected] (Hong Sun)

3. Department of Advanced Interdisciplinary Studies, Institute of Basic Medical Sciences and Tissue Engineering Research Center, Academy of Military Medical Science, Beijing 100850, China. 1

ACS Paragon Plus Environment


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Tel.: +86-10-68166874; Fax: +86-10-68166874 E-mail address: [email protected] (Junjie Li)

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ABSTRACT: In general, the design of the scaffold should imitate certain advantageous properties of native extracellular matrix (ECM) in order to operate as a temporary ECM for cells. From this aspect, a biomimetic scaffold was prepared by using poly (vinyl alcohol) (PVA) and carrageenan (CAR), in which axially oriented pore structure can be formed through a facile unidirectional freeze-thaw method. We examined the feasibility of this oriented scaffold, which has better physico-chemical properties compared to non-oriented scaffold fabricated by conventional method. The microenvironment of this oriented scaffold could imitate biochemical and physical cues of natural cartilage ECM for guiding spatial organization and proliferation of cells in vitro, indicating its potential in cartilage repair strategy. Furthermore, the biocompatibility of the scaffold in vivo was demonstrated in a subcutaneous rat model, which revealed uniform infiltration and survival of newly formed tissue into oriented scaffold after 4 weeks, with only a minimal inflammatory response being observed over the course of the experiments. These results together indicated that the present biomimetic scaffold with oriented microarchitecture could be a promising candidate for cartilage tissue engineering. KEYWORDS: poly (vinyl alcohol); carrageenan; oriented scaffold; tissue engineering

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1. INTRODUCTION Tissue engineering, a field applying the principle and technology of life science and engineering, has good prospects in treatment of diseases and provides opportunities to develop biological substitutes that can repair, maintain, and promote the function of tissues and organs.1 Since tissue structure plays an integral role in maintaining tissue function, the scaffold should imitate certain advantageous properties of native extracellular matrix (ECM) to provide suitable microenvironment for guiding cell growth and thus promote tissue growth.2-3 Cartilage tissues possess complex architectures with depth-dependent collagen content and fibril orientation.4 For example, the orientation of collagen fibers in deep zone is perpendicular towards the joint surface.5 The special longitudinal oriented structure is closely related to the mechanical and physiologic properties of the tissue,6-8 which plays a crucial role in load transfer and energy displacement within the joint.9 In this context, the design of scaffolds with these intricate architectural characteristics would be an important target for the research of cartilage tissue engineering.10 Hydrogels have been proven promising as stand-alone tissue scaffolds for a variety of applications in tissue engineering. Because they resemble the native ECM,11 and could operate as penetrable matrix for the diffusion of soluble substances.12 In particular, the freeze-thawed poly (vinyl alcohol) (PVA) hydrogels stand out because of their attractive features such as processing ease, high hydrophilicity and tissue-like mechanical strength.13-15 Moreover, they can be considered as biocompatible in nature and are non-irritating to soft tissues when in contact with them.16 Therefore, PVA hydrogels have been extensively investigated for the applications in tissue engineering, especially cartilage,17-19 as well as drug

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delivery carriers.20-21 In recent years, a promising and novel technique, called solvent unidirectional freezing method, was used to create oriented structure in hydrogel, where the pore structure is templated from the aligned solvent crystals.22-23 For example, Del Monte et al. demonstrated the ability of unidirectional freezing for preparation of monolithic PVA scaffolds with a microchanneled structure for drug-delivery purposes.24 From an applicative point of view, PVA hydrogels fabricated by unidirectional freezing possess architecture with aligned pore structure, mimicking the structural anisotropy in native cartilage, which will be promisingly biomimetic scaffolds. However, the inability of cell adhesion because of their resistance to protein adsorption is a disadvantage existing in PVA hydrogels, which has restricted their applications in tissue engineering.25-27 Recently, polysaccharides are attractive materials due to some excellent properties like desirable biocompatibility and extensive bioactivities.28-30 Therefore, PVA can be combined with polysaccharides to support cell adhesion and proliferation. As a kind of sulfated polysaccharides, carrageenan attracts more attention due to its mild gelation property. Most important, the structure of carrageenan is similar to the natural glycosaminoglycans (GAGs),31 which makes it a potential candidate for tissue engineering.32-33 Previous study in our lab described a composite hydrogel for enhancing cell adhesion ability of PVA hydrogel through incorporating of carrageenan (CAR) in a single step.34 Nevertheless, a suitable scaffold should not merely imitate composition of the native tissue, and structural bionic design is necessary. The respect effect of biomimetic pore structure in PVA-CAR scaffold on cell growth has not been extensively studied, as well as the tissue-scaffold interaction in vivo of such scaffold needs to be further investigated. 5



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In view of the above circumstances, a worthwhile endeavor would be to construct biomimetic scaffold by using hydrophilic PVA and CAR, in which axially aligned pore structure can be formed through a unidirectional freeze-thaw method. For the purpose of investigating the effect of microstructure on physico-chemical properties of scaffolds, the ordinary scaffold (denoted as non-oriented scaffold) was prepared by traditional freeze-thaw method and served as a control group. Moreover, the cell behaviors in both scaffolds were investigated in detail. Then, assessing performance in vivo was used to determine their suitability for tissue engineering.

2. MATERIALS AND METHODS 2.1. Materials PVA (77000g/mol, 97% hydrolysed) was obtained from Guangfu Fine Chemical Co., Ltd (Tianjin, China). ι-Carrageenan was purchased from Kasei Kogyo Co., Ltd (Tokyo, Japan). All other chemicals were of analytical purity and used without further treatment or purification. 2.2. Scaffolds preparation 4 g of PVA powder was dissolved in 50 g of distilled water to formation transparent solution. 1 g of CAR was added to the PVA solution by vigorously stirring under reflux at 90 °C for 6 h. Then the mixed solution was cooled to 80 °C and settled to remove the bubbles generated from mechanical stirring for 2 h. After ensuring homogeneity, this solution was poured into a polypropylene tube with an inner diameter of 10 mm and a height of 30 mm. Subsequently, the tube was vertically placed onto the surface of a metal plate, which was 6


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rested on the top of liquid nitrogen. Due to the presence of a uniaxial temperature gradient, this mixed solution underwent unidirectional freeze from the bottom to the top. The frozen sample was then thawed for 6 h at ambient temperature. Such unidirectional freeze and thaw process was repeated up to 5 times. Finally, the sample was then lyophilized for 48 h using a freeze-dryer (LGJ10-C, China) to obtain anisotropic scaffold with oriented pore structure (denoted as oriented scaffold). As a contrast, the non-oriented scaffold was prepared in a typical experiment. In brief, the above mixed solution was subjected to freeze at -20 °C and subsequently thaw at room temperature. The sample was also further lyophilized for 48 h. Finally, both types of the dried scaffolds were preserved in a desiccator for further experiments. 2.3. Characterization of scaffolds 2.3.1. Morphology observation After fracturing in liquid nitrogen, the scaffolds were mounted on the sample holder and sputtered coating with a layer of gold. Then, the morphology observations of scaffolds were performed under an S-4800 scanning electron microscope (SEM, Hitachi, Japan). 2.3.2. X-ray diffraction analysis Prior to examination, the scaffolds were ground into powders. The samples were analyzed using a D8-Focus X-ray diffractometer (Bruker, Germany) at 36 kV and 200 mA. The Cu Kα radiation was operated in the range of 2θ from 10 to 60° and the scanning rate was 6°/min. The degrees of crystallinity (Xc) of the scaffolds were estimated according to the literature 35 and calculated with the equation described below:

χ c (%)=

A1 × 100 A2

(1) 7



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where A1 is the area of the crystalline region in the range of 2θ from 18 to 21°, and A2 is the area towards X-ray scattering from the whole region. 2.3.3. Porosity The porosity was determined using the ethanol displacement method.36 In brief, the scaffold was placed into a certain volume of ethanol (V1). Subsequently, vacuumizing was carried out in order to force the ethanol into internal pores of the scaffold and the total volume is V2. After the scaffold was taken out, the remnant volume of ethanol is V3. The porosity was calculated as follows: Porosity (%) =

V1 -V3 ×100 V2 -V3

(2)

2.3.4. Swelling test The dried scaffolds were first weighted (initial weight, W0) and then immersed into PBS with a pH 7.4 at 37 °C. After the weight of swollen scaffold became constant (final weight, W), equilibrium swelling ratio (ESR) was calculated as follows:

ESR =

W-W0 × 100% W0

(3)

The swelling kinetics was also investigated by the gravimetric method. At predetermined time intervals, the sample was taken out from PBS, wiped dry with filter paper on the surface and weighted. The swelling ratio at time t was calculated using the following equation:

Swelling ratio =

Wt -W0 × 100% W0

(4)

where W0 and Wt are the weight of the initial dry and the swollen scaffolds at predetermined time t, respectively. The experimental data were obtained from triplicate samples. 2.3.5. Mechanical behavior 8


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The compressive measurements were carried out on the scaffolds along two orthogonal directions in the dry state (at 25 °C) as well as in the swollen state (at 37 °C) using a M350-2208 universal testing machine (Testometric, U.K.) at a compression rate of 1.0 mm/min. The cylinder-shaped scaffolds (Ø 10 × 20 mm, n = 5) were prepared using a scalpel. 2.4. Weight loss of scaffolds This testing was performing by incubating the cylinder-shaped scaffolds (Ø 10 × 10 mm, n = 3) in PBS (pH 7.4) at 37 °C. Prior to the start of the experiment, the dry scaffolds were weighed in advance (W0), and then placed into plastic flasks with 10 mL of PBS. At predetermined time intervals, the scaffolds were removed from PBS, washed with deionized water and lyophilized. Weight remaining of the scaffolds was calculated as follows: Weight remaining ( % ) =

Wt ×100 W0

(5)

where W0 is the initial weight of the scaffold, and Wt is the weight of scaffold at predetermined time t. 2.5. Cell behaviors in vitro 2.5.1. Cell culture ATDC5 cells were procured from Tianjin University of Traditional Chinese of Medicine. The cells were expanded in a culture flask with culture medium (Dulbecco’s modified Eagle’s medium nutrient mixture F-12 HAM (DMEM/F-12) containing 10% FBS (BI, Israel), and 1% antibiotics solution (100 U/mL penicillin and 100 µg/mL streptomycin, (Sigma-Aldrich, U.S.A.) in a 5% incubator at 37 °C until obtaining enough cells for the experiments. 2.5.2. Cell proliferation and morphology on scaffolds

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The scaffolds were subjected to 60Co radiation sterilization before cell experiment. After resuspended, ATDC5 cells were seeded at 5 × 105 cells onto each scaffold. Then the scaffold was placed in 24-well plate and then incubated in a 5% CO2/ 37 °C incubator for 2 h to allow the cells to diffuse into and attach onto the scaffold. Subsequently, 800 µL of fresh culture medium was added to each well. The cell culture media was replaced every 2-3 days. The 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT; Sigma-Aldrich, U.S.A.) colorimetry

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was used to evaluate the cell proliferation within the scaffolds. Meanwhile, in

order to observe the cell morphology within the scaffolds, the cultured cell-scaffolds were removed and fixed with 2.5% glutaraldehyde solution. After dehydrated in gradient ethanol series and dried fully, the morphology of the samples were determined by SEM. 2.5.3. Hematoxylin and eosin (H&E) staining At specific time intervals, the harvested samples were washed in PBS, and fixed with 4% paraformaldehyde overnight. Then the samples were dehydrated through gradient ethanol series, paraffin-embedded and serially cut. After deparaffinized and rehydrated, the obtained sections were stained with H&E for histological analyses. 2.5.4. Immunohistochemical analysis The section was treated with methanol solution containing 3% hydrogen peroxide for 10 min in order to block the endogenous peroxidase activity, and then incubated with anti-collagen II antibody at 4 °C overnight followed by secondary antibody. Finally, the section was color-developed with the peroxidase substrate 3, 3’-diaminobenzidine (DAB) and counterstained with hematoxylin. The positive staining for collagen II was examined under an

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Olympus microscope (BX51, Japan). The section was also stained with toluidine blue to visualize proteoglycan deposition. 2.5.5. Immunofluorescence analysis for collagen II The section was permeabilized with 0.1% Triton X-100, and subsequently incubated with bovine serum blocking solution to quench the non-specific binding. The anti-collagen II antibody was applied to the section, which was then incubated with secondary antibody (FITC-conjugated) for 2 h in dark. After washing with PBS, the section was straining with DAPI for 10min in dark followed by fluorescence imaging. 2.6. Biochemical analysis The sulphated-glcosaminoglycans (GAGs) content was estimated by shark chondroitin sulfate (Sigma-Aldrich, USA) using 1, 9-dimethylmethylene blue (DMMB) (Sigma-Aldrich, USA) method and then measured the absorbance at 525 nm.38 Total collagen content was analyzed by determining the hydroxyproline content after reaction with chloramine-T and p-dimethylaminobenzaldehyde.39 The spectrophotometric absorbance was then measured at 560 nm. 2.7. Evaluation in vivo 2.7.1. Subcutaneous implantation For in vivo evaluation, 3-week-old healthy Sprague-Dawley rats were used. Animal experiments were conducted according to the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals, and the surgical procedures were approved by the Animal Ethics Committee of North China University of Science and Technology. Scaffold samples were sterilized using the same procedure (60Co radioaction sterilization method) as in 11



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vitro study. Before implantation, the rats were anesthetized using barbital. A small dorsum incision (ca. 1.5 cm) was made on the back to create subcutaneous pockets so as to insert the scaffolds (Ø 10 × 2 mm), subsequently closed with surgical suture. At predetermined implantation time point, the animals were sacrificed, and the implanted scaffolds along with surrounding skin tissues were collected, and fixed in 4% paraformaldehyde overnight. In addition, no animals were harmed or premature death result from toxic side effects (e.g. loss of appetite, fever or diarrhea) in the course of the experiment. 2.7.2. Histological and immunochemical analyses The samples were gradient dehydration using ethanol, embedded in paraffin and sectioned. After deparaffinized and rehydrated, the sections were stained with H&E to access the tissue-scaffold interaction in vivo. Masson’s trichrome staining and Van Gieson staining was used to observe the cell distribution and collagen deposition in scaffold. Additionally, immunohistochemistry analysis was performed by staining the tissue sections with antibody CD31 for evaluating luminal structures containing red blood cells. DAPI was used to locate the cells via staining the nuclei of cells. All histological imaging analyses were performed on an Olympus microscope. 2.8. Statistical analysis All quantitative data were reported as mean ± standard deviation (SD). Statistical analyses were performed using one-way analysis of variance (ANOVA). Differences were considered significant if P < 0.05.

3. RESULTS AND DISCUSSION 3.1. Characterization of scaffolds 12


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Figure 1. SEM micrographs of PVA-CAR scaffolds observed from (a1, b1) longitudinal direction and (a2, b2) transverse direction. The oriented scaffold shows obvious anisotropic microstructure, but there is no difference in non-oriented scaffold. (Magnification: 300 ×)

The microstructures of the PVA-CAR scaffolds prepared with different freezing methods were observed by SEM as shown in Figure 1. The results showed that the oriented scaffold had anisotropic porous microstructures, and pores within scaffold were arranged in parallel in the longitudinal direction (Figure 1a1) and random porous structure in the transverse direction (Figure 1a2). However, there is no appreciable structural change within non-oriented scaffold which had nearly the same irregular pore structure in different directions (Figure 1b1, b2). The direction of freezing plays an important role in fabricating a scaffold with desirable pore structure.40 Unidirectional freezing is a simple technique to produce oriented porous structure in materials. Once a high temperature gradient is applied, the solvent crystals could grow 13



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along one direction. The generation of phase separation is an important mechanism for physically crosslinked PVA hydrogels. During the freezing stage, water crystallizes to form aligned ice templates and the polymers are expelled from the frozen water, leading to the aggregation of polymers between the growing ice crystals. Since the hydrogen bonding occurred between PVA chains, PVA crystallites form subsequently and act as join points in polymer network. At the same time, CAR chains come into close contact with PVA chains due to the formation of hydrogen bonds networks. Consequently, this approach could achieve the hydrogel with oriented porous structure that is imprinted by well-aligned ice templates (Scheme 1). After removal of ice crystals by lyophilization, the unidirectional pores are left in PVA-CAR composite scaffold. In contrast, ice crystals grow in all directions without temperature gradient by means of conventional method. Thus, the pores presented within scaffold are irregular and isotropic without temperature gradient.

Scheme 1. Procedure for the preparation of PVA-CAR scaffolds with different pore structures.

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Figure 2. (A) XRD results for PVA-CAR scaffolds. (B) Swelling kinetics of PVA-CAR scaffolds in PBS (pH 7.4) at 37 °C. Compressive stress-strain curves for PVA-CAR scaffolds in the (C) dry state (at 25 °C) and (D) swollen state (at 37 °C) along the different directions: oriented scaffold along the (a) longitudinal and (b) transverse direction; non-oriented scaffold along the (c) longitudinal and (d) transverse direction.

The degrees of crystallinity (χc) of PVA in scaffolds were approximately evaluated from Figure 2A. As shown in Table 1, the χc of oriented scaffold was 18.5%, which was a little higher than that of non-oriented scaffold. The reason may be due to the different fabrication processes. In addition, the porosity of oriented scaffold and non-oriented scaffold was 85.79 ± 4.12% and 86.88 ± 3.88% respectively. After being saturated in PBS (pH 7.4) at 37 °C for 2 days, both scaffolds followed nearly the same swelling tendency. As shown in Figure 2B, a high swelling ratio could be found in 15



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the first 2 h and the swelling ratio slowed down within 2-12 h. Then the swelling curves became level, indicating both scaffolds reached their ESR. No significant swelling rate change was observed between both scaffolds. Besides, the ESR of oriented scaffold was slightly lower in contrast with non-oriented scaffold. As mentioned earlier, the interaction between PVA chains through hydrogen bonding followed by crystallization, which leads to the formation of hydrogel network where PVA crystallites serve as knots. So the degree of crystallinity has an effect on the crosslink density in polymer network.41 As a consequence, relatively high crystallinity of oriented scaffold reveals a more compact network structure in polymeric matrix to accommodate water. Table 1. Crystallinity, porosity and ESR of PVA-CAR scaffolds Samples

Crystallinity (%)

Porosity (%)

ESR

Oriented scaffold

18.5

85.79± 4.12

13.25 ± 0.55

Non-oriented scaffold

16.3

86.88± 3.88

15.34 ± 0.68

Generally, the scaffold should possess the ability to sustain the structural integrity under certain compression, which is necessary to protect the cells or neo-tissue within scaffold from damage. The compressive stress-strain curves for the scaffolds along different directions were presented in Figure 2. As observed in Figure 2C, the stress-strain curves of both scaffolds in dry state showed similar tendency, which can be divided into three stages: the initial elastic stage, a collapse platform stage and the densification stage. However, as shown in Figure 2C, there is an obvious difference in the compressive strength of oriented scaffolds along two orthogonal directions. The compressive strength along the longitudinal direction is much higher than that in transverse direction, indicating the anisotropic mechanical behavior. The 16


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unidirectional freezing process endowed the scaffold with special anisotropic microstructures, in where highly ordered structures in longitudinal direction withstood compression, leading to high compressive strength compared with non-oriented scaffold. However, there was no difference in compressive strength along two orthogonal directions for non-oriented scaffold, resulting from conventional freeze-thaw method. In addition, the compression behaviors of both scaffolds were also investigated in the swollen state (Figure 2D). Since PVA and CAR were hydrophilic polymers, the polymer chains could change their relative position in the wet state, leading to the elastic characteristic. The compressive stress-strain curves were non-linear and the compressive property was related to the pore structure and interstitial fluid. When the hydrous scaffold compresses, the interstitial fluid was forced out of scaffold at first and a small load could produce a significant deformation. With the continuous extrusion of interstitial fluid, the scaffolds became dense under sustained load, and it needed higher stress to deform the scaffolds. Compared to the hydrous non-oriented scaffold, the hydrous oriented scaffold also exhibited better mechanical properties. Articular cartilage is a complex tissue with unique mechanical characteristic features. Collagen fibrils networks in native cartilage are crucial to joint mechanics and exhibit depth-dependent variations.42 Some research shows that the vertical collagen fibrils in deep zone play an important role in protecting native cartilage from large strain, especially at the subchondral junction where peak strains occur.43-44 As a scaffold for tissue engineering, it needs to play the role of temporary mechanical substitution when implanted in vivo. It is a way

to

design

appropriate

scaffolds

by

improving

the

mechanical

feature, 17



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which inspired from the mechanical property of the target tissue. The scaffolds prepared by the unidirectional freeze-thaw method have improved mechanical properties compared to those obtained by conventional freeze-thaw method.45 Most importantly, no matter in the state of dry or swollen, the oriented scaffold could always possess anisotropic mechanical characteristics, which has been biomimetic the mechanical feature of cartilage in compression and will meet the demanding mechanical environment of the native tissue.

3.2. Weight loss of scaffolds

Figure 3. (A) Weight loss curves of PVA-CAR scaffolds in PBS (pH 7.4) at 37 °C. (B) Compressive stress-strain curves of (a, b) oriented scaffold and (c, d) non-oriented scaffold (a, c) before and (b, d) after immersing in PBS at 37 °C for 4 weeks.

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Figure 4. SEM micrographs showing the morphology changes of oriented scaffolds and non-oriented scaffolds, respectively at different soak time. (Magnification: 300 ×)

As shown in Figure 3A, the weight losses of both scaffolds increased as the time goes on in PBS at 37 °C. Moreover, the oriented scaffold exhibited relative slower weight loss than the non-oriented scaffold at the same time point. Although the crystallization contributes to the formation of polymer network, many amorphous regions also exist in hydrogel due to the semicrystalline behavior of PVA.46 Therefore, the degradation and dissolution of CAR and PVA polymer molecules in amorphous regions will be the main cause of mass loss. With ongoing immersing time, all samples maintain original apparent morphology (Figure 4), which is advantageous to the scaffold for maintaining the template action and mechanical support. Furthermore, though both scaffolds had nearly the same porosity, the scaffold with anisotropic microstructure may protect components from hydrolysis to some extent due to its higher crystallinity and slightly lower water absorption ability. These properties may be beneficial to enhance the stability of the network in PBS. On the contrary, more amorphous 19



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species in non-oriented scaffold will be lost during hydration. In addition, as PVA and CAR are nontoxic materials, the PVA-CAR scaffolds and their degradation by-products were also expected to be nontoxic. In addition, both scaffolds after immersing in PBS for 4 weeks still had good mechanical properties (Figure 3B). The reason is that the mechanical performance mainly depends on the polymer network due to the PVA crystallization and was not affected by the dissolution of polymer molecules in amorphous regions. Besides, this result was in agreement with the fact that all samples maintain their original apparent morphology during the immersion period due to the cross-linked network structure, which is advantageous to the scaffold for maintaining the template action and mechanical support in the process of tissue repair.

3.3. Cell behaviors on scaffolds

Figure 5. MTT assays for proliferation of cells cultured with scaffolds (*p < 0.05).

Some material properties, including composition and geometrical structure should be focused on so as to design serviceable biomimetic scaffolds,47 which is critical for cell growth 20


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and maintaining cell phenotype. Following the same rationale, in our previous study, CAR has been selected to optimize PVA hydrogels due to its similarity with GAGs present in cartilage. The scaffold should satisfy certain criteria such as suitable structure and porosity, which will play a better role in structural template for cells.48 Additionally, the homogeneous cell distribution is critical for the distribution of subsequent ECM deposition within scaffold. Thus, the scaffold microstructure will affect cellular behavior and cell fate in vitro. Prior to the application in vivo, the interaction between cell and scaffold should be taken into consideration at first to screen biomaterials.49 Here we investigate the cellular interaction with both scaffolds according to the cell proliferation within the scaffolds (Figure 5). At day 1, almost the same cell proliferation was observed in both scaffolds, which indicated that there is no difference in cell number at initial time. The cell proliferation showed an increased tendency in both scaffold during the process of cultivation. However, cell proliferation was enhanced within oriented scaffolds from day 4 to day 28 compared with non-oriented scaffolds (*p < 0.05). This result manifested that the scaffolds with oriented structure provided a comfortable environment for cell proliferation and survival.

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Figure 6. H&E staining shows the distribution of cells cultured on scaffolds in vitro. (a1-a3) oriented scaffolds and (b1-b3) non-oriented scaffolds 2 weeks after culture; (c1-c3) oriented scaffolds and (d1-d3) non-oriented scaffolds 4 weeks after culture.

Although both scaffolds exhibited porous structure and nearly the same porosity, completely different structures were found. As shown in Figure 6, more cells were visualized within oriented scaffolds than non-oriented scaffolds, and the cells had a noted tendency to attach and proliferate along the axially aligned porous structure. The diverse nature of tissue histoarchitecture endows scaffold fabrication with unique inspiration, providing the 22


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cells with different microenvironments. The scaffold with biomimetic aligned structure provide more favorable space for cell attachment, migration and proliferation, leading to more evenly distributed staining in oriented scaffold (Figure 6a1, b1). In contrast, a decrease in the cell density within non-oriented scaffold corresponding to the lack of suitable permeability structure was revealed. The reason is that the pores may be plugged by cells, which is not conducive to further cellular penetration within scaffold. The spatial distribution of cells will be limited in some pores (Figure 6d2), and this has a negative effect, because the transport of nutrients and cellular waste was greatly hampered by such unfavorable structure. So the cells aggregated at a low density in some part of non-oriented scaffold, resulting in heterogeneous cell distribution (Figure 6d1). Moreover, both scaffolds could maintain their original shapes and kept the cells viable throughout the entire culture period.

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Figure 7. SEM morphologies of the cells cultured within scaffolds at (a1, a2, d1 and d2) 1 week, (b1, b2, e1 and e2) 2 weeks and (c1, c2, f1 and f2) 4 weeks, respectively.

The architecture of scaffolds should emulate the natural design of the ECM and instruct cellular behavior while adequately housing the cells. The cell behavior within the scaffolds was also monitored by SEM (Figure 7). It could be observed that the cells were attached well onto both scaffold surfaces at week 1 (Figure 7a1, d1). With the extension of incubation time, both scaffolds had shown good cell affinity, leading to the formation of many cell colonies (Figure 7c1, f1). Notably, for oriented scaffolds, the cells infiltrated into the scaffolds, attached

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onto the surface of the unique structure and grew within the axial oriented pores (Figure 7a2, b2 and c2). From the macro view, the cells exhibited directional distribution in oriented scaffolds, whereas the cells within non-oriented scaffolds appeared no obvious regularity (Figure 7d2, e2 and f2). Both scaffolds could promote the cells to secrete extracellular matrix (ECM) after 4 weeks culture. SEM results were in accordance with the H&E analysis. Thus, the biomimetic scaffolds provided the cells with comfortable microenvironment, and effectively promoted cells homogeneous distribution within the oriented pores.

Figure 8. Measurement of (A) GAGs and (B) total collagen contents of cell-seeded scaffolds cultured for 2 and 4 weeks (*p < 0.05).

The appropriate scaffold should not only support cell proliferation, but also possess the ability to preserve proper cellular functions. GAGs production is the main component in cartilaginous matrix, which plays an important role in maintaining the chondrocyte phenotype.50 After investigating the influence of scaffold microstructure on cell proliferation and distribution, extracellular secretion within scaffolds was further detected by the biochemical assays. As shown in Figure 8A, an evidence of accumulated GAGs during further 25



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culturing was found in both scaffolds, which indicating the preservation of proper cellular functions. However, the amount of GAGs production in oriented scaffold was significantly enhanced compared with non-oriented scaffold (*p < 0.05), and similar tendency was also found in deposited collagen within scaffolds (Figure 8B). The suitable structure inside oriented scaffold provides the cells with adequate space to survival and development. It demonstrates that the highly organized structure facilitates ATDC5 cells to secrete ECM continuously, and this result was maintained with the extension of cultivate time. The analysis of GAGs and collagen content was well consistent with cell proliferation results, which evidenced that the biomimetic structure could effectively promote cell distribution and support the cellular function.

Figure 9. Collagen II immunostaining (browny hue represents the positive staining of 26


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collagen II) and immunofluorescence staining on sections of cell-seeded scaffolds. Cell nuclei are stained with DAPI (blue) and collagen II with anti-collagen II antibody (green).

The distribution of ECM components is very important in tissue engineering. Collagen II, a primary component in cartilage, was examined by immunochemical staining in order to understand the effect of scaffold structure on spatial distribution of ECM synthesis. The detection of chondrocyte-specific collagen II within scaffolds indicated that the ATDC5 cells express specific markers of chondrocytes. Collagen II accumulation was evident in both kinds of scaffolds on 2 weeks, although staining appeared more intense for oriented scaffold (Figure 9a) compared with non-oriented scaffolds (Figure 9c). Collagen II production were enhanced with culture time, which was significantly higher after 4 weeks culture (Figure 9e, g). As a consequence, more vastly stained collagen II was detected in the section of the oriented scaffold and the matrix substance was mostly present in the surrounding regions of the cells. Similarly, immunofluorescence analysis showed that the oriented scaffolds could enhance cell migration, leading to a uniform spatial distribution of ECM synthesis. Furthermore, the oriented scaffold seemed to regulate the alignment of cells, which could promote the function of ATDC5 cells in the secretion and the deposition of collagen II most effectively. These results were consistent with quantitative analysis of total collagen content (Figure 8B).

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Figure 10. Toluidine blue staining of cells cultured in the (a) oriented scaffold and (b) non-oriented scaffold for 4 weeks.

Moreover, the extracellular matrices in cell-scaffold constructs for chondrocyte phenotype were positively stained by toluidine blue (Figure 10), indicating the formation of chondrocyte-specific proteoglycan. In contract to non-oriented scaffold, the staining color was deeper in oriented scaffold, which revealed increase producing of cartilage matrix proteoglycan. In addition, the stained area in oriented scaffold was larger than that in non-oriented scaffold. This result indicated that more proteoglycan secreted in the matrix for oriented scaffold, which was consistent with preferable cell affinity for cells proliferation in oriented scaffold due to its well interconnected pore structure. Therefore, the appropriate microstructure provides a comfortable microenvironment for cell growth, which promoted structure homogeneity and lead to a homogeneous ECM dispersion over the scaffold. 3.4. In vivo assessment of scaffolds Physico-chemical properties and cell behavior in vitro provide preliminary information with regard to the suitability of oriented scaffolds. To evaluate of tissue-scaffold interactions, inflammatory responses and the effect of scaffold microstructure on the behavior and fate of host cells in vivo, the scaffolds were implanted subcutaneously rats and the rats were healthy 28


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during the period of implantation. Both scaffolds elicited a moderate inflammatory response and did not show any adverse impact on the surrounding tissue after implantation.

Figure 11. H&E histological evaluation of scaffolds with different microstructures after implantation in vivo.

To detect cell infiltration, the scaffolds were stained with H&E after paraffin embedding and serial section (Figure 11). It was shown that both scaffolds were intact and still displayed obviously porous structure when persisted in vivo for 1 week. Many cells were detected in both scaffolds and there was also a substantial penetration of tissue. Due to the short period of subcutaneous implantation, some voids were obvious in sections, revealing the incomplete tissue infiltration within scaffolds at 1 week. Moreover, the cell density in non-oriented 29



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scaffold was relatively lower than that in oriented scaffold. It is worth noting that the cells moved inward and distributed uniformly in oriented scaffold. However, cell still aggregated on the side of non-oriented scaffolds close to the skin and the scaffolds did not effectively support the cell migration into the inside after 2 weeks in vivo. In comparison to the 2 weeks sections, H&E staining of the 3 weeks sections shown that the oriented scaffolds were completely filled with uniform tissue. However, in non-oriented scaffolds, there was no significant difference in the amount of infiltration tissue after implanting 2 and 3 weeks. The ingrowth tissue could be observed clearly in the outer region of non-oriented scaffold with some voids in the sections. After 4 weeks of implantation, both scaffolds did not cause an adverse impact on the tissue around the implant site. In addition, the major viscera were normal and did not exhibit any signs of pathologic changes and necrosis as the implantation of scaffolds (Figure S1). Compared to the corresponding scaffolds at 1 week, both scaffolds presented higher cell densities after 4 weeks (Figure 11). In oriented scaffold, due to the better capacity of mass transportation, uniform cell distribution gave rise to simultaneous the tissue growth in the whole scaffold with the passage of time. However, the infiltration of cells was still distributed unevenly in non-oriented scaffold after 4 weeks implantation. The reason may be ascribed to the lack of permeable structure, which impeded the cell migration towards the internal regions of non-oriented scaffold. More cells on the side of scaffold next to the skin will subsequently cause the formation of relatively compact tissue. This will be an inevitable obstacle for host cells migrating into the scaffold, resulting in an inhomogeneous tissue formation inside the scaffold. Obviously, these results were inseparable from the structural property of scaffolds. Besides, it was found that the 30


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degree in inflammatory reaction surrounding the tissue sites where the scaffolds were implanted gradually ease over time.

Figure 12. Masson's Trichrome and Van Gieson staining of scaffolds with different microstructures after implantation in vivo. Masson's Trichrome staining: nuclei stain black and collagen stains blue; Van Gieson staining: nuclei stain black blue and collagen stain red. S denotes scaffold material, IT denotes the infiltrated tissue and FL denotes the fibrous layer.

Masson's Trichrome and Van Gieson staining revealed that a thin tissue capsule formed around the implanted scaffolds, and integration of the scaffolds into the surrounding tissue was evident after 1 week in vivo (Figure 12). During the period of implantation, the scaffolds could maintain the structures stability without shrinkage as the evidence of minimal change in pore structure. This is primarily due to the good mechanical behavior the scaffolds. For most 31



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tissue engineering applications, it would be advantageous for the implanted scaffold to degrade in accordance with the growth rate of the newly-formed tissue. However, for load-bearing tissues like cartilage on that regularly experience mechanical stress, stronger and more durable support is needed. Hence, the long-term degradation properties of the scaffolds may therefore be more beneficial for the regeneration of these tissues. The oriented scaffold provides an optimal microenvironment for the migration of surrounding host cells, and neo-tissue in growth compared to non-oriented scaffolds. Viewing the stained, collagen deposition was observed in the bulk of the scaffolds implanted from 1 week to 4 weeks, which suggested that the infiltrating cells could secret their own ECM and remain viable and functional, indicating the good biocompatibility of scaffolds. Importantly, the oriented scaffold displayed an enhanced level of cell infiltration at each time points, resulting in increasing the amount of tissue within the scaffolds.

Figure 13. Immumohistochemical staining against CD31 antibody in scaffolds with different 32


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microstructures harvested at 4 weeks post implantation. The arrows indicate the circular and ellipsoid cross section of the vascular structures present in the infiltrating tissues. Actually, the neo-tissue formed inside the scaffold was unpredictable because of an absence of cells with the scaffolds prior to implantation. One aspect of assessing the tissue reaction used in subcutaneous implantation is that the scaffolds are suitable for cellular ingrowth from the surrounding tissue, and ensure the survival of tissue. The newly formed tissue within scaffold needs a sufficient nutrient supply, especially in the center region. Therefore, it is necessary to have vascular system during the scaffold implantation, which played a decisive role on the extent of oxygen, nutrients and metabolic products exchange within the host cell and tissue that infiltrated into the scaffold. The necrosis of cells in the inner region of the scaffolds is inevitable when the nutrient in short supply due to the lack of intrinsic vasculature, which would adversely affect the function of the scaffolds. The achievement of angiogenesis with implanted scaffolds is at least dependent on three factors: the biological activity of the scaffold, the microstructure, and the metabolic activity of infiltrated host tissue.51 As shown in Figure 13, vascularization was clearly apparent from the intensely eosinophilic red blood cells within the vasculature. Immunohistochemistry evaluation was performed to show the presence of blood vessels in both scaffolds after subcutaneous implantation for 4 weeks. Several blood vessels containing erythrocytes were observed, distributing throughout both scaffolds, which is vital for subsequent cell growth and the survival of regenerating tissue. Importantly, the presence of more vascularization in the infiltrated tissue within oriented scaffolds was observed after 4 weeks of implantation, which indicated a faster integration of oriented scaffolds with newly formed tissue as compared to non-oriented scaffolds. 33



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It should provide appropriate hyperoxic environment and adequate nutrient supply for the newly cartilage constructs so as to promote tissue growth. In most time, the convenient nutrient transportation for cartilage constructs played an important role in tissue engineering.52 As a temporary space, the scaffold should provide suitable in vivo microenvironment to support cell migration and tissue regeneration. Taken together the in vivo experimental results showed that both scaffolds had good compatibility. However, the biomimetic scaffold with unique oriented structure is more beneficial to promote cellular and vascular integration within scaffolds, and ensure adequate nutrient supply for the survival of neo-tissue. Therefore, the results of subcutaneous implantation can, from a side, reflect that oriented scaffold has the potential to provide a conducive healing environment which greatly improves cell-material interaction and it can be a favorable candidate for cartilage repair.

4. CONCLUSION In this study, a biomimetic scaffold was successfully constructed by using a unidirectional freeze-thaw approach. This scaffold had axially aligned pore structure and maintained good mechanical property. Noticeably, this scaffold imitated the structure feature of nature cartilage, which operated as a template for cells growth along the oriented pore structure. Furthermore, such oriented scaffold could not only efficiently enhance uniformity distribution of cells in vitro, but also promote the regularity of the infiltration tissue within the scaffold in vivo. Additionally, tissue responses towards the scaffolds in vivo were benign, indicating their suitability for implantation purposes. Based on simple preparation method and the potential to be used as cell-free scaffold, this oriented scaffold could be promising tool for application in tissue engineering. 34


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ASSOCIATED CONTENT Supporting Information (S1) H&E staining of the major viscera obtained from rats after subcutaneous implantation for 4 weeks.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (F. Yao). Tel.: +86-22-27402893. Fax: +86-22-27403389. *E-mail: [email protected] (H. Sun). Tel.: +86-315-3725740. Fax: +86-315-3726552. *E-mail: [email protected] (J. Li). Tel.: +86-10-68166874. Fax: +86-10-68166874. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by National Nature Science Foundation of China (Grant No. 31271016, 51573127 and 81101448) and Hebei Province Scientific Research Foundation for Returned Scholars (Grant No. C201400560).

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A biomimetic poly (vinyl alcohol)-carrageenan composite scaffold with oriented microarchitecture Yabin Zhang †, Lei Ye †, Jing Cui ‡, Boguang Yang †, Hong Sun*, ‡, Junjie Li*, § and Fanglian Yao*, †,⊥

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A Biomimetic Poly(vinyl alcohol)âCarrageenan Composite Scaffold

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