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Applied Microbiology and Biotechnology

, Volume 98, Issue 17, pp 7423–7435 | Cite as

Biocompatibility evaluation of densified bacterial nanocellulose hydrogel as an implant material for auricular cartilage regeneration

  • Héctor Martínez Ávila
  • Silke Schwarz
  • Eva-Maria Feldmann
  • Athanasios Mantas
  • Achim von Bomhard
  • Paul GatenholmEmail author
  • Nicole Rotter
Biotechnological products and process engineering

Abstract

Bacterial nanocellulose (BNC), synthesized by the bacterium Gluconacetobacter xylinus, is composed of highly hydrated fibrils (99 % water) with high mechanical strength. These exceptional material properties make BNC a novel biomaterial for many potential medical and tissue engineering applications. Recently, BNC with cellulose content of 15 % has been proposed as an implant material for auricular cartilage replacement, since it matches the mechanical requirements of human auricular cartilage. This study investigates the biocompatibility of BNC with increased cellulose content (17 %) to evaluate its response in vitro and in vivo. Cylindrical BNC structures (Ø48 × 20 mm) were produced, purified in a built-in house perfusion system, and compressed to increase the cellulose content in BNC hydrogels. The reduction of endotoxicity of the material was quantified by bacterial endotoxin analysis throughout the purification process. Afterward, the biocompatibility of the purified BNC hydrogels with cellulose content of 17 % was assessed in vitro and in vivo, according to standards set forth in ISO 10993. The endotoxin content in non-purified BNC (2,390 endotoxin units (EU)/ml) was reduced to 0.10 EU/ml after the purification process, level well below the endotoxin threshold set for medical devices. Furthermore, the biocompatibility tests demonstrated that densified BNC hydrogels are non-cytotoxic and cause a minimal foreign body response. In support with our previous findings, this study concludes that BNC with increased cellulose content of 17 % is a promising non-resorbable biomaterial for auricular cartilage tissue engineering, due to its similarity with auricular cartilage in terms of mechanical strength and host tissue response.

Keywords

Bacterial cellulose Depyrogenation Endotoxin analysis Biocompatibility Auricular cartilage tissue engineering 

Notes

Acknowledgments

The authors acknowledge ERANET/EuroNanoMed (EAREG-406340-131009/1) for funding this work; Veronika Fuss and Anette Jork at Cell Med AG, Alzenau, Germany, for the support with histopathological evaluation and cytotoxicity testing; Alexander Elsaesser at Ulm University Medical Center, Ulm, Germany, for helping with the animal study; and Johan Sundberg, Anne Wendel, and Professor Vratislav Langer at Chalmers University of Technology, Gothenburg, Sweden, for the SEM analysis, ESCA, and XRD measurements, respectively.

References

  1. Ahrem H, Pretzel D, Endres M, Conrad D, Courseau J, Muller H, Jaeger R, Kaps C, Klemm DO, Kinne RW (2014) Laser-structured bacterial nanocellulose hydrogels support ingrowth and differentiation of chondrocytes and show potential as cartilage implants. Acta Biomater 10(3):1341–1353. doi: 10.1016/j.actbio.2013.12.004 PubMedCrossRefGoogle Scholar
  2. Almeida CR, Serra T, Oliveira MI, Planell JA, Barbosa MA, Navarro M (2014) Impact of 3-D printed PLA- and chitosan-based scaffolds on human monocyte/macrophage responses: unraveling the effect of 3-D structures on inflammation. Acta Biomater 10(2):613–622. doi: 10.1016/j.actbio.2013.10.035 PubMedCrossRefGoogle Scholar
  3. Anderson JM (1993) Mechanisms of inflammation and infection with implanted devices. Cardiovasc Pathol 2(3):33S–41SCrossRefGoogle Scholar
  4. Anderson JM (2001) Biological responses to materials. Annu Rev Mater Res 31(1):81–110. doi: 10.1146/annurev.matsci.31.1.81 CrossRefGoogle Scholar
  5. Anderson JM, Langone JJ (1999) Issues and perspectives on the biocompatibility and immunotoxicity evaluation of implanted controlled release systems. J Control Release 57(2):107–113PubMedCrossRefGoogle Scholar
  6. Anderson JM, Shive MS (1997) Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv Drug Deliv Rev 28(1):5–24PubMedCrossRefGoogle Scholar
  7. Andersson J, Stenhamre H, Bäckdahl H, Gatenholm P (2010) Behavior of human chondrocytes in engineered porous bacterial cellulose scaffolds. J Biomed Mater Res A 94(4):1124–1132. doi: 10.1002/jbm.a.32784 PubMedGoogle Scholar
  8. Andrade FK, Alexandre N, Amorim I, Gartner F, Mauricio AC, Luis AL, Gama M (2013) Studies on the biocompatibility of bacterial cellulose. J Bioact Compat Polym 28(1):97–112. doi: 10.1177/0883911512467643 CrossRefGoogle Scholar
  9. Bäckdahl H, Esguerra M, Delbro D, Risberg B, Gatenholm P (2008) Engineering microporosity in bacterial cellulose scaffolds. J Tissue Eng Regent Med 2(6):320–330. doi: 10.1002/Term.97 CrossRefGoogle Scholar
  10. Bäckdahl H, Helenius G, Bodin A, Nannmark U, Johansson BR, Risberg B, Gatenholm P (2006) Mechanical properties of bacterial cellulose and interactions with smooth muscle cells. Biomaterials 27(9):2141–2149PubMedCrossRefGoogle Scholar
  11. Bermueller C, Schwarz S, Elsaesser AF, Sewing J, Baur N, Av B, Scheithauer M, Notbohm H, Rotter N (2013) Marine collagen scaffolds for nasal cartilage repair: prevention of nasal septal perforations in a new orthotopic rat model using tissue engineering techniques. Tissue Eng A 19(19–20):2201–2214CrossRefGoogle Scholar
  12. Bodin A, Bharadwaj S, Wu SF, Gatenholm P, Atala A, Zhang YY (2010) Tissue-engineered conduit using urine-derived stem cells seeded bacterial cellulose polymer in urinary reconstruction and diversion. Biomaterials 31(34):8889–8901. doi: 10.1016/J.Biomaterials.2010.07.108 PubMedCrossRefGoogle Scholar
  13. Brown RM Jr, Willison JH, Richardson CL (1976) Cellulose biosynthesis in Acetobacter xylinum: visualization of the site of synthesis and direct measurement of the in vivo process. Proc Natl Acad Sci U S A 73(12):4565–4569PubMedCentralPubMedCrossRefGoogle Scholar
  14. Deinema M, Zevenhuizen LPTM (1971) Formation of cellulose fibrils by gram-negative bacteria and their role in bacterial flocculation. Arch Mikrobiol 78(1):42–57. doi: 10.1007/bf00409087 PubMedCrossRefGoogle Scholar
  15. FDA US (June 2012) Guidance for industry—pyrogen and endotoxins testing. In: Services USDoHH (ed). U.S. Food and Drug Administration, Silver Spring, p 11Google Scholar
  16. Feldmann EM, Sundberg J, Bobbili B, Schwarz S, Gatenholm P, Rotter N (2013) Description of a novel approach to engineer cartilage with porous bacterial nanocellulose for reconstruction of a human auricle. J Biomater Appl. doi: 10.1177/0885328212472547 PubMedGoogle Scholar
  17. Fink HP, Purz HJ, Bohn A, Kunze J (1997) Investigation of the supramolecular structure of never dried bacterial cellulose. Macromol Symp 120:207–217. doi: 10.1002/Masy.19971200121 CrossRefGoogle Scholar
  18. Gatenholm P, Berry J, Rojas R, Sano MB, Davalos RV, Johnson K, Rourke LO (2012) Bacterial nanocellulose biomaterials with controlled architecture for tissue engineering scaffolds and customizable implants. In: Gama M, Gatenholm P, Klemm D (eds) Bacterial Nanocellulose: a sophisticated multifunctional material. Perspectives in Nanotechnology. CRC Press, pp 197-216Google Scholar
  19. 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. BioresourTechnol 102(19):9105–9110. doi: 10.1016/j.biortech.2011.04.077 CrossRefGoogle Scholar
  20. 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(e-caprolactone) biomaterials. Int J Polym Mater Polym Biomater 63(10):518–526. doi: 10.1080/00914037.2013.854223 CrossRefGoogle Scholar
  21. Heine H, Rietschel ET, Ulmer AJ (2001) The biology of endotoxin. Mol Biotechnol 19(3):279–296. doi: 10.1385/MB:19:3:279 PubMedCrossRefGoogle Scholar
  22. Helenius G, Bäckdahl H, Bodin A, Nannmark U, Gatenholm P, Risberg B (2006) In vivo biocompatibility of bacterial cellulose. J Biomed Mater Res A 76(2):431–438. doi: 10.1002/jbm.a.30570 PubMedCrossRefGoogle Scholar
  23. Hold GL, Bryant CE (2011) The molecular basis of lipid A and Toll-like receptor 4 interactions. In: Valvano MA (ed) Knirel YA. Bacterial Lipopolysaccharides, Springer Vienna, pp 371–387Google Scholar
  24. Jones KS (2008) Effects of biomaterial-induced inflammation on fibrosis and rejection. Semin Immunol 20(2):130–136. doi: 10.1016/j.smim.2007.11.005 PubMedCrossRefGoogle Scholar
  25. Klechkovskaya VV, Baklagina YG, Stepina ND, Khripunov AK, Buffat PA, Suvorova EI, Zanaveskina IS, Tkachenko AA, Gladchenko SV (2003) Structure of cellulose Acetobacter xylinum. Crystallogr Rep 48(5):755–762. doi: 10.1134/1.1612596 CrossRefGoogle Scholar
  26. Klemm D, Ahrem H, Kramer F, Fried W, Wippermann J, Kinne RW (2012) Bacterial nanocellulose hydrogels designed as bioartificial medical implants. In: Gama M, Gatenholm P, Klemm D (eds) Bacterial Nanocellulose: a sophisticated multifunctional material. Perspectives in Nanotechnology. CRC Press, pp 175-196Google Scholar
  27. Klemm D, Schumann D, Udhardt U, Marsch S (2001) Bacterial synthesized cellulose—artificial blood vessels for microsurgery. Prog Polym Sci 26(9):1561–1603CrossRefGoogle Scholar
  28. Kondo T, Nojiri M, Hishikawa Y, Togawa E, Romanovicz D, Brown RM (2002) Biodirected epitaxial nanodeposition of polymers on oriented macromolecular templates. Proc Natl Acad Sci U S A 99(22):14008–14013. doi: 10.1073/pnas.212238399 PubMedCentralPubMedCrossRefGoogle Scholar
  29. Kotzar G, Freas M, Abel P, Fleischman A, Roy S, Zorman C, Moran JM, Melzak J (2002) Evaluation of MEMS materials of construction for implantable medical devices. Biomaterials 23(13):2737–2750PubMedCrossRefGoogle Scholar
  30. Kowalska-Ludwicka K, Cala J, Grobelski B, Sygut D, Jesionek-Kupnicka D, Kolodziejczyk M, Bielecki S, Pasieka Z (2013) Modified bacterial cellulose tubes for regeneration of damaged peripheral nerves. Arch Med Sci 9(3):527–534. doi: 10.5114/aoms.2013.33433 PubMedCentralPubMedCrossRefGoogle Scholar
  31. Mansikkamäki P, Lahtinen M, Rissanen K (2005) Structure changes of cellulose crystallites induced by mercerization in different solvent system; determined by powder X-ray diffraction method. Cellulose 12:233–242CrossRefGoogle Scholar
  32. Martinez H, Brackmann C, Enejder A, Gatenholm P (2012) Mechanical stimulation of fibroblasts in micro-channeled bacterial cellulose scaffolds enhances production of oriented collagen fibers. J Biomed Mater Res A 100(4):948–957. doi: 10.1002/jbm.a.34035 PubMedCrossRefGoogle Scholar
  33. Martinez-Sanz M, Olsson RT, Lopez-Rubio A, Lagaron JM (2011) Development of electrospun EVOH fibres reinforced with bacterial cellulose nanowhiskers. Part I: Characterization and method optimization. Cellulose 18(2):335–347. doi: 10.1007/S10570-010-9471-1 CrossRefGoogle Scholar
  34. Matsuoka MTT, Matsushita K, Adachi O, Yoshinaga F (1996) A synthetic medium for bacterial cellulose production by Acetobacter xylinum subsp. sucrofermentation. Biosci Biotechnol Biochem 60:575–579CrossRefGoogle Scholar
  35. Mello LR, Feltrin LT, Neto PTF, Ferraz FAP (1997) Duraplasty with biosynthetic cellulose: an experimental study. J Neurosurg 86(1):143–150. doi: 10.3171/Jns.1997.86.1.0143 PubMedCrossRefGoogle Scholar
  36. Moharram MA, Mahmoud OM (2007) X-ray diffraction methods in the study of the effect of microwave heating on the transformation of cellulose I into cellulose II during mercerization. J Appl Polym Sci 105(5):2978–2983. doi: 10.1002/App.26580 CrossRefGoogle Scholar
  37. Nimeskern L, Martínez Ávila H, Sundberg J, Gatenholm P, Müller R, Stok KS (2013) Mechanical evaluation of bacterial nanocellulose as an implant material for ear cartilage replacement. J Mech Behav Biomed 22:12–21. doi: 10.1016/j.jmbbm.2013.03.005 CrossRefGoogle Scholar
  38. O'Sullivan A (1997) Cellulose: the structure slowly unravels. Cellulose 4(3):173–207. doi: 10.1023/a:1018431705579 CrossRefGoogle Scholar
  39. Robitaille R, Dusseault J, Henley N, Desbiens K, Labrecque N, Halle JP (2005) Inflammatory response to peritoneal implantation of alginate-poly-L-lysine microcapsules. Biomaterials 26(19):4119–4127. doi: 10.1016/j.biomaterials.2004.10.028 PubMedCrossRefGoogle Scholar
  40. Rosen CL, Steinberg GK, DeMonte F, Delashaw JB Jr, Lewis SB, Shaffrey ME, Aziz K, Hantel J, Marciano FF (2011) Results of the prospective, randomized, multicenter clinical trial evaluating a biosynthesized cellulose graft for repair of dural defects. Neurosurgery 69(5):1093–1103. doi: 10.1227/NEU.0b013e3182284aca PubMedGoogle Scholar
  41. Sandle T (2011) A practical approach to depyrogenation studies using bacterial endotoxin. J GXP Compliance 15(4):90–96Google Scholar
  42. Sano MB, Rojas AD, Gatenholm P, Davalos RV (2010) Electromagnetically controlled biological assembly of aligned bacterial cellulose nanofibers. Ann Biomed Eng 38(8):2475–2484. doi: 10.1007/S10439-010-9999-0 PubMedCrossRefGoogle Scholar
  43. Segal L, Creely JJ, Martin AE, Conrad CM (1959) An empirical method for estimating the degree of crystallinity of native cellulose using the X-ray diffractometer. Text Res J 29(10):786–794. doi: 10.1177/004051755902901003 CrossRefGoogle Scholar
  44. Silipo A, Molinaro A (2011) Lipid A structure. In: Valvano MA (ed) Knirel YA. Bacterial Lipopolysaccharides, Springer Vienna, pp 1–20Google Scholar
  45. Svensson A, Nicklasson E, Harrah T, Panilaitis B, Kaplan DL, Brittberg M, Gatenholm P (2005) Bacterial cellulose as a potential scaffold for tissue engineering of cartilage. Biomaterials 26(4):419–431PubMedCrossRefGoogle Scholar
  46. White DG, Brown RM (1989) Prospects for the commercialization of the biosynthesis of microbial cellulose. In: Schuerech C (ed) Cellulose and Wood-Chemistry and Technology. John Wiley & Sons, New York, pp 573–590Google Scholar
  47. Williams KL (2007a) Depyrogenation validation, pyroburden and endotoxin removal. In: Williams KL (ed) Endotoxins. Drugs and the Pharmaceutical Sciences. CRC Press, pp 301-327Google Scholar
  48. Williams KL (2007b) Fever and the host response. In: Williams KL (ed) Endotoxins. Drugs and the Pharmaceutical Sciences. CRC Press, pp 47-66Google Scholar
  49. Zaborowska M, Bodin A, Bäckdahl H, Popp J, Goldstein A, Gatenholm P (2010) Microporous bacterial cellulose as a potential scaffold for bone regeneration. Acta Biomater 6(7):2540–2547. doi: 10.1016/j.actbio.2010.01.004 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Héctor Martínez Ávila
    • 1
  • Silke Schwarz
    • 2
  • Eva-Maria Feldmann
    • 2
  • Athanasios Mantas
    • 1
  • Achim von Bomhard
    • 2
  • Paul Gatenholm
    • 1
    Email author
  • Nicole Rotter
    • 2
  1. 1.Biopolymer Technology, Department of Chemical and Biological EngineeringChalmers University of TechnologyGothenburgSweden
  2. 2.Department of OtorhinolaryngologyUlm University Medical CenterUlmGermany

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