J Periodontal Implant Sci.  2013 Dec;43(6):315-322.

Periodontal regeneration with nano-hyroxyapatite-coated silk scaffolds in dogs

• Yang C
• Lee JS
• Jung UW
• Seo YK
• Park JK
• Choi SH

Abstract

PURPOSE
In this study, we investigated the effect of silk scaffolds on one-wall periodontal intrabony defects. We conjugated nano-hydroxyapatite (nHA) onto a silk scaffold and then seeded periodontal ligament cells (PDLCs) or dental pulp cells (DPCs) onto the scaffold.
METHODS
Five dogs were used in this study. Bilateral 4 mmx2 mm (depthxmesiodistal width), one-wall intrabony periodontal defects were surgically created on the distal side of the mandibular second premolar and the mesial side of the mandibular fourth premolar. In each dog, four of the defects were separately and randomly assigned to the following groups: the PDLC-cultured scaffold transplantation group (PDLC group), the DPC-cultured scaffold transplantation group (DPC group), the normal saline-soaked scaffold transplantation group, and the control group. The animals were euthanized following an 8-week healing interval for clinical, scanning electron microscopy (SEM), and histologic evaluations.
RESULTS
There was no sign of inflammation or other clinical signs of postoperative complications. The examination of cell-seeded constructs by SEM provided visual confirmation of the favorable characteristics of nHA-coated silk scaffolds for tissue engineering. The scaffolds exhibited a firm connective porous structure in cross section, and after PDLCs and DPCs were seeded onto the scaffolds and cultured for 3 weeks, the attachment of well-spread cells and the formation of extracellular matrix (ECM) were observed. The histologic analysis revealed that a well-maintained grafted volume was present at all experimental sites for 8 weeks. Small amounts of inflammatory cells were seen within the scaffolds. The PDLC and DPC groups did not have remarkably different histologic appearances.
CONCLUSIONS
These observations indicate that nHA-coated silk scaffolds can be considered to be potentially useful biomaterials for periodontal regeneration.

Keyword

Dental pulp; Periodontal ligament; Silk; Tissue engineering; Tissue scaffolds

MeSH Terms

Animals
Bicuspid
Biocompatible Materials
Dental Pulp
Dogs*
Extracellular Matrix
Inflammation
Microscopy, Electron, Scanning
Periodontal Ligament
Postoperative Complications
Regeneration*
Silk*
Tissue Engineering
Tissue Scaffolds
Transplants
Biocompatible Materials
Silk

Figure

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  Figure 1 One-wall intrabony defect in a dog. (A) One-wall defects were surgically created (4 mm×2 mm, depth×mesiodistal width). (B) Nano-hydroxyapatite-coated scaffolds were transplanted into the bone defects.

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  Figure 2 Scanning-electron-microscopy (SEM) images of nano-hydroxyapatite-coated silk scaffolds before (A and B, ×100) and after (C and D, ×400) cell culture: cross-sectional (A, C) and longitudinal (B, D) views. Arrows: extracellular matrix.

• 

  Figure 3 Representative photomicrographs from control (A) and experimental (B-D) sites. Low-magnification views of the experimental site in the 8-week control group (A), the 8-week normal saline (NS) group (B), the 8-week periodontal ligament cell (PDLC) group (C), and the 8-week dental pulp cell (DPC) group (D) (H&E, ×40). High-magnification views of the framed areas in a-h (H&E, ×100). Control group (A, a, b), NS group (B, c, d), PDLC group (C, e, f), and DPC group (D, g, h).

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  Figure 4 Representative photomicrographs from sites implanted with different cell sources (H&E, ×100). High-magnification views of the experimental site in the 8-week normal saline group (A), the 8-week periodontal ligament cell group (B), and the 8-week dental pulp cell group (C). Arrowheads: fibrous capsules; asterisks: blood vessels.

Reference

1. Needleman IG, Worthington HV, Giedrys-Leeper E, Tucker RJ. Guided tissue regeneration for periodontal infra-bony defects. Cochrane Database Syst Rev. 2006; (2):CD001724.

2. Venezia E, Goldstein M, Boyan BD, Schwartz Z. The use of enamel matrix derivative in the treatment of periodontal defects: a literature review and meta-analysis. Crit Rev Oral Biol Med. 2004; 15:382–402.

3. Wang HL, Greenwell H, Fiorellini J, Giannobile W, Offenbacher S, Salkin L, et al. Periodontal regeneration. J Periodontol. 2005; 76:1601–1622.

4. Bartold PM, McCulloch CA, Narayanan AS, Pitaru S. Tissue engineering: a new paradigm for periodontal regeneration based on molecular and cell biology. Periodontol 2000. 2000; 24:253–269.

5. Srisuwan T, Tilkorn DJ, Wilson JL, Morrison WA, Messer HM, Thompson EW, et al. Molecular aspects of tissue engineering in the dental field. Periodontol 2000. 2006; 41:88–108.

6. Jang YJ, Jung IH, Park JC, Jung UW, Kim CS, Lee YK, et al. Effect of seeding using an avidin-biotin binding system on the attachment of periodontal ligament fibroblasts to nanohydroxyapatite scaffolds: three-dimensional culture. J Periodontal Implant Sci. 2011; 41:73–78.

7. Willerth SM, Sakiyama-Elbert SE. Combining stem cells and biomaterial scaffolds for constructing tissues and cell delivery. Cambridge (MA): Harvard Stem Cell Institute;2008.

8. Hooper KA, Macon ND, Kohn J. Comparative histological evaluation of new tyrosine-derived polymers and poly (L-lactic acid) as a function of polymer degradation. J Biomed Mater Res. 1998; 41:443–454.

9. Sengupta D, Heilshorn SC. Protein-engineered biomaterials: highly tunable tissue engineering scaffolds. Tissue Eng Part B Rev. 2010; 16:285–293.

10. Venugopal J, Prabhakaran MP, Zhang Y, Low S, Choon AT, Ramakrishna S. Biomimetic hydroxyapatite-containing composite nanofibrous substrates for bone tissue engineering. Philos Trans A Math Phys Eng Sci. 2010; 368:2065–2081.

11. Bruder SP, Fox BS. Tissue engineering of bone: cell based strategies. Clin Orthop Relat Res. 1999; 367 Suppl. S68–S83.

12. Minoura N, Aiba S, Gotoh Y, Tsukada M, Imai Y. Attachment and growth of cultured fibroblast cells on silk protein matrices. J Biomed Mater Res. 1995; 29:1215–1221.

13. Li C, Vepari C, Jin HJ, Kim HJ, Kaplan DL. Electrospun silk-BMP-2 scaffolds for bone tissue engineering. Biomaterials. 2006; 27:3115–3124.

14. Gould TR, Melcher AH, Brunette DM. Migration and division of progenitor cell populations in periodontal ligament after wounding. J Periodontal Res. 1980; 15:20–42.

15. Isaka J, Ohazama A, Kobayashi M, Nagashima C, Takiguchi T, Kawasaki H, et al. Participation of periodontal ligament cells with regeneration of alveolar bone. J Periodontol. 2001; 72:314–323.

16. Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, et al. Stem cell properties of human dental pulp stem cells. J Dent Res. 2002; 81:531–535.

17. Wei X, Ling J, Wu L, Liu L, Xiao Y. Expression of mineralization markers in dental pulp cells. J Endod. 2007; 33:703–708.

18. Ivanovski S, Gronthos S, Shi S, Bartold PM. Stem cells in the periodontal ligament. Oral Dis. 2006; 12:358–363.

19. Lee J, Cuddihy MJ, Kotov NA. Three-dimensional cell culture matrices: state of the art. Tissue Eng Part B Rev. 2008; 14:61–86.

20. Naito H, Yoshimura M, Mizuno T, Takasawa S, Tojo T, Taniguchi S. The advantages of three-dimensional culture in a collagen hydrogel for stem cell differentiation. J Biomed Mater Res A. 2013; 101:2838–2845.

21. Kim CS, Choi SH, Chai JK, Cho KS, Moon IS, Wikesjo UM, et al. Periodontal repair in surgically created intrabony defects in dogs: influence of the number of bone walls on healing response. J Periodontol. 2004; 75:229–235.

22. Han MJ, Seo YK, Yoon HH, Song KY, Park JK. Effect of mechanical tension on the human dental pulp cells. Biotechnol Bioprocess Eng. 2008; 13:410–417.

23. Seo YK, Yoon HH, Song KY, Kwon SY, Lee HS, Park YS, et al. Increase in cell migration and angiogenesis in a composite silk scaffold for tissue-engineered ligaments. J Orthop Res. 2009; 27:495–503.

24. Kim CS, Choi SH, Cho KS, Chai JK, Wikesjo UM, Kim CK. Periodontal healing in one-wall intra-bony defects in dogs following implantation of autogenous bone or a coral-derived biomaterial. J Clin Periodontol. 2005; 32:583–589.

25. Wikesjo UM, Guglielmoni P, Promsudthi A, Cho KS, Trombelli L, Selvig KA, et al. Periodontal repair in dogs: effect of rhBMP-2 concentration on regeneration of alveolar bone and periodontal attachment. J Clin Periodontol. 1999; 26:392–400.

26. Park JS, Choi SH, Moon IS, Cho KS, Chai JK, Kim CK. Eight-week histological analysis on the effect of chitosan on surgically created one-wall intrabony defects in beagle dogs. J Clin Periodontol. 2003; 30:443–453.

27. Choi SH, Kim CK, Cho KS, Huh JS, Sorensen RG, Wozney JM, et al. Effect of recombinant human bone morphogenetic protein-2/absorbable collagen sponge (rhBMP-2/ACS) on healing in 3-wall intrabony defects in dogs. J Periodontol. 2002; 73:63–72.

28. Rezwan K, Chen QZ, Blaker JJ, Boccaccini AR. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials. 2006; 27:3413–3431.

29. Abbott A. Cell culture: biology's new dimension. Nature. 2003; 424:870–872.

30. Ong SM, Zhang C, Toh YC, Kim SH, Foo HL, Tan CH, et al. A gel-free 3D microfluidic cell culture system. Biomaterials. 2008; 29:3237–3244.

31. Naito H, Dohi Y, Zimmermann WH, Tojo T, Takasawa S, Eschenhagen T, et al. The effect of mesenchymal stem cell osteoblastic differentiation on the mechanical properties of engineered bone-like tissue. Tissue Eng Part A. 2011; 17:2321–2329.

32. Lam KH, Nijenhuis AJ, Bartels H, Postema AR, Jonkman MF, Pennings AJ, et al. Reinforced poly(L-lactic acid) fibres as suture material. J Appl Biomater. 1995; 6:191–197.

33. Altman GH, Diaz F, Jakuba C, Calabro T, Horan RL, Chen J, et al. Silk-based biomaterials. Biomaterials. 2003; 24:401–416.

34. Heslot H. Artificial fibrous proteins: a review. Biochimie. 1998; 80:19–31.

35. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet. 2004; 364:149–155.

36. Hasegawa M, Yamato M, Kikuchi A, Okano T, Ishikawa I. Human periodontal ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model. Tissue Eng. 2005; 11:469–478.

37. Iwata T, Yamato M, Zhang Z, Mukobata S, Washio K, Ando T, et al. Validation of human periodontal ligament-derived cells as a reliable source for cytotherapeutic use. J Clin Periodontol. 2010; 37:1088–1099.

38. Iwata T, Yamato M, Tsuchioka H, Takagi R, Mukobata S, Washio K, et al. Periodontal regeneration with multi-layered periodontal ligament-derived cell sheets in a canine model. Biomaterials. 2009; 30:2716–2723.

39. Huang GT, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res. 2009; 88:792–806.

40. Park JY, Jeon SH, Choung PH. Efficacy of periodontal stem cell transplantation in the treatment of advanced periodontitis. Cell Transplant. 2011; 20:271–285.