In situ production of bionanocomposite based on bacterial cellulose and collagen

In situ production of bionanocomposite based on bacterial cellulose and collagen

XII International Macromolecular Colloquium 7th International Symposium on Natural Polymers and Composites

Ígor A. N. Donini1* , Denise B. De Salvi1

, Sybele Saska1

, Hernane S. Barud1

, Wilton R. Lustri2

, Sidney J. L. Ribeiro1

Younes Messaddeq1 , Reinaldo Marchetto1

1* Instituto de Química – UNESP, Caixa Postal 355, 14800-900 Araraquara/SP - igordonini@iq.unesp.br

2 Centro Universitário de Araraquara – UNIARA- wrlustri@gmail.com

Bacterial Cellulose (BC) and collagen are biopolymers with important structural properties. The first, produced by

Gluconacetobacter xylinus, is a linear homopolysaccharide of -D-glicopiranose that shows high thermal and mechanical resistance as well as biodegradability and biocompatible features. Collagen is the most abundant animal protein composed of triple helixes of glicine-proline-X sequence repetition (X is any one amino acid), and shows low antigenicity and physicochemical characteristics ideal for tissue adhesion. This work report the development of a bionanocomposite (BC+Col) based on BC and in situ collagen addition in static culture medium. The material properties were analyzed by MEV, FT-IR, XRD and Thermal Analysis (TG). The collagen insertion was indirectly determined by hydroxyproline analysis. The in situ method was successfully in the production of BC+Col which showed high collagen concentration in BC nanofibers network. XRD peaks decrease in the BC intensity, related possibly to the collagen presence in the BC structures. BC+Col showed high thermal resistance, good homogeneity and biological properties in agree with its possible application as biomaterial in tissue engineering.

Introduction

The tissue regeneration targets the major clinical problems through the repair, replacement and ultimate regeneration of tissues and organs using novel technologies emerging from several scientific and clinical studies. Tissue injury presents a clinical and socio-economic burden; consequently, repair and functional recovery of damaged tissues, including the reduction of regeneration time, is a major goal for researchers and clinicians alike. Many new and exciting solutions to tissue injury are made possible by tissue engineering and regenerative medicine, and, in particular, through biomaterial engineering which uses natural and synthetic biomaterials to provide the tissues regeneration [1]. In this context, a new generation of resorbable materials has been developed for tissue regeneration purposes, including bacterial cellulose (BC) [2].

Figure 1 – Bacterial Cellulose produced by static medium with in situ collagen (CB+Col) addition (a), and dried CB+Col membranes (b).

BC is obtained from cultures of Gram-negative bacteria Acetobacter xylinum (Gluconacetobacter xylinus) as highly hydrated membranes and display great elasticity, high wet strength, and conformability. The gelatinous membrane formed in static culture (Figure 1a) is characterized by a 3-D structure consisting of an ultrafine network of cellulose nanofibres (“nanocelluloses”), resulting in a large nano-porous surface area [3]. The unique properties provided by the nanometric structure of BC have lead to a number of commercial products and medical applications for tissue regeneration [4]. Collagen type I (Col I) is the biopolymer predominant in most animals and is found especially in the skin, tendons and bones. Synthetic biomaterials with physical and chemical structure similar to the tissues have been developed for allow rapid penetration of cells, nerves and blood vessels into the biomaterial. This makes it possible regeneration and tissue remodeling natural, while maintaining its biological function [5]. So, we hypothesized that collagen introduction during the bacterial cellulose production in static medium could result in a bionanocomposite with important characteristics such as biocompatibility, biodegradability and selective permeability for future medicinal applications especially for bone and skin regeneration.

Experimental

BC membranes was produced in static medium in 7 days cultivation, at 28o C in Hestrim-Schramm modified medium (HS) containing manitol as carbon source [6]. Collagen type I (Col I) from tail rats (purchased from Sigma-Aldrich Co.) was added at 1 to 5% (v/v) to the medium for producing bionanocomposites called CB+Col. The CB+Col membranes produced were autoclaved by 15 minutes at 121o

C, dried for 5 days at 37o C, and characterized as following below.

XII International Macromolecular Colloquium 7th International Symposium on Natural Polymers and Composites

Biomaterials Characterizations

Thermogravimetric (TG) analysis of the dried samples were recorded using TA SDT 2960 from TA Instruments Co. Samples were heated in open α-alumina pans from 40 to

600°C under nitrogen atmosphere (flow rate: 70 mL min –1 ) at heating rate of 10°C min –1 . X-rays diffraction (XRD) patterns were obtained using a Kristalloflex Simens Diffractometer with a Ni filter and Cu Kα radiation from 2 and 4 θ. Fourier Transform Infrared (FT-IR) spectra were obtained with dried powdered samples on a Perkin Elmer Spectrum 2000 FT-IR Spectrophotometer. Pellets were prepared from mixtures of the samples and KBr (1:100 in weight). Thirty-two scans were accumulated at a resolution of 4 cm −1 . Scanning Electron Microscopy (SEM) images was performed with a JEOL JSM 7500F. Samples were coated a 1 nm thick layer of gold for 60 s with a current of 40 mA.

Results and Discussion

Thermogravimetric (TG) analysis (Figure 2) shows an initial smooth weight loss (5 to 10 %) due the water losses. At 250°C and 300°C, the BC decomposition leads to an important weight loss. For pure BC membrane is observed carbonic residues of around 20%. For the different CB+Col bionanocomposites the residual mass was dependent of the quantity of in situ collagen added (between 20 and 40%). The insertion of the collagen fiber into cellulose caused a decrease in its stability (characterized by Tonset) which may be related to disruption of hydrogen bonds and a decrease in crystallinity of cellulose. Irradiation leads to no change in the characteristic temperatures.

Temperature (oC)

CB Pura CB+Col 1% CB+Col 2% CB+Col 3% CB+Col 4% CB+Col 5%

Figure 2 – Thermo gravimetric comparative analysis between BC produced and BC+Col synthetic biomaterials.

The FT-IR spectra of dried BC membrane and BC+Col bionanocomposites are shown in Figure 3. Characteristic vibrational frequencies assigned to cellulose were observed stretching of CH2 and CH3 groups), 1645 cm involving C–O strecthing) [7]. The pure collagen shows the typical bands for proteins around 1656 and 1547 cm -1 , related to the C = O stretching of amide I and amide I deformation for NH, respectively (Figure 3). This band was observed in all samples of BC+Col, confirming the collagen presence. The observed band around 1430 cm -1 attributed to the OH deformation of cellulose glycopiranosil residues (red arrow – Figure 3) showed a decreased of the intensity with the collagen presence, suggesting the BC+Col composite formation. This intensity decreased is due possibly to the hydrogen bonds formation to the stabilization of the BC+Col composites.

Figure 3 –FTIR spectra of BC (a) and BC+Col bionanocomposites (b-f, respectively, from 1 to 5 % collagen (v/v). The insert black arrows indicated the characteristic protein amide groups at 1650 cm-1.

Typically BC crystalline phases were also observed in BC+Col composites. Diffraction peaks at 15° and 2.5° are assigned to the cellulose Iα and Iβ phases (1001α, 1101β and 0101β planes at 15° and 1101α and 2001β at 2.5°) [8]. Collagen XRD resulted in a broad peak in the 2θ range of 15º-35º, typical XRD pattern of pure collagen and of an amorphous polymer and low cristallinity. The BC+Col diffractogram analysis showed a decrease in the BC intensity peaks, related possibly to the collagen presence in the BC structure (Figure 5).

Figure 5 – X-ray diffraction patterns of BC (a) collagen (b) and BC-Col 1% nanocomposite (c).

2(degree)

(b)

XII International Macromolecular Colloquium 7th International Symposium on Natural Polymers and Composites

Scanning Electron Microscopy (SEM) of a typical dried BC membrane is showed in the Figure 4. An ultrafine network structure is formed by continuous nanofibers about 10-50 nm wide (“nanocelluloses”) can be observed. This nanometric structure leads to a large surface area for particles stabilization [9].

Figure 4 – SEM image of Bacterial Cellulose

The BC+Col composite micrograph (Figure 5) shows that collagen filled and covered homogeneously the BC structure, resulting in a more compressed BC due the pores filling by the collagen. This is an important characteristic for a biomaterial to be employed in tissue regeneration or as mechanical barrier in ROG. Therefore, could help further the cell occlusion function, preventing the undifferentited mesenchymal cells and fibroblasts infiltrations as well as the invagination of connective tissues to the tissue defect more effectively. Another feature is that the surface roughness of the membrane can promote the adhesion and spreading of osteoblastic cells.

Figure 5 – SEM image of the surface of nanocomposite CB + Collagen.

Conclusions

Bacterial cellulose-collagen bionanocomposites were prepared by in situ collagen addition in the static medium BC production and the material analysis showed that the method is appropriated independently of the collagen quantity added. The proposed method is simpler than to modified BC fibers after its production, although not allow the collagen cross-linking. Low crystallinity materials were obtained, due basically to the amorphous collagen presence. The physical-chemical properties and structural characteristics of the bionanomaterials based on bacterial cellulose and collagen obtained by our method makes a great possibility for future medicinal applications especially for bone and skin regeneration.

Acknowledgements

We gratefully acknowledge FAPESP, CNPq and CAPES for financial support.

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