hydroxyapatite (HAP) composites cast using ionic liquid solutions

Journal Pre-proofs Mechanical and biological properties of chitin/polylactide (PLA)/hydroxyapatite (HAP) composites cast using ionic liquid solutions Jayashree Chakravarty, Md Fazlay Rabbi, Vijaya Chalivendra, Tracie Ferreira, Christopher J. Brigham PII: DOI: Reference:

S0141-8130(19)37859-6 https://doi.org/10.1016/j.ijbiomac.2019.10.168 BIOMAC 13669

To appear in:

International Journal of Biological Macromolecules

Received Date: Revised Date: Accepted Date:

27 September 2019 10 October 2019 22 October 2019

Please cite this article as: J. Chakravarty, M.F. Rabbi, V. Chalivendra, T. Ferreira, C.J. Brigham, Mechanical and biological properties of chitin/polylactide (PLA)/hydroxyapatite (HAP) composites cast using ionic liquid solutions, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac. 2019.10.168

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Mechanical and biological properties of chitin/polylactide (PLA)/hydroxyapatite (HAP) composites cast using ionic liquid solutions Jayashree Chakravarty1, Md Fazlay Rabbi2, Vijaya Chalivendra2, Tracie Ferreira1, Christopher J. Brigham1†* 1

2

Department of Bioengineering, University of Massachusetts Dartmouth, 285 Old Westport Road, N. Dartmouth, MA 027474 USA

Department of Mechanical Engineering, University of Massachusetts Dartmouth, 285 Old Westport Road, N. Dartmouth, MA 027474 USA †Current address: Department of Interdisciplinary Engineering, Wentworth Institute of Technology, 550 Huntington Avenue, Boston, MA 02115 USA * To whom correspondence should be addressed (Tel: 16179894892, Fax: 16179894168, email: [email protected])

ABSTRACT This research investigates the potential development of lobster shell waste-derived chitin reinforced with poly(lactic acid) (PLA) and nano-hydroxyapatite (nHAP) into new materials with potentially superior mechanical and thermal properties for biomedical applications.

The ionic liquid 1-ethyl-3-methylimidazolium acetate

([C2mim][OAc]) was used as a solvent to prepare chitin/PLA/nHAP composites. The effect of variation of the polymer concentrations on the conduct of the resulting composite was explored. The detailed physicomechanical, thermal and surface morphology properties were evaluated with different thermal and optical characterization techniques. When the concentration of PLA in the composite was increased from 20 to 80 wt%, the tensile strength improved by ~77% while the elongation at break and the toughness of the material decreased significantly. The addition of hydroxyapatite was observed to improve strength of the composites up to 140% with an increase in elongation at break up to 465%. Cell growth study show that the composite materials support the growth and proliferation of Ocy 454 osteocyte cells. The materials were shown to have no effect on osteocyte gene expression, as well as minimal cytotoxicity and biodegradability. These results reveal that the biocomposites would be suitable candidates for use in bone regeneration that are not exposed to excessive forces. Keywords: Chitin, PLA, Hydroxyapatite, Composite, Ionic liquid, Biodegradable

1. INTRODUCTION The primary focus of tissue engineering studies is the development of materials that coalesce the mechanical properties, chemical stability, and biological features that are crucial for promoting tissue growth.

These materials need to be biocompatible, biodegradable, be able to promote cell proliferation, and the material degradation products must produce minimal immune reaction [1]. In addition, the material must be able to withstand the mechanical demands of the site of injury throughout the healing process [2]. Polymer blending is an appealing method to produce novel materials with custom-made properties. This method is cost-effective and is a versatile way to develop a novel material that can have a combination of the unique properties of each of the component materials. The last decade has witnessed a massive interest in polymers produced from renewable resources that can readily and harmlessly biodegrade in the environment [3]. Among biopolymers, chitin (β-(1→4) N-acetyl-Dglucosamine) is the second most important natural polymer with such well-known properties as non-toxicity, biocompatibility, and biodegradability, and also possesses wound healing properties [4, 5, 6,7]. Chitin has been used in a multitude of applications in many avenues like food and agriculture, textile, cosmetic, wastewater treatment [8, 9, 10, 11]. The main development of chitin as a biomaterial is, however, in medical and pharmaceutical applications as wound-dressing and as a controlled drug release material [12, 13, 14]. Other commonly used biodegradable polymers used for tissue regeneration applications include polyglycolic acid (PGA), polylactic acid (PLA), PGA-PLA copolymers, polyhydroxyalkanoates (PHA) and polycaprolactone (PCL) [15, 16, 17, 18, 19]. PLA, a linear aliphatic thermoplastic polyester, can be derived from renewable resources, with broad availability and ease of processing [19, 20, 21, 22]. Because of the biodegradability, biocompatibility, and osteoconductive properties of PLA, it has been successfully used as surgical implant materials, drug delivery systems and also as films for tissue growth [23, 24, 25]. It has been shown that PLA has a high modulus of elasticity (3.5–3.8 GPa) and tensile strength (48–110 MPa); however, the inherent brittleness and low toughness of the polymer limits its applications [20, 21, 22]. Hydroxyapatite (HAP, Ca10(PO4)6(OH)2) is naturally found as the main inorganic constituent of bone, which is why it has been extensively studied as an artificial bone replacement [26, 27]. Commercially available hydroxyapatite is both chemically and structurally similar to the mineral phase of human bone [28] and is known to increase the mechanical properties of several polymeric materials [29, 30, 31]. Bionanocomposites based on HAP nanoparticles are acknowledged for their biomedical applications and their high potential for new specific uses [32]. Hydroxyapatite has been added to biocomposites stimulate the growth and differentiation of osteoblasts [33, 34, 35]. Nevertheless, pure hydroxyapatite is fragile, lacks flexibility and hardness, is difficult to form into a particular shape, and its strength is too low for tissue replacement applications [36, 37, 38]. Due to the properties described above, blending polymers is an effective way to formulate novel materials with optimized characteristics. PLA and hydroxyapatite are better used in the form of composites or mixtures, and hence blending the natural and easy-to-functionalize polymer chitin with PLA and nano-hydroxyapatite (nHAP) is

a logical combination. Nonetheless, it is a challenge to mix all of these polymers. PLA is traditionally meltprocessed; and chitin, like other biopolymers, cannot be melted to make composites. The preparation of composites with these polymers are thus restricted to traditional methods like casting, nonaqueous solvent dispersion, polymer grafting, freeze-drying and hot-pressing [39]. While using these techniques, care should be taken for uniform distribution of the polymers; non-uniform distribution could compromise the properties of the composite. Many solvent systems have been in use to produce these composite materials such as DMSO [40], DMAc/LiCl [41], chloroform [42, 43], chloroform mixed with acetic acid [44]. These solvents are harsh, causing deacetylation of the material, and also corrosive. Ionic liquids (ILs) can be used as solvents for biopolymers [45] to enhance their solubility and processability and can be seen as an alternative to using harsh chemicals and solvents. Ionic liquids (ILs) are a group of salts that exist in liquid state at low temperatures [46]. Ionic liquids have tunable solubility properties with negligible vapor pressure and excellent thermal stabilities and have been used to dissolve many biopolymers which are otherwise difficult to dissolve such as cellulose, wool keratin and silk fibroin [45, 46]. IL-polymer solutions have been used to prepare fibers [39, 48, 49], hydrogels [49], membranes or films [50, 51, 52, 53]. The ability of ILs to dissolve (and co-dissolve) a number of biopolymers [54, 55, 56, 57] makes it possible to consider using solution techniques for preparation of a variety of useful composite materials. The use of biodegradable polymer-based materials in tissue engineering and other implantation applications eliminates complications like corrosion, release of metal ions associated with metal implants [58, 59]. In addition, the use of biodegradable materials is extremely appealing in biological and medical applications because microorganisms, enzymatic reactions or passive hydrolytic cleavage can break down these materials in physiological settings [59, 60]. Despite the massive interest in blended polymeric materials, we have not found studies where chitin, PLA and hydroxyapatite were simultaneously dissolved and processed using an IL as a solvent. In the present study, we report our efforts to fabricate chitin/PLA/nHAP composites using the IL 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) as a solvent. Chitin used in this work was extracted from lobster shell waste using a previously described biological treatment [61]. We hypothesize that, by simultaneous dissolution of the polymers in the same solvent, we could achieve uniform blending and even tune the properties of the blended material by changing the polymer ratios. The biocomposite material is characterized and described below which can be used for potential biomedical applications.

2. MATERIALS AND METHODS 2.1 Chemicals and materials

Dried and ground lobster shell waste powder was a generous gift from Gloucester Seafood Processing Inc. (Gloucester, MA, USA) and was stored at room temperature until used. Chitin was extracted from lobster shells as described previously [61]. Poly (L-lactic acid) (PLA) was purchased from 3DX Tech Advanced Materials (Michigan, USA). Nano-Hydroxyapatite (nHAP) and the IL 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc], purity >95%) were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Ocy 454 osteocyte cell line was a generous gift from Prof. Lamya Karim, University of Massachusetts Dartmouth. 2.2 Preparation of Chitin/PLA/nHAP composites Solutions of chitin, PLA and nHAP were prepared by thermal co-dissolution of the polymers in IL ([C2mim][OAc]), heating at 90 °C under and stirred constantly in a loosely-capped vial. The concentrations of the polymers were varied to generate different composite materials. The suspension was then sonicated (Ultrasonic Processors, Cole-Parmer, USA) for 15-20 mins to ensure uniform distribution of the polymers. Following homogenization, the solutions were transferred to a flat glass surface, followed by gelation of the system through immersion in DI water. Afterward, the DI water was replaced several times until IL removal was achieved. The samples were incubated at -20 °C for 2 h and then subjected to freeze-drying for 12 h. A Labconco Freeze Dryer was used at high vacuum (0.010 mbar) and a temperature of -89 °C for the drying process. Following drying, the biocomposites were placed in desiccators to remove residual traces of water. 2.3 Composite material characterization The chitin/PLA/nHAP composite materials were studied under SEM for their morphological characteristics. The dried chitin/PLA/nHAP composites (1 cm × 1 cm) were mounted on carbon tape. High resolution images were produced using a highly focused SU5000 FE-SEM (Hitachi Ltd., Tokyo, Japan) scanning electron microscope operated at 2 kV electron beam Thermogravimetric Analysis (TGA) were conducted with a TGA Q500 (TA Instruments, USA) to check the thermal stability of the composites. The chitin/PLA/nHAP composite material samples (5-10 mg) were heated from 20 °C to 800 °C by ramping the temperature input at 10 °C min-1. All TGA measurements were performed in triplicate. Decomposition temperatures are reported as onset (Tonset) and temperature at maximum degradation rate (Tmax) respectively. The phase morphology of the composite material was determined by using differential scanning calorimetry (DSC). A DSC Q2000 (TA Instruments, USA) equipped with a refrigerated cooling system was used. Precisely weighed samples (5 mg) of the composite materials were placed into aluminum cups and sealed. As a reference, an empty cup was used. The samples were heated at a rate of 10 °C min-1 from 30 to 550 °C under the continuous flow of dry nitrogen gas (10 mL min-1). The melting temperatures (Tm, the transition peak maxima) and

glass transitions (Tg) of the materials were recorded. Runs were performed in triplicate and the average mean values are given. An FTS 3000MX Excalibur Series FTIR (BioRad, USA) featuring an attenuated total reflection (ATR) sampler equipped with a diamond crystal was used to record the FT-IR spectra on the composite material. Spectra were obtained in the range of 400–4000 cm−1. An in-house built micro tensile tester was utilized to characterize mechanical properties of the composite materials under a quasi-static tensile condition [53, 62]. An M-230 High-Resolution Linear Actuator (PI, Germany) associated with a 11-N load cell was used to apply displacement to the specimen. Specimens of size 12 mm in length) and 6 mm wide were loaded at a displacement rate of 0.1 mm s-1. LabView (National Instruments, Austin, TX, USA) software was used to capture the load and displacement data. Materials with uniform thickness and no obvious flaws were selected and used for testing. For each type of composite, a minimum of five experiments were conducted at the same loading condition. 2.4 In vitro degradation of the Chitin/PLA/nHAP composites Sterilized chitin/PLA/nHAP composites of known dry weight (W0) were immersed in an aqueous solution of 1.5 µg mL-1 lysozyme suspended in 0.1 M phosphate buffered saline (PBS) at pH 7.4 ± 0.1 and maintained at 37 °C with mild mechanical agitation (100 rpm). The concentration of lysozyme was chosen, in this case, to correspond to the concentration in human serum [63, 64]. To guarantee continued enzyme activity, the lysozyme solution was refreshed weekly. Intermittently over 35 days, samples were removed and dried at 80 °C for 24 h and the weight was recorded as (Wt). Control samples were performed using the same operation mentioned above, except without the addition of lysozyme. The extent of in vitro degradation was expressed as the percentage of the weight of the dried materials after lysozyme treatment using the formula (1): 𝑊𝑒𝑖𝑔ℎ𝑡 𝑙𝑜𝑠𝑠 (%) =

(𝑊0 −𝑊𝑡 ) × 𝑊0

100

(1)

where W0 denotes the initial weight of the membrane (prior to degradation), whereas Wt is the weight of the membrane at time t (up to 35 days). Each biodegradation experiment was performed in triplicate, and the mean value was taken as the percentage of biodegradation. Values were expressed as mean ± standard error. 2.5 Cell viability studies MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue) assay was conducted to assess the cell viability of osteocytes Ocy 454 grown on the chitin/PLA/nHAP composites [65]. The osteocytes were initially cultured in Alpha MEM media (supplemented with 10% fetal bovine serum, 1% Gibco Anti-Anti; hereon referred to as complete Alpha MEM media) at 37 °C in a 5% CO2 atmosphere. Then, 2.5 × 104 cells/well

were seeded onto chitin membranes in a 96-well plate and incubated for 24 h. Following this incubation, 10 µL of 0.5% MTT solution (Sigma-Aldrich, St. Louis, MO, USA) was added to the media and incubated at 37 °C for 4 h. In order to dissolve the precipitates, 0.2 mL of DMSO was added (after discarding the culture medium) and the resulting solution was measured for absorbance at 570 nm. The cell viability percentage was expressed by equation (2). 𝑚𝑒𝑎𝑛 𝑜𝑝𝑡𝑖𝑐𝑎𝑙 𝑑𝑒𝑛𝑠𝑖𝑡𝑦

𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 % = (𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑜𝑝𝑡𝑖𝑐𝑎𝑙 𝑑𝑒𝑛𝑠𝑖𝑡𝑦) × 100

(2)

The osteocytes’ viability on the chitin/PLA/nHAP composites was confirmed by confocal microscopic analysis. Cells cultured for 14 days on chitin/PLA/nHAP composites were washed with PBS buffer (without Mg2+ and CaCl2) and incubated at 37°C for 10 min in 1 mL of 50 μg acridine orange/mL solution (FDA, Wako Pure Chemicals, Japan) dissolved in PBS buffer (without Mg2+ and CaCl) to stain the viable cells. After 10 min, a confocal laser scanning microscope (Carl Zeiss Laser Scanning Microscopy, LSM710, Germany) was used to view the samples. 2.6 Attachment and proliferation of osteocytes on Chitin/PLA/nHAP composites To examine the cytocompatibility of the fabricated membranes, osteocytes Ocy454 were grown on the composite materials. Chitin/PLA/nHAP composite samples (1 cm × 1 cm) were sterilized by a 15-min immersion in 70% alcohol, then rinsed carefully with sterile PBS (without Mg2+ and CaCl2) and presoaked in culture medium for 2 h. The composite materials were then placed in the wells of a 6-well tissue culture plate. Subsequently, 150 µL of cell suspension (in Alpha MEM media) at a density of 3 × 104 cells/mL was added onto the composite materials. Attachment of the cells to the material was facilitated by incubation at 33 °C for 6 h in a humidified atmosphere with 5% CO2 Following this, the cell-seeded composite materials were washed with 2 mL of fresh, complete Alpha MEM media. The washing media was collected and then centrifuged for precipitation of the cells. A volume of 150 μL of complete Alpha MEM media was used to resuspend the cell pellet. A hemocytometer (C-chip DHC-NO1, NanoEnTek; Nikon Eclipse TS 100) was used to count the number of unattached cells in the suspension. By subtracting the number of unattached cells from the initial number of cells seeded on the membranes, the number of cells attached on the composite material was estimated. The cell-seeded membranes were incubated for 3 days in 5 mL complete Alpha MEM medium at 33 °C in a humidified, 5% CO2 incubator (post-plating) and then moved to another humidified, 5% CO2 incubator at 37 °C. Depending on the period of incubation, fresh media was provided to the cells every 2-3 days. The composite materials were removed from the media after every 7 days, washed with PBS to remove the unattached cells. The materials were then immersed in a 2 mL volume of 1X TrypeE (Life Technologies) and then incubated for 10 min at 37 °C to detach the cells from the membranes. The detached cells were then resuspended in complete Alpha

MEM media (2 mL), centrifuged for 10 min at 180 × g. The cell pellet was resuspended using 150 μL of the same media. A hemocytometer (C-chip DHC-NO1, NanoEnTek; Nikon Eclipse TS 100) was used to count the number of unattached cells in the suspension. 2.7 Osteogenic gene expression by qRT-PCR The Ocy 454 cell cultures grown in the presence of various chitin/PLA/nHAP composites for 12 days were collected for the evaluation of osteogenesis-related gene expression. Total RNA was obtained using a RNeasy Mini kit (Qiagen, CA, USA) in accordance with the manufacturer’s protocol. A Nanodrop assay (Nanodrop One, Thermo Scientific, USA) detected the total concentration and purity of the RNA, and the first strand cDNA was synthesized by reverse transcriptase as described in the manufacturer’s protocol (PrimeScript cDNA synthesis kit, Takara). The osteogenic marker expression was quantified by qPCR SYBRGreen kit (Qiagen). The primer sequences used specifically for the target gene SOST were 5’- CTTCAGGAATGATGCCACAGAGGT-3’ (forward) and 5’ATCTTTGGCGTCATAGGGATGGTG-3’ (reverse). The qPCR amplification was performed as follows: initial denaturation at 95 °C for 15 mins, followed by 40 cycles at 95 °C for 15 s, 54 °C for 30 s, 72 °C for 30 s. Cells grown without the composite material were used as controls. All results were quantified using the ΔΔCt relative quantification method [64]. 2.8 Statistical analysis Each preparation and measurement discussed in this work was conducted in triplicate. The experimental data were subjected to an analysis of variance (ANOVA) for a completely random design (CRD) using a Statistical Analysis System (SAS Institute, Inc., 2000). Differences were deemed significant at p≤0.05.

3. RESULTS 3.1 Preparation of Chitin/PLA/nHAP membrane The preparation of chitin/PLA/nHAP biocomposites was achieved by thermal dissolution of the polymers in [C2mim][OAc]. The concentrations of the polymers were varied to generate different composite materials, as listed in Table 1. For samples CPH1, CPH2, CPH3, the ratio of chitin and PLA were varied while nHAP content was kept constant at 0.5% of the total weight of solutes in the IL. It was observed that the solutions seemed to be more viscous with an increase in PLA concentration. For samples CPH4, CPH5, CPH6 and CPH7, the ratio chitin and PLA were kept constant at 50:50, while nHAP concentration was varied at 2.5, 5, 7 and 10% of the total weight of solutes in the IL in order to investigate the effect of nHAP concentration on the composite material. A solution

with nHAP content of 10% of total weight of solutes in the IL produced materials that did not have a uniform surface; therefore, they were not subjected to further characterization studies. Table 1. Different chitin/PLA/nHAP biocomposites prepared using IL Sample name

Chitin (%)

PLA (%)

CPH1 CPH2 CPH3 CPH4 CPH5 CPH6

20 80 50 50 50 50

80 20 50 50 50 50

nHAP (% of total solutes in IL solution) 0.5 0.5 0.5 2.5 5 7

3.2 Morphological characteristics of Chitin/PLA/nHAP composites Following solvent casting, SEM was used to compare the surface morphology of the chitin/PLA/nHAP composites. As can be seen in the selected micrographs in Fig. 1, the composites have a heterogenous, porous formation. This typical morphology is characterized by a porous, sponge-like three-dimensional network structure with pores distributed randomly.

Fig 1. Representative scanning electron micrographs of chitin-PLA-nHAP composites with different ratios of the polymers co-dissolved in IL a) 20/80/0.5 (CPH1), b) 80/20/0.5 (CPH2), c) 50/50/0.5 (CPH3), d) 50/50/2.5 (CPH4), e) 50/50/5 (CPH5), f) 50/50/7 (CPH6). All the composites exhibit a randomly distributed porous structure (pores indicated by the white arrows), produced due to the freeze-drying process. The scale bars are of 500 μm.

3.3 Thermal analysis of Chitin/PLA/nHAP composites Thermal stability of pure nHAP, PLA, the chitin used in this study, and their corresponding blend products was evaluated by TGA. All data for regarding their Tonset (the onset temperature of thermal degradation), Tmax (temperature at maximum degradation rate) determined from the TGA curves (Fig. 2) are summarized in Table 2. The nHAP appears to have shown little change in weight throughout the process. A slight weight loss in the thermograms was found to be in the range of 80–100°C. The second weight loss represents the maximum degradation stage. All chitin/PLA/nHAP blended material had a decomposition temperature lower than that of neat chitin, but higher than that of neat PLA and nHAP, confirming blending of the polymers (Table 2). The value of Tonset depended on the amount of PLA and increased with increasing PLA content. Fig. 2, shows that the mass of nHAP changed slightly during the heating process from 40 to 800 °C.

Fig. 2. Thermogravimetric (TG) curves for chitin (black), PLA (purple), nHAP (dark red) and chitin/PLA/nHAP composites: 20/80/0.5 (CPH1, orange), 80/20/0.5 (CPH2, dark blue), 50/50/0.5 (CPH3, grey), 50/50/2.5 (CPH4, green), 50/50/5 (CPH5, yellow), and 50/50/7 (CPH6, blue).

DSC was used to determine the phase morphology of the blends. The thermal transitions in the chitin, PLA, nHAP and the blended materials were evaluated in the temperature range from 0 to 550 °C, at a heating/cooling rate of 10 °C/min, and the melting temperatures (Tm) and glass transitions (Tg) were recorded. For PLA, two endothermic peaks were observed: Tg of 58 °C and Tm of 153 °C (Table 2). For samples CPH3, CPH4, CPH5, CPH6, the Tm seemed to decrease with an increase in nHAP ratio. It is also worth noting in comparison to the melting of neat PLA, the Tm shifted to higher values for all blends.

Table 2. Thermal analyses of chitin, PLA, nHAP and composite materials. The Tonset (onset temperature of thermal degradation), Tmax (temperature at maximum degradation rate) were determined from the TGA curves. The T m (melting temperatures) and Tg (glass transitions) were recorded using DSC. Sample Chitin PLA CPH1 CPH2 CHP3 CPH4 CPH5 CPH6

TGA Tonset (°C) 267.8 352.5 336.7 310.8 328.9 325.1 321.4 313.9

DSC Tmax (°C) 378.9 363.8 355.6 369.9 361.4 359.9 354.6 350.2

Tg (°C) 226.3 58 110.5 197.4 136.5 143.7 159.2 163.9

Tm (°C) 153.2 205.3 262.8 229.4 194.1 187.7 181.4

Note: The samples are chitin/PLA/nHAP in different ratios: 20/80/0.5 (CPH1), 80/20/0.5 (CPH2), 50/50/0.5 (CPH3), 50/50/2.5 (CPH4), 50/50/5 (CPH5), and 50/50/7 (CPH6).

3.4 Fourier Transform Infrared Spectroscopy (FTIR) The structure and composition of the composites were studied using Fourier-transform infrared (FT-IR) spectroscopy (Fig. 3). Because of O-H bending and stretching vibration, the FT-IR spectra of all the samples show peaks at 3600 - 3200 cm−1. PLA showed two characteristic bands at 1759 and 869 cm−1 respectively, which corresponds to C=O and C- COO stretching [67, 68]. Chitin showed characteristics groups like (-NH-C(O)-CH3) which exhibits a carbonyl C=O stretch (amide-I) divided into two parts (due to hydrogen at 1647 and 1620 cm−1, and an amide-II band at 1539 cm−1 (Fig. 3). PLA’s distinctive peaks at 1759 cm−1 shifted to reduced frequencies, while the split chitin amide-I bands at 1647 and 1620 cm−1, which had distinct intensities, became of nearly equivalent in the blended fibers. With increasing PLA content, the intensity of the PLA-related peaks increases, and they were observed to be significantly smaller as compared to those of chitin. The presence of bands at 1989, 1020-1034, 957, 620 and 590 cm−1 are assigned to orthophosphate in hydroxyapatite. The bands located at 1020-1034 and 590 cm−1 are characteristic of phosphate bending vibration, while the band at 957 cm−1 is ascribed to phosphate stretching vibration [69] which clearly indicate the incorporation of hydroxyapatite into the composite material. As it appears that there is a shift of peaks of any group in the composites, which suggests that a significant chemical reaction has taken place between the individual components. No [C2mim][OAc] or related peaks were observed in the FT-IR spectra of the fibers, thus confirming effective removal of IL during washing.

Fig. 3. FTIR spectra of chitin (black), PLA (purple), chitin (black) and chitin/PLA/nHAP composites: 20/80/0.5 (CPH1, orange), 80/20/0.5 (CPH2, dark blue), 50/50/0.5 (CPH3, grey), 50/50/2.5 (CPH4, green), 50/50/5 (CPH5, yellow), and 50/50/7 (CPH6, blue).

3.5 Mechanical properties of Chitin/PLA/nHAP composites The mechanical properties of chitin/PLA/nHAP composites were investigated to determine the effect of PLA and HAP in the matrix of the composite material. Fig. 4 shows the tensile strength, percentage of elongation at break and toughness (area under the stress-strain curve) of the chitin/PLA/nHAP composites. Tensile strength of CPH1 was 6.97 MPa while the strength of CPH2, which has higher in chitin content, increased up to 12.37 MPa. The strength of the materials also seems to increase with increasing nHAP content, CPH6 has a strength of 20.68 MPa. However, with the rise in PLA concentration, the elongation at break and toughness of the materials decreased significantly. CPH5 showed the highest percentage of elongation at break and toughness, although it had lower tensile strength than that of CPH6.

Fig 4. Mechanical properties of chitin/PLA/nHAP composite material. a) strength b) elongation at break and c) toughness of composite materials. The samples are chitin/PLA/nHAP in different ratios: 20/80/0.5 (CPH1, orange), 80/20/0.5 (CPH2, dark blue), 50/50/0.5 (CPH3, grey), 50/50/2.5 (CPH4, green), 50/50/5 (CPH5, yellow), and 50/50/7 (CPH6, blue). Data represent mean, and error bars represent standard deviations of triplicate tests, p <0.05.

3.6 In vitro degradation of the Chitin/PLA/nHAP composite composites Degradation of composite samples in PBS containing lysozyme was examined over 35 days. To demonstrate mass loss after incubation with lysozyme, the samples were removed and dried, and the mass loss during the degradation process was determined using equation 1, as described earlier in this work. The weight loss for the different composites is shown in Fig. 5.

Fig. 5. In vitro degradation of chitin/PLA/nHAP composite materials with 1.5 μg/mL lysozyme in PBS at pH 7.4, 37 °C with 100 rpm agitation. The degradation was determined based on percentage of mass loss of the material. The different samples are 20/80/0.5 (CPH1, orange), 80/20/0.5 (CPH2, dark blue), 50/50/0.5 (CPH3, grey),

50/50/2.5 (CPH4, green), 50/50/5 (CPH5, yellow), and 50/50/7 (CPH6, blue). Data shown here represent mean, error bars represent standard deviations of triplicate tests, p <0.05.

It can be observed that the proportion of polymers influenced the degradation rate of the composites. After 5 weeks (35 days) of incubation, CPH1 loses only about 27.5%, CPH3 loses 36%, whereas CPH2 loses almost 46% of its initial mass. These data show that increasing the amount of chitin lead to faster degradation rates. The rate weight loss of all the composites decreased with increasing the concentration of nHAP. Subsequently, the degradation rates slow down after 3 weeks of incubation for samples CPH4, CPH5, CPH6.

3.7 Cytotoxicity studies The viability of Ocy 454 osteocyte cells was evaluated in the presence of the biocomposites using the MTT assay to explore the potential of any cytotoxicity of the chitin/PLA/nHAP biocomposites. The MTT assay is based on the ability of a mitochondrial dehydrogenation enzyme (present in viable cells) to cleave the MTT tetrazolium rings to produce formazan crystals. The visible effect is that the assay mixture undergoes a color change from pale yellow to dark blue [65]. The number of still-viable cells is directly proportional to the amount of formazan produced by this reaction. According to Table 3, all of the tested membranes are nontoxic when compared to the control (cells grown without the material). Interestingly, it was noted that the viability of the cells was not significantly affected by the various material compositions tested. These results indicate that there is no cytotoxicity against the cells growing on the biocomposites, so they have excellent biocompatibility. Table 3. Cytotoxicity of chitin/PLA/nHAP composites against Ocy454 cells Sample name

Cell viability %

Control

98.52 ± 1.45

CPH1

94.37 ± 2.38

CPH2

96.53 ± 1.46

CPH3

95.46 ± 1.97

CPH4

95.86 ± 2.51

CPH5

96.03 ± 1.87

CPH6

96.26 ± 2.34

Note: The samples are chitin/PLA/nHAP in different ratios: 20/80/0.5 (CPH1), 80/20/0.5 (CPH2), 50/50/0.5 (CPH3), 50/50/2.5 (CPH4), 50/50/5 (CPH5), 50/50/7 (CPH6). Each value is expressed as mean ± SD (n=3). Means within a column are significantly different (p < 0.05). Control here means cells grown without the composite material.

3.8 Observation of morphology of osteocytes on Chitin/PLA/nHAP composites The adhesion and morphology of the Ocy 454 osteocytes on the chitin/PLA/nHAP composites were studied by confocal microscopy. The resulting images are visualized in Fig. 6. All the materials show a good distribution of cells and a proliferative cell population. The cells seeded on day 0 began to attach and grow from day 1 and showed enhanced multiplication from day 4 (data not shown). The attachment of osteocytes seems to increase with increasing the chitin content in the composites. It is also interesting to note that the adhesion of osteocyte adhesion appears to increase with an increase of hydroxyapatite content in the composite material. The surfaces of the composites are almost completely covered with osteocytes, and this condition is notably maintained until the end of the kinetic assays. The cells adhered on the composites showed a normal cytoskeletal rearrangement with longitudinally distributed actin filaments. The cells were elongated with membrane extensions that enabled them to adhere to the biocomposite surface. These findings indicate that the composite materials can be used as a substrate to culture osteocyte cells.

Fig. 6. Confocal laser microscopic images (20X) of 14-day-old osteocytes grown on chitin/PLA/nHAP composites. The cells were stained with Acridine orange. The different samples are 20/80/0.5 (CPH1, orange), 80/20/0.5 (CPH2, dark blue), 50/50/0.5 (CPH3, grey), 50/50/2.5 (CPH4, green), 50/50/5 (CPH5, yellow), and 50/50/7 (CPH6, blue).

3.9 Attachment and proliferation of mammalian cells on chitin-PLA-nHAP composites Ocy 454 osteocytes were cultured on the different composites of chitin/PLA/nHAP, and cell attachment and proliferation on these membranes was observed using by a direct cell count. Fig. 7 shows the number of cells on all composite materials between 0 and 12 days. Cells were able to attach to all the materials tested, but it was observed that the number of cells attached on the composite structures varied slightly with the composition of the material. It was observed that with an increase in chitin and nHAP content in the materials, there was an increase in cell attachment.

Fig. 7. Densities of Ocy 454 osteocytes on chitin/PLA/nHAP composites. The cells were cultured for 0-12 days on the chitin membranes. The different samples are 20/80/0.5 (CPH1, orange), 80/20/0.5 (CPH2, dark blue), 50/50/0.5 (CPH3, grey), 50/50/2.5 (CPH4, green), 50/50/5 (CPH5, yellow), and 50/50/7 (CPH6, blue). Data shown here represent mean, error bars represent standard deviations of triplicate tests, p <0.05.

3.10 Bone-related gene expression by qRT-PCR It is believed that, on the molecular level, osteocytes can regulate the response of bone to mechanical loading using at least two key molecules, sclerostin (produced by the SOST gene) and the receptor activator of nuclear factor κΒ ligand (RANKL) [70, 71]. The SOST gene produces sclerostin, which is mainly produced by osteocytes postnatally and is an adverse bone formation regulator [71]. Osteocyte-specific gene expression was explored to see the effect of different composition of the composite substrate on which Ocy 454 cells were growing. The expression of SOST gene in Ocy 454 cells after 12 days of culture growing on the chitin/PLA/nHAP composite materials are shown on Fig. 8. When compared to the control (cells grown without the composite material), the cells grown on the composite material did not show any significant difference in their SOST

expression levels. These results also mean that the amount of nHAP in the composite material did not have any effect on the SOST gene expression of the cells.

Fig. 8. Comparison of osteo-related SOST gene expression of Ocy 454 cells grown on chitin/PLA/nHAP composites. Fold change was calculated following ΔΔCt the method to compare gene expressions between cells cultured on the composite materials versus cells cultures without the composite material (control) as 2(-ΔΔCt).

4. DISCUSSION Composite materials based on biodegradable polymers are commonly used in various biomedical applications. In a previous study, Li et al. showed the development of nHAP/collagen/PLLA composites strengthened with chitin nanofibers for the improvement of mechanical strength [72]. Silva et al. [52] reported the use of the ionic liquid 1-butyl-3-methylimidazolium acetate ([bmim][Ac]) to develop chitin and hydroxyapatite biocomposites for bone tissue engineering applications. Another research reported the use of PLA/chitosan/keratin composites for biomedical applications with an improved Youngs modulus and significant increase in hardness [19]. The characteristic properties of the composite materials are generally affected by the constituent polymer components, their relative amounts, and mixing conditions. In the present research, the use of ionic liquid for dissolution of chitin simultaneously with other polymers like PLA and hydroxyapatite is shown as an efficient process to make biocomposites materials with optimized properties. The composites were prepared by thermal dissolution of the polymers in IL [C2mim][OAc] and the concentrations of the polymers were varied to investigate the effect of the concentration of individual polymers on the composite material. Freeze-drying yielded composites that were porous in nature (Fig. 1). Chitin and nHAP cannot interact strongly in solution [52], perhaps with the addition of PLA, the gelation and high viscosity of the solutions were enough to keep the nanoparticles

dispersed uniformly in the polymeric matrixes. No phase separation was observed for any of our polymer-IL solutions, which suggested that the composites were homogenous. Scanning micrographs reveal a heterogenous porous formation. The variation of polymer concentrations associated with incorporation of nHAP, as well as the freeze-drying method could be possible reasons for these morphological features. The gelation of the polymer-IL solution cooled at -20 °C avoids the collapse of the pores during the removal of moisture due to the formation of a rigid polymer which retains its porous structure [53]. The TGA thermograms show a one-step degradation and two-step weight loss in the composite materials. The first weight loss in all the composites was found to be in the range of 80–100°C which may be due to loss of adsorbed water molecule present in the composites. The quantity of water lost ranges from 6% to 9%. It is established that nHAP is an inorganic material with thermal stability below 900 °C and does not lose its molecular structure at higher temperatures [73]. The maximum weight loss at ~364 °C for PLA is associated with the breaking of ester bonds and release of gaseous products such as cyclic oligomers, lactide molecules, acetaldehyde, and carbon monoxide [74]. It is generally accepted that a significant difference in the Tonset and Tmax of the components of a blend shows that no interaction exists between these components [74, 76]. In such a case, the TGA thermogram of the blend would show its degradation in two stages corresponding to the Tonset of each of its components, i.e., the blend would have two Tonset values. As illustrated in Fig. 2 and Table 2, all blends have only one Tonset which suggests that a considerable amount of interactions exist between the components in each blend, which most likely due to the interactions between the hydrogen bonds. Table 2 also indicates that the Tonset of the blends changes measurably with the composition ratio but higher than Tonset of chitin and lower than Tonset of PLA and nHAP. These results indicate that the components are well blended together. In case of the blended composites, the inclusion of PLA and nHAP improved the thermal stability of the composite, probably owing to the interaction between the three constituents of the composite. The glass transition temperature or Tg is a significant parameter used to determine the miscibility of polymers. The miscibility of two polymers in the bulk amorphous phase can be assessed using DSC by the presence of a single, compositionally dependent Tg value that lies between those of the component polymers. If the polymers are only partially miscible, the resulting mix would have two Tg values related to each component, but the Tg value corresponding to one component could be affected by the other one, usually depending on the composition ratio [77]. Table 2 shows the trend of the glass transition temperature Tg . A reduced Tg for the blended composites could be due to the suppression of some hydrogen bonds between the chitin chains. In other words, increasing PLA content increases the chitin chain flexibility [78]. The FTIR spectrum of the composites gather all the characteristic absorption peaks of chitin, PLA and nHAP. Compared to the neat polymers, the characteristic peaks of both PLA and chitin were slightly shifted (Fig. 3). These results indicate similarity to the

analysis of the polymers preciously observed [52, 67, 73, 79, 80] and reveal miscibility of the polymers in the composite material. The mechanical properties of chitin/PLA/nHAP composites depended on the composition of the materials. From Fig. 4, it was observed that the tensile strength improved by ~77% when the concentration of PLA was increased from 20 to 80%. However, elongation at break and the toughness of material decreased significantly with the increase in concentration of PLA. CPH3 showed 72% and CPH2 showed 68% increase in strength as compared to CPH1. CPH5 (5% nHAP) showed 77% and CPH6 (7% nHAP) showed 140% increase in tensile strength as compared to CPH3, which contained only 0.5% of nHAP. The elongation at break also increased tremendously, up to 465% for CPH5 as compared to CPH3. This indicates that an increase in nHAP concentration also seemed to improve the mechanical properties of the composite material. Our previous studies have shown that pure chitin has very low mechanical strength with a higher percentage of elongation to break [53]. PLA, on the other hand, is regarded as a brittle polymer with greater strength but lower elongation at break [81]. According to the rule of mixture, the mechanical properties of the composite material depend on the individual property with their corresponding wt% into the composites [82]. As PLA has a greater strength and lower elongation at break relative to chitin, higher PLA content helps to increase the composite material strength while reducing the elongation at break. Nanoparticles have a large interfacial area; well-dispersed nanoparticles enhance the interfacial adhesion between the matrix and nanoparticles. This further contributes towards improved strength of the material due to efficient transfer of load from the polymer matrix to nanoparticles [83]. Therefore, the addition of nHAP seems to provide a good interfacial adhesion with PLA. As a result, the applied load is effectively transferred to nanoparticles from the polymer matrix and makes the material stronger with higher elongation at break. In addition to that, a higher amount of the nanoparticles increases the crystallinity of the material which could also help to increase the strength of the material [84]. Fig. 5 shows that the in vivo degradation rate is slower for sample CPH1 which has the highest proportion of PLA. However, increasing the proportion of chitin in the composites made them more susceptible to degradation by lysozyme solution. Given the influence of hydroxyapatite content in the composites, increasing the nHAP concentration retarded the biodegradation process. Degradation of chitin is known to occur by hydrolysis of the β-(1-4) linkages between N-acetylglcuosamine and glucosamine which undergo chain scission by the lysozyme present in the body [85]. The morphological features and crystallinity of the biomaterial could also affect the degradation behavior. Indeed, porosity of the materials could also affect the penetration of the lysozyme solution within the structure and make the polymer degradation easier. The physiochemical degradation of the composite comes from (i) the breakdown of electrostatic interactions and intermolecular bonds, and (ii) the hydrolysis of the membrane [86]. There rate of degradation decreased when the nHAP concentration was

increased from 0.5 to 7%. This could be due to an increase in crystallinity of the material with increasing hydroxyapatite content. Since crystallinity is an organizational measure, more crystalline materials have stronger inter- and intramolecular bonding, and thus degrade slowly [87, 88]. The results from the MTT assay in this study (Table 3) suggest that the composites did not show any obvious signs of cytotoxicity and this was not affected by the composition of the materials. It is very important that the biocomposites should exhibit minimal cytotoxicity for the concerns of tissue engineering applications. The MTT assay used in this work is a quick and effective method for testing mitochondrial activity, which correlates quite well with cell proliferation. The composites were also found to support the growth and proliferation of Ocy 454 cells. Confocal microscopy revealed that the osteocytes showed normal signs of growth with the cells extending dendrites to the biocomposites surfaces (Fig. 6). This can be considered a sign of healthy attachment on the supporting material. The cell count data indicates a gradual increase in cell numbers growing on the composites from days 0-14. An increase in chitin content in the composites resulted in increased attachment of cells (Fig. 7). An increase in nHAP content also showed to favor cell attachment to the material surface. Previous studies have also confirmed similar results [33, 34, 35]. A series of intracellular events regulating gene expression is then followed by the initialization and completion of cellular tasks such as differentiation. SOST gene expression was studied because the cell line Ocy454 is known to produce high levels of SOST/sclerostin at early points and in the absence of differentiation factors [69]. The protein produced by the SOST gene acts as an inhibitor to the Wnt signaling pathway and thus plays a key role in the regulation of bone formation [89, 90]. The results from SOST gene (Fig. 8) expression study here show that the composition of the composite material did not have any effect on the SOST gene expression of the osteocytes growing on the material. Even though materials with more HAP content have been shown to favor cell attachment, the amount of nHAP did not show any significant difference in the SOST gene expression levels. HAP is a major component of bone minerals and osteocytes in bone are surrounded by HAP [90]. We consider these results to be important, because in order to use these biocomposites as scaffolds for tissue engineering applications, these materials must support the processes of tissue regeneration and repair, while providing mechanical support and ultimately degrading to non-toxic products that would be ultimately removed from the body.

5. CONCLUSION In summary, new biocomposites were prepared with chitin, PLA and hydroxyapatite by co-dissolution in ionic liquid and their mechanical properties and biocompatibility were evaluated. The thermal and spectroscopical analyses show well-blended composite structures. The addition of PLA and hydroxyapatite demonstrated

improvement in the mechanical properties that would be appropriate for biomedical applications. The composites are non-toxic and showed good biocompatibility and in vitro biodegradability. The composites are appropriate for growth and proliferation of osteocytes, which make them excellent candidates for use in bone regeneration and are, in general, promising materials for medical application. More work is needed in order to obtain the ideal biodegradable chitin-based biomaterial for bone repair and regeneration that will demonstrate vast improvements in the interface between implants and biological tissue based on the knowledge gained from this research.

6. ACKNOWLEDGEMENTS The authors would like to thank Prof. Lamya Karim (Bioengineering Department, University of Massachusetts Dartmouth) for providing the Ocy 454 osteocyte cell line, as well as for facilitating the Ocy 454 gene expression experiments. This research received no specific grant from public, commercial or non-profit funding agencies.

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Highlights: 

Ionic liquid (IL) was used for co-dissolution of polymers like chitin, PLA and nHAP



Thermal and spectroscopical analyses show well-blended composite structures

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Increasing concentration of PLA improved the material tensile strength ~77%

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The addition of hydroxyapatite improved the strength of the composites up to 140%

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The composite materials show the support the growth and proliferation of osteocytes