BC membranes (pellicles) generated by Gluconacetobacter hansenii (G. hansenii) are promising biomaterials owing to their outstanding biocompatible properties. Recently, specific demands for biomedical applications of BC have increased owing to its excellent mechanical properties. Although many techniques have been developed to improve the biofunctional properties of BC pellicles, such modifications remain limited owing to technical difficulties in the modulation of complex biosynthetic processes. Therefore, we previously developed an in vivo modification technique to produce nanocomposite pellicles composed of BC and HA (in vivo BC/HA), which are directly secreted from genetically engineered G. hansenii. In the present study, the HA extractability and content rate, physical characteristics, and cytocompatibility of in vivo BC/HA have been investigated in comparison to conventional in situ BC/HA and native BC pellicle. The results suggested that HA more strongly adsorbed to the solid BC surface of in vivo BC/HA than that of in situ BC/HA, which possibly affected the dynamic viscoelastic characteristics. In vivo BC/HA exhibited a relatively lower value of 7.5 MPa as storage elastic modulus (E’), whereas in situ BC/HA yielded the highest E’ of 15.6 MPa in comparison to 11.4 MPa as E’of native BC. Although the HA content of in vivo BC/HA (95 μg/g) was indicated lower than in situ BC/HA (300 μg/g), the former showed two times higher ability in human epidermal cell adhesion. These results indicate the great potential of in vivo modification to expand the usefulness of BC-based biomaterials.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
Availability of data and material
Abe M, Fukaya Y, Ohno H (2012) Fast and facile dissolution of cellulose with tetrabutylphosphonium hydroxide containing 40 wt% water. Chem Commun 48:1808–1810. https://doi.org/10.1039/c2cc16203b
Bäckdahl H, Esguerra M, Delbro D, Risberg B, Gatenholm P (2008) Engineering microporosity in bacterial cellulose scaffolds. J Tissue Eng Regen Med 2:320–330. https://doi.org/10.1002/term.97
Berti FV, Rambo CR, Dias PF, Porto LM (2013) Nanofiber density determines endothelial cell behavior on hydrogel matrix. Mater Sci Eng C 33:4684–4691. https://doi.org/10.1016/j.msec.2013.07.029
Carter WG, Wayner EA, Bouchard TS, Kaur P (1990) The role of integrins alpha 2 beta 1 and alpha 3 beta 1 in cell-cell and cell-substrate adhesion of human epidermal cells. J Cell Biol 110:1387–1404. https://doi.org/10.1083/JCB.110.4.1387
Dayal MS, Catchmark JM (2016) Mechanical and structural property analysis of bacterial cellulose composites. Carbohydr Polym 144:447–453. https://doi.org/10.1016/J.CARBPOL.2016.02.055
Fang J, Kawano S, Tajima K, Kondo T (2015) In Vivo Curdlan/Cellulose Bionanocomposite Synthesis by Genetically Modified Gluconacetobacter xylinus. Biomacromol 16:3154–3160. https://doi.org/10.1021/acs.biomac.5b01075
Fernandes M, Gama M, Dourado F, Souto AP (2019) Development of novel bacterial cellulose composites for the textile and shoe industry. Microb Biotechnol 12:650–661. https://doi.org/10.1111/1751-7915.13387
Gea S, Reynolds CT, Roohpour N, Wirjosentono B, Soykeabkaew N, Bilotti E, Peijs T (2011) Investigation into the structural, morphological, mechanical and thermal behaviour of bacterial cellulose after a two-step purification process. Bioresour Technol 102:9105–9110. https://doi.org/10.1016/J.BIORTECH.2011.04.077
Gilli R, Kacuráková M, Mathlouthi M, Navarini L, Paoletti S (1994) FTIR studies of sodium hyaluronate and its oligomers in the amorphous solid phase and in aqueous solution. Carbohydr Res 263:315–326. https://doi.org/10.1016/0008-6215(94)00147-2
Gorgieva S (2020) Bacterial cellulose as a versatile platform for research and development of biomedical materials. Processes 8:624. https://doi.org/10.3390/PR8050624
Grobelski B, Wach RA, Adamus A, Olejnik AK, Kowalska-Ludwicka K, Kolodziejczyk M, Bielecki S, Rosiak JM, Pasieka Z (2014) Biocompatibility of Modified Bionanocellulose and Porous Poly(ϵ-caprolactone) Biomaterials. Int J Polym Mater Polym Biomater 63:518–526. https://doi.org/10.1080/00914037.2013.854223
Hall PE, Anderson SM, Johnston DM, Cannon RE (1992) Transformation of Acetobacter xylinum with plasmid DNA by electroporation. Plasmid 28:194–200. https://doi.org/10.1016/0147-619X(92)90051-B
Helenius G, Bäckdahl H, Bodin A, Nannmark U, Gatenholm P, Risberg B (2006) In vivo biocompatibility of bacterial cellulose. J Biomed Mater Res Part A 76:431–438. https://doi.org/10.1002/jbm.a.30570
Hutmacher DW, Woodfield TBF, Dalton PD (2014) Scaffold Design and Fabrication. In: Blitterswijk C Van, Boer J De (eds) Tissue Engineering: Second Edition, 2nd edn. Elsevier Science & Technology, pp 311–346
Igarashi K, Wada M, Samejima M (2007) Activation of crystalline cellulose to cellulose III(I) results in efficient hydrolysis by cellobiohydrolase. FEBS J 274:1785–1792. https://doi.org/10.1111/j.1742-4658.2007.05727.x
Iguchi M, Yamanaka S, Budhiono A (2000) Bacterial cellulose - a masterpiece of nature’s arts. J Mater Sci 35:261–270. https://doi.org/10.1023/A:1004775229149
Jiang F, Yin L, Yu Q, Zhong C, Zhang J (2015) Bacterial cellulose nanofibrous membrane as thermal stable separator for lithium-ion batteries. J Power Sources 279:21–27. https://doi.org/10.1016/j.jpowsour.2014.12.090
Kim Y, Ullah MW, Ul-Islam M, Khan S, Jang JH, Park JK (2019) Self-assembly of bio-cellulose nanofibrils through intermediate phase in a cell-free enzyme system. Biochem Eng J 142:135–144. https://doi.org/10.1016/j.bej.2018.11.017
Klemm D, Cranston ED, Fischer D, Gama M, Kedzior SA, Kralisch D, Kramer F, Kondo T, Lindström T, Nietzsche S, Petzold-Welcke K, Rauchfuß F (2018) Nanocellulose as a natural source for groundbreaking applications in materials science: Today’s state. Mater Today 21:720–748. https://doi.org/10.1016/j.mattod.2018.02.001
Knudson W, Peterson RS (2004) The Hyaluronan Receptor: CD44. In: Garg HG, Hales CA (eds) Chemistry and Biology of Hyaluronan, 1st edn. Elsevier Ltd, pp 83–123
Kondo T (1997) The assignment of IR absorption bands due to free hydroxyl groups in cellulose. Cellulose 4:281–292. https://doi.org/10.1023/A:1018448109214
Ludwicka K, Jedrzejczak-Krzepkowska M, Kubiak K, Kolodziejczyk M, Pankiewicz T, Bielecki S (2016) Medical and Cosmetic Applications of Bacterial NanoCellulose. In: Dourado F, Bielecki S (eds) Gama M. From Biotechnology to Bio-Economy. Elsevier Inc., Bacterial Nanocellulose, pp 145–165
Mohan S, Jose J, Kuijk A, Veen SJ, Van Blaaderen A, Velikov KP (2017) Revealing and quantifying the three-dimensional nano- and microscale structures in self-assembled cellulose microfibrils in dispersions. ACS Omega 2:5019–5024. https://doi.org/10.1021/acsomega.7b00536
Nishi Y, Uryu M, Yamanaka S, Watanabe K, Kitamura N, Iguchi M, Mitsuhashi S (1990) The structure and mechanical properties of sheets prepared from bacterial cellulose—Part 2 Improvement of the mechanical properties of sheets and their applicability to diaphragms of electroacoustic transducers. J Mater Sci 25:2997–3001. https://doi.org/10.1007/BF00584917
Özdemir B, Reski R (2021) Automated and semi-automated enhancement, segmentation and tracing of cytoskeletal networks in microscopic images: a review. Comput Struct Biotechnol J 19:2106–2120. https://doi.org/10.1016/j.csbj.2021.04.019
Pourali P, Yahyaei B, Ajoudanifar H, Taheri R, Alavi H, Hoseini A (2014) Impregnation of the bacterial cellulose membrane with biologically produced silver nanoparticles. Curr Microbiol 69:785–793. https://doi.org/10.1007/s00284-014-0655-z
Rybchyn MS, Biazik JM, Charlesworth J, le Coutre J (2021) Nanocellulose from nata de coco as a bioscaffold for cell-based meat. ACS Omega. https://doi.org/10.1021/ACSOMEGA.1C05235
Sakuragi K, Igarashi K, Samejima M (2018) Application of ammonia pretreatment to enable enzymatic hydrolysis of hardwood biomass. Polym Degrad Stab 148:19–25. https://doi.org/10.1016/j.polymdegradstab.2017.12.008
Sämfors S, Karlsson K, Sundberg J, Markstedt K, Gatenholm P (2019) Biofabrication of bacterial nanocellulose scaffolds with complex vascular structure. Biofabrication 11:045010. https://doi.org/10.1088/1758-5090/AB2B4F
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. https://doi.org/10.1038/nmeth.2019
Schmidt S, Friedl P (2009) Interstitial cell migration: integrin-dependent and alternative adhesion mechanisms. Cell Tissue Res 339:83–92. https://doi.org/10.1007/S00441-009-0892-9
Schnauß J, Schmidt BUS, Brazel CB, Dogan S, Losert W, Anderegg U, Käs JA (2020) Influence of hyaluronic acid binding on the actin cortex measured by optical forces. J Biophotonics 13:e201960215. https://doi.org/10.1002/JBIO.201960215
Shahmohammadi Jebel F, Almasi H (2016) Morphological, physical, antimicrobial and release properties of ZnO nanoparticles-loaded bacterial cellulose films. Carbohydr Polym 149:8–19. https://doi.org/10.1016/J.CARBPOL.2016.04.089
Sunagawa N, Tajima K, Hosoda M, Kawano S, Kose R, Satoh Y, Yao M, Dairi T (2012) Cellulose production by Enterobacter sp. CJF-002 and identification of genes for cellulose biosynthesis. Cellulose 19:1989–2001. https://doi.org/10.1007/s10570-012-9777-2
Tajima K, Fujisawa M, Takai M, Hayashi J (1995) Synthesis of Bacterial Cellulose Composite by Acetobacter xylinum I. Its mechanical strength and biodegradability. J Wood Sci 41:749–757
Tajima K, Imai T, Yui T, Yao M, Saxena I (2021) Cellulose-synthesizing machinery in bacteria. Cellulose 7:21. https://doi.org/10.1007/s10570-021-04225-7
Takahama R, Kato H, Tajima K, Tagawa S, Kondo T (2021) Biofabrication of a hyaluronan/bacterial cellulose composite nanofibril by secretion from engineered gluconacetobacter. Biomacromol 22:4709–4719. https://doi.org/10.1021/ACS.BIOMAC.1C00987
Trappmann B, Gautrot JE, Connelly JT, Strange DGT, Li Y, Oyen ML, Cohen Stuart MA, Boehm H, Li B, Vogel V, Spatz JP, Watt FM, Huck WTS (2012) Extracellular-matrix tethering regulates stem-cell fate. Nat Mater 11:642–649. https://doi.org/10.1038/nmat3339
Tsimbouri PM, Mcnamara LE, Alakpa E V, Dalby MJ, Turner L (2014) Cell – Material Interactions. In: Blitterswijk C Van, Boer J De (eds) Tissue Engineering: Second Edition, 2nd edn. Elsevier Science & Technology, pp 217–251
Wahid F, Huang L-H, Zhao X-Q, Li W-C, Wang Y-Y, Jia S-R, Zhong C (2021) Bacterial cellulose and its potential for biomedical applications. Biotechnol Adv 53:107856. https://doi.org/10.1016/J.BIOTECHADV.2021.107856
Wan Y, Yang S, Wang J, Gan D, Gama M, Yang Z, Zhu Y, Yao F, Luo H (2020) Scalable synthesis of robust and stretchable composite wound dressings by dispersing silver nanowires in continuous bacterial cellulose. Compos Part B Eng 199:108259. https://doi.org/10.1016/J.COMPOSITESB.2020.108259
Wang Y, Wang G, Luo X, Qiu J, Tang C (2012) Substrate stiffness regulates the proliferation, migration, and differentiation of epidermal cells. Burns 38:414–420. https://doi.org/10.1016/j.burns.2011.09.002
Watanabe K, Eto Y, Takano S, Nakamori S, Shibai H, Yamanaka S (1993) A new bacterial cellulose substrate for mammalian cell culture—a new bacterial cellulose substrate. Cytotechnology 13:107–114. https://doi.org/10.1007/BF00749937
Xu T, Vavylonis D, Tsai FC, Koenderink GH, Nie W, Yusuf E, Lee IJ, Wu JQ, Huang X (2015) SOAX: A software for quantification of 3D biopolymer networks. Sci Rep 5:467. https://doi.org/10.1038/srep09081
Yamanaka S, Watanabe K, Kitamura N, Iguchi M, Mitsuhashi S, Nishi Y, Uryu M (1989) The structure and mechanical properties of sheets prepared from bacterial cellulose. J Mater Sci 24:3141–3145. https://doi.org/10.1007/BF01139032
Zarkoob H, Bodduluri S, Ponnaluri SV, Selby JC, Sander EA (2015) Substrate Stiffness Affects Human Keratinocyte Colony Formation. Cell Mol Bioeng 8:32–50. https://doi.org/10.1007/s12195-015-0377-8
We thank Dr. Satomi Tagawa and staff at the Center for Advanced Instrumental and Educational Supports, Faculty of Agriculture, Kyushu University, for assistance with CLSM observations and quantitative HA assay.
Conflict of interest
This study does not include human participants or animal studies.
Consent to participate
All authors (Ryo Takahama, Honami Kato, Go Takayama, Kenji Tajima and Tetsuo Kondo) have approved the manuscript and agreed with submission to Cellulose. The authors have no conflicts of interest to declare.
Consent for publication
We confirm that this manuscript has not been published elsewhere, and is not under consideration in whole or in part by another journal. All authors have agreed with this submission for publication in Cellulose.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Below is the link to the electronic supplementary material.
About this article
Cite this article
Takahama, R., Kato, H., Takayama, G. et al. Physical characteristics and cell-adhesive properties of in vivo fabricated bacterial cellulose/hyaluronan nanocomposites. Cellulose 29, 3239–3251 (2022). https://doi.org/10.1007/s10570-022-04480-2