Skip to main content

Advertisement

SpringerLink
Log in

Spreading, proliferation and differentiation of human dental pulp stem cells on chitosan scaffolds immobilized with RGD or fibronectin

  • Original Article
  • Published:
Cytotechnology Aims and scope Submit manuscript

Abstract

Nowadays, human dental pulp stem cells (hDPSCs) became more attractive for therapeutic purposes because of their high proliferation and differentiation potential. Thus, coupling the desired cellular characteristics of hDPSCs with good biomaterial properties of the chitosan scaffolds provide an interesting approach for tissue engineering applications. On the other hand, scaffold surface modification is also needed to promote stem cell adhesion since chitosan lacks adhesion motifs to support direct cell anchorage. In this study, hDPSCs were isolated from third molars of healthy female individuals (aged 16–25) with enzymatic digestion. For cell culture studies, the chitosan scaffolds which have approximately 9 mm diameter and 2 mm thickness with interconnected structure were prepared by freeze-drying. To support cellular attachment the scaffolds were covalently immobilized with either RGD (arginine-glycine-aspartic acid) or fibronectin (Fn) molecules. Cells were seeded on chitosan scaffolds with or without immobilized RGD and fibronectin. Cell attachment, spreading, adhesion behaviors and proliferation capacity were examined by scanning electron microscopy, immunofluorescence staining and PrestoBlue® assays, respectively. In addition, differentiation potential of hDPSCs on Fn immobilized chitosan scaffolds was determined with real time reverse transcriptase polymerase chain reaction analysis. The results showed that chitosan scaffolds were not able to support stem cell attachment. hDPSCs on chitosan scaffolds formed spheroids more quickly and the size of spheroids were smaller than on chitosan-RGD while Fn-immobilized chitosan scaffolds strongly supported cellular attachment but not odontogenic differentiation. The results suggest that the Fn-immobilized chitosan scaffolds may serve as good three-dimensional substrates for dental pulp stem cell attachment and proliferation. In the case of dental regeneration, they must be supported by appropriate biosignals to induce odontogenic differentiation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Arnold M, Cavalcanti-Adam EA, Glass R, Blummel J, Eck W, Kantlehner M et al (2004) Activation of integrin function by nanopatterned adhesive interfaces. ChemPhysChem 5:383–388

    Article  CAS  Google Scholar 

  • Atari M, Gil-Recio C, Fabregat M, García-Fernández D, Barajas M, Carrasco MA et al (2012) Dental pulp of the third molar: a new source of pluripotent-like stem cells. J Cell Sci 125:3343–3356

    Article  CAS  Google Scholar 

  • Beşkardeş IG, Demirtaş TT, Durukan MD, Gümüşderelioğlu M (2015) Microwave-assisted fabrication of chitosan-hydroxyapatite superporous hydrogel composites as bone scaffolds. J Tissue Eng Regen Med 9:1233–1246

    Article  Google Scholar 

  • Biran R, Webb K, Noble MD, Tresco PA (2001) Surfactant-immobilized fibronectin enhances bioactivity and regulates sensory neurite outgrowth. J Biomed Mater Res 55:1–12

    Article  CAS  Google Scholar 

  • Boncler M, Rozalski M, Krajewska U, Podsedek A, Watala C (2014) Comparison of PrestoBlue and MTT assays of cellular viability in the assessment of anti-proliferative effects of plant extracts on human endothelial cells. J Pharmacol Toxicol Methods 69:9–16

    Article  CAS  Google Scholar 

  • Braut A, Kollar EJ, Mina M (2003) Analysis of the odontogenic and osteogenic potentials of dental pulp in vivo using a Col1a1-2.3-GFP transgene. Int J Dev Biol 47:281–292

    CAS  Google Scholar 

  • Chandrahasa S, Murray PE, Namerow KN (2011) Proliferation of mature ex vivo human dental pulp using tissue engineering scaffolds. J Endod 37:1236–1239

    Article  Google Scholar 

  • Cheng NC, Wang S, Young TH (2012) The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities. Biomaterials 33:1748–1758

    Article  CAS  Google Scholar 

  • Clements LR, Wang PY, Harding F, Tsai WB, Thissen H, Voelcker NH (2011) Mesenchymal stem cell attachment to peptide density gradients on porous silicon generated by electrografting. Phys Status Solidi A 208:1440–1445

    Article  CAS  Google Scholar 

  • Collart-Dutilleul PY, Secret E, Panayotov I, Deville de Periere D, Martin-Palma RJ, Torres-Costa V et al (2014) Adhesion and proliferation of human mesenchymal stem cells from dental pulp on porous silicon scaffolds. ACS Appl Mater Interfaces 6:1719–1728

    Article  CAS  Google Scholar 

  • Custodio CA, Alves CM, Reis RL, Mano JF (2010) Immobilization of fibronectin in chitosan substrates improves cell adhesion and proliferation. J Tissue Eng Regen Med 4:316–323

    Article  CAS  Google Scholar 

  • D’Anto V, Raucci MG, Guarino V, Martina S, Valletta R, Ambrosio L (2016) Behaviour of human mesenchymal stem cells on chemically synthesized HA-PCL scaffolds for hard tissue regeneration. J Tissue Eng Regen Med 10:E147–E154

    Article  Google Scholar 

  • de Mel A, Jell G, Stevens MM, Seifalian AM (2008) Biofunctionalization of biomaterials for accelerated in situ endothelialization: a review. Biomacromolecules 9:2969–2979

    Article  Google Scholar 

  • Dutta PK, Dutta J, Tripathi VS (2004) Chitin and chitosan: chemistry, properties and applications. J Sci Ind Res 63:20–31

    CAS  Google Scholar 

  • Eslaminejad MB, Bordbar S, Nazarian H (2013) Odontogenic differentiation of dental pulp-derived stem cells on tricalcium phosphate scaffolds. J Dent Sci 8:306–313

    Article  Google Scholar 

  • Gao M, Sotomayor M, Villa E, Lee EH, Schulten K (2006) Molecular mechanisms of cellular mechanics. Phys Chem Chem Phys 8:3692–3706

    Article  CAS  Google Scholar 

  • Goy RC, de Britto D, Assis OBG (2009) A Review of the antimicrobial activity of chitosan. Polimeros 19:241–247

    CAS  Google Scholar 

  • Gronthos S, Mankani M, Brahim J, Robey PG, Shi S (2000) Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA 97:13625–13630

    Article  CAS  Google Scholar 

  • Ho MH, Wang DM, Hsieh HJ, Liu HC, Hsien TY, Lai JY et al (2005) Preparation and characterization of RGD-immobilized chitosan scaffolds. Biomaterials 26:3197–3206

    Article  CAS  Google Scholar 

  • Hsu SH, Huang GS, Feng F (2012) Isolation of the multipotent MSC subpopulation from human gingival fibroblasts by culturing on chitosan membranes. Biomaterials 33:2642–2655

    Article  CAS  Google Scholar 

  • Huang GS, Dai LG, Yen BL, Hsu SH (2011) Spheroid formation of mesenchymal stem cells on chitosan and chitosan-hyaluronan membranes. Biomaterials 32:6929–6945

    Article  CAS  Google Scholar 

  • Jongpaiboonkit L, King WJ, Murphy WL (2009) Screening for 3D Environments That support human mesenchymal stem cell viability using hydrogel arrays. Tissue Eng Part A 15:343–353

    Article  CAS  Google Scholar 

  • Karaoz E, Dogan BN, Aksoy A, Gacar G, Akyuz S, Ayhan S et al (2010) Isolation and in vitro characterisation of dental pulp stem cells from natal teeth. Histochem Cell Biol 133:95–112

    Article  CAS  Google Scholar 

  • Kim NR, Lee DH, Chung P-H, Yang H-C (2009) Distinct differentiation properties of human dental pulp cells on collagen, gelatin, and chitosan scaffolds. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 108:e94–e100

    Article  Google Scholar 

  • Kim JJ, Bae WJ, Kim JM, Kim JJ, Lee EJ, Kim HW et al (2014) Mineralized polycaprolactone nanofibrous matrix for odontogenesis of human dental pulp cells. J Biomater Appl 28:1069–1078

    Article  CAS  Google Scholar 

  • Lee J-H, Lee D-S, Choung H-W, Shon W-J, Seo B-M, Lee E-H et al (2011) Odontogenic differentiation of human dental pulp stem cells induced by preameloblast-derived factors. Biomaterials 32:9696–9706

    Article  CAS  Google Scholar 

  • Li J-H, Liu D-Y, Zhang F-M, Wang F, Zhang W-K, Zhang Z-T (2011) Human dental pulp stem cell is a promising autologous seed cell for bone tissue engineering. Chin Med J 124:4022–4028

    CAS  Google Scholar 

  • Li J, Yan M, Wang Z, Jing S, Li Y, Liu G, Yu J, Fan Z (2014) Effects of canonical nf-b signaling pathway on the proliferation and odonto/osteogenic differentiation of human stem cells from apical papilla. BioMed Res Int 2014:1–12

    Google Scholar 

  • Liu D, Che Abdullah CA, Sear RP, Keddie JL (2010) Cell adhesion on nanopatterned fibronectin substrates. Soft Matter 6:5408–5416

    Article  CAS  Google Scholar 

  • Martens W, Sanen K, Georgiou M, Struys T, Bronckaers A, Ameloot M et al (2014) Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue-engineered collagen construct in vitro. FASEB J 28:1634–1643

    Article  Google Scholar 

  • Murphy WL, McDevitt TC, Engler AJ (2014) Materials as stem cell regulators. Nat Mater 13:547–557

    Article  CAS  Google Scholar 

  • Petrie TA, Capadona JR, Reyes CD, García AJ (2006) Integrin specificity and enhanced cellular activities associated with surfaces presenting a recombinant fibronectin fragment compared to RGD supports. Biomaterials 27:5459–5470

    Article  CAS  Google Scholar 

  • Tigli RS, Karakecili A, Gumusderelioglu M (2007) In vitro characterization of chitosan scaffolds: influence of composition and deacetylation degree. J Mater Sci Mater Med 18:1665–1674

    Article  Google Scholar 

  • Tsai W-B, Chen RP-Y, Wei K-L, Tan S-F, Lai J-Y (2010) Modulation of RGD-functionalized polyelectrolyte multilayer membranes for promoting osteoblast function. J Biomat Sci Polym E 21:377–394

    Article  CAS  Google Scholar 

  • Tsai W-B, Chen Y-R, Liu H-L, Lai J-Y (2011) Fabrication of UV-crosslinked chitosan scaffolds with conjugation of RGD peptides for bone tissue engineering. Carbohydr Polym 85:129–137

    Article  CAS  Google Scholar 

  • Tsai W-B, Chen Y-R, Liu H-L (2013) RGD-conjugated crosslinked chitosan scaffolds for culture and osteogenic differentiation of mesenchymal stem cells. J Taiwan Inst Chem Eng 44:1–7

    Article  CAS  Google Scholar 

  • VandeVord PJ, Matthew HW, DeSilva SP, Mayton L, Wu B, Wooley PH (2002) Evaluation of the biocompatibility of a chitosan scaffold in mice. J Biomed Mater Res 59:585–590

    Article  CAS  Google Scholar 

  • Vargas Becerril N, Téllez-Jurado L, Rodriguez-Lorenzo LM (2013) Adsorption of fibronectin on hydroxyapatite functionalized with alendronate. J Aust Ceram Soc 49:112–118

    Google Scholar 

  • Vishwanath VR, Nadig RR, Nadig R, Prasanna JS, Karthik J, Pai VS (2013) Differentiation of isolated and characterized human dental pulp stem cells and stem cells from human exfoliated deciduous teeth: an in vitro study. J Conserv Dent 16:423–428

    Article  Google Scholar 

  • Yeh HY, Liu BH, Hsu SH (2012) The calcium-dependent regulation of spheroid formation and cardiomyogenic differentiation for MSCs on chitosan membranes. Biomaterials 33:8943–8954

    Article  CAS  Google Scholar 

  • Yeh HY, Liu BH, Sieber M, Hsu SH (2014) Substrate-dependent gene regulation of self-assembled human MSC spheroids on chitosan membranes. BMC Genom 15:10

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the Scientific and Technological Research Council of Turkey (TÜBİTAK), Grant No: SBAG-114S755. The authors are grateful to Selin Gümüşderelioğlu for editing of language in the text.

Author information

Authors and Affiliations

  1. Department of Nanotechnology and Nanomedicine, Hacettepe University, Ankara, Turkey

    Farzin Asghari Sana, Arlin S. Kiremitçi & Menemşe Gümüşderelioğlu

  2. Department of Bioengineering, Hacettepe University, Ankara, Turkey

    Merve Çapkın Yurtsever, Gökçe Kaynak Bayrak, Ekin Özge Tunçay, Arlin S. Kiremitçi & Menemşe Gümüşderelioğlu

  3. Department of Restorative Dentistry, Hacettepe University, Ankara, Turkey

    Arlin S. Kiremitçi

  4. Chemical Engineering Department, Hacettepe University, Ankara, Turkey

    Menemşe Gümüşderelioğlu

Authors
  1. Farzin Asghari Sana
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Merve Çapkın Yurtsever
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gökçe Kaynak Bayrak
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ekin Özge Tunçay
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Arlin S. Kiremitçi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Menemşe Gümüşderelioğlu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Menemşe Gümüşderelioğlu.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Asghari Sana, F., Çapkın Yurtsever, M., Kaynak Bayrak, G. et al. Spreading, proliferation and differentiation of human dental pulp stem cells on chitosan scaffolds immobilized with RGD or fibronectin. Cytotechnology 69, 617–630 (2017). https://doi.org/10.1007/s10616-017-0072-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10616-017-0072-9

Keywords

Search

Navigation