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Bacterial cellulose as a potential scaffold for tissue, Notas de estudo de Engenharia de Produção

Bacterial cellulose as a potential scaffold for tissue

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Baixe Bacterial cellulose as a potential scaffold for tissue e outras Notas de estudo em PDF para Engenharia de Produção, somente na Docsity! Biomaterials 26 (2005) 419–431 ARTICLE IN PRESS*Correspondin 418. E-mail addres 0142-9612/$ - see doi:10.1016/j.bioBacterial cellulose as a potential scaffold for tissue engineering of cartilage A. Svenssona,b, E. Nicklassonb,c, T. Harraha, B. Panilaitisa, D.L. Kaplana, M. Brittbergc, P. Gatenholmb,* aDepartment of Chemical and Biological Engineering, Tufts University, Boston, MA 02155, USA bDepartment of Material and Surface Chemistry/Biopolymer Technology, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden cDepartment of Orthopaedics, Cartilage Research Unit, G .oteborg University, Kungsbacka Hospital, SE-434 80 Kungsbacka, Sweden Received 11 June 2003; accepted 19 February 2004Abstract Tissue constructs for cartilage with native mechanical properties have not been described to date. To address this need the bacterial cellulose (BC) secreted by Gluconacetobacter xylinus (=Acetobacter xylinum) was explored as a novel scaffold material due to its unusual material properties and degradability. Native and chemically modified BC materials were evaluated using bovine chondrocytes. The results indicate that unmodified BC supports chondrocyte proliferation at levels of approximately 50% of the collagen type II substrate while providing significant advantages in terms of mechanical properties. Compared to tissue culture plastic and calcium alginate, unmodified BC showed significantly higher levels of chondrocyte growth. Chemical sulfation and phosphorylation of the BC, performed to mimic the glucosaminoglycans of native cartilage, did not enhance chondrocyte growth while the porosity of the material did affect chondrocyte viability. The BC did not induce significant activation of proinflammatory cytokine production during in vitro macrophage screening. Hence, unmodified BC was further explored using human chondrocytes. TEM analysis and RNA expression of the collagen II from human chondrocytes indicated that unmodified BC supports proliferation of chondrocytes. In addition, ingrowth of chondrocytes into the scaffold was verified by TEM. The results suggest the potential for this biomaterial as a scaffold for tissue engineering of cartilage. r 2004 Elsevier Ltd. All rights reserved. Keywords: Cellulose; Cartilage tissue engineering; Chondrocytes; Scaffold1. Introduction The main purposes of articular cartilage, which contains a small number of cells (chondrocytes) in an extra cellular matrix (ECM) mainly composed of water, collagen type II and proteoglycans, are to cover the ends in bones in joints to provide frictionless movement and to distribute loads [1–3]. Ostheoarthritis is a disease of synovial joints resulting in pain and loss of function for the patient [1,4]. Damaged cartilage has limited regen- erative capacity and therefore over 1 million patients in the United States require treatment for cartilage defects each year. However, presently these treatments result in limited pain relief and/or restorative tissue functiong author. Tel.: +46-317-723-407; fax: +46-317-723- s: pg@pol.chalmers.se (P. Gatenholm). front matter r 2004 Elsevier Ltd. All rights reserved. materials.2004.02.049[5–10]. Thus, tissue engineering has potential to provide a supply of functional cartilage for the repair and regeneration of compromised native soft tissues [10,11]. The use of scaffolds in the tissue engineering of cartilage is essential in order to support cell proliferation and maintain their differentiated function in addition to definition of the shape of the new growing tissue [12]. To this end, a variety of scaffold materials have been evaluated including natural polymers like collagen [13–17], alginate [13,16–18], hyaluronic acid [11,13,16,17], fibrin glue [13,16,17] and chitosan [13,16,17] and synthetic polymers including polyglycolic acid (PGA) [5,7,15–17], polylactic acid (PLA) [9,16,17], polyvinyl alcohol (PVA) [13,16,17,19], polyhydroxyethylmethacrylate (pHEMA) [13,16,17] and polyN-isopropylacrylamide (pNIPAA) [13,16,17]. However, tissue constructs with native mechan- ical properties have not yet been described in the literature. It is also desirable to utilize a scaffold material that has the ARTICLE IN PRESS A. Svensson et al. / Biomaterials 26 (2005) 419–431420porosity necessary to support cell ingrowth and effective mass transport while also supplementing the mechanical properties of engineered tissue. This scaffold must also be biocompatible and support native ECM and biopo- lymer production, with degradation rates commensurate with the rate of the new tissue formation. In the present study, we have explored a novel biomaterial, which meets these requirements, bacterial cellulose (BC). BC is secreted by Gluconacetobacter xylinus (=Acetobacter xylinum) and has unique proper- ties including high water holding capacity, high crystal- linity, a fine fiber network, and high tensile strength [20–24]. BC has recently been studied for use as artificial skin and blood vessels [25]. BC has potential to be used as a substrate for tissue engineering of cartilage due to its high strength in the wet state as well as its moldability in situ, biocompatibility and relatively simple, cost- efficient production. We report the response of primary bovine chondro- cytes on native and chemically modified BC. Phophory- lation and sulfation of matrices were explored in order to add surface charges to mimic the glucosaminoglycans in cartilage tissue in vivo. The materials were analyzed for mechanical properties, microstructure and cell– material interactions in order to assess the potential for this matrix as a scaffold for tissue engineering of cartilage. Chondrocyte response to collagen type II, plant-derived cellulose, calcium alginate and tissue culture plastic were also compared to the BC matrices. The unmodified BC was further explored using human chondrocytes. The RNA-expression of ECM markers, including chondrocyte specific collagen II and fibroblast specific collagen I, was analyzed to study if BC supports proliferation of chondrocytes. In addition, the potential of BC to support cell ingrowth was investigated.2. Methods 2.1. Production of BC G. xylinus (ATCC 10245) purchased from the Amer- ican Type Culture Collection was grown in 10 g/l Bactopeptone (Difco), 10 g/l yeast extract (Fisher), 4mm KH2PO4 (Sigma), 6mm K2HPO4 (Sigma) and 20 g/l d-glucose dissolved in deionized water (DI). The pH of the medium was adjusted to 5.1–5.2. Five milliliters media were inoculated with culture (third cultivation from single colonies) over night at 0.6% v/v in 6-well tissue culture plates and incubated statically at 30C for 7 days. BC pellicles were purified by treatment of 4% SDS (Fisher) in DI at 70C for 3 h and then 4% NaOH in DI at 70C for 90min. Samples were rinsed with DI to pH=7 and stored in DI at room temperature prior to use.2.2. Chemical modifications 2.2.1. Phosphorylation Water was removed from samples by solvent ex- change (70% ethanol-100% ethanol-DMF). Sam- ples were phosphorylated at 120–130C with reaction media of 159ml DMF, 83ml Toluene and 8ml 85% H3PO4 for 30min (BC-P1) and 2 h (BC-P2), respectively. Toluene was refluxed during the reaction and water was collected with a Dean–Stark apparatus. Samples were serially rinsed in DMF, ethanol and DI at room temperature. Samples treated by the identical procedure but without the addition of acid were prepared as controls (BC-P1c and BC-P2c). All samples were lyophilized overnight prior to analysis with ESCA. 2.2.2. Sulfation Water was removed as above. Twenty-five grams sulfamic acid (NH2SO3H) (Sigma) was dissolved in 103ml DMF. This solution was added to 147ml DMF at 80C. Samples were added and the reaction was performed at 80C for 90min. Samples were rinsed with DMF, 100% ethanol, 70% ethanol at room temperature and then with DI at 80C. Samples treated by identical procedure without the addition of acid were prepared as controls (BC-Sc). All samples were lyophilized overnight prior to analysis with ESCA. 2.3. Control samples 2.3.1. Collagen Two-hundred and fifty microliters of 0.1% Collagen type II (Davol, Cranston, RI) solution in 0.2% acetic acid was cast in 24-well plates, dried overnight at room temperature in a fume hood and ethanol sterilized (20min contact and 1 h drying at room temperature). 2.3.2. Alginate High viscosity alginate, sodium salt (Macrocystis pyrifera) was purchased from Sigma, thoroughly mixed as a 1% solution in DI and stored overnight at 4C. The resulting gel was pipetted into a Delrin mold of appropriate dimension and allowed to crosslink for 48 h in 1m CaCl2 with stirring. After gelation was complete, disks of appropriate thickness were sectioned, rinsed twice in an excess of DI and immediately autoclaved at 121C for 20min. 2.3.3. Tissue culture plastic Tissue culture plastic refers to the unmodified surface of Falcon (3524) 24-well tissue culture plates. 2.3.4. Plant derived cellulose Whatman Qualitative filter paper (cellulose), rinsed in an excess of DI and autoclaved, was used as a source of plant derived cellulose (cotton linters) surfaces. ARTICLE IN PRESS A. Svensson et al. / Biomaterials 26 (2005) 419–431 4232.9. Statistical analysis Student’s t-test was used to evaluate statistical significance of differences between sample preparation conditions with respect to the controls with po0:05: 3. Results 3.1. Surface chemistry and morphology ESCA analysis (Table 1) showed that the unmodified samples contained oxygen and carbon with a ratio ofFig. 1. Scanning electron micrographs of s Table 1 Surface analysis of modified and unmodified bacterial cellulose by ESCA At% BC BC-P1 BC-P2 BC-S O 45.74 41.64 43.16 45.10 N 0.00 1.46 1.44 4.28 C 52.25 51.87 50.08 44.89 P 0.00 1.48 2.33 0.00 S 0.00 0.00 0.00 4.16 Si 2.01 3.55 2.99 1.56approximately 5:6. Phosphorous and sulfur were not observed for BC. Initial analysis showed that the amount of phosphorous in the sample was low in both BC-P1 and BC-P2. In order to protonate the surface and thereby remove any salt, some phosphorylated samples were subsequently rinsed with hydrochloric acid at pH 3 for 30 s and 3min. These samples showed no phosphor- ous when analyzed with ESCA. BC-S contained about 4.2% sulfur, which corresponds to a degree of substitu- tion of 0.57. According to subsequent results, the sulfate groups are covalently bonded to the cellulose and the nitrogen signal derives from ammonium ions. As can be seen from the SEM images in Fig. 1, the morphology of the cellulose network of the different samples varied depending on the treatment. Chemical processing of BC-P1 and BC-P2 and BC-S appears to have resulted in a compaction of the native structure. The sulfated samples were more compacted than the phosphorylated samples. Mostly a flat and compact surface structure of BC-S was observed. Confocal microscopy analysis showed the same trend as the SEM analysis (see Fig. 2). Fluorescence signals from cellulose chains were seen from all samples excepturface morphology. Scale bar=4mm. ARTICLE IN PRESS Fig. 2. Confocal microscopy images of cellulose network in wet state. Cellulose fibrils are grafted with the fluorescent probe DTAF. Scale bar=4mm. TGA 0.0000 0.1000 0.2000 0.3000 0.4000 0.5000 0.6000 0.7000 0.8000 BC BC-P1 BC-P2 BC-S D ry w ei g h t (% ) Fig. 3. Dry weight of the samples determined by TGA. N ¼ 5: A. Svensson et al. / Biomaterials 26 (2005) 419–431424the sulfated versions. Higher power on the laser was needed to visualize BC-P1 and BC-P2. As expected [28], the water content of all samples was high (Fig. 3). The dry weight of unmodified BC was about 0.4% and statistical analysis showed that there was no significant difference between the water contents of the different samples. 3.2. Mechanical properties The compressive modulus of modified and unmodi- fied BC and alginate are shown in Figs. 4a and b. The chemical treatments increased the compressive modulus of BC. Also, alginate showed a higher compressive modulus than unmodified BC. Samples with a thickness of 3mm showed higher resistance to compressive forces compared to samples with a thickness of 0.3mm. The Young’s modulus and ultimate tensile strength of BC-P1 did not differ significantly from BC. However, BC-P2 and especially BC-S had significantly lower Young’s modulus and lower ultimate tensile strength than unmodified BC (see Fig. 5).3.3. Cell study I Representative SEM images of cell attachment on unmodified and modified BC surfaces are shown in Fig. 6. Chondrocytes adhered on BC but maintained a spherical shape, while the cells on BC-P1, BC-P2 and BC-S appeared to adhere more strongly to the surface and showed a more extended morphology. As can be seen from the phase contrast microscope images in Fig. 7, chondrocytes adhered and proliferated ARTICLE IN PRESS 0.000 2.000 4.000 6.000 8.000 10.000 12.000 14.000 16.000 BC BC BC-P2 BC-S Articular cartilage M P a 0.0 5.0 10.0 15.0 20.0 25.0 BC BC-P1 BC-P2 BC-S M P a Young's modulus Ultimate tensile strength (a) (b) Fig. 5. Young’ modulus and ultimate tensile strength of modified and unmodified BC, compared to articular cartilage [29–31]. N ¼ 10: 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 BC BC-P1 BC-P2 BC-S Alginate kP a (a) 0.00 50.00 100.00 150.00 200.00 250.00 300.00 BC Alginate kP a (b) Confined compression ( Sample thickness=0.3mm) Confined compression (Sample thickness=3mm) Fig. 4. Compressive modulus of modified and unmodified BC and alginate. Sample thickness: (a) 0.3mm and (b) 3mm, respectively. N ¼ 6: Compressive modulus of articular cartilage vary between 0.5 and 1MPa [29–31]. A. Svensson et al. / Biomaterials 26 (2005) 419–431 425on all materials. No signs of dead cells were seen except from the sulfated samples where some of the cells detached from the surface. After 8 days of cultivation, the cells on all materials had grown to or near confluence. However, the cells appeared to proliferatemore rapidly on tissue culture plastic and unmodified BC than on the other materials. The MTT data describe the relative viability of chondrocytes growing on the different materials. The data are comparable since the same number of cells was added to all samples. The viability of chondrocytes growing on BC was significantly higher than on tissue culture plastic, a frequently used substrate for cultiva- tion of cells, as well as on alginate, which is a well- known, biocompatible material commonly used in cartilage-related biomaterials (Fig. 8). The viability of chondrocytes growing on collagen type II, considered a positive control since collagen type II is the predominant extracellular matrix substrate in native cartilage tissue, showed no significant difference in MTT to BC, BC-P1, BC-P2 and BC-S. Samples treated only with the solvent systems used in the chemical modification reactions (BC-P1c, BCP2c, BC-Sc) also showed no significant differences in MTT absorbance. In addition, it was noticed that chondrocytic viability was affected by network density. Chondrocytes growing on a more porous, i.e. less dense network of unmodified BC showed significantly lower viability (data not shown). Lipopolysaccharide (LPS), a natural macrophage activator, was used in the immune response tests for a reliable comparison among samples. The amount of released TNF in the tissue culture plastic sample containing LPS (positive control) was almost 1 ng and this was regarded as a high immune response (Fig. 9). All samples except collagen showed a high degree of macrophage activation. Alginate, which is a well-known biocompatible material [16,33], induced an immune response in the same range as both unmodified and modified BC. Macrophage activation studies of the media supernatants from cultivation of chondrocytes on the different materials were also performed (data not shown). These results correlate well with the results from the macrophage activation of the materials in Fig. 9 except that all native BC sample supernatants showed elevated responses in comparison to those of the chemically treated samples. 3.4. Cell study II As can be seen in Fig. 10, human chondrocytes cultivated on unmodified BC express collagen I and collagen II B. Collagen I was detected in all samples while collagen II B was seen in two-thirds of the samples. Collagen II A could not be detected in any of the samples. From the TEM images (Figs. 11b and c) it can be seen that human chondrocytes growing at the surface of the cartilage matrix obtain an elongated morphology while a more spherical morphology is noted for cells in the bulk of the matrix. Fig. 11a shows that human chondrocytes growing in BC scaffolds demonstrate a ARTICLE IN PRESS Fig. 9. Immune response. Fig. 10. Gene expression of collagen I and II A/B in human chondrocytes culture passage 0 and 1. A. Svensson et al. / Biomaterials 26 (2005) 419–431428compressive forces with increasing thickness. Alginate showed a higher compressive modulus than unmodified BC, but this difference was not statistically significant (Figs. 4a and b). The compressive modulus of both unmodified BC and alginate was lower than the compressive modulus of articular cartilage, which is about 0.5–1MPa [29–31]. The compressive modulus of the phosphorylated samples increased with longer reaction times and were higher than the compressive modulus of BC (Fig. 4a). This result was probably due to the more compact structure of the networks of BC-P1 and BC-P2 in comparison to BC. BC-S showed an even higher resistance to compressive forces, which was likely due to the even more compact network structure. Compression modulus is a material property that is strongly dependent on solid content. BC used in this study had a water content of approximately 99%. It is possible to improve solid content and thus the compressive modulus. Tensile tests showed that the Young’s modulus of BC was in the same range as articular cartilage [29]. As can be seen in Fig. 5, BC-S had significantly lower Young’s modulus than unmodified BC. Apparently the sulfation reaction resulted in a reduction of mechanical integrity. The lower strength of BC-S may be due to prevention of hydrogen bonds between the cellulose chains by covalently bonded sulfate groups, chain scission by acid hydrolysis, or a combination of these changes.4.3. Cell study I The more extended morphology of the chondrocytes growing on modified BC samples (Fig. 6) may depend on that the chondrocytes adhered more strongly to the phosphorylated and sulfated samples due to surface charges. Chondrocytes must adhere to a surface to be able to grow and proliferate but if the adhesion is too strong, growth and proliferation is prevented [32]. However, the MTT results indicate that the chemical modifications did not alter chondrocyte pro- liferation. All native BC sample supernatants showed elevated responses in the macrophage activation studies com- pared to those of the chemically treated samples. This effect may be related to the presence of low molecular weight cellulose cleavage products, which are removed during chemical modification procedures. 4.4. Cell study II Collagen I is a fibroblast marker which is expressed to a greater extent by chondrocytes growing in a mono- layer. Chondrocytes growing in monolayer become fibroblastic and lose their characteristic pattern of matrix protein production [34]. Collagen II A and B are specific markers for cells differentiated into chon- drocytes in articular cartilage. In more mature chon- drocytes collagen II B is more prevalent than collagen II A [35]. According to the results in cell study II (Figs. 10 and 11a), collagen I is probably expressed by chondro- cytes growing at the surface, while collagen II B is most likely expressed by the cells that have migrated into the scaffold and that have obtained a more spherical morphology. This indicates that BC is able to support growth of chondrocytes and does not induce the cells to differentiate into fibroblasts. ARTICLE IN PRESS Fig. 11. Morphology of human chondrocytes growing at the surface and inside the bulk: (a) unmodified BC (scale bar=5 mm); (b) articular cartilage surface (scale bar=2mm); and (c) articular cartilage bulk (scale bar=2mm). (The cartilage images were captured at the Cartilage Laboratory, Sahlgrenska University Hospital, Gothenburg, Sweden.) A. Svensson et al. / Biomaterials 26 (2005) 419–431 429 ARTICLE IN PRESS Fig. 12. Ingrowth of human chondrocytes in unmodified BC. The upper part of the image represents the scaffold surface. Scale bar=10mm. A. Svensson et al. / Biomaterials 26 (2005) 419–431430Chondrocytes obtain a more extended morphology when growing on a two-dimensional surface, while a three-dimensional structure supports chondrocyte pro- liferation and differentiation [12], which explains why the cells obtain a more spherical morphology, typically for chondrocytes, when growing in the bulk of BC. Further important characteristic that make BC suitable as a scaffold for tissue engineering is that it allows cell ingrowth (Fig. 12).5. Conclusions Our data indicate that the unmodified bacterial cellulose (BC) supports chondrocyte proliferation at levels of approximately 50% of the native tissue substrate, collagen type II. However, compared to tissue culture plastic and alginate, unmodified BC showed significantly higher levels of chondrocyte growth at similar levels of in vitro immune response. Phosphor- ylation and sulfation did not enhance chondrocytegrowth on BC. It was also confirmed that chondrocytes maintain their differentiated form when growing on BC and that the scaffold supports cell ingrowth. The Young’s modulus of unmodified BC was in the same range as articular cartilage while the tensile strength of BC was higher than the tensile strength of highly cross- linked collagen [36]. The compressive modulus of unmodified BC was in the same range as alginate but lower than the compressive modulus of articular cartilage. Based on these results, BC appears to have potential as a scaffold for tissue engineering of cartilage. However, further work must be performed in order to study the long-term effects of the BC scaffold material on chondrocyte extracellular matrix-production and proliferation as well as the influence of stress-relaxation during long-term loading.Acknowledgements The financial funding from Chalmers Bioscience Program and the Swedish Research Council is gratefully acknowledged. The authors thank Dr. Lorenz Meinel (Tufts) for the supply of bovine chondrocytes, Aase Bodin for the supply of bacterial cellulose, Catherine Bengtsson and Marianne Jonsson (Cartilage Labora- tory, Sahlgrenska University Hospital) for helping with cell study II, Anders M(artensson, Mattias Goks .or (Chalmers) and Ulf Nanmark (Gothenburgs University) for helping with microscopy, Vassilis Karagerogiou (Tufts) and Anne Wendel (Chalmers) for ESCA analysis. Dr. Mats Stading (SIK), Dr. Ung-Jin Kim (Tufts) and Dr. Jingsong Chen (Tufts) are acknowl- edged for valuable advice.References [1] Freeman M. Adult articular cartilage. London: Grune & Stratton; 1973. [2] Huber M, Trattnig S, Lintner F. Anatomy, biochemistry, and physiology of articular cartilage. Invest Radiol 2000;35:573–80. [3] Wilkins RJ, Browning JA, Ellory JC. Surviving in a matrix: membrane transport in articular chondrocytes. J Membr Biol 2000;177:95–108. [4] Rheumatology, A.C.o. Osteoarthritis (www.rheumatology.org/ patients/factsheet/oa.html, 2000). [5] Ma PX, Langer R. Morphology and mechanical function of long- term in vitro engineered cartilage. J Biomed Mater Res 1999;44: 217–21. [6] Suh JKF, Matthew HWT. Application of chitosan-based poly- saccharide biomaterials in cartilage tissue engineering: a review. Biomaterials 2000;21:2589–98. [7] Schreiber RE, Dunkelman NS, Naughton G, Ratcliffe A. A method for tissue engineering of cartilage by cell seeding on bioresorbable scaffolds. In: Hunkler D, Prokop A, Cherrington AD, Rajotte RV, Sefton M, editors. Bioartificial organs II. Technology, medicine and materials. New York: New York Academy of Science; 1999. p. 398–404.
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