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Biomimetic fabrication of a three-level hierarchical calcium phosphate/collagen/hydroxyapatite scaffold for bone tissue engineering

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Biofabrication Biofabrication 6 (2014) 035013 (12pp)


Biomimetic fabrication of a three-level hierarchical calcium phosphate/collagen/ hydroxyapatite scaffold for bone tissue engineering Changchun Zhou1, Xingjiang Ye1, Yujiang Fan1, Liang Ma2, Yanfei Tan1, Fangzu Qing1 and Xingdong Zhang1 1

National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, 610064, People’s Republic of China 2 Zhejiang California International NanoSystems Institute, Zhejiang University, Hangzhou, 301158, People’s Republic of China E-mail: [email protected] Received 17 December 2013, revised 4 May 2014 Accepted for publication 12 May 2014 Published 30 May 2014 Abstract

A three-level hierarchical calcium phosphate/collagen/hydroxyapatite (CaP/Col/HAp) scaffold for bone tissue engineering was developed using biomimetic synthesis. Porous CaP ceramics were first prepared as substrate materials to mimic the porous bone structure. A second-level Col network was then composited into porous CaP ceramics by vacuum infusion. Finally, a thirdlevel HAp layer was achieved by biomimetic mineralization. The three-level hierarchical biomimetic scaffold was characterized using scanning electron microscopy, energy-dispersive x-ray spectra, x-ray diffraction and Fourier transform infrared spectroscopy, and the mechanical properties of the scaffold were evaluated using dynamic mechanical analysis. The results show that this scaffold exhibits a similar structure and composition to natural bone tissues. Furthermore, this three-level hierarchical biomimetic scaffold showed enhanced mechanical strength compared with pure porous CaP scaffolds. The biocompatibility and osteoinductivity of the biomimetic scaffolds were evaluated using in vitro and in vivo tests. Cell culture results indicated the good biocompatibility of this biomimetic scaffold. Faster and increased bone formation was observed in these scaffolds following a six-month implantation in the dorsal muscles of rabbits, indicating that this biomimetic scaffold exhibits better osteoinductivity than common CaP scaffolds. Keywords: biomimetic material, calcium phosphate, collagen, mesenchcymal stem cell, bone tissue engineering (Some figures may appear in colour only in the online journal) 1. Introduction

biological organisms [2, 3]. Bone is a complex and wellorganized tissue mainly composed of hydroxyapatite (HAp) and collagen (Col). Approximately 30 wt.% to 35 wt.% of bone is composed of organic materials; approximately 95% of these materials are type I Col [1, 4]. Bone can be considered to be an apatite-reinforced Col composite at the molecular level. Furthermore, the basic structure of natural bone comprises HAp crystals embedded in holes inside the Col fibril to

Various biomedical bone implants used to repair bone defects or damage caused by trauma, accidents, tumors and bone diseases have received considerable attention [1]. Biomimetic scaffolds provide a promising approach to regenerate diseased or injured bone tissues. Biomimetic structures demonstrate increased functionality through mimicking the qualities of 1758-5082/14/035013+12$33.00


© 2014 IOP Publishing Ltd Printed in the UK

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biomimetic scaffold were evaluated by conducting in vitro and in vivo tests. The results demonstrate that this scaffold closely resembles the composition and hierarchical structures of natural bone. This scaffold could be a promising material for bone tissue engineering.

develop intrafibrillar minerals [5, 6]. Hence, HAp- and Colcomposited biomimetic structural materials have been extensively studied for bone tissue engineering. Both HAp and Col have their advantages for use in bone tissue engineering. Col, the main organic component of bone tissue and the extracellular matrix (ECM), induces positive effects on cellular attachment, proliferation and differentiation [7]. HAp, the main inorganic bone component, has been widely used in bone regeneration because of its bioactive [8, 9], osteoconductive and osteoinductive properties [10, 11]. However, pure HAp and Col are not suitable for direct use as bone substitutes. Pure HAp ceramics are mainly used as bonefilling materials at non-load-bearing sites because of their inherent brittleness [12]. Pure Col scaffold usually lacks calcium and exhibits high degradation rates; in addition, weak initial compressive strength hinders the successful application of this scaffold in bone tissue engineering [13]. Therefore, the incorporation of Col into HAp may contribute to biomimetic constructions and improve both their mechanical and biological properties. Hierarchical HAp-Col composites show a chemical structure similar to native bones. Therefore, biomimetic HApCol composites have been widely investigated [14, 15]. Three basic techniques are applied to fabricate HAp-Col composite structures: molecular self-assembly, phase separation and electrospinning [16, 17]. The first two techniques normally involve the precipitation of HAp or in situ synthesis of HAp nanoparticles mixed in a Col solution as well as cross-linkage and lyophilization to develop a porous HAp-Col composite scaffold [8, 18]. Electrospinning utilizes a high electric field to drive the Col solution as a continuous jet from a thin nozzle to form a porous scaffold [19]. However, these methods yield relatively poor biomechanical properties for the prepared scaffolds. Sintering is an important step to fabricate and enhance the mechanical strength of CaP ceramics. The scaffolds fabricated by the above techniques are mostly Col-based and are not suitable for sintering. Furthermore, the obtained scaffolds mimic relatively simple hierarchical bone tissue structures. Achieving multilevel hierarchical structures resembling natural bone is indeed difficult. This study developed a novel, three-level hierarchical biomimetic scaffold resembling natural bone’s hierarchical structures and compositions. A porous CaP ceramic was initially designed to mimic the porous bone structure, provide a sufficient calcium source, and achieve the desired mechanical function and mass transport properties. A secondlevel Col network was composited into the porous structures with the mechanically strong CaP ceramic as a template, further improving the mechanical strength of the composite according to the ‘brick-and-mortar’ reinforcement theory. A third-level nano-HAp layer was achieved by biomimetic mineralization on the second-level Col framework. The biomimetic scaffold microstructure was studied using scanning electron microscopy (SEM) and porosity measurements. The composition of this scaffold was analyzed by energy-dispersive x-ray spectra (EDX), x-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The biocompatibility, osteoconductivity and osteoinductivity of the

2. Materials and methods 2.1. Materials

CaP powders (hydroxyapatite/β-tricalcium phosphate = 30:70) with a particle size of approximately 200 μm were produced by the National Engineering Research Center for Biomaterials of Sichuan University, China. Type I Col was prepared using the hydrogel formation method, in which Col was extracted from bovine skin and telopeptide was removed. The Col cross-linking agent glutaraldehyde (GA) with 99.5% chemical purity was purchased from Kelong Chemical Reagent Co., Ltd, China. Simulated supersaturated body fluid (1.5 SBF) solution, which has a 1.5 times higher ion concentration than normal SBF solution (1.0 SBF), was prepared in accordance with Kokubo’s method [20]. The reagents used to prepare SBF included NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, HCl, CaCl2·2H2O, Na2SO4·10H2O and NH2C(CH2OH)3 (Tris Buffer, Sigma, USA). All of the reagents were purchased from Sinopharm Chemical Reagent Co., Ltd, China. 2.2. Preparation of three-level biomimetic scaffolds

The fabrication of the three-level hierarchical structure of the CaP/Col/HAp biomimetic scaffold is shown in figure 1. The precursor CaP powders (hydroxyapatite/β-tricalcium phosphate = 30:70) were synthesized using the wet chemical method according to the following equation [21]: 5Ca ( NO3) + 3 ( NH4) HPO4 + 4NH4OH 2


→ Ca5 ( OH) ( PO4) + 10 ( NH4) NO3 + 3H 2O. 3


Five liters of 0.85 mol l−1 Ca(NO3)2 solution was dropped slowly into 5 l of 0.55 mol l−1 (NH4)2HPO4 solution under stirring at room temperature. The pH of this solution was maintained at 8 by addition of ammonium hydroxide solution. The reaction solution was stirred for 6 h and aged for 24 h. The reaction precipitate was then collected, washed with deionized water, dried and crushed into a powder with a particle size of about 200 μm. The porous structures of the CaP ceramics were produced and designed using the H2O2 foaming method to form the first-level porous ceramics matrix. Approximately 200 g of CaP powder, 15 ml of polyvinyl alcohol (PVA), 15 ml of cellulose, 50 ml of H2O2 and 120 ml of deionized water were mixed to form a ceramic slurry. This slurry was heated for 2 min in a microwave to generate gas and then molded in a wooden mold to obtain the porous ceramics green body. Afterward, the green body was dried at 80 °C for 12 h and sintered at a heating rate of 5 °C min−1 for 6 h until 1200 °C 2

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Figure 1. Schematic of the three-level hierarchical structure of CaP/Col/HAp biomimetic scaffolds.

was reached. After heating, we cooled the ceramics in the furnace until room temperature was reached. The porosity of the sintered CaP scaffolds was measured using the Mercury intrusion method. The porosity of the bulk ceramics was 75 ± 10%, and the pore size ranged from 200 μm to 400 μm; the pores were interconnected by micropores. Samples were cut into Φ3 mm × 5 mm cylinders. To form a second-level fibrous Col layer, we prepared a Col solution with a concentration ranging from 10 g ml−1 to 20 g ml−1 by dispersing type-I Col in a 1.5 SBF solution and adjusting with 5% w/v acetic acid to a pH ranging from 4.0 to 6.5. The CaP porous ceramics were then fully immersed in the prepared Col solution with pH = 4.0–7.5 and sealed in a high-pressure vessel. The Col solution was filled in the porous CaP ceramics matrix by vacuum infusion. Vacuum infusion was conducted at a pressure of 10 Pa and sustained for 2 h to allow full saturation of the samples. Ultrasonic vibration and a repeated process were conducted for vacuum infusion. The suspension was then adjusted to a pH ranging from 6.5 to 7.5 by using Na2HPO4 and allowed to settle for 2 d at 5 °C; the samples were then lyophilized at different freezing rate from − 1 °C min−1 to −5 °C min−1 to allow the calcium and phosphate ions to deposit onto the Col template and form the thirdlevel nano-HAp layer. After freeze-drying, the scaffolds were cross-linked with 1.5% w/v GA.

2.4. Mechanical property testing

The mechanical properties of the scaffolds were evaluated using dynamic mechanical analysis (DMA; Precision universal Tester Autograph AG-X, Japan). The measurements were carried out at 20 °C. The samples were prepared into cylinders, each with a diameter of 5 mm and a thickness of 10 mm. The dimensions of the samples were measured accurately using a digital micrometer with a precision of 0.001 mm prior to the test. Each group comprised three parallel samples. The experiments were performed at a constant strain amplitude of 70 μm. A small preload was applied to each sample to ensure that the entire scaffold surface was in contact with the compression plates before the experiment was conducted and that the distance between plates was equal in all of the test scaffolds. The compressive modulus of the scaffolds was determined on the basis of dynamic compressive curves in the elastic region with strain ranging from 2% to 6%; this parameter was obtained according to the finite difference method and calculated using the following equation [23]: σ − σ −Δε Eε = ε + Δε (2) 2Δε where Eε is the compressive modulus of the material at the compression strain ratio value of ε, and Δε was set at 1%. 2.5. In vitro experiments

Human bone marrow mesenchymal stem cells (MSCs) were used to assess the in vitro biocompatibility of the biomimetic scaffolds. Cell viability and proliferation were continually monitored for 14 d. Unless stated otherwise, all of the experiments were repeated independently at least three times. The MSCs were incubated in RPMI1640 culture medium (GIBCO, USA) supplemented with 10% fetal calf serum, penicillin and streptomycin in an incubator with 5% CO2 at 37 °C. The medium was changed at an interval of 2 d. The cells were harvested with 0.25% trypsin after confluence was reached. The samples were placed into a 96-well plate, in which 100 μl of 1 × 106 cells ml−1 suspension was seeded in each well. Fluorescent microscopy was performed to determine the presence and proliferation of the cells on the scaffolds. MSC-containing scaffolds were observed at each

2.3. Structural and morphological characterization

SEM (JSE-5900LV, Japan) was used to observe the scaffold microstructures. Local elemental analysis was carried out by EDX (Oxford, IE250, UK) to assist with phase identification. The crystalline phase was analyzed using XRD (Philips X’Pert 1 x-ray diffractometer, Netherlands) with CuKa radiation at a current of 20 mA and voltage 30 kV. Scans were performed with 2θ values from 20° to 60° at a rate of 0.05° sec−1. The obtained peaks were compared with standard references in the JCPDS file available in the software for HAp (09-0432) and β-TCP (09-0169) [22]. The functional groups of the scaffolds were identified by FTIR (Perkin-Elmer Spectrum one B, USA). The FTIR spectrum was scanned from 4000 cm−1 to 500 cm−1. 3

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experimental point and stained by fluorescein diacetate (2 μl, 0.1%, FDA, Sigma, USA) for live cells (green) and propidiumiodide (2 μl, 0.1%, PI, Sigma, USA) for dead cells (red). After 2 min, the cells were observed using a fluorescent microscope (Leica-TCS SPS, Germany). For comparative studies, three different scaffolds, namely pure CaP scaffolds, two-level CaP/Col scaffolds, and three-level biomimetic scaffolds, were used for the in vitro cell culture.

altering the freezing rate and pH. These factors are then used to control the nucleation and growth rate of HAp crystals. During precipitation, the HAp nanocrystallites deposited onto the Col fibril template formed a Col/nano-HAp layer, which forms the third-level structure of the biomimetic scaffolds (figures 2(e), (f)). SEM images reveal that HAp nanocrystallites were deposited onto the Col fibrils and filled part of the void space in the Col matrix (figure 2(f)). This process resulted in an increase in the mechanical strength of the scaffolds. The precipitated HAp nanocrystallites agglomerated to form layers with petal-and needle-like morphologies.

2.6. In vivo experiments

Eight New Zealand white rabbits were obtained from the Sichuan University Laboratory Animal Center (body weight: 2.2 kg to 2.5 kg; gender: male). All of the experiments were approved by the Animal Care and Use Committee of Sichuan University. The animals were anesthetized by intraperitoneally injecting 0.02 g ml−1 pentobarbital sodium before implantation (40 mg kg−1 body weight; Sigma Chemical, St. Louis, MO, USA). The scaffolds were implanted in the dorsal muscles of the rabbits. To eliminate possible variations in individual rabbits, we implanted each rabbit with a total of eight different specimens. After implantation, the rabbits were continuously injected with penicillin at 105 U kg−1 per day for 3 d to prevent infection. The specimens were harvested after 1 to 6 months of implantation by sacrificing the rabbits. The implanted specimens were extracted from the surrounding tissues and washed with phosphate-buffered saline (PBS). The attached tissues were washed with a mixture of PBS (90 wt%) and pepsin (10 wt%); the harvested implants were then fixed in 10% formaldehyde in PBS solution for one week and then histologically examined. The samples were decalcified using a fast-decalcifying fluid (50 ml of hydrochloric acid, 100 ml of methanoic acid, 40 g of AlCl3 and 850 ml of 10% formaldehyde solution) for 7 d. The decalcified samples were washed with PBS, dehydrated with a gradient of ethanol solutions (70%, 80%, 90%, 95%, and 100%), and embedded in polymethylmethacrylate (PMMA). The paraffin-embedded specimens were cut into 5 μm sections using Leica PolycutE (Leica, SM 2500E, Germany) and transferred to glass slides. The sections were stained with hematoxylin and eosin (HE) for histological analysis.

3.2. Effect of pH and lyophilization on the precipitated HAp morphology

The effect of pH and lyophilization on the third-level HAp morphology is shown in figure 3. Figures 3(a)–(e) show that the morphological characteristics of the precipitated HAp layers were varied at different pH values but at the same freezing rate. These precipitated HAp layers decreased in terms of individual volume and density when pH was increased from 4.0 to 7.5. The HAp flakes were stacked layer by layer via self-assembly. Each HAp flake exhibited a size of 1 μm to 2 μm and a thickness of 20 nm to 50 nm. Figures 3(f)–(j) show that the morphological characteristics of the precipitated HAp layers changed from petal-like to needle-like structures when the freezing rate was increased from −1 °C min−1 to −5 °C min−1 at pH 7.0. Figures 3(f)–(h) reveal the petal-like morphologies of the precipitated HAp layers at a low freezing rate. However, the morphological characteristics of the precipitated HAp layers resembled a needle-like structure when a high freezing rate was used. The formed HAp flakes obtained at a low freezing rate (−1 °C min−1) were larger than those obtained at a high freezing rate (−5 °C min−1). 3.3. Chemical composition of the scaffolds

The chemical composition of the three-level biomimetic scaffolds was analyzed using EDX, XRD and FTIR. The EDX results (figure 4) revealed strong peaks of calcium (Ca) and phosphorus (P) and weak peaks of oxygen in these scaffolds. Elemental micro-analysis of the EDX spectrum from three specimens yielded an average Ca/P ratio of 1.69 ± 0.31, which is close to that of the stoichiometric HAp of 1.67. Figure 5 shows the x-ray diffraction pattern of the threelevel biomimetic scaffolds. The curve a reveals a positive correlation with the diffraction peaks found in the HAp diffraction standard 09-0432 (curve c), diffraction peaks at 25.9°, 31.7°, 32.2°, 32.9°, 39.9°, 46.7° and 49.5° (2θ) were identified, indicating the HAp crystalline material. The peaks in the curve a are broad and several peaks overlapped with one another compared with those of the standard HAp pattern. This result indicates the considerably low crystallinity of the third-level HAp phase and small crystallite size, closely resembling those of natural bone tissue [24]. Curve b shows the XRD of the CaP scaffold, the five highest peaks coexist at

3. Results 3.1. Microstructure of the scaffolds

The morphological characteristics and microstructure of the scaffolds are shown in figure 2. The biomimetic scaffolds displayed a classic three-level hierarchical structure. The firstlevel structure of the scaffold was formed by a porous CaP ceramic with 75 ± 10% porosity and 200 μm to 400 μm pore size (figures 2(a), (b)). This three-dimensional interconnected structure allows the infusion of Col to form the second-level layer. The second-level scaffold structure is formed by a Col fibrous network. The vacuum infusion process forces Col to fill the ceramics’ porous matrix and create a Col fibril network (figures 2(c), (d)). The Col fibrous network can be adjusted by 4

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Figure 2. Morphology and microstructure of the three-level biomimetic scaffolds. (a), (b) show the first-level structures of CaP; (c), (d) show the microstructures of second-level hierarchical structure of the Col layer; and (e), (f) show the microstructures of third-level hierarchical structure, which is a HAp layer induced by mineralization on the Col fibrous network.

Figure 3. Effect of pH and lyophilization on precipitated HAp layers. (a)–(e) The morphologies of HAp layer varied with different pH value

when samples were lyophilized with a freezing rate of −3 °C min−1; (f)–(j) The morphologies of HAp layer changed from petal-like to needlelike structure when improving the freezing rate of lyophilization, pH = 7.0. 5

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Figure 6. FTIR spectra of (a) three-level biomimetic scaffold; (b)

CaP scaffold; and (c) natural bone.

Figure 4. An EDX chart of three-level biomimetic scaffolds.

natural bone. The absorption bands at 1089, 1044, 962, 601 and 570 cm−1 were caused by PO3− 4 , and those at 3571 and 633 cm−1 were caused by OH−. The NH2 groups of the Col in the biomimetic scaffold and the bone produced absorption peaks at 1680 cm−1 to 1630 cm−1 and 1570 cm−1 to 1510 cm−1. An absorption peak shift at 1339 cm−1 in Col was observed and represented the carboxyl group; this result is attributed to the absorption peak generated by the covalent bond formation of Ca2+ ions in HAp nano-crystals with Col macromolecules [26, 27]. The third-level HAp nanocrystals were considered to be self-assembled and self-organized at active nucleation sites of the Col matrix by covalent bonding; HAp was linked to Col by the formation of −Ca2+–[COO−]. This linkage may yield a positive effect on the interface behavior and the mechanical properties of the scaffold. In this reaction, Ca2+ ions bind to the carboxyl site of Col because of ionic attraction, and nucleation is initiated from these active sites as a heterogeneous reaction. PO3− 4 ions then accumulate at calcium complexes and grow to the critical nucleation size; as a result, stable HAp layers are formed. The characteristic absorption bands of the three-level biomimetic scaffold are also found in the IR spectrum of the bone (figure 6(c)). This result further indicates that this biomimetic scaffold exhibits similar compositions to natural bone.

β - TCP HA

Three-level biomimetic scaffold




CaP scaffold



JCPS 09-0432 HAp


JCPS 09-0169 β− TCP





40 2θ/degree





Figure 5. (a) XRD pattern of the three-level biomimetic scaffold; (b) CaP scaffold; (c) HAp standard pattern; and (d) β-TCP standard pattern.

2θ of 27.9°, 31.1°, 34.5°, 48.4° and 53.2°, matching those of the β-TCP diffraction standard 09-0169 (curve d) [16, 25]. However, peaks from the HAp materials are also present in the XRD pattern, indicating that the CaP scaffold contains HAp and β-TCP crystalline material. High and separate peaks in the XRD pattern of sintering CaP substrate indicate high crystallinity. The three-level biomimetic scaffolds were further analyzed by FTIR and the FTIR spectra are presented in figure 6. The spectrum of these scaffolds displays typical spectral features in the range of 4000 and 500 cm−1 similar to those of

3.4. Mechanical and in vitro biocompatibility properties

The pure porous CaP ceramics (one-level structure materials), CaP/Col composites (two-level structure materials) and CaP Col HAp scaffolds (three-level biomimetic structure materials) were tested. Table 1 shows the mechanical properties of these scaffolds. Different stress–strain behaviors of these three scaffolds were revealed and these behaviors indicated different mechanical properties. The pure porous CaP ceramics showed an average maximum stress of 37.68 ± 12 N, indicating the inherent brittleness of this 6

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Table 1. Mechanical properties of different scaffolds.

Scaffold Pure porous CaP ceramics (One-level structure) CaP/Col composites (Two-level structure) CaP/Col/HAp scaffolds (Three-level structure )

Maximum stress (N)

Strain (%)

Maximum compressive strength (MPa)

Compressive modulus (MPa)

37.68 ± 12

0.8 − 1.5

1.92 ± 0.61

104 ± 32

83.41 ± 11

1.4 − 2.5

4.25 ± 0.55

232 ± 28

121.09 ± 12

2.0 − 3.5

6.17 ± 0.64

352 ± 37

material. The maximum stress of CaP/Col composites improved to 83.41 ± 11 N after Col was formed. For the threelevel biomimetic structure CaP/Col/HAp scaffolds, the maximum stress was 121.09 ± 12 N, which is approximately 3.2 times higher than that of pure porous CaP ceramics. The strain of these scaffolds varied because the properties were enhanced after Col was formed. Before the materials were damaged, the maximum strain of CaP/Col composites improved from 0.8–1.5% to 1.4–2.5%, whereas the threelevel biomimetic structure CaP/Col/HAp scaffolds improved to 2.0–3.5%. The maximum compressive strength of the pure porous CaP ceramics and the two-level structure of CaP/Col composited scaffolds were 1.92 ± 0.61 MPa, and 4.25 ± 0.55 MPa. For the three-level CaP/Col/HAp scaffolds, the maximum compression strength reached 6.17 ± 0.64 MPa, 3.2 times higher than that of the pure CaP ceramics. This result indicates that this structure can reinforce the mechanical strength of the scaffold. The compressive modulus indicates the ability of the scaffolds to resist deformation under external force. The results also show that the compressive modulus of the three-level biomimetic scaffolds improved from one-level porous CaP ceramics, that is, from 104 ± 32 MPa to 352 ± 37 MPa or an increase of approximately 3.4 times. This mechanical characteristic is important for bone tissue applications. Human bone usually bears compressive stress under normal activities. A higher compressive modulus can protect the bones against damage caused by deformation under relatively high loading forces. Human bone marrow MSCs were used to assess the in vitro biocompatibility of the biomimetic scaffolds. Figure 7 shows the MSC culture results under different experimental times. The morphology of the MSCs on the three scaffold types exhibits no significant difference after culturing for 3 d (figures 7(a)–(c)). After 7 d (figures 7(d)–(f)), the proliferation of MSCs shows a minor difference on these scaffolds, in which these cells formed extensive cell–cell interactions and were gradually connected to the fibrous structure. However, this morphology in the three-level biomimetic scaffolds reflects the porous scaffold structure and was different from the results in the pure CaP or CaP/Col scaffolds. These phenomena indicated better cellular proliferation and migration in the three-level biomimetic scaffolds. More cells were spread in the scaffolds, which indicates a continuous proliferation of MSCs on day 14 (figures 7(g)–(i)). The morphology of the MSCs in the three-level biomimetic

scaffolds displayed a three-dimensional spherical structure. These scaffolds also exhibited a higher growth rate after the culture time was prolonged, although all of the scaffold types in this study showed good biocompatibility. Therefore, the change in biomimetic scaffolds yielded better cellular proliferation and differentiation. The cells were able to come in contact with either collagen fibrils or apatite flakes when these cells were cultured in the three-level biomimetic scaffold. This biomimetic scaffold also provided a rougher surface for cell adsorption. Furthermore, the increasing surface area at the interface of the biomimetic scaffold is favorable for cellbiomaterial interactions. These reasons may provide enhanced cell signaling in biomimetic scaffolds.

3.5. Osteoinductivity

The implanted specimens were harvested after 3, 4.5 and 6 months post-operation. None of the implanted sites of the rabbits manifested any infection after the operation; each implant was wrapped in muscle. After 3 months of implantation, a substantial amount of active, newly formed bone tissue were found in these implanted scaffolds. These tissues were observed at the pore edge region in the scaffolds (the region indicated by yellow arrows, figures 8(a)–(c)). More newly formed bone regions were observed in the three-level biomimetic scaffold, indicating the better osteoinductivity of this scaffold. The three implantation periods were compared. The results revealed that the inflammatory reaction appeared first and fibroblasts could be found after three months of implantation after which bone formation occurred. Only minor inflammatory responses were observed (dark arrows indicate the inflammatory cells in figures 8(a)–(c)); however, these cells gradually diminished as the implantation time was prolonged. In fact, the inflammatory cells after 4.5 and 6 months of implantation can hardly be observed, confirming the excellent biocompatibility of this scaffold. Figures 8(d)–(f) and figures 8(h)–(j) show the scaffolds after 4.5 and 6 months of implantation, respectively. New bone tissues are present in all of the three scaffold types, with neither cartilage nor chondrocytes found. During osteogenesis, capillaries were found close to the developing front of the osseous nidi, and the newly formed bones were found to be in direct contact with the scaffolds. Cubic osteoblasts were found in the developing matrices. The osteocytes in almondshaped lacunae were embedded randomly in the newly 7

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Figure 7. Fluorescent MSC images in different scaffolds after culturing for 3, 7, and 14 d. The scaffolds containing MSCs cells were stained by fluorescein diacetate (2 μl, 0.1%, FDA, Sigma, USA) for live cells (green) and propidiumiodide (2 μl, 0.1%, PI, Sigma, USA) for dead cells (red).

three-level biomimetic scaffold composited with Col provided a beneficial effect on osteoinduction.

formed bones. Some of the osteocytes were laid on the scaffold interfaces, indicating that their precursor osteoblasts were initially aggregated directly on the material surface. This phenomenon further indicated that the condition of the scaffold surface is essential for new bone formation. Bone tissue formation is best in the three-level biomimetic scaffolds. Figure 9 shows the statistical analysis of the bone formation region in the different scaffolds. After 3 months of implantation, less than 10% of newly formed bone area was observed. After 4.5 months of implantation, this area increased, and the bone formation area in the three-level biomimetic scaffolds increased to 22%. After six months of implantation, the bone formation area in the three-level biomimetic scaffolds reached 55%. The osteoinduction of these scaffolds was better than those of the other two scaffolds. The

4. Discussion The mechanical strength of the three-level biomimetic scaffold was a significantly improvement (table 1). The overall porosity of this three-level biomimetic scaffold decreased to 68 ± 7% compared with the initial 75 ± 10% porosity of the pure porous CaP scaffold. However, micropores from 200 μm to 400 μm in size were still present, and allowed cell ingrowth. Good mechanical behavior depends on the special three-level hierarchical structure, comprising a hard CaP ceramics matrix, elastic Col fibrils and stiff HAp crystals. This unique structure enhanced the elastic properties of this 8

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Figure 8. HE staining of three different scaffold types harvested from the rabbit dorsal muscles. Histological characteristics of image (a)–(c)

are observed after 3 months of implantation; Images (d)–(f) are observed after 4.5 months of implantation; Images (h)–(j) are observed after 6 months of implantation. Legend: M = decalcified materials; NB = new bone (yellow arrow); IC = inflammatory cells (dark arrows). Magnification: 400×.

and compressive modulus. Furthermore, the third-level stiff HAp crystals embedded in the Col layer forming a compact three-dimensional network causes classic brick-and-mortar reinforcement [28]. Here, the basic scaffold matrices comprise highly porous hard CaP ceramics, and the second-level Col networks are formed by the intertexture of Col in the CaP matrices with a brick-and-mortar arrangement. The dendritic growth of the third-level HAp crystals aggregated and locked the Col microfibrils; as a result, the scaffold was further reinforced. Various layers were held together by specific interactions between components and operated in a synergistic manner. This process forms hierarchical structures comprising inorganic–organic–inorganic components and resembling the biomimetic structures of natural bone. The formation of the third-level HAp layers may be attributed to the following points. The abundance of Ca2+ and PO34− ions leads to precipitate formation. These ions are possibly the products of the dissolution of the ceramic substrate or the concentrated SBF solution. The growth of new HAp crystals on the Col fibrous template is possibly induced by higher Ca2+ and PO3− 4 concentrations than the concentration threshold of crystallization. This high supersaturation of these ions triggers nucleation and crystal growth. The precipitation under acid conditions (pH < 7.0) was more likely to

Figure 9. A histological statistical analysis of the bone formation

region in different scaffolds. (*: p < 0.05).

biomimetic scaffold. The three-dimensional network structure in the scaffold contains substantial free spaces. These free spaces enable the Col components to respond rapidly to external forces and the forces can be absorbed by Col components simultaneously, resulting in a relatively high strain 9

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Figure 10. Self-assembly in the formation of three-level hierarchical biomimetic scaffolds. (a) Initial stage of HAp layers formation; (b) intermediate stage of HAp layers formation; (c) final morphology of HAp layer; (d) magnified image of (c). Red arrows reveal the selfassembly of HAp crystals and blue arrows reveal the Col fibrous networks.

formation of three-level biomimetic scaffolds. Initially, (figure 10(a)), supersaturated Ca2+ and PO3− 4 ions trigger HAp nucleation and stimulated crystal growth on the Col nanofibril template. Here, the blue and red arrows indicate newly formed nano-HAp crystals and CaP matrix minerals. Figure 10(b) shows the intermediate stage of HAp layer formation. Many HAp crystals were formed and grew, with each Col fibril covered by a layer of HA nanocrystals. Figure 10(c) illustrates the final stage of HAp layer formation. The morphology of the HAp layer demonstrates a preferential alignment of HAp crystals. These crystals eventually grew with the Col fibril longitudinal axis, forming an oriented petal-like morphology. Figure 10(d) shows the magnified image of figure 10(c), revealing the self-assembly of HAp crystals (red arrows) on the Col fibrous networks (blue arrows). These findings provide direct evidence to support the theory that a three-level hierarchical biomimetic scaffold can be constructed using a self-assembly model. Previous studies reported that three-dimensional porous structures with appropriate interconnected pores can be used as biomimetic scaffolds for bone repair or regeneration; these structures can deliver nutrients and excrete metabolic wastes [32]. Therefore, the interconnectivity of scaffolds is critical for osteoinductivity. Figure 11 shows the newly generated tissues grown along the interconnected porous structures in the scaffolds. The overall situation of newly generated tissues growing along the porous material template is shown in figure 11(a). Newly generated tissues (deep red area) gradually extended to the material center. Figure 11(b) shows the

form petal-like morphology than that under neutral conditions (pH 7.0) for the morphology of the HAp layer (figure 10). This result indicates that Ca2+ and PO3− 4 ions were partially produced from the dissolution of the ceramics matrix. Neutral conditions provided fewer available ions to form more needle-like precipitates. The other factor is the presence of the second-level Col layer that provided a favorable template for precipitates. This Col matrix is likely to induce the crystallization of a HAp layer. The new crystals grew along the Col fiber because the Col template provided numerous active crystallization sites. Under acidic conditions, the ionization of Col revealed an amino group with positive sites; as a result, favorable active sites are provided for negatively charged phosphate groups and the deposition of HAp layers on Col fibers is promoted. The SEM results revealed the formation of a three-level hierarchical biomimetic scaffold resembling a self-assembly. Previous studies have reported that hierarchical structures and composition scaffolds can be mimicked through in vitro Col and HAp self-assembly. Glimcher et al [29] reported a biomimetic scaffold in which HAp is self-assembled on the Col matrix surface by applying simulated body fluid induction. Bradt et al [30] obtained a homogeneous three-dimensional HAp-Col composite scaffold by combining Col fibril assembly and HAp formation in a one-step process. Goissis et al [31] reported the biomimetic mineralization of charged Col self-assembled with CaP deposition. In this study, a threelevel biomimetic scaffold was synthesized using the selfassembly model. Figure 10 shows the self-assembly in the 10

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Figure 11. Newly generated tissues grown along the biomimetic scaffold. (a) Decalcified light micrograph after 1 month of implantation (HE,

amplification: 100×); (b) decalcified light micrograph after 1 month of implantation (HE, amplification: 600×); (c) decalcified light micrograph after 6 months of implantation (HE, amplification: 400×).

level hierarchical CaP/Col/HAp scaffold was prepared biomimetically. A designed porous CaP ceramics matrix was prepared using the foaming process to strictly mimic natural bone structures. Vacuum infusion of the second-level Col network enhanced the mechanical strength and facilitated the formation of the third-level HAp layer. The composition, microstructure and mechanical properties of these biomimetic scaffolds can be adjusted using the process parameters. The in vitro MSC culturing study and the in vivo animal test indicated that these scaffolds exhibited good biocompatibility and better osteoinductivity than pure CaP ceramics and twolevel CaP/Col composites. Therefore, biomimetic scaffolds are promising materials for bone tissue engineering because of their optimal biological and mechanical properties.

amplified region of the porous scaffold. The newly generated tissues grew in the interconnected pores (along the blue arrows). Figure 11(c) reveals that the new bone is induced by the scaffold structures grown along the interconnected porous structures (indicated by blue arrows). This result further indicates that this biomimetic scaffold could be used as a guiding template in osteoconductivity. The precipitation of HAp on different substrates has been well documented in previous studies. Different methods have been used by different researchers to deposit apatite on various scaffolds, including inorganic and organic forms. Chen et al [33] coated bonelike apatite on poly (L-lactic acid) films and poly (glycolic acid) scaffolds by employing an accelerated biomimetic process. Honda et al [34] deposited bone-like apatite in a collagen matrix. Deng et al [35] immersed dense HAp and HAp/TCP ceramics in a fast calcified solution to obtain apatite on the surface of ceramics. Yousefpour et al [36] deposited HAp coating on a pure titanium substrate using a hydrothermal–electrochemical deposition method. The scaffolds fabricated using these techniques are mostly twolayer structures, i.e. organic plus apatite or inorganic plus apatite. As such, multilevel hierarchical structures resembling natural bone are indeed difficult to construct. The proposed biomimetic method in this study provides advantages compared with previously described methods. For instance, the proposed method could be used to prepare an apatite layer on a substrate without using sophisticated equipment and complex processes. This method could also be applied to form hierarchical structures comprising inorganic–organic–inorganic components; as a result, the structures of natural bone are mimicked. Furthermore, the three-level hierarchical CaP/ Col/HAp scaffold provides a facile structural design for bone tissue engineering.

Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81190131), the National Basic Research Program of China (‘973’ Program, No. 2011CB606201) and the National Sci & Tech Support Plan of China (2012BAI17B01, 2012BAI42G00).

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5. Conclusions Biomimetic synthesis is a promising approach for repairing damaged bone tissues. An ideal bone tissue engineering scaffold should resemble the composition and hierarchical structures of natural bone tissues. In this study, a novel three11

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hydroxyapatite scaffold for bone tissue engineering.

A three-level hierarchical calcium phosphate/collagen/hydroxyapatite (CaP/Col/HAp) scaffold for bone tissue engineering was developed using biomimetic...
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