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Abstract Background Small extracellular vesicles (sEVs) derived from mesenchymal stem cells (MSCs) are recognized for their therapeutic potential in immune modulation and tissue repair, especially in veterinary medicine. This study introduces an innovative sequential stimulation (IVES) technique, involving low-oxygen gas mixture preconditioning using in vitro fertilization gas (IVFG) and direct current electrical stimulation (ES20), to enhance the anti-inflammatory properties of sEVs from canine adipose-derived MSCs (cAD-MSCs). Initial steps involved isolation and comprehensive characterization of cAD-MSCs, including morphology, gene expression, and differentiation potentials, alongside validation of the electrical stimulation protocol. IVFG, ES20, and IVES were applied simultaneously with a control condition. Stimulated cAD-MSCs were evaluated for morphological changes, cell viability, and gene expressions. Conditioned media were collected and purified for sEV isolation on Day1, Day2, and Day3. To validate the efficacy of IVES for sEV production, various analyses were conducted, including microscopic examination, surface marker assessment, zeta-potential measurement, protein quantification, nanoparticle tracking analysis, and determination of anti-inflammatory activity. Results We found that IVES demonstrated non-cytotoxicity and induced crucial genotypic changes associated with sEV production in cAD-MSCs. Interestingly, IVFG influenced cellular adaptation, while ES20 induced hypoxia activation. By merging these stimulations, IVES enhanced sEV stability and quality profiles. The cAD-MSC-derived sEVs exhibited anti-inflammatory activity in lipopolysaccharide-induced RAW264.7 macrophages, emphasizing their improved effectiveness without cytotoxicity or immunogenicity. These effects were consistent across day 3 collection, indicating the establishment of an effective protocol for sEV production. Conclusions This research established an innovative sequential stimulation method with positive impact on sEV characteristics including stability, quality, and anti-inflammatory activity. This study not only contributes to the enhancement of sEV production but also sheds light on their functional aspects for therapeutic interventions.

Abstract 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.

Abstract 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

IntroductionType 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.MethodThis review explores the physiological and genetic evidence underlying T2D symptoms associated with circadian clock disturbances, including insulin secretion and glucose metabolism.Results and DiscussionNotably, 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.

AbstractImmune 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.

Abstract 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 PVNT50 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.

Abstract 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.

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.

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.

Abstract 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.

Abstract 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.

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.

Abstract 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.

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.

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.

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.

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.

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). Keywords: Periodontal ligament, Mechanical force, RNA sequencing

Establishing the intervention to enhance proliferation and differentiation potential is crucial for the clinical translation of stem cell-based therapy. In this study, the effects of simvastatin on these regards were explored. Canine bone marrow-derived mesenchymal stem cells (cBM-MSCs) were treated with 4 doses of simvastatin, 0.1, 1, 10, and 100 nM. Simvastatin in low-dose range, 0.1 and 1 nM, enhanced dose-dependent cell proliferation at day 5 and 7. Exploration of the mechanisms revealed that simvastatin in low-dose range dose-dependently upregulated sets of cell cycle regulators, Cyclin D1 and Cyclin D2; proliferation marker, Ki-67; and anti-apoptotic gene; Bcl-2. Interestingly, pluripotent markers, Rex1 and Oct4, were dramatically increased upon the low-dose treatment. Contrastingly, treatment with high-dose simvastatin suppressed the expression of those genes. Thus, the results suggested beneficial effects of simvastatin on cBM-MSCs proliferation and expansion. Further study regarding differentiation potential and underlying mechanisms will accelerate the clinical application of the molecule on veterinary stem cell-based therapy.

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.

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. [ABSTRACT FROM AUTHOR]