Materials Science and Engineering C 49 (2015) 174–182

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Pore architecture and cell viability on freeze dried 3D recombinant human collagen-peptide (RHC)–chitosan scaffolds Jing Zhang a, Aimei Zhou a, Aipeng Deng a, Yang Yang b, Lihu Gao a, Zhaocai Zhong a, Shulin Yang a,⁎ a b

School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, China Faculty of Engineering, University of Nottingham, Nottingham NG7 2RD, United Kingdom

a r t i c l e

i n f o

Article history: Received 22 September 2014 Received in revised form 19 November 2014 Accepted 22 December 2014 Available online 24 December 2014 Keywords: 3D scaffold Freezing regime Pore architecture Cell viability

a b s t r a c t Pore architecture of 3D scaffolds used in tissue engineering plays a critical role in the maintenance of cell survival, proliferation and further promotion of tissue regeneration. We investigated the pore size and structure, porosity, swelling as well as cell viability of a series of recombinant human collagen-peptide–chitosan (RHCC) scaffolds fabricated by lyophilization. In this paper, freezing regime containing a final temperature of freezing (Tf) and cooling rates was applied to obtain scaffolds with pore size ranging from 100 μm to 120 μm. Other protocols of RHC/chitosan suspension concentration and ratio modification were studied to produce more homogenous and appropriate structural scaffolds. The mean pore size decreased along with the decline of Tf at a slow cooling rate of 0.7 °C/min; a more rapid cooling rate under 5 °C/min resulted to a smaller pore size and more homogenous microstructure. High concentration could reduce pore size and lead to thick well of scaffold, while improved the ratio of RHC, lamellar and fiber structure coexisted with cellular pores. Human umbilical vein endothelial cells (HUVECs) were seeded on these manufactured scaffolds, the cell viability represented a negative correlation to the pore size. This study provides an alternative method to fabricate 3D RHC–chitosan scaffolds with appropriate pores for potential tissue engineering. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Porous three-dimensional (3D) scaffolds have been extensively utilized in tissue engineering for playing an important role in manipulating cell function and guidance of new tissue formation [1,2] as well as transporting nutrients and removing wastes [3]. In this field, the natural polymer porous scaffolds serve as an analog of the extracellular matrix (ECM) for in vitro study of cell–cell and cell–scaffold interactions and tissue synthesis and in vivo study of induced tissue and organ regeneration [4–6]. Scaffold architecture has a decisive effect in tissue engineering [7]. Ideally, the porosity of the scaffolds, mean pore size and the pore structure should be appropriate for cell adhesion, proliferation, migration, differentiation and extracellular matrix regeneration. The materials should be biocompatible without inflammation or toxicity in vivo and reproducibly processable into 3D structure [1,8]. Collagen is a significant constituent of the natural ECM, and scaffolds made from collagen have been used in a variety of tissue engineering such as skin [9], vessel [10], nerve [11], bone [12] and cartilage [13] due to its biocompatibility and low antigenicity [14]. Additionally, collagen scaffolds have been observed to promote cell attachment and growth [15,16]. However, most commercially available collagen and

⁎ Corresponding author. E-mail address: [email protected] (S. Yang).

http://dx.doi.org/10.1016/j.msec.2014.12.076 0928-4931/© 2014 Elsevier B.V. All rights reserved.

gelatin are derived from animal species whose major drawbacks are possible disease transmission and allergic reactions [17,18]. As a result, many efforts have been made in the field of recombinant protein production to utilize expression systems in a consistent, efficient, and safe manner [19]. With the development of molecular biology and gene technique, recombinant human collagen and gelatin have been produced by insect cells, yeast or many other transgenic systems, and any collagen type can be expressed using the multigen expression technology. Polysaccharide is another extensively used material in tissue engineering because of its numerous benefits [15,20]. Chitosan, a natural polysaccharide produced from the N-deacetylation of chitin, has the ability to foster adequate granulation tissue formation accompanied by angiogenesis and regular deposition of thin collagen fibers which are necessary in wounded skin, nerve, and cartilage tissue regeneration [21]. Therefore, chitosan is a feasible choice to fabricate scaffolds for tissue engineering. A common view had been reached that porosity, pore size and structure have a significant impact in promoting tissue regeneration [2]. High porosity (generally greater than 90%) and a large pore size (about 100 μm–200 μm) as well as highly interconnected pore structure are necessary for the transport of cells and metabolites [22,23]. Whereas the specific situation is dependent on the cell species [3], previous reports have indicated that adhesion, proliferation, and phenotype of cells were affected by the pore size distribution and pore shape of scaffolds [24,25].

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The freeze-dry process is a representative technique for the fabrication of porous materials avoiding the drying stresses and shrinkage that may lead to cracks and warping during normal drying, rather than salt leaching and gas foaming processes [26] which should choose proper porogen [27,28] or need particular instrument [29]. For freeze drying, the pore structure obtained is a replica of entangled dendrites of ice crystals; the diameter, shape and interconnectivity of the pores are regulated by the control of ice crystal growth [30]. Rapid, uncontrolled freezing process results in space and time variable heat transfer through the suspension, leading to non-uniform nucleation and growth of ice crystals, and ultimately, scaffold heterogeneity [31]. It has been reported that freezing at temperatures of −80 °C and below was observed to occur wherein the scaffold invariably cracked [32]. Slowly cooling rate provides a uniform atmosphere allowing the formation of large ice crystals which resulted in a macroporous and isotropic structure [7], with more collapsed pores or sheet-like structure than continuous channels and open cellular structure [2,32,33]. We describe here how cooling regime under moderate conditions (slow cooling rate, high final temperature of freezing), RHC/chitosan suspension concentration and material ratio affect the RHCC scaffold characteristics such as mean pore size, pore microstructure, porosity, swelling, further the influence on cell viability in vitro. 2. Materials and methods 2.1. Materials Recombinant human collagen-peptide (RHC) with powerful hydrophily owing to collagen gene modification was obtained in our laboratory as described previously [34,35]. RHC fermentation supernatant was separated from Pichia pastoris cells by centrifugation at 6000 rpm for 10 min. Subsequently, the supernatant was precipitated by ammonium sulfate in ice-water bath. Afterwards, the sediment was resuspended using deionized water and executed Sephadex G-100 (Amersham Biosciences, Sweden) by gel filtration chromatography. The effluent was detected at 220 nm. Chitosan (deacetylation N 90.0%, viscosity b 100 cps) was purchased from Lanji (Shanghai, China). Primary HUVECs were kindly supplied by Prof. Liang (School of Pharmacy, Whenzhou Medical University, Zhejiang, China), and maintained at 37 °C in a 5% CO2 humidified atmosphere (Thermo Scientific Series 8000 WJ, USA), using Dulbecco's Modified Eagle's Medium-high glucose (DMEM-H, HyClone, Logan, USA) with 1% penicillin/streptomycin and 10% fetal calf serum (Sijiqing, Hangzhou, China). Proanthocyanidin (PA) was obtained from Jianfeng (Tianjin, China). All chemicals of analytical quality were purchased from Sinopharm Chemical Reagent (Shanghai, China). Deionized water was used throughout this study. 2.2. Fabrication of RHCC scaffolds with different pore sizes RHCC scaffolds with different pore sizes were fabricated by freezedrying methods with different protocols. RHC and chitosan were dissolved in 0.12 mol/l acetic acid to 1%, 1.5%, and 2% (wt.%). The RHCC suspension was stirred for 30 min at room temperature (20 °C) and degassed in a vacuum evaporator (Christ, RVC 2-18, German) to remove air bubbles. A 96 well polystyrene plate was used as a mold to fabricate 5.5 mm (diameter) × 5 mm (height) scaffolds. RHCC suspension of 1.5% at a ratio of 1:1 was frozen in preconcerted cooling regime. Different final temperatures of freezing (Tf): The plate containing 200 μl RHCC suspension per well was placed into the uncooling chamber of a freeze dryer (LGJ-10D, Sihuan, China); the temperature of freeze dryer shelf was cooled to −20 °C, −40 °C and −60 °C individually along with the chamber from 20 °C to −65 °C. The temperature of the shelf and chamber was then held at Tf for 6 h to complete the freezing process. Different cooling rates: In the precooling regime,

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the chamber was precooled to − 65 °C and the temperature of the shelf was maintained at −20 °C, −40 °C and −60 °C with instrumental heat compensation, then the plate was placed into the shelf to cool the RHCC suspension. After completely frozen and the temperature of RHCC suspension reduced to −20 °C, the plate was transferred into a refrigerator for 6 h. The solid phase was then sublimated under vacuum to obtain porous scaffolds with different pore sizes. RHC and chitosan suspension of 1% and 2% was mixed at a ratio of 1:1; the suspension of 1.5% was blended at different ratios of 1:4 and 4:1. The degassed RHCC slurry was freeze dried at −20 °C precooling regime. The temperature of the RHCC suspension was monitored during the freezing process at 1 min intervals.

2.3. RHCC scaffold crosslinking All RHCC scaffolds were crosslinked in order to stiffen the interconnected network using PA. 0.5% (wt.%) PA was obtained in PBS (0.3 mol/l, pH 6.8) containing 30% ethanol. RHCC scaffolds were immersed in PA solution for 48 h at room temperature and washed in deionized water (15 min × 4 times), then freeze dried again with the same cooling regime of each scaffold.

2.4. Microstructure observation for pore analysis Micromorphology observation of precrosslinking and postcrosslinking RHCC scaffolds were carried out on a Scanning Electron Microscope (JEOL JSM-6380LV, JEOL, Japan) with an accelerating voltage of 30 kV. Freezedried scaffolds were transversely sectioned then sputter coated with an ultrathin layer of gold. The mean pore size was determined by randomly measuring at least 3 SEM micrographs with 20 pores using ImageJ program. The effective pore diameter (d) was calculated by the relationship: pffiffiffiffiffiffiffiffiffiffi d ¼ l  s, where l is pore long axis length and s is pore short axis length. 2.5. Porosity 2.5.1. Open porosity Open porosity of the prepared scaffolds was determined using the liquid displacement method [36]. The scaffold was immersed in a known volume (V1) of ethanol for 5 min. The total volume of ethanolimpregnated scaffold was recorded (V2). Then the volume of ethanol in the vessel after the removal of ethanol-impregnated scaffold was recorded (V3). Finally, the percentage of porosity (P) of the scaffold was calculated by P(%) = (V1 − V3) / (V2 − V3) × 100 [37]. 2.5.2. Total porosity Total porosity of these scaffolds was evaluated by the following equation: P(%) = (1 − ρ′/ρ) × 100 = (1 −w/ρ/v) × 100, where ρ′ is the density of RHCC scaffold and ρ is the intrinsic density of the mixed materials (1.342 g/cm3 for chitosan [20] and nearly 1.3 g/cm3 for RHC [38]). ρ is calculated as the same ratio of RHC and chitosan in different scaffolds. w and v are the mass and volume of RHCC scaffolds, and the diameter and thickness of the scaffolds were measured to estimate porosity [20,37].

2.6. Swelling Dry scaffolds were weighed (W0) and then immersed in phosphate buffer solution (PBS, pH 7.4) at room temperature for 5 min and weighed (W). After removing the water, scaffolds were immersed in PBS again. The weights were recorded, and this procedure was repeated until equilibrium stage was reached. The swelling ratio was calculated as the wet weight increase to initial weight: S = (W − W0)/W0.

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2.7. Cell viability HUVECs at 5 × 104 cells in 2 ml culture medium were seeded in 35 mm culture dishes for 24 h. The culture medium was replaced by extraction of postcrosslinking RHCC scaffold of 1.5%, 1:1 ratio, − 20 °C precooling regime, and complete culture medium as control. The scaffold leaching liquor was extracted with a surface area/extracting vehicle ratio of 1.25 cm2/ml according to the ISO 10993 guideline; the culture medium was replaced every two days. At defined time, cells were washed by PBS and stained in a mixed dye reagent containing 100 μg/ml AO (Acridine Orange) and 100 μg/ml EB (Ethidium Bromide). After incubation, specimens were imaged at the WU module using a Fluorescent Microcopy (Olympus IX81, Japan). 3-D scaffolds were placed in a 48 well plate and presterilized with UV exposure for 24 h. Equal numbers of HUVEC cells (1 × 105) were seeded on each scaffold. In brief, total cells were suspended in 50 μl medium for efficient seeding with minimum spillage outside the scaffold matrix. After 2 h of initial cell attachment, 1 ml of complete medium was added to each well. Fresh medium was replenished every two days for 3 days. Cell proliferation on 3D scaffold constructs was monitored by AO/EB staining. All fluorescence staining images were processed by Image Pro Plus 6.4 as well as quantitative analysis of live cell count through fluorescence density. 2.8. Statistical analysis All scaffolds fabricated in different protocols were run no less than triplicate and the characteristics are expressed as mean ± SEM. Error is reported in the mean pore sizes as the standard deviation (SD) and as the coefficient of variance (CV = standard deviation/mean). Statistical analysis of data was performed by Student t test and one-way analysis of variance (ANOVA). Differences between groups of p ≤ 0.05 were considered statistically significant. 3. Results 3.1. Cooling rate and frozen time of RHCC suspension The temperature of RHCC suspension during cooling process under different cooling regimes was determined as shown in Fig. 1. An initial decrease in the time–temperature curve occurred until − 11 °C in the uncooling regime (with different Tf) and faded in the precooling regime,

Fig. 1. Average temperature of the RHCC suspension during freezing for six different cooling regimes. Uncooling attributed to different final temperatures of −20 °C, −40 °C, and −60 °C. Precooling belonged to different cooling rates represented by temperatures of −20 °C, −40 °C, and −60 °C.

then a sharp rise to 0 °C or − 1 °C where it was holding for several minutes. The average freezing rate of the suspension in °C/min under all freezing protocols was determined between the starting temperature (20 °C) to a lower limit. A lower limit of 5 °C greater than the final temperature of freezing was established for this experiment to obtain a linear mold [31]. The frozen time was defined as the liquid–solid transition time, which corresponds to the time where liquid and solid coexist [31]. It was measured as the summation of ice crystallization time (the suspension temperature remained between 0 °C and −1 °C following the initial supercooling condition) and water-RHC–chitosan crystallization (the suspension temperature maintained around −12 °C until a sharp decline on the time–temperature curve) (Fig. 1). Fig. 2 shows the cooling rate and frozen time in different cooling regimes. In the uncooling regime, the cooling rates of RHCC suspension were nearly identical in the average of 0.7 °C/min so the frozen rate was not affected by Tf significantly. However, in the precooling regime, the cooling rate of RHCC suspension was significantly increased from 1.7 °C/min to 2.9 °C/min and 4.1 °C/min while the frozen time was shorten from 28 min to 9.9 min and 6.1 min individually compared to the average 30 min of the uncooling regime. 3.2. Influence of freezing protocol on pore structure Precrosslinked and postcrosslinked porous RHCC scaffolds fabricated by different freezing protocols were judged by SEM as shown in Fig. 3 while the mean pore sizes were summarized in Table 1. 3.2.1. Cooling regime All the RHCC scaffolds fabricated in different cooling regimes depicted a cellular microstructure in Fig. 3 (A–F, a–f): under the uncooling regime, the largest mean pore size of 120 μm was achieved at the lowest cooling rate and highest Tf. The mean pore size decrement was about 10 μm when the Tf decreased from −20 °C to −60 °C, but the coefficient of variance (CV) of the mean pore sizes was nearly the same. Compared to the uncooling regime, the precooling regime presented a more rapid cooling rate with the same Tf. It showed that in − 20 °C and −40 °C precooling regimes, the mean pore sizes was nearly about 110 μm but CV in the latter was smaller than the former. Similar results could be observed after crosslinking. After the second freeze drying process, the mean pore size was nearly the same compared to the precrosslinking scaffold. However, the CV in uncooling regime was gradually reduced along with the decrease of Tf. Meanwhile, in the postcrosslinking scaffolds of − 20 °C uncooling and − 40 °C uncooling regimes, elongated sheet-like pores appeared (Fig. 3a, b) and this phenomenon was not observed gradually with the decrease of Tf and increase of cooling rate.

Fig. 2. Cooling rate and frozen time under different cooling regimes. Un attributed to different final temperatures of −20 °C, −40 °C, and −60 °C. Pre belonged to different cooling rates represented by temperatures −20 °C, −40 °C, and −60 °C.

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Fig. 3. Transversal SEM micrographs of RHCC scaffolds under different freezing protocols. (A–C) SEM images of precrosslinking RHCC scaffolds (1.5%, 1:1) with different final temperatures of −20 °C, −40 °C, −60 °C uncooling. (D, E) SEM images of precrosslinking RHCC scaffolds (1.5%, 1:1) with different cooling rates of −40 °C and −60 °C precooling. (F–H) SEM images of precrosslinking RHCC scaffolds (1:1) of −20 °C precooling with concentrations of 1.5%, 1% and 2%. (I, J) SEM image of precrosslinking RHCC scaffolds (1.5%) of −20 °C precooling with RHC/ chitosan ratios of 4:1 and 1:4.

3.2.2. RHC/chitosan concentration Fig. 3(G–H, g–h) shows the microstructure of RHCC scaffolds fabricated with different concentrations under − 20 °C precooling regime. Apparently, at a low concentration (1%), variation in pore structure

could be observed. It significantly becomes more homogeneous with the increase of concentration. However, the mean pore size reduced to 104.5 μm when the concentration increased to 2% and the wall (distance between neighboring pores) turned thicker.

Table 1 Mean pore sizes of RHCC scaffolds under different freezing protocols. Cooling regime

Concentration (g/L)

RHC ratio (%)

−20 °C uncooling −40 °C uncooling −60 °C uncooling −20 °C precooling −40 °C precooling −60 °C precooling −20 °C precooling −20 °C precooling −20 °C precooling −20 °C precooling

15 15 15 15 15 15 10 20 15 15

50 50 50 50 50 50 50 50 80 20

Diameter (μm)

CV

Precrosslinking

Postcrossliking

Precrosslinking

Postcrossliking

119.66 ± 7.18 116.43 ± 9.11 111.97 ± 7.64 109.39 ± 7.28 110.78 ± 4.54 104.59 ± 4.63 120.02 ± 14.74 104.52 ± 4.73 105.79 ± 3.79 113.84 ± 5.12

120.18 ± 7.07 116.10 ± 4.97 110.69 ± 3.30 112.88 ± 5.54 110.96 ± 3.68 105.16 ± 4.86 115.57 ± 7.69 105.62 ± 5.39 107.10 ± 5.49 112.08 ± 4.29

0.060 0.078 0.068 0.066 0.041 0.044 0.123 0.045 0.036 0.049

0.059 0.043 0.030 0.049 0.033 0.046 0.067 0.051 0.051 0.038

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The mean pore size of postcrosslinking scaffolds of different concentrations was close to the precrosslinking scaffolds except the 1% scaffold that changed from 120 μm to 115 μm, whereas the CV of postcrosslinking scaffold of 1% decreased. 3.2.3. RHC/chitosan ratio Fig. 3(I–J, i–j) shows the microstructure of RHCC scaffolds fabricated with different RHC/chitosan ratios under −20 °C precooling regime. In the scaffold with an RHC/chitosan ratio of 4:1, a cellular, lamellar, and fiber structure coexisted and it could not maintain the pore structure well after crosslinking. As the RHC ratio in RHCC suspension is higher, the mean pore size is smaller (here the cellular interconnected pores were calculated in low RHC/chitosan ratio scaffolds). The mean pore size of different RHC/chitosan ratio scaffolds was almost not changed after crosslinking. The CV was not linear to the ratio that the largest approached in 1:1, in other words, the purer the RHCC suspension, the lower the CV. 3.2.4. Pore anisotropy Variation is observed in the microstructure but CV couldn't present a significant distinction in pore anisotropy. All of the pore channels have been characterized by two-dimensional parameters: the long axis and the short axis in Figs. 4 and 5. Either with the decrease of Tf or with the increase of cooling rate, the residual of long axis and short axis reduced gradually and contributed to a more homogenous pore structure controlled by the cooling regime. The residual lessened after crosslinking while the trend was the same as precrosslinking. In different concentrations and RHC/chitosan ratio protocols, the residual trend was nearly the same as the CV evaluated to the mean pore

Fig. 5. Length of long axis and short axis and the residual of precrosslinking and postcrosslinking RHCC scaffolds of different concentrations and RHC/chitosan ratio under −20 °C precooling regime.

size in precrosslinking scaffolds. After crosslinking, the residual was decreased in both of the two protocols. 3.3. Porosity Comparing porosities in precrosslinking and postcrosslinking RHCC scaffolds under different freezing protocols in Fig. 6, porosity remained almost constant at each cooling regime although the pore size and anisotropy varied positively. All these precrosslinking scaffolds under different cooling regimes had high porosities exceeding 95%; no significant difference could be observed in different RHC/chitosan ratio scaffolds in the average porosity of 96%. However, in comparison of different concentration precrosslinking scaffolds, the highest porosity of 96.7% was achieved in 1.5%. Whereas after crosslinking, the porosity of all these RHCC scaffolds went down when the lowest was dropped to 90% in 1% scaffold. 3.4. Swelling Fig. 4. Length of long axis and short axis and the residual of precrosslinking and postcrosslinking RHCC scaffolds under different cooling regimes.

As shown in Fig. 7, the swelling property of postcrosslinking scaffolds was influenced distinctly by the freezing protocol. Under different

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Fig. 6. Open porosity (O) and Total porosity (T) of precrosslinking and postcrosslinking RHCC scaffolds under different freezing protocols.

cooling regimes, the − 60 °C precooling scaffold could bind the least about 27 fold of PBS. The scaffolds could bind more PBS with the admission of chitosan for the swelling value of RHC/chitosan scaffold in 1:4 could reach 40. The highest swelling value was achieved at 37 in 1.5% scaffold when the variation trend is similar to the porosity. After evaluating the PBS absorption procedure repeatedly, the swelling value decreased and the equilibrium stage could be reached after 5–6 times. In the comparison of swelling property, the first PBS absorption was analyzed.

3.5. Cell viability To investigate the cell toxicity of RHCC scaffolds degraded substance and cell viability on these scaffolds fabricated under different protocols, AO/EB fluorescence staining was carried out. The cell viability of HUVEC is shown in Fig.8 after seeding on 35 mm plastic culture dishes and exposing directly to RHCC scaffolds (1.5%, 1:1 ratio, -20 °C precooling regime) extraction up to 96 h. After 96 h, no apoptosis (reddish orange) could be observed and cell membrane maintained its integrity. Cell growth on RHCC scaffolds was exhibited in Fig. 9. Under different cooling regimes, profuse cell growth was observed in smaller pore size scaffolds (Fig. 9C, D, E, F). The cells covered the scaffold surface filling up the pores. In higher magnification (Fig. 9b, e), the cells were observed to align and attach onto the scaffold walls for support. It was obvious that the cells grow intensively in the 2% concentration scaffold with smaller mean pore size and isotropic pore structure than in 1%. No visible difference in cell proliferation and spreading was noticed between different ratio scaffolds.

4. Discussion In consideration of previous report, rapid cooling rate causes lots of ice crystal nucleus resulting in the formation of smaller-sized pores, e.g. liquid nitrogen (fast cooling rate and low Tf) can reduce the pore size and orient the ice crystal due to the perpendicular enlarged anisotropic pore structure [30,31,38]. This study concerns about the inerratic and interconnected pore structure resulting from the slow cooling rate, namely, pore size and its structure controlled by a moderate heat transfer rate. When the RHCC suspension temperature decreased to supercooling condition which is required to initiate ice crystal nucleation, a sharp rise in the time–temperature curve due to the heat release during water crystallization is presented. The heat removal rate by the cold air is not sufficiently rapid under the uncooling regime even in − 20 °C precooling, thus the RHCC suspension temperature rises. The sharpness of temperature rise suggests that crystallization begins simultaneously in the entire solution which indicates that a slow cooling rate provides a nearly homogenous cold atmosphere [2]. When the heat removal is efficient enough in the precooling regime, the supercooling point disappeared. Heat transfer rate affects the pore size and inner structure, since the solvent crystallizes before liquid–liquid phase separation occurs; the solid–liquid phase separation was responsible for the final morphology [39], which in this study depended on the crystallization mode of the water. Under the uncooling regime with a nearly constant cooling rate of 0.7 °C/min, low Tf results to a more homogenous microstructure. This is probably because when the cold air is not able the remove the heat released during ice crystallization efficiently, the released heat may melt small ice crystals and allows the growth of large crystals

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Fig. 7. Swelling of postcrosslinking RHCC scaffolds under different freezing protocols.

until neighboring crystals meet [2]. Such behavior may explain the interconnected pore morphology and larger mean pore size in high Tf, so a slow cooling rate could induce large pore size but if the Tf is too high to remove the release heat ineffectively, the structure would be heterogeneous. In comparison of the mean pore size under the precooling regime with the same Tf, a trend of decrease and more homogenous structure could be observed with the increase of cooling rate. There was no measurable jump in temperature along the cooling process in −40 °C and −60 °C precooling regimes, suggesting that the heat removal rate is efficient under these conditions, so the mean pore size noticeably scaled down and a more homogenous microstructure was obtained. Therefore, we assume that under a moderate condition (the cooling rate is managed below 5 °C/min and the Tf exceeds − 60 °C), the slow cooling rate could lead to a large pore size but heterogeneous structure, meanwhile, a small pore and more homogenous structure could be obtained under a rapid cooling rate and appropriate heat compensation. Pore size can be controlled by varying the concentration of the material during dispersion preparation. In different concentration

protocols, 1% scaffold reflected heterogeneous pores with a sheet like microstructure, and high CV and residual also indicated varied mean pore sizes and irregular pore structure. This may be caused by low viscosity dispersion which could result in a fibrous structure [32]. With the increase of concentration, pores become homogenous and the mean pore size decreased significantly which may due to the thick walls [30]. With a high ratio of RHC, the scaffold reflected a varied microstructure which may result from the natural fiber structure of collagen. A small amount of threadlike structure connected the pores or existed in the space among channels. This is due to the nonuniform pore walls containing thin and weak connection of pores. After crosslinking, the mean pore sizes were almost unchanged while the pore structure became more homogenous according to SEM images and residual values. However, in uncooling regime, elongated pores like cracking occurred, and similar phenomenon appeared in 4:1 ratio scaffold. In the second freeze process, the pores of thin walls formatted in the first freeze drying may be cracked with ice crystal growth, and environment for weak heat removal may perforate adjacent pores

Fig. 8. Fluorescence images and live cell count of HUVEC cultured with complete medium and extraction of postcrosslinking RHCC scaffolds (1.5%, 1:1 ratio, −20 °C precooling regime) under AO (Acridine Orange for live cells, green) and EB (Ethidium Bromide for dead cells, red). (A−D) control and (a–d) extraction for 24 h, 48 h, 72 h and 96 h. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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residual. In view of these results, we assume that swelling is not only related to the mean pore size but also correlated to the homogeneity of pore structure. Notably, there might be an appropriate concentration to obtain high swelling property rather than the lowest or highest similar to the influence of suspension ratio [42]. After 5 times repeat procedure, the PBS absorption tended to an equalized value. It may assume that the reusability of these scaffolds are improved for they could uptake water repeatedly. Cell adhesion is the first cellular event that occurs when a cell comes in contact with a material surface, and it has a strong influence on the subsequent cellular events such as proliferation and differentiation [43]. Cell viability assays have indicated that all these RHCC scaffolds are non-toxic to cells and it is suitable for cell culture on these 3D scaffolds with a mean pore size range from 100 μm to 120 μm. There were no significant effects to cell attachment, but the proliferation was better in smaller pore size scaffolds in contrast to the previous hypothesis [44]. This may be achieved attributing to the high porosity of all these cases and endothelial cells bind preferentially to scaffolds with smaller pores [7,45]. Compared to the large pore size scaffolds, cells grew better in 1% scaffold (Fig. 9G) than in 1.5% scaffold (Fig. 9A) whose porosity was even larger. This may resulted from the difference of anisotropy. We assume that HUVECs prefer not only small pores but also homogenous and inerratic pore structure. 5. Conclusion

Fig. 9. Fluorescence images and live cell count of HUVEC cultured on postcrosslinking RHCC scaffolds fabricated with different freezing regimes under AO/EB staining. (A–C) RHCC scaffolds (1.5%, 1:1) with different final temperatures of − 20 °C, − 40 °C, and −60 °C uncooling (10×). (D–F) RHCC scaffolds (1.5%, 1:1) with different cooling rates of −20 °C, −40 °C, −60 °C precooling (10×). (G, H) RHCC scaffolds (1:1) of − 20 °C precooling with concentrations of 1% and 2% (10×). (I, J) RHCC scaffolds (1.5%) of −20 °C precooling with RHC/chitosan ratio of 4:1 and 1:4 (10×). (b, e) Scaffolds as B and E imaged under 20×.

This work shows that homogenous, isotropic RHC–chitosan 3D porous scaffolds with appropriate structure for distinct cells could be controlled by changing the freezing regime, suspension concentration and ratio during freeze drying process. A relatively rapid cooling rate and efficient heat removal environment produced scaffolds of small pores with a uniform and equiaxed microstructure. The cell viability under treatment of RHCC scaffold extraction indicated the materials are safe in bioengineering application. Further investigation of cell seeding on 3D scaffolds indicated that pore architecture plays an important role in cell survival; hypothetically the pore size is negatively correlated to the HUVEC viability but would not be expected to continue for cell activity within porous scaffolds which indicated that there exists an optimal pore architecture for each distinct cell type. This work provides information for RHC–chitosan scaffold application in tissue engineering, whereas different cell types should investigated since specific cell types respond discrepantly to specific tissue systems. Acknowledgments

leading to an elongated sheet like structure. On the other hand, unorganized ice crystal growth may induce irregular sublimation with recrystallization of water vapor and secondary sublimation in the same areas during freeze-drying procedure [32]. The two times freezing might explain in part the partial collapse of pores during freeze drying and formation of a sheet-like structure (Fig. 3a, b). The total porosity is independent on different freezing protocols, so as to the open porosity except in different concentrations. This may because under rapid cooling rate, a large number of small sized ice crystals was produced. The total pore volume is not affected by the pore structure. The porosity of all scaffolds decreased indicating that the matrix was distorted to form a denser structure by cross-linkage [40], with the participation of PA, new hydrogen bond results to tangled structure which alleviates the irregularly [41]. The relationship between porosity and initial suspension concentration was nearly linear as demonstrated [30], however, the open porosity of 1% scaffold dropped off apparently after crosslinking which may be relative to the change of mean pore size and microstructure. The trend of swelling is similar to the mean pore size under different cooling regimes. But in different concentration protocols, the swelling value of 1% scaffold is decreased and the trend is contrary to the

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