A novel method for detection of pancreatic Ductal Adenocarcinoma using explainable machine learning.

BACKGROUND AND OBJECTIVE: Pancreatic Ductal Adenocarcinoma (PDAC) is a form of pancreatic cancer that is one of the primary causes of cancer-related deaths globally, with less than 10 % of the five years survival rate. The prognosis of pancreatic cancer has remained poor in the last four decades, mainly due to the lack of early diagnostic mechanisms. This study proposes a novel method for detecting PDAC using explainable and supervised machine learning from Raman spectroscopic signals. METHODS: An insightful feature set consisting of statistical, peak, and extended empirical mode decomposition features is selected using the support vector machine recursive feature elimination method integrated with a correlation bias reduction. Explicable features successfully identified mutations in Kirsten rat sarcoma viral oncogene homolog (KRAS) and tumor suppressor protein53 (TP53) in the fingerprint region for the first time in the literature. PDAC and normal pancreas are classified using K-nearest neighbor, linear discriminant analysis, and support vector machine classifiers. RESULTS: This study achieved a classification accuracy of 98.5% using a nonlinear support vector machine. Our proposed method reduced test time by 28.5 % and saved 85.6 % memory utilization, which reduces complexity significantly and is more accurate than the state-of-the-art method. The generalization of the proposed method is assessed by fifteen-fold cross-validation, and its performance is evaluated using accuracy, specificity, sensitivity, and receiver operating characteristic curves. CONCLUSIONS: In this study, we proposed a method to detect and define the fingerprint region for PDAC using explainable machine learning. This simple, accurate, and efficient method for PDAC detection in mice could be generalized to examine human pancreatic cancer and provide a basis for precise chemotherapy for early cancer treatment.

Original and liposome-modified indocyanine green-assisted fluorescence study with animal models.

Medical diagnosis heavily relies on the use of bio-imaging techniques. One such technique is the use of ICG-based biological sensors for fluorescence imaging. In this study, we aimed to improve the fluorescence signals of ICG-based biological sensors by incorporating liposome-modified ICG. The results from dynamic light scattering and transmission electron microscopy showed that MLM-ICG was successfully fabricated with a liposome diameter of 100-300 nm. Fluorescence spectroscopy showed that MLM-ICG had the best properties among the three samples (Blank ICG, LM-ICG, and MLM-ICG), as samples immersed in MLM-ICG solution achieved the highest fluorescence intensity. The NIR camera imaging also showed a similar result. For the rat model, the best period for fluorescence tests was between 10 min and 4 h, where most organs reached their maximum fluorescence intensity except for the liver, which continued to rise. After 24 h, ICG was excreted from the rat's body. The study also analyzed the spectra properties of different rat organs, including peak intensity, peak wavelength, and FWHM. In conclusion, the use of liposome-modified ICG provides a safe and optimized optical agent, which is more stable and efficient than non-modified ICG. Incorporating liposome-modified ICG in fluorescence spectroscopy could be an effective way to develop novel biosensors for disease diagnosis.

Decreased CRISPLD2 expression impairs osteogenic differentiation of human mesenchymal stem cells during in vitro expansion.

Human mesenchymal stem cells (hMSCs) are the cornerstone of regenerative medicine; large quantities of hMSCs are required via in vitro expansion to meet therapeutic purposes. However, hMSCs quickly lose their osteogenic differentiation potential during in vitro expansion, which is a major roadblock to their clinical applications. In this study, we found that the osteogenic differentiation potential of human bone marrow stem cells (hBMSCs), dental pulp stem cells (hDPSCs), and adipose stem cells (hASCs) was severely impaired after in vitro expansion. To clarify the molecular mechanism underlying this in vitro expansion-related loss of osteogenic capacity in hMSCs, the transcriptome changes following in vitro expansion of these hMSCs were compared. Cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2) was identified as the most downregulated gene shared by late passage hBMSCs, hDPSCs, and hASCs. Both the secreted and non-secreted CRISPLD2 proteins progressively declined in hMSCs during in vitro expansion when the cells gradually lost their osteogenic potential. We thus hypothesized that the expression of CRISPLD2 is critical for hMSCs to maintain their osteogenic differentiation potential during in vitro expansion. Our studies showed that the knockdown of CRISPLD2 in early passage hBMSCs inhibited the cells' osteogenic differentiation in a siRNA dose-dependent manner. Transcriptome analysis and immunoblotting indicated that the CRISPLD2 knockdown-induced osteogenesis suppression might be attributed to the downregulation of matrix metallopeptidase 1 (MMP1) and forkhead box Q1 (FOXQ1). Furthermore, adeno-associated virus (AAV)-mediated CRISPLD2 overexpression could somewhat rescue the impaired osteogenic differentiation of hBMSCs during in vitro expansion. These results revealed that the downregulation of CRISPLD2 contributes to the impaired osteogenic differentiation of hMSCs during in vitro expansion. Our findings shed light on understanding the loss of osteogenic differentiation in hMSCs and provide a potential therapeutic target gene for bone-related diseases.

Cyanidin inhibits adipogenesis in 3T3-L1 preadipocytes by activating the PLC-IP3 pathway.

Cyanidin is the most abundant anthocyanin found in red-purple plants and possesses anti-obesity properties. However, its mechanism of action in adipocytes remains unknown. The objective of this study was to elucidate how cyanidin inhibits adipocyte formation in 3T3-L1 preadipocytes. Cells were cultured in adipogenic differentiation medium supplemented with cyanidin and examined for adipogenesis, cell viability, and adipocyte gene expression using Oil Red O staining, MTT assay, and RT-qPCR. Real-time Ca2+ imaging analysis was performed in living cells to elucidate cyanidin's mechanism of action. The results demonstrated that cyanidin (1-50 µM) supplementation to the adipogenic medium inhibited adipogenesis by downregulating adipogenic marker gene expression (PPARγ, C/EBPα, adiponectin, and aP2) without affecting cell viability after 4 days of treatment. Stimulation of cells with cyanidin (30-100 µM) increased intracellular Ca2+ in a concentration dependent manner with peak calcium increases at 50 µM. Pretreatment of cells with the phospholipase C (PLC) inhibitor U73122, inositol triphosphate (IP3) receptor blocker 2-APB, and depletion of endoplasmic reticulum Ca2+ stores by thapsigargin abolished the Ca2+ increases by cyanidin. These findings suggested that cyanidin inhibits adipocyte formation by activating the PLC-IP3 pathway and intracellular Ca2+ signaling. Our study is the first report describing the mechanism underlying the anti-obesity effect of cyanidin.

Optimization of adeno-associated virus (AAV) gene delivery into human bone marrow stem cells (hBMSCs).

Background: Efficiently delivering nucleic acid into mammalian cells is essential to overexpress genes for assessing gene functions. Human bone marrow stem cells (hBMSCs) are the most studied tissue-derived stem cells. Adeno-associated viruses (AAVs) have been used to deliver DNA into hBMSCs for various purposes. Current literature reported that transduction efficiencies of up to 65% could be achieved by AAV gene delivery into hBMSCs. Further improvement of efficiency is needed and possible. This study tested a selection of AAV serotypes for high-efficient DNA delivery into hBMSCs. Methods: hBMSCs from different donors were infected with different serotypes of AAVs containing the enhanced green fluorescence protein (eGFP) reporter gene driven by the CMV promoter. Green fluorescence was monitored in the infected cells at five-day intervals. Cells were collected at designated time points after the infection for reverse-transcription polymerase chain reaction (RT-PCR) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to assess eGFP mRNA transcription. Results: The results indicated that the order of transduction efficiency of the AAV serotypes was AAV2 > AAV2.7m8 > AAV6 > AAV6.2 > AAV1 > AAV-DJ. AAV2 could achieve almost 100% transduction at the multiplicity of infection (MOI) greater than 100K. Over 90% of cells could be transduced at 20K to 50K MOI. About 80% transduction was seen at MOIs of 10K and 15K. RT-PCR analysis showed that eGFP mRNA could be detected from day 5 to day 30 post-AAV infection. The differences in the observed transduction efficiencies of the hBMSCs from different patients indicate donor-to-donor variability, and increased eGFP mRNA was generally seen after day 15 post-AAV2 infection. Maximal eGFP transcription was detected on day 30 post-infection. Conclusions: We conclude that AAV2 and AAV2.7m8 at an MOI of 100K or greater can efficiently deliver transgene into hBMSCs with up to near 100% transduction efficiency for sustained expression over one month. However, donor-to-donor variation exists in transduction efficiency and transgene expression, especially at MOIs less than 100K.

Mouthwash as a non-invasive method of indocyanine green delivery for near-infrared fluorescence dental imaging.

SIGNIFICANCE: X-ray imaging serves as the mainstream imaging in dentistry, but it involves risk of ionizing radiation. AIM: This study presents the feasibility of indocyanine green-assisted near-infrared fluorescence (ICG-NIRF) dental imaging with 785-nm NIR laser in the first (ICG-NIRF-I: 700 to 1000 nm) and second (ICG-NIRF-II: 1000 to 1700 nm) NIR wavelengths. APPROACH: Sprague Dawley rats with different postnatal days were used as animal models. ICG, as a fluorescence agent, was delivered to dental structures by subcutaneous injection (SC) and oral administration (OA). RESULTS: For SC method, erupted and unerupted molars could be observed from ICG-NIRF images at a short imaging time (<1 min). ICG-NIRF-II could achieve a better image contrast in unerupted molars at 24 h after ICG injection. The OA could serve as a non-invasive method for ICG delivery; it could also cause the glow-in-dark effect in unerupted molars. For erupted molars, OA can be considered as mouthwash and exhibits outstanding performance for delivery of ICG dye; erupted molar structures could be observed at a short imaging time (<1 min) and low ICG dose (0.05 mg / kg). CONCLUSIONS: Overall, ICG-NIRF with mouthwash could perform in-vivo dental imaging in two NIR wavelengths at a short time and low ICG dose.

Machine-learning-assisted spontaneous Raman spectroscopy classification and feature extraction for the diagnosis of human laryngeal cancer.

The early detection of laryngeal cancer significantly increases the survival rates, permits more conservative larynx sparing treatments, and reduces healthcare costs. A non-invasive optical form of biopsy for laryngeal carcinoma can increase the early detection rate, allow for more accurate monitoring of its recurrence, and improve intraoperative margin control. In this study, we evaluated a Raman spectroscopy system for the rapid intraoperative detection of human laryngeal carcinoma. The spectral analysis methods included principal component analysis (PCA), random forest (RF), and one-dimensional (1D) convolutional neural network (CNN) methods. We measured the Raman spectra from 207 normal and 500 tumor sites collected from 10 human laryngeal cancer surgical specimens. Random Forest analysis yielded an overall accuracy of 90.5%, sensitivity of 88.2%, and specificity of 92.8% on average over 10 trials. The 1D CNN demonstrated the highest performance with an accuracy of 96.1%, sensitivity of 95.2%, and specificity of 96.9% on average over 50 trials. In predicting the first three principal components (PCs) of normal and tumor data, both RF and CNN demonstrated high performances, except for the tumor PC2. This is the first study in which CNN-assisted Raman spectroscopy was used to identify human laryngeal cancer tissue with extracted feature weights. The proposed Raman spectroscopy feature extraction approach has not been previously applied to human cancer diagnosis. Raman spectroscopy, as assisted by machine learning (ML) methods, has the potential to serve as an intraoperative, non-invasive tool for the rapid diagnosis of laryngeal cancer and margin detection.

Increased Expression of miR-7a-5p and miR-592 during Expansion of Rat Dental Pulp Stem Cells and Their Implication in Osteogenic Differentiation.

Dental pulp stem cells (DPSCs) possess strong osteogenic differentiation potential and are promising cell sources in regenerative medicine. However, such differentiation capacity progressively declines during their in vitro expansion. MicroRNAs (miRNAs) play important roles in modulating stem cell differentiation. This study aimed (1) to determine if miR-7a-5p and miR-592 are involved in maintaining and regulating osteogenic differentiation of DPSCs, and (2) to explore their potential regulatory pathways. We found that the expression of miR-7a-5p and miR-592 was significantly upregulated during the expansion of rat DPSCs (rDPSCs). Overexpression of these miRNAs inhibited the osteogenic/odontogenic differentiation of rDPSCs, as evidenced by calcium deposition and osteogenic/odontogenic gene expression. RT-qPCR determined that miR-592 could downregulate heat shock protein B8, whose expression is reduced during the expansion of rDPSCs. Furthermore, RNA-seq and bioinformatics analysis identified significant signaling pathways of miR-7a-5p and miR-592 in regulating osteogenic differentiation, including TNF, MAPK, and PI3K-Akt pathways. We conclude that upregulating miR-7a-5p and miR-592 suppresses the osteogenic differentiation of rDPSCs during their in vitro expansion, likely via TNF, MAPK, and PI3K-Akt pathways. The results may shed light on application of miR-7a-5p and miR-592 for maintaining osteo-differentiation potential in stem cells for bone regeneration and bone-related disease treatment.

Cyanidin-3-rutinoside stimulated insulin secretion through activation of L-type voltage-dependent Ca2+ channels and the PLC-IP3 pathway in pancreatic ß-cells.

Cyanidin-3-rutinoside (C3R) is an anthocyanin with anti-diabetic properties found in red-purple fruits. However, the molecular mechanisms of C3R on Ca2+-dependent insulin secretion remains unknown. This study aimed to identify C3R's mechanisms of action in pancreatic ß-cells. Rat INS-1 cells were used to elucidate the effects of C3R on insulin secretion, intracellular Ca2+ signaling, and gene expression. The results showed that C3R at 60, 100, and 300 µM concentrations significantly increased insulin secretion via intracellular Ca2+ signaling. The exposure of cells with C3R concentrations up to 100 µM did not affect cell viability. Pretreatment of cells with nimodipine (voltage-dependent Ca2+ channel (VDCC) blocker), U73122 (PLC inhibitor), and 2-APB (IP3 receptor blocker) inhibited the intracellular Ca2+ signals by C3R. Interestingly, C3R increased intracellular Ca2+ signals and insulin secretion after depletion of endoplasmic reticulum Ca2+ stores by thapsigargin. However, insulin secretion was abolished under extracellular Ca2+-free conditions. Moreover, C3R upregulated mRNA expression for Glut2 and Kir6.2 genes. These findings indicate that C3R stimulated insulin secretion by promoting Ca2+ influx via VDCCs and activating the PLC-IP3 pathway. C3R also upregulates the expression of genes necessary for glucose-induced insulin secretion. This is the first study describing the molecular mechanisms by which C3R stimulates Ca2+-dependent insulin secretion from pancreatic ß-cells. These findings contribute to our understanding on how anthocyanins improve hyperglycemia in diabetic patients.

Detection of pancreatic cancer by convolutional-neural-network-assisted spontaneous Raman spectroscopy with critical feature visualization.

Pancreatic cancer is the deadliest cancer type with a five-year survival rate of less than 9%. Detection of tumor margins plays an essential role in the success of surgical resection. However, histopathological assessment is time-consuming, expensive, and labor-intensive. We constructed a lab-designed, hand-held Raman spectroscopic system that could enable intraoperative tissue diagnosis using convolutional neural network (CNN) models to efficiently distinguish between cancerous and normal pancreatic tissue. To our best knowledge, this is the first reported effort to diagnose pancreatic cancer by CNN-aided spontaneous Raman scattering with a lab-developed system designed for intraoperative applications. Classification based on the original one-dimensional (1D) Raman, two-dimensional (2D) Raman images, and the first principal component (PC1) from the principal component analysis on the 2D image, could all achieve high performance: the testing sensitivity, specificity, and accuracy were over 95%, and the area under the curve approached 0.99. Although CNN models often show great success in classification, it has always been challenging to visualize the CNN features in these models, which has never been achieved in the Raman spectroscopy application in cancer diagnosis. By studying individual Raman regions and by extracting and visualizing CNN features from max-pooling layers, we identified critical Raman peaks that could aid in the classification of cancerous and noncancerous tissues. 2D Raman PC1 yielded more critical peaks for pancreatic cancer identification than that of 1D Raman, as the Raman intensity was amplified by 2D Raman PC1. To our best knowledge, the feature visualization was achieved for the first time in the field of CNN-aided spontaneous Raman spectroscopy for cancer diagnosis. Based on these CNN feature peaks and their frequency at specific wavenumbers, pancreatic cancerous tissue was found to contain more biochemical components related to the protein contents (particularly collagen), whereas normal pancreatic tissue was found to contain more lipids and nucleic acid (particularly deoxyribonucleic acid/ribonucleic acid). Overall, the CNN model in combination with Raman spectroscopy could serve as a useful tool for the extraction of key features that can help differentiate pancreatic cancer from a normal pancreas.

Creating Structured Hydrogel Microenvironments for Regulating Stem Cell Differentiation.

The development of distinct biomimetic microenvironments for regulating stem cell behavior and bioengineering human tissues and disease models requires a solid understanding of cell-substrate interactions, adhesion, and its role in directing cell behavior, and other physico-chemical cues that drive cell behavior. In the past decade, innovative developments in chemistry, materials science, microfabrication, and associated technologies have given us the ability to manipulate the stem cell microenvironment with greater precision and, further, to monitor effector impacts on stem cells, both spatially and temporally. The influence of biomaterials and the 3D microenvironment's physical and biochemical properties on mesenchymal stem cell proliferation, differentiation, and matrix production are the focus of this review chapter. Mechanisms and materials, principally hydrogel and hydrogel composites for bone and cartilage repair that create "cell-supportive" and "instructive" biomaterials, are emphasized. We begin by providing an overview of stem cells, their unique properties, and their challenges in regenerative medicine. An overview of current fabrication strategies for creating instructive substrates is then reviewed with a focused discussion of selected fabrication methods with an emphasis on bioprinting as a critical tool in creating novel stem cell-based biomaterials. We conclude with a critical assessment of the current state of the field and offer our view on the promises and potential pitfalls of the approaches discussed.

Detection and analysis of enamel cracks by ICG-NIR fluorescence dental imaging.

Cracked teeth are the third most common cause of tooth loss, but there is no reliable imaging tool for the diagnosis of cracks. Here, we demonstrate the feasibility of indocyanine green near-infrared fluorescence (ICG-NIRF) dental imaging for the detection of enamel cracks and enamel-dentin cracks in vitro in the first (ICG-NIRF-I, 700-950 nm) and second (ICG-NIRF-II, 950-1700 nm) imaging windows with transmission excitation light, and compared ICG-NIRF with conventional NIR illumination-II (NIRi-II) and X-ray imaging. Dentin cracks were detected by CT scan, while most enamel cracks, undetectable under X-ray imaging, were clearly visible in NIR images. We found that ICG-NIRF-II detected cracks more effectively than NIRi-II, and that light orientation is an important factor for crack detection: an angled exposure obtained better image contrast of cracks than parallel exposure, as it created a shadow under the crack. Crack depth could be evaluated from the crack shadow in ICG-NIRF and NIRi-II images; from this shadow we could determine crack depth and discriminate enamel-dentin cracks from craze lines. Cracks could be observed clearly from ICG-NIRF images with 1-min ICG tooth immersion, although longer ICG immersion produced images with greater contrast. Overall, our data show that ICG-NIRF dental imaging is a useful tool for diagnosing cracked teeth at an early stage.

Optimal imaging windows of indocyanine green-assisted near-infrared dental imaging with rat model and its comparison to X-ray imaging.

In this study, we used rat animal model to compare the efficiency of indocyanine green (ICG)-assisted dental near-infrared fluorescence imaging with X-ray imaging, and we optimized the imaging window for both unerupted and erupted molars. The results show that the morphology of the dental structures was observed clearly from ICG-assisted dental images (especially through the endoscope). A better image contrast was easily acquired at the short imaging windows (<10 minutes) for unerupted and erupted molars. For unerupted molars, there is another optimized imaging window (48-96 hours) with a prominent glow-in-the-dark effect: only the molars remain bright. This study also revealed that the laser ablation of dental follicles can disrupt the molar development, and our method is able to efficiently detect laser-treated molars and acquire the precise morphology. Thus, ICG-assisted dental imaging has the potential to be a safer and more efficient imaging modality for the real-time diagnosis of dental diseases.

Improvement of cell deposition by self-absorbent capability of freeze-dried 3D-bioprinted scaffolds derived from cellulose material-alginate hydrogels.

Cell-laden printing is the most commonly used approach in 3D bioprinting. One of the major drawbacks of cell-laden printing is that cell viability is highly affected by the extrusion pressure and shear force in the printing process. We present a new cell-deposition method by using the superabsorbent capability of 3D printed scaffolds with four ink formations: 20:10 nanocrystal/alginate (NCA 20/10), 20:10 nanofiber/alginate (NFA 20/10), 20:02 nanocrystal/alginate (NCA 20/02) and 20:02 nanofiber/alginate (NFA 20/02). Limited pores were observed from the surface of inherent NCA and NFA scaffolds, which may limit the numbers of cells to enter into the scaffolds. Therefore, we designed a dual-porous (DP) structure to connect the inherent pores (IPs) to the scaffold surface. Due to these porous structures, NCA and NFA scaffolds exhibit an excellent capability to absorb cell suspension, which may be used for depositing cells to 3D-printed scaffolds, namely self-absorbent (SA) deposition. Compared to the conventional top-loading (TL) method, the SA method had more uniform cell distributions in the entire 3D-printed scaffolds and higher efficiency of cell deposition. For the TL method, DP scaffold exhibited a more uniform cell distribution, which may provide a better microenvironment for the cells in comparison to the IP scaffold. For both cell loading methods, a rapid increase of cell number was observed in the first 4 days of culture in the 3D-printed NCA and NFA structures. NFA 20/02 exhibits the best cell viability compared to the other three inks. In conclusion, the SA method may serve as a new approach for loading cells in cell-free 3D-bioprinting, and DP design could improve the efficiency of the cell deposition.

Adenosine triphosphate enhances osteoblast differentiation of rat dental pulp stem cells via the PLC-IP3 pathway and intracellular Ca 2+ signaling.

Intracellular Ca2+ signals are essential for stem cell function and play a significant role in the differentiation process. Dental pulp stem cells (DPSCs) are a potential source of stem cells; however, the mechanisms controlling cell differentiation remain largely unknown. Utilizing rat DPSCs, we examined the effect of adenosine triphosphate (ATP) on osteoblast differentiation and characterized its mechanism of action using real-time Ca 2+ imaging analysis. Our results revealed that ATP enhanced osteogenesis as indicated by Ca 2+ deposition in the extracellular matrix via Alizarin Red S staining. This was consistent with upregulation of osteoblast genes BMP2, Mmp13, Col3a1, Ctsk, Flt1, and Bgn. Stimulation of DPSCs with ATP (1-300 µM) increased intracellular Ca 2+ signals in a concentration-dependent manner, whereas histamine, acetylcholine, arginine vasopressin, carbachol, and stromal-cell-derived factor-1α failed to do so. Depletion of intracellular Ca 2+ stores in the endoplasmic reticulum by thapsigargin abolished the ATP responses which, nevertheless, remained detectable under extracellular Ca 2+ free condition. Furthermore, the phospholipase C (PLC) inhibitor U73122 and the inositol triphosphate (IP 3 ) receptor inhibitor 2-aminoethoxydiphenyl borate inhibited the Ca 2+ signals. Our findings provide a better understanding of how ATP controls osteogenesis in DPSCs, which involves a Ca 2+ -dependent mechanism via the PLC-IP 3 pathway. This knowledge could help improve osteogenic differentiation protocols for tissue regeneration of bone structures.

Improvement of osseointegration by recruiting stem cells to titanium implants fabricated with 3D printing.

Slow and incomplete osseointegration and loss of osseointegration are major problems in dental and bone implants. We designed implants with interconnected 3D-tubulous structures and hypothesized that such interconnecting 3D (I3D) structures would serve as a repository for chemoattractants to recruit stem cells to promote osseointegration. A concept Laser Mlab-cusing-R laser-powder-bed-fusion (LPBF) 3D printing system was used to produce titanium implants with designed features. The implants were loaded (coated) with stromal cell-derived factor-1 alpha (SDF-1α), and subjected to stem cell recruitment. Implants were then surgically transplanted into the rabbit skull bone. After 12 weeks, osseointegration was analyzed by reverse-torque test and the implants were examined for calcium deposition by Alizarin Red staining. The I3D implants attracted significantly more stem cells than solid implants when coated (loaded) with SDF-1α. Greater torque force was needed to extract the I3D implants with 200 and 300 µm I3D structures than to extract solid implants from the skull. Generally, more calcium deposition was observed on the I3D implants than on the solid counterparts. LPBF 3D printing can be used to fabricate implants with complex structures. I3D-tubulous structures of implants can retain chemoattractant for recruitment of stem cells to enhance osseointegration.

Expression of microRNAs targeting heat shock protein B8 during in vitro expansion of dental pulp stem cells in regulating osteogenic differentiation.

OBJECTIVE: The objectives of this study were (a) to determine the differentially expressed microRNAs that can target heat shock protein B8 (HspB8) during in vitro expansion of dental pulp stem cells (DPSCs); (b) to identify microRNAs involved in posttranscriptional regulation of HspB8 expression; and (c) to determine if HspB8-targeting microRNAs play roles on osteogenic differentiation of DPSCs. DESIGN: DPSCs were established from rat first molars and expanded in vitro until the passage that cells lost osteogenic potential. TargetScan was used to predict the microRNAs that target HspB8 mRNA. Stem-loop quantitative RT-PCR was conducted to identify the HspB8-targeting microRNAs that were upregulated in late passages. The microRNAs mimics were transfected into DPSCs to assess their effects on HspB8 expression and on osteogenic differentiation. RESULTS: let-7b-5p, miR-98-5p, miR-215, miR-219a-1-3p and miR-295-5p were found to consistently increase expression in DPSCs after expansion. HspB8 mRNA and/or protein were significantly decreased in the DPSCs after transfection of miR-215 and miR-219a-1-3p mimics; whereas no significant reduction was seen after transfecting let-7b-5p, miR-98-5p and miR-295-5p mimics. When subjecting the transfected DPSCs to osteogenic induction, reduction of calcium deposition or osteogenic marker expression were observed with miR-215, miR-219a-1-3p and miR-295-5p transfection. CONCLUSIONS: Increased expression of miR-215 and miR-219a-1-3p downregulates HspB8 expression, which contributes to the reduction of osteogenic capability of DPSCs. Increased expression of miR295-5p also causes a reduction of osteogenic differentiation, but not involved in HspB8.

Ultrastructure Morphological Characterization of Different Passages of Rat Dental Follicle Stem Cells at In vitro Culture.

INTRODUCTION: Stem cells play important roles in tissue renewal and repair. Tissue-derived stem cells have been demonstrated for their applications in tissue engineering and regenerative medicine. Expansion of primary stem cells isolated from tissues to a large quantity through in vitro culture is needed for application of the stem cells. However, it is known that tissue stem cells commonly reduce or lose their stemness properties during in vitro culture. In this study, we assessed ultrastructural changes of rat dental follicle stem cells (DFSCs) during in vitro culture. It is our attempt to explain the loss of stemness properties in cultured tissue-stem cells at the ultrastructural level. METHOD: DFSCs was isolated from first molars of Sprague Dawley rat pups and cultured in medium consisting of alpha-MEM plus 20% FBS. Cells were passaged at 1 to 3 ratio at 90% confluence, and collected at passages 3, 6, 7 and 9 for assessment of ultrastructure morphology by transmission electron microscopy. RESULTS: Of the four passages (3, 6, 7, and 9) examined, dilated rough endoplasmic reticulum (RER) was abundant in Passage 3 but less so in Passages 6, 7, and 9. The dilated RER contained lipid in Passages 3, 7, and 9. The mono- and polyribosomes in Passages 3 and 6 were located between the mitochondria and the RER. Mono- and polyribosomes were abundant in Passage 7, although mainly monoribosomes were present in Passage 9. Membrane-bound glycogen granules were in vacuoles bulging off the cells in Passage 3. Some glycogen granules were grouped in the periphery of a stem cell in Passage 9. Nuclei shapes were irregular and mainly euchromatic in Passages 6, 7, and 9. The mitochondria were dark and scarce in Passage 9; irregular, small, and dark in Passage 7; and small and rounded in Passage 6, and they were spread in the cytoplasm away from the nucleus in Passage 3. Cell contacts were seen in Passages 6, 7, and 9. The ultrastructure morphology of the examined DFScs was not very different from the morphology criteria of the undifferentiated cells. Large vacuoles in Passage 3 were mainly at the periphery of the cell, with the small vacuoles in the cell center. Small vacuoles were scattered in the cell center of Passage 6 and the larger ones were observed at the cell's periphery. CONCLUSIONS: We observed the following ultrastructural changes: decreases of fine cell cytoplasmic processes, dilated cytoplasmic vacuoles, cytoplasmic pinocytotic vesicles, and nuclear heterochromatin with increasing cell passage number. Conversely, mean ratios of lipid globules, nuclear euchromatin, irregular nuclear shape, and cell contact between cells were increased with passage number. The observations may suggest an increase in committed cells among the population after long-term culture of DFSCs.

Indocyanine-green-assisted near-infrared dental imaging - the feasibility of in vivo imaging and the optimization of imaging conditions.

X-ray-based imaging, including computed tomography, plays a crucial role in the diagnosis and surgery of impacted teeth that affects over 25% of the human population. But the greatest disadvantage of this technique is ionizing radiation risk to the patients. Here we describe a completely ionizing-radiation-free in vivo near-infrared (NIR) fluoresence dental imaging with indocyanine green (ICG) agent that has rarely been applied in dental imaging. Our method can acquire dental structure images within a short period (only 10 minutes after injection) without ionizing radiation risk. NIR enables the observation of dental structures that are not distinguishable under visible conditions. At prolonged 72 hours, only molar regions remained highlighted; the contrast between molar regions and surrounding tissues was prominent; this is particularly useful for in vivo dental imaging. Using the quantitative spectral analysis, we found the peak wavelengths of ICG fluorescence shifted along with the injection time: the peak wavelength shifted 8 nm (from 819 nm to 811 nm) in 0~72 hours. The injection methods of tail vein v.s. intradermal injections caused ~3 nm shift. ICG-assisted NIR fluorescence imaging can serve as a useful tool for in vivo real-time diagnosis in dental clinics and surgeries without ionizing radiation risk.