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In obstructive airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), the extracellular matrix (ECM) protein amount and composition of the airway smooth muscle (ASM) is often remodelled, likely altering tissue stiffness. The underlying mechanism of how human ASM cell (hASMC) mechanosenses the aberrant microenvironment is not well understood. Physiological stiffnesses of the ASM were measured by uniaxial compression tester using porcine ASM layers under 0, 5 and 10% longitudinal stretch above in situ length. Linear stiffness gradient hydrogels (230 kPa range) were fabricated and functionalized with ECM proteins, collagen I (ColI), fibronectin (Fn) and laminin (Ln), to recapitulate the above-measured range of stiffnesses. Overall, hASMC mechanosensation exhibited a clear correlation with the underlying hydrogel stiffness. Cell size, nuclear size and contractile marker alpha-smooth muscle actin (αSMA) expression showed a strong correlation to substrate stiffness. Mechanosensation, assessed by Lamin-A intensity and nuc/cyto YAP, exhibited stiffness-mediated behaviour only on ColI and Fn-coated hydrogels. Inhibition studies using blebbistatin or Y27632 attenuated most mechanotransduction-derived cell morphological responses, αSMA and Lamin-A expression and nuc/cyto YAP (blebbistatin only). This study highlights the interplay and complexities between stiffness and ECM protein type on hASMC mechanosensation, relevant to airway remodelling in obstructive airway diseases.
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Hidrogéis , Miócitos de Músculo Liso , Hidrogéis/química , Hidrogéis/farmacologia , Humanos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Animais , Mecanotransdução Celular/fisiologia , Suínos , Matriz Extracelular/metabolismo , Células CultivadasRESUMO
Decellularised extracellular matrix (dECM) produced by mesenchymal stromal cells (MSCs) is a promising biomaterial for improving the ex vivo expansion of MSCs. The dECMs are often deposited on high modulus surfaces such as tissue culture plastic or glass, and subsequent differentiation assays often bias towards osteogenesis. We tested the hypothesis that dECM deposited on substrates of varying modulus will produce cell culture environments that are tailored to promote the proliferation and/or lineage-specific differentiation of MSCs. dECM was produced on type I collagen-functionalised polyacrylamide hydrogels with discrete moduli (â¼4, 10, and 40 kPa) or in a linear gradient of modulus that spans the same range, and the substrates were used as culture surfaces for MSCs. Fluorescence spectroscopy and mass spectrometry characterization revealed structural compositional changes in the dECM as a function of substrate modulus. Softer substrates (4 kPa) with dECM supported the largest number of MSCs after 7 days (â¼1.6-fold increase compared to glass). Additionally, osteogenic differentiation was greatest on high modulus substrates (40 kPa and glass) with dECM. Nuclear translocation of YAP1 was observed on all surfaces with a modulus of 10 kPa or greater and may be a driver for the increased osteogenesis on the high modulus surfaces. These data demonstrate that dECM technology can be integrated with environmental parameters such as substrate modulus to improve/tailor MSC proliferation and differentiation during ex vivo culture. These results have potential impact in the improved expansion of MSCs for tailored therapeutic applications and in the development of advanced tissue engineering scaffolds. STATEMENT OF SIGNIFICANCE: Mesenchymal stromal cells (MSCs) are extensively used in tissue engineering and regenerative medicine due to their ability to proliferate, differentiate, and modulate the immune environment. Controlling MSC behavior is critical for advances in the field. Decellularized extracellular matrix (dECM) can maintain MSC properties in culture, increase their proliferation rate and capacity, and enhance their stimulated differentiation. Substrate stiffness is another key driver of cell function, and previous reports have primarily looked at dECM deposition and function on stiff substrates such as glass. Herein, we produce dECM on substrates of varying stiffness to create tailored environments that enhance desired MSC properties such as proliferation and differentiation. Additionally, we complete mechanistic studies including quantitative mass spec of the ECM to understand the biological function.
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Mitigating inflammation associated with the foreign body response (FBR) remains a significant challenge in enhancing the performance of implantable medical devices. Current anti-inflammatory approaches aim to suppress implant fibrosis, the major outcome of the FBR, but also inadvertently inhibit beneficial immune signalling necessary for tissue healing and vascularization. In a previous study, we demonstrated the feasibility of 'selective' immunosuppression targeting the NLRP3 inflammasome using the small molecule inhibitor MCC950, leading to reduced implant fibrosis without compromising healing and leading to enhanced vascularization. However, the clinical potential of MCC950 is severely limited due to its failure to pass Phase I clinical safety trials. This has triggered substantial efforts to develop safer analogues of NLRP3 inhibitors. Dapansutrile (OLT1177) is emerging as a leading candidate amongst current NLRP3 inhibitors, demonstrating both safety and effectiveness in a growing number of clinical indications and Phase 2 trials. While the anti-inflammatory effects of OLT1177 have been shown, validation of these effects in the context of implanted materials and the FBR have not yet been demonstrated. In this study, we show OLT1177 possesses beneficial effects on key cell types which drive FBR outcomes, including macrophages, fibroblasts, and smooth muscle cells. Evaluation of OLT1177 in a 28 day subcutaneous implantation model showed OLT1177 reduced fibrotic capsule formation while promoting implant vascularization. Mechanistic studies revealed that this occurred through activation of early pro-angiogenic markers while suppressing late-stage anti-angiogenic markers. These findings establish OLT1177 as a promising therapeutic approach for mitigating implant fibrosis while supporting vascularisation, suggesting a highly promising selective immunosuppressive strategy for the FBR warranting further research to explore its optimal integration into medical materials and devices.
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Reação a Corpo Estranho , Inflamação , Inflamação/tratamento farmacológico , Humanos , Animais , Furanos/química , Furanos/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Camundongos , Indenos/farmacologia , Indenos/química , Próteses e Implantes , Sulfonas/química , Sulfonas/farmacologia , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Sulfonamidas/farmacologia , Sulfonamidas/química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologiaRESUMO
Current biofabrication strategies are limited in their ability to replicate native shape-to-function relationships, that are dependent on adequate biomimicry of macroscale shape as well as size and microscale spatial heterogeneity, within cell-laden hydrogels. In this study, a novel diffusion-based microfluidics platform is presented that meets these needs in a two-step process. In the first step, a hydrogel-precursor solution is dispersed into a continuous oil phase within the microfluidics tubing. By adjusting the dispersed and oil phase flow rates, the physical architecture of hydrogel-precursor phases can be adjusted to generate spherical and plug-like structures, as well as continuous meter-long hydrogel-precursor phases (up to 1.75 m). The second step involves the controlled introduction a small molecule-containing aqueous phase through a T-shaped tube connector to enable controlled small molecule diffusion across the interface of the aqueous phase and hydrogel-precursor. Application of this system is demonstrated by diffusing co-initiator sodium persulfate (SPS) into hydrogel-precursor solutions, where the controlled SPS diffusion into the hydrogel-precursor and subsequent photo-polymerization allows for the formation of unique radial stiffness patterns across the shape- and size-controlled hydrogels, as well as allowing the formation of hollow hydrogels with controllable internal architectures. Mesenchymal stromal cells are successfully encapsulated within hollow hydrogels and hydrogels containing radial stiffness gradient and found to respond to the heterogeneity in stiffness through the yes-associated protein mechano-regulator. Finally, breast cancer cells are found to phenotypically switch in response to stiffness gradients, causing a shift in their ability to aggregate, which may have implications for metastasis. The diffusion-based microfluidics thus finds application mimicking native shape-to-function relationship in the context of tissue engineering and provides a platform to further study the roles of micro- and macroscale architectural features that exist within native tissues.
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Hidrogéis , Microfluídica , Engenharia Tecidual , Hidrogéis/química , Humanos , Microfluídica/métodos , Microfluídica/instrumentação , Células-Tronco Mesenquimais/citologiaRESUMO
In recent decades, the role of tumor biomechanics on cancer cell behavior at the primary site has been increasingly appreciated. However, the effect of primary tumor biomechanics on the latter stages of the metastatic cascade, such as metastatic seeding of secondary sites and outgrowth remains underappreciated. This work sought to address this in the context of triple negative breast cancer (TNBC), a cancer type known to aggressively disseminate at all stages of disease progression. Using mechanically tuneable model systems, mimicking the range of stiffness's typically found within breast tumors, it is found that, contrary to expectations, cancer cells exposed to softer microenvironments are more able to colonize secondary tissues. It is shown that heightened cell survival is driven by enhanced metabolism of fatty acids within TNBC cells exposed to softer microenvironments. It is demonstrated that uncoupling cellular mechanosensing through integrin ß1 blocking antibody effectively causes stiff primed TNBC cells to behave like their soft counterparts, both in vitro and in vivo. This work is the first to show that softer tumor microenvironments may be contributing to changes in disease outcome by imprinting on TNBC cells a greater metabolic flexibility and conferring discrete cell survival advantages.
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Ácidos Graxos , Neoplasias de Mama Triplo Negativas , Microambiente Tumoral , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Humanos , Feminino , Ácidos Graxos/metabolismo , Camundongos , Linhagem Celular Tumoral , Animais , Fenômenos Biomecânicos , Modelos Animais de Doenças , Metástase NeoplásicaRESUMO
Present understandings of cardiomyocyte mechanobiology have primarily been developed using 2-dimensional, monocellular cell cultures, however the emergence of 3-dimensional (3D) multicellular cardiac constructs has enabled us to develop more sophisticated recapitulations of the cardiac microenvironment. Several of these strategies have illustrated that incorporating elements of the extracellular matrix (ECM) can promote greater maturation and enhance desirable cardiac functions, such as contractility, but the responses of these cardiac constructs to biophysically aberrant conditions, such as in the post-infarct heart, has remained relatively unexplored. In our study, we employ a stiffness gradient gelatin methacryloyl (GelMA) hydrogel platform to unpack the mechanobiology of cardiac spheroids. We encapsulated neonatal rat cardiac cell spheroids in a 4.4-18.7 kPa linear stiffness gradient up to 120 h. We found the proportion of viable cells within the spheroids increased over time, but the cell number per spheroid decreased. Spheroids expand more in softer matrices while stiffer matrices promote larger nuclei without changing nuclei shape. Volume expansion came primarily from cells expressing vimentin. We did not observe any correlations between stiffness and mechanomarker expression, however we found that after 120 h post-encapsulation, the localization of YAP, the localization of MRTF-A and the expression of Lamin-A was correlated with spheroid morphology. The same trends were not observed 24 h post-encapsulation, indicating that volume adaptation can take a relatively long time. Our data demonstrates that cardiac spheroids are mechanosensitive and that their capacity to respond to ECM-based cues depends on their capacity to adapt their volume with a 3D microenvironment.
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Gelatina , Miócitos Cardíacos , Animais , Ratos , Gelatina/metabolismo , Metacrilatos , Matriz Extracelular/metabolismo , Esferoides Celulares , Hidrogéis/metabolismoRESUMO
Hypertrophic cardiomyopathy is an inherited disorder due to mutations in contractile proteins that results in a stiff, hypercontractile myocardium. To understand the role of cardiac stiffness in disease progression, here we create an in vitro model of hypertrophic cardiomyopathy utilizing hydrogel technology. Culturing wild-type cardiac myocytes on hydrogels with a Young's Moduli (stiffness) mimicking hypertrophic cardiomyopathy myocardium is sufficient to induce a hypermetabolic mitochondrial state versus myocytes plated on hydrogels simulating healthy myocardium. Significantly, these data mirror that of myocytes isolated from a murine model of human hypertrophic cardiomyopathy (cTnI-G203S). Conversely, cTnI-G203S myocyte mitochondrial function is completely restored when plated on hydrogels mimicking healthy myocardium. We identify a mechanosensing feedback mechanism between the extracellular matrix and cytoskeletal network that regulates mitochondrial function under healthy conditions, but participates in the progression of hypertrophic cardiomyopathy pathophysiology resulting from sarcomeric gene mutations. Importantly, we pinpoint key 'linker' sites in this schema that may represent potential therapeutic targets.
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Cardiomiopatia Hipertrófica , Camundongos , Humanos , Animais , Retroalimentação , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Citoesqueleto/metabolismo , Miócitos Cardíacos/metabolismo , Troponina I/genética , Troponina I/metabolismo , Matriz Extracelular/metabolismo , HidrogéisRESUMO
Correction for 'Interrogating cardiac muscle cell mechanobiology on stiffness gradient hydrogels' by Ian L. Chin et al., Biomater. Sci., 2021, 9, 6795-6806, https://doi.org/10.1039/D1BM01061A.
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With the adoption of 3-dimensional (3D) cell culture for in vitro modelling of cardiac function and regenerative medicine applications, there is an increased need to understand cardiomyocyte mechanosensation in 3D. With existing studies of cardiomyocyte mechanosensation primarily focussed on the behaviour of individual cells in a 2-Dimensional context, it is unclear whether mechanosensation is the same in a 3D, multicellular context. In this study, H9C2 cardiac-derived myoblasts were encapsulated as individual cells and as cell spheroids within stiffness gradient gelatin methacryloyl (GelMA) hydrogels to investigate individual and collective cardiac cell mechanosensation in 3D. Over a 3.68-17.52 âkPa stiffness range, it was found that H9C2 cells have a limited capacity to adapt their volume to increasing substrate stiffness, demonstrated by the lack of changes in cell volume and shape across the stiffness gradient. Morphological trends were reflected by the expression of the mechanomarkers YAP, MRTF-A and Lamin-A, which were better correlated with cell and nuclear volume than with substrate stiffness. The localisation of YAP and MRTF-A were dependent on the relative volumes of the cytoplasm and nucleus while Lamin-A expression was elevated with increasing cytoplasmic and nuclear volumes. When cultured as spheroids rather than as individual cells, H9C2 cells adopted a distinct morphology with comparably smaller nuclei than individually cultured cells, while retaining the same overall cell volume. As spheroids, H9C2 cells were sensitive to stiffness cues, shown by decreasing YAP and MRTF-A nuclear localisation, increasing Lamin-A expression, and increasing vinculin expression with increasing substrate stiffness. Like the individually cultured H9C2 cells, mechanomarker expression was correlated to volume adaptation. With increasing cytoplasmic volume, YAP and MRTF-A became less nuclear localised, vinculin expression was increased, and with increasing nuclear volume, the Lamin-A expression fincreased. Together, these data suggest that cardiac cell volume adaptation may be enhanced by cell-cell interactions.
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Extracellular matrix (ECM) remodeling is a major facet of cardiac development and disease, yet our understanding of cardiomyocyte mechanotransduction remains limited. To enhance our understanding of cardiomyocyte mechanosensation, we studied stiffness-driven changes to cell morphology and mechanomarker expression in H9C2 cells and neonatal rat cardiomyocytes (NRCMs). Linear stiffness gradient polyacrylamide hydrogels (2-33 kPa) coated with ECM proteins including Collagen I (Col), Fibronectin (Fn) or Laminin (Ln) were used to represent necrotic, healthy, and infarcted cardiac tissue on a continuous stiffness gradient. Cell size, cell shape and nuclear size were found to be mechanosensitive in H9C2 cells, as was the expression or nuclear translocalization of the mechanomarkers Lamin-A, YAP, and MRTF-A. Minor differences were observed between the different ECM coatings, with the same overarching stiffness-dependent trends being observed across Col, Fn and Ln coated hydrogels. Inhibition of mechanotransduction in H9C2 cells using blebbistatin or Y27632 resulted in disruptions to cell shape, nuclear shape, and nuclear size, however, trends in cell size and mechanomarker expression were not significantly attenuated. Mechanosensation in NRCMs was much less marked, with no significant changes in cell morphology being detected, although YAP did become increasingly nuclear localized with increasing stiffness. In α-actinin positive cells, striations formed with regular structure and frequency at all stiffnesses for Col and Fn coated hydrogels, but not Ln coated gels. In this study, we used our stiffness gradient hydrogels to comprehensively map the relationship between ECM stiffness and cardiac cell phenotype and found that less mature H9C2 cardiac cells are more sensitive to ECM changes than the more developed neonatal cardiomyocytes.
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Hidrogéis , Miócitos Cardíacos , Animais , Biofísica , Matriz Extracelular , Mecanotransdução Celular , RatosRESUMO
BACKGROUND: Fibroblasts found in keloid tissues are known to present an altered sensitivity to microenvironmental stimuli. However, the impact of changes in extracellular matrix stiffness on phenotypes of normal fibroblasts (NFs) and keloid fibroblasts (KFs) is poorly understood. OBJECTIVES: Investigation the impact of matrix stiffness on NFs and KFs mainly via detecting yes-associated protein (YAP) expression. METHODS: We used fibronectin-coated polyacrylamide hydrogel substrates with a range from physiological to pathological stiffness values with or without TGF-ß (fibrogenic inducer). Atomic force microscopy was used to measure the stiffness of fibroblasts. Cellular mechanoresponses were screened by immunocytochemistry, Western blot and Luminex assay. RESULTS: KFs are stiffer than NFs with greater expression of α-SMA. In NFs, YAP nuclear translocation was induced by increasing matrix stiffness as well as by stimulation with TGF-ß. In contrast, KFs showed higher baseline levels of nuclear YAP that was not responsive to matrix stiffness or TGF-ß. TGF-ß1 induced p-SMAD3 in both KFs and NFs, demonstrating the pathway was functional and not hyperactivated in KFs. Moreover, blebbistatin suppressed α-SMA expression and cellular stiffness in KFs, linking the elevated YAP signaling to keloid phenotype. CONCLUSIONS: These data suggest that whilst normal skin fibroblasts respond to matrix stiffness in vitro, keloid fibroblasts have elevated activation of mechanotransduction signaling insensitive to the microenvironment. This elevated signaling appears linked to the expression of α-SMA, suggesting a direct link to disease pathogenesis. These findings suggest changes to keloid fibroblast phenotype related to mechanotransduction contribute to disease and may be a useful therapeutic target.
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Fibroblastos/metabolismo , Queloide/patologia , Mecanotransdução Celular , Pele/patologia , Actinas/metabolismo , Adolescente , Adulto , Idoso , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Transdução de Sinais , Pele/citologia , Fator de Crescimento Transformador beta1/metabolismo , Proteínas de Sinalização YAP/metabolismoRESUMO
We present a series of synthetic polymer hydrogels which break the traditional correlation between pore size and mechanical properties. The hydrogels are prepared from a dendronised polymer architecture based on a methacrylate copolymer to which poly(amido amine) dendrons are attached. Our approach will be useful in tailoring hydrogels for tissue engineering, controlled drug release, and flexible electronics.
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Mechanobiology-a cell's interaction with its physical environment-can influence a myriad of cellular processes including how cells migrate, differentiate and proliferate. In many diseases, remodeling of the extracellular matrix (ECM) is observed such as tissue stiffening in rigid scar formation after myocardial infarct. Utilizing knowledge of cell mechanobiology in relation to ECM remodeling during pathogenesis, elucidating the role of the ECM in the progression-and perhaps regression-of disease is a primary focus of the field. Although the importance of mechanical signaling in the cardiac cell is well-appreciated, our understanding of how these signals are sensed and transduced by cardiomyocytes is limited. To overcome this limitation, recently developed tools and resources have provided exciting opportunities to further our understandings by better recapitulating pathological spatiotemporal ECM stiffness changes in an in vitro setting. In this review, we provide an overview of a conventional model of mechanotransduction and present understandings of cardiomyocyte mechanobiology, followed by a review of emerging tools and resources that can be used to expand our knowledge of cardiomyocyte mechanobiology toward more clinically relevant applications.
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3D culture of stem cells can improve therapeutic effects. However, there is limited research on how to deliver cultured stem cell spheroids to the desired target. Here, we developed lotus seedpod-inspired hydrogel (LoSH) containing microwells for culture and delivery of stem cell spheroids. Human adipose-derived stem cells (hADSCs) inside the square microwells (200 or 400⯵m in width with various depths) spontaneously formed spheroids with high viability (94.08⯱â¯1.56%), and fibronectins conjugated to the hydrogel successfully gripped the spheroids, similar to the funiculus gripping seeds in the lotus seedpod. The spheroids slightly bound to the LoSH surface at 37⯰C were detached by the expansion of LoSH at lower temperature of 4⯰C. After spheroid formation, LoSH was placed on the target substrate upside-down, expanded at 4⯰C for 10â¯min, and removed from the target. As a result, the spheroids within the microwell were successfully transferred to the target substrate with high transfer efficiency (93.78⯱â¯2.30%). A delivery of spheroids from LoSH to full-thickness murine skin wound with chimney model showed significant enhancement of the number of SMA-positive vessels at day 21 compared to the group received the same number of spheroids by injection. Together, our findings demonstrate LoSH as a one-step platform that can culture and deliver spheroids to a large target area, which will be useful for various biomedical applications.
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Materiais Biomiméticos/farmacologia , Técnicas de Cultura de Células/métodos , Hidrogéis/farmacologia , Lotus/química , Sementes/química , Esferoides Celulares/transplante , Células-Tronco/citologia , Animais , Adesão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Camundongos , Regeneração/efeitos dos fármacos , Pele/patologia , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Transplante de Células-Tronco , Células-Tronco/efeitos dos fármacosRESUMO
Recent studies have found discordant mechanosensitive outcomes when comparing 2D and 3D, highlighting the need for tools to study mechanotransduction in 3D across a wide spectrum of stiffness. A gelatin methacryloyl (GelMA) hydrogel with a continuous stiffness gradient ranging from 5 to 38 kPa was developed to recapitulate physiological stiffness conditions. Adipose-derived stem cells (ASCs) were encapsulated in this hydrogel, and their morphological characteristics and expression of both mechanosensitive proteins (Lamin A, YAP, and MRTFa) and differentiation markers (PPARγ and RUNX2) were analyzed. Low-stiffness regions (â¼8 kPa) permitted increased cellular and nuclear volume and enhanced mechanosensitive protein localization in the nucleus. This trend was reversed in high stiffness regions (â¼30 kPa), where decreased cellular and nuclear volumes and reduced mechanosensitive protein nuclear localization were observed. Interestingly, cells in soft regions exhibited enhanced osteogenic RUNX2 expression, while those in stiff regions upregulated the adipogenic regulator PPARγ, suggesting that volume, not substrate stiffness, is sufficient to drive 3D stem cell differentiation. Inhibition of myosin II (Blebbistatin) and ROCK (Y-27632), both key drivers of actomyosin contractility, resulted in reduced cell volume, especially in low-stiffness regions, causing a decorrelation between volume expansion and mechanosensitive protein localization. Constitutively active and inactive forms of the canonical downstream mechanotransduction effector TAZ were stably transfected into ASCs. Activated TAZ resulted in higher cellular volume despite increasing stiffness and a consistent, stiffness-independent translocation of YAP and MRTFa into the nucleus. Thus, volume adaptation as a function of 3D matrix stiffness can control stem cell mechanotransduction and differentiation.
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Adipogenia/genética , Diferenciação Celular/efeitos dos fármacos , Mecanotransdução Celular/genética , Osteogênese/genética , Citoesqueleto de Actina/genética , Actomiosina/genética , Aciltransferases , Adipogenia/efeitos dos fármacos , Amidas/farmacologia , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Encapsulamento de Células/métodos , Núcleo Celular/química , Tamanho Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Gelatina/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Lamina Tipo A/genética , Células-Tronco Mesenquimais/citologia , Miosina Tipo II/genética , PPAR gama/genética , Piridinas/farmacologia , Transativadores/genética , Fatores de Transcrição/genética , Quinases Associadas a rho/genéticaRESUMO
Noise exposures that result in fully reversible changes in cochlear neural threshold can cause a reduced neural output at supra-threshold sound intensity. This so-called "hidden hearing loss" has been shown to be associated with selective degeneration of high threshold afferent nerve fiber-inner hair cell (IHC) synapses. However, the electrophysiological function of the IHCs themselves in hidden hearing loss has not been directly investigated. We have made round window (RW) measurements of cochlear action potentials (CAP) and summating potentials (SP) after two levels of a 10â¯kHz acoustic trauma. The more intense acoustic trauma lead to notch-like permanent threshold changes and both CAP and SP showed reductions in supra-threshold amplitudes at frequencies with altered thresholds as well as from fully recovered regions. However, the interpretation of the results in normal threshold regions was complicated by the likelihood of reduced contributions from adjacent regions with elevated thresholds. The milder trauma showed full recovery of all neural thresholds, but there was a persistent depression of the amplitudes of both CAP and SP in response to supra-threshold sounds. The effect on SP amplitude in particular shows that occult damage to hair cell transduction mechanisms can contribute to hidden hearing loss. Such damage could potentially affect the supra-threshold output properties of surviving primary afferent neurons.