J Periodontal Implant Sci.  2017 Dec;47(6):402-415. 10.5051/jpis.2017.47.6.402.

Evaluation of the periodontal regenerative properties of patterned human periodontal ligament stem cell sheets

• Kim JH
• Ko SY
• Lee JH
• Kim DH
• Yun JH

• KMID: 2399746
• DOI: http://doi.org/10.5051/jpis.2017.47.6.402

Abstract

PURPOSE
The aim of this study was to determine the effects of patterned human periodontal ligament stem cell (hPDLSC) sheets fabricated using a thermoresponsive substratum.
METHODS
In this study, we fabricated patterned hPDLSC sheets using nanotopographical cues to modulate the alignment of the cell sheet.
RESULTS
The hPDLSCs showed rapid monolayer formation on various surface pattern widths. Compared to cell sheets grown on flat surfaces, there were no significant differences in cell attachment and growth on the nanopatterned substratum. However, the patterned hPDLSC sheets showed higher periodontal ligamentogenesis-related gene expression in early stages than the unpatterned cell sheets.
CONCLUSIONS
This experiment confirmed that patterned cell sheets provide flexibility in designing hPDLSC sheets, and that these stem cell sheets may be candidates for application in periodontal regenerative therapy.

Keyword

Extracellular matrix; Periodontal ligament; Regeneration; Stem cells; Tissue engineering

MeSH Terms

Cues
Extracellular Matrix
Gene Expression
Humans*
Periodontal Ligament*
Pliability
Regeneration
Stem Cells*
Tissue Engineering

Figure

• 

  Figure 1 (A) The PNIPAM-functionalized thermoresponsive substratum with nanopatterns. Capillary force lithography was used for the fabrication of the substratum, with subsequent modification using amine-terminated PNIPAM on the PET film. (B) Representative macroscopic image of a large-area, scalable substratum, and (C) an SEM image of the 800-nm nanopatterned surface. (D) A microscopic view of hPDLSCs cultured on the 800-nm width nanopatterned surface incubated at 37°C for 48 hours following cell seeding. The yellow arrow designates the direction in which the hPDLSCs were aligned (scale bar=200 μm). (E) Cell sheet detachment from the substratum after low-temperature treatment. An aligned and interconnected hPDLSC sheet with intact cell-to-cell junctions was slowly detached from the substratum as a single monolayer sheet upon water penetration from the periphery after being maintained at 22°C for 30 minutes. The white arrow indicates the direction of detachment (scale bar=200 μm). PNIPAM: poly(N-isopropyl-acrylamide), SEM: scanning electron microscopy, hPDLSC: human periodontal ligament stem cell, PUA: poly(urethane acrylate), GMA: glycidyl methacrylate, PET: polyethylene terephthalate, PGMA, poly(glycidyl methacrylate).

• 

  Figure 2 Characterization of primary cultured hPDLSCs. (A) Representative images of crystal violet staining of CFUs of primary cultured hPDLSCs at day 14 (scale bars=10 mm) and quantitative measurement of the CFUs (32.3±4.0, mean±SD). (B) Representative images of alizarin red staining of mineralized nodules after osteogenic differentiation (scale bar=100 μm). (C) Representative images of oil red O staining of lipid-rich vacuoles after adipogenic differentiation (scale bar=100 μm). (D) Immunophenotypical analyses of hPDLSCs. CD44, CD105, CD146, and STRO-1 were used as positive markers and CD19 was used as a negative marker. hPDLSC: human periodontal ligament stem cell, CFU: colony-forming unit, SD: standard deviation, STRO-1: stromal cell surface marker-1, FITC: fluorescein isothiocyanate, PE: phycoerythrin.

• 

  Figure 3 Microscopic images of hPDLSC sheet formation from the flat, 200-, 400-, 800-, and 1,600-nm nanopatterns at 48 hours. The hPDLSCs demonstrated varying degrees of confluent cell sheet formation depending on the culture plate surface condition. The 800-nm surface resulted in the optimal conditions for cell patterning (scale bars=200 μm). A histogram of the orientation of the hPDLSCs shows the quantified initial cellular adhesion angle on the flat surface and 800-nm width nanopatterned surface. The cells were patterned almost parallel to the direction of the grooves (n=4). hPDLSC: human periodontal ligament stem cell.

• 

  Figure 4 Cell proliferation was evaluated for up to 10 days (n=4, P<0.05). Microscopic image sets of cell growth on the flat and nanopatterned surfaces on days 1, 4, 7, and 10. On the nanopatterned surfaces, hPDLSCs extended and patterned in the direction of groove orientation with a spindle-like shape, and grew parallel to the direction of the grooves (scale bars=200 μm). hPDLSC: human periodontal ligament stem cell.

• 

  Figure 5 Differences in hPDLSC gene expression between patterned and unpatterned cell sheets as assessed by quantitative RT-PCR. The expression of the following genes was upregulated on the patterned cell sheet; RUNX2 on both days 4 and 7, and POSTN, ALP, and α-SMA on day 7. α-SMA and ALP showed decreased expression on day 4. There was no significant change in the expression of CEMP1, SCLX, and OCN. hPDLSC: human periodontal ligament stem cell, RT-PCR: real-time polymerase chain reaction, RUNX2: runt-related transcription factor 2, POSTN: periostin, ALP: alkaline phosphatase, α-SMA: α-smooth muscle actin, CEMP1: cementum protein 1, SCLX: scleraxis, OCN: osteocalcin. a)Statistical significance (P<0.05).

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