Carboxylated Agarose (CA)-Silk Fibroin (SF) Dual Confluent Matrices

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Carboxylated Agarose (CA)-Silk Fibroin (SF) Dual Confluent Matrices Containing Oriented Hydroxyapatite (HA) Crystals: Biomimetic Organic/Inorganic Composites for Tibia Repair Jingxiao Hu, Jiabing Ran, Si Chen, Pei Jiang, Xinyu Shen, and Hua Tong Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00587 • Publication Date (Web): 17 Jun 2016 Downloaded from http://pubs.acs.org on June 18, 2016

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Carboxylated Agarose (CA)-Silk Fibroin (SF) Dual Confluent

Matrices

Hydroxyapatite

(HA)

Containing Crystals:

Oriented Biomimetic

Organic/Inorganic Composites for Tibia Repair Jing-Xiao Hu, Jia-Bing Ran, Si Chen, Pei Jiang, Xin-Yu Shen and Hua Tong* Key Laboratory of Analytical Chemistry for Biology and Medicine, Ministry of Education, College of Chemistry and Molecular Sciences, Wuhan University, 430072, PR China

ACS Paragon Plus Environment

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ABSTRACT: By in situ combining the dual crosslinking matrices of the carboxylated agarose (CA) and the silk fibroin (SF) with the hydroxyapatite (HA) crystals, the CA-SF/HA composites with optimal physicochemical and biological properties were obtained, which were designed to meet the clinical needs of load-bearing bone repair. With the synergistic modulation of the dual organic matrices, the HA nanoparticles presented sheet and rod morphologies due to the preferred orientation, which successfully simulated the biomineralization in nature. The chemical reactivity of the native agarose (NA) was significantly enhanced via carboxylation, and the CA exhibited higher thermal stability than the NA. In the presence of SF, the composites showed optimal mechanical properties which could meet the standard of bone repair. The degradation of the composites in the presence of CA and SF was significantly delayed such that the degradation rate of the implant could satisfy the growth rate of the newly formed bone tissue. The in vitro tests confirmed that the CA-SF/HA composite scaffolds enabled the MG63 cells to well proliferate and differentiate, and the CA/HA composite presented greater capability of promoting the cell behaviors than the NA/HA composite. After 24-day of implantation, newly formed bone was observed at the tibia defect site and around the implant. Extensive osteogenesis was presented in the rats treated with the CA-SF/HA composites. In general, the CA-SF/HA composites prepared in this work had the great potential to be applied for repairing large bone defect.

KEYWORDS: Carboxylated agarose; Silk fibroin; Biomineralization; Hydroxyapatite; Biocompatibility.


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1. Introduction Therapies for the repair or replacement of load-bearing biological tissues remain a great challenge for researchers, as this requires artificial biomaterials with desirable biocompatibility and sufficient mechanical properties.1-3 All the biomaterials for bone tissue engineering should follow the principles of mimicking the structure and function of natural bone tissues. Biomacromolecules are mostly employed to synthesize artificial biomaterials, including those of natural source and other synthetic polymers.4-10 Of particular interest are various natural polymers which have emerged as optimal candidates for materials science, due to their low toxicity, availability, favorable bioactivity, and other desirable properties.8, 11-13 At present, more and more researchers devote to endowing the natural polymers with more specific functionalities to tailor the biological, mechanical, degradation properties for special applications, such as modification with specific active groups, grafting polymers, and so on.14-17 For bone tissue engineering, the biomaterials should be designed to have almost the same structure and properties with those of the natural bone tissues, especially for load-bearing bone tissues. Besides biocompatibility, the implants must have sufficient mechanical strength to support the human body.18-20 The main organic component of the natural bone is collagen, and inorganic minerals are hydroxyapatite (HA) crystals. Therefore, the researchers typically select proper macromolecules as the organic template, and the HA particles as the inorganic phase, to mimic the composition with accurate weight ratio of the natural bone.21 However, the morphology of the HA particles is difficult to regulate and control without well-designed methods. The HA particles that are at nanoscale with homogeneous dispersion among the organic matrix in the natural bone present specific morphologies. In the biomineralization process of bone, the uniaxially oriented nanocrystals of HA are preferentially aligned with (001)


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along the long collagen fibril axes, due to the reactive residues on the chains of collagen.21 Therefore, the crystal nucleation and growth can be directed by the organic matrix. This has inspired many researchers to regulate and control the morphology of HA crystals via various methods. Akkus and co-workers systematically carried out a comparative study of effects of typical polyelectrolytic peptides on regulating the size and morphology of HA crystals, and concluded that the polymers of greater affinity had the most significant effect on modulating the crystal growth.22 Grondahl and co-workers concluded that peptides with functional groups had stronger capability of controlling the crystal growth and the resultant morphology of the HA particles than the polysaccharides carrying the same functional groups.23 Huang and co-workers successfully synthesized the water-dispersible nano-HA regulated by the silk fibroin (SF), which proved again that the protein with active groups could guide the crystal nucleation and growth efficiently.24 These studies provide cues to prepare HA-based composites with specific morphology which can be more analogous to the natural. In the current study, native agarose (NA) was selected to be the source of organic matrix, and SF acted as the morphology regulator and network reinforcement. HA crystals were formed in situ in the organic network. Agarose, a neutral polysaccharide extracted from marine algea, can form a stable hydrogel under physiological conditions in the absence of any chemical crosslinking agent.25, 26 Due to its desirable biocompatibility and high availability of preparing hydrogel, agarose has attracted increasing attention. However, the high hydrophilicity and lack of functional groups absorbing cell adhesive proteins to the NA gel makes cell adhesion and growth difficult, which is attributed to the lack of the functional groups absorbing the cell adhesive proteins.27 NA can’t be directly employed in many delicate chemical or biological reactions. Modification is an effective way of introducing functional groups to the native


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polymer chains. Herein, carboxylated agarose (CA) was successfully prepared to crosslink with SF via carbodiimide chemistry; and the inorganic precursor salts were immobilized within the three dimensional network to mimic the natural biomineralization. The main goals of this work were to: (1) strengthen the reaction activity of agarose via modification to improve biological properties; (2) investigate how the modified organic matrices regulate and control the morphology of HA crystals; (3) evaluate the physicochemical and biological properties of the CA-SF/HA composites designed for bone repair. 2. Materials and methods 2.1.

Materials

Agarose (Mw = 306.26 KDa, gel strength > 600 g/cm2, Biological Reagent), was purchased from Regal Biotechnology Company (Shanghai, China). TEMPO ((2, 2, 6, 6-Tetramethylpiperidin-1yl) oxyl), EDC-HCl, and NaClO solution (Analytical Reagent) were purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O)), diammonium

hydrogen

phosphate ((NH4)2HPO4), LiBr, acetic acid and ammonia were

purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). FITC-Phalloidin was provided by Yeasen Co. (Shanghai, China). Cell counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Japan). Alkaline phosphatase (ALP) assay kit was purchased from Yeasen Co. (Shanghai, China). Commercial QuantiChromTM Calcium Assay Kit (Bioassay Systems, USA) was purchased. All chemicals were used without any further purification. All the aqueous solutions were prepared using ultra-pure water with a resistance of 18.2 MΩ.cm. 2.2. Methods


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2.2.1. Preparation of carboxylated agarose (CA) The preparation method was reported previously.27, 28 Firstly, the NA (1.0 g) was dissolved in ultra-purified water (200 mL) with vigorous stirring at 93ºC. Then TEMPO and NaBr were added to the above agarose solution with vigorous stirring at 4ºC. After dissolving, the NaClO solution was added dropwise to the solution. The pH value was adjusted to 10.8 and maintained throughout the following 2 h-reaction. The oxidation reaction was quenched by adding 500 mL ethanol. The precipitates were collected via centrifugation, and impurities were removed via rotary evaporation at 40ºC. Then the purified precipitates were dissolved in ultra-purified water with heating, and the pH value of the solution was adjusted to 7.0. The products were dialyzed against ultra-purified water for 5 days, and the water was changed every 12 h. Finally, the oxidized agarose was freeze-dried for 48 h, the spongy solid was obtained. 2.2.2. Preparation of the SF solution The preparation method has been reported earlier.29, 30 In brief, the cocoons were cut into small pieces, and degummed in NaCO3 (0.5w/v %) solution with boiling for 20 min, which was repeated three times. After air drying, the degummed silk was then dissolved in LiBr (9.0 M) solution. Then the silk fibroin solution was dialyzed against ultra-purified water for 3 days with water change every 12 h. 2.2.3. Preparation of the CA-SF/HA composites Firstly, the CA was dissolved in 20 mL ultra-purified water with vigorous stirring at 90ºC. Acetic acid (400 µL) was then added to the CA solution. After that, Ca (NO3)2·4H2O and (NH4)2HPO4 (Ca/P = 1.67) were dissolved in the solution with vigorous stirring, and EDC-HCl was then


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added to activate the carboxyl groups for 30 min. Then the SF solution was added dropwise with gentle stirring for 30 min at room temperature. A stable hydrogel was then formed, and the ammonia liquid was permeated throughout the hydrogel to complete the mineralization process. The CA/HA and NA/HA composites were set as the control groups. The starting content of all reagents was scaled according to the final organic/inorganic weight ratio of 60/40. The specific usage was shown in Table 1.

Table 1. The usage of the chemical reagents.

2.2.3. Preparation of the composite scaffolds The composite scaffolds were prepared by the freeze-drying method. In brief, the composites were first processed by liquid nitrogen for 10 min, and then kept in refrigerator at –20ºC for 24 h, the hierarchical porous scaffolds were prepared by lyophilizing for 48 h (–50ºC, 0.03–0.05 mBar). 2.3.

Characterizations

2.3.1. Compositional determination


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Fourier transform infrared spectrometry (FTIR; Nicolet5700, USA) and energy dispersive X-ray analysis (EDS) were employed to investigate the compositional characteristics. 2.3.2. Crystal phase The crystal phase was characterized by wide-angle X-ray diffraction analysis (XRD, X’pert Pro, PANalytical, Holland). The working conditions of the X-ray diffractometer were CuK0 radiation via a rotating anode at 40 kV and 40 mA. The data were collected in steps of 0.026° s-1 with the scattering angles ranging from 10° to 80°. 2.3.3. Morphological investigations The morphology of the composites was investigated by field emission scanning electron microscope (SEM, Sigma, Zeiss, Germany) and Transmission Electron Microscope (TEM, 2010FEF, JOEL, Japan). 2.3.4. Thermal stability The thermal stability of the composites was investigated by thermogravimetric analysis (TGA) and differential scanning calorimeter (DSC) with a thermo analyzer (NETSCH, Germany). Samples were placed in the aluminum cells and heated from 20ºC to 600ºC at a heating rate of 10ºC/min using nitrogen as purge gas. 2.3.5. Mechanical properties Compression strength tests were performed at room temperature using a universal testing machine (SHIMADZU, AGS-J, Japan) at a cross-head speed of 0.5 mm min-1. The dry specimens were shaped into circular discs of approximately 5 mm diameter and 5 mm height by


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polishing with a sand paper. The mechanical properties were evaluated by calculating the elastic modulus of the samples, using the slope of the initial linear portion of the stress–strain curve. Three specimens of each group were tested, and the data were presented as the means ± standard deviations. 2.3.6. Degradability The degradation of the composites in aqueous environment was investigated. The gravimetric method was used to analyze the degradation. Firstly, the dry weights of the samples were measured (W0). The samples were then immersed in PBS and incubated at 37ºC for three weeks. The dry weights of the samples were measured (Wt) on each day. Then the degradation (%) was obtained using the following equation: Degradation (%) =

ௐ଴ିௐ௧ ௐ଴

× 100%

2.4. In vitro tests 2.4.1. Cell culture In this work, MG63 osteoblast-like cells were selected as the culture model, which were obtained from the Zhongnan Hospital of Wuhan University. MG63 cells have been widely used as an ideal culture model for characterizing cell adhesion, proliferation, and differentiation. Herein, MG63 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. The medium was refreshed every 2 days. The cells were treated with trypsin prior to seeding onto the composites. 2.4.2. Cell adhesion


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The MG63 cells were seeded onto the thin composite films and cultured for 24 h at 37°C in a 5% CO2 incubator. After removing the old medium, the cells were treated by fixation with 4% paraformaldehyde at room temperature for 15 min. The cell membrane was permeablized with 0.5% Triton-X-100 for 10 min. Cells were then processed with 3% FBS for 30 min. Each preparation step was followed by rinsing with PBS. The samples were subsequently incubated with FITC-Phalloidin (1:800) for 45 min at room temperature in the dark. The images were photographed with an inverted fluorescence microscopy (DSY5000X, Chongqing, China). 2.4.3. Cell morphology The MG63 osteoblast-like cells were seeded on the composite scaffolds for 24 h. After cultivation for 24 h, composite scaffolds grown with cells were washed twice with PBS, and cells were fixed with 2.5% glutaraldehyde at 4°C overnight. The cells were dehydrated in a graded ethanol series (30, 50, 70, 90, 95 and 100 vol %), freeze-dried and sputter-coated with gold prior to SEM observation. 2.4.4. Cell proliferation The MG63 osteoblast-like cells (2.0×103 cells/well) were seeded on each composite scaffold (n=4) placed in a 24-well plate (Corning Life Sciences, USA). The plate was incubated in DMEM containing 10% FBS at 37°C in a 5% CO2 incubator for 7 days, and the cell proliferation was studied using a cell counting kit-8 assay (CCK-8; Dojindo Laboratories, Japan) according to the manufacturer’s instructions. Briefly, 450 µL serum-free culture medium and 50 µL CCK-8 solutions were added to each sample. After incubation at 37°C for 3 h, the supernatant was transferred to a 96-well plate and the optical density was measured at 450 nm using an ELX808


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Ultra Microplate Reader (Bio-Tek Instruments, Inc., USA). The cell proliferation was measured on days 1, 4, and 7. 2.4.5. Cell differentiation The alkaline phosphatase (ALP) enzymatic activity was measured to evaluate the early osteogenic differentiation of MG63 osteoblast-like cells on the composite scaffolds. The cells were seeded on the composite scaffolds in a 24-well plate at a density of 2×104 cells/well. The ALP activity was measured with a commercial kit (YEASEN, Shanghai, China) on days 7 and 14. At each time point, the cell-seeded disks were rinsed three times with PBS, and treated with RIPA lysis buffer (YEASEN, Shanghai, China) for 2 s. After removing the sediment by centrifugation, a 50-µL aliquot of each supernatant was transferred to the 96-well plate, followed by addition of 50 µL of assay buffer and 50 µL of p-nitrophenyl phosphate (pNPP) solution. After incubation for 30 min at 37°C, 100 µL of stop buffer was added to the samples and the absorbance was read in the microplate reader at 405 nm. 2.4.6. Calcium deposition The MG63 cells were seeded onto the composite scaffolds in a 24-well plate and cultured for 21 days. On day 21, 0.5 M HCl was added to each well with samples, the supernatants were collected and transferred to a 96-well plate, and the calcium deposition was measured using a commercial QuantiChromTM Calcium Assay Kit (Bioassay Systems, USA). According to the manufacturer’s instructions, quantitative determination of calcium ions by the colorimetric (612 nm) method was used. A microplate reader was employed to determine the optical density at 612 nm.


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2.5. In vivo transplantation of CA-SF/HA composites SD male rats at the age of 5 months were used for the in vivo transplantation study of CA-SF/HA composites. Sprague−Dawley (SD) rats were purchased from the Laboratory Animal Center of Zhongnan Hospital of Wuhan University (China), and all of the animal experiments were approved by the Animal Research Committee of Zhongnan Hospital of Wuhan University and conducted in accordance with the guidelines of the Experimental Animals Management Committee (Hubei Province, China).The surgical procedures are illustrated in Figure 1. The rats were adapted to the cage life for 7 days prior to the surgery. During this period, the weight of the animal was monitored to ensure stability and adaptability. Before the surgery, the animals were anesthetized with pentobarbital sodium solution, following the approved ethical protocol. During the surgical procedures, the animal was placed on a warm plate to maintain body temperature. Skin around the tibia was carefully shaved and disinfected with 5% povidone-iodine (Changdao, Shanghai, China). The tibia was exposed by making a skin incision. The defect of tibia was created by cutting the end-portion of the tibia. Then the composite pillar was inserted into the site of the defect, and the surrounding periosteum as well as the underlying fibrous tissues were wrapped around and sutured with nylon threads. The rats were given an appropriate dose of prophylactic antibiotics. They were housed in spacious cages to allow ambulation throughout the postoperative period. The animals were fed with water and lab chow regularly. At the end of the 3-week evaluation period, the rats were sacrificed with CO2 and the tibia of each rat was carefully articulated and retrieved for histological investigation. The rats were randomly assigned to the treatment groups (n=10).


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Figure 1. The surgical procedure (the arrow indicates the implant site). 2.6. Histological analysis The tibia of each rat was carefully excised in its entirety. The samples were fixed in buffered, neutral 10% formalin solution for 10 days and then decalcified in 10% formic acid for 10 days. The specimens were trimmed so as to include the implant site and the adjacent bone tissue. After rinsing in PBS, the specimens were dehydrated in a graded ethanol series (70 to 100%). They were embedded in the extra-large paraffin blocks. Then the sections were fixed on poly-L-lysine coated glass slides, and stained with hematoxylin and eosin (H&E). 3. Results and discussions 3.1. Compositional analysis The compositional analysis was determined by FTIR and EDS. The results in Figure 2A showed that the CA was successfully prepared due to the presence of characteristic band at the wavenumber of 1750 cm−1 assigned to the vibration of C=O.27 In the spectra of the CA-SF/HA composites, the bands at 1095，1033，961，603 and 567 cm-1 were assigned to the different


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vibration modes of phosphate groups of HA, and the bands at 3570 and 633 cm-1 were assigned to the stretching and bending vibration of hydroxyl group of HA, which indicated the presence of HA in the composites.31,

32

The bands at 1265 cm-1 (amide Ⅲ ) and 1625 cm-1 (amide Ⅰ )

corresponding to silkⅡsuggested the structural transition from random coils to β-sheets.33, 34 In general, the carboxylation significantly improved the functionality of agarose, which easily allowed the SF to be introduced into the system by crosslinking. Therefore, the number of the active sites in the organic matrices was increased due to the presence of SF and CA. The EDS results (Figure 2B (a)) indicated that the Ca/P molar ratio of the obtained inorganic phase exhibited a close resemblance to the stoichoimetric HA.35, 36 The element distribution of calcium and phosphorus in the composites was investigated by EDS mapping (Figure 2B (b)). The results ascertained that both of the elements assigned to the inorganic phase were uniformly distributed in the organic matrices, thus, the HA crystals proved to be well-dispersed in the gel media.


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Figure 2. A. The FTIR of the composites. B. (a) The EDS result of the typical sample; (b) the result of the EDS mapping for the CA-SF/HA sample. C. The XRD patterns of the samples. 3.2. Crystal phase The XRD patterns of all the samples were shown in Figure 2C. Obviously, the diffraction peaks around 26º and 32º were assigned to the crystal planes (002) and (211), respectively.36, 37 The samples with different CA/SF weight ratio presented similar diffraction patterns, however, the


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intensity of some bands varied, suggesting the different crystallinity of the inorganic minerals guided by the different organic matrices. In other words, the organic matrices had a significant effect on guiding and regulating the inorganic crystallites, thus the characteristics of the inorganic phase could vary. 3.3. Morphological investigation The morphological details of the composites were investigated by SEM (Figure 3) and TEM (Figure 4). The HA nanoparticles were well-dispersed in the organic matrices. In the presence of SF, the HA nanoparticles presented the rod-like and sheet-like morphologies. The width and length of the nano-sheets were about 100 nm and 200 nm, respectively, which were consistent with the size of HA nano-sheets in the natural bone.21,

37

In addition, as the content of SF

decreased, the HA nano-sheets became narrower, and only a small amount of nano-rods appeared, suggesting that the increasing content of carboxyl groups and decreasing content of amino groups determined the morphological changes of the HA nanoparticles. The TEM micrographs exhibited the morphological details of the nanocomposites, which were consistent with the morphology showed in the SEM micrographs. The diffraction pattern of the selected area revealed the amorphous spots and the polycrystalline rings, which were assigned to the polymers and the HA crystallites, respectively. The polycrystalline rings corresponded to the (002) and (210) planes.38 The micrographs with the high magnification showed the crystalline details, and the spacing of one lattice fringe corresponded to the crystal plane (112) of the HA crystallites.


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Figure 3. The SEM images of (a) the CA-SF/HA (1#) composite; (b) the CA-SF/HA (2#) composite; (c) the CA-SF/HA (3#) composite; (d)-(f) the magnified images of the CA-SF/HA (1#)-(3#) composites correspondingly; (g) the CA/HA composite; (h) the NA/HA composite.


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Figure 4. (a) The TEM image of the CA-SF/HA (2#) composite. (b) The HRTEM image of the CA-SF/HA (2#) composite. (c) The diffraction pattern of the selected area.


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The morphological characteristics of the HA nanoparticles in the composites verified the intensity difference of the diffraction peaks shown in XRD. The preferred orientation determined the diffraction patterns and the morphology of the HA crystals. The HA nanoparticles precipitated in the CA-SF matrices presented the distinct rod and sheet morphologies. The dual organic matrices with active groups allowed for the efficient regulation and control on the nucleation and growth of the HA crystals. SEM results showed the HA nanoparticles in the NA matrix presented spherical morphology, while the HA nanoparticles in the CA matrix presented irregular sheet morphology. This fact indicated the CA played a key role in regulating and controlling the nucleation and growth of HA crystals. However, the crystals grown in the NA matrix were mainly attributed to the size effect of the gel network without active groups. Both the carboxyl groups and amino groups had a close affinity with calcium ions so that the oriented nanostructured HA crystals were achieved in the CA-SF matrices.37, 39 Therefore, in the presence of SF and CA, the inorganic phase was regulated and controlled in a comprehensive and delicate manner. In particular, the β-sheet conformation of the gelling SF molecules allowed for the ordered alignment of the active sites. The specific sites order induced the crystals to form certain morphology so that the HA nano-sheets and nano-rods were produced. That is, the active groups preferentially adsorbed on the certain crystal planes of the premature crystals during the initial stage of crystallization. However, the spherical crystals were obtained in the way that the active sites were randomly dispersed on the premature crystal nucleus. Besides, the size effect of the gel network limited the average size of the HA nanoparticles, preventing the crystals from being oversized. This hypothesis was illustrated in Figure 5.


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Figure 5. The illustration of the mechanism hypothesis. 3.4. Degradability and swelling ratio The curves obtained in Figure 6a showed the weight losses of the samples at each time point throughout the 21-day period. The CA-SF/HA composites displayed the better swelling property and degradability. Moreover, the CA-SF/HA (3#) containing the lowest content of SF had the highest swelling ratio. As the content of SF decreased in the CA-SF/HA composites, the samples showed an increasing degradation. The CA/HA composites exhibited lower degradation rate than the NA/HA composites, whereas, the swelling ratio of the NA/HA composites was higher than


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that of the CA/HA composites. In general, the carboxylation delayed the degradation, and the composites containing SF displayed much slower degradation rate than the composites without SF. It has been reported that the crystalline β-sheet content of the SF was the main factor that directly determined the degradation rate.40 The processing methods of SF could also induce the hydrophobic domains to form the β-sheet regions.41 That is, the presence of β-sheets had a protective effect on the degradation rate. In this work, sufficient β-sheets were provided via the crosslinking and mineralization processes such that the CA-SF/HA composites presented relatively slow degradation rate compared with the other samples. The final average weight loss of the CA-SF/HA (1#) composites was even below 5%, suggesting the degradation rate of the CA-SF/HA composites might fit well with the load-bearing bone reconstruction which was typically accomplished within six months or even more.


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Figure 6. (a) The degradation curves (n=3). (b) The DSC curves. (c) The TG curves; (d) DTG curves of the composites. 3.5. Thermal properties The thermal properties of the composites were intimately correlated with the structural or compositional changes in the composites. The DSC curves shown in Figure 6b reflected the thermal characteristics of the composites. The exothermic peaks at 212.5°C assigned to the CA/HA and NA/HA composites were worth noting, that is, the peak area of the CA/HA composite was significantly larger than that of the NA/HA composite, indicating that the thermal stability of the CA was elevated because of the carboxylation. Although the CA-SF/HA composites with different CA/SF weight ratio presented thoroughly different thermal behaviors due to the complex physicochemical changes during the crosslinking and the subsequent mineralization procedures, no thermal event was detected around 212.5°C, suggesting the good polymer compatibility.42 The TG/DTG curves of the samples reflected the decomposition of the polymers (Figure 6c and d). The curves obtained for CA-SF/HA (1#) and (3#) composites displayed two main mass losses. However, the curve for CA-SF/HA (2#) composites showed three mass losses. For CA-SF/HA (1#) and (3#) composites, the first stage occurred from room temperature to 65°C, and the second mass loss began at about 170°C, with the maximum decomposition at about 310°C. For CA-SF/HA (2#), the second mass loss occurred in the range of 150-225°C, and the third stage began at 235°C, ended at the same maximum decomposition temperature with the other CA-SF/HA composites. In conclusion, the composites containing SF showed the main decomposition at 281°C. Apparently, the curves obtained for CA/HA and NA/HA composites presented different mass losses in the range of 150-316°C. The hydrogenbonded water desorption caused the early weight loss of all the samples in the range of 20-


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150°C; the subsequent weight losses in the range of 150-600°C were attributed to the decompositions of polysaccharides and SF. Besides, the TG curves showed that the residue mass of the NA/HA composites was the lowest of all the samples, which was much lower than that of the CA/HA composites, further suggesting that the carboxylation was conducive to improve the thermal stability. 3.6. Mechanical properties All of the samples presented the same mechanical behaviors in the stress-strain curves (Figure 7a). The elastic modulus (Figure 7b) and compressive strength (Figure 7c) were calculated from the stress-strain curves.43 The content of CA or NA in the CA/HA or NA/HA composites was equivalent to the total content of CA and SF in the CA-SF/HA composites. Interestingly, the composites containing SF could form stable and strong gel even the weight content of CA was below 50%, which demonstrated the great effect of the chemical bonds because of the carboxylation. The CA/HA composites showed the lowest elastic modulus and compressive strength. For the CA-SF/HA composites, the mechanical strength increased as the content of SF increased. The CA-SF/HA composites in this work showed higher mechanical strength than the composites reported previously.44 The composites also had better mechanical strength than the agarose/HA composites reported previously by our research.26 In comparison with the natural bone tissues, the mechanical strength of the CA-SF/HA composites was even higher than the cancellous bone of human body 45, and was equivalent to that of the femur of rats, yet slightly lower than that of the proximal tibia of rats.46 According to the structural analysis of SF in the composites, the sufficient crystalline β-sheets in the fibroin could significantly increase the stability and mechanical properties of SF.41 Therefore, the composites containing SF showed


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great compressive strengths and elastic moduli, suggesting the SF actually reinforced the organic matrices and even the whole composite system.

Figure 7. (a) The stress-strain curves of the composites. (b) The elastic moduli of the samples. (c) The compressive strengths of the samples (n=3). P-values of < 0.05 (*) and < 0.01 (**) were considered statistically significant. 3.7. In vitro tests 3.7.1. Cell adhesion


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Cell adhesion was the first important index for the implants in contact with cells. As shown in Figure 8, the cell adhesion on the composites was investigated by the inverted fluorescence microscopy. After 24-h culture, the cells on the composite films displayed well-defined cytoskeletal organization and fibrous structure with fine cell density. Apparently, the cells on the CA-SF/HA composite films showed greater spreading and more robust actin filaments than the cells on the other samples. Besides, the CA/HA composites were more capable of supporting the cell adhesion than the NA/HA composites. The modification made the CA more appealing for cell adhesion than the NA. Moreover, the desirable biocompatibility of SF was mainly attributed to the tripeptide sequence of Arg-Gly-Asp (RGD) which acted as the biological recognition signal such that the CA-SF/HA composites exhibited the greatest cell spreading and the highest cell density.


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Figure 8. The MG63 osteoblast-like cells on the composite disks after 24-h culture (a) CASF/HA (1#); (b) CA-SF/HA (2#); (c) CA-SF/HA (3#); (d) CA/HA; (e) NA/HA; (f) cover glass slip. Figure 9 shows the hierarchical porous structure of the composite scaffolds, both the porosity and the pore size could meet the prerequisite of the bone tissue engineering. The MG63 cells showed strong adhesion on the pore wells of the CA-SF/HA composite scaffolds after 24-h culture (Figure 10).

Figure 9. The porous architecture of the CA-SF/HA composite scaffolds ((2#) as the example).


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Figure 10. The MG63 cells adhering on the CA-SF/HA composite scaffolds after 24-h culture: (a) CA-SF/HA (1#); (b) CA-SF/HA (2#); (c) CA-SF/HA (3#); (d)-(f) the SEM images of the morphological details of the cells on the (1#)-(3#) scaffolds. 3.7.2. Cell proliferation and differentiation The cell proliferation in the composite scaffolds was evaluated by the CCK-8 kit assay (Figure 11A). In general, the cell growth and proliferation on all the composite scaffolds were significantly promoted throughout the duration of cell culturing. Cell cultured on the CA-SF/HA composite scaffolds showed the greatest viability. Apparently, the CA was more capable of enhancing the cell viability than the NA. Besides, the cell viability was significantly enhanced when the SF content in the organic matrices increased. The cell differentiation in the early stage can be evaluated by the ALP activity. As shown in Figure 11B, the cells cultured on the CA-SF/HA composite scaffolds displayed the greater


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activity, and the CA/HA composite scaffolds showed greater ability to enhance the cells to differentiate than the NA/HA composite scaffolds. The cell differentiation in the late stage can be evaluated by the calcium depositions. As shown in Figure 11C, the CA-SF/HA (2#) group showed the highest concentration of calcium deposition, and the CA/HA group showed higher calcium deposition than the NA/HA group, indicating the good biocompatibility of the CA.

Figure 11. A. The MG63 cell proliferation results of different groups through CCK-8 assay (n=5). B. The ALP activity of the MG63 osteoblast-like cells seeded onto the composite scaffolds (n=5). C. The calcium deposition of each group (n=5) after 21-day culture, the number


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in each column indicated the concentration of calcium ions (mg/dL). P-values of < 0.05 (*) and < 0.01 (**) were considered statistically significant. D. (a) Histological evaluation of bone regeneration in rats after transplantation of the CA-SF/HA (2#) composites with H&E staining (n=10); (b) magnified morphology of the bone regeneration treated with the CA-SF/HA (2#) composites; (c) the bone defect of the rat in control group without treatment; (d) the magnified image of the control group. The arrows denote the osteoblast-like cells. HB: host bone; NB: new bone. 3.8. Histological analysis After comprehensively evaluating the in vitro results, we chose the CA-SF/HA (2#) composites to be used for the subsequent in vivo tests. As shown in Figure 11D (a) and (b), immature newly formed bone and inflammatory cells were observed after 24 days. A mild innate immune response can often be beneficial as it activates many healing processes.41 Numerous osteoblastlike cells that were also found on the bone surface presented flat or cylindrical shape. Meanwhile, the mesenchymal cells also showed great proliferation. In general, the histological examinations demonstrated that the rats treated with the CA-SF/HA composites exhibited extensive osteogenesis in and nearby the defect site. The composite did not cause any serious inflammatory response. In contrast, no new bone formation was found in the rats treated without the composite (Figure 11D (c-d)). Only the lamella of the mature host bone and sparse osteoblast-like cells were presented, and there was serious inflammatory cell infiltration which can be detrimental causing destruction of local tissue or even systematic problems. According to the in vivo result, it can be shown that the CA-SF/HA composites have a great potential to be applied in the clinical treatments.


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4. Conclusions In this work, crosslinked CA-SF dual confluent matrices provided the homogeneous compartments for the nucleation and growth of the HA crystals. The unique nucleation site order of the organic matrices allowed for the oriented growth of the crystals such that the versatile morphologies of the HA nanoparticles were presented in an interesting template-regulating pattern, providing a new model of mimicking the microstructure of natural bone. The mechanical and thermal investigations both confirmed that the carboxylation improved the reaction capability of the polymer. Therefore, the CA-SF/HA composites exhibited optimal properties by comprehensively combining the merits of both polymers. The degradability results revealed that the carboxylation delayed the degradation, allowing the degradation procedure to be smooth. Due to the presence of SF, the CA-SF/HA composites exhibited a much slower degradation rate. The stable β-sheet conformation made the molecular chains more compact and stronger so that the SF molecules could withstand the intervention and pressure from outside. Therefore, the CASF/HA composites showed similar mechanical strengths to the natural bone. The in vitro and in vivo tests confirmed that the CA-SF/HA composites had desirable biocompatibility and enabled the large bone defect to be repaired. In conclusion, the CA-SF/HA composites can be potentially used for repairing the injured load-bearing bone tissues. Besides, it has been reported that crystal sizes and shapes play critical roles in biological responses47, so how about the mechanical properties? Apparently, the mechanical properties and the biological responses investigated in this work were attributed to the synergistic effect of the organic matrices and the crystals. The question mentioned above is of great importance for biomimetic research, so in the next, we will explore whether the morphologies have specific correspondence with the mechanical properties and the biological responses.


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AUTHOR INFORMATION Corresponding Author *Hua Tong (E-mail address: [email protected]; Tel: +86 02768764510; Fax: +86 02768752136) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources The National Science Foundation of China (Nos.31071265, 30900297) and the National Basic Research Program of China (2012CB725302). Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the National Science Foundation of China (Nos.31071265, 30900297) and the National Basic Research Program of China (2012CB725302). REFERENCES 1.

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Carboxylated Agarose (CA)-Silk Fibroin (SF) Dual Confluent Matrices

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