International Journal of Biological Macromolecules 65 (2014) 1–7

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Preparation and evaluation of collagen-silk fibroin/hydroxyapatite nanocomposites for bone tissue engineering Li Chen, Jingxiao Hu, Jiabing Ran, Xinyu Shen ∗ , Hua Tong Key Laboratory of Analytical Chemistry for Biology and Medicine, Ministry of Education, College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China

a r t i c l e

i n f o

Article history: Received 27 September 2013 Received in revised form 31 December 2013 Accepted 2 January 2014 Available online 9 January 2014 Keywords: Collagen Hydroxyapatite Silk fibroin

a b s t r a c t A new in situ precipitation technique was developed to synthesize collagen-silk fibroin/hydroxyapatite nanocomposites. The componential properties and morphological of nanocomposites were investigated. It was revealed that the inorganic phase in the nanocomposite was carbonate-substituted hydroxyapatite with low crystallinity. Morphology studies showed that hydroxyapatite particles with size ranging from 30 to 100 nm were distributed uniformly in the polymer matrix. According to the TEM micrographs, inorganic particles were composed of more fine sub-particles whose diameters were between 2 and 5 nm in size without regular crystallographic orientation. The mechanical properties of the composites were evaluated by measuring their elastic modulus. The data indicated that the elastic modulus of nanocomposites was improved by the addition of silk fibroin. Finally, the cell biocompatibility of the composites was tested in vitro, which showed that they have good biocompatibility. These results suggest that the collagen-silk fibroin/hydroxyapatite nanocomposites are promising biomaterials for bone tissue engineering. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Bone is a dynamic and highly vascularized tissue that is formed from a composite of 70% mineral (mostly nanoscale HA crystals) and 30% organics (including collagen, glycoproteins, proteoglycans, and sialoproteins) by dry weight [1]. Bone repair or regeneration is a key problem in orthopedic surgery. Traditional therapeutic approaches in treating large bone defects include bone grafts [2] and transplants [3] (autologous – from the iliac bone or fibular grafts, allograft – fresh or frozen after cleaning, or xenografts). Autografts have achieved various degrees of success in treating bone defects. However, the autograft is limited by the donor site morbidity, prolonged rehabilitation, increased risk of deep infection and restricted availability. Moreover, allografts might cause potential risks of transmitted diseases such as HIV or contamination [4,5]. Thus, more and more researchers have focused on organic/inorganic artificial biomaterials for treating bone defects in recent years. Recently, various synthetic polymers, such as poly(glycolic acid) (PGA) [6], poly-l-lactic acid (PLLA) [7] and poly-(lactic-co-glycolic acid) (PLGA) [8], have been thought as potential bone grafts. However, the low strength and inflammatory responses caused by release of degraded acidic products limit their applications in bone repairing. Consequently, some natural polymers, containing

∗ Corresponding author. Tel.: +86 27 68764510; fax: +86 27 68752136. E-mail address: [email protected] (X. Shen). 0141-8130/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2014.01.003

polysaccharides (alginate, chitosan and cellulose) and proteins (collagen, gelatin, and silk), have been considered as the most important substrates for bone grafts. Collagen (Col), one of the most important natural polymers and the main organic component of bone tissue and extracellular matrix (ECM), has been widely applied in tissue engineering owing to its excellent bioactivity and degradability. However, the weak mechanical properties and fast degradability of Col may limit its applications. In the most biomaterials applications, the collagen matrix is usually combined with a reinforcing phase such as hydroxyapatite (HA), which should improve both the mechanical properties and the bioactivity of the material [9–11]. More than that, Col/HA composites bear a very close compositional resemblance to natural bone. Hydroxyapatite, the major inorganic mineral of natural bone, is a biologically active calcium phosphate ceramic that is used in surgery to replace and mimic bone. Currently, there are some techniques concerning preparing Col/HA composite materials, including co-precipitation [12–14], alternate soaking [15,16] and mechanical mixing [17]. Among these methods, there is a common shortcoming that inorganic particles cannot be distributed homogeneously in the organic matrices at nanolevel, which leads to poor mechanical properties and limits their applications. Moreover, some researchers attempted to add other components to strengthen mechanical strength of Col/HA composite. Song et al. [18] applied physical crosslinking method to obtain poly(vinyl alcohol)/collagen/hydroxyapatite hydrogel. Poly(vinyl alcohol) (PVA) endowed mechanical strength and safety [19,20]. However, the network of PVA hydrogel prepared through

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physical crosslinking method is not strong enough and easily influenced by external conditions, such as temperature, and so on. Besides, polylactic acid (PLA) [21] was introduced into Col/HA system, which inevitably damaged the biocompatibility of Col/HA composites. Not only to enhance mechanical strength of Col/HA composite, but also to maintain its good biocompatibility, silk fibroin was introduced into the Col/HA system. Nevertheless, there are few reports on the preparation of Col-SF/HA composites. Wang [22] et al. synthesized the Col-SF/HA composite by co-precipitation method. Resulting from mechanical mixing during the pressure casting process through the co-precipitation, inorganic particles couldn’t be distributed homogeneously within the organic matrices at nanolevel. Silk fibroin (SF), a fibrillar protein derived from the silkworm Bombyx mori, is composed of 17 amino acids. SF with ␤-sheet structure has several unique properties such as advantageous processability, long-term biodegradability, superior mechanical strength as well as biocompatibility and good oxygen permeability [23–26]. Thus, it has been widely applied in drug-delivery [27,28], bone tissue engineering [29], wound dressing [30], skin tissue [31,32], and so on. In our previous work [33,34], a novel in situ precipitation method was developed to endow synthesized composites with unique morphology ultrafine nano-HA particles dispersed in organic template homogenously. The aim of this work is to fabricate homogeneous Col-SF/HA composites by the in situ precipitation approach, which is totally different from the traditional ones and rarely reported in the synthesis of Col-SF/HA composites. In this work, the growth of HA in Col-SF hydrogel was compared with that of HA in a single protein (collagen or silk fibroin) hydrogel through in situ precipitation method. The approaches currently used to obtain SF/HA composite materials are based on co-precipitation [35,36], alternate soaking [37,38] and mechanical mixing [39,40]. The mechanical performance and the compositional properties of as-synthesized nanocomposites were investigated. The cell biocompatibility with the composites was also tested in vitro. MG63 osteoblast-like cells were selected to evaluate the biocompatibility of the composites architecture with cells such as cell adhesion, cell spreading, cell proliferation and cell viability. The resulting composite, which combines good biocompatibility with high strength, provides a promising material in bone tissue engineering. 2. Materials and methods Soluble type-I collagen from porcine dermis was purchased from National Engineering Research Center for Biomaterials of Sichuan university (Sichuan, China). Bombyx mori cocoons were obtained from Jingwei silk Co., Ltd. (Hubei, China). Dialysis membranes were obtained from Shenshi Co., Ltd. (Wuhan, China). Calcium nitrate tetrahydrate (Ca(NO3 )2 ·4H2 O), diammonium hydrogen phosphate ((NH4 )2 HPO4 ), glutaraldehyde, acetic acid and ammonia were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All the reagents used in this work were of analytical grade (AR) and used without any further purification. Deionized ultrapure water was used throughout the experiment. 2.1. Preparation of Bombyx mori silk fibroin solution Silk fibroin solution was prepared according to a previously reported method [41]. Bombyx mori cocoons were boiled for 10 min in 0.02 mol/L Na2 CO3 solution for three times and rinsed thoroughly with distilled water to extract the glue-like sericin protein. The extracted silk was dissolved in 9.3 mol/L LiBr solution at 60 ◦ C. The solution was dialyzed against water with a dialysis membrane

(MW = 3500) for 3 days. The final concentration of the silk fibroin in aqueous solution was about 5.0% (w/v), which was determined by weighing the solid after drying. Silk fibroin solution was stored at 4 ◦ C. 2.2. Synthesis of Col-SF/HA nanocomposites by in situ precipitation Col solution was made by dissolving type-I collagen in 8 ml of acetic acid solution (1.5 vol.%) with continuously stirring at room temperature for 12 h. Then Ca(NO3 )2 ·4H2 O and (NH4 )2 HPO4 (Ca/P = 1.67) were together added to the Col solution under agitation until the salts were entirely dissolved. SF solution was dropped slowly into the Col solution of Ca2+ and PO4 3− with gently stirring at ambient temperature. Subsequently 0.3 ml glutaraldehyde (25 wt.%) was added to the previous mixed solution as a crosslinking agent. The solution was gently stirred until a transparent hydrogel formed. The resulting hydrogel was then stored under ambient conditions for 24 h to reach complete crosslinking. It was then added with ammonia solution for 24 h at room temperature. Under this alkaline condition, HA precipitated within the hydrogel gradually. The in situ precipitation method can be represented by the following chemical reaction: 10Ca2+ + 6HPO4 2− + 8OH− + Ca10 (PO4 )6 (OH)2 (↓) + 6H2 O(pH > 10) The nanocomposite was finally washed with distilled water until the pH of eluate was about 7. The starting content of all reagents was scaled according to the final organic/HA weight ratio of 40/60, and the initial amounts of the reagents used in this work are listed in Table 1. The weights of HA, Ca(NO3 )2 ·4H2 O and (NH4 )2 HPO4 were calculated according to above equation. 2.3. Synthesis of Col/HA nanocomposites by in situ precipitation The preparing procedures are the same as described in Section 2.2, but without the addition of SF. An opaque composite of Col/HA was produced. The starting content of all reagents was scaled according to the final Col/HA weight ratio of 40/60. 2.4. Synthesis of SF/HA nanocomposites by in situ precipitation The preparing procedures are the same as described in Section 2.2, but without the addition of Col. An opaque composite of SF/HA was produced. The starting content of all reagents was scaled according to the final SF/HA weight ratio of 40/60. 2.5. Characterization Morphology of inorganic/organic composite was observed using Environmental Scanning Electron Microscopy (SEM, Quanta200, FEI, Holland) and field emission transmission electron microscope (2010FEF, JOEL, Japan). The crystalline phase and component of obtained products were identified using wide angle X-ray diffraction analysis (XRD, X’pert PRO, Panalytical, Holland) and Fourier Transform Infrared Spectrometer (FT-IR, Nicolet5700, America). Mechanical properties tests were measured at room temperature by a universal testing machine (SHIMADZU, AGS-J, Japan) at a crosshead speed of 0.5 mm min−1 . Elastic modulus was calculated as the slope of the initial linear portion of the stress–strain curve. Samples of Col-SF/HA nanocomposites were made into circular discs suitably sized (diameter 5 mm, height 2 mm). The MG63 cells (2.0 × 104 cells/well) were seeded on each discs placed in the 96-well plates (Corning Life Sciences). Cells cultivated in the

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Table 1 The dosage of the reagents. Sample

SF (g)

Col (g)

Organic (SF + Col) (g)

HA (g)

Ca(NO3 )2 ·4H2 O (g)

(NH4 )2 HPO4 (g)

Col/HA-1 SF/HA-2 Col-SF/HA-3 Col-SF/HA-4 Col-SF/HA-5

0 0.100 0.025 0.050 0.075

0.100 0 0.075 0.050 0.025

0.1

0.15

0.36

0.12

same wells without samples were used as a control. Plates were incubated in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 ◦ C in a 5% CO2 incubator for 9 days, and the cell viability was studied using cell counting kit-8 assay (CCK-8; Dojindo Laboratories, Japan) according to the manufacturer’s instructions. After 1, 3 and 7 days of culture, nanocomposites were gently washed with PBS and then 2 ml of DMEM containing 10% CCK-8 was added per well. The disks were incubated at 37 ◦ C for 3 h. After incubation, the supernatant was transferred to a 96-well plate and the optical density (O.D.) was measured at 450 nm using an ELX808 Ultra Microplate Reader (BioTek Instruments, Inc., America). Behaviors of MG63 cells on various Col-SF/HA nanocomposites were studied by SEM. After cultivation for 2 days, composites grown with cells were washed twice with PBS, and cells were fixed with 2.5 wt.% glutaraldehyde under 4 ◦ C overnight. Fixed samples were dehydrated by ethanol in an increasing concentration gradient (30, 50, 70, 90, 95 and 100 vol.%), followed by lyophilization. The dried samples were glued onto copper stubs, and sputter coated with gold prior to SEM observation.

Fig. 1b-d shows the FTIR spectra of Col/HA (Fig. 1b), SF/HA (Fig. 1c) and Col-SF/HA (Fig. 1d) composite. The bands at 1092, 1035, 961, 603 and 566 cm−1 corresponded to different vibration modes of phosphate group in HA, while the bands at 3570 and 632 cm−1 represented hydroxyl group as stretching and bending vibration. Bands assigned to carbonate group at 1482, 1452, 1424 and 874 cm−1 were also observed. This agreed with the fact that

HA crystals prepared using the precipitation method contained carbonate ions [42]. As shown in Fig. 1a, the typical amide peaks of the type I Col appear at 1639 cm−1 (amide I), 1551 cm−1 (amide II), 1238 cm−1 (amide III). The peak at 3080 cm−1 belongs to the stretching vibration of amide hydrogen bonding (NH), but it weakened in the Col/HA composite system (Fig. 1b), which result from that amino groups of Col react with glutaraldehyde via Schiff’s base linkage. It is well known that FTIR is a powerful tool for the study of secondary and tertiary structure and conformational transitions of polypeptides and proteins [43]. In 1985, Asakura et al. [44] studied the conformation of solid mulberry SF under different conditions. The spectrum for the solid mulberry SF showed absorbance at 1660 cm−1 (amide I), 1540 cm−1 (amide II), 1235 cm−1 (amide III) and 650 cm−1 (amide V), which were ascribed to the typical peaks of random coils. The absorption bands at 1630 cm−1 (amide I), 1530 cm−1 (amide II), 1265 cm−1 (amide III) and 700 cm−1 (amide V) were assigned to the characteristic peaks of ␤-sheet. In 1990, Venyaminov and Kalnin [45] investigated the IR absorption bands of liquid mulberry SF, and the signals at 1652–1654 cm−1 (amide I) and 1546–1548 cm−1 (amide II) were attributed to the characteristic absorption peaks of random-coil conformation, while the signals at 1623 cm−1 (amide I), 1530 cm−1 (amide II) and 1699 cm−1 were associated with ␤-sheet conformation. Fig. 2a–c represents FT-IR spectra of liquid SF, self-crosslinking SF hydrogel and SF/HA composite, respectively. In comparison with liquid SF (Fig. 2a), a random-coil/␣-helix structure of silk molecules in self-crosslinking SF hydrogel (Fig. 2b) was shown to transform into a ␤-sheet structure. Generally speaking, the filamentization of silk is essentially due to the conformation transformation of SF from random coils/␣helix to ␤-sheet. In the SF/HA composite system, SF may participate in the intermolecular crosslinking reaction after the addition of glutaraldehyde. The formation of the intermolecular crosslinking network structure may limit the intramolecular crosslinking of SF

Fig. 1. FTIR spectra of (a) pure collagen; (b) the Col/HA; (c) the SF/HA composite; (d) the Col-SF/HA composite.

Fig. 2. FTIR spectra of (a) liquid SF; (b) self-crosslinking SF hydrogel; (c) the SF/HA composite. The black real line marks the characteristic peak structure for the silk I (random coils/␣-helix), the black dotted line marks the characteristic peak structure for the silk II (␤-sheet).

3. Results and discussion 3.1. Compositional properties and morphology of Col-SF/HA nanocomposites

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to a certain extent. According to the IR spectrum (Fig. 2c), there was silk I (random coils/␣-helix) structure still in SF/HA composite. The structure of SF molecules in the hydrogel should transform into ␤sheet when intramolecular cross-linking occurred, which has been proven by IR results (Fig. 2b). However, the ␤-sheet and random coil structure exist in SF/HA composite together, indicating that SF molecules partially involved in the intermolecular cross-linking while a part of SF molecules were concerned in intramolecular cross-linking as a result of hydrogen bond interaction. Fig. 1d shows the FTIR spectra of Col-SF/HA nanocomposite. It is hard to distinguish the characteristic peaks of Col and SF, the amino acids component of which are all mainly glycine and alanine [46,47], and they both belong to structural protein [48]. However, based on above FTIR analysis concerning SF/HA and Col/HA composite, it was inferred that SF probably involved in the intermolecular crosslinking reaction of Col molecules after mixed with glutaraldehyde, of which Tyr, His and Lys residues cross-linked with Col molecules to form the intermolecular cross-linking network. Moreover, intramolecular cross-linking reaction may occur in a part of SF molecules derived from hydrogen bonding. XRD spectra of Col/HA (Fig. 3a), SF/HA (Fig. 3b) and Col-SF/HA (Fig. 3c) nanocompositea were verified by the Powder Diffraction File (PDF Card No. 9-432). It is evident that three intense peaks of crystal phases at 25.9◦ , 32◦ and 39.7◦ (2), which are assigned to (0 0 2), (2 1 1) and (3 1 0) of crystalline HA, respectively. The XRD patterns of the three samples showed broad peaks with poor crystallinity around the characteristic diffraction region near 32◦ (2), which suggested low crystallinity of HA in all samples. This crystallographic structure of three samples was more similar to natural bone mineral (biological apatite) [49]. Therefore, Col/HA, SF/HA and Col-SF/HA composites have more similarities with natural bone mineral in terms of the carbonated hydroxyapatite, which has been

Fig. 3. XRD pattern of (a) the Col/HA composite; (b) the SF/HA composite and (c) the Col-SF/HA composite.

also confirmed by FTIR analysis (Fig. 1b–d). The reason for the low crystallinity of precipitated HA might be the size effect owing to the three-dimensional network microstructure provided by the crosslinked protein hydrogel, in which the growth of inorganic crystal was limited. Fig. 4a-c shows lower magnification SEM micrographs of Col/HA, SF/HA and Col-SF/HA nanocomposites. In comparison with Col/HA (Fig. 4a) and Col-SF/HA nanocomposites (Fig. 4c), the surface of SF/HA (Fig. 4b) nanocomposites is not smooth enough, which may result from the less amino groups and lower crosslinking density of silk fibroin than that of collagen. Through the observation of higher magnification SEM micrographs (Fig. 4d–f), the morphological features revealed that inorganic crystals of HA had

Fig. 4. Lowly magnified SEM micrograghs of (a) the Col/HA composite; (b) the SF/HA composite; (c) the Col-SF/HA composite; highly magnified SEM micrograghs of (d) the Col/HA composite; (e) the SF/HA composite; (f) the Col-SF/HA composite and (g) TEM micrographs of the Col-SF/HA composites; the inset shows polycrystall diffraction ring

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Fig. 5. Scheme of the formation mechanism of Col-SF/HA nanocomposites.

high-affinity with organic matrices in all samples. The inorganic particles with size ranging from 30 to 100 nm distributed within Col, SF or Col-SF matrix homogenously. Thus, this close bonding between inorganic particles and organic matrix may enhance the mechanical properties of the composites. However, it is hard to get this kind of decentralization effect by conventional mechanical mixing or co-precipitation. In the case of Col/HA, SF/HA and Col-SF/HA nanocomposites, they have similar morphology. The similar structure of Col and SF may have the similar mechanism on regulating the HA crystal growth. In this work, protein (Col, SF or Col-SF) hydrogel played an important role in the superfine interaction of effect of its interior. The decentralization of inorganic nano-particles within organic matrices was improved significantly. According to the analysis of FTIR, there was a small shift of absorption peaks of the amides I by comparison between pure proteins (Col, SF) and composites (Col-HA, SF-HA, Col-SF/HA). Also, the peak of amide III almost disappeared in the composites. These changes suggest that the amides could serve as the initial nucleation site of crystals. Moreover, fibroin mainly composed of repeating (Gly-Ser-Gly-Ala-Gly-Ala)n amino acid chain has few free carboxylic acid groups and collagen has more acid groups. In our previous research [50], it has been proposed the formation mechanism of homogeneous Col/HA nanocomposite through in situ precipitation technique. Hence, the biomineralization process may be described as following (Fig. 5): with the increase of pH after the addition of ammonia solution, residue groups of amino acid in collagen or silk fibroin, such as carboxyl and amino groups, may begin to act as nucleation center for hydroxyapatite formation. These negatively charged residue groups can interact with Ca2+ and thus form a large scale of local supersaturation microenvironment. Besides, strong electric field resulted from the high concentration of negatively charged carboxyl groups were in favor of the interaction with the most positively charged crystalline plane. Thus, many nucleation sites formed in the interior network of hydrogel matrix, and each point of nucleation sites can result in microcrystal. A large number

of nucleation sites favor the formation of fine crystallites. Furthermore, the compartment effect of crosslinking protein hydrogel (Col, SF or Col-SF) which was provided with three dimensional network microstructure limited the excessive growth of HA particles, so the inorganic nano-particles were limited to aggregate in the compartment of the protein hydrogel (Col, SF or Col-SF) template according to orientation of preferential growth of crystal plane. Furthermore, through the observation of highly magnified TEM image of crystal lattice (Fig. 4g), it indicated that composites had more precise bonding at 2–5 nm level, and nano-scale subcrystallites in organic matrices had no uniform crystallographic orientation. The polycrystal diffraction ring and amorphous spots shown in the inset of TEM selected area electron diffraction pattern also accorded with the structure in Fig. 4g. It can be believed that the strong combination of two phases from nano-sized to submicron level would benefit to ideal stress impress and increase of mechanical strength, while the random crystallographic orientation of the nanoparticles may be responsible for the isotropic character of the composite. 3.2. Mechanical properties of Col-SF/HA nanocomposites In dry state, the mechanical properties of the Col/HA and ColSF/HA nanocomposites with different SF/Col weight ratios were tested by a universal testing machine. Fig. 6a–d shows the stressstrain data of samples with different SF/Col weight ratios ranging from 0 to 3:1. All the tests were conducted under a compressive load at 0.5 mm/min. The results indicated that all the samples presented similar stress-strain behavior. The stress increased sharply at initial stage and then reduced in slope. As expected, Col-SF/HA nanocomposites showed higher stress than Col/HA at the same strain. With increasing SF/Col weight ratio the elastic modulus increased sharply from 481.6 to 723.1 MPa (Fig. 6e). Nevertheless, there are few reports on the preparation of Col-SF/HA composites. Mou et al. [51] utilized mechanical mixing method to prepare silk fibroin/collagen/hydroxyapatite composite. Wang [22] et al.

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Fig. 6. Mechanical properties curves of Col/HA and Col-SF/HA composites: (a) compressive stress-strain curve of Col/HA composites; (b) compressive stress-strain curve of Col-SF/HA composites with an SF/Col weight ratio of 1:3; (c) compressive stress-strain curve of Col-SF/HA composites with an SF/Col weight ratio of 1:1; (d) compressive stress-strain curve of Col-SF/HA composites with an SF/Col weight ratio of 3:1; and (e) elastic modulus-SF/Col weight ratio bar graph (n = 3).

synthesized the Col-SF/HA composite through co-precipitation method. In these methods, inorganic particles could not be distributed homogeneously within the organic matrices at nanolevel, so the mechanical properties of composite materials were weakened correspondingly. Compared with the Col/HA nanocomposites, the Col-SF/HA nanocomposites obviously exhibited higher elastic modulus. Some of the factors leading to the enhancement in mechanical properties of Col-SF/HA nanocomposites could be identified: (1) the strong interfacial interaction between inorganic and organic phase based on the in situ precipitation method; (2) the addition of SF, which has a high mechanical property; (3) the suitable SF/Col ratio.

3.3. Cell morphology and proliferation on Col-SF/HA nanocomposites A study on the cell biocompatibility of Col-SF/HA composites is especially interesting in determining possible uses of these composites as bone repairing material. In this study, MG63 cells were employed to test the biocompatibility of the Col-SF/HA nanocomposites. Cell morphology on the composites was evaluated by SEM. Fig. 7a–c represents micrographs of MG63 cells cultured on the Col-HA nanocomposite (Fig. 7a), SF/HA nanocomposite (Fig. 7b) and Col-SF/HA nanocomposite (Fig. 7c) after 2 days. The overall results indicate that the cells were spread out and had some filopods. Meanwhile, the cells exhibit fusiform and polygonal morphology. Cell proliferation on Col-SF/HA nanocomposites was quantified by using CCK-8 assay. Fig. 7d presents the proliferation of MG63 cells on Col/HA, SF/HA and Col-SF/HA nanocomposites after 1, 3 and 7 days of culture. Cell quantity on the Col-SF/HA nanocomposites was significantly higher than that on the SF/HA nanocomposites in different days (Fig. 7d). The data proved that the Col-SF/HA nanocomposites has better biocompatibility than the SF/HA nanocomposites. This result is consistent with the previous research demonstrating that collagen promoted cell spreading and proliferation [52–54]. Some researchers [55] have shown that three-dimensional fibroin nets support the attachment, spread and growth of varieties of tissue cells. Silk fibroin has fulfilled many essential requirements for an optimal biomaterial. In this study, we found that the growth of MG63 cells on the Col-SF/HA nanocomposites was better than on SF/HA nanocomposites. Considering that collagen has more cell-recognition signals than silk fibroin, the increase of the biocompatibility should be due to the abundance of cell-recognition signals of collagen. Therefore, the favorable cell

Fig. 7. SEM micrographs of MG63 cell morphology on composites after incubation for 2 days: (a) the Col/HA; (b) the SF/HA composite; (c) the Col-SF/HA composite and (d) CCK-8 assay of the proliferation of MG63 cells cultured on the Col/HA, the SF/HA composite and the Col-SF/HA composite (n = 3).

growth morphology and proliferation observed on the composites mentioned above suggest the possible use of the Col-SF/HA composites for bone tissue engineering. 4. Conclusions Collagen-silk fibroin/hydroxyapatite nanocomposite was obtained via a new in situ precipitation method. The inorganic component in the composite was identified as monophase low crystalline HA containing carbonate ions. The inorganic particles with size ranging from 30 to 100 nm were distributed within organic matrix homogenously. The chemical interactions between the inorganic and organic constituents in the nanocomposite, take place via the electrostatic adsorption between Ca2+ and the carboxyl or amino bands of protein template. The involvement of SF endows the composite with a higher elastic modulus compared to Col/HA. The culture of MG63 cells indicates that the ColSF/HA nanocomposite has good biocompatibility. Based on above research, we make a conclusion that the Col-SF/HA nanocomposites are promising biomaterials for bone tissue engineering.

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Acknowledgements This research was supported by the National Natural Science Foundation of China (Nos. 31071265 and 30900297) and the Research Fund for the Doctoral Program of Higher Education (No. 20090141120055). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26]

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hydroxyapatite nanocomposites for bone tissue engineering.

A new in situ precipitation technique was developed to synthesize collagen-silk fibroin/hydroxyapatite nanocomposites. The componential properties and...
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