Nano Research

, Volume 11, Issue 7, pp 3658–3677 | Cite as

Promoting osteogenic differentiation in pre-osteoblasts and reducing tibial fracture healing time using functional nanofibers

  • Gu Cheng
  • Jiajia Chen
  • Qun Wang
  • Xuewen Yang
  • Yuet Cheng
  • Zhi Li
  • Hu Tu
  • Hongbing DengEmail author
  • Zubing LiEmail author
Research Article


Various composite materials are now used as artificial tissue substitutes, and are defining new frontiers in tissue engineering. In the present study, composite membranes based on silk fibroin (SF) were fabricated to form a synthetic periosteum. The fabricated membranes were physicochemically characterized by their morphology, porosity, biocompatibility, biodegradability, chemical structure, and mechanical properties. Following the addition of polycaprolactone (PCL) to the silk fibers, there was a 3–5-fold increase in the elongation at break compared with the pure silk membranes, and surface wettability was retained. The degradation time of the SF within the membranes was also prolonged by adding PCL. Compared with pure PCL membranes or plastic culture plates, the SF-based membranes significantly enhanced the cellular viability and osteogenic differentiation capability of MC3T3-E1 cells. Higher expression levels of osteogenic differentiation markers (runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteopontin (OP)) further supported the use of the SF component in bone-related applications. A non-rigid internal fixation (non-RIF) fracture model that healed via endochondral bone formation was created, and fracture callus samples were collected to perform micro-computed tomography, histology, and immunohistochemistry analyses at 8 weeks after surgery. A smaller bone volume accompanied by a mineralized bony callus was observed in SF/PCL membrane-treated rats. Immunohistochemistry also indicated that the SF/PCL membrane-treated rats exhibited increased osteocalcin expression but reduced collagen type X expression. These findings could lead to an alternative strategy for treating comminuted fractures with enhanced intramembranous ossification and reduced endochondral ossification.


silk fibroin polycaprolactone scaffold bone tissue engineering 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.



This work was supported by the National Key Research and Development Program of China (No. 2016YFB0303303), the National High-tech R&D Program of China (863 program, No. 2015AA020313), and the National Natural Science Foundation of China (No. 81271107), and was partially supported by the Natural Science Foundation of Hubei Province of China (Team Project, No. 2015CFA017).


  1. [1]
    Tabisz, B.; Schmitz, W.; Schmitz, M.; Luehmann, T.; Heusler, E.; Rybak, J. C.; Meinel, L.; Fiebig, J. E.; Mueller, T. D.; Nickel, J. Site-directed immobilization of BMP-2: Two approaches for the production of innovative osteoinductive scaffolds. Biomacromolecules 2017, 18, 695–708.CrossRefGoogle Scholar
  2. [2]
    Gu, Z.; Aimetti, A. A.; Wang, Q.; Dang, T. T.; Zhang, Y. L.; Veiseh, O.; Cheng, H.; Langer, R. S.; Anderson, D. G. Injectable nano-network for glucose-mediated insulin delivery. ACS Nano 2013, 7, 4194–4201.CrossRefGoogle Scholar
  3. [3]
    Yu, J. C.; Zhang, Y. Q.; Sun, W. J.; Kahkoska, A. R.; Wang, J. Q.; Buse, J. B.; Gu, Z. Insulin-responsive glucagon delivery for prevention of hypoglycemia. Small 2017, 13, 16030.28.Google Scholar
  4. [4]
    Evans, S. F.; Parent, J. B.; Lasko, C. E.; Zhen, X. W.; Knothe, U. R.; Lemaire, T.; Tate, M. L. K. Periosteum, bone’s “smart” bounding membrane, exhibits direction-dependent permeability. J. Bone Mineral Res. 2013, 28, 608–617.CrossRefGoogle Scholar
  5. [5]
    Hutmacher, D. W. Scaffolds in tissue engineering bone and cartilage. Biomaterials 2000, 21, 2529–2543. asp 18 Nano Res.CrossRefGoogle Scholar
  6. [6]
    Frohbergh, M. E.; Katsman, A.; Botta, G. P.; Lazarovici, P.; Schauer, C. L.; Wegst, U. G. K.; Lelkes, P. I. Electrospun hydroxyapatite-containing chitosan nanofibers crosslinked with genipin for bone tissue engineering. Biomaterials 2012, 33, 9167–9178.CrossRefGoogle Scholar
  7. [7]
    Hoffman, M. D.; Xie, C.; Zhang, X. P.; Benoit, D. S. W. The effect of mesenchymal stem cells delivered via hydrogelbased tissue engineered periosteum on bone allograft healing. Biomaterials 2013, 34, 8887–8898.CrossRefGoogle Scholar
  8. [8]
    Kang, Y. Q.; Ren, L. L.; Yang, Y. Z. Engineering vascularized bone grafts by integrating a biomimetic periosteum and β-TCP scaffold. ACS Appl. Mater. Interfaces 2014, 6, 9622–9633.CrossRefGoogle Scholar
  9. [9]
    Zhou, Y. F.; Chen, F. L.; Ho, S. T.; Woodruff, M. A.; Lim, T. M.; Hutmacher, D. W. Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts. Biomaterials 2007, 28, 814–824.CrossRefGoogle Scholar
  10. [10]
    Chen, K.; Lin, X. F.; Zhang, Q.; Ni, J. H.; Li, J. M.; Xiao, J.; Wang, Y.; Ye, Y. H.; Chen, L.; Jin, K. K. et al. Decellularized periosteum as a potential biologic scaffold for bone tissue engineering. Acta Biomater. 2015, 19, 46–55.CrossRefGoogle Scholar
  11. [11]
    Zhao, X.; Sun, X. M.; Yildirimer, L.; Lang, Q.; Lin, Z. Y.; Zheng, R. L.; Zhang, Y. G.; Cui, W. G.; Annabi, N.; Khademhosseini, A. Cell infiltrative hydrogel fibrous scaffolds for accelerated wound healing. Acta Biomater. 2017, 49, 66–77.CrossRefGoogle Scholar
  12. [12]
    Xu, Y.; Cui, W. G.; Zhang, Y. X.; Zhou, P. H.; Gu, Y.; Shen, X. F.; Li, B.; Chen, L. Hierarchical micro/nanofibrous bioscaffolds for structural tissue regeneration. Adv. Healthcare Mater. 2017, 6, 16014.57.Google Scholar
  13. [13]
    Lee, J. M.; Chae, T.; Sheikh, F. A.; Ju, H. W.; Moon, B. M.; Park, H. J.; Park, Y. R.; Park, C. H. Three dimensional poly(ε-caprolactone) and silk fibroin nanocomposite fibrous matrix for artificial dermis. Mater. Sci. Eng. C 2016, 68, 758–767.CrossRefGoogle Scholar
  14. [14]
    Shao, Z. Z.; Vollrath, F. Materials: Surprising strength of silkworm silk. Nature 2002, 418, 741..CrossRefGoogle Scholar
  15. [15]
    Chlapanidas, T.; Faragò, S.; Mingotto, F.; Crovato, F.; Tosca, M. C.; Antonioli, B.; Bucco, M.; Lucconi, G.; Scalise, A.; Vigo, D. et al. Regenerated silk fibroin scaffold and infrapatellar adipose stromal vascular fraction as feederlayer: A new product for cartilage advanced therapy. Tissue Eng. Part A 2011, 17, 1725–1733.CrossRefGoogle Scholar
  16. [16]
    Chen, K.; Sahoo, S.; He, P. F.; Ng, K. S.; Toh, S. L.; Goh, J. C. H. A hybrid silk/RADA-based fibrous scaffold with triple hierarchy for ligament regeneration. Tissue Eng. Part A 2012, 18, 1399–1409.CrossRefGoogle Scholar
  17. [17]
    Lee, O. J.; Ju, H. W.; Kim, J. H.; Lee, J. M.; Ki, C. S.; Kim, J. H.; Moon, B. M.; Park, H. J.; Sheikh, F. A.; Park, C. H. Development of artificial dermis using 3D electrospun silk fibroin nanofiber matrix. J. Biomed. Nanotechnol. 2014, 10, 1294–1303.CrossRefGoogle Scholar
  18. [18]
    Wang, Y. Z.; Rudym, D. D.; Walsh, A.; Abrahamsen, L.; Kim, H. J.; Kim, H. S.; Kirker-Head, C.; Kaplan, D. L. In vivo degradation of three-dimensional silk fibroin scaffolds. Biomaterials 2008, 29, 3415–3428.CrossRefGoogle Scholar
  19. [19]
    Acharya, C.; Ghosh, S. K.; Kundu, S. C. Silk fibroin film from non-mulberry tropical tasar silkworms: A novel substrate for in vitro fibroblast culture. Acta Biomater. 2009, 5, 429–437.CrossRefGoogle Scholar
  20. [20]
    Benfenati, V.; Toffanin, S.; Capelli, R.; Camassa, L. M. A.; Ferroni, S.; Kaplan, D. L.; Omenetto, F. G.; Muccini, M.; Zamboni, R. A silk platform that enables electrophysiology and targeted drug delivery in brain astroglial cells. Biomaterials 2010, 31, 7883–7891.CrossRefGoogle Scholar
  21. [21]
    Melke, J.; Midha, S.; Ghosh, S.; Ito, K.; Hofmann, S. Silk fibroin as biomaterial for bone tissue engineering. Acta Biomater. 2016, 31, 1–16.CrossRefGoogle Scholar
  22. [22]
    Meinel, L.; Hofmann, S.; Betz, O.; Fajardo, R.; Merkle, H. P.; Langer, R.; Evans, C. H.; Vunjak-Novakovic, G.; Kaplan, D. L. Osteogenesis by human mesenchymal stem cells cultured on silk biomaterials: Comparison of adenovirus mediated gene transfer and protein delivery of BMP-2. Biomaterials 2006, 27, 4993–5002.CrossRefGoogle Scholar
  23. [23]
    Mi, R. X.; Liu, Y. X.; Chen, X.; Shao, Z. Z. Structure and properties of various hybrids fabricated by silk nanofibrils and nanohydroxyapatite. Nanoscale 2016, 8, 20096–20102.CrossRefGoogle Scholar
  24. [24]
    Mcclure, M. J.; Sell, S. A.; Ayres, C. E.; Simpson, D. G.; Bowlin, G. L. Electrospinning-aligned and random polydioxanone-polycaprolactone-silk fibroin-blended scaffolds: Geometry for a vascular matrix. Biomed. Mater. 2009, 4, 05501.0.CrossRefGoogle Scholar
  25. [25]
    Lee, H.; Kim, G. Biocomposites electrospun with poly(ε-aprolactone) and silk fibroin powder for biomedical applications. J. Biomater. Sci. 2010, 21, 1687–1699.CrossRefGoogle Scholar
  26. [26]
    Zhang, F.; You, X. R.; Dou, H.; Liu, Z.; Zuo, B. Q.; Zhang, X. G. Facile fabrication of robust silk nanofibril films via direct dissolution of silk in CaCl2-formic acid solution. ACS Appl. Mater. Interfaces 2015, 7, 3352–3361.CrossRefGoogle Scholar
  27. [27]
    Guan, X. F.; Avci-Adali, M.; Alarçin, E.; Cheng, H.; Kashaf, S. S.; Li, Y. X.; Chawla, A.; Jang, H. L.; Khademhosseini, A. Development of hydrogels for regenerative engineering. Biotechnol. J. 2017, 12, 16003.94.CrossRefGoogle Scholar
  28. [28]
    Chen, X.; Wang, W. D.; Cheng, S.; Dong, B.; Li, C. Y. Mimicking bone nanostructure by combining block copolymer self-assembly and 1D crystal nucleation. ACS Nano 2013, 7, 8251–8257.CrossRefGoogle Scholar
  29. [29]
    Cheng, Z. Y.; Teoh, S. H. Surface modification of ultra thin poly (ε-caprolactone) films using acrylic acid and collagen. Biomaterials 2004, 25, 1991–2001.CrossRefGoogle Scholar
  30. [30]
    Huang, R.; Li, W. Z.; Lv, X. X.; Lei, Z. J.; Bian, Y. Q.; Deng, H. B.; Wang, H. J.; Li, J. Q.; Li, X. Y. Biomimetic LBL structured nanofibrous matrices assembled by chitosan/ collagen for promoting wound healing. Biomaterials 2015, 53, 58–75.CrossRefGoogle Scholar
  31. [31]
    Colnot, L. Skeletal cell fate decisions within periosteum and bone marrow during bone regeneration. J. Bone Mineral Res. 2009, 24, 274–282.CrossRefGoogle Scholar
  32. [32]
    Roberts, S. J.; Van Gastel, N.; Carmeliet, G.; Luyten, F. P. Uncovering the periosteum for skeletal regeneration: The stem cell that lies beneath. Bone 2015, 70, 10–18.CrossRefGoogle Scholar
  33. [33]
    van Gastel, N.; Torrekens, S.; Roberts, S. J.; Moermans, K.; Schrooten, J.; Carmeliet, P.; Luttun, A.; Luyten, F. P.; Carmeliet, G. Engineering vascularized bone: Osteogenic and proangiogenic potential of murine periosteal cells. Stem Cells 2012, 30, 2460–2471.CrossRefGoogle Scholar
  34. [34]
    Li, M. Z.; Ogiso, M.; Minoura, N. Enzymatic degradation behavior of porous silk fibroin sheets. Biomaterials 2003, 24, 357–365.CrossRefGoogle Scholar
  35. [35]
    Horan, R. L.; Antle, K.; Collette, A. L.; Wang, Y. Z.; Huang, J.; Moreau, J. E.; Volloch, V.; Kaplan, D. L.; Altman, G. H. In vitro degradation of silk fibroin. Biomaterials 2005, 26, 3385–3393.CrossRefGoogle Scholar
  36. [36]
    Zellin, G.; Gritlilinde, A.; Linde, A. Healing of mandibular defects with different biodegradable and non-biodegradable membranes: An experimental study in rats. Biomaterials 1995, 16, 601–609.CrossRefGoogle Scholar
  37. [37]
    Yuan, W. Z.; Tang, X. Z.; Huang, X. B.; Zheng, S. X. Synthesis, characterization and thermal properties of hexaarmed star-shaped poly(ε-caprolactone)-b-poly(D,L-lactide-co-glycolide) initiated with hydroxyl-terminated cyclotriphosphazene. Polymer 2005, 46, 1701–1707.CrossRefGoogle Scholar
  38. [38]
    Ghasemi-Mobarakeh, L.; Prabhakaran, M. P.; Morshed, M.; Nasr-Esfahani, M. H.; Ramakrishna, S. Electrospun poly(ε-caprolactone)/gelatin nanofibrous scaffolds for nerve tissue engineering. Biomaterials 2008, 29, 4532–4539.CrossRefGoogle Scholar
  39. [39]
    Wilson, D.; Valluzzi, R.; Kaplan, D. Conformational transitions in model silk peptides. Biophys J. 2000, 78, 2690–2701.CrossRefGoogle Scholar
  40. [40]
    Li, Y. P.; Xiao, W. W.; Xiao, K. K.; Berti, L.; Luo, J. T.; Tseng, H. P.; Fung, G.; Lam, K. S. Well-defined, reversible boronate crosslinked nanocarriers for targeted drug delivery in response to pH and cis-diols. Angew. Chem., Int. Ed. 2012, 51, 2864–2869.CrossRefGoogle Scholar
  41. [41]
    Declercq, H. A.; Desmet, T.; Berneel, E. E. M.; Dubruel, P.; Cornelissen, M. J. Synergistic effect of surface modification and scaffold design of bioplotted 3-D poly-ε-caprolactone scaffolds in osteogenic tissue engineering. Acta Biomater. 2013, 9, 7699–7708.CrossRefGoogle Scholar
  42. [42]
    Wang, L.; Wu, Y. B.; Guo, B. L.; Ma, P. X. Nanofiber yarn/hydrogel core–shell scaffolds mimicking native skeletal muscle tissue for guiding 3D myoblast alignment, elongation, and differentiation. ACS Nano 2015, 9, 9167–9179.CrossRefGoogle Scholar
  43. [43]
    Lin, A. S. P.; Barrows, T. H.; Cartmell, S. H.; Guldberg, R. E. Microarchitectural and mechanical characterization of oriented porous polymer scaffolds. Biomaterials 2003, 24, 481–489.CrossRefGoogle Scholar
  44. [44]
    Ghezzi, C. E.; Marelli, B.; Donelli, I.; Alessandrino, A.; Freddi, G.; Nazhat, S. N. Multilayered dense collagen-silk fibroin hybrid: A platform for mesenchymal stem cell differentiation towards chondrogenic and osteogenic lineages. J. Tissue Eng. Regener. Med. 2017, 11, 2046–2059.CrossRefGoogle Scholar
  45. [45]
    Kim, H. K.; Park, K. S.; Lee, J. S.; Kim, J. H.; Park, D. S.; Shin, J. W.; Yoon, T. R. Salicylideneamino-2-thiophenol enhances osteogenic differentiation through the activation of MAPK pathways in multipotent bone marrow stem cell. J. Cell. Biochem. 2012, 113, 1833–1841.CrossRefGoogle Scholar
  46. [46]
    Hoppe, A.; Güldal, N. S.; Boccaccini, A. R. A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. Biomaterials 2011, 32, 2757–2774.CrossRefGoogle Scholar
  47. [47]
    Maeno, S.; Niki, Y.; Matsumoto, H.; Morioka, H.; Yatabe, T.; Funayama, A.; Toyama, Y.; Taguchi, T.; Tanaka, J. The effect of calcium ion concentration on osteoblast viability, proliferation and differentiation in monolayer and 3D culture. Biomaterials 2005, 26, 4847–4855.CrossRefGoogle Scholar
  48. [48]
    Mosekilde, L.; Bak, B. The effects of growth hormone on fracture healing in rats: A histological description. Bone 1993, 14, 19–27.CrossRefGoogle Scholar
  49. [49]
    van Gastel, N.; Stegen, S.; Stockmans, I.; Moermans, K.; Schrooten, J.; Graf, D.; Luyten, F. P.; Carmeliet, G. Expansion of murine periosteal progenitor cells with fibroblast growth factor 2 reveals an intrinsic endochondral ossification program mediated by bone morphogenetic protein 2. Stem Cells 2014, 32, 2407–2418.CrossRefGoogle Scholar
  50. [50]
    Wohl, G. R.; Towler, D. A.; Silva, M. J. Stress fracture healing: Fatigue loading of the rat ulna induces upregulation in expression of osteogenic and angiogenic genes that mimic the intramembranous portion of fracture repair. Bone 2009, 44, 320–330.CrossRefGoogle Scholar
  51. [51]
    Alexander, K. A.; Chang, M. K.; Maylin, E. R.; Kohler, T.; Müller, R.; Wu, A. C.; van Rooijen, N.; Sweet, M. J.; Hume, D. A.; Raggatt, L. J. et al. Osteal macrophages promote in vivo intramembranous bone healing in a mouse tibial injury model. J. Bone Mineral Res. 2011, 26, 1517–1532.CrossRefGoogle Scholar
  52. [52]
    Mi, M.; Jin, H. T.; Wang, B. L.; Yukata, K.; Sheu, T.; Ke, Q. H.; Tong, P. J.; Im, H. J.; Xiao, G. Z.; Chen, D. Chondrocyte BMP2 signaling plays an essential role in bone fracture healing. Gene 2013, 512, 211–218.CrossRefGoogle Scholar
  53. [53]
    Wixted, J. J.; Fanning, P. J.; Gaur, T.; O’ Connell, S. L.; Silva, J.; Mason-Savas, A.; Ayers, D. C.; Stein, G. S.; Lian, J. B. Enhanced fracture repair by leukotriene antagonism is characterized by increased chondrocyte proliferation and early bone formation: A novel role of the cysteinyl LT-1 receptor. J. Cell. Physiol. 2009, 221, 31–39.CrossRefGoogle Scholar
  54. [54]
    Studer, D.; Millan, C.; Öztürk, E.; Maniura-Weber, K.; Zenobi-Wong, M. Molecular and biophysical mechanisms regulating hypertrophic differentiation in chondrocytes and mesenchymal stem cells. Eur. Cells Mater. 2012, 24, 118–135.CrossRefGoogle Scholar
  55. [55]
    You, M.; Jing, J.; Tian, D.; Qian, J.; Yu, G. Dioscin stimulates differentiation of mesenchymal stem cells towards hypertrophic chondrocytes in vitro and endochondral ossification in vivo. Am. J. Transl.Res. 2016, 8, 3930–3938.Google Scholar
  56. [56]
    Grant, W. T.; Wang, G. J.; Balian, G. Type X collagen synthesis during endochondral ossification in fracture repair. J. Biol. Chem. 1987, 262, 9844–9849.Google Scholar

Copyright information

© Tsinghua University Press and Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  • Gu Cheng
    • 1
  • Jiajia Chen
    • 2
  • Qun Wang
    • 3
  • Xuewen Yang
    • 1
  • Yuet Cheng
    • 1
  • Zhi Li
    • 1
  • Hu Tu
    • 2
  • Hongbing Deng
    • 2
    Email author
  • Zubing Li
    • 1
    Email author
  1. 1.The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology & Department of Oral and Maxillofacial Trauma and Plastic Surgery, Wuhan University Stomatological HospitalWuhan UniversityWuhanChina
  2. 2.Hubei International Scientific and Technological Cooperation Base of Sustainable Resource and Energy, Hubei Key Lab of Biomass Resource Chemistry and Environmental Biotechnology, School of Resource and Environmental ScienceWuhan UniversityWuhanChina
  3. 3.Department of Chemical and Biological EngineeringIowa State UniversityAmesUSA

Personalised recommendations