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\u27 response. Activating TRPV4 with the agonist GSK101 resulted in a C

Skeletal dysplasia-causing TRPV4 mutations suppress the hypertrophic differentiation of human iPSC-derived chondrocytes

Mutations in the TRPV4 ion channel can lead to a range of skeletal dysplasias. However, the mechanisms by which TRPV4 mutations lead to distinct disease severity remain unknown. Here, we use CRISPR-Cas9-edited human-induced pluripotent stem cells (hiPSCs) harboring either the mild V620I or lethal T89I mutations to elucidate the differential effects on channel function and chondrogenic differentiation. We found that hiPSC-derived chondrocytes with the V620I mutation exhibited increased basal currents through TRPV4. However, both mutations showed more rapid calcium signaling with a reduced overall magnitude in response to TRPV4 agonist GSK1016790A compared to wildtype (WT). There were no differences in overall cartilaginous matrix production, but the V620I mutation resulted in reduced mechanical properties of cartilage matrix later in chondrogenesis. mRNA sequencing revealed that both mutations up-regulated several anterio

Hydrogel encapsulation of genome-engineered stem cells for long-term self-regulating anti-cytokine therapy

Biologic therapies have revolutionized treatment options for rheumatoid arthritis (RA) but their continuous administration at high doses may lead to adverse events. Thus, the development of improved drug delivery systems that can sense and respond commensurately to disease flares represents an unmet medical need. Toward this end, we generated induced pluripotent stem cells (iPSCs) that express interleukin-1 receptor antagonist (IL-1Ra, an inhibitor of IL-1) in a feedback-controlled manner driven by the macrophage chemoattractant protein-1 (Ccl2) promoter. Cells were seeded in agarose hydrogel constructs made from 3D printed molds that can be injected subcutaneously via a blunt needle, thus simplifying implantation of the constructs, and the translational potential. We demonstrated that the subcutaneously injected agarose hydrogels containing genome-edited Ccl2-IL1Ra iPSCs showed significant therapeutic efficacy in the K/BxN model of inflammatory arthritis, with nearly complete abolishment of disease severity in the front paws. These implants also exhibited improved implant longevity as compared to the previous studies using 3D woven scaffolds, which require surgical implantation. This minimally invasive cell-based drug delivery strategy may be adapted for the treatment of other autoimmune or chronic diseases, potentially accelerating translation to the clinic

A synthetic mechanogenetic gene circuit for autonomous drug delivery in engineered tissues

Mechanobiologic signals regulate cellular responses under physiologic and pathologic conditions. Using synthetic biology and tissue engineering, we developed a mechanically responsive bioartificial tissue that responds to mechanical loading to produce a preprogrammed therapeutic biologic drug. By deconstructing the signaling networks induced by activation of the mechanically sensitive ion channel transient receptor potential vanilloid 4 (TRPV4), we created synthetic TRPV4-responsive genetic circuits in chondrocytes. We engineered these cells into living tissues that respond to mechanical loading by producing the anti-inflammatory biologic drug interleukin-1 receptor antagonist. Chondrocyte TRPV4 is activated by osmotic loading and not by direct cellular deformation, suggesting that tissue loading is transduced into an osmotic signal that activates TRPV4. Either osmotic or mechanical loading of tissues transduced with TRPV4-responsive circuits protected constructs from inflammatory degradation by interleukin-1α. This synthetic mechanobiology approach was used to develop a mechanogenetic system to enable long-term, autonomously regulated drug delivery driven by physiologically relevant loading

Mechanosensitive Ion Channels as Therapeutics Targets for Osteoarthritis

Osteoarthritis (OA) is a common degenerative joint disease characterized by the degenerative changes in joint cartilage and underlying bone. It commonly occurs in weight-bearing joints like the knees, hips, and spine but can affect any joint in the body, resulting in symptoms such as pain, stiffness, and reduced range of motion. Post-traumatic osteoarthritis (PTOA) is a subtype of OA that occurs after a joint injury, such as a fracture, dislocation, or ligament tear, causing damage to the cartilage and other joint tissues leading to OA over time. PTOA accounts for a significant proportion of all OA cases. OA is a major cause of disability worldwide and poses a significant economic burden on both individuals and healthcare systems, including direct medical costs like doctor visits, medications, and surgeries, and indirect costs like lost productivity due to absenteeism or disability. The total cost of OA is estimated to be several percent of a country\u27s GDP. Epidemiologically, OA is one of the most common chronic diseases, affecting millions worldwide. Its prevalence increases with age and is more common in women than in men. Other risk factors include obesity, joint injury, and repetitive use of certain joints. PTOA can affect individuals of any age and is often associated with sports injuries or accidents. It is estimated that about 12% of all OA cases are post-traumatic. Despite its prevalence and associated costs, there is no cure for OA or PTOA, and current treatments primarily focus on managing symptoms and improving joint function. These treatments include pain relievers (such as acetaminophen or nonsteroidal anti-inflammatory drugs), physical therapy, joint injections (such as corticosteroids or hyaluronic acid), and in severe cases, joint replacement surgery. However, these treatments often have limitations in terms of efficacy, side effects, and the fact that they do not stop the progression of the disease. For example, pain relievers can cause gastrointestinal issues, kidney or liver damage, and an increased risk of heart attack or stroke; injections may provide temporary relief but do not address the underlying cause of the disease, and joint replacement surgery is a major procedure with associated risks and a long recovery time. Preventive measures, such as maintaining a healthy weight, staying active, and avoiding joint injuries, can help reduce the risk of developing OA. For PTOA, prompt and appropriate treatment of joint injuries can help reduce the risk of developing OA later on. Therefore, finding new targets for drug development in OA is critically important to develop more effective and safer treatments that can stop or slow the progression of the disease, rather than just managing the symptoms. Therefore, we first sought to investigate the role of different mechanosensors is translating different mechanical cues such as hydrostatic pressure and mechanical compression. We used primary porcine chondrocytes and either encapsulated them in agarose hydrogel or plated them on coverslip to apply hydrostatic pressure and mechanical compression on them respectively. First, we showed that TRPV1 channel is the mechanosensory of hydrostatic pressure since blocking this channel using its specific inhibitor increased the production of sGAG that was induced by hydrostatic pressure. On the other hand, we demonstrated that PIEZO1 channel, is the only channel in the PIEZO family that responds to high magnitudes of mechanical strain and injurious loads. We also showed that PIEZO1 is sensitive to membrane tension and manipulating the membrane can regulate the sensitivity of the PIEZO1 channel to both chemical and mechanical stimuli. Additionally, we observed that PIEZO1 channel is sensitive to rate of loading, and they need intracellular and extracellular Ca2+ sources to be able to response to mechanical compression. Lastly, we took a novel approach and combined our experimental data with finite element modeling and determined the membrane strain threshold required for the PIEZO1 channel to get activated. For the purpose of finding how regulating the PIEZO channels sensitivity can affect the progression of OA and PTOA, we investigated the role of polyunsaturated fatty acids including 3 and 6 fatty acids in regulating the functionality of the PIEZO channels. We showed that both 3 and 6 fatty acids were able to reduce the sensitivity of the PIEZO channels to both chemical and mechanical stimuli. Furthermore, we showed that supplementation of 6 fatty acids increase the level of inflammatory biomarker IL-6 and senescence marker MMP3 compared to the control. However, treating the chondrocytes with 3 decreased the level of IL-6, MMP3, and other senescence factor P53 showing the effect of 3 fatty acids on reducing inflammation and cellular aging. Lastly, we assessed the role of voltage gated Ca2+ channels (VGCCs) in regulating the PIEZO channels activity and see if the downstream effect of inhibiting the VGCCs on PIEZOs can be used as a therapeutic for OA and PTOA. We showed that blocking the L-type VGCCs activity using their specific antagonist Nifedipine can reduce the sensitivity of the PIEZO channels. However, blocking the T-type VGCCs activity using NNC-55 would significantly increase the sensitivity of the PIEZO channels. Moreover, we demonstrated that treating cartilage explants with Nifedipine would rescue the cartilage under injury, although, blocking the T-type channels induce more cell death in an injured cartilage explant. Overall, understanding the mechanisms by which chondrocytes respond to physiologic or pathological cartilage loading is crucial for the development of new pharmacologic therapies to treat mechanically-regulated conditions such as PTOA. Our research has shown that dietary polyunsaturated fatty acids (PUFAs) can reduce the mechanosensitivity of PIEZO channels in chondrocytes in response to deformation, thereby elucidating the role of mechanobiology in cartilage health and disease. Moreover, 3 PUFAs were able to decrease the inflammatory and senescence markers in chondrocytes elucidating their positive effect on cell health. Additionally, we found that hypo-osmotic conditions, which may occur in early OA, can sensitize PIEZO activation of chondrocytes by increasing the apparent membrane tension. Furthermore, intracellular Ca2+ activation is sensitive to the rate of loading, potentially due to the viscoelasticity of the cell or membrane. Lastly, we demonstrated that inhibiting the activity of the L-type VGCCs can rescue the cartilage under injury through regulating the activity of the PIEZO channels. These findings not only provide new insights into developing future OA therapeutics but also underscore the importance of understanding the intersection of different mechanisms involved in chondrocyte mechanotransduction. This knowledge can open new pathways for the development of pharmacologic therapies to treat mechanically-regulated conditions such as PTOA

An immortalized human adipose-derived stem cell line with highly enhanced chondrogenic properties

Human adipose-derived stem cells (ASCs) are a commonly used cell type for cartilage tissue engineering. However, donor-to-donor variability, cell heterogeneity, inconsistent chondrogenic potential, and limited expansion potential can hinder the use of these cells for modeling chondrogenesis, in vitro screening of drugs and treatments for joint diseases, or translational applications for tissue engineered cartilage repair. The goal of this study was to create an immortalized ASC line that showed enhanced and consistent chondrogenic potential for applications in cartilage tissue engineering as well as to provide a platform for investigation of biological and mechanobiological pathways involved in cartilage homeostasis and disease. Starting with the ASC52telo cell line, a hTERT-immortalized ASC line, we used lentivirus to overexpress SOX9, a master regulator of chondrogenesis, and screened several clonal populations of SOX9 overexpressing cells to form a new stable cell line with high chondrogenic potential. One clonal line, named ASC52telo-SOX9, displayed increased GAG and type II collagen synthesis and was found to be responsive to both mechanical and inflammatory stimuli in a manner similar to native chondrocytes. The development of a clonal line such as ASC52telo-SOX9 has the potential to be a powerful tool for studying cartilage homeostasis and disease mechanisms in vitro, and potentially as a platform for in vitro drug screening for diseases that affect articular cartilage. Our findings provide an approach for the development of other immortalized cell lines with improved chondrogenic capabilities in ASCs or other adult stem cells