SHED′s Response to Various Pulpotomy Materials: Cytotoxicity and Gene Expression Analysis

Purpose : To examine the cytotoxicity and genetic expression of SHEDs cultured in eluates of various calcium silicate based pulpotomy materials. Study design : MTT assay, flow cytometry, alizarin red staining and scratch assay was used to assess the cellular viability, apoptosis, calcium matrix deposits and cell migration respectively. The gene expression of ALP, OCN and BMP -2, were measured with rtPCR. One way ANNOVA and Bonferroni post test was used for statistical analysis. Results : MTT assay analysis reported that all the test specimen had no cytotoxic effects. The highest number of live cells [ % ] was found in RetroMTA. The highest percentage of cell migration was observed in SHEDs cultured in EndoCem Zr. The mean absorbance for calcium matrix deposition was higher or similar in all test specimens, when compared to control groups. The expression of BMP -2 and OCN were significantly higher in cells exposed to RetroMTA and NeoMTA respectively after 24 hrs of incubation. After 72 hrs of incubation the mRNA expression of ALP was significantly higher in MTA. Conclusions: SHEDs cultured in eluates of various calcium silicate based cements exhibited cytocompatibility and maintained odontogenic like phenotype differentiation in SHEDs.

Corneal epithelial differentiation of human pluripotent stem cells generates ABCB5+ and ∆Np63α+ cells with limbal cell characteristics and high wound healing capacity

Background Differentiation of functional limbal stem cells (LSCs) from human pluripotent stem cells (hPSCs) is an important objective which can provide novel treatment solutions for patients suffering from limbal stem cell deficiency (LSCD). Yet, further characterization is needed to better evaluate their immunogenicity and regenerative potential before clinical applications. Methods Human PSCs were differentiated towards corneal fate and cryopreserved using a clinically applicable protocol. Resulting hPSC-LSC populations were examined at days 10–11 and 24–25 during differentiation as well as at passage 1 post-thaw. Expression of cornea-associated markers including PAX6, ABCG2, ∆Np63α, CK15, CK14, CK12 and ABCB5 as well as human leukocyte antigens (HLAs) was analyzed using immunofluorescence and flow cytometry. Wound healing properties of the post-thaw hPSC-LSCs were assessed via calcium imaging and scratch assay. Human and porcine tissue-derived cultured LSCs were used as controls for marker expression analysis and scratch assays at passage 1. Results The day 24–25 and post-thaw hPSC-LSCs displayed a similar marker profile with the tissue-derived LSCs, showing abundant expression of PAX6, ∆Np63α, CK15, CK14 and ABCB5 and low expression of ABCG2. In contrast, day 10–11 hPSC-LSCs had lower expression of ABCB5 and ∆Np63α, but high expression of ABCG2. A small portion of the day 10–11 cells coexpressed ABCG2 and ABCB5. The expression of class I HLAs increased during hPSC-LSCs differentiation and was uniform in post-thaw hPSC-LSCs, however the intensity was lower in comparison to tissue-derived LSCs. The calcium imaging revealed that the post-thaw hPSC-LSCs generated a robust response towards epithelial wound healing signaling mediator ATP. Further, scratch assay revealed that post-thaw hPSC-LSCs had higher wound healing capacity in comparison to tissue-derived LSCs. Conclusions Clinically relevant LSC-like cells can be efficiently differentiated from hPSCs. The post-thaw hPSC-LSCs possess functional potency in calcium responses towards injury associated signals and in wound closure. The developmental trajectory observed during hPSC-LSC differentiation, giving rise to ABCG2+ population and further to ABCB5+ and ∆Np63α+ cells with limbal characteristics, indicates hPSC-derived cells can be utilized as a valuable cell source for the treatment of patients afflicted corneal blindness due to LSCD. Graphical Abstract

Peri-Prostatic Adipocyte-Released TGFβ Enhances Prostate Cancer Cell Motility by Upregulation of Connective Tissue Growth Factor

Periprostatic adipose tissue (PPAT) has emerged as a key player in the prostate cancer (PCa) microenvironment. In this study, we evaluated the ability of PPAT to promote PCa cell migration, as well as the molecular mechanisms involved. Methods: We collected conditioned mediums from in vitro differentiated adipocytes isolated from PPAT taken from PCa patients during radical prostatectomy. Migration was studied by scratch assay. Results: Culture with CM of human PPAT (AdipoCM) promotes migration in two different human androgen-independent (AI) PCa cell lines (DU145 and PC3) and upregulated the expression of CTGF. SB431542, a well-known TGFβ receptor inhibitor, counteracts the increased migration observed in presence of AdipoCM and decreased CTGF expression, suggesting that a paracrine secretion of TGFβ by PPAT affects motility of PCa cells. Conclusions: Collectively, our study showed that factors secreted by PPAT enhanced migration through CTGF upregulation in AI PCa cell lines. These findings reveal the potential of novel therapeutic strategies targeting adipocyte-released factors and TGFβ/CTGF axis to fight advanced PCa dissemination.

Repetitive Treatment with Volatile Anesthetics Does Not Affect the In Vivo Plasma Concentration and Composition of Extracellular Vesicles in Rats

Background: Anesthetic-induced preconditioning (AIP) with volatile anesthetics is a well-known experimental technique to protect tissues from ischemic injury or oxidative stress. Additionally, plasmatic extracellular vesicle (EV) populations and their cargo are known to be affected by AIP in vitro, and to provide organ protective properties via their cargo. We investigated whether AIP would affect the generation of EVs in an in vivo rat model. Methods: Twenty male Sprague Dawley rats received a repetitive treatment with either isoflurane or with sevoflurane for a duration of 4 or 8 weeks. EVs from blood plasma were characterized by nanoparticle tracking analysis, transmission electron microscopy (TEM) and Western blot. A scratch assay (H9C2 cardiomyoblast cell line) was performed to investigate the protective capabilities of the isolated EVs. Results: TEM images as well as Western blot analysis indicated that EVs were successfully isolated. The AIP changed the flotillin and CD63 expression on the EV surface, but not the EV concentration. The scratch assay did not show increased cell migration and/or proliferation after EV treatment. Conclusion: AIP in rats changed the cargo of EVs but had no effect on EV concentration or cell migration/proliferation. Future studies are needed to investigate the cargo on a miRNA level and to investigate the properties of these EVs in additional functional experiments.

Insight Into the Beneficial Role of Lactiplantibacillus plantarum Supernatant Against Bacterial Infections, Oxidative Stress, and Wound Healing in A549 Cells and BALB/c Mice

Lactiplantibacillus plantarum MTCC 2621 is a well-characterized probiotic strain and is reported to possess many health benefits. However, the wound healing potential of this probiotic is yet to be explored. Here, we have assessed the antibacterial, antioxidant, and wound healing activities of cell-free supernatant of Lactiplantibacillus plantarum MTCC 2621 (Lp2621). Lp2621 exhibited excellent antibacterial activity against the indicator bacteria in the agar well diffusion assay. Lp2621 did not show any hemolytic activity. The safety of Lp2621 gel was established using the skin irritation assay in BALB/c mice, and no dermal reactions were observed. The supernatant showed 60–100% protection of A549 cells against H2O2-induced stress. In the scratch assay, Lp2621 accelerated wound healing after 24 h of treatment. The percent wound healing was significantly higher in cells treated with Lp2621 at 18–24 h posttreatment. In an excision wound healing in mice, topical application of Lp2621 gel showed faster healing than the vehicle- and betadine-treated groups. Similar wound healing activity was observed in wounds infected with Staphylococcus aureus. Histological examination revealed better wound healing in Lp2621-treated mice. Topical treatment of the wounds with Lp2621 gel resulted in the upregulation of pro-inflammatory cytokine IL-6 in the early phase of wound healing and enhanced IL-10 expression in the later phase. These findings unveil a protective role of Lp2621 against bacterial infection, oxidative stress, and wound healing.

Interaction between Macrophages and Human Mesenchymal Stromal Cells Derived from Bone Marrow and Wharton’s Jelly—A Comparative Study

Despite intensive clinical research on the use of mesenchymal stromal cells (MSCs), further basic research in this field is still required. Herein, we compared human bone marrow MSCs (BM-MSCs, n = 6) and Wharton’s jelly MSCs (WJ-MSCs, n = 6) in their ability to interact with human primary macrophages. Evaluation of secretory potential revealed that under pro-inflammatory stimulation, WJ-MSCs secreted significantly more IL-6 than BM-MSCs (2-fold). This difference did not translate into the effect of MSCs on macrophages: both types of MSCs significantly directed M1-like macrophages toward the M2 phenotype (based on CD206 expression) to a similar extent. This observation was consistent both in flow cytometry analysis and immunocytochemical assessment. The effect of MSCs on macrophages was sustained when IL-6 signaling was blocked with Tocilizumab. Macrophages, regardless of polarization status, enhanced chemotaxis of both BM-MSCs and WJ-MSCs (p < 0.01; trans-well assay), with WJ-MSCs being significantly more responsive to M1-derived chemotactic signals than BM-MSCs. Furthermore, WJ-MSCs increased their motility (scratch assay) when exposed to macrophage-conditioned medium while BM-MSCs did not. These results indicate that although both BM-MSCs and WJ-MSCs have the ability to reciprocally interact with macrophages, the source of MSCs could slightly but significantly modify the response under clinical settings.

Cartilage Acidic Protein a Novel Therapeutic Factor to Improve Skin Damage Repair?

Fish skin has been gaining attention due to its efficacy as a human-wound-treatment product and to identify factors promoting its enhanced action. Skin fibroblasts have a central role in maintaining skin integrity and secrete extra cellular matrix (ECM) proteins, growth factors and cytokines to rapidly repair lesions and prevent further damage or infection. The effects on scratch repair of the ubiquitous but poorly characterized ECM protein, cartilage acidic protein 1 (CRTAC1), from piscine and human sources were compared using a zebrafish SJD.1 primary fibroblast cell line. A classic in vitro cell scratch assay, immunofluorescence, biosensor and gene expression analysis were used. Our results demonstrated that the duplicate sea bass Crtac1a and Crtac1b proteins and human CRTAC-1A all promoted SJD.1 primary fibroblast migration in a classic scratch assay and in an electric cell impedance sensing assay. The immunofluorescence analysis revealed that CRTAC1 enhanced cell migration was most likely caused by actin-driven cytoskeletal changes and the cellular transcriptional response was most affected in the early stage (6 h) of scratch repair. In summary, our results suggest that CRTAC1 may be an important factor in fish skin promoting damage repair.

In-vitro Wound Healing Properties of Commiphora swynnertonii Resinous Extracts

Wound healing is a complex multicellular process involving many cell types which include; inflammatory cells, endothelial cells, fibroblasts and keratinocytes. The process involves an orderly sequence of events with four overlapping phases namely; haemostasis, inflammatory, proliferation and remodeling phases. The process can be facilitated by the use of wound healing agents including herbal remedies from plants. In this study the main objective was to evaluate the in vitro wound healing activity of the resin obtained from Commiphora swynnertonii (C.swynnertonii). First the NIH -3T3 cells viability were evaluated using (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl Tetrazolium Bromide (MTT) assay. Then the wound scratch assay model was used to evaluate cellular proliferation, closure of the wound and release of matrix metalloproteinase enzymes. Results indicate differences in mean cell viability between different concentrations within 24 hours of incubation. The highest viability was recorded at the concentration of 1% (v/v). The in-vitro wound scratch assay showed positive NIH - 3T3 cells proliferation on the wound area and cells migration when compared with control group (without treatment) at 0 and 24 hours. In addition, C. swynnertonii was able to stimulate secretion of MMP-2 release from NIH - 3T3 cells. MMP-2 is an important enzyme for extracellular matrix remodeling during wound healing suggesting that C. swynnertonii promotes wound healing by stimulating cell proliferation and production of MMP-2 in a mechanism that is currently not known.

Effects of platelet-rich plasma on in vitro proliferation and migration of fibroblasts from human chronic refractory wound granulation tissue

Objective: To observe the effects of platelet-rich plasma (PRP) on in vitro proliferation and migration of fibroblasts from human chronic refractory wound granulation tissue.Methods: Fibroblasts were separated from human chronic refractory wound granulation tissue and then were identified. The obtained fibroblasts were divided into fetal bovine serum (FBS) group, hydrogel group and PRP group, and the three groups were cultured with culture mediums containing FBS, hydrogel and PRP respectively, in order to observe the growth of fibroblasts. The wound scratch assay was used to observe the migration of fibroblasts.Results: PRP group had more fibroblasts than FBS group and hydrogel group since Day 5 of culture, and exhibited greater fibroblast scratch migration area than FBS group on 48 h and 72 h of wound scratch assay (all p < .05).Conclusions: Compared with FBS, human fibroblasts cultured by PRP can more effectively promote the proliferation and migration of fibroblasts.