bovine bone powder porous biocomposite

    Development, characterization and biocompatibility of chondroitin sulfate/poly(vinyl alcohol)/bovine bone powder porous biocomposite Gabriela T. da Silva, Guilherme T. Voss, Vanessa Kaplum, Celso V. Nakamura, Ethel A. Wilhelm, Cristiane Luchese, Andr´e R. Fajardo PII: DOI: Reference:

S0928-4931(16)32280-9 doi:10.1016/j.msec.2016.11.069 MSC 7123

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

26 July 2016 14 November 2016 21 November 2016

Please cite this article as: Gabriela T. da Silva, Guilherme T. Voss, Vanessa Kaplum, Celso V. Nakamura, Ethel A. Wilhelm, Cristiane Luchese, Andr´e R. Fajardo, Development, characterization and biocompatibility of chondroitin sulfate/poly(vinyl alcohol)/bovine bone powder porous biocomposite, Materials Science & Engineering C (2016), doi:10.1016/j.msec.2016.11.069

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Development, characterization and biocompatibility of chondroitin sulfate/poly(vinyl alcohol)/bovine bone powder porous biocomposite

PT

Gabriela T. da Silvaa, Guilherme T. Vossb, Vanessa Kaplumc, Celso V. Nakamurac,

a

SC RI

Ethel A. Wilhelmb, Cristiane Lucheseb and André R. Fajardoa* Laboratório de Tecnologia e Desenvolvimento de Materiais Poliméricos e Compósitos – LaCoPol. Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Universidade Federal de Pelotas (UFPel), 96010-900, Pelotas-RS, Brazil.

Grupo de Pesquisa em Neurobiotecnologia (GPN). Centro de Ciências Químicas, Farmacêuticas e de

NU

b

Alimentos, Universidade Federal de Pelotas (UFPel), 96010-900, Pelotas-RS, Brazil. c

Laboratório de Microbiologia Aplicada aos Produtos Naturais e Sintéticos and Laboratório de Inovação

MA

Tecnológica no Desenvolvimento de Fármacos e Cosméticos, Universidade Estadual de Maringá (UEM), 87020-900 - Maringá, Paraná, Brazil.

ED

Abstract

Chondroitin sulfate (ChS), a sulfated glycosaminoglycan, poly(vinyl alcohol) (PVA) and

PT

bovine bone powder (BBP) were blended to form a novel eco-friendly biocomposite through cyclic freeze-thawing under mild conditions. The systematic investigation reveals that the

CE

content of BBP has a remarkable effect on the pore size, porosity, mechanical and liquid uptake properties and biodegradability. At 10 wt.% BBP the biocomposite exhibited enhanced

AC

mechanical properties and biodegradability rate as compared to the pristine sample. Further, different properties of the biocomposite can be tailored according to the content of BBP. In vitro assays showed that ChS/PVA-BBP does not exert cytotoxicity against healthy cells. In vivo and ex vivo experiments revealed that ChS/PVA-BBP biocomposites are biocompatibility materials without exert pro-inflammatory responses. The biocomposite was completely biodegraded and bioresorbed after 15 days of treatment. Taken together, BBP is a low-cost source of hydroxyapatite and collagen, which are insurance. All these results suggest that the biocomposite designed in this study is a promising biomaterial for potential skin tissue engineering. Keywords: porous materials; biocomposites; composites; chondroitin sulfate; biomaterial; tissue engineering. (*) Corresponding author. E-mail: [email protected] - Phone: +55 53 3274-7356 - Fax: +55 53 32757354.

1

ACCEPTED MANUSCRIPT 1. Introduction Innovative and versatile scaffolds had been fabricated in the last years for the most

PT

varied purposes (e.g. cell engineering and tissue and bone regeneration) [1, 2]. The success of

SC RI

these systems in biotechnological uses is ascribed to the possibility of to mimic a typical in vivo cellular environment [3]. Scaffolds provide nutrients and other factors that allow cells and tissues growing in vitro conditions. Herein, different types of stem cell are seeded in

NU

scaffolds to grow and divide as autonomous units. However, in this approach, the cell anchorage on scaffold matrix is required. That means cells will stop growing, dividing and die

MA

without anchoring centers. For that reason, among various requirements for an ideal scaffold (adequate architecture, cyto- and tissue-compatibility, bioactivity and good mechanical

ED

properties, for instance), the good attachment of anchorage-dependent cells is essential [4]. Scaffolds made of synthetic or natural or both polymers typically provide the necessary

PT

structural support for cell attachment and development [1, 2]. Plenty of works report on the

CE

literature original scaffolds fabricated from several polymers (pure or blended) using different processing techniques [5, 6]. In this sense, the glycosaminoglycans (GAGs) are a prominent

AC

class of biopolymers used to fabricate scaffolds with controllable chemical, mechanical, and micro structural properties [7]. Chondroitin sulfate (ChS), a sulfated GAG composed of alternating units of (β-1,4)linked N-acetyl-galactosamine (GalNAc) and (β-1,3) glucuronic acid (GlcUA), has captured considerable attention in biotechnological uses due to its numerous biological activities and enhanced physico-chemical properties [8, 9]. One of the most important functions of ChS in tissue regeneration is related to its signaling functions of various growth factors and chemokines (i.e. fibroblast and epidermal growth factors) [10]. ChS contributes to stabilization and modulation of the active conformation of these growth factors against fast degradation, for instance. These properties are closely associated with the sulfate groups of

2

ACCEPTED MANUSCRIPT ChS [10]. The associations of ChS with other polymers results in reliable scaffolds materials that can absorb growth factors, show good cell adhesion, and avoid undesirable inflammatory

PT

reactions at application sites [11, 12]. Moreover, ChS allows performing different processing

SC RI

techniques to fabricate scaffolds (i.e. polyelectrolyte complexation, freezing-thawing, chemical and physical crosslinking, etc.) [13, 14]. Taking into account these aforementioned aspects, here ChS was blended with poly(vinyl alcohol) (PVA) to fabricate an eco-friendly

NU

hydrogel using the freezing-thawing technique. PVA, a water-soluble synthetic polymer, is commonly used to manufacture biomaterials due to its good mechanical properties,

biomaterial for tissue engineering.

MA

nontoxicity and biocompatibility [15, 16]. Thus, ChS/PVA can be considered a potential

ED

To further enhance the mechanical properties of ChS/PVA we have explored the addition of bovine bone powder (BBP) into the same resulting in a biocomposite material.

PT

BBP, a waste material from bovine slaughterhouse, is an eco-friendly and low-cost source of

CE

hydroxyapatite and collagen fibrils. Several works make use of collagen or hydroxyapatite in scaffold formulation; in order to endow some required property (i.e. adhesion,

AC

biocompatibility, biodegradability, mechanical properties, and so on) [17, 18]. To the best of our knowledge a porous biocomposite based on ChS/PVA-BBP has not been explored till date for scaffolding. Here, biocomposites were fabricated using mild conditions and fully characterized in terms of physico-chemical, morphological and water uptake properties; then in vitro tests were carried out to evaluate the cytotoxicity of ChS/PVA-BBP biocomposite against healthy cells. In order to demonstrate the biocompatibility and security of the biocomposites, in vivo and ex vivo experiments were performed.

2. Materials and Methods 2.1. Materials

3

ACCEPTED MANUSCRIPT Chondroitin sulfate (ChS) (Mw ~20,000 g/mol) was kindly donated by Solabia (Maringá-PR, Brazil). Poly(vinyl alcohol) (PVA) (Mw 84,000 – 124,000 g/mol, 99%

PT

hydrolyzed) and hyaluronidase from bovine testes (type I, 103 units/mg) were purchased from

SC RI

Sigma-Aldrich (St. Louis, USA). Slices of adult femoral bovine cortical bones were purchased from the local butcher shop (Pelotas-RS, Brazil). Bovine bone powder was prepared according to the method described by Alves et al. [23]. Briefly, slices of bovine

NU

bones were freed from macroscopic fragments manually and by immersion in boiling distilled water. After cooling, they were sonicated in a bath filled with acetone (~15 min). Cleaned

MA

bovine bone was oven-dried (100 °C for 24 h) and then, ball milling treated at room temperature for 10 min (Marconi, model MA350, Brazil). The white powder obtained, labeled

ED

as BBP, was grounded and sieved through an 80-mesh sieve before use. All other chemicals

PT

of analytical grade were used as received without further purification.

CE

2.2. Preparation of ChS/PVA-BBP biocomposites PVA solution (3.75 wt.%) was prepared by dissolving the polymer in deionized water

AC

at 90 °C for 4 h. After the complete solubilization the solution was cooled to 60 °C. ChS solution (8 wt.%) was prepared in deionized water at room temperature for 4 h. PVA and ChS solutions were gently blended and homogenized for 4 h to yield 1:2 PVA/CS weight ratio. Then, different BBP mass contents (0, 1, 5, and 10 wt.%) with respect to ChS/PVA total weight were added in feed solutions, which were stirred for 1 h to homogeneous the system. Afterward, the resulting solutions were cast into plates, which were immediately deep-frozen (-20 °C) overnight and slowly thawed (~5 h). Six freeze-thaw cycles were carried out to physically crosslink the biocomposites, which after the last freeze-thaw cycle were immersed in distilled water overnight prior to eliminate some impurity. After this procedure, the

4

ACCEPTED MANUSCRIPT biocomposites (marked here as ChS/PVA-BBP0, ChS/PVA-BBP1, ChS/PVA-BBP5, and

PT

ChS/PVA-BBP10, respectively) were recovered and oven-dried at 40 °C for 48h.

SC RI

2.3. Characterization techniques

The biocomposites and the neat components (BBP, ChS, and PVA) were characterized by Fourier-Transformed Infra Red (FTIR) spectroscopy, X-ray diffraction (XRD) and

NU

Thermogravimetry (TGA) analysis. FTIR spectra were recorded in a Shimadzu spectrometer (model Affinity, Japan) operating in the spectral region of 4000-600 cm-1 with resolution of 4

MA

cm-1. The samples were ground with spectroscopic grade KBr and pressed into disks. XRD patterns were measured using a powder diffractometer Siemens (model D500, Germany),

ED

with Cu-Kα radiation source (λ = 0.154178 nm) at 30 kV and 20 mA. The scanning range was 5–50° with a scanning rate of 1 °/min. TGA curves were obtained using a Shimadzu Analyzer

PT

(model DTG60, Japan) with a scanning rate of 10 ◦ C min-1 under N2(g) atmosphere with flow

CE

of 20 mL min-1in a range of temperature of 25–500 ◦C. Mechanical properties were investigated by compressive testing using a Texturometer (Stable Micro System, Model

AC

TA.TXT2, UK). For this, set of hydrogels samples (cubic shape - edges about 10 mm) were swollen up to equilibrium (~24 h) and then tested in unconfined compressive mode (maximum strain of 95% and crosshead speed of 1 mm/s) at room temperature. SEM images were recorded using a JEOL scanning electron microscope (model JSM6610LV, USA) coupled with an energy dispersive X-ray (EDX) analyzer. Prior to the SEMimage record, samples were immersed in PBS (pH 7.4) at room temperature up to the equilibrium swelling (~24 h). Later, these swollen samples (rectangular shape, 1.5 x 0.5 cm) were frozen using liquid nitrogen (5 min of immersion), then, they were removed and carefully fractured (manually) at the middle. The fractured samples were lyophilized in a freeze dryer (Terroni, model LS3000, Brazil) at -55 °C for 24 h. Next, the dried samples were

5

ACCEPTED MANUSCRIPT mounted on a stub sample holder and gold-coated by sputtering before SEM/EDX visualization. The microscope was operated at a working distance of around 10 mm and an

PT

acceleration voltage of 10–11 kV.

SC RI

2.4 Porosity

The porosity of each biocomposite was evaluated using a simple liquid displacement method [11]. In brief, a cubic shaped sample was immersed into known initial volume (V1) of

NU

acetone for 24 h at room temperature. After acetone impregnation, the volume of the set

MA

hydrogel-ethanol was determined (V2). Finally, the acetone-impregnated sample was set off and the remaining liquid volume was determined (V3). The total porosity was calculated using

ED

the following equation:

( 𝑉1 − 𝑉3 ) 𝑥 100 ( 𝑉2 − 𝑉3 )

(1)

CE

PT

𝑃𝑜𝑟𝑜𝑠𝑖𝑡𝑦 (%) =

AC

2.5 Uptake and retention capacities Here maximum uptake capacity at equilibrium (Weq) was evaluated as function of the percentage of BBP incorporated into the biocomposites formulation. For this, a conventional gravimetric procedure was conducted in each experiment. Briefly, known amounts of each dried hydrogel sample (ca. 100 mg) were pre-weighed and put in a glass vial filled with 100 mL of PBS pH (7.4). Each vial was kept under constant slow stirring at 37 °C for 24 h to reach the swelling equilibrium. The swollen samples were taken from the vials and gently drained to remove the liquid excess. Afterward, those samples were weighed and the Weq parameter was calculated from the following equation:

6

ACCEPTED MANUSCRIPT 𝑊𝑒𝑞 (%) = [

𝑤𝑠 − 𝑤𝑜 ] 𝑤𝑜

𝑥 100

(2)

PT

where ws is the swollen sample weight and wo is the dried sample weight.

SC RI

Liquid retention capacity of swollen samples was measured using the follow procedure. Samples were swollen in PBS (pH 7.4) up to equilibrium (~24 h), weighed (ws) then inserted in a dry centrifuge tube containing filter paper at the bottom. This set was

NU

centrifuged at 10,000 rpm for 5 min; the sample was recovered and weighed again (ws´).

MA

Retention capacity (Wret) was calculated using the follow equation:

𝑤𝑠

] 𝑥 100

(3)

PT

2.5 Biodegradation assays

𝑤𝑠 ´

ED

𝑊𝑟𝑒𝑡 (%) = [

In vitro biodegradation assays were carried out in two conditions: (i) in PBS (pH 7.4)

CE

and (ii) in PBS (pH 7.4) supplemented with hyaluronidase from bovine tests (103 units/mg).

AC

Hyaluronidase acts at the initial stage of the degradation process of ChS in vivo [19]. For both assays, pre-weighed samples were put in sealed tubes filled with 10 mL of PBS or PBS supplemented with hyaluronidase. At desired time intervals (1, 2, and 3 weeks), the samples were withdrawn, washed with ultrapure water to remove undesired compounds and dried to a constant weight in an oven (50 °C). The percentage of degradation was calculated using the following equation:

𝑤0 − 𝑤𝑡

𝐷𝑒𝑔𝑟𝑎𝑑𝑎𝑡𝑖𝑜𝑛 (%) = [

𝑤0

] 𝑥 100

(4)

where w0 is the sample initial weight and wt is the weight after soaking.

7

ACCEPTED MANUSCRIPT 2.9. Biological assays 2.9.1. In vitro cytotoxic test

PT

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric

SC RI

assays were carried out to investigate the cytotoxicity of ChS/PVA-BBP0 and ChS/PVABBP10 against healthy cells [20]. Briefly, Vero cells (epithelial cells from the kidney of African green monkey - Cercopithecus aethiops, ATCC@-CCL-81TM) were cultured in

NU

Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10 % FBS – Fetal Bovine Serum (Gibco, USA) and 50 µg/mL gentamicin (oven, 37 °C, 5% CO2). They

MA

were stored in sealed plastic bottles for cell culture. For the cytotoxicity tests, the biocomposites samples (cubic shape 5 x 5 x 5 mm) were placed in culture microplate (24-

ED

wells) and 500 μL of culture media was added and incubated for 4 h (oven, 37 °C, 5% CO2). The cells (in DMEM with 10 % FBS and 50 µg/mL getamicin) were seeded at a seeding

PT

density of 2.5 x 105 cells/mL onto the top of the samples and incubated in a humidity oven at

CE

37 °C. Fresh culture medium was replaced every other day. After incubation time (24 or 48 h), each media on the control (wells without test samples) and on the samples were removed

AC

and the wells were washed with PBS (500 μL). Then, MTT solution (2 mg per mL of PBS) was added to each well and the microplate was incubated for 4 h (dark oven, 37 °C, 5% CO2). Finally, the unreacted MTT was removed and 500 μL of DMSO was added to dissolve the formazan product. The optical density of living cells was measured on a micro-plate reader (Bio-Tek Power Wave XS, USA) at 570 nm.

2.9.2. Animals All experiments were performed using male adult Swiss mice (25–35 g). Mice were kept in a separate animal room, on a 12 h light/dark cycles, with lights on at 7:00 a.m., at room temperature (22 ± 1°C), with free access to food and water. The experiments were

8

ACCEPTED MANUSCRIPT performed according to the guidelines of the Committee on Care and Use of Experimental Animal Resources, Federal University of Pelotas, Brazil (CEEA 1289-2016). The number of

SC RI

consistent effects of the ChS/PVA-BBP biocomposites.

2.9.3. In vivo and ex vivo experiments order

to

verify

the

ChS/PVA-BBP0

and

ChS/PVA-BBP10

potential

NU

In

PT

animals and intensities of noxious stimuli used were the minimum necessary to demonstrate

biocompatibility to the skin, mice were anaesthetized with isoflurane by inhalation (0.08 ‐

MA

1.5%), and then their backs were shaved. 0.5 cm wounds were created on the back of each mouse with a biopsy puncher (diameter of 0.5 cm). ChS/PVA-BBP0 and ChS/PVA-BBP10

ED

were placed on the wound site, whereas the negative control remained untreated. The change in the wound area was verified at 0, 3, 9 and 15 days. On the 15th day, mice were killed and

PT

the wound area was removed to the macroscopic observation and myeloperoxidase (MPO)

CE

activity determination.

AC

Neutrophil infiltration in wound area was estimated indirectly by MPO activity, as previously described by Suzuki et al. (1983) with some modifications. The skin samples were homogenized

in

potassium

phosphate

buffer

(20

mmol/L,

pH

7.4)

containing

ethylenediaminetetraacetic acid (0.1 mmol/L). After the homogenization, samples were centrifuged at 900 rpm at 4°C for 10 min to yield a low speed supernatant fraction (S1). Then, S1 fraction was centrifuged again at 20,000 rpm at 4°C for 15 min to yield a final pellet (P2) that was resuspended in medium containing potassium phosphate buffer (50 mmol/L, pH 6.0) and hexadecyltrimethyl ammonium bromide (0.5%). Samples were finally frozen (-20 °C) and thawed three times for the posterior enzymatic assay. For the MPO activity measurement, an aliquot of resuspended P2 (100 μL) was added to a medium containing the medium of resuspension and N,N,N′,N′-tetramethylbenzidine (1.5 mmol/L). The kinetic analysis of MPO

9

ACCEPTED MANUSCRIPT was started after H2O2 (0.01%) addition, and color reaction was measured at 655 nm at 37°C. Results were expressed as optic density (OD)/mg protein/min. Protein concentration was

PT

measured by the method of Bradford [21], using bovine serum albumin (1 mg/mL) as the

SC RI

standard.

2.10. Statistical analysis

NU

All data were expressed here as means ± standard deviation of replicates (n ≥ 3) for each sample in each experiment. The statistical analysis was performed by using OriginPro

MA

8.5 software. Data of ex vivo experiments were analyzed by one-way analysis of variance (ANOVA) followed by the Newman-Keuls test when appropriated and it was expressed as

ED

mean ± S.E.M. Probability values less than 0.05 (P < 0.05) were considered statistically

PT

significant.

CE

3. Results and Discussion 3.1. Characterization

AC

ChS/PVA hydrogel and ChS/PVA-BBP biocomposites were prepared via ionic gelation by using freeze-thaw cycles. The neat components (ChS, PVA, and BBP) and ChS/PVA-BBP0 and ChS/PVA-BBP10 were characterized using FTIR and XRD (Figure 1a,b). The BBP spectrum (see Figure 1a) presents characteristic bands of collagen and hydroxyapatite, its two major components [22]. The bands assigned to the collagen are observed at 1645 cm-1 (amide I band), 1238 cm-1 (amide III band), in the ranges of 1450–1350 cm-1 (C-H symmetric deformation) and 1080–980 cm-1 (C-O and C-O-C stretching from carbohydrate residues) [23, 24]. The hydroxyapatite exhibits a quite intense broad band centered at 3287 cm-1 (hydroxyl stretching from hydrogen-phosphate group or bonded water), bands at 1447 cm-1 and 872 cm-1 (CO32- groups) and intense bands that ranges from 1080 cm-1

10

ACCEPTED MANUSCRIPT to 990 cm-1 (PO43- groups) [23, 24]. PVA exhibits a broad band centered at 3453 cm-1 (hydroxyl groups involved in intra- and intermolecular interactions), bands in the range 2940–

PT

2910 cm-1 (C-H stretching from alkyl groups), at 1715 cm-1 (C=O stretching from residual

SC RI

acetate groups), at 1090 cm-1 (C-O stretching) [25]. ChS spectrum shows a broad band centered at 3435 cm-1 (hydroxyl stretching and overlapped N-H stretching), bands at 1640 cm1

(C=O stretching), 1569 cm-1 (N-H bending) 1421 cm-1 (C-O stretching and O-H angular

NU

vibration coupling), 1240 cm-1 (S=O stretching), 924 cm-1 (α-1,4 glycosidic bond) and bands

MA

in the range 1135-1030 cm-1 (symmetric and asymmetric C-O-C stretching) [26, 27].

ED

Figure 1

In the ChS/PVA/BBP-0 spectrum, the hydroxyl band got shifted to 3270 cm-1 due to

PT

the formation of hydrogen bonds among the PVA chains [28]. Furthermore, the appearance of

CE

a band at 1141 cm-1 was observed. Colin et al. [29] reports that this band is related to the crystallinity of PVA after the gelling process. Such band is assigned to C-C and C-O

AC

stretching of the chain at the point where an intramolecular hydrogen bond is formed from two hydroxyl groups on the same side of the PVA backbone [29]. Characteristic bands of ChS at 1240 cm-1 (S=O stretching) and 924 cm-1 (α-1,4 glycosidic bond) were observed in ChS/PVA/BBP-0 spectrum, which confirms the presence of ChS in the hydrogel. The FTIR analysis suggests that the hydrogel matrix is formed due to the physical crosslinking of PVA chains, while the ChS chains remain in their linear form [30]. This kind of crosslinking procedure favors the use of such material in further biomedical applications since any toxic crosslinking agent or auxiliary molecule was utilized. Moreover, this approach highlights the eco-friendly characteristic o this hydrogel. The incorporation of 10 wt.% BBP into ChS/PVA matrix caused the enlargement and shifting of the band assigned to the hydroxyl stretching to

11

ACCEPTED MANUSCRIPT 3400 cm-1. Moreover, the intensity of the band at 1645 cm-1 (assigned to amide I) increased and the bands in the range of 1100-900 cm-1 (C-O and C-O-C stretching and PO43- groups)

PT

have showed some enlargement. The appearing of the band at 1454 cm-1 (CO32- groups) was

SC RI

also observed. Similar observations were reported by Alves et al. regarding a composite based on chitosan/PVA filled with BBP [23]. Overall, the FTIR results confirm the insertion of BBP and the composite formation.

NU

The structure investigation (Figure 1b) showed that the crystallinity of the ChS/PVA/BBP-0 hydrogel is higher than that observed for the neat materials (PVA and ChS).

MA

The XRD pattern of ChS/PVA/BBP-0 shows the appearing of an intense and sharp diffraction peak at 2θ ≈ 28.39º and additional diffraction peaks at 2θ ≈ 31.41º, 40.51º, 45.43º, 50.29º, and

ED

66.49º. Such increasing regarding the crystallinity can be associated with the freezing-thawing process that allows forming novel ordered regions into the hydrogel matrix [31]. In this sense,

PT

the ordering is promoted by the secondary forces (e.g. hydrogen bonds) among the PVA

CE

chains. The enlargement of the halo close to 2θ ≈ 22.90º suggests that the amount of amorphous domains into the hydrogel matrix increases likely due to ChS that has a

AC

predominantly amorphous structure [26]. The incorporation of BBP (at 10 wt.%) into the ChS/PVA matrix shows an obvious effect on the crystallinity of such composite regarding the pristine hydrogel. The relative intensity of some diffraction peaks is reduced, while other peaks disappear completely after the BBP adding. Such evidences suggest that BBP disrupts the ordering of the PVA chains during the gelling process. Moreover, the appearing of peaks at 2θ ≈ 25.87º and 31.90º attributed to the reflection of (002) and (211) crystalline planes of hydroxyapatite (inorganic moiety of BBP) confirm the biocomposite formation. TGA-DTG analyses were used to investigate the thermal behavior of the neat materials (ChS, PVA, and BBP), as well as the composites. As demonstrated in Figure 2a,c all samples showed different stages of weight loss. The first stage (ranging from 30º to 150

12

ACCEPTED MANUSCRIPT ºC) is ascribable to the moisture and volatile compounds evolution. The second stage is related to the thermal degradation of the hydrogel/biocomposite matrices. The thermal

PT

degradation of ChS/PVA hydrogel occurred at 372 ºC (see Figure 2d). This result reveals that

SC RI

ChS/PVA hydrogel has higher thermal stability than the neat polymers, which can be attributed to its higher structural organization (i.e. crystallinity) obtained due to the gelling process. ChS is thermally degraded at 246 ºC, while PVA may shows two degradation stages

NU

(see Figure 2b). The first at 262 ºC occurs due to the elimination of its hydroxyl side groups and the second at 431 ºC due to polyene chain degradation [32]. On the other hand, the

MA

addition of BBP decreases the thermal stability of the ChS/PVA matrix when the amount of BBP is equal or higher than 5 wt.%. As evidenced, the thermal degradation temperature

ED

decreases from 372 ºC for the ChS/PVA-BBP0 to 363 ºC for the ChS/PVA-BBP10 sample (Figure 2d). As above discussed, BBP affects the crystallinity of the ChS/PVA matrix and

PT

increases the content of amorphous domains into the biocomposite. For this reason, the

CE

biocomposites at 5 and 10 wt.% BBP showed poor thermal stability in comparison to the pristine sample. This result is in agreement with the XRD analysis. On other notes, the

AC

increasing of the amount of BBP incorporated into the biocomposites increases the residues at 500 ºC. At this temperature, the residues are composed basically of the inorganic moiety of BBP (i.e. hydroxyapatite). Residues at 500 ºC increase from 11.2% for the ChS/PVA-BBP0 up to 26.3% for the ChS/PVA-BBP10 sample (Figure 2c).

Figure 2

The mechanical properties are a very important issue regarding the application of the scaffold in cell/tissue regeneration. Such properties are associated with the cell adhesion and proliferation and biosynthesis [33]. Further, mechanical properties help to define in vitro and

13

ACCEPTED MANUSCRIPT in vivo applications [33]. Herein, compression tests were carried out on the ChS/PVA-BBP0 and ChS/PVA-BBP10 samples in order to evaluate different mechanical properties. The

SC RI

PT

results are listed in Table 1.

Table 1

NU

As observed, the incorporation of different amounts of BBP into the ChS/PVA matrix has a clear effect on the mechanical properties. The pristine sample provided a value of

MA

Young´s modulus similar to that evaluated for ChS/PVA-BBP1 (~ 9.1 KPa), the sample filled with the lowest content of BBP. On the other hand, the samples with 5 and 10 wt.% of BBP

ED

showed an increment in Young´s modulus at about 25 and 52% as compared to ChS/PVABBP0. Such result suggests that from 5 wt.% BBP acts as a reinforcement agent. As assessed,

PT

this reinforcement effect depends on the content of BBP into the hydrogel matrix. Interactions

CE

between BBP-ChS/PVA restrict the mobility of the polymer chains, which affect the dispersion of compressive forces through the biocomposite matrix. As consequence, the

AC

composites with high amounts of BBP show higher rigidity than the pristine sample. Moreover, these interactions (i.e. physical interaction and hydrogen bonding) [34] cause changes in the structure and thermo properties of the biocomposites, as assessed by XRD and TGA analyses. Alves et al. observed this reinforcement effect caused by BBP on a chitosan/PVA matrix [23]. In addition, the mechanical analyses showed that the rupture force increased at about 25% for ChS/PVA-BBP5 and 36% for the ChS/PVA-BBP10 in comparison to the ChS/PVABBP1 and ChS/PVA-BBP0 samples. The additional interaction points into the biocomposite matrix due to the incorporation of high amounts of BBP make harder breaking up these samples. On contrary, other mechanical properties such as brittleness and deformation at

14

ACCEPTED MANUSCRIPT rupture point were slightly affected by BBP, as illustrated in Table 1. Mechanical properties exhibited by the pristine and composites samples are comparable to other porous scaffolds

PT

described elsewhere [35-37]. As compared to other composite materials previously reported

SC RI

on the literature, all the samples fabricated in this study showed enhanced mechanical properties. Chang et al. synthesized a chitin/hydroxyapatite hybrid hydrogel that exhibited porous structure showed Young´s modulus at about 0.3 MPa [38]. Gantar et al. reinforced

NU

gellan-gum gels with a nanoparticulate glass, which exhibited a Young´s modulus of 1.2 MPa [39]. Guha et al. fabricated a series of PVA hydrogels filled with different amounts of

MA

hydroxyapatite via freeze-thawing method. As assessed, the amount of hydroxyapatite into the PVA matrix changes the Young´s modulus, which varies from 11 to 20 MPa [40].

ED

Furthermore, for all tested samples the average values for the Young´s modulus are in good agreement with the reported values for human skin [41, 42].

PT

The morphology of the ChS/PVA-BBP0 and ChS/PVA-BBP10 samples and their

CE

elemental composition were assessed by SEM images and EDX analyses. Representative low

3(a-d).

AC

and high magnification SEM images recorded for the fractured samples are shown in Figure

Figure 3

As evidenced in SEM images, both samples are characterized by a highly porous structure. Typically, such structures are formed due to the phase separation between the polymeric compounds and ice water during the freezing-thawing cycles [16]. Both samples showed a heterogeneous pore distribution with an obvious large porous size distribution. The average pore size calculated for ChS/PVA-BBP0 is 119.6 ± 21.1 μm. At 10 wt.% BBP the average pore size decreased to 87.3 ± 13.9 μm. This finding can be explained by the BBP-

15

ACCEPTED MANUSCRIPT ChS/PVA interactions, which allow a close entanglement of BBP and the ChS/PVA chains. As a result, the free water has less empty spaces to form big ice nuclei during the freezing-

PT

thawing process and the pore size decreases [16]. On other notes, ChS/PVA-BBP0 and

SC RI

ChS/PVA-BBP10 samples show a noticeable interconnectivity between the pores, which is a desirable characteristic of a scaffold [43]. Furthermore, SEM images confirm that BBP particles were uniformly incorporated into the ChS/PVA matrix likely due to the good

NU

interaction between compounds that form the biocomposite. No phase separation or clusters was observed in the SEM images recorded from the composite. This inference corroborates

MA

with the other above discussed analyses.

Figure 3(e,f) displays the EDX spectra recorded for the ChS/PVA-BBP0 and

ED

ChS/PVA-BBP10 samples. As clearly demonstrated, the EDX analysis showed the presence of calcium (Ca) and phosphorous (P) elements on the ChS/PVA-BBP10 sample. These

PT

elements are the main components of hydroxyapatite [Ca5(PO4)3(OH)] proceeding from BBP.

CE

In comparison, the EDX spectrum recorded for the pristine sampled did not demonstrate the presence of such elements. These results confirm the presence of BBP in the composite

AC

composition and corroborate with the FTIR and XRD analyses.

3.2. Porosity In this study, the total porosity of the ChS/PVA-BBP(0-10) samples was estimated using a simple liquid displacement method [11]. According to the data listed in Table 2 is noticed that ChS/PVA-BBP1 and ChS/PVA-BBP5 show similar porosity as compared to the pristine sample. This suggests that the incorporation up to 5 wt.% BBP does not have an effect on total porosity. Nonetheless, the porosity calculated for ChS/PVA-BBP10 decreases as compared to the other samples. Such result strengthens the suggestion that physical interactions may be occurring between BBP and ChS/PVA matrix, which result in lower

16

ACCEPTED MANUSCRIPT porosity. Venkatesan et al. [11] have noticed similar behavior regarding the porosity of scaffolds

based

on

chitosan-amylopectin/hydroxyapatite

and

chitosan-chondroitin-

PT

sulfate/hydroxyapatite composites. Other studies infer that scaffolds with high porosity

SC RI

(>60%) are able to provide adequate fluid flow to deliver nutrients and bioactive compounds (i.e. growth factors) and remove degradation byproducts [11, 44]. In this sense, it is possible to infer that all the original biocomposites described in this study could potentially be used as

MA

3.3. Uptake and retention capacities

NU

a scaffold.

A biomaterial designed to act as a scaffold should exhibit the ability of to absorb and

ED

retain physiological fluids as well as the ability of to transfer nutrients and bioactive compounds through its matrix. According to Yan et al. [14] a high uptake capacity is

PT

desirable to maintain the shape of scaffolds and transit the cell/tissue nutrients and wastes

CE

inherent from the regeneration process. The maximum uptake capacity at equilibrium (Weq) and retention ability (Wret) calculated for the ChS/PVA-BBP(0-10) samples are shown in

AC

Table 2. Despite ChS and PVA have a considerable hydrophilic character, especially PVA due to its great number of hydroxyl groups, the hydrogel ChS/PVA-BBP0 showed the lowest Weq value as compared to the other samples. The freezing-thawing process consumes part of the hydroxyl groups of PVA due to the formation of hydrogen bond among them [31]. In addition, the higher crystallinity of ChS/PVA-BBP0 affects negatively the uptake ability [45]. On the other hand, adding BBP increases the uptake capacity of the biocomposites samples as compared to the pristine sample. At 1 wt.% BBP, the uptake capacity of ChS/PVA-BBP1 was enhanced ~24%. As assessed, such enhancement caused by BBP in the ChS/PVA matrix decreases as the amount of BBP increases. As above discussed, BBP is composed mainly of collagen and hydroxyapatite. Since collagen has a hydrophobic nature the enhancement of the

17

ACCEPTED MANUSCRIPT uptake capacity can be attributed to the hydroxyapatite, which have a large number of hydrophilic groups that can easily hydrate. The decreasing of uptake capacity calculated for

PT

ChS/PVA-BBP5 and ChS/PVA-BBP10 can be ascribed to the increment of the interactions

SC RI

between BBP-ChS/PVA matrixes. Therefore, the biocomposite matrix became rigid and the porosity and pore size decreased preventing liquid penetration. Such effect of BBP on the decreasing of the uptake capacity was markedly higher for BBP-based composite films [23].

NU

Liquid retention capacities calculated for the ChS/PVA-BBP(0-10) samples (Table 2) showed that the increasing of the content of BBP into the ChS/PVA matrix decreases the

MA

retention capacity. Conversely, even the biocomposite with the highest BBP content was capable to retain more than 45% of the liquid absorbed previously. This result suggests that

ED

the ChS/PVA-BBP biocomposites have considerable retention capacity due to their higher water-binding ability and allow confirming their hydrophilic nature. Hydrophilicity is an

PT

important property presented by a suitable scaffold for tissue engineering. Hydrophilic

CE

material allows the transport of waste and nutrients at the time of cell growth as well as helps

AC

to accumulate growth factors and provides compressive characteristics of the tissue.

3.4. In vitro biodegradability Biodegradability is a crucial property requested to a potential polymeric scaffold. It controls the long-term performance of the scaffold; regulates cellular growth rate and acts as mechanical/structural support while the new cells/tissue regenerates the treated area. In addition, the biodegradation of the polymeric scaffold assures that its fragments will be absorbed or eliminated by the body leaving no trace. Implantable polymeric scaffolds that have biodegradability under the biological conditions reduce the need for additional surgical intervention for explant at the end of their functional life or eventual reimplant [11]. Here, in vitro biodegradation tests were carried out with the ChS/PVA-BBP(0-10) samples using two

18

ACCEPTED MANUSCRIPT soaking conditions; PBS (pH 7.4) that mimics the intracellular human body fluids and tissues

degrade ChS [46]. The results are presented in Table 2.

PT

and PBS (pH 7.4) supplemented with hyaluronidase, a non-specific enzyme capable to

SC RI

From the biodegradation tests, we found that ChS/PVA-BBP0 sample was degraded ~13% after being soaked in PBS for 3 weeks. ChS/PVA-BBP1 showed a slight increasing of degradation (~14%), while the samples ChS/PVA-BBP5 and ChS/PVA-BBP10 showed the

NU

highest degradation values (about 50% and 48%, respectively). This increment regarding the degradation rate of the biocomposites with more than 5 wt.% of BBP as compared to the

MA

pristine sample can be attributed to the incorporation of BBP, which affects the ChS/PVA matrix. Such effects were also observed in the structure and mechanical, morphological and

ED

uptake properties of the biocomposites, especially for ChS/PVA-BBP5 and ChS/PVABBP10. These findings allow inferring that the addition of BBP into the polymeric matrix

PT

might control the degradation rate of the biocomposite. Such results differ from those reported

CE

by Alves et al., where the BBP did not influence the degradation rate of a chitosan/PVA-BBP composite [23]. This discrepancy may be due to the different morphology presented by that

AC

composite, which was fabricated as a film. Therefore, it can be inferred that the degradation rate of BBP-based composites depends on some parameters (e.g. BBP content, morphology of the composite, starting materials, etc.). Here, XRD and thermal analyses reveled that BBP affects the interactions within the ChS/PVA matrix, which towards degradation due to the solubilization (erosion) and removal of chain fragments outward the biocomposites. This process is favored likely due to the porous morphology of ChS/PVA-BBP composites. Overall, the incorporation of hydroxyapatite and/or collagen into the scaffold matrices causes an increment of the degradation rate, as reported elsewhere [47, 48]. The degradation rates calculated for the samples soaked in PBS supplemented with hyaluronidase for 21 days were quite similar to those calculated for the samples soaked in PBS (see Table 2). In this sense,

19

ACCEPTED MANUSCRIPT such results suggest that the enzyme does not affect degradability of the ChS/PVA-BBP(0-10)

PT

samples.

SC RI

3.5. In vitro cytotoxic test

In vitro tests were carried out to investigate the cytotoxic effect of ChS/PVA-BBP0 and ChS/PVA-BBP10 against healthy cells. As showed in Figure 4, any statistically

NU

significant variation in cell viability was verified for ChS/PVA-BBP0 and ChS/PVA-BBP10 as compared to control after 24 or 48 h of incubation. Up to 48 h of incubation, the samples

MA

did not affect the cell proliferation as compared to control. These findings suggest that both scaffolds are non-cytotoxic materials. It should be highlighted the absence of chemical

ED

crosslink agents and the use of mild conditions to fabricate the ChS/PVA-BBP biocomposite, which reduces the potential cytotoxicity of this potential biomaterial. This aspect represents

PT

an important advantage over other chemical crosslinked scaffold materials [51]. These

CE

preliminary results have demonstrated that both ChS/PVA-BBP0 and ChS/PVA-BBP10

AC

shows potential to be applied as a scaffold.

Figure 4

3.6. In vivo and ex vivo experiments After

subcutaneous

implantation

in

mice,

ChS/PVA-BBP

superporous

biocomposites were resorbable within 15 days and did not elicit a local inflammatory response (Figure 5). The results presented in Figure 6 showed ChS/PVA-BBP0 and ChS/PVA-BBP10 did not alter MPO activity in wound area when compared to the control group.

20

ACCEPTED MANUSCRIPT Figure 5

PT

The results presented in Figure 6 showed ChS/PVA-BBP0 and ChS/PVA-BBP10

SC RI

samples did not alter MPO activity in wound area when compared to the control group. An inflammatory cascade is initiated after implantation of a foreign in the body and enzymes released by neutrophils mediate it. Indeed, if the material is not enzymatically degraded, the

NU

inflammation process becomes chronic [52]. Smaller particles are phagocytized by macrophages, which are recruited at the beginning of the process. To phagocytose larger

MA

molecules, macrophages fuse to form foreign-body giant cells [53]. Additionally, if the materials were similar to the macromolecules in the body, they are degraded by

Figure 6

CE

PT

ED

macrophage-mediated phagocytosis, with limited inflammatory infiltration [54].

An interesting result found in the present study was that ChS/PVA-BBP

AC

biocomposites were biodegraded into two weeks and in this time the MPO activity was not altered in the wound area. It is well established that MPO activity is a marker of neutrophil influx into tissue [55]. This finding demonstrated that ChS/PVA-BBP biocomposites are biocompatibility materials without exert pro-inflammatory responses. It is important to highlight that this result is in agreement with results obtained in the cytotoxicity assay, supporting the ChS/PVA-BBP biocomposites security. These findings corroborate with the results previously reported by Alves et al. [23]. Using a mice model, the authors demonstrated that a chitosan/PVA-BBP based composite film was efficient to attenuate atopic dermatitis (AD)-like inflammatory symptoms by suppressing the increase of MPO activity [23]. Regarding the ChS/PVA-BBP composites, it should be mentioned that ChS

21

ACCEPTED MANUSCRIPT shows anti-inflammatory activity, which contributes to absence of cytotoxicity or adverse responses to the treated area [56].

PT

Taken together, the results presented here demonstrated that these porous

SC RI

biocomposites are future promising to wound healing treatments, especially as implantable devices. Further studies can be performed to investigate the cell/tissue regeneration process in this case, the biological role of the ChS/PVA-BBP biocomposite and the safety of BBP

NU

for biomedical applications.

MA

4. Conclusion

Biocomposites based on chondroitin sulfate (ChS), a sulfated glycosaminoglycan,

ED

poly(vinyl alcohol) (PVA) and bovine bone powder (BBP) were prepared through cyclic freeze-thawing method using mild conditions. The biocomposite formation was confirmed by

PT

FTIR, XRD and EDX analysis. SEM images demonstrated that all the biocomposites showed

CE

high porosity with an interconnected morphology. BBP is well dispersed into the biocomposite, which suggest a good interaction between the filler and the polymeric matrix.

AC

The systematic characterization of the biocomposites revealed that the content of BBP into the ChS/PVA matrix affects several physico-chemical and morphological properties. High content of BBP decreases the pore size to 87.3 ± 13.9 μm; however at 10 wt.% BBP the mechanical properties and the biodegradability rate are markedly enhanced as compared to the pristine sample. In vitro tests showed that ChS/PVA-BBP biocomposite does not exert a cytotoxic effect on healthy cells. In vivo and ex vivo experiments revealed that ChS/PVA-BBP biocomposites are biocompatibility materials without exert pro-inflammatory response; therefore, they are safe for use. All these results suggest that the eco-friendly biocomposite designed in this study is a promising biomaterial for potential skin tissue engineering. Furthermore, these results described in this study complement other results previously

22

ACCEPTED MANUSCRIPT reported by our research group. All these collected data strengthen the suggestion that BBP can be utilized as an eco-friendly and low-cost source of hydroxyapatite and collagen to

SC RI

PT

design original biomaterials.

Acknowledgments

NU

The authors thank to CAPES for a master fellowship to G.T.S and to CNPq for their

AC

CE

PT

ED

MA

financial support (Universal grant. - Process 441888/2014-3).

23

ACCEPTED MANUSCRIPT References [1] R.J. Wade, J.A. Burdick, Advances in nanofibrous scaffolds for biomedical applications:

PT

From electrospinning to self-assembly, Nano Today 9 (2014) 722-742. [2] S. Bose, M. Roy, A. Bandyopadhyay, Recent advances in bone tissue engineering

SC RI

scaffolds, Trend. Biotechnol. 30 (2012) 546-554.

[3] H. Geckil, F. Xu, X.H. Zhang, S. Moon, U. Demirci, Engineering hydrogels as extracellular matrix mimics, Nanomedicine 5 (2010) 469-484.

NU

[4] F. Khan, S.R. Ahmad, Polysaccharides and their derivatives for versatile tissue engineering application, Macromol. Biosci. 13 (2013) 395-421.

MA

[5] M.R. Fan, M. Gong, L.C. Da, L. Bai, X.Q. Li, K.F. Chen, J. Li-Ling, Z.M. Yang, H.Q. Xie, Tissue engineered esophagus scaffold constructed with porcine small intestinal submucosa and synthetic polymers, Biomed. Mater. 9 (2014) 015012.

ED

[6] S.H. Oh, J.H. Lee, Hydrophilization of synthetic biodegradable polymer scaffolds for improved cell/tissue compatibility, Biomed. Mater. 8 (2013) 014101.

PT

[7] E. Quinlan, S. Partap, M.M. Azevedo, G. Jell, M.M. Stevens, F.J. O'Brien, Hypoxiamimicking bioactive glass/collagen glycosaminoglycan composite scaffolds to enhance

CE

angiogenesis and bone repair, Biomaterials 52 (2015) 358-366. [8] S. Mathews, S.A. Mathew, P.K. Gupta, R. Bhonde, S. Totey, Glycosaminoglycans

AC

enhance osteoblast differentiation of bone marrow derived human mesenchymal stem cells, J. Tissue Eng. Regenerat. Med. 8 (2014) 143-152. [9] N.B. Shelke, R. James, C.T. Laurencin, S.G. Kumbar, Polysaccharide biomaterials for drug delivery and regenerative engineering, Polym. Adv. Technol. 25(5) (2014) 448-460. [10] W.H. Liang, B.L. Kienitz, K.J. Penick, J.F. Welter, T.A. Zawodzinski, H. Baskaran, Concentrated collagen-chondroitin sulfate scaffolds for tissue engineering applications, J. Biomed. Mater. Res. A 94 (2010) 1050-1060. [11] J. Venkatesan, R. Pallela, I. Bhatnagar, S.K. Kim, Chitosan-amylopectin/hydroxyapatite and chitosan-chondroitin sulphate/hydroxyapatite composite scaffolds for bone tissue engineering, Int. J. Biol. Macromol. 51 (2012) 1033-1042.

24

ACCEPTED MANUSCRIPT [12] R.A.A. Muzzarelli, F. Greco, A. Busilacchi, V. Sollazzo, A. Gigante, Chitosan, hyaluronan and chondroitin sulfate in tissue engineering for cartilage regeneration: A

PT

review, Carbohyd. Polym. 89 (2012) 723-739. [13] A.R. Fajardo, L.C. Lopes, A.O. Caleare, E.A. Britta, C.V. Nakamura, A.F. Rubira, E.C.

SC RI

Muniz, Silver sulfadiazine loaded chitosan/chondroitin sulfate films for a potential wound dressing application, Mater. Sci. Eng. C 33 (2013) 588-595. [14] S.Q. Yan, Q. Zhang, J.N. Wang, Y. Liu, S.Z. Lu, M.Z. Li, D.L. Kaplan, Silk fibroin/chondroitin sulfate/hyaluronic acid ternary scaffolds for dermal tissue

NU

reconstruction, Acta Biomater. 9 (2013) 6771-6782.

[15] A. Tocchio, M. Tamplenizza, F. Martello, I. Gerges, E. Rossi, S. Argentiere, S.

MA

Rodighiero, W.W. Zhao, P. Milani, C. Lenardi, Versatile fabrication of vascularizable scaffolds for large tissue engineering in bioreactor, Biomaterials 45 (2015) 124-131.

ED

[16] R.X. Hou, G.H. Zhang, G.L. Du, D.X. Zhan, Y. Cong, Y.J. Cheng, J. Fu, Magnetic nanohydroxyapatite/PVA composite hydrogels for promoted osteoblast adhesion and

PT

proliferation, Colloid Surface B 103 (2013) 318-325. [17] W.H. Wang, M. Zhang, W. Lu, X.J. Zhang, D.D. Ma, X.M. Rong, C.Y. Yu, Y. Jin,

CE

Cross-linked collagen-chondroitin sulfate-hyaluronic acid imitating extracellular matrix as scaffold for dermal tissue engineering, Tissue Eng. C 16 (2010) 269-279.

AC

[18] A. Matsiko, T.J. Levingstone, F.J. O'Brien, Advanced strategies for articular cartilage defect repair, Materials 6 (2013) 637-668. [19] T. Kaneiwa, S. Mizumoto, K. Sugahara, S. Yamada, Identification of human hyaluronidase-4 as a novel chondroitin sulfate hydrolase that preferentially cleaves the galactosaminidic linkage in the trisulfated tetrasaccharide sequence, Glycobiology 20 (2010) 300-309. [20] M. Vittekova, J. Dragunova, P. Kabat, M. Bozikova, D. Bakos, J. Koller, Cytotoxicity testing of scaffolds potentially suitable for the preparation of three-dimensional skin substitutes, Cell Tissue Bank. 15 (2014) 345-355. [21] M.M. Bradford, A rapid and sensitive method for the quantification of microgram quantitites of protein utilizing the principles of protein-dye binding, Anal. Biochem. 72 (1976) 6.

25

ACCEPTED MANUSCRIPT [22] M.M. Figueiredo, G. J.A.F., M. A.G., Characterization of bone and bone-based graft materials using ftir spectroscopy, in: T. Theophanides (Ed.), Infrared Spectroscopy - Life

PT

and Biomedical Sciences, InTech (2012). [23] N.O. Alves, G.T. da Silva, D.M. Webber, C. Luchese, E.A. Wilhem, A.R. Fajardo,

SC RI

Chitosan/poly(vinyl alcohol)/bovine bone powder biocomposites: A potential biomaterial for the treatment of atopic dermatitis-like skin lesions, Carbohyd. Polym. 148 (2016) 115-124.

[24] T. Thitiset, S. Damrongsakkul, T. Bunaprasert, W. Leeanansaksiri, S. Honsawek,

NU

Development of Collagen/demineralized bone powder scaffolds and periosteum-derived cells for bone tissue engineering application, Int. J. Mol. Sci. 14 (2013) 2056-2071.

MA

[25] E.G. Crispim, J.F. Piai, A.R. Fajardo, E.R.F. Ramos, T.U. Nakamura, C.V. Nakamura, A.F. Rubira, E.C. Muniz, Hydrogels based on chemically modified poly(vinyl alcohol) (PVA-GMA) and PVA-GMA/chondroitin sulfate: Preparation and characterization,

ED

Express Polym. Lett. 6 (2012) 383-395. [26] A.R. Fajardo, L.C. Lopes, A.G.B. Pereira, A.F. Rubira, E.C. Muniz, Polyelectrolyte

PT

complexes based on pectin-NH2 and chondroitin sulfate, Carbohyd. Polym. 87 (2012)

CE

1950-1955.

[27] K. Pietrucha, Physicochemical properties of 3D collagen-CS scaffolds for potential use

AC

in neural tissue engineering, Int. J. Biol. Macromol. 80 (2015) 732-739. [28] M. Huang, D.D. Cai, Y.H. Liu, J. Sun, J.J. Wang, C.X. Qin, L.X. Dai, Y. Kazuo, Investigation of a-PVA/s-PVA hydrogels prepared by freezing-thawing method, Fiber Polym. 13 (2012) 955-962. [29] C. Curley, J.C. Hayes, N.J. Rowan, J.E. Kennedy, An evaluation of the thermal and mechanical properties of a salt-modified polyvinyl alcohol hydrogel for a knee meniscus application, J. Mech. Behav. Biomed. Mater. 40 (2014) 13-22. [30] M.M. Liu, L.L. Wang, H.J. Su, H. Cao, T.W. Tan, pH-sensitive IPN hydrogel based on poly(aspartic acid) and poly(vinyl alcohol) for controlled release, Polym. Bull. 70 (2013) 2815-2827. [31] C.M. Hassan, N.A. Peppas, Structure and applications of poly(vinyl alcohol) hydrogels produced

by

conventional

crosslinking

or

by

freezing/thawing

methods,

26

ACCEPTED MANUSCRIPT Biopolymers/PVA Hydrogels/Anionic Polymerisation Nanocomposites, Springer Berlin Heidelberg 153 (2000) 37-65.

PT

[32] N.A. El-Zaher, W.G. Osiris, Thermal and structural properties of poly(vinyl alcohol) doped with hydroxypropyl cellulose, J. Appl. Polym. Sci. 96 (2005) 1914-1923.

SC RI

[33] A. Salerno, M. Fernandez-Gutierrez, J.S.R. del Barrio, C.D. Pascual, Macroporous and nanometre scale fibrous PLA and PLA-HA composite scaffolds fabricated by a bio safe strategy, RSC Adv. 4 (2014) 61491-61502.

[34] A.K. Gaharwar, S.A. Dammu, J.M. Canter, C.J. Wu, G. Schmidt, Highly Extensible,

NU

Tough, and Elastomeric Nanocomposite Hydrogels from Poly(ethylene glycol) and Hydroxyapatite Nanoparticles, Biomacromolecules 12 (2011) 1641-1650.

MA

[35] J.T. Podichetty, S.V. Madihally, Modeling of porous scaffold deformation induced by medium perfusion, J. Biomed. Mater. Res. B 102 (2014) 737-748.

ED

[36] A.M. Martins, G. Eng, S.G. Caridade, J.F. Mano, R.L. Reis, G. Vunjak-Novakovic, Electrically conductive chitosan/carbon scaffolds for cardiac tissue engineering,

PT

Biomacromolecules 15 (2014) 635-643. [37] A. Sergeeva, N. Feoktistova, V. Prokopovic, D. Gorin, D. Volodkin, Design of porous

CE

alginate hydrogels by sacrificial caco3 templates: Pore formation mechanism, Adv. Mater. Interface 2 (2015) 10-19.

AC

[38] C. Chang, N. Peng, M. He, Y. Teramoto, Y. Nishio, L. Zhang, Fabrication and properties of chitin/hydroxyapatite hybrid hydrogels as scaffold nano-materials, Carbohyd. Polym. 91 (2013) 7-13. [39] A. Gantar, L.P. da Silva, J.M. Oliveira, A.P. Marques, V.M. Correlo, S. Novak, R.L. Reis, Nanoparticulate bioactive-glass-reinforced gellan-gum hydrogels for bone-tissue engineering, Mater. Sci. Eng. C 43 (2014) 27-36. [40] A. Guha, A. Sinha, A Biomimetic approach to synthesized 3-dimensional poly(vinyl alcohol) hydroxyapatite scaffolds, Trend Nanotech. Mater. Sci. 1 (2016) 1-7. [41] C. Pailler-Mattei, S. Bec, H. Zahouani, In vivo measurements of the elastic mechanical properties of human skin by indentation tests, Med. Eng. Phys. 30 (2008) 599-606. [42] C. Pailler-Mattei, H. Zahouani, Study of adhesion forces and mechanical properties of human skin in vivo, J. Adhesion Sci.Technol. 18 (2004) 1739-1758.

27

ACCEPTED MANUSCRIPT [43] P.X. Ma, J.W. Choi, Biodegradable polymer scaffolds with well-defined interconnected spherical pore network, Tissue Eng. 7 (2001) 23-33.

PT

[44] C.M. Agrawal, J.S. McKinney, D. Lanctot, K.A. Athanasiou, Effects of fluid flow on the in vitro degradation kinetics of biodegradable scaffolds for tissue engineering,

SC RI

Biomaterials 21 (2000) 2443-2452.

[45] T. Takigawa, H. Kasihara, T. Masuda, Swelling and mechanical-properties of polyvinylalcohol hydrogels, Polym. Bull. 24 (1990) 613-618.

[46] A.R. Fajardo, A. Guerry, E.A. Britta, C.V. Nakamura, E.C. Muniz, R. Borsali, S. Halila,

NU

Sulfated glycosaminoglycan-based block copolymer: preparation of biocompatible chondroitin sulfate-b-poly(lactic acid) micelles, Biomacromolecules 15 (2014) 2691-

MA

2700.

[47] J.R. Ramya, K.T. Arul, P. Sathiamurthi, K. Asokan, S.N. Kalkura, Novel gamma

ED

irradiated agarose-gelatin-hydroxyapatite nanocomposite scaffolds for skin tissue regeneration, Ceramic Int. 42 (2016) 11045-11054.

PT

[48] H. Cao, M.M. Chen, Y. Liu, Y.Y. Liu, Y.Q. Huang, J.H. Wang, J.D. Chen, Q.Q. Zhang, Fish collagen-based scaffold containing PLGA microspheres for controlled growth factor

CE

delivery in skin tissue engineering, Colloid Surface B 136 (2015) 1098-1106. [49] P. Thangavel, B. Ramachandran, V. Muthuvijayan, Fabrication of chitosan/gallic acid

AC

3D microporous scaffold for tissue engineering applications, J. Biomed. Mater. Res 104 (2016) 750-760. [50] C.W. Lou, S.P. Wen, J.H. Lin, Chitosan/gelatin porous bone scaffolds made by crosslinking treatment and freeze-drying technology: Effects of crosslinking durations on the porous structure, compressive strength, and in vitro cytotoxicity, J. Appl. Polym. Sci. 132 (2015) 41851. [51] H.P. Tan, K.G. Marra, Injectable, biodegradable hydrogels for tissue engineering Applications, Materials 3 (2010) 1746-1767. [52] A.B. Gardner, S.K.C. Lee, E.C. Woods, A.P. Acharya, Biomaterials-based modulation of the immune system, Biomed. Res. Int. 2013 (2013) 1-7. [53] J.M. Anderson, A. Rodriguez, D.T. Chang, Foreign body reaction to biomaterials, Seminar Immunol. 20 (2008) 86-100.

28

ACCEPTED MANUSCRIPT [54] P.R. Chang, P.W. Zheng, B.X. Liu, D.P. Anderson, J.G. Yu, X.F. Ma, Characterization of magnetic soluble starch-functionalized carbon nanotubes and its application for the

PT

adsorption of the dyes, J. Hazard. Mater. 186 (2011) 2144-2150. [55] J.E. Krawisz, P. Sharon, W.F. Stenson, Quantitative assay for acute intestinal

SC RI

inflammation based on myeloperoxidase activity - assessment of inflammation in rat and hamster models, Gastroenterology 87 (1984) 1344-1350.

[56] F. Ronca, L. Palmieri, P. Panicucci, G. Ronca, Anti-inflammatory activity of chondroitin

AC

CE

PT

ED

MA

NU

sulfate, Osteoarthr. Cartilage 6 (1998) 14-21.

29

ACCEPTED MANUSCRIPT Figure captions

Figure 1. FTIR spectra (a) and XRD diffraction patterns (b) of ChS, PVA, BBP, ChS/PVA-

PT

BBP0 and ChS/PVA-BBP10.

SC RI

Figure 2. TGA (a,c) and DTG (b,d) curves of ChS, PVA, BBP and ChS/PVA-BBP(0-10) biocomposites.

NU

Figure 3. SEM images and EDX analysis of ChS/PVA-BBP0 (a,c,e) and ChS/PVA-BBP10 (b,d,f) cross-section. [(a,b) magnification x50; (c,d) magnification x300].

MA

Figure 4. Cytotoxic effect of ChS/PVA-BBP0 and ChS/PVA-BBP10 on Vero cells evaluated by MTT assay. Data represent the mean ± S.E.M.

ED

Figure 5. Representative photographic images of the extent of wound healing on days 0, 3, 9, and 15 for the developed ChS/PVA-BBP0 and ChS/PVA-BBP10 dressings as well as the

PT

untreated negative control (Control).

CE

Figure 6. Effect of ChS/PVA-BBP0 and ChS/PVA-BBP10 on MPO activity in wound area of

AC

mice. Data represent the mean ± S.E.M.

30

AC

Fig. 1a

CE

PT

ED

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

31

ED

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

Fig. 1b

32

ED

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

Fig. 2a

33

ED

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

Fig. 2b

34

ED

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

Fig. 2c

35

ED

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

Fig. 2d

36

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

Fig. 3a

37

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

Fig. 3b

38

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

Fig. 3c

39

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

Fig. 3d

40

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

Fig. 4a

41

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

Fig. 4b

42

ED

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

Fig. 5

43

NU

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

Fig. 6

44

ED

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

Fig. 7

45

ACCEPTED MANUSCRIPT Table 1. Mechanical properties of ChS/PVA-BBP0 and ChS/PVA-BBP10.

ChS/PVA-BBP0

70.1 ± 16.7

Young´s Modulus (KPa) 9.0 ± 2

ChS/PVA-BBP1

68.7 ± 12.4

9.1 ± 6

ChS/PVA-BBP5

87.5 ± 8.1

ChS/PVA-BBP10

95.7 ± 18.3

Brittleness (mm)

Deformation at rupture (%)

9.2 ± 0.2

93

8.9 ± 0.6

93

12.2 ± 7

9.2 ± 0.3

92

17.3 ± 2

9.3 ± 0.1

94

AC

CE

PT

ED

MA

NU

SC RI

PT

Sample

Rupture Force (N)

46

ACCEPTED MANUSCRIPT Table 2. Comparative analysis of total porosity, water uptake capacity at equilibrium, percentage of biodegradation in different media for ChS/PVA-BBP(0-10) biocomposites. Sample

Porosity (%)

Weq (%)

Wret (%)

ChS/PVA-BBP0

87.4 ± 0.7

252 ± 12

ChS/PVA-BBP1

88.1 ± 0.4

312 ± 21

ChS/PVA-BBP5

88.5 ± 0.6

269 ± 8

ChS/PVA-BBP10

65.6 ± 4.4

268 ± 9

Biodegradability (%) PBS + enzime

56.0 ± 12.4

12.9 ± 0.8

15.44 ± 1.4

59.0 ± 6.7

14.4 ± 0.7

12.9 ± 0.1

51.8 ± 2.0

50.5 ± 1.9

46.6 ± 3.6

45.2 ± 1.8

47.7 ± 3.1

40.5 ± 3.5

AC

CE

PT

ED

MA

NU

SC RI

PT

PBS

47

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

Graphical abstract

48

ACCEPTED MANUSCRIPT

Highlights > ChS/PVA/BBP biocomposite was designed through cyclic freeze-thaw method.

PT

> The biocomposite showed high porosity, water uptaking and biodegradability. > BBP is a low-cost source of the bioactive compounds; hydroxyapatite and collagen.

SC RI

> ChS/PVA/BBP biocomposites are non-cytotoxic and did not cause inflammatory processes.

AC

CE

PT

ED

MA

NU

> Tests showed that the biocomposites can be applied in skin tissue regeneration.

49