Accepted Manuscript Title: Bio-inspired aloe vera sponges for biomedical applications Author: S.S. Silva M.B. Oliveira J.F. Mano R.L. Reis PII: DOI: Reference:

S0144-8617(14)00512-8 http://dx.doi.org/doi:10.1016/j.carbpol.2014.05.042 CARP 8909

To appear in: Received date: Revised date: Accepted date:

27-11-2013 6-5-2014 14-5-2014

Please cite this article as: Silva, S. S., Oliveira, M. B., Mano, J. F., & Reis, R. L.,Bioinspired aloe vera sponges for biomedical applications, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.05.042 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.

1

Bio-inspired aloe vera sponges for biomedical applications

2

S S Silva a,b, M B Oliveira a,b, JF Mano a,b, RL Reis a,b

ip t

3 4

a

5

University of Minho, Headquarters of the European Institute of Excellence on

6

Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas,

7

Guimarães, Portugal

8

b

9

Portugal

us

cr

3B’s Research Group - Biomaterials, Biodegradables and Biomimetics,

an

ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães,

M

10

Corresponding-Author:

14

3B´s

15

Department of Polymer Engineering, University of Minho, Headquarters of the

16

European Institute of

17

Medicine – AvePark, Zona Industrial da Gandra – Caldas das Taipas – 4806-

18

909 Guimarães- Portugal – Fax: +351 253510909; ICVS/3B´s- PT Government

19

Associate Laboratory, Braga/Guimarães, Portugal

20

[email protected]

te

Simone S. Silva

Research Group- Biomaterials,

Biodegradables and Biomimetics,

Ac ce p

21

d

11 12 13

Excellence on Tissue Engineering and Regenerative

22 23

Highlights

24

- Sponges showed heterogeneous pore formation and interconnected pores

25

- Swelling behavior of the sponges was dependent on the pH 1 Page 1 of 26

26

- The sponges provided the sustained release of BSA-FTIC over 3 weeks

27

ip t

28 29

Abstract

31

Chemical composition and biological properties of Aloe vera (AV), a tropical

32

plant, explain its potential use for cosmetic, nutritional and biomedical

33

applications.

34

nutrients and polysaccharides. This work proposes using AV gel complex

35

structure and chemical composition, associated with freeze-drying, to produce

36

sponges. To increase the structures stability in aqueous media, a thin coating of

37

gellan gum (GG), was applied onto AV gel. AV-based sponges showed a

38

heterogeneous porous formation, interconnected pores and good porosity (72-

39

77%). The coating with a GG layer onto AV influenced the stability, swelling

40

behavior and mechanical properties of the resulting sponges. Moreover,

41

sponges provided the sustained release of BSA-FTIC, used as a model protein,

42

over 3 weeks. Also, in vitro cell culture studies evidenced that sponges are not

43

cytotoxic for a mouse fibroblast-like cell line. Therefore, developed AV-based

44

sponges have potential use in biomedical applications.

us

cr

30

Ac ce p

te

d

M

an

AV gel present in AV leaves is rich in several compounds,

45 46

Keywords: aloe vera gel; sponges; gellan gum; regenerative medicine

47 48 2 Page 2 of 26

49 50

ip t

51 52

1. Introduction

cr

53

Worldwide, there is a need for the search for new therapies and drugs due to

55

the incidence of diseases such as cancer and diabetes. As such, the use of

56

plants as a natural resource of bioactive phytochemical compounds may be

57

useful for developing new formulations and strategies to enhance life quality. It

58

is recognized that some plant extracts contain compounds and polysaccharides,

59

which display anticancer, antioxidant, antibacterial and antiviral activities. Aloe

60

vera (AV), a tropical plant belonging to the liliaceae family, is known to be the

61

oldest medicinal plant in Nature (Gage, 1996). AV plant per se is an unique

62

resource of the natural compounds, nutrients and polysaccharides with several

63

bioactivities (Boudreau & Beland, 2006; Hamman, 2008; Reynolds & Dweck,

64

1999). This chemical diversity has encouraged its utilization in cosmetic

65

formulations, food supplements and medical devices (Cole & Heard, 2007;

66

Hamman, 2008; Rodriguez Rodriguez, Darias Martin & Diaz Romero, 2010;

67

Silva, Caridade, Mano & Reis, 2013; Silva et al., 2013). By its turn, the

68

properties of AV gel, contained in the inner parts of the fresh AV leaves

69

(Hamman, 2008) have anti-inflammatory, antioxidant, antiviral and antibacterial

70

action. These properties are associated to its complex composition. AV gel has

71

also been useful to design and create functional films, microspheres and

72

sponges. Most of these biomaterials have been prepared by combining AV gel

Ac ce p

te

d

M

an

us

54

3 Page 3 of 26

with bacterial cellulose, chitosan, alginate, and gelatin (Chen, Wang & Weng,

74

2010; Pereira, Tojeira, Vaz, Mendes & Bartolo, 2011; Saibuatong &

75

Phisalaphong, 2010; Silva, Caridade, Mano & Reis, 2013; Silva et al., 2013).

76

For instance, Silva et al (Silva et al., 2013) reported that a small portion of AV

77

gel into chitosan provided blended membranes with adequate roughness,

78

degradation rate, wettability and mechanical properties to be used as wound

79

dressing materials. Due to the wide use of AV, a high number of possibilities are

80

still open to investigation. Therefore, in the present work, the exploitation of AV

81

gel, in the development of sponges using freeze-drying technique was

82

evaluated. It is well known that freeze drying is one of the most popular

83

technique for producing three dimensional (3D) porous structures based on

84

natural or synthetic polymers (Reys et al., 2011; Silva et al., 2008). This

85

technique is based on the formation of ice crystals that induce porosity through

86

ice sublimation and desorption (Mano et al., 2007); that could be useful for

87

increasing the shelf-life of Aloe components (Krokida, Pappa & Agalioti, 2011).

88

To increase the stability of the structures in aqueous media, a thin coating layer

89

of gellan gum (GG), was applied onto AV gel. GG is a well-known water soluble,

90

biocompatible polysaccharide that could be used as an adhesive to apply

91

seasoning to the surfaces of foods (Oliveira et al., 2010b). In addition, GG has

92

been proposed for tissue engineering applications (Oliveira et al., 2010a;

93

Oliveira et al., 2010b; Silva-Correia et al., 2013). We hypothesized that the

94

developed AV-based sponges could represent a natural therapeutic strategy,

95

where the natural biological activity of AV may act in different stages of the

96

tissue regeneration. To evaluate the potential use of AV-based sponges as a

97

carrier for therapeutic molecules, the sponges were labelled with a model

Ac ce p

te

d

M

an

us

cr

ip t

73

4 Page 4 of 26

protein, namely bovine serum albumin labelled with fluorescein isothiocyanate

99

(BSA-FTIC). Furthermore, the studies presented in this article focus on

100

understanding the morphological features, mechanical properties and cellular

101

response of the AV-based sponges.

ip t

98

cr

102 103

2. Materials and methods

105

2.1. Materials

106

Fresh whole aloe vera (Aloe barbadensis Miller) leaves, obtained from a

107

Portuguese botanic shop, were used as the raw material in all experiments. The

108

studied leaves, between 30 and 40 cm of length, corresponded to 4-year old

109

plants. Low-acyl gellan gum (GG, GelzanTM) was purchased from Sigma-Aldrich

110

Co., USA. All other reagents were obtained from Sigma Aldrich Co. (USA),

111

unless otherwise indicated.

112

2.2. Methods

113

2.2.1. Preparation of the aloe vera-based sponges

114

All whole aloe vera leaves were washed with distilled water to remove dirt from

115

the surface. The skin was separated from the parenchyma using a scalpel-

116

shaped knife. The AV filets were extensively washed with distilled water to

117

remove the exudates from their surfaces. AV fillets were cut in small cubes

118

(diameter 0.8 cm). Gellan gum (GG) powder was mixed with distilled water

119

under constant stirring at room temperature to obtain a concentration of 1%

Ac ce p

te

d

M

an

us

104

5 Page 5 of 26

(w/v). The solution was heated to 90C during 20-30 minutes. Afterwards, the

121

temperature was progressively decreased to 50C (Oliveira et al., 2010a).

122

Further, the molded AV gels were immersed into GG solution during 30

123

seconds, in order to create a thin layer on the material. After that, AVG matrices

124

were rinsed with phosphate buffer solution (PBS). Then, both matrices (AV and

125

AVG) were stabilized at 65C for 15-20 minutes, and at 4C for 5 minutes,

126

followed by freezing

127

sponges were also prepared without any GG coating. The identification of the

128

AV-based sponges was AVG and AV, for those prepared with and without GG

129

coating, respectively.

130

2.2.2. Bovine serum albumin (BSA) incorporation

131

The AV-based sponges were incubated into bovine serum albumin labeled with

132

fluorescein isothiocyanate (BSA-FTIC) at a concentration of 2 mg/ml overnight

133

at 4C to avoid the gel oxidation. Afterwards, the samples were frozen at -80 C

134

and submitted to freeze-drying for 3 days.

135

3. Characterization

136

3.1. Scanning electron microscopy (SEM)

137

Samples of the freeze-dried hydrogels were observed by a Leica Cambridge

138

S360 Scanning Electron Microscope. The materials were fixed by mutual

139

conductive adhesive tape on aluminum stubs and covered with gold palladium

140

using a sputter coater.

cr

ip t

120

Ac ce p

te

d

M

an

us

at -80C overnight and freeze-drying for 3 days. AV

141 142

3.2. Micro-computed tomography (µ-CT)

6 Page 6 of 26

The microstructure of the sponges was evaluated using a high-resolution μ-CT

144

Skyscan 1072 scanner (Skyscan,Kontich, Belgium) with a resolution pixel size

145

of 6.59 μm and integration time of 1.9 s. The x-ray source was set at 32 keV

146

and 143 μA. Approximately 500 projections were acquired over a rotation range

147

of 180◦with a rotation step of 0.45◦. Data sets were reconstructed using

148

standardized cone-beam reconstruction software (NRecon v1.4.3, SkyScan).

149

The output format for each sample was 500 serial 1024 ×1024 bitmap images.

150

A representative data set of 500 slices was segmented into binary images with

151

a dynamic threshold of 57–255 (grey values). The same representative volume

152

of interest (VOI) was analysed for all the samples. These data sets were used

153

for morphometric analysis (CT Analyser, v1.5.1.5, SkyScan) and to build the 3D

154

models (ANT 3D creator, v2.4, SkyScan).

155

3.3. Swelling behavior

156

Swelling tests were performed by immersing the sponges in different buffer

157

solutions (pH 5, 7.4 and 10) up to 360 minutes. The swollen sample weights

158

were measured after removing excess surface water by gently tapping the

159

surface with filter paper. The percentage of water uptake was calculated using

160

equation 1, where ws is the weight of the swollen sample and wd is the weight of

161

the dry sample. Each experiment was repeated three times, and the average

162

value was considered to be the water uptake value.

Ac ce p

te

d

M

an

us

cr

ip t

143

Equation 1

163

3.4. Stability

164

The AVG matrices (6.5 mm diameter, 4.5 mm height) were immersed in

7 Page 7 of 26

phosphate buffer solution (PBS) at 37 C for up to 30 days. Samples were

166

collected at different time points, washed gently with distilled water, dried at

167

40C and weighed. Percentage loss in weight at respective time points was

168

calculated.

ip t

165

169

3.5. In vitro BSA release

171

BSA-FTIC-loaded sponges were weighted and suspended in 3 ml of PBS. The

172

samples were immersed in a thermostatic water bath at 37C and 60 rpm.

173

Aliquots of 300 µl were withdrawn at predetermined time intervals, and the

174

same volume of fresh buffer was added to the suspension. The samples were

175

analyzed by UV-spectroscopy at 280 nm and 495 nm to eliminate the

176

contribution of the FITC absorption at 280 nm in a microplate reader (Synergy

177

HT, Bio-Tek Instruments, USA). The results presented are the average of three

178

measurements. Calculations on the amount of drug released took into account

179

the replacement of aliquots with fresh medium. The concentration of BSA-FTIC

180

was calculated using a standard curve (concentrations ranging from 0.0 to 1

181

mg/mL), relating the amount of BSA-FTIC with the intensity of light absorbance.

us

an

M

d

te

Ac ce p

182

cr

170

183

3.6. Dynamical mechanical analysis (DMA)

184

The viscoelastic measurements of the AV sponges before and after GG coating

185

were performed using a TRITEC2000B DMA from Triton Technology (UK),

186

equipped with a compression mode. The matrices were always analyzed in

187

hydrated conditions through immersion of the samples in a Teflon reservoir

188

containing PBS, used during all analysis period. After equilibration at 37C, the

8 Page 8 of 26

DMA spectra were obtained during a frequency scan between 0.1 and 10 Hz.

190

The experiments were performed under constant strain amplitude (30 µm). The

191

average value of four tests was reported for each sample.

192

3.7. Cytotoxicity tests

193

Before testing, the samples were sterilized under an ethylene oxide

194

atmosphere. To assess the possible cytotoxicity of the samples, extracts

195

(leachables) of both sponges (AV and AVG) were evaluated following MEM

196

extraction tests (72 h), according to ISO-10993-5 guidelines (ISO/10993, 1992).

197

In MEM extraction tests, both materials and the control (latex as positive

198

control) were extracted by incubation in culture medium (the same used for cell

199

culture) for 24 h at 37C and 60 rpm (in thermostatic bath) (Gomes, Reis,

200

Cunha, Blitterswiijk & de Bruijn, 2001). After the defined period of incubation,

201

the resulting solution was removed and filtrated, obtaining the extraction fluid.

202

For these tests a mouse fibroblast-like cell line, L929 cells, (European

203

Collection of Cell Cultures-ECACC, UK) at passage 16 was used. L929 cells

204

were cultured in basic medium: Dulbecco’s Modified Eagle’s Medium (DMEM,

205

Sigma-Aldrich, USA) with phenol red and supplemented with 10% foetal bovine

206

serum, FBS (Gibco, UK) and 1% antibiotic/antimycotic (A/B, Gibco, UK) solution

207

and incubated at 37◦C in an atmosphere containing 5% CO2 until achieving

208

90% confluence. Then, a cell suspension was prepared with a concentration of

209

1×10

210

h. The metabolic activity of L929 cells in contact with the extracts of the

211

sponges was assessed using an AlamarBlue assay. For this assay, an Alamar

212

Blue solution (AlamarBlueCell Viability Assay Protocol, Life Technologies)

213

was prepared, added to the medium and incubated for 4 h at 37C. The optical

Ac ce p

te

d

M

an

us

cr

ip t

189

4

cells ml–1 and seeded on extracts in 24-well plates and incubated for 72

9 Page 9 of 26

density (OD) was read at 570 and 600 nm in a multiwell microplate reader

215

(Synergy HT, BioTek Instruments). The L929 cell viability (%) was determined

216

for each extract and compared to tissue culture polystyrene (TCPS) wells, used

217

as a negative control of cell death. Latex extracts were considered as a positive

218

control of cellular death.

ip t

214

cr

219

3.8 Statistical analysis

221

All quantitative experiments were run in triplicate and results were expressed

222

as mean ± standard deviation for n=3. Statistical analysis of the data was

223

conducted using two-way ANOVA with Bonferroni´s posthoc comparisons by

224

means of using the GraphPadPrism version 5.0 for Windows (GraphPad

225

Software, San Diego, http://www.graphpad.com). Differences between the

226

groups with p0.05 were considered to be statistically significant.

an

M

d te

227

us

220

4. Results and discussion

229

4.1. Morphological features

230

For construction of AV–based sponges (AV and AVG), the AV gel was molded

231

in cylindrical shapes (0.8 cm diameter, Figure 1a). Even though the matrices

232

showed good mechanical stability, the stabilization of AV gel should be

233

considered before use. Then, the stabilization of AV-based structures (AV and

234

AVG) was performed by applying high temperature (65C) for a short period of

235

time (15-20 min). This process will preserve the biological activity of the AV gel.

236

GG is a linear anionic polysaccharide that forms thermoreversible gels, and it

Ac ce p

228

10 Page 10 of 26

has demonstrated non-cytotoxicity in different studies (Oliveira et al., 2010b). In

238

our work, a thin layer of GG solution (1 wt%) was applied on molded AV gel.

239

Since the gelation of GG solutions is influenced by the presence of cations

240

(Oliveira et al., 2010a), the matrices were rinsed with PBS which allowed the

241

formation of a three dimensional network on the AV gels, increasing their

242

stability and mechanical properties.

243

Both developed AV an AVG sponges showed a heterogeneous porous

244

formation distributed in the matrix (Figure 1b-1d). Moreover, the freeze-drying

245

process allowed retaining properly the biological activity of the gel and

246

preserving the cell wall polysaccharides network of AV. It is also increased the

247

shelf-life of the produced sponges (Krokida, Pappa & Agalioti, 2011).

Ac ce p

te

d

M

an

us

cr

ip t

237

248 11 Page 11 of 26

249

Figure 1. Photographs of the AV gel (A), AVG sponge (B) and representative

250

SEM image of the surface of the AV (C) and AVG sponges (D).

251

Micro-CT analysis was used to obtain quantitative information of the 3D

253

architecture of the aloe-based sponges. The 3D micro-CT images, obtained

254

from digital geometry processing from a series of two-dimensional x-ray images,

255

are illustrated in Figure 2. From the 3D AV image we observed a close pore

256

structure as compared to AVG. The values of interconnectivity (Table 1)

257

indicated that all structures have interconnected pores, which may help

258

homogeneous cell distribution and transferring of nutrients effectively, as well as

259

to facilitate cell growth within the 3D porous structures (Madihally & Matthew,

260

1999). The pore size distribution of the samples (Figures 3) presented values

261

ranging between 50 and 300 µm. The AV sponges have also a wide pore size

262

distribution, while AVG sponges have a narrow pore size distribution. In one

263

hand, AV gel is a nature made sponge, possessing high values of porosity (76.9

264

± 4.6 %) and interconnectivity (79.3 ± 10.1%) that could be adequate for

265

regenerative medicine approaches. On the other hand, the morphological

266

features of AV sponges were affected by the GG coating, where the AVG

267

sponges have values of porosity and interconnectivity lower than AV (Table 1).

Ac ce p

te

d

M

an

us

cr

ip t

252

12 Page 12 of 26

ip t cr us an M d te

268

Figure 2. 3D image reconstructions of the sponges (AV and AVG) obtained by

270

µ-CT seen on a lateral and top views.

271

Table 1: Values of porosity and interconnectivity for AV and AVG sponges.

Ac ce p

269

Sample

AV

AVG

Porosity (%)

76.9 ± 4.6

72.3 ± 9.4

Interconnectivity (%)

79.3 ± 10.1

74.6 ± 4.9

272 273

13 Page 13 of 26

ip t cr an

us 277 278 279

d te

276

Figure 3. Porosity distribution of AV and AVG.

Ac ce p

275

M

274

280

Swelling and stability behavior

281

Depending on the site of implantation, the biomaterials are subjected to different

282

pH environments, which could affect their degradation, mechanical and swelling

283

properties. To determine how the pH affects the swelling behavior, AVG

284

sponges were immersed in different buffer solutions at pH 5, 7.4 and 10. AVG

285

samples immersed at pH 10 and pH 7.4 showed an appreciable swelling

286

compared to the ones immersed at pH 5. Statistical differences (p< 0.05) were 14 Page 14 of 26

observed for pH 5 in relation to pH 10 and pH 7.4 along the swelling time. The

288

AV gel is rich in hydroscopic polysaccharides such as acemannan (Hamman,

289

2008), which together with the acid resistance of the coating layer of GG at low

290

pH (Picone & Cunha, 2011), could explain the greater swelling in high pH and

291

its decreasing at pH 5. GG is able to form gels through an ionotropic gelation

292

mechanism (Oliveira et al., 2010b); where an increase in pH leads to the

293

formation of a reduced number of junction regions (Picone & Cunha, 2011).

294

Other studies on aloe vera/alginate films demonstrated that an increase in AV

295

content implied an increase in the water absorption ability at pH 7.4 (Pereira,

296

Tojeira, Vaz, Mendes & Bartolo, 2011), as result of the hydrophilicity of AV.

an

us

cr

ip t

287

298

Ac ce p

te

d

M

297

299

Figure 4: Swelling profiles of AVG sponges in different buffer pH´s (A) and

300

weight loss (B) in function of immersion time on PBS at 37C. Data represent

301

the mean ± standard deviation (p< 0.05, two-way ANOVA).

302

In tissue engineering approaches, the 3D structures are expected to degrade

303

during tissue regeneration. Thus, it is important to characterize the structural

304

stability of AV-based sponges to understand their potential for application in 15 Page 15 of 26

tissue engineering strategies. The structural stability of the AV and AVG

306

sponges was investigated by weight loss after their incubation in PBS up to 30

307

days in order to simulate physiological conditions. During the assay, the AV

308

sponges gradually dissolved, leading to the formation of gels and loss of their

309

integrity after a few days (data not shown). Compared with AV, AVG sponges

310

showed a delayed disintegration and increased structural stability. This result

311

could be attributed to the presence of the GG coating on AV. In the presence of

312

salt ions, contained in PBS, the number of junction zones in GG is enhanced

313

(Picone & Cunha, 2011). This leads to an increase in the gel rigidity, increasing

314

its stability to aqueous medium. Furthermore, the AVG sponges showed a

315

weight loss of AVG was about 30 wt% along the degradation period (Figure 4B)

316

that can be related with the soluble compounds.

M

an

us

cr

ip t

305

d

317

Mechanical properties and in vitro release studies

319

Almost all biological tissues show native viscoelastic properties (Sasaki, 2012).

320

Those are conferred by constituents such as cells, extracellular matrix and

321

proteins. These properties are of uttermost importance for the correct function

322

of the tissue that must be able to stand the physiological load, as well as be

323

capable of dissipating mechanical energy during cyclic solicitation. Upon the

324

implantation of a biomaterial, it is important to make sure that the structure is

325

compatible also with the tissue mechanical properties, so that it allows for

326

correct biomaterial integration. Moreover, it was shown that the mechanical

327

properties of the biomaterials influence the proliferation and even differentiation

328

phenomena of cells (Engler, Sen, Sweeney & Discher, 2006). In order to

Ac ce p

te

318

16 Page 16 of 26

investigate possible applications of the AVG sponges in the regenerative

330

medicine field, we assessed the viscoelastic properties of the structures by

331

dynamic mechanical analysis (DMA). Mechanical analysis of the AV sponge

332

was not conducted as the sponge disintegrate along the tests. The AVG

333

sponges developed in this work present a storage modulus (E’) – a measure of

334

the material’s stiffness - varying from average values of 5 kPa to 10 kPa (Figure

335

5A), over a biological relevant frequency range (0.1 Hz to 10 Hz, respectively).

336

These E’ values are in the range of elastic modulus measured for several soft

337

tissues present in the human body. For example, skeletal muscle was reported

338

to have an elastic modulus of 7 kPa (Chino, Akagi, Dohi, Fukashiro &

339

Takahashi, 2012) and subcutaneous fat tissue a modulus varied from 0.12 kPa

340

to 30 kPa, according to the applied measuring method (Geerligs, van Breemen,

341

Peters, Ackermans, Baaijens & Oomens, 2011; Hendriks, Brokken, Van

342

Eemeren, Oomens, Baaijens & Horsten, 2003). Skin tissue in wet state was

343

evaluated in 10 kPa–100 MPa (Derler & Gerhardt, 2012). Although in this case

344

the E’ reported value is higher than the one of the biomaterial proposed herein,

345

it was previously shown that the formation of extracellular matrix by cells

346

seeded in biomaterials increase their stiffness (Popa, Caridade, Mano, Reis &

347

Gomes, 2013; Silva-Correia et al., 2013), and the final tissue may show

348

properties similar to the native tissue (Silva-Correia et al., 2013). Soft biological

349

tissues play an important role in the mechanical integrity of the body. Indeed,

350

these tissues transfer loads between bones to ligaments, or between muscles

351

and bones to the tendons (Silva, Mano & Reis, 2010). They have an important

352

viscous component in their behavior. The loss factor or damping (herein

353

presented as tan ) is a measure of the viscoelasticity of materials, as it

Ac ce p

te

d

M

an

us

cr

ip t

329

17 Page 17 of 26

correlates the elastic and viscous components. Regarding the loss factor of the

355

AVG sponges, the value 0.5 indicates that the material showed a marked

356

viscoelastic behavior (Figure 5A). Moreover, this value is also close to the loss

357

factors of native tissues, such as skin, which showed in in vivo testing a

358

damping of 0.6-0.8 (Sandford, Chen, Hunter, Hillebrand & Jones, 2013).

359

Moreover, brain tissue showed loss factor values of 0.4-0.6 (Fallenstein, Hulce

360

& Melvin, 1969). Considering the results obtained in the analysis of the

361

viscoelastic properties of this new biomaterial, we conclude that it may have

362

potential in tissue engineering of soft tissues, as its properties resemble those

363

from the native tissues.

364

Ac ce p

te

d

M

an

us

cr

ip t

354

365

Figure 5: Evaluation of the viscoelastic properties of AVG sponges by DMA.

366

Frequency scans were performed in the range 0.1-10 Hz under wet conditions

367

at 37C (A), and BSA-FTIC release profile of the samples (▲) AV and (*) AVG

368

(B). Data represent the mean ± standard deviation (p< 0.05, two-way ANOVA.

369 370

In vitro release studies

18 Page 18 of 26

The characteristics of AV-based sponges could be interesting in the

372

construction of a drug delivery system. In this sense, BSA-FTIC was used as a

373

model to analyze their profile release from the AV-based sponges. The release

374

profile of the samples was evaluated in PBS for a time period up to 3 weeks.

375

We hypothesized that the presence of GG coating onto AV (AVG) will act as a

376

barrier to BSA release. In Figure 5B the release profile of BSA from AV and

377

AVG is shown. It seems that AVG sponges have a faster release than AV,

378

suggesting that the protein was dispersed in the sponge. Although the

379

encapsulated protein on AV could be easily released in the aqueous

380

environment, the findings suggested that the interactions between BSA-FTIC

381

and AV were stronger than AVG, controlling the protein release. Typically,

382

conventional drug delivery systems provide a sharp increase in the

383

concentration of the drug, followed by a rapid decrease in subsequent time

384

periods, until new administration. In controlled delivery systems designed for

385

long term administration the concentration should increase slowly in the

386

beginning of the process, followed by a steady release. The findings obtained

387

for both AV and AVG suggest that a controlled drug release system for

388

therapeutic molecules could be developed from them.

cr

us

an

M

d

te

Ac ce p

389

ip t

371

390

Cytotoxicity assays

391

Considering the potential use of these materials in regenerative medicine, a

392

cytotoxicity assessment of both AV and AVG sponges was carried out as a

393

preliminary approach to determine the toxicity potential the sponges. The cell

394

viability values found for AV, AVG extracts and tissue culture polystyrene were

19 Page 19 of 26

166.9 ± 3.9, 155.8 ± 6.4% and 151.4 ± 8.3%, respectively. The obtained data

396

indicates that both AV and AVG do not exert any cytotoxic effect on L929 cells,

397

which supports the concept that these materials could be used in contact with

398

the body. In fact, the complex composition of AV gel could enhance the wound

399

healing, influencing the wound vascularization and contraction due to the

400

presence of specific active compounds such as beta-sitosterol (Yadav, Kumar,

401

Basha, Deshmukh, Gujjula & Santhamma, 2012). For instance, AV gel extracts

402

have been shown to stimulate and speed up the production of hyaluronic acid

403

and dermatan sulphate (Chithra, Sajithlal & Chandrakasan, 1998). Therefore, it

404

is likely that the multiple active phytochemicals present in the AV gel may be

405

required to address different aspects of the wound healing. Further in vitro

406

direct cell contact assays will be performed to gain a better understanding of the

407

cellular behavior of AV-based sponges.

408

te

d

M

an

us

cr

ip t

395

5. Conclusions

410

Versatile 3D sponges were successful developed from native aloe vera gel

411

using the freeze-drying technique. Both AV-based sponges (AV and AVG) have

412

a heterogeneous porous formation distribution in the matrix, with an

413

interconnected pore structure and good porosity. Moreover, the presence of a

414

coating layer of gellan gum onto aloe vera matrices improved the stability and

415

mechanical properties of AV sponges. The swelling of AVG was dependent on

416

the pH at which the sponges were prepared. Also, the sponges provided the

417

sustained release of BSA-FTIC, used as a release model for over 3 weeks.

418

Moreover, the low cytotoxicity levels of the sponge extracts are encouraging for

Ac ce p

409

20 Page 20 of 26

419

further in vitro and in vivo studies. All findings suggest that the AV-based

420

sponges can be promising candidates for biomedical applications, including in

421

the regenerative medicine field.

ip t

422

Acknowledgments

424

The authors acknowledge financial support from Portuguese Foundation for

425

Science

426

SFRH/BD/64601/2009),

427

Diferencial de Potencial Humano-POPH". This work was partially supported by

428

European Union Seventh Framework Programme (FP7/2007-2013) under grant

429

agreement no. KBBE-2010-266033 (project SPECIAL).

-FCT

(Grant

SFRH/BPD/45307/2008

and

us

Technology

“Fundo Social Europeu”-

FSE,

and “Programa

M

an

and

cr

423

te

References

d

430

Boudreau, M. D., & Beland, F. A. (2006). An evaluation of the biological and

432

toxicological properties of Aloe barbadensis (miller), Aloe vera. Journal of

433

Environmental Science and Health Part C - Environmental Carcinogenesis &

434

Ecotoxicology Reviews, 24(1), 103-154.

435

Chen, C. P., Wang, B. J., & Weng, Y. M. (2010). Physiochemical and

436

antimicrobial properties of edible aloe/gelatin composite films. International

437

Journal of Food Science and Technology, 45(5), 1050-1055.

438

Chino, K., Akagi, R., Dohi, M., Fukashiro, S., & Takahashi, H. (2012). Reliability

439

and Validity of Quantifying Absolute Muscle Hardness Using Ultrasound

440

Elastography. PLoS ONE, 7(9), e45764.

Ac ce p

431

21 Page 21 of 26

Chithra, P., Sajithlal, G. B., & Chandrakasan, G. (1998). Influence of Aloe vera

442

on the glycosaminoglycans in the matrix of healing dermal wounds in rats.

443

Journal of Ethnopharmacology, 59(3), 179-186.

444

Cole, L., & Heard, C. (2007). Skin permeation enhancement potential of Aloe

445

Vera and a proposed mechanism of action based upon size exclusion and pull

446

effect. International Journal of Pharmaceutics, 333(1-2), 10-16.

447

Derler, S., & Gerhardt, L. (2012). Tribology of Skin: Review and Analysis of

448

Experimental Results for the Friction Coefficient of Human Skin. Tribology

449

Letters, 45, 1-27.

450

Engler, A., Sen, S., Sweeney, H., & Discher, D. (2006). Matrix Elasticity Directs

451

Stem Cell Lineage Specification. Cell, 126, 677-689.

452

Fallenstein, G. T., Hulce, V. D., & Melvin, J. W. (1969). Dynamic mechanical

453

properties of human brain tissue. Journal of biomechanics, 2(3), 217-226.

454

Gage, D. (1996). Aloe Vera: Nature's Soothing Healer. Rochester: Healing Arts

455

Press.

456

Geerligs, M., van Breemen, L., Peters, G., Ackermans, P., Baaijens, F., &

457

Oomens, C. (2011). In vitro indentation to determine the mechanical properties

458

of epidermis. Journal of biomechanics, 44, 1176-1181.

459

Gomes, M. E., Reis, R. L., Cunha, R., Blitterswiijk, C., & de Bruijn, J. (2001).

460

Cytocompatibility and response of osteoblastic-like cells to starch-based

461

polymers: effect of several additives and processing conditions. Biomaterials,

462

22(13), 1911-1917.

463

Hamman, J. H. (2008). Composition and applications of Aloe vera leaf gel.

464

Molecules, 13(8), 1599-1616.

Ac ce p

te

d

M

an

us

cr

ip t

441

22 Page 22 of 26

Hendriks, F., Brokken, D., Van Eemeren, J., Oomens, C., Baaijens, F., &

466

Horsten, J. (2003). A numerical-experimental method to characterize the non-

467

linear mechanical behaviour of human skin. Skin Research and Technology., 9,

468

274-283.

469

ISO/10993. (1992). Biological evaluation of medical devices Part 5: Tests for in

470

vitro cytotoxicity, (ISO 10993-5:2009).

471

Krokida, M., Pappa, A., & Agalioti, M. (2011). Effect of drying on Aloe's

472

functional components. 11th International Congress on Engineering and Food

473

(Icef11), 1, 1523-1527.

474

Madihally, S. V., & Matthew, H. W. T. (1999). Porous chitosan scaffolds for

475

tissue engineering. Biomaterials, 20(12), 1133-1142.

476

Mano, J., Silva, G., Azevedo, H. S., Malafaya, P., Sousa, R., Silva, S., Boesel,

477

L., Oliveira, J. M., Santos, T., & Marques, A. (2007). Natural origin

478

biodegradable systems in tissue engineering and regenerative medicine:

479

present status and some moving trends. Journal of the Royal Society Interface,

480

4(17), 999-1030.

481

Oliveira, J., Martins, L., Picciochi, R., Malafaya, P., Sousa, R., Neves, N., Mano,

482

J., & Reis, R. (2010a). Gellan gum: a new biomaterial for cartilage tissue

483

engineering applications. Journal of Biomedical Material Research A, 93(3),

484

852-863.

485

Oliveira, J., Santos, T., Martins, L., Picciochi, R., Marques, A. P., Castro, N.,

486

Mano, J. F., & Reis, R. L. (2010b). Gellan Gum Injectable Hydrogels for

487

Cartilage Tissue Engineering Applications: In Vitro Studies and PreliminaryIn

488

Vivo Evaluation. Tissue Engineering Part A, 16, 343-353.

Ac ce p

te

d

M

an

us

cr

ip t

465

23 Page 23 of 26

Pereira, R., Tojeira, A., Vaz, D. C., Mendes, A., & Bartolo, P. (2011).

490

Preparation and Characterization of Films Based on Alginate and Aloe Vera.

491

International Journal of Polymer Analysis and Characterization, 16(7), 449-464.

492

Picone, C., & Cunha, R. (2011). Influence of pH on formation and properties of

493

gellan gels. Carbohydrate Polymers, 84, 662-668.

494

Popa, E. G., Caridade, S. G., Mano, J. F., Reis, R. L., & Gomes, M. E. (2013).

495

Chondrogenic

496

encapsulated adipose stem cells for cartilage tissue-engineering applications.

497

Journal

498

10.1002/term.1683.

499

Reynolds, T., & Dweck, A. C. (1999). Aloe vera leaf gel: a review update.

500

Journal of Ethnopharmacology, 68(1-3), 3-37.

501

Reys, L. L., Silva, S. S., Oliveira, J. M., Frias, A. M., Mano, J. F., Silva, T. H., &

502

Reis, R. L. (2011). Valorization of Chitosan from Squid Pens and Further Use

503

on the Development of Scaffolds for Biomedical Applications. International

504

Journal of Artificial Organs, 34(8), 704-704.

505

Rodriguez Rodriguez, E., Darias Martin, J., & Diaz Romero, C. (2010). Aloe

506

vera as a functional ingredient in foods. Critical Reviews in Food Science and

507

Nutrition, 50(4), 305-326.

508

Saibuatong, O.-a., & Phisalaphong, M. (2010). Novo aloe vera–bacterial

509

cellulose composite film from biosynthesis. Carbohydrate Polymers, 79(2), 455-

510

460.

511

Sandford, E., Chen, Y., Hunter, I., Hillebrand, G., & Jones, L. (2013). Capturing

512

skin properties from dynamic mechanical analyses. Skin Research and

513

Technology, 19(1), e339-e348.

cr

Engineering

and

hydrogel

us

injectableκ-carrageenan

Regenerative

Medicine,

with

DOI:

an

Tissue

of

Ac ce p

te

d

M

of

potential

ip t

489

24 Page 24 of 26

Sasaki, N. (2012). Viscoelastic Properties of Biological Materials. In D. Vicente

515

(Ed.). Viscoelasticity - From Theory to Biological Applications: In Tech.

516

Silva-Correia, J., Gloria, A., Oliveira, M. B., Mano, J. F., Oliveira, J. M.,

517

Ambrosio, L., & Reis, R. L. (2013). Rheological and mechanical properties of

518

acellular and cell-laden methacrylated gellan gum hydrogels. Journal of

519

Biomedical Materials Research Part A, 101(12), 3438–3446.

520

Silva, S. S., Caridade, S. G., Mano, J. F., & Reis, R. L. (2013). Effect of

521

crosslinking

522

applications. Carbohydrate Polymers, 98(1), 581–588.

523

Silva, S. S., Mano, J. F., & Reis, R. L. (2010). Potential applications of natural

524

origin polymer-based systems in soft tissue regeneration. Critical Reviews in

525

Biotechnology, 30(3), 200-221.

526

Silva, S. S., Motta, A., Rodrigues, M. T., Pinheiro, A. F., Gomes, M. E., Mano, J.

527

F., Reis, R. L., & Migliaresi, C. (2008). Novel Genipin-Cross-Linked

528

Chitosan/Silk

529

Biomacromolecules, 9(10), 2764-2774.

530

Silva, S. S., Popa, E. G., Gomes, M. E., Cerqueira, M., Marques, A. P.,

531

Caridade, S. G., Teixeira, P., Sousa, C., Mano, J. F., & Reis, R. L. (2013). An

532

investigation of the potential application of chitosan/aloe-based membranes for

533

regenerative medicine. Acta Biomaterialia, 9(6), 6790-6797.

534

Yadav, K., Kumar, J., Basha, S., Deshmukh, G., Gujjula, R., & Santhamma, B.

535

(2012). Wound healing activity of topical application of aloe vera gel in

536

experimental animal models International Journal of Pharma and Bio Sciences,

537

Vol 3/Issue 2/April – June2012(2), 63-72.

cr

vera-based

membranes

for

us

chitosan/aloe

biomedical

te

d

M

an

in

ip t

514

Sponges

for

Cartilage

Engineering

Strategies.

Ac ce p

Fibroin

538 25 Page 25 of 26

539 540 541 542

Graphical abstract

ip t

543 544

an

us

cr

545

M

546

Ac ce p

te

d

547

26 Page 26 of 26

Bio-inspired Aloe vera sponges for biomedical applications.

Chemical composition and biological properties of Aloe vera (AV), a tropical plant, explain its potential use for cosmetic, nutritional and biomedical...
971KB Sizes 0 Downloads 8 Views