Preparation, in vitro degradability, cytotoxicity, and in vivo biocompatibility of porous hydroxyapatite whisker-reinforced poly(L-lactide) biocomposite scaffolds.

Journal of Biomaterials Science, Polymer Edition

ISSN: 0920-5063 (Print) 1568-5624 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsp20

Preparation, in vitro degradability, cytotoxicity, and in vivo biocompatibility of porous hydroxyapatite whisker-reinforced poly(L-lactide) biocomposite scaffolds Lu Xie, Haiyang Yu, Weizhong Yang, Zhuoli Zhu & Li Yue To cite this article: Lu Xie, Haiyang Yu, Weizhong Yang, Zhuoli Zhu & Li Yue (2016): Preparation, in vitro degradability, cytotoxicity, and in vivo biocompatibility of porous hydroxyapatite whisker-reinforced poly(L-lactide) biocomposite scaffolds, Journal of Biomaterials Science, Polymer Edition, DOI: 10.1080/09205063.2016.1140613 To link to this article: http://dx.doi.org/10.1080/09205063.2016.1140613

Accepted author version posted online: 10 Jan 2016.

Submit your article to this journal

Article views: 7

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tbsp20 Download by: [Gazi University]

Date: 25 January 2016, At: 23:27

Publisher: Taylor & Francis Journal: Journal of Biomaterials Science, Polymer Edition DOI: http://dx.doi.org/10.1080/09205063.2016.1140613

Preparation, in vitro degradability, cytotoxicity, and in vivo biocompatibility of porous hydroxyapatite whisker-reinforced poly(L-lactide) biocomposite scaffolds

Downloaded by [Gazi University] at 23:27 25 January 2016

Lu Xie1, Haiyang Yu1,*, Weizhong Yang2,*, Zhuoli Zhu1, Li Yue1 1

State Key Laboratory of Oral Diseases, West China College of Stomatology, Sichuan University,

Chengdu 610065, China 2

School of Materials Science and Engineering, Sichuan University, Chengdu 610064, China

Corresponding authors: Prof. Haiyang Yu, State Key Laboratory of Oral Diseases, Sichuan University, No.24 South Section 1, Yihuan Road, Chengdu 610065, China, Email: [email protected]; Prof. Weizhong Yang, School of Materials Science and Engineering, Sichuan University, No.24 South Section 1, Yihuan Road, Chengdu 610065, China, Email: [email protected]

1

Abstract: Biodegradable and bioactive scaffolds with interconnected macroporous structures, suitable biodegradability, adequate mechanical property, and excellent biocompatibility have drawn increasing attention in bone tissue engineering. Hence, in this work, porous hydroxyapatite whisker-reinforced poly(L-lactide) (HA-w/PLLA) composite scaffolds with different ratios of HA and PLLA were successfully developed through compression molding and particle leaching. The microstructure, in vitro mineralization, cytocompatibility, hemocompatibility and in vivo biocompatibility of the porous


HA-w/PLLA were investigated for the first time. The SEM results revealed that these HA-w/PLLA scaffolds possessed interconnected pore structures. Compared with porous HA powder-reinforced PLLA (HA-p/PLLA) scaffolds, HA-w/PLLA scaffolds exhibited better mechanical property and in vitro bioactivity, as more formation of bone-like apatite layers were induced on these scaffolds after mineralization in SBF. Importantly, in vitro cytotoxicity displayed that porous HA-w/PLLA scaffold with HA/PLLA ratio of 1:1 (HA-w1/PLLA1) produced no deleterious effect on human mesenchymal stem cells (hMSCs), and cells performed elevated cell proliferation, indicating a good cytocompatibility. Simultaneously, well-behaved hemocompatibility and favorable in vivo biocompatibility determined from acute toxicity test and histological evaluation were also found in the porous HA-w1/PLLA1 scaffold. These findings may provide new prospects for utilizing the porous HA whisker-based biodegradable scaffolds in bone repair, replacement and augmentation applications. Keywords: hydroxyapatite whisker, poly(L-lactide), porous scaffold, degradability, bone tissue engineering.

3

1. Introduction One of the actual and much-needed demands in orthopedics is the clinical availability of biodegradable artificial implants.[1,2,3,4] In some clinical applications, such as fracture treatment, permanent metal and polymer biomaterials are not necessary or even disadvantageous, and a temporary implant would be much more suitable. Temporary implantable biomaterials made of biodegradable materials are destined to degrade and dissolve postoperatively matching with the bone healing period,


so that a second surgery for implant removal is escapable, eliminating surgery and associated expenditure, as well as unnecessary health risks for patients. Therefore, biodegradable materials have been developed and widely used in bone tissue engineering utilizing magnesium alloys [4,5] as metallic materials; poly(L-lactide) (PLLA),[6] poly(lactide-co-glycolide) (PLGA) [7,8] and polycaprolactone (PCL) [9] as polymer materials; hydroxyapatite (HA) [10,11] and tricalcium phosphate [12] as ceramic materials. Among these biodegradable polymers, PLLA and its derivatives have gained extensive attention owing to their excellent processability, biocompatibility, and tunable biodegradability with molecular weight.[13,14,15] The scaffolds consisting of only PLLA, however, have defective bioactivity and poor mechanical strength, hampering their further applications in bone tissue engineering.[16] Furthermore, the degraded acidic monomers from PLLA may cause inflammatory and allergic reactions, which impede their adoption in clinic.[17] To address the above concerns for bone repair and provide a better environment for cell attachment/proliferation, a variety of bioactive fillers, especially bioactive calcium phosphate-based biomaterials and bioceramics [18,19] have been frequently used to reinforce the common

biopolymers with mechanical property and bioactivity. In addition, composite scaffolds

usually have better osteoconductivity than single-ingredient scaffolds. Hydroxyapatite 4

(Ca10(PO4)6(OH)2, HA), among these bioceramics, has attracted considerable interest due to its preeminent biocompatibility, bone-bonding ability, osteoconductivity, and moderate absorbability.[20,21] The addition of HA into PLLA matrix, moreover, could buffer the acidic degradation products with the purpose of enhancing the bioactivity of PLLA.[22,23] Another motivation to develop HA/polymer composites is based upon the biomimetic strategy since natural bone considered as an example of a biocomposite is composed mainly of HA crystallites in the


collagen-rich organic matrix.[24] So HA is often regarded as a reinforced phase with different morphologies such as whisker and powder. It is reported that HA whisker reinforcement resulted in an improvement of mechanical property and work-to-failure compared with HA powder reinforcement due to its high tensile strength, oriented morphology and other remarkable characteristics.[25] Furthermore, HA whisker reinforcement also contributes to elastic anisotropy similar to that found in human cortical bone tissue.[26] An idea biomaterial scaffold for bone tissue engineering should possess highly macroporous and interconnected pore structures, adequate mechanical stability, and excellent bioactivity, as well as proper biodegradability.[27,28] Porous structure in biomaterials is proved to play a key role in the exchange of waste, nutrients, water, ions and regulatory molecules between cells, and provides favorable environment for bone cell proliferation and succedent new bone formation.[29] Therefore, an increasing attraction has being given to three-dimensional (3D) porous biodegradable materials for use in orthopedic applications, including bone ingrowth scaffolds for implant fixation, and synthetic bone graft substitutes for tissue regeneration. Guided by these considerations, as aforementioned, we develop a novel porous HA whisker-reinforced PLLA (HA-w/PLLA) biocomposite scaffold possessing major features of ideal biomaterial scaffolds for bone tissue engineering. Despite the attractive advantages 5

and progress in preparation of innovative porous biodegradable materials, to our best knowledge, a porous HA-w/PLLA scaffold and its in vitro cellular response and in vivo biocompatibility has not been reported and investigated. Hence, in the present study, the HA whisker-reinforced PLLA porous scaffold was created via a process of compression molding and particle leaching. The degradability, in vitro blood compatibility, and in vitro/vivo biocompatibility of the porous HA-w/PLLA scaffold, with the porous HA powder-reinforced PLLA (HA-p/PLLA) scaffold as control group in some experiments,


were detailedly evaluated as a critical step towards its use for biomedical applications. Compared with HA-p/PLLA, the HA-w/PLLA biocomposite with the HA/PLLA ratio of 1:1 exhibited deferred degradability, better mechanical property, superior bioactivity, well-behaved hemocompatibility, and good in vivo biocompatibility, presenting a bona fide potential as a bioactive bone substitute material for tissue engineering.

2. Materials and methods 2.1. Materials Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O, analytical reagent (AR)), diammonium hydrogen phosphate ((NH4)2HPO4, AR), nitric acid (HNO3, AR), ammonium hydroxide (NH3·H2O, AR), absolute ethanol (CH3CH2OH, AR), acetone (CH3COCH3, AR), trichloromethane (CHCl3, AR), sodium chloride (NaCl, AR), carbamide (NH2CONH2, AR) and D-sorbitol (C6H14O6, biochemical reagent (BR)) were purchased from ChengDu Kelong Chemical Co., Ltd. (China). Poly(L-lactide) (PLLA, i.v. 2.67 dL/g) was obtained from the Institute of Medical Devices of Shandong Province (Jinan, China). All chemicals were used as received without any purification, and all aqueous solutions were prepared with de-ionized water (D.I. water). 6

2.2. Synthesis of HA powders and HA whiskers HA powders (HA-p) were synthesized based on the previous literature with some modification.[30,31] Firstly, Ca(NO3)2 and (NH4)2HPO4 were dissolved in the D.I. water separately according to a Ca/P molar ratio of 1.67/1. The pH of each solution was adjusted to 10 by adding NH3·H2O. Then, Ca(NO3)2 solution was dropped into (NH4)2HPO4 solution with continuous stirring. Crystal growth occurred when kept at 40 °C for 1 h, and the pH value of the supernatant was


maintained in the range of 10-10.5 using NH3·H2O. After reaction, HA slurry was kept at ambient temperature for 0.5 h, and the precipitate was filtered, and rinsed with D.I. water and absolute ethanol, successively. Finally, the HA powders were dried in an oven at 80 °C for 24 h. The synthesis procedure for HA whiskers (HA-w) through hydrothermal homogeneous precipitation approach was described as follows: 75 mL of aqueous solution containing calcium and phosphate was prepared by dissolving Ca(NO3)2 and (NH4)2HPO4 in 1 mol/L HNO3 solution, respectively, with the Ca/P ratio fixed at 1.67 throughout. Ca(NO3)2 was added into (NH4)2HPO4 solution drop-wise with continuous stirring followed by the addition of 5 g carbamide. Afterwards, 0.9715 g D-sorbitol was introduced as a template into a mixed solution of Ca(NO3)2 and (NH4)2HPO4, and the supernatant was adjusted to pH= 3 via dropping NH3·H2O. The hydrothermal processing was achieved by loading the prepared solution in an oil-bath, refluxed at 98 °C for 48 h, and then cooled to room temperature. The precipitation was then filtered and cleaned with D.I. water and absolute ethanol at least three times. The product was finally dried at 60 °C to obtain HA whiskers. 2.3. Fabrication of compact and porous HA/PLLA biocomposites PLLA was first dissolved in CHCl3 under constant stirring until the 2 % w/v solution was clarified. Predetermined amounts of HA whiskers with different mass ratios of HA and PLLA (HA:PLLA= 1:1, 7

1:2, and 2:1) were suspended in the PLLA solution with 20-min high-speed stirring and 40-min ultrasonically dispersing until homogeneous. Then, the above dispersion was slowly poured into a mixed solution of ethanol and acetone and kept for 10 min to obtain cotton-shaped granule precipitates via diffusion and phase separation. Afterwards, the precipitates were vacuum-filtered, washed with ethanol, and dried at 60 °C for 24 h to remove the residual solvent and moisture. The mixture was then ground to powders by hand using a mortar, and an appropriate amount (corresponded to 30 % by mass)


of the NaCl porogen with size of 120 µm-380 µm was added to the precipitates and evenly mixed. Composite scaffolds were prepared by compression molding and particle leaching. In order to find the optimum temperature for hot-pressing, the dry powder mixture (HA:PLLA= 1:1) was densified at 15 MPa at various temperatures varying from 100 °C to 180 °C for 30 min in a cylindrical die (WY-98-A, Tianjin Keqi High & New Technology Corp., China). After determining the optimal temperature, the composite was hot-pressed at that temperature (140 °C) using a manual hydraulic platen press (SSP-10A, SHIMADZU, Japan). The molded composite scaffolds were 10 mm in diameter and 1 mm in height. After cooling to ambient temperature, the molded composite was ejected from the die and placed in D.I. water for at least 7 days to dissolve the NaCl. The D.I. bath was changed daily. After immersion, the composites were dried at 60 °C to constant weight to attain the porous HA/PLLA scaffolds. The resulting HA whiskers/PLLA composite prepared from different volume ratios of HA/PLLA were denoted as HA-w1/PLLA1, HA-w1/PLLA2, and HA-w2/PLLA1, respectively. The HA powders/PLLA composite was fabricated via the same method as per the aforementioned protocol and was named as HA-p1/PLLA1, HA-p1/PLLA2, and HA-p2/PLLA1, respectively. 2.4. Characterization 8

The crystalline phase of HA powders and whiskers was examined by X-ray diffraction analysis (XRD, DX-100, PHILIPS, Netherlands) using a Cu target as radiation source (λ= 1.540598 Å). The diffraction angles (2θ) were set between 10 o and 70 o, incremented with a step size of 0.03 o/s. The identification of phases was achieved by comparing the obtained sample diffraction pattern with standard cards in ICDD-JCPDS database. The particle size distributions were measured by a laser particle size analyzer (JL-1177, Chengdu, China) at a concentration of approximately 0.1 mg


particle/mL water, after the particles were homogeneously dispersed in D.I. water. The morphological structures of pure HA powders, HA whiskers and different porous HA-w/PLLA composites were characterized by a field emission scanning electron microscope (FE-SEM, JSM-7500F, JEOL, Japan) at an accelerating voltage of 20 kV. HA samples for SEM imaging were prepared by placing a drop of the aged HA powders and whiskers suspensions (the suspensions were diluted in D.I. waters and dispersed by ultrasonic waves before use) onto a clean silicon wafer, and dried in air. All samples were coated by gold for 1 min before SEM observation. The compressive strength of the HA-w1/PLLA1 biocomposite scaffolds fabricated under different temperatures was evaluated by a mechanical tester (XDL-50000N, Jingdu Xinhong Test Factory, Yangzhou, China) at room temperature. 2.5. In vitro degradability and bioactivity in SBF 2.5.1. Weight loss The in vitro degradation characteristics and bioactivity were evaluated in a simulated body fluid (SBF, nearly equal to that of human blood plasma). The 1×SBF (Table S1) was prepared by dissolving the following chemicals in the sequence of NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, CaCl2, and Na2SO4 in distilled water and buffering to pH 7.4 with Tris ((CH2OH)3CNH2) and 1mol/L HCl at 37 °C. Compact and porous HA-p1/PLLA1 and HA-w1/PLLA1 samples were immersed in SBF at 37 °C 9

in a static condition for 21 days, and the SBF was changed every 2 days. After 1, 3, 5, 7, 14, and 21 days, the specimens were retrieved from the given solution, gently rinsed several times with D.I. water, then dried at room temperature before being subjected to weight loss analysis. The weight loss of HA/PLLA biocomposites with time (δ, %) was determined by the equation δ= (W-W0)/W0, where W is the initial weight of the HA/PLLA biocomposite before immersion, and W0 is the current weight of the HA/PLLA biocomposite after immersion in SBF.


2.5.2. Scaffold microstructure and mechanical testing during degradation After 5, 14, and 21 days of soaking, the microstructures of HA-p1/PLLA1 and HA-w1/PLLA1 samples were examined by FE-SEM equipped with energy dispersive X-ray spectroscopy (EDS) after coated with gold for 1 min. At the same time, the compressive strength of these HA/PLLA biocomposite scaffolds was also tested by the mechanical tester (XDL-50000N) at ambient temperature. 2.6. Cell culture Human mesenchymal stem cells (hMSCs, obtained from ChengDu Military General Hospital, China) were cultured in F12 cell medium (Gibco, Carlsbad, Canada) containing 10 % fetal calf serum (Gibco), 100 µg/mL streptomycin (Beyotime Biotechnology, Shanghai, China) and 100 µg/mL penicillin (Beyotime Biotechnology) at 37 °C in a humidified atmosphere of 5 % CO2. hMSCs at passage three were used for the in vitro experiments. The culture medium was changed every 2 days. All samples were sanitized under ultraviolet for at least 4 h and rinsed twice with sterile PBS before cell culture. 2.7. Cytotoxicity assay 2.7.1. Indirect cytotoxicity on various extracts Test on extracts was carried out according to the instruction of ISO 10993 in the study. The 10

extraction media of the porous HA-w/PLLA biocomposite was prepared using serum-free cell culture medium (F12), with the extraction ratio (the ratio of specimen weight to extraction medium) of 0.1 g/mL, then incubated in a water-bathing constant temperature vibrator (SYP, Hongyiyuhua Instrument Co., Ltd, China) at 37 °C for 72 h, and sterilized using the 0.22 µm filter membrane before cell culture. Then, a dilution series of extract culture media with different volume ratio of extraction medium and total medium (= extraction medium+F12 medium) including extraction medium/total medium ratio of


1/3, 2/3, and 3/3 (pure extraction medium) were implemented to detect the cytotoxicity of these HA-w/PLLA biocomposite. Pure F12 medium was used as a negative control. Cells were incubated in 96-well cell culture plates at 3 × 103 cells per 100 μL in each well and incubated for 12 h to allow attachment. Then culture media were substituted by various dilutions of extract medium obtained from the studied materials. Cell were cultured for 24 h, 48 h and 72 h for assessing cell proliferation on these porous HA-w/PLLA groups and cultured for 1 h-6 h for testing cell attachment on the porous HA1-w/PLLA1 biocomposite, respectively. The cell counting kit-8 (CCK-8, Dojindo, Japan) assay was used to evaluate the viability of cells. After the cultivation period, 10 μL of CCK-8 was added into each well for further 2 h incubation at 37 °C, and then 100 μL of supernatant from each well was transferred to new 96-well cell culture plates. The absorbance value of supernatant optical density was measured with a microplate reader (Model 680, Bio-Rad, USA) at 450 nm wavelength. Four parallel measurements were used to provide an average and standard deviation. Simultaneously the cell morphology was shown by an inverted optical microscope (BX60, Olympus, Japan). The cell viability was expressed as a percentage as following:

Cell viability (%) =

OD(test)－OD(blank) OD(negative control)－OD(blank)

2.7.2. Direct cell contact on samples 11

×100%

The HA-w1/PLLA1 specimen was placed in a well followed by cell seeding (3 × 104/mL) for light microscope observation. The specimen was left undisturbed in an incubator for 2 h to allow the attachment of cells. Then, the cell/specimen construct was cultured for 1 and 3 days under the conditions. The cell morphology was examined microscopically using the inverted optical microscope (Olympus) for cellular response at the edges of samples since the materials were not transparent. Tissue culture plate (TCP) was used as a negative control.


2.8. Hemocompatibility assessments 2.8.1. Hemolysis test Healthy New Zealand white rabbit blood (provided from West China Center of Medical Sciences, Sichuan University, Chengdu, China) containing sodium citrate (3.8 wt%) in the ratio of 9:1 was taken and diluted with normal saline (4:5 ratio by volume). The porous HA-w1/PLLA1 biocomposite (Ф10 mm × 1 mm) was dipped in a standard tube containing 10 mL of normal saline that was previously incubated at 37 °C for 30 min. Then 0.2 mL of diluted blood was added to this standard tube, and the mixtures were incubated at 37 °C for 60 min. As a negative control, 0.2 ml of diluted blood was again diluted with normal saline, and distilled water was added to a standard tube containing diluted blood, which served as a positive control. After the incubation, all the tubes were centrifuged for 5 min at 2500 rpm (LDZ4-0.8A, Beijing Medical Centrifuge Factory, China), and the supernatant was carefully removed and transferred to a new 96-well plate for spectroscopic analysis by an ultraviolet-visible (UV-VIS) spectrophotometer (WFJ7200, UNIC, France) at 545 nm. Four parallel measurements were used to provide an average and standard deviation.

Hemolysis (%) =

OD(test )－OD(negative control) OD(positive control)－OD(negative control)

2.8.2. Dynamic clotting time assay 12

×100%

Dynamic blood coagulation tests measured the release of hemoglobin from residual erythrocytes that remained free from entrapment during clot formation. Assays were conducted by recalcifying anticoagulated whole blood, as described previously with several modifications. Briefly, the porous HA-w1/PLLA1 biocomposite was placed at the bottom of individual 100 mL beakers and then pre-warmed in a water bath at 37 °C for 5 min. Subsequently, anticoagulated whole blood (0.2 mL) was dripped onto the surface of the biocomposite and incubated at 37°C for a further 5 min, after which


CaCl2 solution (25 μL of 0.2 mol/L) was dripped into the blood to initiate the coagulation cascade (time 0). The beakers were shaken for 1 minute to mix the CaCl2 uniformly with the blood. Heating of the covered beakers was continued at 37 °C for a predetermined time (5, 10, 20, 30, 40, 50, and 60 min). At the designated termination point, the beakers were removed from the water bath and shaken for 10 min following addition of 100 mL D.I. water to lyse the free erythrocytes. The absorbance of the supernatants at 540 nm using a visible spectrophotometer (UNIC) was determined. The control groups involved the use of silylated glass as negative control and unmodified glass as positive control. Since the signal was derived from that proportion of erythrocytes remaining free of clot entrapment, absorbance was inversely proportional to the size of the clots.[32] The absorbance-time curve was constructed using the average values from four replicate experiments. 2.9. In vivo biocompatibility evaluation 2.9.1. Preparation of extracts of HA-w1/PLLA1 The extracts of the porous HA-w1/PLLA1 biocomposite scaffold (Ф10 mm × 1 mm) were also performed in accordance with the instruction of ISO 10993. The extraction media was prepared using normal saline with the extraction ratio (the ratio of specimen weight to extraction medium) of 0.1 g/mL, and then incubated in the water-bathing constant temperature vibrator (SYP) at 37 °C for 72 h. 13

The experimental protocol was all approved by the Institutional Animal Care and Use Committee of Sichuan University. All the animals were maintained on a normal, solid lab diet and regular tap water. 2.9.2. Skin irritation test Ten male rats (weight 160-180 g, provided from ChengDu Military General Hospital) were randomly divided into two groups: HA-w1/PLLA1 scaffold, and normal saline (negative control). After under general anesthesia (0.5 % pentobarbital sodium, intravenous injection, 1 mL/100g), the rats were


dehaired on the back, then 0.1 mL of extracts and normal saline solutions were injected into the back subcutaneous area as shown in Fig. S1. After 24, 48, and 72 h of the injection, the erythema and dropsy area of rats were observed. 2.9.3. Acute toxicity evaluation Twenty rats were randomly assigned to two groups (n= 10) after one week of accommodation. Animals in the HA-w1/PLLA1 group were injected intraperitoneally (50 mg/kg) with the extract media of the porous HA-w1/PLLA1 biocomposite. Negative controls were injected with an equal volume of physiological saline. The general conditions (the activity, energy, feces, behavior pattern, and other clinical signs), body weight, and mortality of all rats after administration were continuously recorded during the experiments. At the end of 72 h, all animals were killed through air embolism, and their livers and kidneys were excised and kept in 10 % formalin for histopathology examination. Sections (about 5 μm) of liver and kidney tissues embedded in paraffin blocks were stained with hematoxylin-eosin (H&E) and then observed using an optical microscope (TE2000-U, Nikon, Japan). 2.9.4. Subcutaneous implant test After under general anesthesia, a skin incision was made to create a subcutaneous pocket at the back of the rat. Three samples of the porous HA-w1/PLLA1 biocomposite scaffold (Ф10 mm × 1 mm) were 14

placed aseptically into the dorsal subcutaneous area of the rats for in vivo study. Then the incision was closed with surgical staples, and the rats after surgery received subcutaneous injection of penicillin for 3 consecutive days. At 14 days after surgery, the implants were harvested together with their surrounding tissues (3 implants). The tissues with the implants were fixed in 10 % formalin in phosphate buffer, dehydrated in a series of solutions with different ethanol concentrations (70 %, 80 %, 90 %, 95 %, and 100 %), and transferred to a methylmethacrylate solution at 37 °C. Afterwards, the


embedded samples were cut into sections by LEICA SP1600 (Germany), and hard tissue slices (5 μm/section) were observed under the light microscope (TE2000-U) after toluidine blue staining. 2.10. Statistical analysis All quantitative data expressed as mean ± standard deviations were derived from experiments. Statistical analysis was carried out with the Origin software. Student's t-test was used to identify the significant differences among the experimental groups, and a p-value less than 0.05 was considered statistically significant.

3. Results and discussion 3.1. Characterization of HA whiskers To examine whether HA was the primary calcium phosphate phase, we performed XRD analysis. The powder XRD patterns under wide angle (10 °-70 °) diffractions of both HA powders and whiskers were shown in Fig. 1. Despite some variation in peak intensity, the Bragg diffraction peaks of the products matched quite well with those of the standard HA (PDF # 09-0432, and PDF # 46-0905) at 2θ values of 25.9 °, 31.7 °, 32.2 °, 32.8 °, 46.6 °, 49.5 ° and 53.1°, which were indexed to (002), (211), (112), (300), (222), (213), and (004) planes, respectively, indicating that both samples were 15

predominantly HA.[33,34] It was obvious that the relative intensity of diffraction peaks of HA whiskers, particularly the three most intense peaks of HA, corresponding to (211), (112), and (300) planes, was stronger than those of HA powders, suggesting a higher HA crystallinity for HA whiskers. In addition, sharper and higher diffraction peaks were present in HA whiskers, which might be ascribed to bigger sized crystals Fig. 1


The morphologies of the HA powders and whiskers were observed by SEM. HA powders in Fig. 2(a) displayed quantities of irregular agglomerates of particles with a size of 5-25 μm. The synthesized nano-HA particles were more likely to aggregate and form isolated large grains because of the high specific surface energy of nano-particles. However, the morphologies of HA whiskers were in a typical long stick-like shape with a length of about 50-300 μm (Fig. S2) and a width of about 1 μm (Fig. 2(d)). Moreover, the HA whiskers were dispersive, quite different from the HA powders, which was thought to enhance the dispersion of fillers in subsequent composite preparation. Fig. 2 The size distributions of the HA powders and HA whiskers were also measured and the results were shown in Fig. 3. It could be seen that the HA powders followed normal distribution patterns with the average diameter of 5-30 μm, consistent with the SEM observation. Nevertheless, there were two obvious peaks in the size distribution pattern of HA whiskers, suggesting that the main concentrations of HA whisker’s length were in approximately 50 μm and 350 μm. In the current experiment, the particle dispersion coefficient (PDC) could be expressed using following equation:

PDC =

D90－D10 D50

where D90, D50, and D10 were the corresponding diameter of particles when cumulative distribution of 16

volume reached to 90 %, 50 %, and 10 %, respectively. After calculation, the PDC of HA whiskers was about 5.538, higher than that of HA powders with a PDC of 2.840, suggesting HA powders displayed a more narrow size distribution pattern than HA whiskers. Fig. 3 3.2. Morphology of HA-w/PLLA biocomposites To determine the optimum temperature for the composite to reach the maximum mechanical property,


the compressive property and the morphological structure of the prepared compact HA-w/PLLA composite (at the HA/PLLA ratio of 1:1) densified at various temperatures varied from 100 °C to 180 °C were investigated. Fig. 4 reflected that the compressive strength of the compact HA-w1/PLLA1 increased from 90.04 MPa to 109.52 MPa with the increase of hot-pressing temperature from 100 °C to 140 °C. However, the compressive strength decreased dramatically to 96.04 MPa, when temperature elevated to 180 °C. At the same time, the differences in surface topologies of these HA-w1/PLLA1 composite fabricated at different temperatures were also observed. For instance, under 100 and 120 °C, SEM images of the composites exhibited rough surfaces with exposed HA whiskers and obvious voids, showing that the PLLA matrix cannot envelop all HA whiskers, so some HA whiskers were extended from the PLLA matrix and hanged on the surface. While at 140 °C, smooth surface was present on the surface of the HA-w1/PLLA1, and all HA whiskers were enveloped in the PLLA matrix. Up to higher temperature (180 °C), the cracks were re-founded on the surface. Because the temperature of 140 °C was close to the melting point of PLLA about 155-170 °C,[35] under 140 °C the HA whiskers were well mixed in the fully-melting PLLA to obtain a homogeneous HA/PLLA mixture, contributing to the enhancement of compressive property. However, excessive temperature could give rise to partial vaporization of PLLA matrix, generating bubbles inside and cracks on the surface of the composite. 17

This possibly was the reason why excessive temperature (180 °C) led to the reduction of compressive strength for HA-w/PLLA composite. Therefore, according to the mechanical and morphological data, the optimal hot-pressing temperature for the composite was about 140 °C, and the HA-w1/PLLA1 composite had a maximum compressive strength at 109.52 MPa. Fig. 4 After determining the temperature, different weight ratios of HA whiskers/PLLA polymer (1:1, 1:2,


and 1:2) were mixed to prepare the various porous HA-w/PLLA biocomposite scaffolds through compression molding and particle leaching. The scaffolds were cut in the middle and the incision observed by SEM, as depicted in Fig. 5. The surface morphology of all HA-w/PLLA composites revealed a uniform distribution of the spherical macropores and the interconnections with their neighbors. The HA-w1/PLLA1 and HA-w1/PLLA2 had a homogeneous pore size of 300 ± 100 μm. However, the HA-w2/PLLA1 scaffolds with higher proportion of HA whiskers exhibited looser and rougher surfaces with irregular macropores, owing to the difficulty in shaping with higher content of inorganic fillers. The porogen leaching process in the mixture of HA/PLLA and NaCl particles led to the formation of pores, and the macropore size and distribution of the pore could be tailored by the particle size and amount of NaCl crystals. Besides, from the enlarged image, it could be found that the interior surface of macropores was comprised of the agglomerated HA whiskers crystallites. All HA whiskers were dispersed and embedded well in the PLLA matrix, and no significant fracture of whisker was detected. Previous work has proved that macropore structures in bone tissue engineering scaffold are not only helpful to ion exchange and functional proteins absorption/loading, but also contribute to osteoblast proliferation, as well as promote bone ingrowth.[36,37,38] Furthermore, the exposure of HA on surface appeared to provide anchoring sites for cell attachment and proliferation.[39] Therefore, the 18

HA-w/PLLA with abundant macropores would have a promising application as scaffold materials in bone tissue engineering, especially for HA-w1/PLLA1 and HA-w1/PLLA2. Fig. 5 3.3. In vitro degradation and bioactivity of HA/PLLA biocomposite The bioactivity and degradability are directly related to the chemical stability and durability of scaffolds, and it depends on the chemical and crystallographic composition and microstructure of the


materials as well as the host environment.[40] To be used as the biomedical application, it is necessary to probe the bioactivity and degradability of the HA/PLLA biocomposite. The HA-p1/PLLA1 and HA-w1/PLLA1 scaffolds were subjected to the in vitro bioactivity and degradability tests by examining the formation capability of bone-like apatite layer on their surfaces after immersion in SBF under normal physiological condition. Figs. 6-7 exhibited the surface morphologies of the porous HA-p1/PLLA1 and HA-w1/PLLA1 specimens under SEM after preconcerted times in SBF. There was no apparent observation of Ca-P deposition on the surface of both porous HA-p1/PLLA1 and HA-w1/PLLA1 in the first several days. After 2 weeks immersion in SBF, the aggregation of large amount of rounded nodulus started to emerge on the surface of porous HA/PLLA biocomposites. The mineral layer formed was very thin and the original contour of the specimen could still be seen. With longer immersion time to 21 days, the number of particles on the porous HA-w1/PLLA1 surfaces substantially increased and a thick layer of sphere-shaped and dense apatite covered the entire surface of the composite, which was more than that on the porous HA-p1/PLLA1. From higher magnification of SEM, we could see that the formed apatite possessed a flower-like microstructure, consisting of poorly crystallized plate-like crystals, and particles became interconnected on the surface of scaffolds. Similar apatite formation was observed on both compact HA-w1/PLLA1 and HA-p1/PLLA1 biocomposite (Fig. 19

S3). On the other hand, slight dissolution at the surface and edges of the porous HA/PLLA scaffolds were observed. Furthermore, the EDS acquired from the immersed specimens revealed that the formed inorganic phase on the porous HA/PLLA was composed mainly of Ca and P as major constituents with some minor element of O, which was similar to natural bone apatite and was also found on bioactive glass and HA coating after they were immersed in SBF. The Ca/P ratio of deposit sediment formed on the porous HA-w1/PLLA1, whereas, determined from EDS was about 1.59, higher than that on the


porous HA-p1/PLLA1, suggesting HA-w1/PLLA1 had a superior in vitro bioactivity. Therefore, the SEM analysis combined with EDS analysis revealed that the dissolution and precipitation process resulted in calcium phosphate globule formation. The mechanism of apatite formation [41,42,43] on the HA/PLLA biocomposites can be explained in terms of the electrostatic interaction of the Ca2+ on the surface of HA with the ions in the SBF. Based on the previous studies, the HA phase of the composite possesses abundant Ca2+ ions on the surface, and can induce heterogeneous nucleation and growth of apatite. Considering the ionic nature, the electrostatic interaction triggers initial nucleation, and the Ca2+ cation presents on the surface may play a pivotal part in anchoring phosphate and hydroxyl ions. Consequently, negatively-charged ions (HPO42- and OH-) in the SBF are incorporated onto the surfaces leading to the formation of a hydrated precursor cluster consisting of calcium hydrogen phosphate. Since this phase is metastable, it grows spontaneously and transforms into a stable bone-like apatite crystal by consuming Ca2+, HPO42- and OH- ions from SBF. The more formation of a bone-like apatite layer in SBF confirmed that the in vitro bioactivity of the porous HA-w1/PLLA1 composite was better than that its HA-p1/PLLA1 counterpart. Fig. 6 and Fig. 7 Then, the degradability based on weight-loss and mechanical property measurements were further 20

carried out. Gravimetric evaluation of the loss of these compact and porous HA-w1/PLLA1 and HA-p1/PLLA1 biocomposites during incubation was plotted in Fig. 8(a). It showed that for all samples the weight loss steadily increased as the extension of immersion time in SBF. But the degradation rate slowed down at 21 days, possibly because the apatite layer recovered the whole specimen surface. It was noted that the mass loss of porous samples was much faster than their compact counterparts over the incubation period, in accordance with previous works that porous structure in biomaterials could


remarkably facilitate the degradation performance due to its larger surface area.[44] In addition, we found that HA-w1/PLLA1 displayed slower degradation rate and lower total weight loss (= -1.23 % and -2.82 %, respectively) than HA-p1/PLLA1 (=-1.49 % and -4.16 %, respectively) regardless of compact and porous types, suggesting HA whiskers could retard the degradation of HA/PLLA scaffolds. The results of compressive strength for the compact and porous HA-w1/PLLA1 and HA-p1/PLLA1 in SBF at different time points showed that compressive strength of all scaffolds reduced with the soaking time (Fig. 8(b)), proving that degradation would contribute to compromised mechanical properties. Similar to weight loss, porous HA/PLLA scaffolds exhibited a lower compressive strength compared with compact samples before and during SBF immersion due to their anisotropic structure. It was worth that higher compressive strength were always detected in HA-w/PLLA biocomposite than HA-p/PLLA, and the compressive strength of porous HA-w1/PLLA1 (= 78.40 MPa ) was about 2.6-fold than that of porous HA-p1/PLLA1 (= 30.66 MPa) before immersion (0 days), demonstrating that the addition of HA whiskers into PLLA matrix could effectively boost the mechanical property of the binary composite. Recently, HA whiskers has been applied to replace HA particles fillers and to reinforce many polymers to develop HA whiskers/polymer matrix composites, like HA whiskers/polyetherketoneketone (PEEK) [45,46] and HA whiskers/high-density polyethylene (HDPE) 21

biocomposites,[26,47] for hard tissue repairing due to their excellent mechanical property and well-crystallized and controlled morphology. It is essential, in fact, to create appropriate materials to build up a fixation or scaffold with the desirable mechanical properties and suitable biodegradation rate to match those of bones and meet the clinical need. Thus, the porous HA-w1/PLLA1 scaffold with good mechanical property and applicable degradation behavior is expected to be an alternative for orthopedic implantable material.


Fig. 8 3.3. Cytotoxicity of HA-w/PLLA scaffolds The cytotoxicity of the as-prepared HA-w/PLLA porous scaffolds to hMSCs, a type of stem cells abundantly existing in bone marrow, is another critical factor that should be carefully evaluated when the novel HA-w/PLLA porous scaffolds is employed in bone tissue engineering. The results of the indirect cytotoxicity test for various HA-w/PLLA scaffolds in a dilution series of extract culture media for 1-3 days were depicted in Fig. 9(a). Clearly, at the same culture time, greater cell viability of hMSCs was detected when cultured with HA-w1/PLLA1 and HA-w2/PLLA1 extraction than that with HA-w2/PLLA1 in three diluted extract culture media, and the cell proliferation of HA-w1/PLLA1 group throughout the test dominated. Furthermore, the in vitro cytotoxicity of the HA-w1/PLLA1 did not exhibit obvious concentration-dependent characteristics. This was quite different from that of HA-w1/PLLA2 and HA-w2/PLLA1 which displayed a dose-dependent cytotoxicity, and the cell viability decreased with the increase of extraction concentration, suggesting HA-w1/PLLA2 and HA-w2/PLLA1 exerted obviously detrimental effects on cell proliferation/growth. Moreover, the suppression of cell proliferation with time went on for all three HA-w2/PLLA1 extract medium and pure HA-w1/PLLA2 (3/3) extract medium, featuring toxicity to cell. As for HA-w1/PLLA1, the cell viability cultured in all 22

extraction concentrations was higher than 85 % at each time point, and apparent time-dependence behavior could be seen, indicating HA-w1/PLLA1 could remarkably promote the hMSCs proliferation. These phenomena were further confirmed by optical microscopic observation. The morphologies of cells cultured in extraction media from HA-w1/PLLA1 groups displayed healthy spindle-like shape, but in HA-w2/PLLA1 group, some cells had round-like shape. Besides, the amount of hMSCs responding to HA-w1/PLLA1 groups was much higher than those of both HA-w1/PLLA2 and HA-w2/PLLA1 groups at


72 h (Fig. 10), consistent with the results from the indirect method. It is reported that proper amount of HA is beneficial to cell proliferation, and overdosage of HA would inhibit the growth of bone cells and be harmful for body tissues in a concentration-dependent manner.[48,49] These results demonstrated that HA-w1/PLLA1 had clear advantages in in vitro biocompatibility over HA-w1/PLLA2 and HA-w2/PLLA1. Fig. 9 and Fig. 10 Next, the in vitro cell adhesion and direct cell contact of the selected HA-w1/PLLA1 scaffold were further investigated. As shown in Fig. 9(b), the cell cultivated in the extract medium of HA-w1/PLLA1 kept a continuous growing in viability for 6 h, similar to that of TCP (negative control) which was regarded as the most suitable culture for cell adhesion and proliferation, representing excellent cell attachment-promoting property. With regard to direct cell contact, as the HA-w1/PLLA1 scaffold was not transparent under inverted microscope, cell image around the sample was presented. Fig. S4 showed a representative light micrograph of hMSCs at the edge of the HA-w1/PLLA1 composite after 3 days culture. Note that large amount of proliferated cells were observed at the edge of the HA-w1/PLLA1 scaffold, and it was hard to find a significant difference in cell morphology and density between the HA-w1/PLLA1 biocomposite and control group (TCP), indicating that HA-w1/PLLA1 had 23

non-toxicity on cell proliferation. These preliminary cell studies attest that the HA-w1/PLLA1 scaffold does not have negative effect on the cell adhesion, proliferation, and morphology, and it is suitable for cell proliferation. But yet, more detailed investigations on in vivo responses should be carried out, if the HA-w1/PLLA1 scaffold is utilized as artificial bone substitution. 3.4. Blood compatibility of HA-w1/PLLA1 biocomposite Hemocompatibility assessments including hemolysis test and dynamic blood coagulation testing


were also evaluated in the present study, because for potential blood-contacting bone implant, the interplay between blood and biomaterials could affect bone formation and healing. The degree of hemolysis is a sensitive indicator of the extent of damage to erythrocytes. Absorbances in the hemolysis test for the samples exposed to HA-w1/PLLA1 scaffold, the positive control, and the negative control were 0.014 ± 0.0023, 0.747 ± 0.0255, and 0.013 ± 0.0021, respectively. The degree of hemolysis in the presence of HA-w1/PLLA1 was therefore 1.79 ± 0.39 %, way below the safety-threatening threshold of 5 %. The data suggested that HA-w1/PLLA1 scaffold provided an acceptable level of hemolysis. The dynamic blood clotting time is an in vitro test that measures the degree of activation of intrinsic coagulation factors when surfaces interact with blood (“contact activation”). Activation is measured by the absorbance-time curve for the release of hemoglobin from erythrocytes that avoid being entrapped during clot formation. A curve providing a gentle slope usually implied low procoagulant properties in the implanted material. Fig. 11 presented that the similar curves for HA-w1/PLLA1 biocomposite and silylated glass (negative control), with both presenting slow and smooth downward inclination with coagulation time beyond 60 min; however, unmodified glass as positive control displayed sharp blood coagulation cascade with procoagulant time at 20 min. Endosseous implants initially come into contact with blood. The interactions between blood and bone 24

implants have a tremendous influence on subsequent bone healing events in the peri-implant healing compartment.[50] The findings of the study indicated that HA-w1/PLLA1 scaffold with superb hemocompatibility could have better potential to be used for bone implants. Fig. 11 3.5. In vivo biocompatibility evaluation As mentioned earlier, to verify the biocompatibility of the HA-w1/PLLA1 scaffold for clinical use, it


is crucial to know its in vitro responses; therefore, the in vivo assessments including skin irritation test, acute toxicity evaluation, and subcutaneous implant experiment were performed in the present work. First, a skin irritation test was conducted to determine whether HA-w1/PLLA1 scaffold induced inflammatory and allergic reactions. Although there was a little erythema on both dorsal areas of rats at the 1st day after injection of HA-w1/PLLA1 extraction and normal saline solution (Fig. 12), after 3 days, the erythema disappeared and none of the tested parts of white rats treated with the extract medium of HA-w1/PLLA1 displayed edema reactions. It indicated that HA-w1/PLLA1 was not irritant or traumatic to the skin. Fig. 12 No death occurred during the whole 3-day observation period, and no toxic response was found in mice. The animals displayed normal energy, normal behavior, free movement, and shining hair. The animals’ feces were in regular form and normal color, without mucus, pus, or blood. Body weight of the mice was monitored during the 3-day period. According to Fig. 13 (a), weight gain of mice treated with extracts of the HA-w1/PLLA1 biocomposite did not show significant difference compared with the physiological saline control group. After sacrificing the rats, no macroscopic pathological alterations and histopathological lesions attributed to the extracts of the HA-w1/PLLA1 biocomposite were found 25

in any rats at necropsy. Fig. 13(b) presented the light microscopic image of the liver and kidney treated with or without the extraction of HA-w1/PLLA1. The classic structure of liver lobule with central vein was delineated, and no hepatocellular degeneration or necrosis was observed. From the light micrograph of rat kidney, many renal tubes with normal shape were observed, with no degeneration, bleeding, or necrosis. Overall, these results showed that the HA-w1/PLLA1 scaffold had no short-term acute systemic toxicity, indicating it might serve as a safe candidate for its potential applications in


biomedicine fields. Fig. 13 In order to further explore biocompatibility in vivo, subcutaneous implant experiments were designed and carried out. As shown in Fig. 14, the histological analysis through toluidine blue staining of the surrounding tissue of implanted HA-w1/PLLA1 scaffold showed that no significant signs of macrophage activation, inflammation, encapsulation, hemorrhage, necrosis, or discoloration after 14 days’ implantation. Furthermore, only a small number of fibrocytes existed at the implant-tissue interface in a fast healing process. A further comprehensive in vivo bone formation assessment of the porous HA-w/PLLA biocomposite is currently underway in the laboratory. As a consequence, in vivo tests clearly indicated our HA-w1/PLLA1 implant as a bioactive implantable material, possessed the high in vivo biocompatibility with host tissue thereby boding well to orthopedic applications. Fig. 14

4. Conclusion Taken together, it has been demonstrated that the novel HA whisker-reinforced PLLA biocomposite with well-defined interconnected macropore structure was facilely developed. The prepared porous 26

HA-w/PLLA scaffolds were used as matrices for the biomineralization in SBF. In comparison to porous HA-p/PLLA counterparts, the porous HA-w/PLLA scaffolds displayed an increased mechanical property, in vitro bioactivity, and slower degradation behavior. At 3 days incubation, the porous HA-w1/PLLA1 induced no in vitro cytotoxicity toward hMSCs, and the cytotoxicity had no concentration-dependence for the composites’ extraction but had good time-dependent property, indicating its excellent cytocompatibility. Moreover, in vivo preclinical evaluation revealed that the


porous HA-w1/PLLA1 scaffolds exhibited no irritant to skin, no short-term acute systemic toxicity, and good tissue biocompatibility without causing significant haemolysis and clotting. These results have paved the way for the porous HA-w/PLLA biocomposite to be used as orthopedic implantable material in many challenging bone tissue engineering applications.

Acknowledgments This work was supported by Sichuan Province Science and Technology Innovation Team Program (2011JTD0006).

References

[1] Willbold E, Gu X, Albert D, et al. Effect of the addition of low rare earth elements (lanthanum, neodymium, cerium) on the biodegradation and biocompatibility of magnesium. Acta Biomater. 2015;11:554-562.

[2] Holzapfel BM, Reichert JC, Schantz J-T, et al. How smart do biomaterials need to be? A translational science and clinical point of view. Adv. Drug Deliv. Rev. 2013;65:581-603.

27

[3] Higashi S, Yamamuro T, Nakamura T, et al. Polymer-hydroxyapatite composites for biodegradable bone fillers. Biomaterials. 1986;7:183-187.

[4] Witte F. The history of biodegradable magnesium implants: A review. Acta Biomater. 2010;6:1680-1692.

[5] Han P, Cheng P, Zhang S, et al. In vitro and in vivo studies on the degradation of high-purity Mg (99.99wt.%) screw with femoral intracondylar fractured rabbit model. Biomaterials. 2015;64:57-69.


[6] Schofer MD, Roessler PP, Schaefer J, et al. Electrospun PLLA nanofiber scaffolds and their use in combination with BMP-2 for reconstruction of bone defects. Plos One. 2011;6:e25462.

[7] Zhao S. Biological augmentation of rotator cuff repair using bFGF-loaded electrospun poly(lactide-co-glycolide) fibrous membranes. Int. J. Nanomedicine. 2014;9:2373-2385.

[8] Aubert-Pouëssel A, Venier-Julienne M-C, Clavreul A, et al. (2004) In vitro study of GDNF release from biodegradable PLGA microspheres. J. Control. Release. 2004;95:463-475.

[9] Kang YM, Lee SH, Lee JY, et al. (2010) A biodegradable, injectable, gel system based on MPEG-b-(PCL-ran-PLLA) diblock copolymers with an adjustable therapeutic window. Biomaterials. 2010;31:2453-2460.

[10] Gonda Y, Ioku K, Shibata Y, et al. (2009) Stimulatory effect of hydrothermally synthesized biodegradable hydroxyapatite granules on osteogenesis and direct association with osteoclasts. Biomaterials. 2009;30:4390-4400.

[11] Kilian O, Fuhrmann R, Alt V, et al. (2005) Plasma transglutaminase factor XIII induces microvessel ingrowth into biodegradable hydroxyapatite implants in rats. Biomaterials. 2005;26:1819-1827. 28

[12] Yuan H, De Bruijn JD, Li Y, et al. (2001) Bone formation induced by calcium phosphate ceramics in soft tissue of dogs: a comparative study between porous α-TCP and β-TCP. J. Mater. Sci. Mater. Med. 2001;12:7-13.

[13] Zhang Q, Mochalin VN, Neitzel I, et al. Mechanical properties and biomineralization of multifunctional nanodiamond-PLLA composites for bone tissue engineering. Biomaterials. 2012;33:5067-5075.


[14] Hu Y, Zou S, Chen W, et al. Mineralization and drug release of hydroxyapatite/poly(l-lactic acid) nanocomposite scaffolds prepared by Pickering emulsion templating. Colloids Surf. B Biointerfaces. 2014;122:559-565.

[15] Li J, Chen Y, Mak AFT, et al. A one-step method to fabricate PLLA scaffolds with deposition of bioactive hydroxyapatite and collagen using ice-based microporogens. Acta Biomater. 2010;6:2013-2019.

[16] Ronca A, Ambrosio L, Grijpma DW. Preparation of designed poly(d,l-lactide)/nanosized hydroxyapatite composite structures by stereolithography. Acta Biomater. 2013;9:5989-5996.

[17] Suganuma J, Alexander H. Biological response of intramedullary bone to poly-L-lactic acid. J. Appl. Biomater. 1993;4:13-27.

[18] Lou T, Wang X, Song G, et al. Fabrication of PLLA/β-TCP nanocomposite scaffolds with hierarchical porosity for bone tissue engineering. Int. J. Biol. Macromol. 2014;69:464-470.

[19] Shikinami Y, Okuno M. Bioresorbable devices made of forged composites of hydroxyapatite (HA) particles and poly-L-lactide (PLLA): Part I. Basic characteristics. Biomaterials. 1999;20:859-877.

29

[20] Sun Y, Deng Y, Ye Z, et al. Peptide decorated nano-hydroxyapatite with enhanced bioactivity and osteogenic differentiation via polydopamine coating. Colloids Surf. B Biointerfaces. 2013;111:107-116.

[21] Woodard JR, Hilldore AJ, Lan SK, et al. (2007) The mechanical properties and osteoconductivity of hydroxyapatite bone scaffolds with multi-scale porosity. Biomaterials. 2007;28:45-54.

[22] Kim S-S, Sun Park M, Jeon O, et al. Poly(lactide-co-glycolide)/hydroxyapatite composite scaffolds for bone tissue engineering. Biomaterials. 2006;27:1399-1409.


[23] Jung Y, Kim S-S, Kim YH, et al. A poly(lactic acid)/calcium metaphosphate composite for bone tissue engineering. Biomaterials. 2005;26:6314-6322.

[24] Olszta MJ, Cheng X, Jee SS, et al. Bone structure and formation: A new perspective. Mater. Sci. Eng. R. 2007;58:77-116.

[25] Converse GL, Yue W, Roeder RK. Processing and tensile properties of hydroxyapatite-whisker-reinforced polyetheretherketone. Biomaterials. 2007;28:927-935.

[26] Roeder RK, Sproul MM, Turner CH. Hydroxyapatite whiskers provide improved mechanical properties in reinforced polymer composites. J. Biomed. Mater. Res. A. 2003;67A:801-812.

[27] Zhang P, Hong Z, Yu T, et al. (2009) In vivo mineralization and osteogenesis of nanocomposite scaffold of poly(lactide-co-glycolide) and hydroxyapatite surface-grafted with poly(l-lactide). Biomaterials. 2009;30:58-70.

[28] Sowjanya JA, Singh J, Mohita T, et al. Biocomposite scaffolds containing chitosan/alginate/nano-silica for bone tissue engineering. Colloids Surf. B Biointerfaces. 2013;109:294-300.

30

[29] Murphy CM, Haugh MG, O'Brien FJ. The effect of mean pore size on cell attachment, proliferation and migration in collagen–glycosaminoglycan scaffolds for bone tissue engineering. Biomaterials. 2010;31:461-466.

[30] Deng Y, Sun Y, Chen X, et al. Biomimetic synthesis and biocompatibility evaluation of carbonated apatites template-mediated by heparin. Mater. Sci. Eng. C Mater. Biol. Appl. 2013;33:2905-2913.


[31] Liu X, Zhao M, Lu J, et al. Cell responses to two kinds of nanohydroxyapatite with different sizes and crystallinities. Int. J. Nanomedicine. 2012;7:1239-1250.

[32] Liu Y, Cai D, Yang J, et al. In vitro hemocompatibility evaluation of poly (4-hydroxybutyrate) scaffold. Int. J. Clin. Exp. Med. 2014;7:1233-1243.

[33] Li Q, Li M, Zhu P, et al. In vitro synthesis of bioactive hydroxyapatite using sodium hyaluronate as a template. J. Mater. Chem. 2012;22:20257-20265.

[34] Gunawidjaja PN, Izquierdo-Barba I, Mathew R, et al. Quantifying apatite formation and cation leaching from mesoporous bioactive glasses in vitro: a SEM, solid-state NMR and powder XRD study. J. Mater. Chem. 2012;22:7214-7223.

[35] Lai W-C, Liau W-B, Lin T-T. The effect of end groups of PEG on the crystallization behaviors of binary crystalline polymer blends PEG/PLLA. Polymer. 2004;45:3073-3080.

[36] Gauthier O, Bouler J-M, Aguado E, et al. Macroporous biphasic calcium phosphate ceramics: influence of macropore diameter and macroporosity percentage on bone ingrowth. Biomaterials. 1998;19:133-139.

31

[37] Hideo K, Noboru K, Ryoichiro U, et al. Bone ingrowth into the parallel cylindrical tubes with different sizes of porous hydroxyapatite implanted into the rabbits. J. Hard. Tissue. Biol. 2012;21:307-314.

[38] Vrana NE, Dupret A, Coraux C, et al. Hybrid titanium/biodegradable polymer implants with an hierarchical pore structure as a means to control selective cell movement. Plos One. 2011;6:e20480.

[39] Rizzi SC, Heath DJ, Coombes AGA, et al. Biodegradable polymer/hydroxyapatite composites:


Surface analysis and initial attachment of human osteoblasts. J. Biomed. Mater. Res. 2001;55:475-486.

[40] Shuai C, Zhuang J, Hu H, et al. In vitro bioactivity and degradability of β-tricalcium phosphate porous scaffold fabricated via selective laser sintering. Biotechnol. Appl. Biochem. 2013;60:266-273.

[41] He J, Yang X, Mao J, et al. Hydroxyapatite–poly(l-lactide) nanohybrids via surface-initiated ATRP for improving bone-like apatite-formation abilities. Appl. Surf. Sci. 2012;258:6823-6830.

[42] Nga NK, Hoai TT, Viet PH. Biomimetic scaffolds based on hydroxyapatite nanorod/poly(d,l) lactic acid with their corresponding apatite-forming capability and biocompatibility for bone-tissue engineering. Colloids Surf. B Biointerfaces. 2015;128:506-514.

[43] Kokubo T, Takadama H. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials. 2006;27:2907-2915.

[44] Wu L, Ding J. In vitro degradation of three-dimensional porous poly(d,l-lactide-co-glycolide) scaffolds for tissue engineering. Biomaterials. 2004;25:5821-5830.

[45] Converse GL, Conrad TL, Merrill CH, et al. Hydroxyapatite whisker-reinforced polyetherketoneketone bone ingrowth scaffolds. Acta Biomater. 2010;6:856-863.

32

[46] Converse GL, Conrad TL, Roeder RK. Mechanical properties of hydroxyapatite whisker reinforced polyetherketoneketone composite scaffolds. J. Mech. Behav. Biomed. Mater. 2009;2:627-635.

[47] Kane RJ, Converse GL, Roeder RK. Effects of the reinforcement morphology on the fatigue properties of hydroxyapatite reinforced polymers. J. Mech. Behav. Biomed. Mater. 2008;1:261-268.

[48] Wang L, Zhou G, Liu H, et al. Nano-hydroxyapatite particles induce apoptosis on MC3T3-E1


cells and tissue cells in SD rats. Nanoscale. 2012;4:2894-2899.

[49] Fu Q, Zhou N, Huang W, et al. Effects of nano HAP on biological and structural properties of glass bone cement. J. Biomed. Mater. Res. A. 2005;74A:156-163.

[50] Park JY, Gemmell CH, Davies JE. Platelet interactions with titanium: modulation of platelet activity by surface topography. Biomaterials. 2001;22:2671-2682.

33

Captions of figures

Fig. 1. XRD patterns of the synthetic HA powders (a) and HA whiskers (b). The JCPDS patterns of standard HA crystals were shown for reference. Fig. 2. SEM micrographs of HA powders (a-b) and HA whiskers (c-d) with different magnifications. Fig. 3. Size distribution of the HA powders (a) and HA whiskers (b) analyzed using a laser particle size analyzer.


Fig. 4. Compressive strength curve and surface morphology versus hot-pressing temperature for the compact HA-w1/PLLA1 biocomposites. Fig. 5. SEM micrographs of three HA-w/PLLA scaffolds containing HA-w1/PLLA1, HA-w1/PLLA2, and HA-w2/PLLA1with different magnifications. Fig. 6. SEM images of the porous HA-w1/PLLA1 scaffold acquired after soaking in SBF for 5, 14 and 21 days, and EDS spectra of the particle sediments on the surface at 21 days. Fig. 7. SEM images of the porous HA-p1/PLLA1 scaffold acquired after soaking in SBF for 5, 14 and 21 days, and EDS spectra of the particle sediments on the surface at 21 days. Fig. 8. The weight loss and compressive strength of different HA/PLLA scaffolds in SBF at each prescribed time. Fig. 9. Cell proliferation of hMSCs cultured a series of diluted extract culture media from different porous HA-w/PLLA scaffolds (a), and cell attachment of hMSCs responding to pure extract media (3/3) of porous HA-w1/PLLA1 scaffolds (b) evaluated by CCK-8 assay at scheduled time intervals. # represents p< 0.05, ## represents p< 0.01, and ** represents p< 0.05 compared with HA-w1/PLLA1 and HA-w1/PLLA2. Fig. 10. The optical microscope images of hMSCs cultured in extract media (3/3) of porous 34

HA-w1/PLLA1 scaffolds. Fig. 11. Curve of dynamic clotting time of HA-w1/PLLA1 scaffold with silylated glass and unmodified glass as negative and positive controls, respectively. * represents p< 0.05, ** represents p< 0.01 compared with HA-w1/PLLA1 and negative group. Fig. 12. Photographs of skin irritation reaction of rats treated with the extract medium of HA-w1/PLLA1 scaffold at 1 day (a) and 3 days (c) and normal saline solution at 1 day (b). And the


photograph of the injection of solutions into the back subcutaneous area is shown in (d). Fig. 13. The acute systemic toxicity of rats treated with the extract of HA-w1/PLLA1 scaffold and normal saline: weight gain of each group during the observation period (a); Photograph of liver and kidney for each group at 3 days. Green arrows in (b) point to the liver lobules.

Fig. 14. Toluidine blue staining images of surrounding tissue after implantation of the porous HA-w1/PLLA1 scaffold in the rat for 14 days.

35


Fig. 1. XRD patterns of the synthetic HA powders (a) and HA whiskers (b). The JCPDS patterns of standard HA crystals were shown for reference.

36


Fig. 2. SEM micrographs of HA powders (a-b) and HA whiskers (c-d) with different magnifications.

37


Fig. 3. Size distribution of the HA powders (a) and HA whiskers (b) analyzed using a laser particle size analyzer.

38


Fig. 4. Compressive strength curve and surface morphology versus hot-pressing temperature for the compact HA-w1/PLLA1 biocomposites.

39


Fig. 5. SEM micrographs of three HA-w/PLLA scaffolds containing HA-w1/PLLA1, HA-w1/PLLA2, and HA-w2/PLLA1with different magnifications.

40


Fig. 6. SEM images of the porous HA-w1/PLLA1 scaffold acquired after soaking in SBF for 5, 14 and 21 days, and EDS spectra of the particle sediments on the surface at 21 days.

41


Fig. 7. SEM images of the porous HA-p1/PLLA1 scaffold acquired after soaking in SBF for 5, 14 and 21 days, and EDS spectra of the particle sediments on the surface at 21 days.

42


Fig. 8. The weight loss and compressive strength of different HA/PLLA scaffolds in SBF at each prescribed time.

43


Fig. 9. Cell proliferation of hMSCs cultured a series of diluted extract culture media from different porous HA-w/PLLA scaffolds (a), and cell attachment of hMSCs responding to pure extract media (3/3) of porous HA-w1/PLLA1 scaffolds (b) evaluated by CCK-8 assay at scheduled time intervals. # represents p< 0.05, ## represents p< 0.01, and ** represents p< 0.05 compared with HA-w1/PLLA1 and HA-w1/PLLA2.

44


Fig. 10. The optical microscope images of hMSCs cultured in extract media (3/3) of porous HA-w1/PLLA1 scaffolds.

45


Fig. 11. Curve of dynamic clotting time of HA-w1/PLLA1 scaffold with silylated glass and unmodified glass as negative and positive controls, respectively. * represents p< 0.05, ** represents p< 0.01 compared with HA-w1/PLLA1 and negative group.

46


Fig. 12. Photographs of skin irritation reaction of rats treated with the extract medium of HA-w1/PLLA1 scaffold at 1 day (a) and 3 days (c) and normal saline solution at 1 day (b). And the photograph of the injection of solutions into the back subcutaneous area is shown in (d).

47


Fig. 13. The acute systemic toxicity of rats treated with the extract of HA-w1/PLLA1 scaffold and normal saline: weight gain of each group during the observation period (a); Photograph of liver and kidney for each group at 3 days. Green arrows in (b) point to the liver lobules.

48


Fig. 14. Toluidine blue staining images of surrounding tissue after implantation of the porous HA-w1/PLLA1 scaffold in the rat for 14 days.

49

Degradability, biocompatibility, and osteogenesis of biocomposite scaffolds containing nano magnesium phosphate and wheat protein both in vitro and in vivo for bone regeneration.

multi-(amino acid) copolymer: in vitro degradability and osteoblast biocompatibility.

Fabrication of porous hydroxyapatite scaffolds as artificial bone preform and its biocompatibility evaluation.

In vitro and in vivo biocompatibility and osteogenesis of graphene-reinforced nanohydroxyapatite polyamide66 ternary biocomposite as orthopedic implant material.

polyetheretherketone biocomposite as orthopedic implant material.

silk fibroin nanocomposite scaffolds.

poly(L-lactide) composite.

In vitro and in vivo study of additive manufactured porous Ti6Al4V scaffolds for repairing bone defects.

Degradability, bioactivity, and osteogenesis of biocomposite scaffolds of lithium-containing mesoporous bioglass and mPEG-PLGA-b-PLL copolymer.

In vivo biocompatibility assessment of poly (ether imide) electrospun scaffolds.

In vivo high-content evaluation of three-dimensional scaffolds biocompatibility.

Degradability, cytocompatibility, and osteogenesis of porous scaffolds of nanobredigite and PCL-PEG-PCL composite.

Potential use of porous titanium-niobium alloy in orthopedic implants: preparation and experimental study of its biocompatibility in vitro.

Ectopic osteogenesis and angiogenesis regulated by porous architecture of hydroxyapatite scaffolds with similar interconnecting structure in vivo.

Biocompatibility of new nanostructural materials based on active silicate systems and hydroxyapatite: in vitro and in vivo study.

Preparation, in vitro characterization, and in vivo pharmacokinetic evaluation of respirable porous microparticles containing rifampicin.

Biocomposite of hydroxyapatite-titania rods (HApTiR): physical properties and in vitro study.

Preparation of Porous Chitosan-Siloxane Hybrids Coated with Hydroxyapatite Particles.

chitosan scaffolds for bone regeneration in calvarial defects.

pearl powder composite scaffolds.

Biocompatibility of MG-63 cells on collagen, poly-L-lactic acid, hydroxyapatite scaffolds with different parameters.

Biocomposite scaffolds based on electrospun poly(3-hydroxybutyrate) nanofibers and electrosprayed hydroxyapatite nanoparticles for bone tissue engineering applications.

Effects of cell-attachment and extracellular matrix on bone formation in vivo in collagen-hydroxyapatite scaffolds.

poly-amino acid complex biomaterials.