preparation and characterization of bionic bone structure...

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Preparation and characterizationof bionic bone structure chitosan/hydroxyapatite scaffold for bone tissueengineeringJiazhen Zhang a , Jingyi Nie a , Qirong Zhang a , Youliang Li a ,Zhengke Wang a & Qiaoling Hu aa MOE Key Laboratory of Macromolecular Synthesis andFunctionalization, Department of Polymer Science andEngineering , Zhejiang University , Hangzhou , 310027 , ChinaPublished online: 23 Sep 2013.

To cite this article: Jiazhen Zhang , Jingyi Nie , Qirong Zhang , Youliang Li , Zhengke Wang& Qiaoling Hu , Journal of Biomaterials Science, Polymer Edition (2013): Preparation andcharacterization of bionic bone structure chitosan/hydroxyapatite scaffold for bone tissueengineering, Journal of Biomaterials Science, Polymer Edition, DOI: 10.1080/09205063.2013.836950

To link to this article: http://dx.doi.org/10.1080/09205063.2013.836950

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Preparation and characterization of bionic bone structure chitosan/hydroxyapatite scaffold for bone tissue engineering

Jiazhen Zhang, Jingyi Nie, Qirong Zhang, Youliang Li, Zhengke Wang*Qiaoling Hu*

MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department ofPolymer Science and Engineering, Zhejiang University, Hangzhou 310027, China

(Received 4 May 2013; accepted 20 August 2013)

Three-dimensional oriented chitosan (CS)/hydroxyapatite (HA) scaffolds wereprepared via in situ precipitation method in this research. Scanning electronmicroscopy (SEM) images indicated that the scaffolds with acicular nano-HA hadthe spoke-like, multilayer and porous structure. The SEM of osteoblasts which werepolygonal or spindle-shaped on the composite scaffolds after seven-day cell cultureshowed that the cells grew, adhered, and spread well. The results of X-ray powderdiffractometer and Fourier transform infrared spectrometer showed that the mineralparticles deposited in the scaffold had phase structure similar to natural bone andconfirmed that particles were exactly HA. In vitro biocompatibility evaluation indi-cated the composite scaffolds showed a higher degree of proliferation of MC3T3-E1cell compared with the pure CS scaffolds and the CS/HA10 scaffold was the highestone. The CS/HA scaffold also had a higher ratio of adhesion and alkaline phosphateactivity value of osteoblasts compared with the pure CS scaffold, and the ratioincreased with the increase of HA content. The ALP activity value of compositescaffolds was at least six times of the pure CS scaffolds. The results suggested thatthe composite scaffolds possessed good biocompatibility. The compressive strengthof CS/HA15 increased by 33.07% compared with the pure CS scaffold. This novelporous scaffold with three-dimensional oriented structure might have a potentialapplication in bone tissue engineering.

Keywords: chitosan; hydroxyapatite; three-dimensional oriented scaffold; bonetissue engineering; in situ precipitation

1. Introduction

Natural bone, similar to other calcified tissues, is a kind of complex inorganic–organicnanocomposite material and has an intricate hierarchical architecture. Bone is formedby a series of complex events involving the mineralization of extracellular matrixproteins rigidly orchestrated by cells with specific functions of maintaining the integrityof the bone. Actually, it is assembled through the orderly deposition of hydroxyapatite(HA) along the type I collagen organic matrix. The unique composition and hierarchicalstructure endow the natural bone’s good mechanical properties and biocompatibility.Therefore, the development of materials by mimicking the structure and composition ofthe bone attracts more and more attention in the field of bone tissue engineering.

*Corresponding authors. Email: [email protected] (Z. Wang); [email protected] (Q. Hu)

Journal of Biomaterials Science, Polymer Edition, 2013http://dx.doi.org/10.1080/09205063.2013.836950

� 2013 Taylor & Francis

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Chitosan (CS), well known to be excellent in biocompatibility, biodegradability,antimicrobial property, wound-healing, cell proliferation, and tissue regeneration, is alinear polysaccharide composed of glucosamine and N-acetyl glucosamine with β,1–4glycosidic linkages; the latter is a moiety of glycosaminoglycans.[1,2] CS is insolublein NaOH aqueous solution but can be easily soluble in dilute acids (pH < 6). The reasonis that the free amino groups can be protonated in dilute acids. The pH-dependent solu-bility provides a convenient mechanism for preparation. Due to the excellent ability tobe processed, CS can be processed into various forms such as zero-dimensional micro-sphere, one-dimensional nanofiber, two-dimensional membrane, and three-dimensionalscaffold.[3–5] As scaffold materials in bone tissue engineering, CS scaffold has beenproven to be a potential candidate for bone regeneration because of its good biologicaland physical properties.[6]

Calcium phosphate ceramics are the most common biomaterials studied in bonetissue engineering because their chemical compositions are similar to the mineral phaseof bone. They can form a chemical bond with surrounding tissues through a layer ofbone-like apatite on their surface in vivo. HA is an excellent candidate for bone repairand regeneration due to its bioactivity and osteoconductivity.[7,8] However, themechanical properties of the pure HA are inadequate, which limit its use in bonerepair.

In order to combine the favorable biocompatibility of CS with the osteoconductivityof HA, CS/HA composites with favorable properties have been prepared by directmechanical mixing,[9] by co-precipitation, [10,11] or by an alternate soaking process.[12,13] Some researchers have reported that CS/HA composites show goodbiocompatibility and favorable bonding with the surrounding host tissues, and canfurther enhance tissue regenerative efficacy and osteoconductivity.[9,14–16] However,within all these methods, homogeneous distribution of HA in the scaffold at a micro/nano level cannot be achieved. Chen et al. [17,18] prepared the nanohybrid scaffold viain situ crystallization of HA in CS matrix. The nano-HA distributed homogeneously inthe CS organic matrix. But the CS matrix did not have the bone-like hierarchicalstructure. Although Ma et al. [7], Nitzsche et al. [19], and Jiang et al. [20] obtainedcomposite scaffolds with the bone-like hierarchical structure and nano-HA particles, ourpresent work still possessed novelty with its distinct characteristics. Compared to thecomposite scaffolds mentioned above that consisted of konjac glucomannan [7] orcollagen [19] or carboxymethyl cellulose [20] except for CS, the composite scaffoldsprepared in the present work incorporated nano-HA in the bone-like polymeric matrixonly containing CS. In our previous researches, three-dimensional oriented porous CSscaffolds with multilayer structure were successfully prepared via in situ precipitationmethod.[21,22] The oriented CS scaffolds with connective pores had spoke-likeframework in the cross-section and multilayer structure in the vertical section. In thisresearch, in view of the CS’s pH-dependent solubility and HA’s precipitation condition,we prepared homogenous and bionic CS/HA composite scaffolds, in which the filler ofHA crystallized simultaneously by in situ hybridization with the matrix CS precipitated.

2. Materials and methods

2.1. Materials

Biomedical grade CS was supplied by Zhejiang Golden-Shell Biochemical Co. Ltd,Taizhou, China. The degree of deacetylation was 85% and the viscosity averagemolecular weight (Mη) was 5.63� 105. All the solvents used were of analytical

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quality. Distilled water and deionized water were used throughout this study. Forcell culture, α-minimum essential medium (α-MEM), fetal bovine serum (FBS), andpenicillin–streptomycin–amphotericin were purchased from Gibco, Invitrogen Corpora-tion Co. Ltd. Alkaline phosphatase detection kit was purchased from Chinese MedicalBlood Institute. Bicinchoninic acid reagents were purchased from Sigma.

2.2. Preparation of CS/HA composite

The CS/HA composite scaffolds were prepared by in situ precipitation and solid–liquidphase separation. Scaffolds were prepared as follows. The CaCl2 and K2HPO4 weredissolved in 2% (v/v) acetic acid solution according to the Ca/P= 1.67 (Table 1).

Then the CS powders were added to the mixture solution. After 3 h agitation,transparent and yellow solution was obtained, and then the solution was transferred tothe beaker to remove the air bubbles. The final mixture solution was added into themold with a semipermeable membrane in the inner wall, and then the solution wrappedby a semipermeable membrane was soaked in 5% (w/v) NaOH aqueous solution for8 h. The CS/HA gel rods were constructed via in situ precipitation. The obtained gelrods were rinsed in distilled water until the pH of rinsed water turned to neutral. In thefinal stage, the gel rods were cut into 1.5mm height pieces, and then lyophilized usinga freeze-dryer (LGJ-18A, Sihuan, China) at 0.001mbar and at freeze-drying temperatureof �70 °C for two days. The obtained porous scaffold samples were stored undervacuum.

2.3. Characterization

2.3.1. Scanning electron microscopy (SEM)

The morphology of the scaffolds and the spatial distribution of apatite were studied bySEM (SIRION-100, FEI Inc., USA; JSM5600LV, JEOL Co., Japan). The osteoblastsadhesion and distribution on the composite scaffolds after cell culture were alsoobserved by SEM.

2.3.2. Porosity measurement

The porosity of the scaffolds was measured with a mercury porosimeter (Autopore IV9500; Micromeritics® Instrument Corp., Norcross, GA).

2.3.3. Fourier transform infrared spectrometer (FTIR)

FTIR (Vector 22, Bruker, Germany) was used to collect the FTIR spectra over the rangeof 4000–400 cm�1 by using a KBr disk technique.

Table 1. HA precursor content in the CS solution.

Sample CS/HA CS (g) HA (g) CaCl2 (g) KH2PO4 (g)

CS/HA5 100/5 10.00 0.50 0.59 0.43CS/HA10 100/10 10.00 1.00 1.20 0.86CS/HA15 100/15 10.00 1.50 1.80 1.30

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2.3.4. X-ray powder diffractometer (XRD)

To investigate the components of the composite scaffolds, the samples were analyzedby an XRD (Rigaku Co., model; DMAX-2200, Japan) using a monochromatic Cu Kαradiation.

2.4. In vitro biocompatibility evaluation

2.4.1. Cell culture

Cell studies were conducted using Mouse calvarial preosteoblasts (MC3T3-E1). Celllines were cultured in alpha minimum essential medium (α-MEM) supplemented with10% (v/v) FBS, 100U/mL penicillin–streptomycin. Prior to cell seeding, scaffolds weresterilized by ethanol/UV treatment and pre-wetted with the culture medium for 1 h at37 °C in a humidified incubator with 5% CO2 and 85% humidity. Cells were detachedfrom the culture plate at 80–85% confluence, centrifuged, and resuspended in a knownamount of α-MEM, and then counted and diluted to concentrations of 1.0� 106 cells/ml.Aliquots of 100 μl of cell suspensions were seeded dropwise onto the top of pre-wettedscaffolds. The scaffolds placed in 24-well tissue culture plates were left in an incubatorfor 3 h under standard culturing conditions to allow the cells to distribute throughout thescaffolds and then attach to the plates. After 3 h, an additional 1ml of culture mediumwas added to each well. Culture medium was changed every two days.

2.4.2. Cytocompatability of the scaffolds

The viability of MC3T3-E1 on the scaffolds was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. The assay is based on theprinciple of reduction of the tetrazolium component MTT by living cells. Therefore, thelevel of the reduction of the pale yellow MTT into dark blue formazan is directlyproportional to the number of metabolically active cells. Briefly, at the set time, 100 μlof MTT solution (5mg MTT/ml PBS) was added to each well. Following incubationfor 4 h under standard culture conditions, MTT was reduced to insoluble purpleformazan granules in the mitochondria by active cells. And then, the medium wasdiscarded and the intracellular formazan crystals were dissolved in DMSO. The opticaldensity of the solution was measured in a microplate reader (Biotek) at a wavelength of570 nm. The analytical assays were performed at 1, 4, 7, 10, and 14 days.

Morphology and spreading pattern of cells on the scaffolds was evaluated using aconfocal laser scanning microscope (CLSM) with double staining by fluoresceindiacetate (FDA) and propidium iodide (PI). FDA stains viable cells green by energy-dependent endocytosis into them; while PI stains nuclear DNA of necrotic andsecondary apoptotic cells red. Briefly, cell-scaffold constructs were washed in phos-phate-buffered saline (PBS) and stained with diluting 10 μl� 5mg FDA/ml acetone in10ml PBS for 10min at room temperature in the dark. Samples were washed again inPBS and counterstained with 200 μl� 1mg/ml PI in 10ml PBS in the above-mentionedconditions. The stained samples were immediately observed using a CLSM withexcitation at 488 nm and detection at 530 nm after rinsing them in PBS twice.

2.4.3. Alkaline phosphatase activities of osteogenic cells

Cells were inoculated on each kind of scaffolds for differentiation detection and werecultured for 21 days. Three parallel samples were taken from each kind of scaffolds,

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respectively. Freezing–thawing process was repeated several times to promote cell lysiswhen cell lysate was added to every sample. Alkaline phosphate activities (ALP) of celllysates was determined according to the kit instruction. BAC assay was performed inthe determination of total protein concentration, which was used in the calculation ofALP activity per unit mass of total protein.

2.4.4. Statistical analysis

All quantitative results were obtained from triplicate samples. Quantitative data werepresented as mean ± standard deviation. Student’s two-tailed t-test was used to determinethe statistical significance between experimental groups. A value of p< 0.05 wasconsidered to be statistically significant.

2.5. Compressive strength

The composite scaffolds prepared with all CS/HA ratios were cut into identicalcylindrical samples with a height of 15mm and a diameter of 8mm. The compressivestrength tests of the composite scaffolds were carried out on Shenzhen RegerCompany’s universal materials testing machine at room temperature, with maximumcompression ratio of 50% and a cross head speed of 2mm/min. For each ratio ofscaffold, at least three samples were conducted.

3. Results and discussion

3.1. Characterization

Topographies of extracellular environments can influence cellular responses fromattachment and migration to differentiation and production of a new tissue.[23] So, thepore morphology and porosity of scaffolds are the important parameters for supportingthe invasion of cells from surrounding tissues and contributing to angiogenesis.[3,9,24]The freeze-drying method provides a straightforward way of introducing pores in apolymer structure by the ice crystals nucleating and growing along the lines of thermalgradients.[25,26] Although pore orientation can be limited by controlling the geometryof the thermal gradients in the mold during freezing, it is not complete. In our previousresearches,[21,22] protonated CS molecules would reassemble via the electrostatic forcebetween CS–NHþ3 and OH

� during the process of CS multilayer gel rod formation.And then the porous oriented CS scaffolds were obtained by lyophilization. In addition,the HA precursors, which distributed homogeneously in the CS organic matrix, wouldcrystallize in situ in the alkaline environment (see Figure 1). In this study, to producethe CS/HA composite scaffolds, the two methods, lyophilization and in situprecipitation were combined.

3.2. Morphology of the scaffolds

The morphology of the composite scaffolds was shown in Figure 2 by SEM. Thein situ scaffolds exhibited a uniform interconnected open pore microstructure and aspoke-like, multilayer, porous structure. Compared with the former studies of CS-basedporous scaffolds,[22] the composite scaffolds were similar in the microscopic morphol-ogy, which indicated that the addition of HA did not influence the porous structure.Moreover, the HA particles were seen on the pore walls of the composite scaffolds and


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were homogeneously dispersed in the matrix. It can be observed from Figure 3 that thesize of the HA crystal is about 50–500 nm with acicular shape, and increased with thecontent of HA precursor. The bulge of the HA might increase the mechanical propertyand the biocompatibility of the composite scaffolds because of the well-known goodbiocompatibility of HA and more contacting areas available for bone cells.

Figure 1. Scheme of the formation mechanism of CS/HA composite scaffolds.

Figure 2. The SEM micrographs of cross-section (a–c) and vertical section (d) of the CS/HA10composite scaffolds prepared by lyophilization method: (a) CS/HA5, cross-section of centralregion; (b) CS/HA10, cross-section of peripheral region; (c) CS/HA15, cross-section of peripheralregion; and (d) CS/HA10.

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However, the SEM images of Figure 3 also indicated that when the HA concentrationreached a certain level like the CS/HA15 scaffolds, the clustering phenomenon began toappear. The CS/HA10 scaffolds showed the best dispersibility. Previous research [27]reported that the porosity needs to be >30% to achieve interconnection and the minimumrecommended and more ideal pore size for a scaffold is 100 μm and 6300 μm. In thisresearch, the porosity of the in situ scaffolds examined by a mercury porosimeter wasabove 85% (see Figure 4) and had a decreasing trend with increased content of HA. Thistrend might be because HA-situ precipitation process affected the formation of the porousstructure, which could be proved by the clustering phenomenon in the SEM photograph ofFigure 3(d).

The main pore diameter was in the range of 100–300 μm from Figure 4, which wasmore ideal pore size and related to the suitable heat and mass transfer rates.[3]Therefore, the porous composite scaffolds were sufficient for exchange of nutrition, highoxygenation, and vascularization. In addition, the pore size can be regulated throughcontrolling the heat transfer rate and the freezing temperature. And the porous compositescaffolds with appropriate three-dimensional geometry are able to bind and concentrateendogenous bone morphogenetic proteins in circulation, and may become osteoinductive(capable of osteogenesis), and can be effective carriers of bone cell seeds.

Figure 3. SEM micrographs of CS/HA composite scaffolds illustrating distribution of HAparticles (marked by the red coil) in the composite scaffolds: (a) CS/HA0; (b) CS/HA5;(c) CS/HA10; and (d) CS/HA15. (Please see the online article for the colour version of thisfigure: http://dx.doi.org/10.1080/09205063.2013.836950.)


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3.3. Phase of the scaffolds

The X-ray diffractograms of the raw CS powder, pure CS scaffold, and the compositescaffolds with different content of HA in the matrix were shown in Figure 5.

Two broad diffraction peaks of the raw CS powder around 10° and 20° wereobserved, which were attributed to hydrogen bond effect within the intermolecular orinner molecular CS and the amorphous structure, respectively. The weaker peakof the pure CS scaffold around 10° was seen, indicating that the forming processof the scaffolds prevented the yield of hydrogen bonds. And the phenomenon appearedin the diffraction patterns of all composite scaffolds. From the Figure 5 (right), thediffraction characteristic peaks of the composite scaffolds around 31.8° and 25.7° corre-sponded to the peaks of HA (31.86°, 25.94°), showing that the HA was exactly formedin the CS matrix. It has been reported that the peak around 31° in the compositescaffolds is a summed contribution of (2 11), (1 1 2), and (3 0 0) lattice planes of HA,and the appearance of (2 11) and (11 2) peaks indicates the interaction of the CS poly-mer backbone with apatite crystals,[28] and the two peaks of the composite scaffoldsbecame separated and sharp without the treatment of heating with the increase of the

Figure 5. XRD patterns of raw material, pure CS scaffold, and CS/HA composite scaffolds:(a) CS/HA5; (b) CS/HA10; (c) CS/HA15; and (d) HA (homemade).

Figure 4. Porosity and pore size diameter of CS/HA composite scaffolds.

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HA content. The existence of the HA phase in the scaffolds will improve thebiomineralization and bioactivity.[29]

In order to further study the relationship between CS and HA, FTIR was introduced,and the spectra were shown in Figure 6. The assignments of characteristic peaks of CSare listed in Table 2.

The spectra showed the strong absorption bands at 561, 603, and 1040 cm�1

assigned to the stretching and bending vibrations of the PO3�4 ion of the apatite. And, itwas easy to observe the C–O stretching vibration at 1040 and 1070 cm�1 to be a bigband, the amide II vibration at 1597 cm�1, and the symmetrical deformation vibrationat 1380 cm�1 to be a small band. The reason may be that, with the increase of the HAcontent, the vibrations were limited by the HA filling in the space between CS mole-cules, but the C–O stretching vibration incorporated with the P–O band more and more.As such, it can be inferred that the HA formed in the CS scaffolds had interaction withthe –OH (corresponding to the third C of CS structure), –CH2–OH (corresponding tothe sixth C of CS structure), and CS’s amino groups. In addition, the peak around1418 cm�1, along with the peaks of phosphate groups, may come from the CO2�3 groupof carbonated HA.[17] The presence of CO2�3 ions into HA ceramic played an impor-tant role in the bone metabolism and they occupy about 8wt% of the calcified tissueand may vary depending on the age factor.[30]

4000 3500 3000 2500 2000 1500 1000 500

561603

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Figure 6. FTIR spectra of (a) CS/HA0; (b) CS/HA5; (c) CS/HA10; and (d) CS/HA15 scaffolds.

Table 2. The assignments of FTIR peaks of CS.

Peak value (cm�1) Assignment

1656–1658 Amide І (C–O)1591–1599 Amide II (–NH2)1380 –CH3 symmetrical deformation1120 C3–OH1013 C6–OH


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3.4. Cell study

Cell culture studies using MC3T3-E1 cell line were introduced to assess thebiocompatibility of the composite scaffolds. The initial cell attachment studies werecarried out after 12 h incubation at 37 °C in a humidified incubator with 5% CO2 and 85%humidity. The adhesion rate was calculated with the formula: En = [(N0�N)/N0]� 100%.Here, N0 and N stand for the number of seeded cells and the number of unattached cells,respectively.

As shown in Figure 7, the adhesion ratio of osteoblasts on the composite scaffoldswas all above 80%, which also showed that composite scaffolds had the higher ratio ofadhesion compared with the pure CS scaffold, and the ratio increased with the increaseof HA content. This may be due to the change of roughness of the surface with HAcontent. From the photographs of SEM (see Figure 3) in the high magnification, a largenumber of HA nanoparticle bulges formed on the surface of scaffolds, which couldaffect cell adhesion on the implants. From Figure S1, the SEM micrographs ofosteoblasts on the CS/HA10 composite scaffolds after seven-day cell culture showedthat the osteoblasts grew, adhered, and spread well. The osteogenic cells were polygonalor spindle-shaped and it was known that the larger surface area, another key parameter,also played an important role in the increasing protein adsorption, especially adhesiveproteins. Finally, the two effects resulted in an increase of adhesion rate of thecomposite scaffolds.

Cell proliferation studies on the composite scaffolds were examined by CLSM andMTT assay. As shown in Figure 8, CLSM pictures of the cells that cultured on thescaffolds for one day showed that shutter-like and polygon-shaped cells spread activelyon the composite scaffolds and round-like cells grew on the pure CS scaffolds. After14 days, the cells on the two kinds of scaffolds already attached onto the walls of thepores with their pseudopodia. From Figure S2, the ALP activities also showed that theosteogenic cell differentiates better on composite scaffolds than the pure CS scaffolds.After a 21-day cell culture, the ALP activity value of the composite scaffolds was allabove 1:2 μ/mg, which increased six times compared with the pure CS scaffold. And,the ALP activity value increased with the increase of HA content. This is because the

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Figure 7. Adhesion of osteoblasts on CS/HA composite scaffolds.

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additional HA improved the cell affinity and cell differentiation on composite scaffolds.And over cultivation, only a few dead cells were observed. Figure 9 shows theabsorbance obtained from the MTT assay of MC3T3-E1 cells that were cultured withthe scaffolds.

A higher degree of proliferation of MC3T3-E1 was observed on the CS/HA10(standing for containing 10% HA in the CS solution) scaffolds as compared to pure CSscaffolds and other composite scaffolds. The reason is that, with the increase of the HAprecursor, we have found some chlor-HA forms by XRD. That apatite will affect thegrowth of cells on the surface of composite scaffolds. However, the CS/HA10 scaffold iscytocompatible and nontoxic to MC3T3-E1. Throughout the culture process, the state ofcell growth on composite scaffolds kept always good, showing good cell compatibility.

Figure 8. Confocal micrographs illustrating proliferation of osteoblasts on the CS/HA scaffolds.The green points in pictures were living osteoblasts. The red points were dead osteoblasts. All themicrographs had the same scale bars. (Please see the online article for the colour version of thisfigure: http://dx.doi.org/10.1080/09205063.2013.836950.)

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Figure 9. Viability of osteoblasts on CS/HA composite scaffolds as a function of time measuredby MTT assay.


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Therefore, this novel porous scaffold with three-dimensional oriented structure might bea promising scaffold in bone tissue engineering.

The scaffolds for bone tissue engineering not only need microporous structure andgood cell compatibility, which will be beneficial to the growth of the seed cells, butalso should have a certain mechanical strength before the cell function successfullyreconstructed.

From Figure 10, the compressive strength of the scaffolds was improved to0.680MPa from 0.511MPa with the increase of the content of HA. Although the CSmolecules and calcium ions had complexation reaction, which resulted in the decreaseof CS crystallinity and affected the strength of the scaffold, a large number of hydroxylgroups were brought into in the composite scaffold because of the formation of HA,which generated a large number of hydrogen bonds that made CS and HA more closelyintegrated. Moreover, HA, as commonly used as inorganic ceramic materials, especiallyin the form of human bone with acicular shape crystals of HA, had high strength. Socompared with the pure CS scaffold, the CS/HA composite scaffold had higher strength.Even the compressive strength of CS/HA15 increased by 33.07%. At the same time,many experiments and documents proved that HA precursor solution could not exist inthe CS solution and produced a kind of white precipitate of calcium phosphate,[21] ifthe concentration of HA continued to increase. So this approach cannot be used toenhance the compressive strength of the composite scaffolds by improving the contentof HA.

4. Conclusion

The three-dimensional oriented CS/HA scaffolds were prepared by in situ precipitationand lyophilization. SEM images indicated that the porous scaffolds had the spoke-likeframework in cross-section and multilayer structure in vertical section. The nano-HAparticles with acicular shape formed on the surface of the composite scaffolds and scat-tered homogeneously in them. XRD and FTIR were used to investigate the fabricationstructure of the hybrid scaffold. The results showed that the in situ deposited mineral(nano-HA) in the scaffold had the phase structure similar to natural bone and confirmedthat particles were exactly HA. This effect would affect the crystallization of CS, and

CS/HA0 CS/HA5 CS/HA10 CS/HA15

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Pa)

Figure 10. The compressive strengths of composite CS scaffolds.

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the CS’s structure and functional groups regulated the HA crystallization process. Thesescaffolds were also evaluated by the in vitro cell studies. The CS/HA scaffold also hada higher ratio of adhesion and ALP activity value of osteoblasts compared with the pureCS scaffold, and the ratio increased with the increase of HA content. The ALP activityvalue of composite scaffolds was at least six times that of the pure CS scaffolds.The composite scaffold also showed a higher degree of proliferation of MC3T3-E1 ascompared to the pure CS scaffold and the CS/HA10 scaffold was the highest one in thecomposite scaffolds. SEM micrographs of osteoblasts on the CS/HA10 compositescaffolds after seven-day cell culture also showed that the osteoblasts grew, adhered,and spread well. The osteogenic cells were polygonal or spindle shape. The resultssuggested that the composite scaffolds possessed good biocompatibility. Compared withthe pure CS scaffold, the compressive strength of CS/HA15 increased by 33.07%. Thisnovel porous scaffold with three-dimensional oriented structure might be a promisingscaffold in bone tissue engineering.

FundingThis work was partially funded by National Natural Science Foundation of China [Grant Nos.21104067 and 21274127], the Key Basic Research Development Plan (Project 973) of China[Grant Nos. 2011CB606203 and 2009CB930104], Grand Science and Technology Special Projectof Zhejiang Province [Grant No. 2008C11087], and Fundamental Research Funds for the Central20 Universities (2013QNA4048).

Supplemental data

Supplemental data for this article can be accessed here http://dx.doi.10.1080/09205063.2013.836950.

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