Membrane stretch as the mechanism of activation of PIEZO1 ion channels in chondrocytes.

Osteoarthritis is a chronic disease that can be initiated by altered joint loading or injury of the cartilage. The mechanically sensitive PIEZO ion channels have been shown to transduce injurious levels of biomechanical strain in articular chondrocytes and mediate cell death. However, the mechanisms of channel gating in response to high cellular deformation and the strain thresholds for activating PIEZO channels remain unclear. We coupled studies of single-cell compression using atomic force microscopy (AFM) with finite element modeling (FEM) to identify the biophysical mechanisms of PIEZO-mediated calcium (Ca2+) signaling in chondrocytes. We showed that PIEZO1 and PIEZO2 are needed for initiating Ca2+ signaling at moderately high levels of cellular deformation, but at the highest strains, PIEZO1 functions independently of PIEZO2. Biophysical factors that increase apparent chondrocyte membrane tension, including hypoosmotic prestrain, high compression magnitudes, and low deformation rates, also increased PIEZO1-driven Ca2+ signaling. Combined AFM/FEM studies showed that 50% of chondrocytes exhibit Ca2+ signaling at 80 to 85% nominal cell compression, corresponding to a threshold of apparent membrane finite principal strain of E = 1.31, which represents a membrane stretch ratio (λ) of 1.9. Both intracellular and extracellular Ca2+ are necessary for the PIEZO1-mediated Ca2+ signaling response to compression. Our results suggest that PIEZO1-induced signaling drives chondrocyte mechanical injury due to high membrane tension, and this threshold can be altered by factors that influence membrane prestress, such as cartilage hypoosmolarity, secondary to proteoglycan loss. These findings suggest that modulating PIEZO1 activation or downstream signaling may offer avenues for the prevention or treatment of osteoarthritis.

Mechanobiology of Hyaluronan: Connecting Biomechanics and Bioactivity in Musculoskeletal Tissues.

Hyaluronan (HA) plays well-recognized mechanical and biological roles in articular cartilage and synovial fluid, where it contributes to tissue structure and lubrication. An understanding of how HA contributes to the structure of other musculoskeletal tissues, including muscle, bone, tendon, and intervertebral discs, is growing. In addition, the use of HA-based therapies to restore damaged tissue is becoming more prevalent. Nevertheless, the relationship between biomechanical stimuli and HA synthesis, degradation, and signaling in musculoskeletal tissues remains understudied, limiting the utility of HA in regenerative medicine. In this review, we discuss the various roles and significance of endogenous HA in musculoskeletal tissues. We use what is known and unknown to motivate new lines of inquiry into HA biology within musculoskeletal tissues and in the mechanobiology governing HA metabolism by suggesting questions that remain regarding the relationship and interaction between biological and mechanical roles of HA in musculoskeletal health and disease.

The chondrocyte "mechanome": Activation of the mechanosensitive ion channels TRPV4 and PIEZO1 drives unique transcriptional signatures.

The mechanosensitive ion channels Transient Receptor Potential Vanilloid 4 (TRPV4) and PIEZO1 transduce physiologic and supraphysiologic magnitudes of mechanical signals in the chondrocyte, respectively. TRPV4 activation promotes chondrogenesis, while PIEZO1 activation by supraphysiologic deformations drives cell death. The mechanisms by which activation of these channels discretely drives changes in gene expression to alter cell behavior remain to be determined. To date, no studies have contrasted the transcriptomic response to activation of these channels nor has any published data attempted to correlate these transcriptomes to alterations in cellular function. This study used RNA sequencing to comprehensively investigate the transcriptomes associated with activation of TRPV4 or PIEZO1, revealing that TRPV4 and PIEZO drive distinct transcriptomes and also exhibit unique co-regulated clusters of genes. Notably, activation of PIEZO1 through supraphysiologic deformation induced a transient inflammatory profile that overlapped with the interleukin (IL)-1-responsive transcriptome and contained genes associated with cartilage degradation and osteoarthritis progression. However, both TRPV4 and PIEZO1 were also shown to elicit anabolic effects. PIEZO1 expression promoted a pro-chondrogenic transcriptome under unloaded conditions, and daily treatment with PIEZO1 agonist Yoda1 significantly increased sulfated glycosaminoglycan deposition in vitro. These findings emphasize the presence of a broad "mechanome" with distinct effects of TRPV4 and PIEZO1 activation in chondrocytes, suggesting complex roles for PIEZO1 in both the physiologic and pathologic responses of chondrocytes. The identification of transcriptomic profiles unique to or shared by PIEZO1 and TRPV4 (distinct from IL-1-induced inflammation) could inform future therapeutic designs targeting these channels for the management and treatment of osteoarthritis.

Modulation of TRPV4 protects against degeneration induced by sustained loading and promotes matrix synthesis in the intervertebral disc.

While it is well known that mechanical signals can either promote or disrupt intervertebral disc (IVD) homeostasis, the molecular mechanisms for transducing mechanical stimuli are not fully understood. The transient receptor potential vanilloid 4 (TRPV4) ion channel activated in isolated IVD cells initiates extracellular matrix (ECM) gene expression, while TRPV4 ablation reduces cytokine production in response to circumferential stretching. However, the role of TRPV4 on ECM maintenance during tissue-level mechanical loading remains unknown. Using an organ culture model, we modulated TRPV4 function over both short- (hours) and long-term (days) and evaluated the IVDs' response. Activating TRPV4 with the agonist GSK101 resulted in a Ca2+ flux propagating across the cells within the IVD. Nuclear factor (NF)-κB signaling in the IVD peaked at 6 h following TRPV4 activation that subsequently resulted in higher interleukin (IL)-6 production at 7 days. These cellular responses were concomitant with the accumulation of glycosaminoglycans and increased hydration in the nucleus pulposus that culminated in higher stiffness of the IVD. Sustained compressive loading of the IVD resulted in elevated NF-κB activity, IL-6 and vascular endothelial growth factor A (VEGFA) production, and degenerative changes to the ECM. TRPV4 inhibition using GSK205 during loading mitigated the changes in inflammatory cytokines, protected against IVD degeneration, but could not prevent ECM disorganization due to mechanical damage in the annulus fibrosus. These results indicate TRPV4 plays an important role in both short- and long-term adaptations of the IVD to mechanical loading. The modulation of TRPV4 may be a possible therapeutic for preventing load-induced IVD degeneration.

Aging and obesity prime the methylome and transcriptome of adipose stem cells for disease and dysfunction.

The epigenome of stem cells occupies a critical interface between genes and environment, serving to regulate expression through modification by intrinsic and extrinsic factors. We hypothesized that aging and obesity, which represent major risk factors for a variety of diseases, synergistically modify the epigenome of adult adipose stem cells (ASCs). Using integrated RNA- and targeted bisulfite-sequencing in murine ASCs from lean and obese mice at 5- and 12-months of age, we identified global DNA hypomethylation with either aging or obesity, and a synergistic effect of aging combined with obesity. The transcriptome of ASCs in lean mice was relatively stable to the effects of age, but this was not true in obese mice. Functional pathway analyses identified a subset of genes with critical roles in progenitors and in diseases of obesity and aging. Specifically, Mapt, Nr3c2, App, and Ctnnb1 emerged as potential hypomethylated upstream regulators in both aging and obesity (AL vs. YL and AO vs. YO), and App, Ctnnb1, Hipk2, Id2, and Tp53 exhibited additional effects of aging in obese animals. Furthermore, Foxo3 and Ccnd1 were potential hypermethylated upstream regulators of healthy aging (AL vs. YL), and of the effects of obesity in young animals (YO vs. YL), suggesting that these factors could play a role in accelerated aging with obesity. Finally, we identified candidate driver genes that appeared recurrently in all analyses and comparisons undertaken. Further mechanistic studies are needed to validate the roles of these genes capable of priming ASCs for dysfunction in aging- and obesity-associated pathologies.

Inflammatory signaling sensitizes Piezo1 mechanotransduction in articular chondrocytes as a pathogenic feed-forward mechanism in osteoarthritis.

Osteoarthritis (OA) is a painful and debilitating condition of synovial joints without any disease-modifying therapies [A. M. Valdes, T. D. Spector, Nat. Rev. Rheumatol. 7, 23-32 (2011)]. We previously identified mechanosensitive PIEZO channels, PIEZO1 and PIEZO2, both expressed in articular cartilage, to function in chondrocyte mechanotransduction in response to injury [W. Lee et al., Proc. Natl. Acad. Sci. U.S.A. 111, E5114-E5122 (2014); W. Lee, F. Guilak, W. Liedtke, Curr. Top. Membr. 79, 263-273 (2017)]. We therefore asked whether interleukin-1-mediated inflammatory signaling, as occurs in OA, influences Piezo gene expression and channel function, thus indicative of maladaptive reprogramming that can be rationally targeted. Primary porcine chondrocyte culture and human osteoarthritic cartilage tissue were studied. We found that interleukin-1α (IL-1α) up-regulated Piezo1 in porcine chondrocytes. Piezo1 expression was significantly increased in human osteoarthritic cartilage. Increased Piezo1 expression in chondrocytes resulted in a feed-forward pathomechanism whereby increased function of Piezo1 induced excess intracellular Ca2+ at baseline and in response to mechanical deformation. Elevated resting state Ca2+ in turn rarefied the F-actin cytoskeleton and amplified mechanically induced deformation microtrauma. As intracellular substrates of this OA-related inflammatory pathomechanism, in porcine articular chondrocytes exposed to IL-1α, we discovered that enhanced Piezo1 expression depended on p38 MAP-kinase and transcription factors HNF4 and ATF2/CREBP1. CREBP1 directly bound to the proximal PIEZO1 gene promoter. Taken together, these signaling and genetic reprogramming events represent a detrimental Ca2+-driven feed-forward mechanism that can be rationally targeted to stem the progression of OA.

Adipose tissue is a critical regulator of osteoarthritis.

Osteoarthritis (OA), the leading cause of pain and disability worldwide, disproportionally affects individuals with obesity. The mechanisms by which obesity leads to the onset and progression of OA are unclear due to the complex interactions among the metabolic, biomechanical, and inflammatory factors that accompany increased adiposity. We used a murine preclinical model of lipodystrophy (LD) to examine the direct contribution of adipose tissue to OA. Knee joints of LD mice were protected from spontaneous or posttraumatic OA, on either a chow or high-fat diet, despite similar body weight and the presence of systemic inflammation. These findings indicate that adipose tissue itself plays a critical role in the pathophysiology of OA. Susceptibility to posttraumatic OA was reintroduced into LD mice using implantation of a small adipose tissue depot derived from wild-type animals or mouse embryonic fibroblasts that undergo spontaneous adipogenesis, implicating paracrine signaling from fat, rather than body weight, as a mediator of joint degeneration.

The role of PIEZO ion channels in the musculoskeletal system.

PIEZO1 and PIEZO2 are mechanosensitive cation channels that are highly expressed in numerous tissues throughout the body and exhibit diverse, cell-specific functions in multiple organ systems. Within the musculoskeletal system, PIEZO1 functions to maintain muscle and bone mass, sense tendon stretch, and regulate senescence and apoptosis in response to mechanical stimuli within cartilage and the intervertebral disc. PIEZO2 is essential for transducing pain and touch sensations as well as proprioception in the nervous system, which can affect musculoskeletal health. PIEZO1 and PIEZO2 have been shown to act both independently as well as synergistically in different cell types. Conditions that alter PIEZO channel mechanosensitivity, such as inflammation or genetic mutations, can have drastic effects on these functions. For this reason, therapeutic approaches for PIEZO-related disease focus on altering PIEZO1 and/or PIEZO2 activity in a controlled manner, either through inhibition with small molecules, or through dietary control and supplementation to maintain a healthy cell membrane composition. Although many opportunities to better understand PIEZO1 and PIEZO2 remain, the studies summarized in this review highlight how crucial PIEZO channels are to musculoskeletal health and point to promising possible avenues for their modulation as a therapeutic target.

Leptin mediates the regulation of muscle mass and strength by adipose tissue.

Adipose tissue secretes numerous cytokines (termed 'adipokines') that have known or hypothesized actions on skeletal muscle. The majority of adipokines have been implicated in the pathological link between excess adipose and muscle insulin resistance, but approximately half also have documented in vitro effects on myogenesis and/or hypertrophy. This complexity suggests a potential dual role for adipokines in the regulation of muscle mass in homeostasis and the development of pathology. In this study, we used lipodystrophic 'fat-free' mice to demonstrate that adipose tissue is indeed necessary for the development of normal muscle mass and strength. Fat-free mice had significantly reduced mass (∼15%) and peak contractile tension (∼20%) of fast-twitch muscles, a slowing of contractile dynamics and decreased cross-sectional area of fast twitch fibres compared to wild-type littermates. These deficits in mass and contractile tension were fully rescued by reconstitution of ∼10% of normal adipose mass, indicating that this phenotype is the direct consequence of absent adipose. We then showed that the rescue is solely mediated by the adipokine leptin, as similar reconstitution of adipose from leptin-knockout mice fails to rescue mass or strength. Together, these data indicate that the development of muscle mass and strength in wild-type mice is dependent on adipose-secreted leptin. This finding extends our current understanding of the multiple roles of adipokines in physiology as well as disease pathophysiology to include a critical role for the adipokine leptin in muscle homeostasis. KEY POINTS: Adipose-derived cytokines (adipokines) have long been implicated in the pathogenesis of insulin resistance in obesity but likely have other under-appreciated roles in muscle physiology. Here we use a fat-free mouse to show that adipose tissue is necessary for the normal development of muscle mass and strength. Through add-back of genetically modified adipose tissue we show that leptin is the key adipokine mediating this regulation. This expands our understanding of leptin's role in adipose-muscle signalling to include development and homeostasis and adds the surprising finding that leptin is the sole mediator of the maintenance of muscle mass and strength by adipose tissue.

Transient receptor potential vanilloid 4 as a regulator of induced pluripotent stem cell chondrogenesis.

Transient receptor potential vanilloid 4 (TRPV4) is a polymodal calcium-permeable cation channel that is highly expressed in cartilage and is sensitive to a variety of extracellular stimuli. The expression of this channel has been associated with the process of chondrogenesis in adult stem cells as well as several cell lines. Here, we used a chondrogenic reporter (Col2a1-GFP) in murine induced pluripotent stem cells (iPSCs) to examine the hypothesis that TRPV4 serves as both a marker and a regulator of chondrogenesis. Over 21 days of chondrogenesis, iPSCs showed significant increases in Trpv4 expression along with the standard chondrogenic gene markers Sox9, Acan, and Col2a1, particularly in the green fluorescent protein positive (GFP+) chondroprogenitor subpopulation. Increased gene expression for Trpv4 was also reflected by the presence of TRPV4 protein and functional Ca2+ signaling. Daily activation of TRPV4 using the specific agonist GSK1016790A resulted in significant increases in cartilaginous matrix production. An improved understanding of the role of TRPV4 in chondrogenesis may provide new insights into the development of new therapeutic approaches for diseases of cartilage, such as osteoarthritis, or channelopathies and hereditary disorders that affect cartilage during development. Harnessing the role of TRPV4 in chondrogenesis may also provide a novel approach for accelerating stem cell differentiation in functional tissue engineering of cartilage replacements for joint repair.

Single-cell RNA sequencing reveals the induction of novel myeloid and myeloid-associated cell populations in visceral fat with long-term obesity.

Macrophages and other immune cells are important contributors to obesity-associated inflammation; however, the cellular identities of these specific populations remain unknown. In this study, we identified individual populations of myeloid cells found in mouse epididymal/visceral adipose tissue by single-cell RNA sequencing, immunofluorescence, and flow cytometry. Multiple canonical correlation analysis identified 11 unique myeloid and myeloid-associate cell populations. In obese mice, we detected an increased percentage of monocyte-derived pro-inflammatory cells expressing Cd9 and Trem2, as well as significantly decreased percentages of multiple cell populations, including tissue-resident cells expressing Lyve1, Mafb, and Mrc1. We have identified and validated a novel myeloid/macrophage population defined by Ly6a expression, exhibiting both myeloid and mesenchymal characteristics, which increased with obesity and showed high pro-fibrotic characteristics in vitro. Our mouse adipose tissue myeloid cell atlas provides an important resource to investigate obesity-associated inflammation and fibrosis.

Regulation of chondrocyte biosynthetic activity by dynamic hydrostatic pressure: the role of TRP channels.

INTRODUCTION: Chondrocytes perceive and respond to mechanical loading as signals that regulate their metabolism. Joint loading exposes chondrocytes to multiple modes of mechanical stress, including hydrostatic pressure; however, the mechanisms by which chondrocytes sense physiologically relevant levels of hydrostatic pressure are not well understood. We hypothesized that hydrostatic pressure is transduced to an intracellular signal through mechanosensitive membrane ion channels of chondrocytes. The goals of this study were to examine the effect of hydrostatic loading on the development of engineered cartilage tissue and the contribution of mechanosensitive ion channels on these hydrostatic loading effects. METHODS: Using a 3D model of porcine chondrocytes in agarose, we applied specific chemical inhibitors to determine the role of transient receptor potential (TRP) ion channels TRPV1, TRPV4, TRPC3, and TRPC1 in transducing hydrostatic pressure. RESULTS: Hydrostatic loading caused a frequency and magnitude-dependent decrease in sulfated glycosaminoglycans (S-GAG), without changes in DNA content. Inhibiting TRPC3 and TRPV4 decreased S-GAG content; however, only the inhibition of TRPV1 partially attenuated the hydrostatic loading-induced reduction in S-GAG content. CONCLUSIONS: Our findings indicate that TRPV1 may serve as a transducer of hydrostatic pressure in chondrocytes, and provide further support for the role of TRPV4 in regulating chondrocyte anabolism, as well as initial evidence implicating TRPC3 in chondrogenesis. These findings add to our further understanding of the chondrocyte "channelome" and suggest that a range of ion channels mediate the transduction of different biophysical stimuli such as hydrostatic pressure, membrane stretch, or osmotic stress.

TRPV4-mediated calcium signaling in mesenchymal stem cells regulates aligned collagen matrix formation and vinculin tension.

Microarchitectural cues drive aligned fibrillar collagen deposition in vivo and in biomaterial scaffolds, but the cell-signaling events that underlie this process are not well understood. Utilizing a multicellular patterning model system that allows for observation of intracellular signaling events during collagen matrix assembly, we investigated the role of calcium (Ca2+) signaling in human mesenchymal stem cells (MSCs) during this process. We observed spontaneous Ca2+ oscillations in MSCs during fibrillar collagen assembly, and hypothesized that the transient receptor potential vanilloid 4 (TRPV4) ion channel, a mechanosensitive Ca2+-permeable channel, may regulate this signaling. Inhibition of TRPV4 nearly abolished Ca2+ signaling at initial stages of collagen matrix assembly, while at later times had reduced but significant effects. Importantly, blocking TRPV4 activity dramatically reduced aligned collagen fibril assembly; conversely, activating TRPV4 accelerated aligned collagen formation. TRPV4-dependent Ca2+ oscillations were found to be independent of pattern shape or subpattern cell location, suggesting this signaling mechanism is necessary for aligned collagen formation but not sufficient in the absence of physical (microarchitectural) cues that force multicellular alignment. As cell-generated mechanical forces are known to be critical to the matrix assembly process, we examined the role of TRPV4-mediated Ca2+ signaling in force generated across the load-bearing focal adhesion protein vinculin within MSCs using an FRET-based tension sensor. Inhibiting TRPV4 decreased tensile force across vinculin, whereas TRPV4 activation caused a dynamic unloading and reloading of vinculin. Together, these findings suggest TRPV4 activity regulates forces at cell-matrix adhesions and is critical to aligned collagen matrix assembly by MSCs.

The Use of Biomarkers in the Early Diagnosis of Septic Arthritis and Osteomyelitis-A Pilot Study.

BACKGROUND: The diagnosis of septic arthritis (SA) and osteomyelitis (OM) has remained challenging in the pediatric population, often accompanied by delays and requiring invasive interventions. The purpose of this pilot study is to identify a novel panel of biomarkers and cytokines that can accurately differentiate SA and OM at initial presentation using serum alone. METHODS: Twenty patients below 18 years old whose working diagnosis included SA (n=10) and OM (n=10) were identified. Serum was collected at initial evaluation. Each sample underwent seven ELISA [C1-C2, COMP, CS-846, hyaluronan, procalcitonin, PIIANP, C-terminal telopeptide of type II collagen (CTX-II)] and 65-plex cytokine panels. Principal component and Lasso regression analysis were performed to identify a limited set of predictive biomarkers. RESULTS: Mean age was 4.7 and 9.5 years in SA and OM patients, respectively (P=0.029). 50% of SA patients presented within 24 hours of symptom onset, compared with 0% of OM patients (P=0.033). 30% of SA patients were discharged home with an incorrect diagnosis and re-presented to the emergency department days later. At time of presentation: temperature ≥38.5°C was present in 10% of SA and 40% of OM patients (P=0.12), mean erythrocyte sedimentation rate (mm/h) was 51.6 in SA and 44.9 in OM patients (P=0.63), mean C-reactive protein (mg/dL) was 55.8 in SA and 71.8 in OM patients (P=0.53), and mean white blood cells (K/mm3) was 12.5 in SA and 10.4 in OM patients (P=0.34). 90% of SA patients presented with ≤2 of the Kocher criteria. 100% of SA and 40% of OM patients underwent surgery. 70% of SA cultures were culture negative, 10% MSSA, 10% Kingella, and 10% Strep pyogenes. 40% of OM cultures were culture negative, 50% MSSA, and 10% MRSA. Four biomarkers [CTx-II, transforming growth factor alpha (TGF-α), monocyte chemoattractant protein 1 (MCP-1), B cell-attracting chemokine 1] were identified that were able to classify and differentiate 18 of the 20 SA and OM cases correctly, with 90% sensitivity and 80% specificity. CONCLUSIONS: This pilot study identifies a panel of biomarkers that can differentiate between SA and OM at initial presentation using serum alone. LEVEL OF EVIDENCE: Level II-diagnostic study.

Engineering Hyaluronic Acid for the Development of New Treatment Strategies for Osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease that is characterized by inflammation of the joints, degradation of cartilage, and the remodeling of other joint tissues. Due to the absence of disease-modifying drugs for OA, current clinical treatment options are often only effective at slowing down disease progression and focus mainly on pain management. The field of tissue engineering has therefore been focusing on developing strategies that could be used not only to alleviate symptoms of OA but also to regenerate the damaged tissue. Hyaluronic acid (HA), an integral component of both the synovial fluid and articular cartilage, has gained widespread usage in developing hydrogels that deliver cells and biomolecules to the OA joint thanks to its biocompatibility and ability to support cell growth and the chondrogenic differentiation of encapsulated stem cells, providing binding sites for growth factors. Tissue-engineering strategies have further attempted to improve the role of HA as an OA therapeutic by developing diverse modified HA delivery platforms for enhanced joint retention and controlled drug release. This review summarizes recent advances in developing HA-based hydrogels for OA treatment and provides additional insights into how HA-based therapeutics could be further improved to maximize their potential as a viable treatment option for OA.

Cartilage from human-induced pluripotent stem cells: comparison with neo-cartilage from chondrocytes and bone marrow mesenchymal stromal cells.

Cartilage has little intrinsic capacity for repair, so transplantation of exogenous cartilage cells is considered a realistic option for cartilage regeneration. We explored whether human-induced pluripotent stem cells (hiPSCs) could represent such unlimited cell sources for neo-cartilage comparable to human primary articular chondrocytes (hPACs) or human bone marrow-derived mesenchymal stromal cells (hBMSCs). For this, chondroprogenitor cells (hiCPCs) and hiPSC-derived mesenchymal stromal cells (hiMSCs) were generated from two independent hiPSC lines and characterized by morphology, flow cytometry, and differentiation potential. Chondrogenesis was compared to hBMSCs and hPACs by histology, immunohistochemistry, and RT-qPCR, while similarities were estimated based on Pearson correlations using a panel of 20 relevant genes. Our data show successful differentiations of hiPSC into hiMSCs and hiCPCs. Characteristic hBMSC markers were shared between hBMSCs and hiMSCs, with the exception of CD146 and CD45. However, neo-cartilage generated from hiMSCs showed low resemblances when compared to hBMSCs (53%) and hPACs (39%) characterized by lower collagen type 2 and higher collagen type 1 expression. Contrarily, hiCPC neo-cartilage generated neo-cartilage more similar to hPACs (65%), with stronger expression of matrix deposition markers. Our study shows that taking a stepwise approach to generate neo-cartilage from hiPSCs via chondroprogenitor cells results in strong similarities to neo-cartilage of hPACs within 3 weeks following chondrogenesis, making them a potential candidate for regenerative therapies. Contrarily, neo-cartilage deposited by hiMSCs seems more prone to hypertrophic characteristics compared to hPACs. We therefore compared chondrocytes derived from hiMSCs and hiCPCs with hPACs and hBMSCs to outline similarities and differences between their neo-cartilage and establish their potential suitability for regenerative medicine and disease modelling.

Transcriptomic analysis of bone and fibrous tissue morphogenesis during digit tip regeneration in the adult mouse.

Humans have limited regenerative potential of musculoskeletal tissues following limb or digit loss. The murine digit has been used to study mammalian regeneration, where stem/progenitor cells (the "blastema") completely regenerate the digit tip after distal, but not proximal, amputation. However, the molecular mechanisms responsible for this response remain to be determined. Here, we evaluated the spatiotemporal formation of bone and fibrous tissues after level-dependent amputation of the murine terminal phalanx and quantified the transcriptome of the repair tissue. Distal (regenerative) and proximal (non-regenerative) amputations showed significant differences in temporal gene expression and tissue regrowth over time. Genes that direct skeletal system development and limb morphogenesis are transiently upregulated during blastema formation and differentiation, including distal Hox genes. Overall, our results suggest that digit tip regeneration is controlled by a gene regulatory network that recapitulates aspects of limb development, and that failure to activate this developmental program results in fibrotic wound healing.

The miRNA-mRNA interactome of murine induced pluripotent stem cell-derived chondrocytes in response to inflammatory cytokines.

Osteoarthritis (OA) is a degenerative joint disease, and inflammation within an arthritic joint plays a critical role in disease progression. Pro-inflammatory cytokines, specifically IL-1 and TNF-α, induce aberrant expression of catabolic and degradative enzymes and inflammatory cytokines in OA and result in a challenging environment for cartilage repair and regeneration. MicroRNAs (miRNAS) are small noncoding RNAs and are important regulatory molecules that act by binding to target messenger RNAs (mRNAs) to reduce protein synthesis and have been implicated in many diseases, including OA. The goal of this study was to understand the mechanisms of miRNA regulation of the transcriptome of tissue-engineered cartilage in response to IL-1ß and TNF-α using an in vitro murine induced pluripotent stem cell (miPSC) model system. We performed miRNA and mRNA sequencing to determine the temporal and dynamic responses of genes to specific inflammatory cytokines as well as miRNAs that are differentially expressed (DE) in response to both cytokines or exclusively to IL-1ß or TNF-α. Through integration of mRNA and miRNA sequencing data, we created networks of miRNA-mRNA interactions which may be controlling the response to inflammatory cytokines. Within the networks, hub miRNAs, miR-29b-3p, miR-17-5p, and miR-20a-5p, were identified. As validation of these findings, we found that delivery of miR-17-5p and miR-20a-5p mimics significantly decreased degradative enzyme activity levels while also decreasing expression of inflammation-related genes in cytokine-treated cells. This study utilized an integrative approach to determine the miRNA interactome controlling the response to inflammatory cytokines and novel mediators of inflammation-driven degradation in tissue-engineered cartilage.

Single Cell Omics for Musculoskeletal Research.

PURPOSE OF REVIEW: The ability to analyze the molecular events occurring within individual cells as opposed to populations of cells is revolutionizing our understanding of musculoskeletal tissue development and disease. Single cell studies have the great potential of identifying cellular subpopulations that work in a synchronized fashion to regenerate and repair damaged tissues during normal homeostasis. In addition, such studies can elucidate how these processes break down in disease as well as identify cellular subpopulations that drive the disease. This review highlights three emerging technologies: single cell RNA sequencing (scRNA-seq), Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), and Cytometry by Time-Of-Flight (CyTOF) mass cytometry. RECENT FINDINGS: Technological and bioinformatic tools to analyze the transcriptome, epigenome, and proteome at the individual cell level have advanced rapidly making data collection relatively easy; however, understanding how to access and interpret the data remains a challenge for many scientists. It is, therefore, of paramount significance to educate the musculoskeletal community on how single cell technologies can be used to answer research questions and advance translation. This article summarizes talks given during a workshop on "Single Cell Omics" at the 2020 annual meeting of the Orthopedic Research Society. Studies that applied scRNA-seq, ATAC-seq, and CyTOF mass cytometry to cartilage development and osteoarthritis are reviewed. This body of work shows how these cutting-edge tools can advance our understanding of the cellular heterogeneity and trajectories of lineage specification during development and disease.

Dynamics and mechanisms of intracellular calcium waves elicited by tandem bubble-induced jetting flow.

One of the earliest events in cellular mechanotransduction is often an increase in intracellular calcium concentration associated with intracellular calcium waves (ICWs) in various physiologic or pathophysiologic processes. Although cavitation-induced calcium responses are believed to be important for modulating downstream bioeffects such as cell injury and mechanotransduction in ultrasound therapy, the fundamental mechanisms of these responses have not been elucidated. In this study, we investigated mechanistically the ICWs elicited in single HeLa cells by the tandem bubble-induced jetting flow in a microfluidic system. We identified two distinct (fast and slow) types of ICWs at varying degrees of flow shear stress-induced membrane deformation, as determined by different bubble standoff distances. We showed that ICWs were initiated by an extracellular calcium influx across the cell membrane nearest to the jetting flow, either primarily through poration sites for fast ICWs or opening of mechanosensitive ion channels for slow ICWs, which then propagated in the cytosol via a reaction-diffusion process from the endoplasmic reticulum. The speed of ICW (CICW ) was found to correlate strongly with the severity of cell injury, with CICW in the range of 33 µm/s to 93 µm/s for fast ICWs and 1.4 µm/s to 12 µm/s for slow ICWs. Finally, we demonstrated that micrometer-sized beads attached to the cell membrane integrin could trigger ICWs under mild cavitation conditions without collateral injury. The relation between the characteristics of ICW and cell injury, and potential strategies to mitigate cavitation-induced injury while evoking an intracellular calcium response, may be particularly useful for exploiting ultrasound-stimulated mechanotransduction applications in the future.