Extracellular matrix-mediated differentiation of periodontal progenitor cells

Abstract

The periodontal ligament (PDL) is a specialized connective tissue that connects the surface of the tooth root with the bony tooth socket. The healthy PDL harbors stem cell niches and extracellular matrix (ECM) microenvironments that facilitate periodontal regeneration. During periodontal disease, the PDL is often compromised or destroyed, reducing the life-span of the tooth. In order to explore new approaches toward the regeneration of diseased periodontal tissues, we have tested the effect of periodontal ECM signals, fibroblast growth factor 2 (FGF2), connective tissue growth factor (CTGF), and the cell adhesion peptide Arg-Gly-Asp (RGD) on the differentiation of two types of periodontal progenitor cells, PDL progenitor cells (PDLPs) and dental follicle progenitor cells (DFCs). Our studies documented that CTGF and FGF2 significantly enhanced the expression of collagens I & III, biglycan and periostin in tissue engineered regenerates after 4 weeks compared to untreated controls. Specifically, CTGF promoted mature PDL-like tissue regeneration as demonstrated by dense periostin localization in collagen fiber bundles. CTGF and FGF2 displayed synergistic effects on collagen III and biglycan gene expression, while effects on mineralization were antagonistic to each other: CTGF promoted while FGF2 inhibited mineralization in PDL cell cultures. Incorporation of RGD peptides in hydrogel matrices significantly enhanced attachment, spreading, survival and mineralization of the encapsulated DFCs, suggesting that RGD additives might promote the use of hydrogels for periodontal mineralized tissue engineering. Together, our studies have documented the effect of three key components of the periodontal ECM on the differentiation of periodontal progenitor populations.

2009 International Society of Differentiation. Published by Elsevier Ltd.

Figures

Fig 1. ECM components of the mouse periodontium

Collagens I and III as well as biglycan were localized in the interstitial extracellular matrix (ECM) of the periodontal ECM. Periodontal fibers were intensely stained for periostin (asterisk). AB = alveolar bone, PDL = periodontal ligament, Root = tooth root including dentin, predentin, and cementum.

Fig 2. Time course comparison of the expression levels of collagens (I & III) in DFCs and PDLPs and the effect of growth factors and their combinations on PDL morphology and ECM gene expression

Figs. 2A and B illustrate elongated, parallel arrangement of DFCs (2A) and radially symmetric colonies of PDLPs after one week of monolayer culture (2B). When cultured over several weeks, there was a distinct difference in the expression of collagen I (Fig. 2C) and collagen III (Fig. 2D) between PDLPs and DFCs as revealed by real time RT-PCR analysis (Figs. 2C and D). In both studies cell-gel constructs were harvested after 1d, 1 week (Wk) or 2 weeks (Wks) of in vitro culture (Fig. 2C, D). Fig. 2E illustrates formation of dense colonies of PDLPs after one week of culture. In contrast, treatment with a combination of CTGF and FGF2 growth factors during this time period resulted in the formation of elongated and thin individual cells (Fig. 2F). In Figs. 2G–J, gene expression collagen type I (2G), collagen type III (2H), periostin (2I) and biglycan (2J) genes in cell-gel constructs was determined by Real time RT-PCR analysis and cells were harvested after 1 day, 2 weeks (Wks) or 4 weeks (Wks) of subcutaneous implantation in nude mice. While in general, gene expression levels increased after two or four weeks implantation when treated with FGF2 and/or CTGF growth factors, individual ECM gene responses to growth factors were distinctly different and growth factor combinations had either synergistic or antagonistic effects (Fig. 2G–F). In all bar graphs, gene expression levels were displayed relative to the house keeping gene GAPDH.

Fig 3. Immunohistochemical and Western blot analysis of key ECM molecules in tissue engineered PDL regenerates

Fig. 3A–E are paraffin sections of PDLP/collagen tissue engineered constructs subcutaneously implanted in nude mice for 4 weeks. Prior to implantation, PDLPs were treated for ten days with various growth factors such as FGF2 (10ng/ml, Fig. 3B), CTGF (50ng/ml, Fig. 3C), FGF2+CTGF (10/50ng/ml Fig. 3D), or left untreated (controls, Fig. 3A). The strongest staining intensity for periostin was found in the CTGF-treated group (Fig. 3C), followed by the group treated with CTGF + FGF2 (Fig. 3D). There was little or no periostin localization in untreated controls (Fig. 3A) and in the FGF2 treated group (Fig. 3B). Fig. 3E is the negative control that was not subjected to primary antibody. In Fig. 3F we have used Western blot analysis to determine levels of collagen III, biglycan, and periostin in our growth factor-treated tissue engineered constructs; lane 1 was the control group, lane 2 treated with FGF2, lane 3 treated with CTGF, and lane 4 treated with FGF2 plus CTGF (for concentrations see above). The results of our Western blot analysis were quantified using the Kodak 1D Image analysis software, and graphs were plotted by normalizing the value of collagen III, biglycan and periostin with GAPDH as control (Fig. 3G–I). The results of this study illustrate that all three growth factor treatment modalities (FGF2, CTGF, and FGF2+CTGF) resulted in a significant increase in the expression level of ECM matrix proteins (Collagen III, Biglycan, and Periostin). Moreover, CTGF alone or combinations with CTGF and FGF2 had a stronger effect on matrix proteins than FGF2 alone.

Fig 4. Effects of FGF2 and CTGF treatment on matrix mineralization in PDLPs

Fig.(4 A–F) is a photograph of a culture plate containing PDLPs cultured at a density of 5 × 104 cells/well for a total of 10 days in osteogenic differentiation media with or without growth factors stained with Alizarin Red S to visualize relative levels of calcium. Cells were cultured in the following conditions: (i) continuous treatment with FGF2 for 10 days (Fig. 4A), (ii) continuous treatment with CTGF for 10 days (Fig. 4B), (iii) continuous treatment with FGF2 and CTGF (Fig. 4C), (iv) sequential treatment with FGF2 for 4 days followed by CTGF for the next 6 days (Fig. 4D), (v) sequential treatment with CTGF for 4 days followed by FGF2 for the next 6 days (Fig. 4E), and (vi) without growth factor (Fig. 4F). In Fig. 4G, relative calcium levels were calculated based on the o-cresolphthalein complexone method and spectrophotometric evaluation. Note that both CTGF treatment alone and CTGF followed by FGF2 treatment resulted in the most significant increases in calcium levels while simple addition of FGF2 and CTGF to the medium significantly decreased calcium levels (Fig. 4G).

Fig 5. Attachment, spreading, viability and mineralization of DFCs depending on RGD-modification of PEGDA hydrogels

Fig. 5A. Cell density of attached DFCs after 2 and 24 hrs on 10% PEGDA without Acr-PEG-RGD or containing 2mM Acr-PEG-RGD. Values are reported as an average of three random fields times three hydrogel wells at each time point. There was a statistical difference (p2) over three random fields per composition. Fig. 5C. Viability of DFCs in 10% PEGDA with or without 2mM of Acr-PEG-RGD after 3 days culture in osteogenic differentiation medium, PDGF supplemented medium, and control medium. Values are reported as the average of three constructs per composition per treatment. There was no statistical difference (p>0.05) in the viability of DFCs between the modified and unmodified hydrogels and their treatment groups. Fig. 5D. Von Kossa stained sections of untreated (control) and treated (osteogenic, PDGF) DFCs encapsulated in unmodified (upper row) and RGD-modified (lower row) hydrogels after 2 weeks of in vitro culture. Mineralization was significantly increased in the RGD-modified group as seen by dense mineralization nodules.