Bioactive fiber-reinforced hydrogel to tailor cell microenvironment for structural and functional regeneration of myotendinous junction.

Myotendinous junction (MTJ) injuries are prevalent in clinical practice, yet the treatment approaches are limited to surgical suturing and conservative therapy, exhibiting a high recurrence rate. Current research on MTJ tissue engineering is scarce and lacks in vivo evaluation of repair efficacy. Here, we developed a three-dimensional-printed bioactive fiber-reinforced hydrogel containing mesenchymal stem cells (MSCs) and Klotho for structural and functional MTJ regeneration. In a rat MTJ defect model, the bioactive fiber-reinforced hydrogel promoted the structural restoration of muscle, tendon, and muscle-tendon interface and enhanced the functional recovery of injured MTJ. In vivo proteomics and in vitro cell cultures elucidated the regenerative mechanisms of the bioactive fiber-reinforced hydrogel by modulating oxidative stress and inflammation, thus engineering an optimized microenvironment to support the survival and differentiation of transplanted MSCs and maintain the functional phenotype of resident cells within MTJ tissues, including tendon/muscle cells and macrophages. This strategy provides a promising treatment for MTJ injuries.

Stiffness-dependent dynamic effect of inflammation on keratocyte phenotype and differentiation.

Although extensive studies have evaluated the regulation effect of microenvironment on cell phenotype and cell differentiation, further investigations in the field of the cornea are needed to gain sufficient knowledge for possible clinical translation. This study aims to evaluate the regulation effects of substrate stiffness and inflammation on keratocyte phenotype of corneal fibroblasts, as well as the differentiation from stem cells towards keratocytes. Soft and stiff substrates were prepared based on polydimethylsiloxane. HTK and stem cells were cultured on these substrates to evaluate the effects of stiffness. The possible synergistic effects between substrate stiffness and inflammatory factor IL-1ßwere examined by qPCR and immunofluorescence staining. In addition, macrophages were cultured on soft and stiff substrates to evaluate the effect of substrate stiffness on the synthesis of inflammatory factors. The conditioned medium of macrophages (Soft-CM and Stiff-CM) was collected to examine the effects on HTK and stem cells. It was found that inflammatory factor IL-1ßpromoted keratocyte phenotype and differentiation when cells were cultured on soft substrate (∼130 kPa), which were different from cells cultured on stiff substrate (∼2 × 103kPa) and TCP (∼106kPa). Besides, macrophages cultured on stiff substrates had significantly higher expression ofIL-1ßandTnf-αas compared to the cells cultured on soft substrates. And Stiff-CM decreased the expression of keratocyte phenotype markers as compared to Soft-CM. The results of our study indicate a stiffness-dependent dynamic effect of inflammation on keratocyte phenotype and differentiation, which is of significance not only in gaining a deeper knowledge of corneal pathology and repair, but also in being instructive for scaffold design in corneal tissue engineering and ultimate regeneration.

Material Stiffness in Cooperation with Macrophage Paracrine Signals Determines the Tenogenic Differentiation of Mesenchymal Stem Cells.

Stiffness is an important physical property of biomaterials that determines stem cell fate. Guiding stem cell differentiation via stiffness modulation has been considered in tissue engineering. However, the mechanism by which material stiffness regulates stem cell differentiation into the tendon lineage remains controversial. Increasing evidence demonstrates that immune cells interact with implanted biomaterials and regulate stem cell behaviors via paracrine signaling; however, the role of this mechanism in tendon differentiation is not clear. In this study, polydimethylsiloxane (PDMS) substrates with different stiffnesses are developed, and the tenogenic differentiation of mesenchymal stem cells (MSCs) exposed to different stiffnesses and macrophage paracrine signals is investigated. The results reveal that lower stiffnesses facilitates tenogenic differentiation of MSCs, while macrophage paracrine signals at these stiffnesses suppress the differentiation. When exposed to these two stimuli, MSCs still exhibit enhanced tendon differentiation, which is further elucidated by global proteomic analysis. Following subcutaneous implantation in rats for 2 weeks, soft biomaterial induces only low inflammation and promotes tendon-like tissue formation. In conclusion, the study demonstrates that soft, rather than stiff, material has a greater potential to guide tenogenic differentiation of stem cells, which provides comprehensive evidence for optimized bioactive scaffold design in tendon tissue engineering.

Silk Fibroin and Sericin Differentially Potentiate the Paracrine and Regenerative Functions of Stem Cells Through Multiomics Analysis.

Silk fibroin (SF) and sericin (SS), the two major proteins of silk, are attractive biomaterials with great potential in tissue engineering and regenerative medicine. However, their biochemical interactions with stem cells remain unclear. In this study, multiomics are employed to obtain a global view of the cellular processes and pathways of mesenchymal stem cells (MSCs) triggered by SF and SS to discern cell-biomaterial interactions at an in-depth, high-throughput molecular level. Integrated RNA sequencing and proteomic analysis confirm that SF and SS initiate widespread but distinct cellular responses and potentiate the paracrine functions of MSCs that regulate extracellular matrix deposition, angiogenesis, and immunomodulation through differentially activating the integrin/PI3K/Akt and glycolysis signaling pathways. These paracrine signals of MSCs stimulated by SF and SS effectively improve skin regeneration by regulating the behavior of multiple resident cells (fibroblasts, endothelial cells, and macrophages) in the skin wound microenvironment. Compared to SS, SF exhibits better immunomodulatory effects in vitro and in vivo, indicating its greater potential as a carrier material of MSCs for skin regeneration. This study provides comprehensive and reliable insights into the cellular interactions with SF and SS, enabling the future development of silk-based therapeutics for tissue engineering and stem cell therapy.

Hydroxycamptothecin and Substratum Stiffness Synergistically Regulate Fibrosis of Human Corneal Fibroblasts.

Corneal fibrosis is a common outcome of inappropriate repair associated with trauma or ocular infection. Altered biomechanical properties with increased corneal stiffness is a feature of fibrosis that cause corneal opacities, resulting in severe visual impairment and even blindness. The present study aims to determine the effect of hydroxycamptothecin (HCPT) and matrix stiffness on transforming growth factor-ß1 (TGF-ß1)-induced fibrotic processes in human corneal fibroblasts (HTK cells). HTK cells were cultured on substrates with different stiffnesses ("soft", ∼261 kPa; "stiff", ∼2.5 × 103 kPa) and on tissue culture plastic (TCP, ∼106 kPa) and simultaneously treated with or without 1 µg/mL HCPT and 10 ng/mL TGF-ß1. We found that HCPT induced decreased cell viability and antiproliferative effects on HTK cells. TGF-ß1-induced expression of fibrosis-related genes (FN1, ACTA2) was reduced if the cells were simultaneously treated with HCPT. Substrate stiffness did not affect the expression of fibrosis-related genes. The TGF-ß1 induced expression of FN1 on both soft and stiff substrates was reduced if cells were simultaneously treated with HCPT. However, this trend was not seen for ACTA2, i.e., the TGF-ß1 induced expression of ACTA2 was not reduced by simultaneous treatment of HCPT in either soft or stiff substrate. Instead, HCPT treatment in the presence of TGF-ß1 resulted in increased gene expression of keratocyte phenotype makers (LUM, KERA, AQP1, CHTS6) on both substrate stiffnesses. In addition, the protein expression of keratocyte phenotype makers LUM and ALDH3 was increased in HTK cells simultaneously treated with TGF-ß1 and HCPT on stiff substrate as compared to control, i.e., without HCPT. In conclusion, we found that HCPT can reduce TGF-ß1-induced fibrosis and promote the keratocyte phenotype in a substrate stiffness dependent manner. Thus, HCPT stimulation might be an approach to stimulate keratocytes in the appropriate healing stage to avoid or reverse fibrosis and achieve more optimal corneal wound healing.