Biocompatible, Ultralight, Strong Hydroxyapatite Networks Based on

Feb 27, 2017 - In the past decade, ultralight materials such as aerogels have become one of the hottest research topics owing to their unique properti...
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Biocompatible, Ultralight, Strong Hydroxyapatite Networks Based on Hydroxyapatite Microtubes with Excellent Permeability and Ultralow Thermal Conductivity Yong-Gang Zhang, Ying-Jie Zhu,* Feng Chen,* and Tuan-Wei Sun State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, P. R. China University of Chinese Academy of Sciences, Beijing, 100049, China S Supporting Information *

ABSTRACT: In the past decade, ultralight materials such as aerogels have become one of the hottest research topics owing to their unique properties. However, most reported ultralight materials are bioinert. In this work, by using biocompatible, monodisperse, single-crystalline hydroxyapatite (HAP) microtubes as the building blocks, ultralight, strong, highly porous, three-dimensional (3-D) HAP networks have been successfully fabricated through a facile freeze-drying method and subsequent sintering at 1300 °C for 2 h. The as-prepared ultralight, strong, highly porous 3-D HAP microtube networks exhibit superior properties, such as ultrahigh porosity (89% to 96%), low density (94.1 to 347.1 mg/cm3), high compressive strength that can withstand more than 6400 times of their own weight without any fracture and is higher than aerogels with similar densities, and ultralow thermal conductivity (0.05 W/mK). Owing to their high porosity, ultralight, and good mechanical properties and high biocompatibility, the HAP microtube networks reported herein are promising for applications in various fields. KEYWORDS: hydroxyapatite, microtubes, networks, ultralight, porous applications, especially in biomedical fields. Therefore, the challenge remains for the preparation of biocompatible, ultralight, strong, highly porous, thermally, and mechanically stable structures with novel properties by simple, environmentally friendly, low-cost methods which can be put into practical use. Herein, biocompatible, monodisperse, single-crystalline hydroxyapatite (HAP) microtubes have been successfully prepared by a simple one-step solvothermal strategy. By using the as-prepared HAP microtubes as the building blocks, ultralight, strong, highly porous, three-dimensional HAP microtube networks have been successfully prepared through the freeze-drying method and sintering at a relatively low temperature of 1300 °C under air atmosphere without using any additives. The as-prepared HAP microtube networks exhibit high porosity (89% to 96%), low density (94.1 to 347.1 mg/cm3), high compressive strength (up to 1 MPa) that is larger than those of the aerogels with similar densities, ultralow thermal conductivity (0.05 W/mK), and high biocompatibility. The HAP microtube networks reported herein have promising applications not only in the traditional

1. INTRODUCTION In the past decade, ultralight materials such as aerogels and microlattices have been of great interest owing to their superior properties including high porosity, low density, and low thermal conductivity; thus, they are promising for applications in many advanced technologies from catalysis to thermal/acoustic insulation to environmental protection, etc.1−9 Up to now, different kinds of ultralight materials have been successfully prepared including Au aerogel, Cu aerogel, graphene aerogel, carbon nanotube aerogel, silica aerogel, alumina aerogel, lightweight ceramics, nanolattices/microlattices, etc. 9−19 Although some progress has been made, the dimension, porosity, density, and mechanical properties of those materials usually cannot meet the requirements for practical applications. For example, aerogels exhibit high porosity but tend to be low in mechanical strength.18,19 Nanolattices and microlattices possess both high porosity and outstanding mechanical properties, but their dimensions are usually small.14,16 Recently, ultralight, strong, three-dimensional SiC structures with high porosity and ultralow density (300 mg/cm3) were fabricated using SiC microfibers, which exhibited advantages such as low thermal conductivity and acoustic impedance;10 however, the sintering temperature was very high nearly up to 2000 °C, and sintering aids and a special atmosphere were needed for the synthesis. It should be noted that most reported ultralight materials are bioinert, which has greatly constrained their © XXXX American Chemical Society

Received: October 19, 2016 Accepted: February 16, 2017

A

DOI: 10.1021/acsami.6b13328 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces fields such as thermal insulation and shock energy absorption but also in various biomedical fields such as bone defect repair.

seeded and cultured on the HAP microtube networks in the 24 well microassay plate at 37 °C under a 5% CO2 humidified atmosphere for 7 h. Then, the samples were withdrawn and rinsed with phosphate buffered saline (PBS), and adherent cells were fixed in 4% paraformaldehyde and then washed with PBS three times. The samples were treated with 0.1% Triton X-100 in PBS for 2 min before washing with PBS three times, and nonspecific binding sites were blocked using 1% bovine serum albumin (BSA) in PBS for 20 min. The actin cytoskeletons were labeled green by incubation with phalloidin−FITC (Sigma) for 1 h. After rinsing with PBS three times, the cell nuclei were stained blue by 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma) for 10 min and washed with PBS three times. Finally, the morphology of bMSCs on the surface of HAP microtube networks was observed using a confocal laser scanning microscope (Leica, SP8, Germany) and scanning electron microscope (SEM). After 24 h of culture, the cells were fixed with glutaraldehyde at 4 °C overnight. Then, the cells were gradually dehydrated with ethanol solutions with gradient concentrations (0%, 30%, 50%, 70%, 95%, and 100%). Then, the surface of samples was sputter-coated with gold for SEM observation. 2.6. Characterization. The as-prepared HAP microtubes and HAP microtube networks were characterized using X-ray powder diffraction (XRD, Rigaku D/max 2550 V, Cu Kα radiation, λ = 1.54178 Å), transmission electron microscopy (TEM, JEOL JEM 2100F), and scanning electron microscopy (SEM, Hitachi TM-3000, Japan). The pore size distribution and total pore surface area of the samples were measured using the mercury porosimetry (AutoPore IV 9510, USA). The thermal conductivity of the HAP ceramics (100 × 40 × 10 mm3) was measured by a hot wire method using a thermal conductivity meter (QTM 500, Japan). The compressive strength of the HAP ceramics (10 × 10 mm2) was tested by a universal testing machine (DRK 101A, China).

2. EXPERIMENTAL SECTION 2.1. Materials. Anhydrous calcium chloride (CaCl2), sodium hydroxide (NaOH), sodium hexametaphosphate ((NaPO3)6), and oleic acid were purchased from Sinopharm Chemical Reagent Co. AlamarBlue was purchased from AbD Serotec (UK). HAP nanoparticles were purchased from Aladdin Industrial Co. Rat bone marrow stromal cells (bMSCs) were obtained from the Cell Resource Center, Shanghai Institutes for Biological Sciences, China. Other reagents used in the cell viability and cell adhesion experiments were purchased from Sigma-Aldrich (USA). All reagents were used as received without further purification. 2.2. Preparation of Hydroxyapatite (HAP) Microtubes. The typical synthetic procedure for HAP microtubes is as follows: First, a ternary solvent of deionized water (5 mL), ethanol (7 mL), and oleic acid (8 mL) was prepared by vigorous stirring for 5 min. Then, the aqueous solutions of CaCl2 (10 mL, 0.198 M), NaOH (10 mL, 1.650 M), and sodium hexametaphosphate (NaPO3)6 (10 mL, 0.032 M) were separately and dropwise added into the above mixed solvent. The resulting reaction suspension was poured into a 100 mL Teflon-lined stainless steel autoclave, sealed, and put into an oven which was preheated to 180 °C and maintained at that temperature for 25 h. Finally, the product was washed with ethanol and deionized water twice, respectively. After centrifugation, the product was dried at 60 °C for further use. 2.3. Preparation of Ultralight, Strong, Three-Dimensional Highly Porous HAP Microtube Networks. A certain amount of powdered HAP microtubes was mixed with a 0.5 wt % chitosan solution which was obtained by dissolving 0.500 g of chitosan in 99.500 g of 1 vol % acetic acid solution at a certain solid loading to form a slurry. The solid loading is defined as the weight of powdered HAP microtubes/the total weight of the slurry ×100%. After stirring for 1 h, the white slurry was added into a cylindrical mold with a diameter of 10 mm and was frozen in a refrigerator. Subsequently, the frozen samples were dried by freeze-drying. Finally, the green bodies were sintered in a muffle furnace at 1300 °C for 2 h at a heating rate of 10 °C/min. Ultralight, strong, highly porous three-dimensional (3-D) HAP microtube networks were obtained after the furnace cooled to room temperature. The control sample was prepared with the same procedure as mentioned above except for using the commercial HAP nanoparticles instead of HAP microtubes. 2.4. Characterization of Density, Porosity, Water Absorption, Pore Surface Area, and Pore Size Distribution of HAP Microtube Networks. The density and porosity were measured in water by the Archimedes’ method. The total pore surface area and pore size distribution were measured by the mercury porosimetry (AutoPore IV 9510, USA). Mercury was forced into the pores under the applied pressure. The pore size distribution was calculated by combining the applied pressure with the intrusion volume. The water absorption ability was calculated by weighing the weight of HAP microtube networks before and after water absorption. 2.5. Cell Culture and in Vitro Cell Viability of HAP Microtube Networks. The biocompatibility of the HAP microtube networks was characterized by the alamarBlue (AbD Serotec Ltd., UK) assay. The rat bone marrow stromal cells (bMSCs) were seeded in a 24 well plate at a density of 5 × 104 cells per well with the HAP microtube networks. After 1, 4, and 7 day(s) of culture, the HAP microtube networks were washed with phosphate buffer solution twice and then transferred to a new 24 well plate, and 500 μL of alamarBlue (10%) was added in each well. The plate was put into the incubator to culture for 2 h. Then, 100 μL of solution of each well was transferred into a new 96 well plate for measurement. The fluorescence intensity of reduced alamarBlue was determined by an enzyme-labeling instrument (BIO-TEK, ELX 800) at wavelengths of 530 nm (extinction) and 590 nm (emission). The experimental procedure and analysis of cell proliferation conformed to the instruction of the alamarBlue assay. The adhesion of bMSCs on the HAP microtube networks was also tested. The typical procedure is as follows: 5 × 104 bMSCs were

3. RESULTS AND DISCUSSION The morphology of the as-prepared HAP microtubes is shown in Figure 1. The product is composed of HAP microtubes with

Figure 1. SEM micrographs (a−c) and TEM micrograph (d) of the asprepared HAP microtubes with needle-like ends.

lengths from several tens of microns up to ∼100 μm and diameters of several microns. Moreover, the SEM and TEM micrographs show that many HAP microtubes have sharp needle-like ends. In addition, the HAP microtubes are well dispersed without any aggregation, indicating the excellent dispersibility of HAP microtubes. B

DOI: 10.1021/acsami.6b13328 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 2. Schematic illustration for the preparation of the HAP microtube networks using HAP microtubes as the building blocks.

Figure 3. SEM micrographs of the as-prepared biocompatible, ultralight, highly porous 3-D HAP microtube networks with different solid loadings of 6.5 wt % (sample I, a1−a4), 12.3 wt % (sample II, b1−b4), and 16.7 wt % (sample III, c1−c4) synthesized by freeze-drying at −20 °C and sintering at 1300 °C for 2 h at a heating rate of 10 °C/min.

formed as the precursor via chemical reactions 1 and 2. Subsequently, the nucleation of hydroxyapatite occurs under solvothermal conditions through chemical reactions 3, 4, and 5. Then, the crystal growth of hydroxyapatite takes place to form HAP microtubes under the solvothermal conditions in a relatively long period of time at a relatively high temperature and high pressure. Calcium oleate acts as both the calcium source and the precursor for the formation of HAP microtubes. The chitosan solution is used to prepare the stable HAP microtube slurry considering hydroxyl and amine groups exist in the repeating unit of chitosan chain.20 When the HAP microtubes are mixed with the chitosan solution, the hydroxyl groups on HAP microtubes can form hydrogen bonds with amine groups and hydroxyl groups of chitosan, resulting in the formation of the stable and homogeneous HAP microtube slurry. By using the HAP microtube slurry, ultralight, strong, highly porous 3-D HAP microtube networks can be prepared by the freeze-drying method and subsequent sintering at a relatively low temperature of 1300 °C, which is illustrated in Figure 2. The effect of the freeze-drying temperature on the product was investigated. The SEM images of the samples with different

We propose a possible mechanism for the formation of HAP microtubes. The following chemical reactions take place in the reaction system: CH3(CH 2)7 CHCH(CH 2)7 COOH + NaOH → CH3(CH 2)7 CHCH(CH 2)7 COONa + H 2O

(1)

2CH3(CH 2)7 CHCH(CH 2)7 COONa + CaCl 2 → [CH3(CH 2)7 CHCH(CH 2)7 COO]2 Ca + 2NaCl (2)

3(NaPO3)6 + 4NaOH + 10H 2O → 2Na5P3O10 + 12NaH 2PO4 (3)

H 2PO4 − + 2OH− → PO4 3 − + 2H 2O

(4)

10[CH3(CH 2)7 CHCH(CH 2)7 COO]2 Ca + 6PO4 3 − + 2OH− → Ca10(OH)2 (PO4 )6 + 20CH3(CH 2)7 CHCH(CH 2)7 COO−

(5)

From the above-mentioned chemical reactions, we propose that at the early stage of the formation process calcium oleate is C

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Figure 4. SEM micrographs of the as-prepared biocompatible, ultralight, highly porous 3-D HAP microtube networks with a layered structure and different solid loadings of 6.5 wt % (sample I, a1−a4), 12.3 wt % (sample II, b1−b4), and 16.7 wt % (sample III, c1−c4) synthesized by freeze-drying at −80 °C and sintering at 1300 °C for 2 h at a heating rate of 10 °C/min.

solid loadings synthesized by the freeze-drying method at different temperatures of −20 and −80 °C are shown in Figures 3 and 4, respectively. From Figures 3 and 4, one can see that the structures of the samples synthesized at varied freeze-drying temperatures are different. The reason is that water can form ice crystals with different morphologies at different temperatures.21 When the freeze-drying temperature is −20 °C, ice spheres are formed and HAP microtubes are expelled out from water to form the walls of the HAP microtube networks. After freeze-drying and sintering, the spherical ice crystals and chitosan are removed, and the HAP microtube networks with the honeycomb structure are obtained. However, when the freeze-drying temperature is −80 °C, water forms layered crystals and HAP microtubes are expelled out from water and form parallel layers. After freeze-drying and sintering, ice and chitosan are removed and HAP microtube networks with a lamella structure are obtained.22−24 In both cases, the thickness of the walls of HAP microtube networks increases with increasing solid loading. However, the samples synthesized through the freeze-drying method at both −20 and −80 °C exhibit an interconnected highly porous structure and are composed of the relatively uniform stacking of individual HAP microtubes, forming the 3-D highly porous network of HAP microtubes. In addition to the micropores derived from the freeze-drying process, there are also nanosized pores existing in the walls of the HAP microtube network. Interestingly, the tubular structure of HAP microtubes can be well preserved even after sintering, as marked in Figure 3a4,b4,c4. These characteristics endow the HAP microtube networks with advantages such as high biocompatibility, high porosity, ultralight weight, high compressive strength, and excellent permeability. The crystal phase of the as-prepared HAP microtubes and HAP microtube networks was characterized by XRD. The crystal phase of the HAP microtubes can be determined to be single-phase HAP (JCPDS No. 09-0432), indicating that the synthesized product is composed of single-phase hydroxyapatite microtubes (Figure 5). However, the crystal phases of the HAP microtube networks can be indexed to HAP (JCPDS No. 09-0432) as the major product and a small amount of β-

Figure 5. XRD patterns of HAP microtubes and the HAP microtube network (solid loading 16.7 wt %, freeze-drying −20 °C, sintering 1300 °C).

Ca3(PO4)2 (JCPDS No. 09-0169) as the minor product. For the HAP microtube networks, two small peaks of β-Ca3(PO4)2 appear at 2θ = 27.77° and 2θ = 31.02°,25 indicating the partial phase transformation of HAP takes place during the sintering process. The densities, porosities, and water absorption capacities of the HAP microtube networks with different solid loadings obtained at different freeze temperatures were measured. As shown in Figure 6 and Table 1, the porosity, density, and water absorption capacity of the samples obtained at the freeze temperature of −20 °C are similar to those of the samples obtained at the freeze temperature of −80 °C with the same solid loading. The densities of the as-prepared HAP microtube networks range from 94.1 to 347.1 mg/cm3, and the porosities of the HAP microtube networks range from 89% to 97%. The highest porosity of the HAP microtube network is as high as 97%, and its water absorption capacity is about 10-fold of its own weight. The HAP microtube network (solid loading 16.7 wt %, freeze-drying −20 °C, sintering 1300 °C) has a low density of 249.9 mg/cm3, which is so light that a very thin stem of a flower can even withstand its weight (∼140 mg), as shown in Figure 7a; in addition, this HAP microtube network (∼140 D

DOI: 10.1021/acsami.6b13328 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 7. Digital images of the demonstration of the HAP microtube network sample (solid loading 16.7 wt %, freeze-drying −20 °C, sintering 1300 °C; ∼140 mg) with a density of 249.9 mg/cm3 and porosity of 92%: (a) the HAP microtube network sample on a thin stem of a flower; (b−d) the HAP microtube network sample can even withstand a weight of ∼900 g of a 50 mL stainless steel autoclave without any fracture, which is more than 6400 times as heavy as the HAP microtube network sample.

freezing at the temperature of −20 °C with different solid loadings of 6.5, 12.3, and 16.7 wt % is 59.2, 42.1 and 10.3 m2/g, respectively. The solid loading has an effect on the pore size distribution and the total pore surface area because larger solid loading makes thicker walls of the HAP microtube networks. The mechanical properties of the HAP microtube networks are dependent on the solid loading of HAP microtubes. The compressive strength of the HAP microtube networks increases with the solid loading of HAP microtubes, as shown in Figure 8a−c. When the solid loading is 16.7 wt % (Figure 8c), the compressive strength of the HAP microtube network obtained by the freeze-drying at −20 °C and sintering at 1300 °C is about 0.45 MPa. When its sintering temperature increases to 1400 °C, the compressive strength increases to nearly 1 MPa (Figure 8d) with a slight decrease in porosity from 92% to 89% and an increase in density from 249.9 to 347.1 mg/cm3. The crystal phase of the HAP microtube network sintered at 1400 °C was characterized by XRD (Figure S2), which is similar to the XRD pattern of the HAP microtube network sintered at

Figure 6. (a) Porosity, (b) density, and (c) water absorption capacity of the HAP microtube networks with different solid loadings of 6.5 wt % (sample I), 12.3 wt % (sample II), and 16.7 wt % (sample III) obtained at different freeze temperatures of −80 °C and −20 °C and sintered at 1300 °C.

mg) can even withstand a weight of about 900 g of a 50 mL stainless steel autoclave without any fracture, which is more than 6400 times as heavy as the HAP microtube network itself, as shown in Figure 7b−d, indicating that the HAP microtube networks exhibit not only ultrahigh porosity and ultralight weight but also high compressive strength. The pore size distributions of the HAP microtube networks with different solid loadings of 6.5, 12.3, and 16.7 wt % obtained by freezing at the temperature of −20 °C are narrow, mainly ranging from 50 to 110 μm (Figure S1). The total pore surface area of the HAP microtube networks obtained by

Table 1. Density, Porosity, and Water Absorption Capacity of the HAP Microtube Networks with Different Solid Loadings of 6.5 wt % (I), 12.3 wt % (II), and 16.7 wt % (III) Obtained at Different Sintering Temperatures sample

I (−20 °C)

I (−80 °C)

II (−20 °C)

II (−80 °C)

III (−20 °C)

III (−80 °C)

III (−20 °C)

solid loading (wt %) sintering temperature (°C) density (mg/cm3) porosity (%) water absorption capacity (wt %)

6.5 1300 94.1 97 1021

6.5 1300 100.4 96.8 927

12.3 1300 170.2 94.6 542

12.3 1300 177.7 94.4 495.2

16.7 1300 249.9 92 358

16.7 1300 248.2 92.1 342.3

16.7 1400 347.1 89 220

E

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Figure 8. (a−c) Compressive strength−strain curves of the HAP microtube networks with different solid loadings of 6.5 wt % (sample I), 12.3 wt % (sample II), and 16.7 wt % (sample III) fabricated at freezing temperatures of −80 and −20 °C and sintered at 1300 °C. (d) Compressive strength− strain curves of the HAP microtube network with a solid loading of 16.7 wt % obtained at the freezing temperature of −20 °C and sintered at 1400 °C. Three parallel samples were measured for each measurement.

1300 °C except for the formation of α-Ca3(PO4)2. According to the literature,26 the phase transformation of β-Ca3(PO4)2 to αCa3(PO4)2 was considered to be deleterious to mechanical properties. Therefore, the enhancement of the mechanical properties of the HAP microtube network sintered at 1400 °C is attributed to other reasons. In addition, the morphology of the HAP microtube network sintered at 1400 °C was also characterized. As shown in Figure S3, the HAP microtube network becomes denser and its density is larger after sintering at 1400 °C, which contributes to the increase of the mechanical strength. However, the compressive strengths of the HAP microtube networks prepared at −80 °C are much lower than those of the HAP microtube networks obtained at −20 °C; the reason is that the HAP microtube networks prepared at −80 °C have a layered structure. However, not all layers are parallel to each other, as shown in Figure 4a1, when the load is applied, and the contact regions of the different oriented layers easily collapse. Moreover, the critical compression strain of the HAP microtube networks with different solid loadings prepared by freeze-drying at the temperature of −20 °C and sintering at 1300 °C was measured. As shown in Figure 9, the HAP microtube networks do not collapse suddenly even at a relatively high load until densification occurs, indicating the HAP microtube networks can effectively absorb energy. Therefore, the HAP microtube networks may be used as shock energy absorption material. HAP microtubes play an important role in the fabrication of the highly porous ultralight HAP microtube networks with superior properties, compared with the control sample fabricated under the same conditions using HAP nanoparticles (Figure S4) instead of HAP microtubes. The water absorption capacity of the HAP microtube network with a solid loading of

Figure 9. Critical compressive strain of the HAP microtube networks with different solid loadings of 6.5 wt % (sample I), 12.3 wt % (sample II), and 16.7 wt % (sample III) prepared at a freezing temperature of −20 °C and sintered at 1300 °C.

16.7 wt % prepared at −20 °C and sintered at 1300 °C is almost 3 times as large as that of the control sample, and the density of the HAP microtube network is only ∼40% of that of the control sample (Figure 10). The steric hindrance among the HAP microtubes is much stronger than the HAP particles during the sintering process, leading to smaller shrinkage. Moreover, HAP microtubes stack on each other to form a 3-D highly porous network with a large number of tubular channels, resulting in a high porosity and ultralow density. In contrast, HAP particles grow into a whole bulk with a low porosity and a relatively high density. Compared with other porous HAP ceramics (Figure 11a,b) and other biomaterials (Figure 11c,d) fabricated by the freezedrying method, the HAP microtube networks reported in this work have obvious advantages, such as good mechanical F

DOI: 10.1021/acsami.6b13328 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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those of SiO2 aerogels with similar densities, as shown in Figure 11e. The HAP microtube network with the solid loading of 16.7 wt % fabricated at the freezing temperature of −20 °C and sintered at 1300 °C also exhibits ultralow thermal conductivity at room temperature (0.05 W/mK); this value is smaller than the traditional porous Al2O3, porous ZrO2, and mullite and is comparable to the aerogels, as shown in Figure 11f. Moreover, the thermal conductivity of the HAP microtube network still remains very low even at a high temperature of 900 °C, as shown in Figure 12. The thermal insulating property of the HAP microtube network was investigated, as shown in Figure 13. A piece of cotton was directly placed on a copper plate which was heated by an alcohol burner, and the cotton was totally carbonized and turned to black after 1 min. However, when a piece of HAP microtube network (2 mm in thickness) was placed in between the cotton and the Cu plate, the cotton was well protected even when the heating time was up to 2 min, indicating that the HAP microtube networks have very low thermal conductivity and excellent thermal insulating property, which is promising for the application as the thermal insulator material.

Figure 10. Comparison of the porosity, density, and water absorption capacity between the HAP microtube network (solid loading 16.7 wt %, freezing temperature −20 °C, sintering 1300 °C) and the control sample.

properties, high porosity, and ultralow density. The compressive strengths of the HAP microtube networks are larger than

Figure 11. (a, b) Comparison of the compressive strengths of the HAP microtube networks in this work with other porous HAP ceramics27−35 obtained by the freeze-drying method. (c, d) Comparison of the compressive strengths of the HAP microtube networks in this work with other biomaterials36−49 prepared by the freeze-drying method. (e) Comparison of the compressive strengths of the HAP microtube networks in this work with other ultralight materials.7,16,50,51 (f) Comparison of the room-temperature thermal conductivity of the HAP microtube network (solid loading 16.7 wt %, freezing temperature −20 °C, sintering 1300 °C) with other porous thermal insulating materials.10,18,19,52−60 G

DOI: 10.1021/acsami.6b13328 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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microtube network with long filopodia. These results indicate that the HAP microtube networks are biocompatible and favorable for cell attachment and growth. All these results indicate that the highly porous ultralight HAP microtube networks exhibit excellent biocompatibility and high water/ nutrients absorption ability; they are a novel candidate for application in tissue engineering. The high porosity, favorable mechanical properties, and good permeability of the highly porous ultralight HAP microtube networks are promising for the nutrient absorption and transportation, tissue infiltration and, ultimately, vascularization when they are used as biomedical scaffolds. Figure 12. Thermal conductivities at different temperatures of the HAP microtube network (solid loading 16.7 wt %, freezing temperature −20 °C, sintering 1300 °C).

4. CONCLUSIONS In this work, single-phase, monodisperse, and biocompatible hydroxyapatite (HAP) microtubes have been successfully synthesized by a simple one-step solvothermal strategy using CaCl2, NaOH, and (NaPO3)6 in a mixed ternary solvent of deionized water, ethanol, and oleic acid. Furthermore, we have successfully made full use of the unique tubular structure of HAP microtubes to overcome the difficulties in fabricating highly porous, ultralight HAP microtube networks with high biocompatibility, high porosity, interconnected tubular channels, high thermal stability, excellent permeability, low thermal conductivity, and excellent mechanical properties through freeze-drying and sintering at a relative low temperature of 1300 °C under air atmosphere. The as-prepared highly porous ultralight HAP microtube networks exhibit high porosities ranging from 89% to 96%, ultralow densities ranging from 94.1 to 349.9 mg/cm3, good compressive strengths (up to 1 MPa) that are larger than those of aerogels with similar densities, very low thermal conductivity (0.05 W/mK), and high biocompatibility. The unique tubular structure of HAP microtubes can be well preserved in the as-prepared highly porous ultralight HAP

On the other hand, the HAP microtube network (solid loading 16.7 wt %, freezing temperature −20 °C, sintering 1300 °C) has a high absorption capacity for the Dulbecco’s Modified Eagle’s Medium, as shown in Figure 14a, indicating that the HAP microtube network can effectively absorb nutrients from nearby tissues if it is implanted in vivo as the bone scaffold. The biocompatibility of the HAP microtube network was characterized by cell proliferation and cell adhesion tests. As shown in Figure 14b−d, the cell viability of the rat bone marrow stromal cells (bMSCs) cultured on the HAP microtube network increases with the culture time, indicating that bMSCs cells can proliferate on the HAP microtube network and that the HAP microtube network is biocompatible and nontoxic. The bMSCs can adhere and spread well on the surface of the HAP microtube network after culturing for 7 h, as shown in Figure 14c. From the SEM image in Figure 14d, one can clearly see that the bMSCs spread well on the surface of the HAP

Figure 13. Thermal insulation property of the HAP microtube network (solid loading 16.7 wt %, freezing temperature −20 °C, sintering 1300 °C). (a1−a3) The cotton placed on the copper plate on the flame of an alcohol lamp is burnt in a short period of time (