Preparation and Characterization of Caffeic Acid-Grafted Electrospun Poly(l-Lactic Acid) Fiber Mats for Biomedical Applications

Caffeic acid (CA) was chemically immobilized onto the surfaces of the individual electrospun poly­(l-lactic acid) (PLLA) fibers to enhance the hydrophilicity and impart the antioxidant activity to the obtained fibrous membranes. This was done in two sequential steps. First, amino groups were covalently introduced onto the surfaces through the reaction with 1,6-hexamethylenediamine (HMD). In the second step, the amino moieties reacted with CA, which had been preactivated sequentially with 1-ethyl-3-(3-dimethylaminopropyl)­carbodiimide (EDC) and <i>N</i>-hydroxysuccinimide (NHS). The success of the reactions was confirmed by the ninhydrin assay and X-ray photoelectron spectroscopic analysis (XPS). Indirect cytotoxicity evaluation with murine dermal fibroblasts (L929) and human dermal fibroblasts (HDFa) revealed that the neat and the modified PLLA fibrous matrices released no substances in the levels that were harmful to the cells. Direct culturing of HDFa on these fibrous substrates indicated that they supported the proliferation of the cells on days 2 and 3 very well and that the CA-immobilized substrates exhibited the highest cell viability. Lastly, the antioxidant activity of the CA-immobilized substrates, as revealed by the 1,1-diphenyl-2-picryldrazyl (DPPH) radical scavenging assay, was as high as 88% on average

Improvement of dual-leached polycaprolactone porous scaffolds by incorporating with hydroxyapatite for bone tissue regeneration

<div><p>Polycaprolactone (PCL)/hydroxyapatite (HA) composite scaffolds were prepared by combining solvent casting and salt particulate leaching with a polymer leaching technique. The hydrophilicity of the dual-leached scaffold was improved by alkaline (NaOH) treatment. Well-defined interconnected pores were detected by scanning electron microscopy. The water absorption capacity of the NaOH-treated PCL/HA dual-leached scaffold increased greatly, confirming that the hydrophilicity of the scaffold was improved by NaOH treatment. The compressive modulus of the PCL/HA dual-leached scaffold was greatly increased by the addition of HA particles. An indirect evaluation of the cytotoxicity of all PCL dual-leached scaffolds with mouse fibroblastic cells (L929) and mouse calvaria-derived pre-osteoblastic cells (MC3T3-E1) indicated that the PCL dual-leached scaffolds are non-toxic to cells. The ability of the scaffolds to support mouse calvaria-derived pre-osteoblastic cell (MC3T3-E1) attachment, proliferation, differentiation, and mineralization was also evaluated. Although the viability of cells was lower on the PCL/HA dual-leached scaffold than on the tissue-culture polystyrene plates (TCPS) and on the other substrates at early time points, both the PCL and NaOH-treated PCL/HA dual-leached scaffolds supported the attachment of MC3T3-E1 at significantly higher levels than TCPS. During the proliferation period (days 1–3), all of the PCL dual-leached scaffolds were able to support the proliferation of MC3T3-E1 at higher levels than the TCPS; in addition, the cells grown on NaOH-treated PCL/HA dual-leached scaffolds proliferated more rapidly. The cells cultured on the surfaces of NaOH-treated PCL/HA dual-leached scaffolds had the highest rate of mineral deposition.</p></div

Osteoblastic Phenotype Expression of MC3T3-E1 Cultured on Electrospun Polycaprolactone Fiber Mats Filled with Hydroxyapatite Nanoparticles

Electrospun (e-spun) fiber mats of polycaprolactone (PCL; Mn = 80 000 g mol-1) with or without the presence of hydroxyapatite (HAp) nanoparticles (at 1% w/v based on the volume of the PCL solution) were successfully fabricated. The potential for use of these e-spun fiber mats as bone scaffolds was assessed by mouse calvaria-derived pre-osteoblastic cells, MC3T3-E1, in terms of attachment, proliferation, differentiation, and mineralization. Despite the lower number of cells attached at early time points, both the fibrous scaffolds supported the proliferation of MC3T3-E1 at similar levels to tissue-culture polystyrene plate (TCPS), with the cells growing on the PCL/HAp fiber mat (i.e., PCL/HAp-FS) showing the greatest proliferation rate on day 3 after the initial attachment period of 16 h. Alkaline phosphatase (ALP) activity of the cells grown on TCPS was the greatest on day 3 after cell culturing, while that of the cells grown on PCL/HAp-FS reached a maximum on day 5. On the other hand, the ALP activity of the cells grown on the neat PCL fiber mat (i.e., PCL-FS) was the lowest at any given time point. MC3T3-E1 cultured on the surface of PCL/HAp-FS expressed the greatest amount of osteocalcin (OC) gene on day 14 after cell culturing and OC protein on day 21 after cell culturing, respectively, when compared with those cultured on the surfaces of PCL-FS and TCPS. This corresponded to the greatest extent of mineralization for the cells grown on the surface of PCL/HAp-FS on day 21, followed by that for the cells grown on PCL-FS and TCPS, respectively

Supplementary Information 2: Characterisation of cells adhering on PDL cells from IFNγ-primed periodontal ligament cells regulate T cell responses via IFNγ-inducible mediators and ICAM-1-mediated direct cell contact

Periodontal ligament (PDL) cells help maintain tissue homeostasis by balancing PDL tissue inflammation and regeneration. However, the mechanisms by which interferon γ (IFNγ) modulate this process are not yet fully understood. The present study aimed to examine the effect of primed and non-primed PDL cells with IFNγ on the viability and differentiation of T lymphocytes and its functional consequences. The results showed that IFNγ-primed PDL cells possessed enhanced immunosuppression by suppressing T lymphocyte viability and directing T lymphocyte differentiation towards a higher Th2/Th1 ratio. Suppression of T cell viability was mainly mediated by IFNγ-inducible secreted mediators, which was prevented in the presence of direct cell contact, likely by intercellular adhesion molecule-1 (ICAM-1)-induced PI3 K-mediated TGFβ1 expression in PDL cells. By contrast, ICAM-1 activation augmented IFNγ-induced IFNγ and IL-6 expression in PDL cells, which in turn modulated T cell differentiation. The resulting interaction between these two cell types activated macrophage and suppressed osteoclast differentiation. In conclusion, the results have shown, for the first time, that primed and non-primed PDL cells with IFNγ differentially control T cell responses via IFNγ-inducible mediators and ICAM-1-mediated direct cell contact, suggesting the role of PDL cells in shifting an inflammatory phase towards a regenerative phase

Supplementary Information 1: Characterisation of T cells and their activation from IFNγ-primed periodontal ligament cells regulate T cell responses via IFNγ-inducible mediators and ICAM-1-mediated direct cell contact

Periodontal ligament (PDL) cells help maintain tissue homeostasis by balancing PDL tissue inflammation and regeneration. However, the mechanisms by which interferon γ (IFNγ) modulate this process are not yet fully understood. The present study aimed to examine the effect of primed and non-primed PDL cells with IFNγ on the viability and differentiation of T lymphocytes and its functional consequences. The results showed that IFNγ-primed PDL cells possessed enhanced immunosuppression by suppressing T lymphocyte viability and directing T lymphocyte differentiation towards a higher Th2/Th1 ratio. Suppression of T cell viability was mainly mediated by IFNγ-inducible secreted mediators, which was prevented in the presence of direct cell contact, likely by intercellular adhesion molecule-1 (ICAM-1)-induced PI3 K-mediated TGFβ1 expression in PDL cells. By contrast, ICAM-1 activation augmented IFNγ-induced IFNγ and IL-6 expression in PDL cells, which in turn modulated T cell differentiation. The resulting interaction between these two cell types activated macrophage and suppressed osteoclast differentiation. In conclusion, the results have shown, for the first time, that primed and non-primed PDL cells with IFNγ differentially control T cell responses via IFNγ-inducible mediators and ICAM-1-mediated direct cell contact, suggesting the role of PDL cells in shifting an inflammatory phase towards a regenerative phase

Additional file 1: of Basic fibroblast growth factor regulates phosphate/pyrophosphate regulatory genes in stem cells isolated from human exfoliated deciduous teeth

Table S1. Primer sequences for QPCR. (DOCX 18 kb

Short-term NaCl rinsing enhanced the migration of hGFs.

<p>A) Migrated cells were stained with crystal violet after transwell migration assay. Migrated cells B) and cell area C) were measured and compared by one-way ANOVA vs. 0% NaCl, scale bar 100 μm.</p

Chloride involved in the migration of hGFs.

<p>A) Scratch-test assay. B) Remaining wound area was normalized with the time point 0 h. C) NaCl, KCl rinsing did not alter the proliferation of hGFs by MTT assay while NaH₂PO₄, KH₂PO₄ did. D) NaCl, KCl rinsing increased migrated cells by transwell migration assay. One-way ANOVA vs. 0% NaCl, scale bar 100 μm. E) Gene expression of COL1, Fn, FAK with and without NaCl, KCl, NaH₂PO₄ and KH₂PO₄ rinsing by RT-PCR.</p

Rinsing with Saline Promotes Human Gingival Fibroblast Wound Healing <i>In Vitro</i>

<div><p>Rinsing the mouth with sodium chloride (NaCl) solution is believed to promote healthy gum and improve oral ulcer healing. Scientific evidence to support this assumption is, however, lacking. This study aims to investigate the effect and clarify underlying mechanisms of short-term rinsing with NaCl on human gingival fibroblast (hGFs) wound healing. Isolated primary hGFs and human normal oral keratinocytes (hNOKs) were rinsed with 0–7.2% NaCl for 2 min, 3 times a day. Scratch-tests, trans-well migration assays and MTT activity were performed. mRNA expression was assessed of type-I collagen, fibronectin and FAK. Changes in FAK and F-actin were detected by immunofluorescence. KCl, NaH₂PO₄, KH₂PO₄ were used to clarify the molecules involved. Rinsing with 0.9–1.8% NaCl significantly promoted hGFs cell migration but not proliferation. However, it had no effect on hNOKs. Rinsing with 1.8% NaCl significantly up-regulated the expression of type-I collagen and fibronectin. FAK and F-actin, molecules responsible for cytoskeleton re-organization and cell migration, were also up-regulated. Cl^- seemed to be essential since rinsing with KCl resulted in a similar effect as noted with NaCl. In conclusion, short-term rinsing with NaCl promoted hGFs migration, and increased the expression of extracellular matrix as well as cytoskeletal proteins. These data strongly support the long held belief in the benefits of using NaCl mouth-rinse.</p></div

Short-term NaCl rinsing enhanced wound healing in hGFs.

<p>A) Scratch-test assay. B) Remaining wound area was normalized with the time point 0 h. C) NaCl rinsing did not alter the proliferation of hGFs by MTT assay. One-way ANOVA vs. 0% NaCl, *p< 0.05, **p<0.01, ***p<0.001, scale bar 100 μm.</p