Introduction

Hydroxyapatite (HAp, Ca10(PO4)6(OH)2, HAp) has attracted great attention because of its structural and chemical similarities to the mineral components of bones [1,2,3], and over the past decade, particular focus has been placed on the fabrication of nanocrystalline HAp. Several studies have indicated that biomaterials containing nanocrystalline HAp exhibit a significant increase in protein adsorption and osteoblast adhesion compared to micrometer-sized Hap [4,5,7]. Moreover, natural bones and teeth contain HAp nanocrystals bound to the arranged type I collagen nanofibrous matrix [1, 8, 9]. During the bone formation process, a fibrous matrix of collagen is generated, followed by the precipitation of HAp minerals to form a compact bone. Therefore, fabrication of HAp-based hybrids by mimicking the biomineralization procedure has recently gained an increasing amount of attention [9,10,11,12,13,14,15,16,17,18,19,20,21,22,23].

On the other hand, carbon nanotubes (CNTs) are attractive in the context of material science due to not only their good mechanical, electrical, and physicochemical properties but also their similar size to triple-helical collagen fibrils, which can act as scaffolds for the induction of the nucleation and crystallization of HAp as long as they contain nucleation sites. Recently, the use of covalently [24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41] and non-covalently functionalized CNTs [42,43,44,45] as scaffolds for the growth of artificial bone material has been reported. Moreover, Bouropoulos and coworkers [25] have reported the in vitro formation of calcium phosphate on citrate-treated CNT buckypaper. These types of films represent attractive candidates for the self-repair of bone, as these sheets can persist in the body.

In this context, we have reported the synthesis of various CNT/inorganic hybrids under mild reaction conditions by biomineralization [46, 47] or sol–gel condensation reactions [48, 49]. Our method uses a water-soluble dendrimer that contains affinity sites for CNTs (C60 or 1,10-bis(decyloxy)decane)), while poly(amidoamine) (PAMAM) dendrimers with terminal carboxylate groups function as a dispersant for the CNTs [47, 50]. These dendritic dispersants act as an effective “glue” between the CNTs and the inorganic materials, which enables the facile formation of CNT/inorganic hybrids, such as CNT/CaCl2 [47], CNT/CaCO3 [46, 47], CNT/SiO2 [48], and CNT/TiOx [49]. These results prompted us to investigate the fabrication of CNT/dendrimer sheet scaffolds and their subsequent use for the mineralization of HAp. In this paper, we report the fabrication of CNT/dendrimer sheet scaffolds, i.e., dendrimers that are attached to the surface of multi-walled carbon nanotube (MWCNT) buckypaper [51]. The subsequent mineralization of HAp on the MWCNT/dendrimer sheet scaffolds was investigated via an alternate soaking process (ASP) [52] in simulated body fluid (SBF) [53].

Experimental procedure

Instruments

Fourier transform infrared spectroscopy (FT-IR) analysis was carried out using a Shimadzu IRAffinity-1 IR spectrometer (Shimadzu, Kyoto, Japan). Scanning electron microscopy (SEM) was carried out on a Hitachi S-3500N microscope (Hitachi, Tokyo, Japan). X-ray diffraction (XRD) patterns were recorded using Cu-Kα radiation on a Rigaku RAD (Rigaku, Tokyo, Japan) at room temperature. Thermogravimetric analysis (TGA) was carried out on a Shimadzu DTG-60 in air. Quantitative energy-dispersive X-ray spectroscopy (EDX) analysis was carried out on an image obtained by a high-angle annular dark-field scanning transmission electron microscope. Transmission electron microscopy (TEM) measurements were recorded using a JEM02100 transmission electron microscope (200 kV). Water was deionized using a Millipore Milli-Q water purification system. Calcium chloride (CaCl2) was purchased from Kanto Kagaku Co., Ltd. (Tokyo, Japan). Dibasic sodium phosphate (Na2HPO4) was purchased from Nacalai Tesque Co., Ltd. (Kyoto, Japan). MWCNTs (diameter: 20–40 nm; length: 1–2 μm; purity: 95% w/w) were purchased from Tokyo Kasei Co., Ltd. (Tokyo, Japan). Dendrimer was prepared according to previously reported procedures [47]. Buckypaper was prepared according to a previously reported method [51]. The SBF solution at pH 7.4 was prepared at 36.5 °C according to a method reported elsewhere [54].

Preparation of the MWCNT buckypaper

MWCNTs (30 mg) were suspended in N-methyl-2-pyrrolidone (10 mL) and sonicated for 4 h at 17–25 °C using a bath-type ultrasonic cleaner (Honda Electronics Co., Ltd., vs-D100, 110 W, 24 kHz). The resulting suspension was filtered through a membrane filter with a pore size of 0.45 μm (diameter: 2.5 cm, Merck Millipore Ltd.) using an all-glass filtration apparatus [55]. The prepared film was stored for 2 days in a vacuum desiccator containing freshly activated silica gel. The film (29.7 mg, diameter: 2 cm) was gently handled and carefully peeled off the surface of the membrane filter.

Preparation of MWCNT/dendrimer sheet scaffolds that contain 9 wt% dendrimer (MWCNT-D9wt% sheet)

The protocol for the formation of MWCNT-D9wt% sheets is as follows: pristine buckypaper (5 mg, 5 mm × 5 mm) cuttings were soaked for 13 h at room temperature in an aqueous solution (2 mL) of dendrimer (5 mg, 2.74 μmol). After storing the MWCNT/dendrimer sheet scaffolds for 2 days in a vacuum desiccator containing P2O5, MWCNT-D9wt% sheets (5.8 mg) were obtained. The content of dendrimer in the MWCNT/dendrimer sheet scaffold was estimated based on TGA measurements.

Preparation of MWCNT-D14wt% sheets

MWCNTs (30 mg) were suspended in an aqueous solution (10 mL) of dendrimer (30 mg, 16.5 µmol) and sonicated for 4 h at 17–25 °C using a bath-type ultrasonic cleaner (Honda Electronics Co., Ltd., vs-D100, 110 W, 24 kHz). The resulting suspension was filtered through a membrane filter (pore size: 0.45 μm; diameter: 2.5 cm; Merck Millipore Ltd.) using an all-glass filtration apparatus [55]. The sheet obtained in this manner was stored for 2 days in a vacuum desiccator containing P2O5 before the sheet (25.3 mg) was carefully peeled off from the surface of the membrane filter. The content of dendrimer in the MWCNT/dendrimer sheet scaffolds was estimated based on TGA measurements (14 wt%).

Fabrication of MWCNT/HAp (ASP)

An ASP was performed to promote the formation of HAp on the surface of the buckypaper. Initially, the MWCNT/dendrimer sheet scaffold (5 mg; 5 mm × 5 mm) was soaked in an aqueous solution of CaCl2 (10 mL, 0.2 M, pH = 7–8) at 38 °C. After 30 min, the sheet was washed with acetone and dried. Then, the MWCNT/dendrimer sheet scaffold was soaked for 30 min in an aqueous solution of Na2HPO4 (10 mL, 0.12 M, pH = 8–9) at 38 °C. After washing with small portions of water and acetone, the film was dried. These steps represent one cycle. The ASP was repeated for a variable number of cycles, as outlined in the individual experiments. The content of HAp was estimated based on the weight of the residue at 700 °C from TGA measurements.

Fabrication of MWCNT/HAp (SBF process)

The MWCNT/dendrimer sheet scaffold (MWCNT-D9wt%, 5.0 mg, 5 mm × 5 mm) was immersed in SBF (prepared with pH = 7.4 at 36.5 °C) and kept at 60 °C for 14 days. After washing with small portions of water and acetone, the resulting film was stored in a vacuum desiccator containing P2O5 to afford the MWCNT/HAp hybrid (6.3 mg).

Results and discussion

PAMAM dendrimers with a 1,10-bis(decyloxy)decane core [48] (Fig. 1a) were used as a glue to form MWCNT/HAp hybrids. MWCNT/dendrimer sheets were prepared by two different methods. In method A, MWCNT buckypaper, which was fabricated by vacuum filtration of MWCNTs dispersed in N-methylpyrrolidone, was soaked in an aqueous solution of dendrimer (Fig. 1b, method A). The dendrimer contents in the MWCNT/dendrimer sheets obtained in this manner (9 wt%; MWCNT-D9wt%) were determined by TGA in air (Figure S1). In method B, MWCNT/dendrimer sheets were directly fabricated by vacuum filtration of an aqueous solution of the MWCNT/dendrimer composite (Fig. 1b, method B), which afforded MWCNT/dendrimer sheets containing 14 wt% dendrimer (MWCNT-D14wt%).

Fig. 1
figure 1

a Schematic illustration of the dendrimer used in this study. b Fabrication of MWCNT/dendrimer sheets

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HAp was mineralized on the MWCNT-D9wt% sheets using an ASP, which is a well-established and facile method for the formation of HAp on materials [52, 56]. For that purpose, MWCNT-D9wt% sheets were alternately soaked in solutions that contain either calcium or phosphate ions, whereby one cycle lasts 60 min, i.e., 30 min in the calcium-containing solution and 30 min in the phosphate-containing solution, to fabricate the MWCNT/HAp hybrids. Figure 2 shows SEM images of the MWCNT-D9wt%/HAp hybrids that were obtained by subjecting MWCNT-D9wt% sheets to 1, 2, 3, or 5 soaking cycles. After 1 or 2 soaking cycles, the obtained sheets retained the fibrous morphology of the MWCNTs. Interestingly, small spheres attached to the MWCNT-D9wt% sheet surface were clearly observable, and the number of spheres increased with the number of soaking cycles. These results suggest that HAp mineralizes on the MWCNT-D9wt% sheets. Figure S2 shows XRD patterns of the fabricated MWCNT/HAp hybrids. The main diffraction peak at 26.08° can be attributed to the graphite plane (002) of the MWCNTs. The peaks at 31.64° (211) and 39.28° (310) are a close match with the diffraction from HAp (JCPD card number: 34-0010). These peaks sharpen and increase in intensity with increasing number of soaking cycles, which suggests that the amount of crystalline HAp increases with the number of soaking cycles. To characterize the as-prepared nanohybrids, their chemical composition was analyzed by EDX, and the corresponding EDX spectra are shown in Figure S3. The pronounced Ca and P signals on the buckypaper surface suggest the formation of HAp. The Ca/P ratio determined by EDX reached 1.62 after 5 soaking cycles, which is in good agreement with the value for Ca10(PO4)6(OH)2 (HAp). Figure 3a shows a cross-sectional SEM image of the MWCNT/HAp hybrids. The mapping of the elements on the sample cross-section revealed homogeneous deposition of Ca (Fig. 3b), P (Fig. 3c), and C (Fig. 3d) on the surface of the MWCNT-D9wt% sheet. The chemical structure of HAp on the MWCNT-D9wt% sheet was examined by FT-IR. Characteristic peaks at approximately 1034 and 590 cm–1 (Figure S4) are assignable to the P–O stretching of the phosphate groups [57]. These results strongly support the formation of HAp on the surface of the MWCNT-D9wt% sheets.

Fig. 2
figure 2

SEM images of the MWCNT-D9wt%/HAp hybrids obtained by exposing MWCNT-D9wt% to 1, 2, 3, or 5 soaking cycles

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Fig. 3
figure 3

Cross-sectional SEM images of a MWCNT-D9wt%/HAp hybrid obtained from 5 soaking cycles. Mapping of Ca, P, and C in the sample cross-section

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To investigate the influence of the dendrimer content, we also investigated the mineralization of HAp on MWCNT-D14wt% and pristine buckypaper (MWCNT-D0wt%). Small spheres similar to those attached to the surface of the MWCNT-D9wt% sheets were also observed in the SEM images of the MWCNT-D14wt%/HAp hybrids (Figure S5). However, it seems that the amount of Hap on the MWCNT-D14wt%/HAp hybrids is lower than that on the MWCNT-D9wt%/HAp hybrids. Figure S6 depicts TGA thermographs of the MWCNT-D0wt%/HAp, MWCNT-D9wt%/HAp, and MWCNT-D14wt%/HAp hybrids, wherein weight loss in the range of 300–450 °C is considered to signify decomposition of the dendrimer. A more substantial weight loss caused by the decomposition of the MWCNTs was observed in the range of 500–600 °C (600–700 °C for MWCNT-D0wt%/HAp). The weight of the residue from each run at 700 °C was assigned exclusively to HAp. For the MWCNT-D0wt%/HAp, MWCNT-D9wt%/HAp, and MWCNT-D14wt%/HAp hybrids, HAp contents of 0.7, 12.5, and 5.5 wt%, respectively, were estimated. This result indicates that dendrimers provide nucleation sites and effectively act as a glue between the CNTs and HAp, even though the amount of Hap decreases with increasing dendrimer content. This trend is most likely due to differences associated with the nucleation sites on the surface of the MWCNTs. Higher amounts of dendrimers may cause higher binding levels of calcium ions, corresponding to a depletion of these ions in solution, which could inhibit nucleation.

To probe potentially beneficial aspects and the utility of the physical modifications of buckypaper with dendrimers, we carried out biomimetic crystallization of calcium phosphate on a MWCNT/dendrimer sheet in SBF. For this purpose, MWCNT-D9wt% sheets were immersed into SBF and kept at 60 °C for 14 days under static conditions in order to crystallize HAp. TEM analysis of the resulting buckypaper film revealed that the MWCNT backbone was covered in scaly crystals (Fig. 4). In the case of the pristine buckypaper (MWCNT-D0wt%), only very few particles were observed on the MWCNTs (Figure S7), although EDX analysis revealed significant adsorption of calcium and phosphate ions from the SBF (Ca:P = 1.40). Even though this ratio is smaller than the ideal ratio of HAp (1.67), the result indicates that the Ca2+ and PO43– ions at least interact with the surface of the buckypaper with the help of the dendrimer scaffold. Another important biomaterial in this context is β-tricalcium phosphate (TCP), which shows in vivo resorbability during bone tissue ingrowth [58]. In addition, the interaction between β-TCP and body fluids results in HAp formation after implantation [59]. Therefore, we investigated the sintering of the MWCNT/apatite hybrid (Ca:P = 1.40) obtained from SBF to potentially fabricate MWCNT/TCP sheets. After sintering of the MWCNT/apatite hybrid (Ca:P = 1.40) at 1000 °C, MWCNT/β-TCP sheets were obtained. The XRD pattern of the sintered MWCNT/apatite hybrid (Ca:P = 1.40) is shown in Figure S8. The sharp peaks of the recorded XRD pattern are consistent with the characteristics of β-TCP according to the Joint Committee on Powder Diffraction Standards card (JCPDS) PDF No. 9–169.

Fig. 4
figure 4

TEM image of a dendrimer-modified buckypaper (MWCNT-D9wt%) after immersion for 14 days in SBF at 60 °C

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Conclusions

The crystallization of HAp on buckypaper, whose surface was modified with PAMAM dendrimers that contain a 1,10-bis(decyloxy)decane core, using an ASP was investigated. Moreover, the biomimetic crystallization of HAp on dendrimer-modified buckypaper from SBF was investigated. SEM and TEM analyses of the MWCNT/HAp hybrids revealed that HAp successfully mineralizes on the surface of these MWCNTs. These CNT/dendrimer sheets should become useful for the production of synthetic HAp as a bone replacement and for the investigation at the nano-bio interface on the growth of nerve cells.