CRISPR innovations in tissue engineering and gene editing.

The CRISPR/Cas9 system is a powerful tool for genome editing, utilizing the Cas9 nuclease and programmable single guide RNA (sgRNA). However, the Cas9 nuclease activity can be disabled by mutation, resulting in catalytically deactivated Cas9 (dCas9). By combining the customizable sgRNA with dCas9, researchers can inhibit specific gene expression (CRISPR interference, CRISPRi) or activate the expression of a target gene (CRISPR activation, CRISPRa). In this review, we present the principles and recent advancements of these CRISPR technologies, as well as their delivery vectors. We also explore their applications in stem cell engineering and regenerative medicine, with a focus on in vitro stem cell fate manipulation and in vivo treatments. These include the prevention of retinal and muscular degeneration, neural regeneration, bone regeneration, cartilage tissue engineering, and the treatment of blood, skin, and liver diseases. Furthermore, we discuss the challenges of translating CRISPR technologies into regenerative medicine and provide future perspectives. Overall, this review highlights the potential of CRISPR in advancing regenerative medicine and offers insights into its application in various areas of research and therapy.

Neurodevelopmental functions and activities of the KAT3 class of lysine acetyltransferases.

The human lysine acetyltransferases KAT3A (CREBBP) and KAT3B (EP300) are essential enzymes in gene regulation in the nucleus. Their ubiquitous expression in metazoan cell types controls cell proliferation and differentiation during development. This comprehensive review delves into the biological roles of KAT3A and KAT3B in neurodevelopment, shedding light on how alterations in their regulation or activity can potentially contribute to a spectrum of neurodegenerative diseases (e.g., Huntington's and Alzheimer's). We explore the pathophysiological implications of KAT3 function loss in these disorders, considering their conserved protein domains and biochemical functions in chromatin regulation. The discussion also underscores the crucial role of KAT3 proteins and their substrates in supporting the integration of key cell signaling pathways. Furthermore, the narrative highlights the interdependence of KAT3-mediated lysine acetylation with lysine methylation and arginine methylation. From a cellular perspective, KAT3-dependent signal integration at subnuclear domains is mediated by liquid-liquid phase separation in response to KAT3-mediated lysine acetylation. The disruption of these finely tuned regulatory processes underscores their pathological roles in neurodegeneration. This review also points to the exciting potential for future research in this field, inspiring further investigation and discovery in the area of neurodevelopment and neurodegenerative diseases.

Comparative analysis of vision transformers and convolutional neural networks in osteoporosis detection from X-ray images.

Within the scope of this investigation, we carried out experiments to investigate the potential of the Vision Transformer (ViT) in the field of medical image analysis. The diagnosis of osteoporosis through inspection of X-ray radio-images is a substantial classification problem that we were able to address with the assistance of Vision Transformer models. In order to provide a basis for comparison, we conducted a parallel analysis in which we sought to solve the same problem by employing traditional convolutional neural networks (CNNs), which are well-known and commonly used techniques for the solution of image categorization issues. The findings of our research led us to conclude that ViT is capable of achieving superior outcomes compared to CNN. Furthermore, provided that methods have access to a sufficient quantity of training data, the probability increases that both methods arrive at more appropriate solutions to critical issues.

Cancer Cell-Type-Dependent Modifications of Metastatic Parameters by SLIT2-ROBO1 and RHOA cAMP Signaling in Response to TGFß1 and FGF2.

The epithelial to mesenchymal transition (EMT) is a multistep process involving structural and functional alterations that are required for cancer metastasis, as well as loss of epithelial markers (e.g., E-cadherin/CDH1) and gain of mesenchymal markers (e.g., N-cadherin/CDH2, vimentin/VIM). Pathological events modify cell-cell interactions, cell-matrix adhesion and extra cellular matrix integrity leading to cell migration, evasion from the primary tumor and augmented invasiveness in the metastatic niche. This transformation is modulated by multiple paracrine factors (e.g., chemokines, growth factor), as well as SLIT2-ROBO1 signaling that collectively regulate expression of RHO GTPases (e.g., RHOA) and EMT marker genes. Yet, the roles of SLIT proteins in cancer remain enigmatic. In some cancer types, SLIT2 is anti-tumorigenic, while in other cancers it contributes towards the metastatic phenotype. Here we investigated the ambivalent metastatic activity of SLIT2 by analyzing how cAMP/RHOA signal transduction modulates SLIT-ROBO controlled metastatic parameters in response to the phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) and paracrine factors (TGF-ß/TGFß1 and FGF2). Upon SLIT2 administration cell migration and proliferation increases in colon cancer cells and decreases in cervical cancer cells, while altering cell morphology and proliferation in both cancer types. These effects are reinforced by TGF-ß/TGFß1 and FGF2, but attenuated by elevation of cAMP with IBMX, depending on the cancer cell type. Our data indicate that SLIT2 represents a potential biomarker for cancer diagnosis, prognosis, and therapy.

Inhibition of EZH2 Reduces Aging-Related Decline in Interstitial Cells of Cajal of the Mouse Stomach.

BACKGROUND & AIMS: Restricted gastric motor functions contribute to aging-associated undernutrition, sarcopenia, and frailty. We previously identified a decline in interstitial cells of Cajal (ICC; gastrointestinal pacemaker and neuromodulator cells) and their stem cells (ICC-SC) as a key factor of gastric aging. Altered functionality of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) is central to organismal aging. Here, we investigated the role of EZH2 in the aging-related loss of ICC/ICC-SC. METHODS: klotho mice, a model of accelerated aging, were treated with the most clinically advanced EZH2 inhibitor, EPZ6438 (tazemetostat; 160 mg/kg intraperitoneally twice a day for 3 weeks). Gastric ICC were analyzed by Western blotting and immunohistochemistry. ICC and ICC-SC were quantified by flow cytometry. Gastric slow wave activity was assessed by intracellular electrophysiology. Ezh2 was deactivated in ICC by treating KitcreERT2/+;Ezh2fl/fl mice with tamoxifen. TRP53, a key mediator of aging-related ICC loss, was induced with nutlin 3a in gastric muscle organotypic cultures and an ICC-SC line. RESULTS: In klotho mice, EPZ6438 treatment mitigated the decline in the ICC growth factor KIT ligand/stem cell factor and gastric ICC. EPZ6438 also improved gastric slow wave activity and mitigated the reduced food intake and impaired body weight gain characteristic of this strain. Conditional genomic deletion of Ezh2 in Kit-expressing cells also prevented ICC loss. In organotypic cultures and ICC-SC, EZH2 inhibition prevented the aging-like effects of TRP53 stabilization on ICC/ICC-SC. CONCLUSIONS: Inhibition of EZH2 with EPZ6438 mitigates aging-related ICC/ICC-SC loss and gastric motor dysfunction, improving slow wave activity and food intake in klotho mice.

Transcriptomic responses of peripheral blood leukocytes to cardiac surgery after acute inflammation, and three months recovery.

Traumatic perioperative conditions may trigger early systemic responses, activate leukocytes and reprogram the immune system. We hypothesize that leukocyte activation may not revert to pre-surgical states, and that protracted activation may emerge with increased risks of comorbidities. We tested this concept by examining the transcriptomes of monocytes and T cells in a representative observational cohort of patients (n = 13) admitted for elective cardiac surgery. Transcriptomes in T cells and monocytes were compared from before surgery (t0), and monocytes were analyzed longitudinally after acute (t24hr), and convalescent (t3m) time points. Monocytes and T cells expressed distinct transcriptomes, reflected by statistically significant differential expression of 558 T cell related genes. Monocytes expressed genes related to protein degradation and presented atypical activation of surface markers and cytoplasmic functions over time. Additionally, monocytes exhibited limited transcriptomic heterogeneity prior to surgery, and long-term patterns of gene expression associated with atherosclerosis showed three temporally distinct signatures. These data establish that post-cardiac surgery transcriptomes of monocytes differ even at three months compared to baselines, which may reflect latent ('smoldering') inflammation and persistent progression of tissue degenerative processes that should inform clinical care.

Inflammatory Markers Involved in the Pathogenesis of Dupuytren's Contracture.

Dupuytren's disease is a common fibroproliferative disease that can result in debilitating hand deformities. Partial correction and return of deformity are common with surgical or clinical treatments at present. While current treatments are limited to local procedures for relatively late effects of the disease, the pathophysiology of this connective tissue disorder is associated with both local and systemic processes (e.g., fibrosis, inflammation). Hence, a better understanding of the systemic circulation of Dupuytren related cytokines and growth factors may provide important insights into disease progression. In addition, systemic biomarker analysis could yield new concepts for treatments of Dupuytren that attenuate circulatory factors (e.g., anti-inflammatory agents, neutralizing antibodies). Progress in the development of any disease modifying biologic treatment for Dupuytren has been hampered by the lack of clinically useful biomarkers. The characterization of nonsurgical Dupuytren biomarkers will permit disease staging from diagnostic and prognostic perspectives, as well as allows evaluation of biologic responses to treatment. Identification of such markers may transcend their use in Dupuytren treatment, because fibrotic biological processes fundamental to Dupuytren are relevant to fibrosis in many other connective tissues and organs with collagen-based tissue compartments. There is a wide range of potential Dupuytren biomarker categories that could be informative, including disease determinants linked to genetics, collagen metabolism, as well as immunity and inflammation (e.g., cytokines, chemokines). This narrative review provides a broad overview of previous studies and emphasizes the importance of inflammatory mediators as candidate circulating biomarkers for monitoring Dupuytren's disease.

Fundamentals and Translational Applications of Stem Cells and Biomaterials in Dental, Oral and Craniofacial Regenerative Medicine.

Regenerative dental medicine continuously expands to improve treatments for prevalent clinical problems in dental and oral medicine. Stem cell based translational opportunities include regenerative therapies for tooth restoration, root canal therapy, and inflammatory processes (e.g., periodontitis). The potential of regenerative approaches relies on the biological properties of dental stem cells. These and other multipotent somatic mesenchymal stem cell (MSC) types can in principle be applied as either autologous or allogeneic sources in dental procedures. Dental stem cells have distinct developmental origins and biological markers that determine their translational utility. Dental regenerative medicine is supported by mechanistic knowledge of the molecular pathways that regulate dental stem cell growth and differentiation. Cell fate determination and lineage progression of dental stem cells is regulated by multiple cell signaling pathways (e.g., WNTs, BMPs) and epigenetic mechanisms, including DNA modifications, histone modifications, and non-coding RNAs (e.g., miRNAs and lncRNAs). This review also considers a broad range of novel approaches in which stem cells are applied in combination with biopolymers, ceramics, and composite materials, as well as small molecules (agonistic or anti-agonistic ligands) and natural compounds. Materials that mimic the microenvironment of the stem cell niche are also presented. Promising concepts in bone and dental tissue engineering continue to drive innovation in dental and non-dental restorative procedures.

Natural selection and evolution: evolving concepts.

Many recent studies in evolutionary biology have expanded and refined definitions of biological evolution and natural selection. Current evolutionary models incorporate different adaptive and non-adaptive processes based on molecular genetic changes and how DNA is modified over time in unicellular species, or in germline versus somatic cells in metazoan species. Cogent arguments can be raised for the view that natural selection should be considered a biological law, consistent with quantitative mathematical equations that describe the fitness of individuals, as well as variations within and among populations. Evolution is an overarching framework that incorporates the laws of natural selection and clarifies why phenotypic variation can increase in prevalence and result in species adaptations. The conceptual framework for biological evolution incorporates many cohesive principles that collectively have a predictive value. This framework will continue to evolve with improvements in high-resolution technologies that enable us to examine both adaptive and non-adaptive changes that drive biological phenotypes.

Obesity-driven mitochondrial dysfunction in human adipose tissue-derived mesenchymal stem/stromal cells involves epigenetic changes.

Obesity exacerbates tissue degeneration and compromises the integrity and reparative potential of mesenchymal stem/stromal cells (MSCs), but the underlying mechanisms have not been sufficiently elucidated. Mitochondria modulate the viability, plasticity, proliferative capacity, and differentiation potential of MSCs. We hypothesized that alterations in the 5-hydroxymethylcytosine (5hmC) profile of mitochondria-related genes may mediate obesity-driven dysfunction of human adipose-derived MSCs. MSCs were harvested from abdominal subcutaneous fat of obese and age/sex-matched non-obese subjects (n = 5 each). The 5hmC profile and expression of nuclear-encoded mitochondrial genes were examined by hydroxymethylated DNA immunoprecipitation sequencing (h MeDIP-seq) and mRNA-seq, respectively. MSC mitochondrial structure (electron microscopy) and function, metabolomics, proliferation, and neurogenic differentiation were evaluated in vitro, before and after epigenetic modulation. hMeDIP-seq identified 99 peaks of hyper-hydroxymethylation and 150 peaks of hypo-hydroxymethylation in nuclear-encoded mitochondrial genes from Obese- versus Non-obese-MSCs. Integrated hMeDIP-seq/mRNA-seq analysis identified a select group of overlapping (altered levels of both 5hmC and mRNA) nuclear-encoded mitochondrial genes involved in ATP production, redox activity, cell proliferation, migration, fatty acid metabolism, and neuronal development. Furthermore, Obese-MSCs exhibited decreased mitochondrial matrix density, membrane potential, and levels of fatty acid metabolites, increased superoxide production, and impaired neuronal differentiation, which improved with epigenetic modulation. Obesity elicits epigenetic changes in mitochondria-related genes in human adipose-derived MSCs, accompanied by structural and functional changes in their mitochondria and impaired fatty acid metabolism and neurogenic differentiation capacity. These observations may assist in developing novel therapies to preserve the potential of MSCs for tissue repair and regeneration in obese individuals.

CpG methylation changes in human mesenchymal and neural stem cells in response to in vitro niche modifications.

Stem cell therapies hold promise in addressing the burden of neurodegenerative diseases with human embryonic neural stem cells (hNSC-H9s) and bone marrow-derived human mesenchymal stem cells (hMSCs) as viable candidates. The induction of hMSC neurospheres (hMSC-IN) generate a more lineage-restricted common neural progenitor-like cell population, potentially tunable by heparan sulfate proteoglycans (HSPGs). We examined CpG (5 mC) site methylation patterns using Illumina Infinium 850 K EPIC arrays in hNSC-H9, hMSCs and hMSC-IN cultures with HSPG agonist heparin at early and late phases of growth. We identified key regulatory CpG sites in syndecans (SDC2; SDC4) that potentially regulate gene expression in monolayers. Unique hMSC-IN hypomethylation in glypicans (GPC3; GPC4) underscore their significance in neural lineages with Sulfatase 1 and 2 (SULF1 &2) CpG methylation changes potentially driving the neurogenic shift. hMSC-INs methylation levels at SULF1 CpG sites and SULF2:cg25401628 were more closely aligned with hNSC-H9 cells than with hMSCs. We further suggest SOX2 regulation governed by lncSOX2-Overall Transcript (lncSOX2-OT) methylation changes with preferential activation of ENO2 over other neuronal markers within hMSC-INs. Our findings illuminate epigenetic dynamics governing neural lineage commitment of hMSC-INs offering insights for targeted mechanisms for regenerative medicine and therapeutic strategies.

Cellular Enhancement of Frozen Meniscus Allograft Combining Native Meniscus and Mesenchymal Stromal Cell Injections.

OBJECTIVE: This proof-of-concept study investigated an improved cell-based injection therapy combining mesenchymal stem cells (MSCs) and meniscus cells (MCs) to support superior meniscus allograft repopulation and early revival compared to injecting MSCs alone. DESIGN: In this controlled laboratory study, frozen meniscus allograft samples were injected vertically with a cell suspension containing different ratios of MSCs and MCs or control (lactated ringers) and cultured for 28 days. Samples were analyzed weekly for cell viability, migration, and metabolism using histological and biochemical assays. Tissue medium was analyzed for matrix metalloproteinase (MMP) expression using zymography. RESULTS: Cellular repopulation of frozen allografts injected with different cell suspensions was validated by immunohistochemistry. Significant higher DNA content was evidenced in grafts treated with suspensions of MCs or MC:MSC (1:4 ratio). Cell metabolic activity was significantly different between all treated groups and control group after 1 week. Allografts injected with MCs showed significantly more cell proliferation than injections with MSCs. MMP2 activity was detected in medium of all grafts cellularized with MCs with or without MSCs. Scanning electron microscopy (SEM) analysis showed resolution of the needle puncture, but not in the control group. Cell labeling of MCs upon injection of mixed MC:MSC suspensions revealed a gradual increase in the cell ratio. CONCLUSIONS: The findings of this study establish that injection of MCs with or without MSCs enhances the cellularity of meniscus allograft to support early graft revival and remodeling.

Epigenetic regulators controlling osteogenic lineage commitment and bone formation.

Bone formation and homeostasis are controlled by environmental factors and endocrine regulatory cues that initiate intracellular signaling pathways capable of modulating gene expression in the nucleus. Bone-related gene expression is controlled by nucleosome-based chromatin architecture that limits the accessibility of lineage-specific gene regulatory DNA sequences and sequence-specific transcription factors. From a developmental perspective, bone-specific gene expression must be suppressed during the early stages of embryogenesis to prevent the premature mineralization of skeletal elements during fetal growth in utero. Hence, bone formation is initially inhibited by gene suppressive epigenetic regulators, while other epigenetic regulators actively support osteoblast differentiation. Prominent epigenetic regulators that stimulate or attenuate osteogenesis include lysine methyl transferases (e.g., EZH2, SMYD2, SUV420H2), lysine deacetylases (e.g., HDAC1, HDAC3, HDAC4, HDAC7, SIRT1, SIRT3), arginine methyl transferases (e.g., PRMT1, PRMT4/CARM1, PRMT5), dioxygenases (e.g., TET2), bromodomain proteins (e.g., BRD2, BRD4) and chromodomain proteins (e.g., CBX1, CBX2, CBX5). This narrative review provides a broad overview of the covalent modifications of DNA and histone proteins that involve hundreds of enzymes that add, read, or delete these epigenetic modifications that are relevant for self-renewal and differentiation of mesenchymal stem cells, skeletal stem cells and osteoblasts during osteogenesis.

Nanoparticle-based inhibition of vascular endothelial growth factor receptors alleviates osteoarthritis pain and cartilage damage.

Osteoarthritis (OA) is characterized by cartilage damage, inflammation, and pain. Vascular endothelial growth factor receptors (VEGFRs) have been associated with OA severity, suggesting that inhibitors targeting these receptors alleviate pain (via VEGFR1) or cartilage degeneration (via VEGFR2). We have developed a nanoparticle-based formulation of pazopanib (Votrient), an FDA-approved anticancer drug that targets both VEGFR1 and VEGFR2 (Nano-PAZII). We demonstrate that a single intraarticular injection of Nano-PAZII can effectively reduce joint pain for a prolonged time without substantial side effects in two different preclinical OA rodent models involving either surgical (upon partial medial meniscectomy) or nonsurgical induction (with monoiodoacetate). The injection of Nano-PAZII blocks VEGFR1 and relieves OA pain by suppressing sensory neuronal ingrowth into the knee synovium and neuronal plasticity in the dorsal root ganglia and spinal cord. Simultaneously, the inhibition of VEGFR2 reduces cartilage degeneration. These findings provide a mechanism-based disease-modifying drug strategy that addresses both pain symptoms and cartilage loss in OA.

Osmolarity-Induced Altered Intracellular Molecular Crowding Drives Osteoarthritis Pathology.

Osteoarthritis (OA) is a multifactorial degenerative joint disease of which the underlying mechanisms are yet to be fully understood. At the molecular level, multiple factors including altered signaling pathways, epigenetics, metabolic imbalance, extracellular matrix degradation, production of matrix metalloproteinases, and inflammatory cytokines, are known to play a detrimental role in OA. However, these factors do not initiate OA, but are mediators or consequences of the disease, while many other factors causing the etiology of OA are still unknown. Here, it is revealed that microenvironmental osmolarity can induce and reverse osteoarthritis-related behavior of chondrocytes via altered intracellular molecular crowding, which represents a previously unknown mechanism underlying OA pathophysiology. Decreased intracellular crowding is associated with increased sensitivity to proinflammatory triggers and decreased responsiveness to anabolic stimuli. OA-induced lowered intracellular molecular crowding could be renormalized via exposure to higher extracellular osmolarity such as those found in healthy joints, which reverse OA chondrocyte's sensitivity to catabolic stimuli as well as its glycolytic metabolism.

Serotonin Transporter (5-Hydroxytryptamine Transporter, SERT, SLC6A4) and Sodium-dependent Reuptake Inhibitors as Modulators of Pain Behaviors and Analgesic Responses.

The serotonin transporter (5-hydroxytryptamine transporter [5-HTT], Serotonin Transporter (SERT), SLC6A4) modulates the activity of serotonin via sodium-dependent reuptake. Given the established importance of serotonin in the control of pain, 5-HTT has received much interest in studies of pain states and as a pharmacological target for serotonin reuptake inhibitors (SRIs). Animal models expressing varying levels of 5-HTT activity show marked differences in pain behaviors and analgesic responses, as well as many serotonin-related physiological effects. In humans, functional nucleotide variations in the SLC6A4 gene, which encodes the serotonin transporter 5-HTT, are associated with certain pathologic pain conditions and differences in responses to pharmacological therapy. These findings collectively reflect the importance of 5-HTT in the intricate physiology and management of pain, as well as the scientific and clinical challenges that need to be considered for the optimization of 5-HTT-related analgesic therapies. PERSPECTIVE: The serotonin transporter 5-HTT/SCL6A4 is sensitive to pharmacological SRIs. Experimental studies on the physiological functions of serotonin, as well as genetic mouse models and clinical phenotype/genotype correlations of nucleotide variation in the human 5-HTT/SCL6A4 gene, provide new insights for the use of SRIs in chronic pain management.

Loss of PKCδ/Prkcd prevents cartilage degeneration in joints but exacerbates hyperalgesia in an experimental osteoarthritis mouse model.

Pain is the prime symptom of osteoarthritis (OA) that directly affects the quality of life. Protein kinase Cδ (PKCδ/Prkcd) plays a critical role in OA pathogenesis; however, its significance in OA-related pain is not entirely understood. The present study investigated the functional role of PKCδ in OA pain sensation. OA was surgically induced in control (Prkcdfl/fl), global- (Prkcdfl/fl; ROSACreERT2), and sensory neuron-specific conditional knockout (cKO) mice (Prkcdfl/fl; NaV1.8/Scn10aCreERT2) followed by comprehensive analysis of longitudinal behavioral pain, histopathology and immunofluorescence studies. GlobalPrkcd cKO mice prevented cartilage deterioration by inhibiting matrix metalloproteinase-13 (MMP13) in joint tissues but significantly increased OA pain. Sensory neuron-specificdeletion of Prkcd in mice did not protect cartilage from degeneration but worsened OA-associated pain. Exacerbated pain sensitivity observed in global- and sensory neuron-specific cKO of Prkcd was corroborated with markedly increased specific pain mediators in knee synovium and dorsal root ganglia (DRG). These specific pain markers include nerve growth factor (NGF) and vascular endothelial growth factor (VEGF), and their cognate receptors, including tropomyosin receptor kinase A (TrkA) and vascular endothelial growth factor receptor-1 (VEGFR1). The increased levels of NGF/TrkA and VEGF/VEGFR1 were comparable in both global- and sensory neuron-specific cKO groups. These data suggest that the absence of Prkcd gene expression in the sensory neurons is strongly associated with OA hyperalgesia independent of cartilage protection. Thus, inhibition of PKCδ may be beneficial for cartilage homeostasis but could aggravate OA-related pain symptoms.

AdipoRon reduces TGFß1-mediated collagen deposition in vitro and alleviates knee stiffness in vivo.

Arthrofibrosis, which causes joint motion restrictions, is a common complication following total knee arthroplasty (TKA). Key features associated with arthrofibrosis include myofibroblast activation, knee stiffness, and excessive scar tissue formation. We previously demonstrated that adiponectin levels are suppressed within the knee tissues of patients affected by arthrofibrosis and showed that AdipoRon, an adiponectin receptor agonist, exhibited anti-fibrotic properties in human mesenchymal stem cells. In this study, the therapeutic potential of AdipoRon was evaluated on TGFß1-mediated myofibroblast differentiation of primary human knee fibroblasts and in a mouse model of knee stiffness. Picrosirius red staining revealed that AdipoRon reduced TGFß1-induced collagen deposition in primary knee fibroblasts derived from patients undergoing primary TKA and revision TKA for arthrofibrosis. AdipoRon also reduced mRNA and protein levels of ACTA2, a key myofibroblast marker. RNA-seq analysis corroborated the anti-myofibrogenic effects of AdipoRon. In our knee stiffness mouse model, 6 weeks of knee immobilization, to induce a knee contracture, in conjunction with daily vehicle (DMSO) or AdipoRon (1, 5, and 25 mg/kg) via intraperitoneal injections were well tolerated based on animal behavior and weight measurements. Biomechanical testing demonstrated that passive extension angles (PEAs) of experimental knees were similar between vehicle and AdipoRon treatment groups in mice evaluated immediately following immobilization. Interestingly, relative to vehicle-treated mice, 5 mg/kg AdipoRon therapy improved the PEA of the experimental knees in mice that underwent 4 weeks of knee remobilization following the immobilization and therapy. Together, these studies revealed that AdipoRon may be an effective therapeutic modality for arthrofibrosis.

LINC01638 sustains human mesenchymal stem cell self-renewal and competency for osteogenic cell fate.

The skeleton forms from multipotent human mesenchymal stem cells (hMSCs) competent to commit to specific lineages. Long noncoding RNAs (lncRNAs) have been identified as key epigenetic regulators of tissue development. However, regulation of osteogenesis by lncRNAs as mediators of commitment to the bone phenotype is largely unexplored. We focused on LINC01638, which is highly expressed in hMSCs and has been studied in cancers, but not in regulating osteogenesis. We demonstrated that LINC01638 promotes initiation of the osteoblast phenotype. Our findings reveal that LINC01638 is present at low levels during the induction of osteoblast differentiation. CRISPRi knockdown of LINC01638 in MSCs prevents osteogenesis and alkaline phosphatase expression, inhibiting osteoblast differentiation. This resulted in decreased MSC growth rate, accompanied by double-strand breaks, DNA damage, and cell senescence. Transcriptome profiling of control and LINC01638-depleted hMSCs identified > 2000 differentially expressed mRNAs related to cell cycle, cell division, spindle formation, DNA repair, and osteogenesis. Using ChIRP-qPCR, molecular mechanisms of chromatin interactions revealed the LINC01638 locus (Chr 22) includes many lncRNAs and bone-related genes. These novel findings identify the obligatory role for LINC01638 to sustain MSC pluripotency regulating osteoblast commitment and growth, as well as for physiological remodeling of bone tissue.