Design of an Electrospun Collagen Scaffold for the Regeneration of Periodontal Tissues

Creber, Kendal I (Biomedical Engineering , The University of Western, London, ON)
Kim, Shawna (Anatomy and Cell Biology, The University of Western Ontario, London, ON)
Guan, Jianjun (Materials Science and Engineering, The Ohio State University, Columbus, OH)
Hamilton, Douglas W (Division of Oral Biology, The University of Western Ontario, London, ON)

Introduction

Periodontitis affects over 25% of adults in Canada and is the leading cause of tooth loss due to severe, irreversible damage to the periodontium (1). Though current treatments are able to impede disease progression, tissue-engineering strategies for the periodontium are still in their infancy. Consequently, restoration of a fully functional periodontium has yet to be achieved and tooth loss is a frequent outcome. The recent identification of mesenchymal progenitor cells within the human periodontal ligament (PDL) and periosteum, which are capable of differentiating into fibroblastic, cementoblastic and osteoblastic cells, has sparked new interest in the development of regenerative periodontal treatments (2). We have designed a novel electrospun scaffold to facilitate the regeneration of periodontal tissues affected by periodontitis through the recruitment and activation of these progenitor cells. The type I collagen scaffold is co-spun with periostin, a matricellular protein which is highly expressed in the healthy PDL and periosteum. Periostin is expected to enhance the recruitment of progenitor cells and induce osteogenic differentiation to promote tissue regeneration.

Materials and Methods

Type I collagen, dissolved in hexafluoroisopropanol was combined with a solution of recombinant human periostin and electrospun to produce a scaffold mat (Fig. 1) with approximately 1:22500 periostin to collagen by weight. The scaffold was then cross-linked in a solution of 5% glutaraldehyde in ethanol. Scaffolds containing collagen alone were used as controls. The attachment and spreading of human PDL cells on each scaffold was observed with scanning electron microscopy (SEM). Cellular adhesion and proliferation were assessed with CyQuant® GR fluorescent dye to quantify cell number based on DNA content. Deposition and organization of the extracellular matrix proteins fibronectin and tenascin C on the scaffold surface were visualized with immunocytochemical staining. For in vivo studies, fenestration defects were created in the second molar of rats, and scaffolds were implanted along the side of the tooth. 10 days, post surgery, immunohistochemical staining was used to visualize the extent of regeneration.

Results

Scanning electron microscopy revealed that cells attach to both the control and periostin scaffolds in as little as 15 minutes and begin to spread within 30 minutes. Lamellipodia were observed as early as 30 minutes post-seeding (Fig. 2a) and within 1 hour, filopodia had extended (Fig. 2b). Immunocytochemical staining was used to visualize the secretion of extracellular matrix proteins by PDL cells on the scaffolds in vitro. By day 3 post-seeding, the deposition of fibronectin and tenascin C, two glycoproteins abundant in the healthy periodontal ligament, was observed. Cell proliferation was assessed up to 10 days, and confirmed that both scaffolds were able to support cellular proliferation in vitro. The rate of proliferation on collagen scaffolds was comparable to that of cells on tissue culture plastic, whereas the rate of proliferation on the periostin scaffold was significantly lower at 7 and 10 days (p 0.05). This decrease in proliferation may indicate that cells are undergoing differentiation in response to the periostin scaffold. Preliminary in vivo studies suggested that the conditions with scaffolds implanted had less inflammation than an untreated defect. Furthermore, the periostin scaffold condition appeared to stimulate new bone formation.

Discussion and Conclusion

Human PDL cells seeded on both the control and recombinant periostin electrospun type I collagen scaffolds support cellular attachment, the secretion of extracellular matrix proteins, and cell proliferation, indicating that the scaffolds provide a suitable environment for cellular growth and development. Future studies will assess the extent of regeneration and the effect of recombinant periostin scaffold on osteogenic differentiation

Fig.1 SEM images of control type I collagen scaffold. Randomly aligned fibers of roughly 1μm in diameter (a) with scaffold thickness of approximately 30 μm (b).

Fig. 2 SEM images of human PDL cells seeded on control type I collagen scaffold. At 30 minutes post-seeding, lamelipodia are observed (a). At 1 hour, filopodia have extended (b).

References

1. Health Canada. Summary report on the findings of the oral health component of the Canadian Health Measures Survey 2007 - 2009. Health Reports [Internet]. 2010 Jun 10. Available from: http://www.fptdwg.ca/assets/PDF/CHMS/CHMS-E-summ.pdf 2. Seo B-M, Miura M, Gronthos S, Mark Bartold P, Batouli S, Brahim J, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. The Lancet. Elsevier; 2004 Jul;364(9429):149–55.

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