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Background: The integration of stem cells, signaling molecules, and biomaterial scaffolds is fundamental for the successful engineering of functional bone tissue. Currently, the development of composite scaffolds has emerged as an attractive approach to meet the criteria of ideal scaffolds utilized in bone tissue engineering (BTE) for facilitating bone regeneration in bone defects. Recently, the incorporation of polycaprolactone (PCL) with hydroxyapatite (HA) has been developed as one of the suitable substitutes for BTE applications owing to their promising osteogenic properties. In this study, a three-dimensional (3D) scaffold composed of PCL integrated with HA (PCL/HA) was prepared and assessed for its ability to support osteogenesis in vitro. Furthermore, this scaffold was evaluated explicitly for its efficacy in promoting the proliferation and osteogenic differentiation of canine bone marrow-derived mesenchymal stem cells (cBM-MSCs) to fill the knowledge gap regarding the use of composite scaffolds for BTE in the veterinary orthopedics field., Results: Our findings indicate that the PCL/HA scaffolds substantially supported the proliferation of cBM-MSCs. Notably, the group subjected to osteogenic induction exhibited a markedly upregulated expression of the osteogenic gene osterix (OSX) compared to the control group. Additionally, the construction of 3D scaffold constructs with differentiated cells and an extracellular matrix (ECM) was successfully imaged using scanning electron microscopy. Elemental analysis using a scanning electron microscope coupled with energy-dispersive X-ray spectroscopy confirmed that these constructs possessed the mineral content of bone-like compositions, particularly the presence of calcium and phosphorus., Conclusions: This research highlights the synergistic potential of PCL/HA scaffolds in concert with cBM-MSCs, presenting a multidisciplinary approach to scaffold fabrication that effectively regulates cell proliferation and osteogenic differentiation. Future in vivo studies focusing on the repair and regeneration of bone defects are warranted to further explore the regenerative capacity of these constructs, with the ultimate goal of assessing their potential in veterinary clinical applications., (© 2024. The Author(s).)

Introduction: Type 2 diabetes (T2D) is the predominant form of diabetes mellitus and is among the leading causes of death with an increasing prevalence worldwide. However, the pathological mechanism underlying T2D remains complex and unclear. An increasing number of studies have suggested an association between circadian clock disruption and high T2D prevalence., Method: This review explores the physiological and genetic evidence underlying T2D symptoms associated with circadian clock disturbances, including insulin secretion and glucose metabolism., Results and Discussion: Notably, circadian clock disruption reduces insulin secretion and insulin sensitivity and negatively affects glucose homeostasis. The circadian clock regulates the hypothalamic-pituitary-adrenal axis, an important factor that regulates glucose metabolism and influences T2D progression. Therefore, circadian clock regulation is an attractive, novel therapeutic approach for T2D, and various circadian clock stabilizers play therapeutic roles in T2D. Lastly, this review suggests novel therapeutic and preventive approaches using circadian clock regulators for T2D., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Tran, Kondo, Ashry, Fu, Okawa, Sawangmake and Egusa.)

Nanostructured lipid carriers (NLC) represent the second generation of nanoparticles, offering numerous advantages over conventional delivery systems. These include improved stability, enhanced drug-loading capacity, and controlled release profiles, making them highly attractive candidates for a wide range of therapeutic applications. Their suitability for hydrophobic drugs like a traditional medicinal plant of Thailand as clove oil and alpha-mangostin. We investigated into nanostructured lipid carriers loaded with Alpha-Mangostin and clove oil (NLC-AMCO) into the physicochemical and biological characteristics to identify the formulation with the highest efficacy for treatment. The particle size, charge, polydispersity index, and other characterizations were recorded. The realtime ex vivo penetration was explored using canine gingival tissue. Drug sustained release was assessed by HPLC. Moreover, the antibacterial properties were tested by conventional methods. The NLC-AMCO can be stored at up to 40 °C for 60 days without any alterations in particle characteristics. Gingival tissue penetration and sustained drug release were superior compared to unencapsulated counterparts. It exhibited greater effectiveness in inhibiting bacterial growth than the antibiotics tested, particularly against bacteria from the oral cavities of dogs. Therefore, this alternative treatment approach offers cost-effectiveness and ease of administration for pet owners and reduces discomfort for the animals during restraint.

Surgical treatment of tracheal diseases, trauma, and congenital stenosis has shown success through tracheal reconstruction coupled with palliative care. However, challenges in surgical-based tracheal repairs have prompted the exploration of alternative approaches for tracheal replacement. Tissue-based treatments, involving the cultivation of patient cells on a network of extracellular matrix (ECM) from donor tissue, hold promise for restoring tracheal structure and function without eliciting an immune reaction. In this study, we utilized decellularized canine tracheas as tissue models to develop two types of cell carriers: a decellularized scaffold and a hydrogel. Our hypothesis posits that both carriers, containing essential biochemical niches provided by ECM components, facilitate cell attachment without inducing cytotoxicity. Canine tracheas underwent vacuum-assisted decellularization (VAD), and the ECM-rich hydrogel was prepared through peptic digestion of the decellularized trachea. The decellularized canine trachea exhibited a significant reduction in DNA content and major histocompatibility complex class II, while preserving crucial ECM components such as collagen, glycosaminoglycan, laminin, and fibronectin. Scanning electron microscope and fluorescent microscope images revealed a fibrous ECM network on the luminal side of the cell-free trachea, supporting epithelial cell attachment. Moreover, the ECM-rich hydrogel exhibited excellent viability for human mesenchymal stem cells encapsulated for 3 days, indicating the potential of cell-laden hydrogel in promoting the development of cartilage rings of the trachea. This study underscores the versatility of the trachea in producing two distinct cell carriers-decellularized scaffold and hydrogel-both containing the native biochemical niche essential for tracheal tissue engineering applications., (© 2024. The Author(s) under exclusive licence to Biomedical Engineering Society.)

Because of the time-consuming nature of surgical neutering and the rapid rate of reproduction among domestic cats, it is crucial to investigate alternative, nonsurgical methods of contraception for this species. Sperm protein IZUMO1 and its oocyte receptor JUNO have been proposed as potential targets for nonsurgical contraceptives. This study aimed to demonstrate (1) the protein coding sequence of feline IZUMO1 and JUNO, (2) gene expression in specific organs by measuring mRNA levels in different visceral tissues, and (3) the expression of IZUMO1 and JUNO during sperm maturation and folliculogenesis, respectively. Amplification for sequencing of feline IZUMO1 and JUNO was performed using the RT-PCR method. Levels of gene expression in different tissues were evaluated using real-time PCR. In situ hybridization was performed to localize JUNO mRNA in ovarian tissues. The complete coding sequences of IZUMO1 and JUNO were obtained and analyzed. A comparison between protein orthologs demonstrated the conservation of IZUMO1 and JUNO in Felidae. The real-time PCR results from various visceral organs indicated that IZUMO1 was significantly higher in the testis than in other organs, whereas JUNO was significantly higher in the ovary than in other organs. Expression of IZUMO1 was found to be higher in the testes than in the caput, corpus, and cauda of epididymides. In situ hybridization revealed that JUNO mRNA was in the ooplasm and nucleus of the primordial, primary, secondary, and antral follicles. Importantly, this was the first study to demonstrate the IZUMO1 and JUNO genes in the testis and ovary of cats. The results are useful for future research related to these genes and for developing contraceptives against these targets., (Copyright © 2024 Elsevier Inc. All rights reserved.)

To better understand molecular aspects of equine endometrial function, there is a need for advanced in vitro culture systems that more closely imitate the intricate 3-dimensional (3D) in vivo endometrial structure than current techniques. However, development of a 3D in vitro model of this complex tissue is challenging. This study aimed to develop an in vitro 3D endometrial tissue (3D-ET) with an epithelial cell phenotype optimized by treatment with a Rho-associated protein kinase (ROCK) inhibitor. Equine endometrial epithelial (eECs) and mesenchymal stromal (eMSCs) cells were isolated separately, and eECs cultured in various concentrations of Rock inhibitor (0, 5, 10 µmol) in epithelial medium (EC-medium) containing 10% knock-out serum replacement (KSR). The optimal concentration of Rock inhibitor for enhancing eEC proliferation and viability was 10 µM. However, 10 µM Rock inhibitor in the 10% KSR EC-medium was able to maintain mucin1 (Muc1) gene expression for only a short period. In contrast, fetal bovine serum (FBS) was able to maintain Muc1 gene expression for longer culture durations. An in vitro 3D-ET was successfully constructed using a collagen-based scaffold to support the eECs and eMSCs. The 3D-ET closely mimicked in vivo endometrium by displaying gland-like eEC-derived structures positive for the endometrial gland marker, Fork headbox A2 (FOXA2), and by mimicking the 3D morphology of the stromal compartment. In addition, the 3D-ET expressed the secretory protein MUC1 on its glandular epithelial surface and responded to LPS challenge by upregulating the expression of the interleukin-6 (IL6) and prostaglandin F synthase (PGFS) genes (P < 0.01), along with an increase in their secretory products, IL-6 (P < 0.01) and prostaglandin F2alpha (PGF2α) (P < 0.001) respectively. In the future, this culture system can be used to study both normal physiology and pathological processes of the equine endometrium., (© 2024. The Author(s).)

Oral and maxillofacial (OMF) defects are not limited to humans and are often encountered in other species. Reconstructing significant tissue defects requires an excellent strategy for efficient and cost-effective treatment. In this regard, tissue engineering comprising stem cells, scaffolds, and signaling molecules is emerging as an innovative approach to treating OMF defects in veterinary patients. This review presents a comprehensive overview of OMF defects and tissue engineering principles to establish proper treatment and achieve both hard and soft tissue regeneration in veterinary practice. Moreover, bench-to-bedside future opportunities and challenges of tissue engineering usage are also addressed in this literature review., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Purbantoro, Taephatthanasagon, Purwaningrum, Hirankanokchot, Peralta, Fiani, Sawangmake and Rattanapuchpong.)

Immune escape is the hallmark of carcinogenesis. This widely known mechanism is the overexpression of immune checkpoint ligands, such as programmed cell death protein 1 and programmed death-ligand 1 (PD-1/PD-L1), leading to T cell anergy. Therefore, cancer immunotherapy with specific binding to these receptors has been developed to treat human cancers. Due to the lack of cross-reactivity of these antibodies in dogs, a specific canine PD-1/PD-L1 antibody is required. The aim of this study is to develop mouse anti-canine PD-L1 (cPD-L1) monoclonal antibodies and characterize their in vitro properties. Six mice were immunized with recombinant cPD-L1 with a fusion of human Fc tag. The hybridoma clones that successfully generated anti-cPD-L1 antibodies and had neutralizing activity were selected for monoclonal antibody production. Antibody properties were tested by immunosorbent assay, surface plasmon resonance, and immunohistochemistry. Four hybridomas were effectively bound and blocked to recombinant cPD-L1 and cPD-1-His-protein, respectively. Candidate mouse monoclonal antibodies worked efficiently on formalin-fixed paraffin-embedded tissues of canine cancers, including cutaneous T-cell lymphomas, mammary carcinomas, soft tissue sarcomas, squamous cell carcinomas, and malignant melanomas. However, functional assays of these anti-cPD-L1 antibodies need further investigation to prove their abilities as therapeutic drugs in dogs as well as their applications as prognostic markers.

An HPMC-based nasal spray solution containing human IgG1 antibodies against SARS-CoV-2 (nasal antibody spray or NAS) was developed to strengthen COVID-19 management. NAS exhibited potent broadly neutralizing activities against SARS-CoV-2 with PVNT 50 values ranging from 0.0035 to 3.1997 μg/ml for the following variants of concern (ranked from lowest to highest): Alpha, Beta, Gamma, ancestral, Delta, Omicron BA.1, BA.2, BA.4/5, and BA.2.75. Biocompatibility assessment showed no potential biological risks. Intranasal NAS administration in rats showed no circulatory presence of human IgG1 anti-SARS-CoV-2 antibodies within 120 h. A double-blind, randomized, placebo-controlled trial (NCT05358873) was conducted on 36 healthy volunteers who received either NAS or a normal saline nasal spray. Safety of the thrice-daily intranasal administration for 7 days was assessed using nasal sinuscopy, adverse event recording, and self-reporting questionnaires. NAS was well tolerated, with no significant adverse effects during the 14 days of the study. The SARS-CoV-2 neutralizing antibodies were detected based on the signal inhibition percent (SIP) in nasal fluids pre- and post-administration using a SARS-CoV-2 surrogate virus neutralization test. SIP values in nasal fluids collected immediately or 6 h after NAS application were significantly increased from baseline for all three variants tested, including ancestral, Delta, and Omicron BA.2. In conclusion, NAS was safe for intranasal use in humans to increase neutralizing antibodies in nasal fluids that lasted at least 6 h., (© 2023. Springer Nature Limited.)

Periodontal ligament stem cells (PDLSCs) play a significant role on periodontal tissue and alveolar bone homeostasis. During inflammation, interleukin (IL)-6 serves as one of key cytokine players controlling tissue reaction as well as alveolar bone tissue remodeling. It is believed that periodontal tissue inflammation causes periodontium degradation, especially alveolar bone. However, in this study, we show that an inflammatory mediator, IL-6, may serve another direction on alveolar bone homeostasis during inflammatory condition. We found that, IL-6 at 10 and 20 ng/mL was not cytotoxic and dose-dependently exerted beneficial effects on osteogenic differentiation of human PDLSCs (hPDLSCs), as demonstrated by increased alkaline phosphatase activity, mRNA expression of osteogenic markers, and matrix mineralization. The presence of physiological and inflammatory level of IL-6, the osteogenic differentiation potential by hPDLSCs was enhanced by several possible mechanisms including transforming growth factor (TGF), Wnt, and Notch pathways. After in-depth and thorough exploration, we found that Wnt pathway serves as key regulator controlling osteogenic differentiation by hPDLSCs amid the IL-6 presentation. Surprisingly, apart from other mesenchymal stem cells, distinct Wnt components are employed by hPDLSCs, and both canonical and non-canonical Wnt pathways are triggered by different mechanisms. Further validation by gene silencing, treatment with recombinant Wnt ligands, and β-catenin stabilization/translocation confirmed that IL-6 governed the canonical Wnt/β-catenin pathway via either WNT2B or WNT10B and employed WNT5A to activate the non-canonical Wnt pathway. These findings fulfill the homeostasis pathway governing periodontal tissue and alveolar bone regeneration and may serve for further therapeutic regimen design for restoring the tissues., (© 2023. The Author(s).)

Veterinary education is the foundation of veterinary services in the country. Starting from the service sector in the army, veterinary education and practice in Thailand have been standardized and progressed toward international veterinary standards. The 6-year Doctor of Veterinary Medicine core curriculum is deployed to develop the curriculum for each Veterinary Education Establishment (VEE). The challenges for veterinary education and practices reflect the country's expectations of veterinary services. With regional and global collaboration, the VEEs have been developing tools and learning platforms for delivering qualified veterinary graduates that fit fast-growing society needs., Competing Interests: The authors declare no conflicts of interest., (© 2022 The Korean Society of Veterinary Science.)

Bone tissue engineering consists of three major components namely cells, scaffolds, and signaling molecules to improve bone regeneration. These integrated principles can be applied in patients suffered from bone resorption diseases, such as osteoporosis and periodontitis. Osteogenic growth peptide (OGP) is a fourteen-amino acid sequence peptide that has the potential to regenerate bone tissues. This study aimed to disseminate the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) with OGP treatment. OGP was elaborated for proliferation, cytotoxicity, osteogenic differentiation effects, and the involvement of osteogenic related signaling pathways in vitro . This study found that OGP at lower concentration shows better effects on cytotoxicity and proliferation. Moreover, OGP at concentration 0.01 nM had the most potential to differentiate hPDLSCs toward osteogenic lineage comparing with higher concentrations of OGP. The phenomenon was mainly involving transforming growth factor-beta (TGF-β), bone morphogenetic protein (BMP), Hedgehog, and Wingless-related (Wnt) pathways. Further, SB-431542 treatment demonstrated the partial involvement of OGP in regulating osteogenic differentiation of hPDLSCs. In conclusion, OGP at low concentration enhances osteogenic differentiation of hPDLSCs by governing TGF-β signaling pathway., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

Canine mesenchymal stem cells (cMSCs) have potential applications for regenerative therapy, including the generation of insulin-producing cells (IPCs) for studying and treating diabetes. In this study, we established a useful protocol for generating IPCs from canine adipose mesenchymal stem cells (cAD-MSCs). Subsequently, in vitro preservation of pluronic F127-coated alginate (ALGPA)-encapsulated cAD-MSC-derived IPCs was performed to verify ready-to-use IPCs. IPCs were induced from cAD-MSCs with the modulated three-stepwise protocol. The first step of definitive endoderm (DE) induction showed that the cooperation of Chir99021 and Activin A created the effective production of Sox17-expressed DE cells. The second step for pancreatic endocrine (PE) progenitor induction from DE indicated that the treatment with taurine, retinoic acid, FGF2, EGF, TGFβ inhibitor, dorsomorphin, nicotinamide, and DAPT showed the significant upregulation of the pancreatic endocrine precursor markers Pdx1 and Ngn3. The last step of IPC production, the combination of taurine, nicotinamide, Glp-1, forskolin, PI3K inhibitor, and TGFβ inhibitor, yielded efficiently functional IPCs from PE precursors. Afterward, the maintenance of ALGPA-encapsulated cAD-MSC-derived IPCs with VSCBIC-1, a specialized medium, enhanced IPC properties. Conclusion, the modulated three-stepwise protocol generates the functional IPCs. Together, the encapsulation of cAD-MSC-derived IPCs and the cultivation with VSCBIC-1 enrich the maturation of generated IPCs., (© 2022. The Author(s).)

The human amniotic membrane has been successfully used in human ocular reconstruction. Several studies have demonstrated its properties, including antimicrobial features. As a result of the restricted availability of human amniotic membrane for veterinary use, canine amniotic membrane has become an attractive alternative. Clinical studies of the application of canine amniotic membrane in animals and the understanding of its biological properties are limited. This study aimed to determine the expression of peptide genes of natural antimicrobials in canine amniotic membrane. Expressions of canine β-defensin 1, 102, and 103, and canine Elafin were determined in healthy puppies by real-time quantitative polymerase chain reaction. Canine β-defensin 1, 103, and Elafin were expressed in all samples, possibly suggesting a role in the innate immune system of normal canine amniotic membrane. Further investigations of protein expression and localization are recommended.

Corneal grafts are the imperative clinical treatment for canine corneal blindness. To serve the growing demand, this study aimed to generate tissue-engineered canine cornea in part of the corneal epithelium and underlying stroma based on canine limbal epithelial stem cells (cLESCs) seeded silk fibroin/gelatin (SF/G) film and canine corneal stromal stem cells (cCSSCs) seeded SF/G scaffold, respectively. Both cell types were successfully isolated by collagenase I. SF/G corneal films and stromal scaffolds served as the prospective substrates for cLESCs and cCSSCs by promoting cell adhesion, cell viability, and cell proliferation. The results revealed the upregulation of tumor protein P63 (P63) and ATP-binding cassette super-family G member 2 (Abcg2) of cLESCs as well as Keratocan (Kera), Lumican (Lum), aldehyde dehydrogenase 3 family member A1 (Aldh3a1) and Aquaporin 1 (Aqp1) of differentiated keratocytes. Moreover, immunohistochemistry illustrated the positive staining of tumor protein P63 (P63), aldehyde dehydrogenase 3 family member A1 (Aldh3a1), lumican (Lum) and collagen I (Col-I), which are considerable for native cornea. This study manifested a feasible platform to construct tissue-engineered canine cornea for functional grafts and positively contributed to the body of knowledge related to canine corneal stem cells., Competing Interests: The authors have declared that no competing interests exist.

Tissue engineering has been extensively investigated and proffered to be a potential platform for novel tissue regeneration. The utilization of mesenchymal stem cells (MSCs) from various sources has been widely explored and compared. In this regard, MSCs derived from bone marrow have been proposed and described as a promising cell resource due to their high yield of isolated cells with colony-forming potential, self-renewal capacity, MSC surface marker expression, and multi-lineage differentiation capacities in vitro . However, there is evidence for bone marrow MSCs (BM-MSCs) both in vitro and in vivo from different species presenting identical and distinct potential stemness characteristics. In this review, the fundamental knowledge of the growth kinetics and stemness properties of BM-MSCs in different animal species and humans are compared and summarized. Finally, to provide a full perspective, this review will procure results of current information studies focusing on the use of BM-MSCs in clinical practice., Competing Interests: The authors declare no conflict of interest., (© 2021 The Korean Society of Veterinary Science.)

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

The trend of regenerative therapy for diabetes in human and veterinary practices has conceptually been proven according to the Edmonton protocol and animal models. Establishing an alternative insulin-producing cell (IPC) resource for further clinical application is a challenging task. This study investigated IPC generation from two practical canine mesenchymal stem cells (cMSCs), canine bone marrow-derived MSCs (cBM-MSCs) and canine adipose-derived MSCs (cAD-MSCs). The results illustrated that cBM-MSCs and cAD-MSCs contain distinct pancreatic differentiation potential and require the tailor-made induction protocols. The effective generation of cBM-MSC-derived IPCs needs the integration of genetic and microenvironment manipulation using a hanging-drop culture of PDX1-transfected cBM-MSCs under a three-step pancreatic induction protocol. However, this protocol is resource- and time-consuming. Another study on cAD-MSC-derived IPC generation found that IPC colonies could be obtained by a low attachment culture under the three-step induction protocol. Further, Notch signaling inhibition during pancreatic endoderm/progenitor induction yielded IPC colonies through the trend of glucose-responsive C-peptide secretion. Thus, this study showed that IPCs could be obtained from cBM-MSCs and cAD-MSCs through different induction techniques. Also, further signaling manipulation studies should be conducted to maximize the protocol's efficiency.

Utilization of canine mesenchymal stem cells (cMSCs) for regenerating incorrigible bone diseases has been introduced. However, cMSCs harvested from different sources showed distinct osteogenicity. To clarify this, comparative proteomics-based systems biology analysis was used to analyze osteogenic differentiation behavior by cMSCs harvested from bone marrow and dental pulp. The results illustrated that canine dental pulp stem cells (cDPSCs) contained superior osteogenicity comparing with canine bone marrow-derived MSCs (cBM-MSCs) regarding alkaline phosphatase activity, matrix mineralization, and osteogenic marker expression. Global analyses by proteomics platform showed distinct protein clustering and expression pattern upon an in vitro osteogenic induction between them. Database annotation using Reactome and DAVID revealed contrast and unique expression profile of osteogenesis-related proteins, particularly on signaling pathways, cellular components and processes, and cellular metabolisms. Functional assay and hierarchical clustering for tracking protein dynamic change confirmed that cBM-MSCs required the presences of Wnt, transforming growth factor (TGF)-beta, and bone-morphogenetic protein (BMP) signaling, while cDPSCs mainly relied on BMP signaling presentation during osteogenic differentiation in vitro. Therefore, these findings illustrated the comprehensive data regarding an in vitro osteogenic differentiation behavior by cBM-MSCs and cDPSCs which is crucial for further mechanism study and the establishment of cMSC-based bone tissue engineering (BTE) for veterinary practice.

Efficiency of the induction protocol is crucial for the generation of insulin-producing cells (IPCs) from human dental pulp stem cells (hDPSCs). Here, we established the integrative induction protocol by merging genetic manipulation technique with our previous published 3-step induction protocol aiming to enhance the pancreatic progenitor commitment and production yield. We found that the overexpression of PDX1 following with 3-step induction protocol were able to generate the 3-dimensional (3D) colony structure of pancreatic progenitors (PPs) with the beneficial trends of pancreatic endoderm commitment and production yield, while other protocols using the prolong maintenance of PDX1-overexpressed hDPSCs and the PDX1 overexpression after definitive endoderm induction were unable to generate and sustain the 3D structure of the colonies. Further Notch signaling manipulation by DAPT treatment showed lesser degree of positive effects on progenitor commitment and production yield. Although the generated PPs from the integrative protocol expressed pancreatic mRNA markers along with pro-insulin and insulin proteins, they still contained the defective glucose-responsive C-peptide secretion. Only basal secreted C-peptide level was observed. In summary, the integrative induction protocol potentially enhanced the PP generation with high colony production yield and could serve as an efficient platform for further hDPSC-derived IPC production and maturation., Competing Interests: Declaration of competing interest The authors have no financial support or personal relationship with individuals or organizations that could inappropriately influence or bias the content of this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Current approach for diabetes treatment remained several adverse events varied from gastrointestinal to life-threatening symptoms. Regenerative therapy regarding Edmonton protocol has been facing serious limitations involving protocol efficiency and safety. This led to the study for alternative insulin-producing cell (IPC) resource and transplantation platform. In this study, evaluation of encapsulated human dental pulp-derived stem cell (hDPSC)-derived IPCs by alginate (ALG) and pluronic F127-coated alginate (ALGPA) was performed., Results: The results showed that ALG and ALGPA preserved hDPSC viability and allowed glucose and insulin diffusion in and out. ALG and ALGPA-encapsulated hDPSC-derived IPCs maintained viability for at least 336 h and sustained pancreatic endoderm marker (NGN3 ), pancreatic islet markers ( NKX6.1, MAF-A, ISL-1, GLUT-2 and INSULIN ), and intracellular pro-insulin and insulin expressions for at least 14 days. Functional analysis revealed a glucose-responsive C-peptide secretion of ALG- and ALGPA-encapsulated hDPSC-derived IPCs at 14 days post-encapsulation., Conclusion: ALG and ALGPA encapsulations efficiently preserved the viability and functionality of hDPSC-derived IPCs in vitro and could be the potential transplantation platform for further clinical application., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Diabetes mellitus (DM) remains a global concern in both human and veterinary medicine. Type I DM requires prolonged and consistent exogenous insulin administration to address hyperglycemia, which can increase the risk of diabetes complications such as retinopathy, nephropathy, neuropathy, and heart disorders. Cell-based therapies have been successful in human medicine using the Edmonton protocol. These therapies help maintain the production of endogenous insulin and stabilize blood glucose levels and may possibly be adapted to veterinary clinical practice. The limited number of cadaveric pancreas donors and the long-term use of immunosuppressive agents are the main obstacles for this protocol. Over the past decade, the development of potential therapies for DM has mainly focused on the generation of effective insulin-producing cells (IPCs) from various sources of stem cells that can be transplanted into the body. Another successful application of stem cells in type I DM therapies is transplanting generated IPCs. Encapsulation can be an alternative strategy to protect IPCs from rejection by the body due to their immunoisolation properties. This review summarizes current concepts of IPCs and encapsulation technology for veterinary clinical application and proposes a potential stem-cell-based platform for veterinary diabetic regenerative therapy., (Copyright © 2020 Kuncorojakti, Srisuwatanasagul, Kradangnga and Sawangmake.)

Bone tissue engineering has been widely studied and proposed as a promising platform for correcting the bone defects. The applications of mesenchymal stem cell (MSC)-based bone tissue engineering have been investigated in various in vitro and in vivo models. In this regard, the promising animal bone defect models have been employed for illustrating the bone regenerative capacity of MSC-based bone tissue engineering. However, most studies aimed for clinical applications in human. These evidences suggest a knowledge gap to fulfill the accomplishment for veterinary implementation. In this review, the fundamental concept, knowledge, and technology of MSC-based bone tissue engineering focusing on veterinary applications are summarized. In addition, the potential canine MSCs resources for veterinary bone tissue engineering are reviewed, including canine bone marrow-derived MSCs, canine adipose-derived MSCs, and canine dental tissue-derived MSCs. This review will provide a basic and current information for studies aiming for the utilization of MSC-based bone tissue engineering in veterinary practice., (© 2019 The Author(s).)

Mechanical force regulates periodontal ligament cell (PDL) behavior. However, different force types lead to distinct PDL responses. Here, we report that pretreatment with an intermittent compressive force (ICF), but not a continuous compressive force (CCF), promoted human PDL (hPDL) osteogenic differentiation as determined by osteogenic marker gene expression and mineral deposition in vitro. ICF-induced osterix (OSX) expression was inhibited by cycloheximide and monensin. Although CCF and ICF significantly increased extracellular adenosine triphosphate (ATP) levels, pretreatment with exogenous ATP did not affect hPDL osteogenic differentiation. Gene-expression profiling of hPDLs subjected to CCF or ICF revealed that extracellular matrix (ECM)-receptor interaction, focal adhesion, and transforming growth factor beta (TGF-β) signaling pathway genes were commonly upregulated, while calcium signaling pathway genes were downregulated in both CCF- and ICF-treated hPDLs. The TGFB1 mRNA level was significantly increased, while those of TGFB2 and TGFB3 were decreased by ICF treatment. In contrast, CCF did not modify TGFB1 expression. Inhibiting TGF-β receptor type I or adding a TGF-β1 neutralizing antibody attenuated the ICF-induced OSX expression. Exogenous TGF-β1 pretreatment promoted hPDL osteogenic marker gene expression and mineral deposition. Additionally, pretreatment with ICF in the presence of TGF-β receptor type I inhibitor attenuated the ICF-induced mineralization. In conclusion, this study reveals the effects of ICF on osteogenic differentiation in hPDLs and implicates TGF-β signaling as one of its regulatory mechanisms.

Mechanical force regulates numerous biological functions. Application of different force types leads to different cell responses. This data article describes RNA sequencing data identifying gene expression of human periodontal ligament cells (hPDLs) treated with the continuous or intermittent compressive force. These data could be further utilized to investigate the controlling pathways that regulate hPDLs' behaviors by the different force types. Raw RNA sequencing data were deposited in the NCBI Sequence Read Archive (SRP136155) and NCBI Gene Expression Omnibus (GSE112122)., (© 2019 The Authors.)

Idiopathic basal ganglia calcification is a brain calcification disorder that has been genetically linked to autosomal dominant mutations in the sodium-dependent phosphate co-transporter, SLC20A2. The mechanisms whereby deficiency of Slc20a2 leads to basal ganglion calcification are unknown. In the mouse brain, we found that Slc20a2 was expressed in tissues that produce and/or regulate cerebrospinal fluid, including choroid plexus, ependyma and arteriolar smooth muscle cells. Haploinsufficient Slc20a2 +/- mice developed age-dependent basal ganglia calcification that formed in glymphatic pathway-associated arterioles. Slc20a2 deficiency uncovered phosphate homeostasis dysregulation characterized by abnormally high cerebrospinal fluid phosphate levels and hydrocephalus, in addition to basal ganglia calcification. Slc20a2 siRNA knockdown in smooth muscle cells revealed increased susceptibility to high phosphate-induced calcification. These data suggested that loss of Slc20a2 led to dysregulated phosphate homeostasis and enhanced susceptibility of arteriolar smooth muscle cells to elevated phosphate-induced calcification. Together, dysregulated cerebrospinal fluid phosphate and enhanced smooth muscle cell susceptibility may predispose to glymphatic pathway-associated arteriolar calcification., (© 2016 International Society of Neuropathology.)

Aims: Vascular smooth muscle cells (SMCs) are major precursors contributing to osteochondrogenesis and calcification in atherosclerosis. Runt-related transcription factor-2 (Runx2) has been found essential for SMC differentiation to an osteochondrogenic phenotype and subsequent calcification in vitro. A recent study using a conditional targeting allele that produced a truncated Runx2 protein in SMCs of ApoE-/- mice showed reduced vascular calcification, likely occurring via reduction of receptor activator of nuclear factor-κB ligand (RANKL), macrophage infiltration, and atherosclerotic lesion formation. Using an improved conditional Runx2 knockout mouse model, we have elucidated new roles for SMC-specific Runx2 in arterial intimal calcification (AIC) without effects on atherosclerotic lesion size., Methods and Results: We used an improved targeting construct to generate LDLr-/- mice with floxed-Runx2 alleles ( LDLr-/- :Runx2 f/f ) such that Cre-mediated recombination ( LDLr-/- :Runx2 ΔSM ) does not produce functional truncated Runx2 protein, thereby avoiding off-target effects. We found that both LDLr-/- :Runx2 f/f and LDLr-/- :Runx2 ΔSM mice fed with a high fat diet developed atherosclerosis. SMC-specific Runx2 deletion did not significantly reduce atherosclerotic lesion size, macrophage number, or expression of RANKL, MCP-1, and CCR2. However, it significantly reduced AIC by 50%. Mechanistically, Sox9 and type II collagen were unaltered in vessels of LDLr-/- :Runx2 ΔSM mice compared to LDLr-/- :Runx2 f/f counterparts, while type X collagen, MMP13 and the osteoblastic marker osteocalcin were significantly reduced., Conclusions: SMC autonomous Runx2 is required for SMC differentiation towards osteoblast-like cells, SMC-derived chondrocyte maturation and AIC in atherosclerotic mice. These effects were independent of systemic lipid metabolism, RANKL expression, macrophage infiltration, and atheromatous lesion progression.

Aims: The present study investigated the surface properties and murine pre-osteoblast cell (MC3T3-E1) responses of phosphoric acid (H3PO4) treated commercially pure titanium., Methods: Titanium discs were treated with various concentration of H3PO4 (5%, 10%, and 20%; v/v) at 90 °C for 30 min. Surface properties were evaluated by profilometer, contact angle meter, and scanning electron microscopy (SEM) with energy dispersive X-rays. MC3T3-E1 attachment and spreading were evaluated by SEM and phalloidin immunohistochemistry staining., Results: Surface roughness and wettability were not statistically difference among all experimental and control groups. Phosphate and oxygen were detected on H3PO4 treated surfaces. At 20 min, cell attachment was significantly higher in 10% and 20% H3PO4 treated groups compared to the control. Cells exhibited orientated-cytoskeleton fibers on 20% H3PO4 modified titanium surface. Though, there was no difference in cell spreading stage among all treatment groups., Conclusion: H3PO4 treatment on titanium may influence early cell response, particularly on attachment and spreading.

Dental tissue-derived mesenchymal stem cells have been proposed as an alternative source for mesenchymal stem cells. Here, we investigated the differentiation ability toward insulin producing cells (IPCs) of human dental pulp stem cells (hDPSCs) and human periodontal ligament stem cells (hPDLSCs). These cells expressed mesenchymal stem cell surface markers and were able to differentiate toward osteogenic and adipogenic lineages. Upon 3 step-IPCs induction, hDPSCs exhibited more colony number than hPDLSCs. The mRNA upregulation of pancreatic endoderm/islet markers was noted. However, the significant increase was noted only for PDX-1, NGN-3, and INSULIN mRNA expression of hDPSCs. The hDPSCs-derived IPCs expressed PRO-INSULIN and released C-PEPTIDE upon glucose stimulation in dose-dependent manner. After IPCs induction, the Notch target, HES-1 and HEY-1, mRNA expression was markedly noted. Notch inhibition during the last induction step or throughout the protocol disturbed the ability of C-PEPTIDE release upon glucose stimulation. The results suggested that hDPSCs had better differentiation potential toward IPCs than hPDLSCs. In addition, the Notch signalling might involve in the differentiation regulation of hDPSCs into IPCs., (Copyright © 2014 Elsevier Inc. All rights reserved.)

D-Glucose serves many roles in cellular functions, but its role in human periodontal ligament-derived mesenchymal stem cells (hPSLSCs) is yet unknown. Here, the roles of high glucose concentration on neurogenic differentiation by hPDLSCs were investigated. Two-stage neurogenic induction protocol was employed. Cells were maintained in normal neurogenic induction medium, high glucose condition, or high mannose condition. The results showed that high glucose attenuated neurosphere formation efficiency by hPDLSCs in terms of morphology, neurogenic marker expression, without a deleterious effect on cell viability. Contrastingly, neurosphere-derived cells matured in high glucose condition exhibited normal neuronal characteristics compared to the control. During neurosphere formation in high glucose, glucose transporters (GLUTs) mRNA levels were significantly decreased, corresponding with the deprivation of cellular glucose uptake. Further, a glucose uptake inhibitor, cytochalasin B, was used to confirm the deleterious effects of glucose uptake deprivation during neurosphere formation. The results demonstrated that deprivation of glucose uptake attenuated neurosphere formation efficiency by hPDLSCs. Together, the results illustrated that high glucose condition attenuated the efficiency of neurosphere formation but not neuronal maturation, which may occur through the downregulation of GLUTs and the reduction of glucose uptake., (© 2013 Wiley Periodicals, Inc.)