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Mesenchymal stem cells (MSCs) have gained considerable attention in tissue engineering due to their remarkable multilineage differentiation potential, recruitment of endogenous cells, and orchestration of a regenerative microenvironment via the secretion of trophic factors. Contemporary research increasingly underscores that, rather than direct differentiation into injured tissue cells, MSCs primarily facilitate tissue repair through the secretion of bioactive paracrine factors. Notably, the physical properties of scaffold materials have emerged as key regulators of MSC paracrine function. This review provides a comprehensive overview of how diverse physical characteristics of scaffold materials modulate the composition and concentration of MSC-derived paracrine factors, thereby shaping the cellular microenvironment to enhance tissue regeneration. By elucidating the mechanistic interplay between scaffold physicality and MSC paracrine function, we aim to inform the rational design of biomaterials for optimized regenerative therapies.

Purpose: This study aimed to determine how different strain intensities-including normal, moderately increased, and high strain-influence protein expression profiles and related biological processes in human corneal stromal keratocytes.

Methods: A well-established in vitro model using the Flexcell FX-5000 Tension System, which replicates the natural corneal curvature and enables precise strain application to keratocytes, was used. Keratocytes were exposed to three strain levels: 3% (normal), 6% (moderately increased), and 12% (high). Following strain application, cells were collected for liquid chromatography-tandem mass spectrometry-based proteomic analysis to generate protein expression profiles. Differentially expressed proteins (DEPs) among the three groups were identified and subjected to biological pathway enrichment to reveal strain-dependent biological processes. Western blot analysis was performed to validate the expression of selected DEPs.

Results: Keratocytes exhibited strain intensity-dependent responses. Three percent strain maintained keratocytes in a quiescent phenotype, consistent with our previous findings. Six percent strain activated protective and adaptive programs to preserve tissue homeostasis under stress. In contrast, 12% strain suppressed immune-related processes and induced extracellular matrix (ECM) remodeling. Notably, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) and cathepsin L (CTSL)-two ECM remodeling-related proteins implicated in fibrotic responses-were significantly upregulated under 12% strain, highlighting a potential link between excessive mechanical stress and stromal fibrosis.

Conclusions: These findings demonstrate that corneal strain regulates keratocyte behavior in an intensity-dependent manner and suggest that high mechanical stress may drive pathologic stromal remodeling and fibrotic responses, offering mechanistic insights that may inspire future therapeutic strategies.

Exercise is well known to promote tendon healing, an effect traditionally attributed to mechanical loading-induced responses within the tendon itself. However, skeletal muscle also functions as a secretory organ, releasing bioactive factors (secretome) during exercise that influence various tissues. We hypothesized that muscle-derived secretome released during exercise may also contribute to tendon healing. To test this, we applied mechanical loading to cultured muscle cells (myoblasts) using the FlexCell tension system to simulate exercise in vitro. Our previous studies, using 2D-cultured tendon cells (tenocytes), have demonstrated that secretome from statically loaded myoblasts, particularly under 2% loading, enhanced tendon healing-related responses. Building upon these findings, we employed a 3D tendon construct model to more closely mimic in vivo healing conditions. We found that secretome derived from statically loaded myoblasts, especially at 2% loading, promoted tendon healing-related processes as compared with the control group, which received no secretome treatment (no conditioned media). These included increased cell-covered area, expression of the tenocyte marker scleraxis (SCX), and elevated production of Type I and III collagens at an early stage (Day 7). Additionally, a reduction in type III collagen production was found at a later stage (Day 14), suggesting a potentially accelerated healing process. These findings highlight the therapeutic potential of the muscle-derived secretome in promoting tendon healing and may inform future strategies for rehabilitation and regenerative medicine.

The dysregulation of the inflammatory microenvironment following tendon injury significantly hinders regeneration. In this study, we developed an all-silk-derived functional scaffold (rKL@MPs-ASF) by integrating silk fibroin (SF) microspheres (MPs) loaded with the anti-inflammatory protein recombinant α-Klotho (rKL) into a biomimetic aligned SF (ASF) scaffold. This scaffold is designed to regulate the inflammatory microenvironment and facilitate tendon regeneration. Proteomic analysis revealed that rKL preserves the tenogenic differentiation potential of tendon stem/progenitor cells (TSPCs) by mitigating the oxidative stress response in a tumor necrosis factor-alpha-induced inflammatory microenvironment in vitro. The rKL@MPs-ASF scaffold demonstrated good drug loading/release capabilities and biocompatibility in vitro. In a rat full-thickness Achilles tendon defect model, the rKL@MPs-ASF scaffold reduced inflammatory cells infiltration and promoted fibroblast infiltration compared to the PBS@MPs-ASF group at 4 weeks post-operation. At 8 weeks post-operation, rKL@MPs-ASF-treated tendons showed increased collagen fiber deposition and reduced heterogeneous ossification, facilitating tendon regeneration and functional recovery. In conclusion, this all-silk-derived functional system creates a conducive microenvironment for tendon regeneration. Statement of significance: Regulation of the inflammatory microenvironment plays a crucial role in modulating the differentiation of TSPCs, thereby promoting tendon tissue regeneration. In this study, we demonstrate that rKL effectively preserves the tenogenic differentiation potential of TSPCs by mitigating oxidative stress within an inflammatory microenvironment. We developed an innovative, all-silk-based functional system (rKL@MPs-ASF), which integrates rKL-loaded SF MPs into an aligned silk fibroin scaffold. This system enables the controlled release of rKL, thereby modulating inflammation, promoting collagen fiber deposition, inhibiting heterotopic ossification, and ultimately improving tendon regeneration and functional recovery. Our findings highlight the potential of the rKL@MPs-ASF system, which combines structural and biological properties with a versatile drug-delivery platform, as a promising strategy for enhancing tendon repair and regenerative outcomes.

Silk fibroin (SF), the core structural protein derived from Bombyx mori silk, is extensively employed in tissue engineering and regenerative medicine due to its exceptional mechanical properties, favorable biocompatibility, tunable biodegradability, and versatile processing capabilities. Despite these advantages, current research predominantly focuses on SF biomaterials as structural scaffolds or drug carriers, often overlooking their potential role in modulating cellular behavior and tissue regeneration. This review aims to present a comprehensive overview of the inherent biological effects of SF biomaterials, independent of any exogenous biomolecules, and their implications for various tissue regeneration. It will cover in vitro cellular interactions of SF with various cell types, including stem cells and functional tissue cells such as osteoblasts, chondrocytes, keratinocytes, endothelial cells, fibroblasts, and epithelial cells. Moreover, it will summarize in vivo immune responses, cellular responses, and tissue regeneration following SF implantation, specifically focusing on vascular, bone, skin, cartilage, ocular, and tendon/ligament regeneration. Furthermore, it will address current limitations and future perspectives in the design of bioactive SF biomaterials. A comprehensive understanding of these cellular interactions and the biological effects of SF is crucial for predicting regenerative outcomes with precision and for designing SF-based biomaterials tailored to specific properties, enabling broader applications in regenerative medicine.

Tendon repair remains challenging due to its poor intrinsic healing capacity, and stem cell therapy has emerged as a promising strategy to promote tendon regeneration. Nevertheless, the inflammatory environment following acute tendon injuries disrupts stem cell differentiation, leading to unsatisfied outcomes. Our study recognized the critical role of NF-κB signaling in activating inflammation and suppressing tenogenic differentiation of stem cells after acute tendon injury via multiomics analysis. TPCA-1, a selective inhibitor of IKKβ/NF-κB signaling, efficiently restored the impaired tenogenesis of stem cells in the inflammatory environment. By developing a microsphere-incorporated hydrogel system for stem cell delivery and controlled release of TPCA-1, we successfully engineered a pro-tenogenic niche to initiate tenogenesis for tendon regeneration. Collectively, we recognize NF-κB signaling as a critical target to tailor a pro-tenogenic niche and propose the combined delivery of stem cells and TPCA-1 as a potential strategy for acute tendon injuries.

Tendons are vital for maintaining integrity and movement, but current treatment options are insufficient for their regeneration after injuries. Previous studies have shown that the secretome from mesenchymal stem cells (MSCs) promoted tendon regeneration. However, limited studies have explored the impact of the physical microenvironment on the secretome's efficacy of MSCs. In this study, it is shown that the topographic orientation regulates the secretome of human adipose-derived stem cells (ADSCs) and promotes tendon regeneration. Conditioned medium (CM) is collected from ADSCs cultured on the scaffolds with different topography. The results show that CM generated from aligned structure group has a potent effect in promoting cell migration and proliferation, tenogenic differentiation, macrophage polarization toward M2 phenotype, tendon structure and mechanical function recovery. Proteomic analysis revealed that the aligned structure can up-regulate the secretion of Extracellular matrix (ECM) proteins while down-regulate proinflammatory factors. This modulation activates the MAPK, GPCR and Integrin signaling pathways which may account for the enhanced effect on tendon regeneration. This study offers a promising and safer non-cell-based treatment option for tendon repair.

Exercise has been shown to promote wound healing, including tendon repair. Myokines released from the exercised muscles are believed to play a significant role in this process. In our previous study, we used an in vitro coculture and loading model to demonstrate that 2% static loading of myoblasts increased the migration and proliferation of cocultured tenocytes─two crucial aspects of wound healing. IGF-1, released from myoblasts in response to 2% static loading, was identified as a contributor to the increased proliferation. However, the factors responsible for the enhanced migration remained unknown. In the current study, we subjected myoblasts in single culture conditions to 2, 5, and 10% static loading and performed proteomic analysis of the cell supernatants. Gene Ontology (GO) analysis revealed that 2% static loading induced the secretion of NBL1, C5, and EFEMP1, which is associated with cell migration and motility. Further investigation by adding exogenous recombinant proteins to human tenocytes showed that NBL1 increased tenocyte migration but not proliferation. This effect was not observed with treatments using C5 and EFEMP1.

The purpose of this study was to examine the nature of the underlying molecular mechanisms of cell death in human keratocytes treated with nigericin, a known pyroptosis inducer. Human keratocytes were exposed to nigericin, and cell death was assessed through morphological analysis and detection of related molecular markers. Proteomic profiling was performed to identify cell death-related proteins, with key findings validated by western blot. Additionally, organelle disruptions were examined using immunostaining techniques. Pyroptosis-like cell death was observed morphologically in cultured keratocytes. Moreover, an elevated release of IL-1beta was detected, accompanied by a significant loss of mitochondrial membrane potential. However, nigericin treatment induced a form of non-inflammatory cell death characterized by extensive vacuolation, resembling paraptosis. This was accompanied by the absence of caspase-3 activation and endoplasmic reticulum (ER) stress markers, along with increased accumulation of the autophagic marker LC3-II. Proteomic analysis revealed the absence of key components of the canonical pyroptosis pathway, including proteins involved in inflammasome assembly and the gasdermin (GSDM) family. These results were further confirmed by western blot. Significant alterations were also observed in the Golgi apparatus, mitochondria, and lysosomes following nigericin treatment. These findings suggest that nigericin triggers a paraptosis-like cell death in human keratocytes, rather than pyroptosis, as keratocytes lack the canonical executors of pyroptosis. This highlights an alternative mechanism of cell death in the cornea, warranting further exploration to understand its role and potential therapeutic implications.

Purpose: To develop an in vitro model that mimics aspects of corneal healing in humans for uncovering key mechanisms involved in the mechanisms involved in the healing and scarring processes.

Methods: As part of the healing matrix, TGF-β1–induced and corneal-derived myofibroblasts were cultured in fibrin hydrogels with configurations that recapitulate the healthy (aligned) and wounded (random) microenvironment of the cornea.

Results: Evaluation of cellular alpha smooth muscle actin (α-SMA) and collagen hybridizing peptide (CHP) showed cell and matrix alignment, respectively. The aligned compared to the random constructs demonstrated an increased ability to synthesize total soluble proteins, including collagen type V, but collagen type I levels were reduced. This finding reveals a differential pattern for these proteins. Additionally, the collagen fibril diameters were larger in the aligned tissue constructs compared to the random constructs. Fibronectin and CHP colocalization patterns did not differ between groups; however, fibronectin and decorin were increased in the aligned group in contrast to tenascin C, which showed no difference.

Conclusions: These findings suggest that the alignment of the healing microenvironment plays a crucial role in modulating the structural properties of the extracellular matrix (ECM) and regulates the synthesis of key proteins that are closely involved in fibrillogenesis and are indicative of the quality of the deposited ECM.

Translational Relevance: We developed a three-dimensional in vitro model that closely mimics in vivo conditions to investigate the role of corneal myofibroblasts in healing and regeneration. Ultimately, this model can help develop targeted antifibrotic therapies to prevent corneal scarring.