A Biomimetic Poly(vinyl alcohol)âCarrageenan ... - ACS Publications

Article pubs.acs.org/journal/abseba

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 and ⊥Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin University, Tianjin 300072, China ‡ Department of Basic Medical Sciences, North China University of Science and Technology, Tangshan 063000, China § Department of Advanced Interdisciplinary Studies, Institute of Basic Medical Sciences and Tissue Engineering Research Center, Academy of Military Medical Science, Beijing 100850, China S Supporting Information *

ABSTRACT: In general, the design of a scaffold should imitate certain advantageous properties of native extracellular matrix (ECM) to operate as a temporary ECM for cells. From this perspective, a biomimetic scaffold was prepared using poly(vinyl alcohol) and carrageenan 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 physicochemical properties than a non-oriented scaffold fabricated by the 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 the 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

1. INTRODUCTION

Hydrogels have proven to be 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 a 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 nonirritating to soft tissues when in contact with them.16 Therefore, PVA hydrogels have been extensively investigated for applications in tissue engineering, especially cartilage,17−19 as well as drug delivery carriers.20,21 In recent years, a promising and novel technique called the solvent unidirectional freezing method has been used to create oriented structure in hydrogels, where the pore structure is templated from aligned solvent crystals.22,23 For example, Del Monte et al. demonstrated the ability for unidirectional freezing in the preparation of monolithic PVA

Tissue engineering, a field applying the principles and technologies of the life sciences and engineering, has good prospects in the treatment of diseases and provides opportunities to develop biological substitutes that can repair, maintain, and promote the function of tissues and organs.1 Because tissue structure plays an integral role in maintaining tissue function, the scaffold should imitate certain advantageous properties of native extracellular matrix (ECM) to provide a 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 to 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 © 2016 American Chemical Society

Received: December 12, 2015 Accepted: March 22, 2016 Published: March 22, 2016 544

DOI: 10.1021/acsbiomaterials.5b00535 ACS Biomater. Sci. Eng. 2016, 2, 544−557

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ACS Biomaterials Science & Engineering

freezing at −20 °C and subsequently thawed at room temperature. The sample was also further lyophilized for 48 h. Finally, both types of dried scaffolds were preserved in a desiccator for further experiments. 2.3. Characterization of Scaffolds. 2.3.1. Morphological Observations. After fracturing in liquid nitrogen, the scaffolds were mounted on the sample holder and sputter coated with a layer of gold. Then, morphological observations of the 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 D8Focus 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 (χc) of the scaffolds were estimated according to the literature35 and calculated with the equation

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 of native cartilage, which is promising for biomimetic scaffolds. However, the inability of PVA hydrogels to partake in cell adhesion because of their resistance to protein adsorption is a disadvantage and has restricted their applications in tissue engineering.25−27 Recently, polysaccharides have been shown to be 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 polysaccharide, carrageenan has attracted more attention because of its mild gelation property. Most importantly, the structure of carrageenan is similar to that of natural glycosaminoglycans (GAGs),31 which makes it a potential candidate for tissue engineering.32,33 A previous study in our lab described a composite hydrogel for enhancing the cell adhesion ability of PVA hydrogels through the incorporation of carrageenan (CAR) in a single step.34 Nevertheless, a suitable scaffold should not merely imitate the composition of the native tissue, and structural bionic design is necessary. The respect effect of biomimetic pore structure in a PVA-CAR scaffold on cell growth has not been extensively studied; thus, that and the in vivo tissue−scaffold interaction of such a scaffold needs to be further investigated. In light of the above circumstances, a worthwhile endeavor would be to construct a biomimetic scaffold by using hydrophilic PVA and CAR in which an axially aligned pore structure could be formed through a unidirectional freeze−thaw method. For the purpose of investigating the effect of microstructure on physicochemical properties of scaffolds, the ordinary scaffold (denoted as non-oriented scaffold) was prepared by the 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.

χc (%) =

A1 × 100 A2

(1)

where A1 is the area of the crystalline region in the range of 2θ from 18 to 21°, and A2 is the area toward 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 is placed into a certain volume of ethanol (V1). Subsequently, vacuumizing is carried out to force the ethanol into internal pores of the scaffold, and the total volume is V2. After the scaffold is taken out, the remaining volume of ethanol is V3. The porosity is calculated as porosity (%) =

V1 − V3 × 100 V2 − V3

(2)

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

ESR =

W − W0 × 100% W0

(3)

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

swelling ratio =

2. MATERIALS AND METHODS

Wt − W0 × 100% W0

(4)

where W0 and Wt are the weights of the initial dry and swollen scaffolds at predetermined time t, respectively. The experimental data were obtained from triplicate samples. 2.3.5. Mechanical Behavior. 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 an M350-2208 universal testing machine (Testometric, UK) 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 in plastic flasks with 10 mL of PBS. At predetermined time intervals, the scaffolds were removed from PBS, washed with deionized water, and lyophilized. The weight remaining of the scaffolds was calculated as

2.1. Materials. PVA (77000 g/mol, 97% hydrolyzed) was obtained from Guangfu Fine Chemical Company, Ltd. (Tianjin, China). ιCarrageenan was purchased from Kasei Kogyo Company, Ltd. (Tokyo, Japan). All other chemicals were of analytical purity and used without further treatment or purification. 2.2. Preparation of Scaffolds. Four grams of PVA powder was dissolved in fifty grams of distilled water to form a transparent solution. One gram 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 rested on the top of liquid nitrogen. Because of 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 a 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 an anisotropic scaffold with oriented pore structure (denoted as oriented scaffold). Contrastingly, a non-oriented scaffold was prepared through a typical experiment. In brief, the above mixed solution was subjected to

weight remaining (%) =

Wt × 100 W0

(5)

where W0 is the initial weight of the scaffold, and Wt is the weight of the 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 Medicine. The cells were expanded in a culture flask with culture 545


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Figure 1. SEM micrographs of PVA-CAR scaffolds observed from the (a1, b1) longitudinal direction and (a2, b2) transverse direction. The oriented scaffold shows an obvious anisotropic microstructure, but there is no difference in the non-oriented scaffold (magnification: 300×). medium (Dulbecco’s modified Eagle’s medium nutrient mixture F-12 HAM (DMEM/F-12)) containing 10% FBS (BI, Israel) and 1% antibiotic solution (100 U/mL of penicillin and 100 μg/mL of streptomycin (Sigma-Aldrich, USA)) in a 5% incubator at 37 °C until enough cells were obtained for the experiments. 2.5.2. Cell Proliferation and Morphology on Scaffolds. The scaffolds were subjected to 60Co radiation sterilization before cell experiments. After resuspension, ATDC5 cells were seeded at 5 × 105 cells on each scaffold. Then, the scaffold was placed in a 24-well plate and 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. 3-(4, 5-Dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT; Sigma-Aldrich, USA) colorimetry37 was used to evaluate cell proliferation within the scaffolds. Meanwhile, for observation of the cell morphology within the scaffolds, the cultured cell-scaffolds were removed and fixed with 2.5% glutaraldehyde solution. After the samples were dehydrated in a gradient ethanol series and dried fully, the morphologies 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 with PBS and fixed with 4% paraformaldehyde overnight. Then, the samples were dehydrated through a gradient ethanol series, paraffin embedded, and serially cut. After being deparaffinized and rehydrated, the obtained sections were stained with H&E for histological analyses. 2.5.4. Immunohistochemical Analysis. The section was treated with a methanol solution containing 3% hydrogen peroxide for 10 min to block the endogenous peroxidase activity and then incubated with anticollagen II antibody at 4 °C overnight followed by the addition of a 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 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 nonspecific binding. The anticollagen II antibody was applied to the section, which was then incubated with secondary antibody (FITC-conjugated) for 2 h in the dark. After being washed with PBS, the section was stained with DAPI for 10 min in the dark followed by fluorescence imaging. 2.6. Biochemical Analysis. The sulfated glycosaminoglycans (GAGs) content was estimated by shark chondroitin sulfate (SigmaAldrich, USA) using the 1,9-dimethylmethylene blue (DMMB) (Sigma-Aldrich, USA) method, and the absorbance was then measured at 525 nm.38 Total collagen content was analyzed by determining the hydroxyproline content after the reaction with chloramine-T and pdimethylaminobenzaldehyde.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 for the in vitro study. Before implantation, the rats were anesthetized using barbital. A small dorsum incision (∼1.5 cm) was made on the back to create subcutaneous pockets so as to insert the scaffolds (Ø 10 × 2 mm), which were subsequently closed with a surgical suture. At predetermined implantation time points, the animals were sacrificed, and the implanted scaffolds along with surrounding skin tissues were collected and fixed in 4% paraformaldehyde overnight. No animals were harmed or suffered from premature death resulting from toxic side effects (e.g., loss of appetite, fever, or diarrhea) over the course of the experiment. 2.7.2. Histological and Immunochemical Analyses. The samples were gradient dehydrated using ethanol, embedded in paraffin, and sectioned. After deparaffinization and rehydration, the sections were stained with H&E to access the tissue−scaffold interaction in vivo. Masson’s Trichrome and Van Gieson staining were used to observe the cell distribution and collagen deposition in the scaffold. Additionally, immunohistochemistry analysis was performed by 546


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ACS Biomaterials Science & Engineering Scheme 1. Procedure for the Preparation of PVA-CAR Scaffolds with Different Pore Structures

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 and non-oriented scaffold along the (c) longitudinal and (d) transverse direction. staining the tissue sections with antibody CD31 for evaluating luminal structures containing red blood cells. DAPI was used to locate the cells through staining of the cell nuclei. All histological imaging analyses were performed on an Olympus microscope. 2.8. Statistical Analysis. All quantitative data are 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.

methods were observed by SEM as shown in Figure 1. The results showed that the oriented scaffold had anisotropic porous microstructures, and pores within the scaffold were arranged in parallel in the longitudinal direction (Figure 1a1) and had a random porous structure in the transverse direction (Figure 1a2). However, there is no appreciable structural change within the 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 an oriented porous structure

3. RESULTS AND DISCUSSION 3.1. Characterization of Scaffolds. The microstructures of the PVA-CAR scaffolds prepared with different freezing 547


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PVA chains through hydrogen bonding was followed by crystallization, which led to the formation of a hydrogel network where PVA crystallites serve as knots. Thus, the degree of crystallinity has an effect on the cross-linking density in the polymer network. 41 As a consequence, relatively high crystallinity of the oriented scaffold reveals a more compact network structure in the polymeric matrix to accommodate water. Generally, the scaffold should possess the ability to sustain the structural integrity under a certain amount of compression, which is necessary to protect the cells or neo-tissue within the scaffold from damage. The compressive stress−strain curves for the scaffolds along different directions are presented in Figure 2. As observed in Figure 2C, the stress−strain curves of both scaffolds in the dry state showed a similar tendency, which can be divided into three stages: the initial elastic stage, the 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 the two orthogonal directions. The compressive strength along the longitudinal direction is much higher than that in the transverse direction, indicating anisotropic mechanical behavior. The unidirectional freezing process endowed the scaffold with special anisotropic microstructures in which highly ordered structures in the longitudinal direction withstood compression, leading to high compressive strength compared with that of the non-oriented scaffold. However, there was no difference in compressive strength along the two orthogonal directions for the nonoriented scaffold resulting from the conventional freeze−thaw method. In addition, the compression behaviors of both scaffolds were also investigated in the swollen state (Figure 2D). Because PVA and CAR are hydrophilic polymers, the polymer chains could change their relative position in the wet state, leading to elastic characteristics. The compressive stress−strain curves were nonlinear, and the compressive property was related to the pore structure and interstitial fluid. When the hydrous scaffold is compressed, the interstitial fluid is forced out of scaffold at first, and a small load can produce a significant deformation. With the continuous extrusion of interstitial fluid, the scaffolds become dense under a sustained load, and higher stress is needed to deform the scaffolds. Compared to the hydrous, nonoriented scaffold, the hydrous, oriented scaffold also exhibited better mechanical properties.

in materials. Once a high temperature gradient is applied, the solvent crystals could grow along one direction. The generation of phase separation is an important mechanism for physically cross-linked 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. Because the hydrogen bonding occurs between PVA chains, PVA crystallites form subsequently and act as join points in the polymer network. At the same time, CAR chains come into close contact with PVA chains due to the formation of hydrogen bond networks. Consequently, this approach could achieve the hydrogel with an 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 the PVA-CAR composite scaffold. In contrast, ice crystals grow in all directions without a temperature gradient by means of the conventional method. Thus, the pores presented within the scaffold are irregular and isotropic without a temperature gradient. The degrees of crystallinity (χc) of PVA in scaffolds were approximately evaluated from Figure 2A. As shown in Table 1, Table 1. Crystallinity, Porosity, and ESR of PVA-CAR Scaffolds sample

crystallinity (%)

porosity (%)

ESR

oriented scaffold non-oriented scaffold

18.5 16.3

85.79 ± 4.12 86.88 ± 3.88

13.25 ± 0.55 15.34 ± 0.68

the χc of the oriented scaffold was 18.5%, which was a little higher than that of the non-oriented scaffold. The reason may be due to the different fabrication processes. In addition, the porosities of the oriented and non-oriented scaffolds were 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 the first 2 h, and the swelling ratio slowed within 2−12 h. Then, the swelling curves became level, indicating that both scaffolds reached their ESR. No significant swelling rate change was observed between both scaffolds. Moreover, the ESR of the oriented scaffold was slightly lower than that of the nonoriented scaffold. As mentioned earlier, the interaction between

Figure 3. (A) Weight loss curves of PVA-CAR scaffolds in PBS (pH 7.4) at 37 °C. (B) Compressive stress−strain curves of the (a, b) oriented scaffold and (c, d) non-oriented scaffold (a, c) before and (b, d) after being immersed in PBS at 37 °C for 4 weeks. 548


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Figure 4. SEM micrographs showing the morphology changes of oriented and non-oriented scaffolds at different soak times (magnification: 300×).

CAR are nontoxic materials, the PVA-CAR scaffolds and their degradation byproducts were also expected to be nontoxic. Furthermore, both scaffolds, after immersion 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 PVA crystallization and is not affected by the dissolution of polymer molecules in amorphous regions. Moreover, 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 for the scaffold for maintaining the template action and mechanical support in the process of tissue repair. 3.3. Cell Behaviors on Scaffolds. Some material properties, including composition and geometrical structure, should be a focus so as to design serviceable biomimetic scaffolds,47 which is critical for cell growth and maintaining cell phenotype. Following the same rationale, in our previous study, CAR was 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 as a structural template for cells.48 Additionally, homogeneous cell distribution is critical for the distribution of subsequent ECM deposition within the 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 first to screen biomaterials.49 Here, we investigate the cellular interaction of 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 the initial time. The cell proliferation showed an increased tendency in both scaffolds during the cultivation process. However, cell proliferation was enhanced within the oriented scaffolds from days 4 to 28 compared with that of the non-oriented scaffolds (*p < 0.05). This result suggests that the scaffolds with oriented structure provide a comfortable environment for cell proliferation and survival.

Articular cartilage is a complex tissue with unique mechanical characteristic features. Collagen fibril networks in native cartilage are crucial to joint mechanics and exhibit depthdependent 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, which is 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 the conventional freeze−thaw method.45 Most importantly, regardless of the dry or swollen state, the oriented scaffold could always possess anisotropic mechanical characteristics, which is biomimetic for the mechanical feature of cartilage under compression and will meet the demanding mechanical environment of the native tissue. 3.2. Weight Loss of Scaffolds. As shown in Figure 3A, the weight loss of both scaffolds increased as the time increased in PBS at 37 °C. Moreover, the oriented scaffold exhibited relative slower weight loss than the non-oriented scaffold at the same time points. Although the crystallization contributes to the formation of a polymer network, many amorphous regions also exist in the 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 immersion time, all of the samples maintain the original apparent morphology (Figure 4), which is advantageous for 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 for enhancing the stability of the network in PBS. On the contrary, more amorphous species in the non-oriented scaffold will be lost during hydration. In addition, as PVA and 549


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had a noted tendency to attach and proliferate along the axially aligned porous structure. The diverse nature of the tissue histoarchitecture endows the scaffold fabrication with unique inspiration, providing the cells with different microenvironments. The scaffold with the biomimetically aligned structure provides more favorable space for cell attachment, migration, and proliferation, leading to more evenly distributed staining in the oriented scaffold (Figure 6a1, b1). In contrast, a decrease in the cell density within the non-oriented scaffold corresponding to the lack of a 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 the 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 an unfavorable structure. Thus, the cells aggregated at a low density in some part of the non-oriented scaffold, resulting in heterogeneous cell distribution (Figure 6d1). Moreover, both scaffolds could maintain their original shapes and keep the cells viable throughout the entire culture period. The architecture of the scaffolds should emulate the natural design of the ECM and instruct cellular behavior while adequately housing the cells. The cell behavior within the

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

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 in non-oriented scaffolds, and the cells

Figure 6. H&E staining shows the distribution of cells cultured on scaffolds in vitro: (a1−a3) oriented and (b1−b3) non-oriented scaffolds 2 weeks after culture and (c1−c3) oriented and (d1−d3) non-oriented scaffolds 4 weeks after culture. 550


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

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

in oriented scaffolds, whereas the cells within non-oriented scaffolds appeared with no obvious regularity (Figure 7d2, e2, and f2). Both scaffolds could promote the cells to secrete extracellular matrix (ECM) after 4 weeks of culture. SEM results were in accordance with the H&E analysis. Thus, the biomimetic scaffolds provided the cells with a comfortable microenvironment and effectively promoted homogeneous cell distribution within the oriented pores.

scaffolds was also monitored by SEM (Figure 7). It could be observed that the cells were attached well onto both scaffold surfaces at 1 week (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 onto the surface of the unique structure, and grew within the axially oriented pores (Figure 7a2, b2, and c2). From a macro view, the cells exhibited directional distribution 551


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Figure 9. Collagen II immunostaining (brown hue represents the positive staining of collagen II) and immunofluorescence staining on sections of cell-seeded scaffolds. Cell nuclei are stained with DAPI (blue), and collagen II is stained with an anticollagen II antibody (green).

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

within scaffolds (Figure 8B). The suitable structure inside the oriented scaffold provides the cells with adequate space to survive and develop. It demonstrates that the highly organized structure facilitates ATDC5 cells to secrete ECM continuously, and this result is maintained with the extension of cultivation time. The analysis of GAGs and collagen content was consistent with the cell proliferation results, which provides evidence that the biomimetic structure could effectively promote cell distribution and support the cellular function. The distribution of ECM components is very important in tissue engineering. Collagen II, a primary component in cartilage, was examined by immunochemical staining to understand the effect of scaffold structure on the spatial distribution of ECM synthesis. The detection of chondrocyte-

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 culturing was found in both scaffolds, which indicating the preservation of proper cellular functions. However, the amount of GAGs production in the oriented scaffold was significantly enhanced compared with that in the non-oriented scaffold (*p < 0.05), and a similar tendency was also found in deposited collagen 552


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

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 at 2 weeks, although staining appeared to be more intense in the oriented scaffold (Figure 9a) compared with that in the non-oriented scaffold (Figure 9c). Collagen II production was enhanced over culture time and was significantly higher after 4 weeks of 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 deposition of collagen II most effectively. These results were consistent with quantitative analysis of total collagen content (Figure 8B). Moreover, the extracellular matrices in the cell-scaffold constructs for the chondrocyte phenotype were positively stained by toluidine blue (Figure 10), indicating the formation of chondrocyte-specific proteoglycans. In contract to the nonoriented scaffold, the staining color was deeper in the oriented scaffold, which revealed increased production of cartilage matrix proteoglycans. In addition, the stained area in the oriented scaffold was larger than that in the non-oriented scaffold. This result indicated that more proteoglycans were secreted in the matrix of the oriented scaffold, which was consistent with preferable cell affinity for cell proliferation in the oriented scaffold due to its well-interconnected pore structure. There-

fore, the appropriate microstructure provides a comfortable microenvironment for cell growth, which promotes structural homogeneity and leads to a homogeneous ECM dispersion over the scaffold. 3.4. In Vivo Assessment of Scaffolds. Physicochemical properties and cell behavior in vitro provide preliminary information with regard to the suitability of oriented scaffolds. For evaluation 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 in rats, and the rats were healthy 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. For cell infiltration to be detected, the scaffolds were stained with H&E after paraffin embedding and serial sectioning (Figure 11). It was shown that both scaffolds were intact and still displayed obviously porous structures when they persisted in vivo for 1 week. Many cells were detected in both scaffolds, and there was also a substantial penetration of tissue. Because of the short period of subcutaneous implantation, some voids were obvious in the sections, revealing incomplete tissue infiltration within scaffolds at 1 week. Moreover, the cell density in the non-oriented scaffold was relatively lower than that in the oriented scaffold. It is worth noting that the cells moved inward and distributed uniformly in the oriented scaffold. However, cells still aggregated on the side of non-oriented scaffolds close to the skin, and the scaffolds did not effectively support cell migration into the inside after 2 weeks in vivo. 553


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

In comparison to the 2 week sections, H&E staining of the 3 week sections showed that the oriented scaffolds were completely filled with uniform tissue. However, in the nonoriented scaffolds, there was no significant difference in the amount of infiltrated tissue after implantation for 2 or 3 weeks. The ingrowth of tissue could clearly be observed in the outer region of the non-oriented scaffold with some voids in the sections. After 4 weeks of implantation, neither scaffold caused 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 or necrosis upon the implantation of the scaffolds (Figure S1). Compared to the corresponding scaffolds at 1 week, both scaffolds presented higher cell densities after 4 weeks (Figure 11). In the oriented scaffold, because of the better capacity of mass transportation, the uniform cell distribution gave rise to simultaneous tissue growth in the whole scaffold over time. However, the infiltration of cells was still unevenly distributed in the nonoriented scaffold 4 weeks after implantation. The reason may be ascribed to the lack of a permeable structure, which impeded the cell migration toward the internal regions of the nonoriented scaffold. More cells on the side of the 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 inhomogeneous tissue formation inside the scaffold. Obviously, these results were inseparable from the structural properties of the scaffolds. Moreover, it was found that the degree of inflammatory

reaction surrounding the tissue sites where the scaffolds were implanted gradually eased over time. 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 structural stability without shrinkage as evidenced by the minimal change in the pore structures. This is primarily because of the good mechanical behavior of the scaffolds. For most 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 that regularly experience mechanical stress, stronger and more durable support is needed. Hence, the longterm 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 those of the non-oriented scaffolds. Viewing the staining, collagen deposition was observed in the bulk of the scaffolds implanted from 1 to 4 weeks, which suggested that the infiltrating cells could secret their own ECM and remain viable and functional, indicating good biocompatibility of the scaffolds. Importantly, the oriented scaffold displayed an enhanced level of cell infiltration at each time point, resulting in an increasing amount of tissue within the scaffolds. 554


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Figure 13. Immumohistochemical staining against CD31 antibody in scaffolds with different microstructures harvested at 4 weeks post implantation. The arrows indicate the circular and ellipsoid cross sections of the vascular structures present in the infiltrating tissues.

the scaffold should provide a 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 the uniquely oriented structure is more beneficial for promoting cellular and vascular integration within scaffolds and for ensuring an adequate nutrient supply for the survival of neo-tissue. Therefore, the results of subcutaneous implantation can, from a side, reflect that the oriented scaffold has the potential to provide a conducive healing environment that greatly improves the cell−material interaction and that it can be a favorable candidate for cartilage repair.

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 the scaffold needs a sufficient nutrient supply, especially in the center region. Therefore, it is necessary to have a vascular system during the scaffold implantation, which plays a decisive role on the extent of oxygen, nutrients, and metabolic product exchange within the host cell and tissue that infiltrates into the scaffold. Necrosis of cells in the inner region of the scaffolds is inevitable when nutrients are 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 dependent on at least three factors: the biological activity of the scaffold, the microstructure, and the metabolic activity of the 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 to be distributed throughout both scaffolds, which are 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 that with non-oriented scaffolds. An appropriate hyperoxic environment and adequate nutrient supply should be provided for the new cartilage constructs so as to promote tissue growth. Most of the time, convenient nutrient transportation for cartilage constructs plays an important role in tissue engineering.52 As a temporary space,

4. CONCLUSIONS In this study, a biomimetic scaffold was successfully constructed by using a unidirectional freeze−thaw approach. This scaffold had an axially aligned pore structure and maintained good mechanical properties. Noticeably, this scaffold imitated the structural features of natural cartilage, which operated as a template for cell growth along the oriented pore structure. Furthermore, such an oriented scaffold could not only efficiently enhance the uniform distribution of cells in vitro but also promote the regularity of the infiltrating tissue within the scaffold in vivo. Additionally, tissue responses toward the scaffolds in vivo were benign, indicating their suitability for implantation purposes. On the basis of the simple preparation method and the potential to be used as a cell-free scaffold, this oriented scaffold could be a promising tool for applications in 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/acsbiomaterials.5b00535. 555


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H&E staining of the major viscera obtained from rats after subcutaneous implantation for 4 weeks (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (H. Sun). Tel.: +86-3153725740. Fax: +86-315-3726552. *E-mail: [email protected] (J. Li). Tel.: +86-10-68166874. Fax: +86-10-68166874. *E-mail: [email protected] (F. Yao). Tel.: +86-2227402893. Fax: +86-22-27403389. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Nature Science Foundation of China (Grant Nos. 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)âCarrageenan ... - ACS Publications

A Biomimetic Poly(vinyl alcohol)âCarrageenan ... - ACS Publications

A Biomimetic Poly(vinyl alcohol)âCarrageenan ... - ACS Publications

A Biomimetic Poly(vinyl alcohol)âCarrageenan ... - ACS Publications