Nano-hydroxyapatite

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Preparation and properties of bamboo fiber/nano-hydroxyapatite/ poly(lactic-co-glycolic) composite scaffold for bone tissue engineering Liuyun Jiang, Ye Li, Chengdong Xiong, Shengpei Su, and Haojie Ding ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15032 • Publication Date (Web): 13 Jan 2017 Downloaded from http://pubs.acs.org on January 19, 2017

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ACS Applied Materials & Interfaces

Preparation and Properties of Bamboo Fiber/Nano-Hydroxyapatite/ Poly(lactic-co-glycolic) Composite Scaffold for Bone Tissue Engineering Liuyun Jiang *1, 2, Ye Li 3, Chengdong Xiong 3, Shengpei Su 1, 2, and Haojie Ding 1, 2

1 Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China 2 National & Local Joint Engineering Laboratory for New Petro-chemical Materials and Fine Utilization of Resources, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China 3 Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041, China

Corresponding Authors *E-mail: [email protected].

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ABSTRACT In

this

study,

bamboo

fiber

nano-hydroxyapatite/poly(lactic-co-glycolic)

was

first

designed

to

to obtain a new composite

incorporate

into

scaffold of bamboo

fiber/nano-hydroxyapatite/poly(lactic-co- glycolic) (BF/n-HA/PLGA) by freeze-drying method. The effect of their components and some factors consisting of different freeze temperatures, concentrations and pore-forming agents on the porous morphology, porosity, compressive properties of the scaffold were investigated by scanning electron microscope (SEM), modified liquid displacement method and electromechanical universal testing machine. The results indicated that the 5% BF/30% n-HA/PLGA composite scaffold, prepared with 5% (w/v) high concentration, frozen at-20℃ without pore-forming agent, had the best ideal porous structure and porosity as well as compressive properties, which is far exceed that of n-HA/PLGA composite scaffold. In addition, the in vitro simulated body fluids (SBF) soaking and cell culture experiment showed the addition of BF into the scaffold accelerated the BF/n-HA/PLGA composite scaffolds degradation and exhibited good cytocompatibility, including attachment and proliferation. All the results of the study show that BF has improved the properties of n-HA/PLGA composite scaffolds, and BF/n-HA/PLGA might have a great potential for bone tissue engineering scaffold.

Keywords: Bamboo fiber, nano-hydroxyapatite, poly(lactic-co-glycolic), degradation, scaffold

2


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INTRODUCTION Bone tissue engineering is a significant strategy for bone repair or replacement through the combination of scaffolds, implanted cells, and biologically active molecules, which has emerged as a new aspect of regenerative medicine.1,

2

Among these factors, scaffold

materials play an essential role in concerning cell-adhesion, spreading, proliferation, differentiation and new bone formation. Considering these aspects, how to select scaffold materials and how to prepare the scaffolds have turned into a key problem in the field of bone tissue engineering.3-5 Poly(lactic-co-glycolic)(PLGA) is a synthetic polymer, and it is widely used in tissue engineering

field

owing

to

its

innoxious

biodegradation

products,

and

good

biocompatibility.6-8 However, its hydrophobicity and the lack of cell recognition signals have restricted its application.9,10 To settle these problems, combining bioactive minerals as fillers with the PLGA polymer matrix is a promising technique.11-13 Thus, the scaffolds containing inorganic materials and polymers may have excellent properties such as mechanical strength, biocompatibility and biodegradation. Based on the biomimetic point of view, it has been suggested that nano-hydroxyapatite (n-HA) may be an ideal filler due to its chemical component similarity and crystal analogy with bone mineral, which has osteoconductive properties and a prominent osseointegration, so that it could potentially improve both biocompatibility and bone integration ability.14-17 Accordingly, n-HA/PLGA composite scaffold is a promising candidate for bone tissue regeneration.18-20 However, the improvement of mechanical strength and degradation of n-HA/PLGA composite scaffold remained as a crucial challenge. To achieve the high mechanical property of n-HA/PLGA 3



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composite scaffold, polymeric composites reinforced with fibres are widening application fields, including construction trade and automotive business.21 In the past years, some researchers have been concentrating on the probing natural fibres as reinforcement in composite, such as kenaf, hemp, jute, and flax, which was combined with biodegradable polymers, so as to obtain a new kind of entirely biodegradable “green” composites.

22-25

Similarly, bamboo fibers (BFs) are longitudinally arranged in the culm, and the mechanical properties produced by BF can rival glass fibres, so BF has the reputation of “natural glass fibres”.

26, 27

Therefore, BF is good for reinforcement of plastic, rubber, and biopolymer

matrices, and it has been widely used in polymer composite industry.28-31 Based on our former study, we concluded that the surface-treated bamboo fiber could enhance the mechanical properties of the n-HA/PLGA composite, and a novel biodegradable BF/n-HA/PLGA ternary composite was achieved.32 However, as far as we know, the BF/n-HA/PLGA composite aimed as bone tissue engineering scaffold has not been reported. Additionally, as we know, pore interconnection and pore size of the scaffold were the pivotal factors for affecting bone formation, which plays a crucial part in harmonizing bone cells activity and their remotion for new bone construction.33 Perfectly, the scaffold should possesse ideal porous structure so as to facilitate cell incursion, tissue growth, fast vascular inbreak and nutrient transmission. To obtain the ideal porous scaffold, extensive efforts have been made to develop the preparation methods of n-HA/PLGA composite scaffold, including solvent molding with or without particle leaching, and thermally induced phase separation (TIPS) associated with freeze-drying method, which seems to be the best way to achieve ceramic/polymer composite scaffolds.34-36 Herein, in this study, a new composite scaffold of 4


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BF/n-HA/PLGA were prepared with freeze-drying method. However, for the novel BF/n-HA/PLGA composite scaffold, whether BF could improve mechanical properties, and the effect of BF on the porous structure, in vitro degradation and biocompatibility of scaffold, which were all expected to be demonstrated. Therefore, the effect of their components and some factors consisting of different freeze temperatures, concentrations and pore-forming agents on the porous morphology, porosity and compressive properties of the scaffold were investigated using scanning electron microscope (SEM), modified liquid displacement method and electromechanical universal testing machine. In addition, the in vitro degradation and the cytocompatibility of the BF/n-HA/PLGA composite scaffold were studied by soaking with simulated body fluid (SBF) and culturing with human osteoblast cells (MG-63).

EXPERIMENTAL SECTION Materials Nano-hydroxyapatite (n-HA ) with the size of 80-100 nm long and 20-40 nm wide was synthesized in our research group. PLGA (95:05) was also prepared in our laboratory, whose intrinsic viscosity was 4.0-4.2 Pa/S. Bamboo fiber (BF) was provided by Zhejiang A&F University, which was treated by NaOH and KH550 32, and the size is 6-10 mm in length and 0.03-0.2 mm in diameter, polymerization degree is approaching 1000, crystallinity is 70 %. All of other agents were analytical reagent.

Preparation of BF/n-HA/PLGA Composite Scaffold Based on the component peculiarities of n-HA, BF and PLGA, and some previous experimental results about BF/n-HA/PLGA system, the appropriate weight ratios of 5:10:85, 5



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5:20:75, 5:30:65, 10:30:60 and 20:30:50 were selected, and the corresponding BF/n-HA/PLGA composite scaffolds were obtained by freeze-drying method. Taking the BF/n-HA/PLGA composite scaffold with weight ratio of 5:10:85 as an example, the detailed preparation was given according to the following procedure. Firstly, PLGA ( 8.5 g) was dissolved in 200 ml of 1,4-dioxane with the addition of BF (0.5 g), and n-HA(1.0 g) was dispersed in 50 mL of 1,4-dioxane by sonication processing at 800 W for 60 min (BILON-500DL, China), which was slowly added into the BF/PLGA mixture solution with or without pore-forming agent of Carboxymethyl cellulose (CMC) or NaCl, and kept it stirring for 4 h with ultrasonic treatment. Afterward, the BF/n-HA/PLGA resulting mixture solution with different concentrations was casted into Petri dishes (9 cm in diameter) and frozen at -20 °C or -196 °C for 12 h, respectively. Eventually, the sample was displaced into a freeze-dryer for more than 30 h to get a porous scaffold. Besides, the n-HA/PLGA composite scaffold was obtained according to the similar preparation procedure without adding BF into PLGA solution.

Properties of BF/n-HA/PLGA Composite Scaffold The porous morphology of BF/n-HA/PLGA composite scaffolds was observed with scanning electron microscopy (SEM) (KYKY-2800 KYKY, China), after being evenly sprayed a gold layer. The porosity was measurated by mean of the modified liquid replacement method.37 The procedure was given as follows. Firstly, the sample with the original weight W1 was immerged into a measuring cylinder with abundant absolute ethanol for 72 h until it reached saturation by absorption at room temperature, and the total volume was noted as V1. Then the 6


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sample was taken out from the cylinder and immediately weighted as W2, and the remaining dehydrated alcohol volume was noted as V2. Finally, the sample porosity was worked out based on the expressions of P= [(W2- W1)/ρ]/(V1-V2), where ρ represents absolute ethanol density. The mean value was given according to the results of three parallel specimens performed for each scaffold. The electromechanical universal testing machine (CMT6000, Sans, China) was used to test the compressive properties of the scaffold, with 1 mm/min compression strain rate until 40 % reduction height of specimen. The scaffold specimen was sliced into cube blocks of 10 mm×10 mm × 10 mm size. The mean value was given based on the five parallel specimens for each scaffold.

In vitro soaking The in vitro degradation of n-HA/PLGA and BF/n-HA/PLGA composite scaffolds was studied by simulated body fluid (SBF) soaking for prearranged time. The ion concentrations of SBF here are very similar to human blood plasma, which was obtained according to the reference.38 Namely, reagent chemicals of NaCl (7.996 g), NaHCO3 (0.350 g), KCl (0.224 g), K2HPO4.3H2O (0.228 g), MgCl2.6H2O (0.305 g), CaCl2 (0.278 g) and Na2SO4 (0.071 g) were dissolved into less than 1000 ml deionized water. The solution was adjusted to physiological pH 7.40 at 37 °C by bufferring with tri-(hydroxymethyl) aminomethane (6.057 g) and hydrochloric acid, and the solution was replenished with deionized water up to 1000 mL. After soaking for 2, 4, 8 and 12 weeks, the samples were removed from SBF at the prescribed time, and mildly swashed with distilled water three times, absorbed the surface water with filter paper, marked wet weight as W1 and dry weight as W2 after being entirely 7



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dried at 40°C in a vacuum drying oven. The water absorption amount was given from the wet weight W1 and final dried weight W2 as followed: Water absorption (%) =

ௐభ ିௐమ

× 100%

ௐమ

Likewise, three parallel specimens for the scaffold were tested and the average value was listed after soaking different periods. The compressive strength reduction of the samples at interval time were tested according to the previous procedure. A pH meter was used for measuring the solution pH value. The surfaces microstructure of n-HA/PLGA and BF/n-HA/PLGA composite scaffolds were examined with SEM.

In vitro cell experiment Human osteoblast cells (MG-63) was purchased from West China School of Stomatology Sichuan University, aimed to primarily evaluate in vitro cells viability. The samples of n-HA/PLGA and BF/n-HA/PLGA composite scaffolds with size of 10 mm × 10 mm × 2 mm were put in a 48-well cell culture plate after being sterilized with ethylene oxide gas. Afterward, samples with seeded about 2.5×104 cells of MG63 were incubated undisturbedly for 3 h. Next, another 1.0 mL of culture medium was introduced into each well, which was cultured at 37 °C in an incubator containing a humidified 5% CO2 atmosphere. Empty wells with no sample were handled with the same process as controls. At 2 and 6 day of incubation, samples were washed with phosphate buffer saline (PBS) two times after being taken out, and two drops 0.1 mg/ml of acridine orange solution was dropped onto sample and dyed for 3~5 min, following washed with PBS twice again, and the morphology and growth of cells was observed by Fluorescence Microscope (Olympus B × 8


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60) . MTT (3-{4,5-dimethylthiazol-2yl}-2,5-diphenyl-2H-tetrazoliumbromide) assay was used to estimate the cell proliferation.39 The medium with cell-loaded sample in the culture plate was thrown off after being cultivated for 1, 3 and 6 days, and 20 µL MTT solution with the concentration of 5 mg/mL was dropped into every well. Then, kept it for 4 h in the incubator with 5% CO2 air atmosphere at 37°C, and the formazan crystals was dissolved with 0.2 ml of DMSO. Finally, the optical densities (OD) were measured with an Elisa microplate reader (ELx 800, BIOTEK) at 545 nm, after 200 µL of solution was added to a 48-well plate with enzyme-linked immunosorbent assay. More than 5 parallel wells for every sample were randomly numerated, thus the mean value as well as standard deviation for every sample were recorded.

Statistical analysis All data were listed as mean ± standard error of at least three or five samples. One-way analysis of variance was used to implement statistical analysis. The value of P

Nano-hydroxyapatite

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