, Volume 23, Issue 2, pp 1263–1282 | Cite as

Preparation and characterization of biodegradable nano hydroxyapatite–bacterial cellulose composites with well-defined honeycomb pore arrays for bone tissue engineering applications

  • Pelagie Marlene Favi
  • Sandra Patricia Ospina
  • Mukta Kachole
  • Ming Gao
  • Lucia Atehortua
  • Thomas Jay Webster
Original Paper


Bacterial cellulose (BC), a nano fibrous hydrogel synthesized from non-pathogenic bacteria, is an excellent candidate scaffold for bone tissue engineering applications due to its biocompatibility, high purity and mechanical strength. However, BC is not biodegradable and possesses small pore sizes, which hinders the ingrowth of cells and thereby limits its potential as a bone tissue engineering scaffold. In this study, microporous BC (termed Porous BC) scaffolds with well-defined honeycomb pore arrays were prepared using a laser patterning technique. The BC scaffolds were modified using periodate oxidation to yield biodegradable oxidized BC scaffolds. In a unique manner, the BC scaffolds were then mineralized with nano hydroxyapatite (nano HA) to mimic the inorganic component of native bone tissue, improve bone cell compatibility, enhance mechanical properties, and control degradation. Results confirmed that sodium periodate oxidation successfully oxidized BC and Porous BC honeycomb pore arrays with 300 μm pore sizes with irregularly shaped 77 ± 15 nm nano HA and aggregated 200–500 nm nano HA were formed. BC and its composites displayed suitable mechanical properties for bone tissue engineering applications. The in vitro degradation study showed a significant 13–25 % loss of their dry mass in the oxidized BC composites thus confirming that the oxidized cellulose can biodegrade. Most importantly, the results also demonstrated that human-derived bone marrow mesenchymal stem cells (hMSCs) adhered to and were viable on the BC and its composites, thus, confirming their potential to serve as improved bone tissue engineering scaffolds. The novelty of the present study includes the precipitation of nano HA onto cellulose to promote hMSCs functions for improving orthopedic applications.


Biodegradable Microporous scaffold 2,3-Dialdehyde bacterial cellulose Biomimetic hydroxyapatite Laser perforation Bone tissue regeneration Nanotechnology 



Bacterial cellulose


Bacterial cellulose–hydroxyapatite


Fourier-transform infrared spectroscopy



Native BC

Native bacterial cellulose


Oxidized native bacterial cellulose


Oxidized native bacterial cellulose–hydroxyapatite

Porous BC

Microporous bacterial cellulose

Porous BC–HA

Microporous bacterial cellulose–hydroxyapatite

Porous OBC

Oxidized microporous bacterial cellulose

Porous OBC–HA

Oxidized microporous bacterial cellulose–hydroxyapatite


Scanning electron microscope



This study was supported by Northeastern University. Dr. Pelagie Favi is grateful for Northeastern University’s ADVANCE Future Faculty Postdoctoral Fellowship Program (sponsored by National Science Foundation Grant #0811170). The authors also thank Mr. William Fowle and Dr. Wentao Liang for assistance with the TEM images and Dr. Roberto Benson for the donation of the Acetobacter xylinus subsp. Sucrofermentans cells.


  1. Ahrem H et al (2014) Laser-structured bacterial nanocellulose hydrogels support ingrowth and differentiation of chondrocytes and show potential as cartilage implants. Acta Biomater 10:1341–1353. doi: 10.1016/j.actbio.2013.12.004 CrossRefGoogle Scholar
  2. Akkouch A, Zhang Z, Rouabhia M (2014) Engineering bone tissue using human dental pulp stem cells and an osteogenic collagen–hydroxyapatite-poly(L-lactide-co-ε-caprolactone) scaffold. J Biomater Appl 28(6):922–936. doi: 10.1177/0885328213486705 CrossRefGoogle Scholar
  3. American Society for Testing and Materials (2010) Standard test method for tensile properties of plastics. ASTM International, West Conshohocken, PAGoogle Scholar
  4. Backdahl H, Esguerra M, Delbro D, Risberg B, Gatenholm P (2008) Engineering microporosity in bacterial cellulose scaffolds. J Tissue Eng Regen Med 2:320–330. doi: 10.1002/term.97 CrossRefGoogle Scholar
  5. 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 27:2141–2149. doi: 10.1016/j.biomaterials.2005.10.026 CrossRefGoogle Scholar
  6. Bansal M, Kaushik M, Khattak BBP, Sharma A (2014) Comparison of nanocrystalline hydroxyapatite and synthetic resorbable hydroxyapatite graft in the treatment of intrabony defects: a clinical and radiographic study. J Indian Soc Periodontol 18:213–219. doi: 10.4103/0972-124X.131329 CrossRefGoogle Scholar
  7. Berner A et al (2012) Biomimetic tubular nanofiber mesh and platelet rich plasma-mediated delivery of BMP-7 for large bone defect regeneration. Cell Tissue Res 347:603–612. doi: 10.1007/s00441-011-1298-z CrossRefGoogle Scholar
  8. Bhardwaj N, Kundu SC (2010) Electrospinning: a fascinating fiber fabrication technique. Biotechnol Adv 28:325–347. doi: 10.1016/j.biotechadv.2010.01.004 CrossRefGoogle Scholar
  9. Bielecki S, Krystoynowicz A, Turkiewicz M, Kalinowska H (2001) Bacterial cellulose. In: Vandamme EJ, De Baets S, Steinb A (eds) Biopolymers. Polysaccharides I, polysaccharides from prokaryotes, vol 5. Wiley, Weinham, pp 37–46Google Scholar
  10. Bose S, Vahabzadeh S, Bandyopadhyay A (2013) Bone tissue engineering using 3D printing. Mater Today 16:496–504. doi: 10.1016/j.mattod.2013.11.017 CrossRefGoogle Scholar
  11. Croisier F, Jérôme C (2013) Chitosan-based biomaterials for tissue engineering. Eur Polym J 49:780–792. doi: 10.1016/j.eurpolymj.2012.12.009 CrossRefGoogle Scholar
  12. Cui D, Daley W, Naftel JP, Lynch JC, Haines DE, Yang G, Fratkin JD (2011) Atlas of histology: with functional and clinical correlations, 1st edn. Wolters Kluwer Health/Lippincott Williams & Wilkins, PhiladelphiaGoogle Scholar
  13. El-Ayoubi R, Degrandpre C, DiRaddo R, Yousefi A-M, Lavigne P (2011) Design and dynamic culture of 3D-scaffolds for cartilage tissue engineering. J Biomater Appl 25:429–444. doi: 10.1177/0885328209355332 CrossRefGoogle Scholar
  14. Engler AJ, Sen S, Sweeney HL, Discher DE (2006) Matrix elasticity directs stem cell lineage specification. Cell 126:677–689. doi: 10.1016/j.cell.2006.06.044 CrossRefGoogle Scholar
  15. Evans FG (1976) Mechanical properties and histology of cortical bone from younger and older men. Anat Rec 185:1–11. doi: 10.1002/ar.1091850102 CrossRefGoogle Scholar
  16. Favi PM, Benson RS, Neilsen NR, Hammonds RL, Bates CC, Stephens CP, Dhar MS (2013) Cell proliferation, viability, and in vitro differentiation of equine mesenchymal stem cells seeded on bacterial cellulose hydrogel scaffolds. Mater Sci Eng C 33:1935–1944. doi: 10.1016/j.msec.2012.12.100 CrossRefGoogle Scholar
  17. Favi PM, Dhar MS, Neilsen NR, Benson RS (2014) Proliferation and osteogenic differentiation of mesenchymal stem cells on biodegradable calcium-deficient hydroxyapatite tubular bacterial cellulose composites. MRS Proc. doi: 10.1557/opl.2014.287 Google Scholar
  18. Fontana J et al (1990) Acetobacter cellulose pellicle as a temporary skin substitute. Appl Biochem Biotechnol 24–25:253–264. doi: 10.1007/bf02920250 CrossRefGoogle Scholar
  19. Harley BA, Lynn AK, Wissner-Gross Z, Bonfield W, Yannas IV, Gibson LJ (2010) Design of a multiphase osteochondral scaffold. II. Fabrication of a mineralized collagen–glycosaminoglycan scaffold. J Biomed Mater Res A 92A:1066–1077. doi: 10.1002/jbm.a.32361 Google Scholar
  20. Helenius G, Bäckdahl H, Bodin A, Nannmark U, Gatenholm P, Risberg B (2006) In vivo biocompatibility of bacterial cellulose. J Biomed Mater Res A 76A:431–438. doi: 10.1002/jbm.a.30570 CrossRefGoogle Scholar
  21. Hersel U, Dahmen C, Kessler H (2003) RGD modified polymers: biomaterials for stimulated cell adhesion and beyond. Biomaterials 24:4385–4415. doi: 10.1016/S0142-9612(03)00343-0 CrossRefGoogle Scholar
  22. Hu J, Zhou Y, Huang L, Liu J, Lu H (2014) Effect of nano-hydroxyapatite coating on the osteoinductivity of porous biphasic calcium phosphate ceramics. BMC Musculoskelet Disord 15:114. doi: 10.1186/1471-2474-15-114 CrossRefGoogle Scholar
  23. Hu J et al (2011) J Biomed Mater Res B, 2011, 97B and US Patent No. 20100172889Google Scholar
  24. Hutchens SA, Benson RS, Evans BR, O’Neill HM, Rawn CJ (2006) Biomimetic synthesis of calcium-deficient hydroxyapatite in a natural hydrogel. Biomaterials 27:4661–4670. doi: 10.1016/j.biomaterials.2006.04.032 CrossRefGoogle Scholar
  25. Hutchens S, Benson R, Evans B, Rawn C, O’Neill H (2009) A resorbable calcium-deficient hydroxyapatite hydrogel composite for osseous regeneration. Cellulose 16:887–898. doi: 10.1007/s10570-009-9300-6 CrossRefGoogle Scholar
  26. Jalota S, Bhaduri SB, Tas AC (2008) Using a synthetic body fluid (SBF) solution of 27 mM HCO3− to make bone substitutes more osteointegrative. Mater Sci Eng C 28:129–140. doi: 10.1016/j.msec.2007.10.058 CrossRefGoogle Scholar
  27. Karageorgiou V, Kaplan D (2005) Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 26:5474–5491. doi: 10.1016/j.biomaterials.2005.02.002 CrossRefGoogle Scholar
  28. Kim B-S, Kang HJ, Lee J (2013) Improvement of the compressive strength of a cuttlefish bone-derived porous hydroxyapatite scaffold via polycaprolactone coating. J Biomed Mater Res B 101:1302–1309. doi: 10.1002/jbm.b.32943 CrossRefGoogle Scholar
  29. Kirkham KD, Roundy CB (2014) Current technology of beam profile measurement. In: Dickey FM (ed) Laser beam shaping: theory and techniques, 2nd edn. CRC Press, Taylor & Francis Group, Boca Raton, FL, pp 463–524Google Scholar
  30. Klemm D, Schumann D, Udhardt U, Marsch S (2001) Bacterial synthesized cellulose—artificial blood vessels for microsurgery. Prog Polym Sci 26:1561–1603. doi: 10.1016/s0079-6700(01)00021-1 CrossRefGoogle Scholar
  31. Kondo T (1998) Hydrogen bonds in cellulose and cellulose derivatives. In: Dumitriu S (ed) Polysaccharides: structural diversity and functional versatility. vol Dumitriu S. Marcel Dekker Inc, New York, NY, pp 131–172Google Scholar
  32. Kuboki Y, Jin Q, Takita H (2001) Geometry of carriers controlling phenotypic expression in BMP-induced osteogenesis and chondrogenesis. J Bone Joint Surg Am 83-A(Suppl 1 (Pt 2)):S105–S115Google Scholar
  33. Kubota T, Sato K, Yamamoto S, Hirano A (1984) Ultrastructural study of the formation of psammoma bodies in fibroblastic meningioma. J Neurosurg 60:512–517. doi: 10.3171/jns.1984.60.3.0512 CrossRefGoogle Scholar
  34. Kucharzewski M, Slezak A, Franek A (2003) Topical treatment of non-healing venous leg ulcers by cellulose membrane. Phlebologie 32:138–169Google Scholar
  35. Lee YM et al (2000) Tissue engineered bone formation using chitosan/tricalcium phosphate sponges. J Periodontol 71:410–417. doi: 10.1902/jop.2000.71.3.410 CrossRefGoogle Scholar
  36. Li J, Wan Y, Li L, Liang H, Wang J (2009) Preparation and characterization of 2,3-dialdehyde bacterial cellulose for potential biodegradable tissue engineering scaffolds. Mater Sci Eng C 29:1635–1642. doi: 10.1016/j.msec.2009.01.006 CrossRefGoogle Scholar
  37. Li K, Wang J, Liu X, Xiong X, Liu H (2012) Biomimetic growth of hydroxyapatite on phosphorylated electrospun cellulose nanofibers. Carbohydr Polym 90:1573–1581. doi: 10.1016/j.carbpol.2012.07.033 CrossRefGoogle Scholar
  38. Liu H, Webster TJ (2011) Enhanced biological and mechanical properties of well-dispersed nanophase ceramics in polymer composites: from 2D to 3D printed structures. Mater Sci Eng C 31:77–89. doi: 10.1016/j.msec.2010.07.013 CrossRefGoogle Scholar
  39. Lynn AK, Best SM, Cameron RE, Harley BA, Yannas IV, Gibson LJ, Bonfield W (2010) Design of a multiphase osteochondral scaffold. I. Control of chemical composition. J Biomed Mater Res A 92A:1057–1065. doi: 10.1002/jbm.a.32415 Google Scholar
  40. Ma J et al (2014) Concise review: cell-based strategies in bone tissue engineering and regenerative medicine. Stem Cells Transl Med 3:98–107. doi: 10.5966/sctm.2013-0126 CrossRefGoogle Scholar
  41. Marolt D, Knezevic M, Novakovic GV (2010) Bone tissue engineering with human stem cells. Stem Cell Res Ther 1:10–20CrossRefGoogle Scholar
  42. Murphy CM, Haugh MG, O’Brien FJ (2010) The effect of mean pore size on cell attachment, proliferation and migration in collagen–glycosaminoglycan scaffolds for bone tissue engineering. Biomaterials 31:461–466. doi: 10.1016/j.biomaterials.2009.09.063 CrossRefGoogle Scholar
  43. Nguyen DT, Burg KJL (2015) Bone tissue engineering and regenerative medicine: targeting pathological fractures. J Biomed Mater Res A 103:420–429. doi: 10.1002/jbm.a.35139 CrossRefGoogle Scholar
  44. Okita Y, Saito T, Isogai A (2010) Entire surface oxidation of various cellulose microfibrils by TEMPO-mediated oxidation. Biomacromolecules 11:1696–1700. doi: 10.1021/bm100214b CrossRefGoogle Scholar
  45. Painter TJ (1988) Control of depolymerisation during the preparation of reduced dialdehyde cellulose. Carbohydr Res 179:259–268. doi: 10.1016/0008-6215(88)84123-5 CrossRefGoogle Scholar
  46. Parenteau-Bareil R, Gauvin R, Berthod F (2010) Collagen-based biomaterials for tissue engineering applications. Materials 3:1863–1887CrossRefGoogle Scholar
  47. Pina S, Oliveira JM, Reis RL (2015) Natural-based nanocomposites for bone tissue engineering and regenerative medicine: a review. Adv Mater. doi: 10.1002/adma.201403354 Google Scholar
  48. Pommerening K, Rein H, Betram D, Muller R (1992) Estimation of dialdehyde groups in 2,3-dialdehyde bead cellulose. Carboohydr Res 233:219–223CrossRefGoogle Scholar
  49. Rambo CR, Recouvreux DOS, Carminatti CA, Pitlovanciv AK, Antônio RV, Porto LM (2008) Template assisted synthesis of porous nanofibrous cellulose membranes for tissue engineering. Mater Sci Eng C 28:549–554. doi: 10.1016/j.msec.2007.11.011 CrossRefGoogle Scholar
  50. Roy-Chowdhury P, Kumar V (2006) Fabrication and evaluation of porous 2,3-dialdehydecellulose membrane as a potential biodegradable tissue-engineering scaffold. J Biomed Mater Res A 76A:300–309. doi: 10.1002/jbm.a.30503 CrossRefGoogle Scholar
  51. Salmen L, Akerholm M, Hinterstoisser B (2005) Two-dimensional Fourier transform infrared spectroscopy applied to cellulose and paper. In: Dumitriu S (ed) Polysaccharides: structural diversity and functional versatility, 2nd edn. CRC Press, Taylor & Francis Group, Boca Raton, FL, pp 159–187Google Scholar
  52. Sanchavanakit N, Sangrungraungroj W, Kaomongkolgit R, Banaprasert T, Pavasant P, Phisalaphong M (2006) Growth of human keratinocytes and fibroblasts on bacterial cellulose film. Biotechnol Prog 22:1194–1199. doi: 10.1021/bp060035o CrossRefGoogle Scholar
  53. Schrarnrn M, Hestrin S (1954) Factors affecting production of cellulose at the air/liquid interface of a culture of Acetobacter xylinum. J Gen Microbiol 11:123–129CrossRefGoogle Scholar
  54. Smith EL et al (2014) Evaluation of skeletal tissue repair, part 1: assessment of novel growth-factor-releasing hydrogels in an ex vivo chick femur defect model. Acta Biomater 10:4186–4196. doi: 10.1016/j.actbio.2014.06.011 CrossRefGoogle Scholar
  55. Suárez-González D et al (2013) Controlled multiple growth factor delivery from bone tissue engineering scaffolds via designed affinity. Tissue Eng A 20:2077–2087. doi: 10.1089/ten.tea.2013.0358 CrossRefGoogle Scholar
  56. 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 26:419–431. doi: 10.1016/j.biomaterials.2004.02.049 CrossRefGoogle Scholar
  57. Torabinejad B, Mohammadi-Rovshandeh J, Davachi SM, Zamanian A (2014) Synthesis and characterization of nanocomposite scaffolds based on triblock copolymer of l-lactide, ε-caprolactone and nano-hydroxyapatite for bone tissue engineering. Mater Sci Eng C 42:199–210. doi: 10.1016/j.msec.2014.05.003 CrossRefGoogle Scholar
  58. Tracy BM, Doremus RH (1984) Direct electron microscopy studies of the bone–hydroxylapatite interface. J Biomed Mater Res 18:719–726. doi: 10.1002/jbm.820180702 CrossRefGoogle Scholar
  59. Tsuruga E, Takita H, Itoh H, Wakisaka Y, Kuboki Y (1997) Pore size of porous hydroxyapatite as the cell-substratum controls BMP-induced osteogenesis. J Biochem 121:317–324CrossRefGoogle Scholar
  60. Ustundag CB, Kaya F, Kamitakahara M, Kaya C, Ioku K (2012) Production of tubular porous hydroxyapatite using electrophoretic deposition. J Ceram Soc Jpn 120:569–573CrossRefGoogle Scholar
  61. Varma AJ, Kulkarni MP (2002) Oxidation of cellulose under controlled conditions. Polym Degrad Stabil 77:25–27CrossRefGoogle Scholar
  62. Wan YZ et al (2007) Biomimetic synthesis of hydroxyapatite/bacterial cellulose nanocomposites for biomedical applications. Mater Sci Eng C 27:855–864. doi: 10.1016/j.msec.2006.10.002 CrossRefGoogle Scholar
  63. Wang J, Chunxi Y, Yizao W, Honglin L, Fang H, Kerong D, Yuan H (2011) Laser patterning of bacterial cellulose hydrogel and its modification with gelatin and hydroxyapatite for bone tissue engineering. Soft Mater 11:173–180. doi: 10.1080/1539445X.2011.611204 Google Scholar
  64. Webster TJ, Ergun C, Doremus RH, Siegel RW, Bizios R (2000a) Enhanced functions of osteoblasts on nanophase ceramics. Biomaterials 21:1803–1810. doi: 10.1016/S0142-9612(00)00075-2 CrossRefGoogle Scholar
  65. Webster TJ, Ergun C, Doremus RH, Siegel RW, Bizios R (2000b) Specific proteins mediate enhanced osteoblast adhesion on nanophase ceramics. J Biomed Mater Res 51:475–483. doi: 10.1002/1097-4636(20000905)51:3<475:aid-jbm23>;2-9 CrossRefGoogle Scholar
  66. Wegman F, Geuze RE, van der Helm YJ, Cumhur Öner F, Dhert WJA, Alblas J (2014) Gene delivery of bone morphogenetic protein-2 plasmid DNA promotes bone formation in a large animal model. J Tissue Eng Regen Med 8:763–770. doi: 10.1002/term.1571 CrossRefGoogle Scholar
  67. Wu J, Zheng Y, Yang Z, Lin Q, Qiao K, Chen X, Peng Y (2014) Influence of dialdehyde bacterial cellulose with the nonlinear elasticity and topology structure of ECM on cell adhesion and proliferation. RSC Adv 4:3998–4009. doi: 10.1039/C3RA45407J CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Pelagie Marlene Favi
    • 1
  • Sandra Patricia Ospina
    • 2
  • Mukta Kachole
    • 3
  • Ming Gao
    • 4
  • Lucia Atehortua
    • 5
  • Thomas Jay Webster
    • 1
    • 6
  1. 1.Department of Chemical EngineeringNortheastern UniversityBostonUSA
  2. 2.Institute of Biology, University Research HeadquartersUniversity of AntioquiaMedellínColombia
  3. 3.Department of Chemistry and Chemical BiologyNortheastern UniversityBostonUSA
  4. 4.Department of Pharmaceutical Sciences, School of Pharmacy, Bouvé College of Health SciencesNortheastern UniversityBostonUSA
  5. 5.Instituto de BiologíaSede de Investigación Universidad de Antioquia-SIU.MedellínColombia
  6. 6.Center of Excellence for Advanced Materials ResearchKing Abdulaziz UniversityJeddahSaudi Arabia

Personalised recommendations