Matrix stiffness regulates the immunomodulatory effects of mesenchymal stem cells on macrophages via AP1/TSG-6 signaling pathways.

It is well-recognized that the matrix stiffness as an important stem cell niche can mediate stem cell behavior such as attachment, proliferation and differentiation, but how matrix stiffness affects the immunomodulatory efficacy of stem cells has been little explored, which, however, is of significant importance in determining the outcomes of stem cell-based therapies and engineered tissue mimics. We herein studied the immunomodulatory efficacy of mesenchymal stem cells (MSCs) in response to matrix stiffness by the evaluation of macrophage polarization in vitro and inflammatory response in vivo by subcutaneous implantation of MSC-laden hydrogels. Remarkably, we found that soft matrix enabled MSCs to produce significantly higher levels of immunomodulatory factors compared to stiff matrix, and induced the presence of more anti-inflammatory macrophages in vitro and attenuated macrophages-mediated inflammatory response in vivo. More importantly, we revealed stiffness-mediated immunoregulatory effect of MSCs was mainly attributed to tumor necrosis factor-α-stimulated protein 6 (TSG-6), which was mechanosensitively regulated by the MAPK and Hippo signaling pathway and downstream AP1 complex, and which in turn exerted an effect on macrophages through CD44 receptor to inhibit NF-κB pathway. To conclude, our results for the first time identify TSG-6 as the key factor in regulating immunomodulatory efficacy of MSCs in mechanical response, and can be potentially utilized to empower stem cell-based therapy and tissue engineering strategy in regenerative medicine. STATEMENT OF SIGNIFICANCE: Matrix stiffness as an important stem cell niche can mediate stem cell behavior such as attachment and differentiation, but how matrix stiffness affects the immunomodulatory efficacy of stem cells has been little explored, which, however, is of significant importance in determining the outcomes of stem cell-based therapies and engineered tissue mimics. Our results for the first time identify TSG-6 as the key factor in regulating the immunomodulatory efficacy of MSCs in mechanical response, which was regulated by the MAPK and Hippo signaling pathways and downstream AP1 complex, and which in turn exerted an effect on macrophages through CD44 receptor to inhibit NF-κB pathway, and can be potentially utilized to empower stem cell-based therapy and tissue engineering strategy in regenerative medicine.

Gelatin-Based Colloidal Versus Monolithic Gels to Regulate Macrophage-Mediated Inflammatory Response.

Unlike conventional monolithic hydrogels with covalent cross-linkage that are typically elastic, colloidal gels assembled by reversibly assembled particles as building blocks have shown fascinating viscoelastic properties. They follow a gel-sol transition upon yielding and recover to the initial state upon the release of the shear force (so-called shear-thinning and self-healing behavior); this makes them an ideal candidate as injectable and moldable biomaterials for tissue regeneration. The immune response provoked by the implantation of the colloidal gels with special viscoelastic and structural features is critical for the successful integration of the implants with the host tissues, which, however, remains little explored. Since macrophages are known as the primary immune cells in determining the inflammatory response against the implants, we herein investigated in vitro macrophage polarization and in vivo inflammatory response induced by gelatin-based colloidal gels as compared to monolithic gels. Specifically, self-healing colloidal gels composed of pure gelatin nanoparticles, or methacrylate gelatin (GelMA) nanoparticles to allow secondary covalent cross-linkage were compared with GelMA bulk hydrogels. We demonstrated that hydrogel's elasticity plays a more dominant role rather than the structural feature in determining in vitro macrophage polarization evidenced by the stiffer gels inducing pro-inflammation M2 macrophage phenotype as compared to soft gels. However, subcutaneous implantation revealed a significantly alleviated immune response characterized by less fibrous capsule formation for the colloidal gels as compared to bulk gels of similar matrix elasticity. We speculated this can be related to the improved permeability of the colloidal gels for cell penetration, thereby leading to less fibrosis. In general, this study provided in-depth insight into the biophysical regulator of hydrogel materials on macrophage behavior and related inflammatory response, which can further direct future implant design and predict biomaterial-host interactions for immunotherapy and regenerative medicine. Impact statement Macrophages response to implanted biomaterials is a highly regulated process that influences device functionality and clinical outcome. Nowadays, the viscoelastic properties of colloidal versus monolithic hydrogels on macrophage phenotype in vitro and the host inflammatory response are not known. Our study found that colloidal hydrogels composed of nanoparticles of gelatin and methacrylate gelatin (GelMA) led to more anti-inflammatory polarization especially on soft colloidal gel (5.9 KPa) compared to bulk GelMA hydrogels. It suggested that macrophage response can be mechanically regulated by the viscoelastic signals of the hydrogels, which could be a promising strategy for the future design and application of novel biomaterials.

Species-independent stimulation of osteogenic differentiation induced by osteoclasts.

The coupling of bone resorption and bone formation is well-recognized in the bone remodeling process, in which osteoblasts and osteoclasts are key players. However, the anabolic effect of human primary osteoclasts has rarely been reported as mouse and cell line derived osteoclasts were mostly used in previous reports. Therefore, a comprehensive comparison of mouse and human osteoclasts and their corresponding functions is needed to study cell-cell interactions between osteoclasts and osteoblasts. Osteoclasts from mouse and human origin were generated, characterized and compared, after which their anabolic effects on the osteogenic differentiation of mouse and human MSCs were assessed. Both murine RAW264.7 derived osteoclasts (mOCs) and primary human osteoclasts (hOCs) derived from buffy coats characteristically displayed multinuclearity, marked integrin ß3 expression and enhanced TRAP activity. Despite comparable cell size, mOCs showed higher osteoclast density (number of osteoclasts per cm2 culture dish) and osteoclast nuclearity (average number of nuclei per osteoclast), but lower TRAP activity compared to hOCs. Culturing primary rat and human bone marrow MSCs with the conditioned medium of mOCs or hOCs showed anabolic effects regarding the osteogenic differentiation of MSCs with superiority of hOCs over mOCs. We conclude that despite morphological and functional differences between mouse and human osteoclasts, their secretory factors evoke similar anabolic effects on MSC osteogenic differentiation.

An All-in-One Tannic Acid-Containing Hydrogel Adhesive with High Toughness, Notch Insensitivity, Self-Healability, Tailorable Topography, and Strong, Instant, and On-Demand Underwater Adhesion.

Hydrogels that are mechanically tough and capable of strong underwater adhesion can lead to a paradigm shift in the design of adhesives for a variety of biomedical applications. We hereby innovatively develop a facile but efficient strategy to prepare hydrogel adhesives with strong and instant underwater adhesion, on-demand detachment, high toughness, notch-insensitivity, self-healability, low swelling index, and tailorable surface topography. Specifically, a polymerization lyophilization conjugation fabrication method was proposed to introduce tannic acid (TA) into the covalent network consisting of polyethylene glycol diacrylate (PEGDA) of substantially high molecular weight. The presence of TA facilitated wet adhesion to various substrates by forming collectively strong noncovalent bonds and offering hydrophobicity to allow water repellence and also provided a reversible cross-link within the binary network to improve the mechanical performance of the gels. The long-chain PEGDA enhanced the efficacy and stability of TA conjugation and contributed to gel mechanics and adhesion by allowing chain diffusion and entanglement formation. Moreover, PEGDA/TA hydrogels were demonstrated to be biocompatible and capable of accelerating wound healing in a skin wound animal model as compared to commercial tissue adhesives and can be applied for the treatment of both epidermal and intracorporeal wounds. Our study provides new, critical insight into the design principle of all-in-one hydrogels with outstanding mechanical and adhesive properties and can potentially enhance the efficacy of hydrogel adhesives for wound healing.

Control of Matrix Stiffness Using Methacrylate-Gelatin Hydrogels for a Macrophage-Mediated Inflammatory Response.

The successful tissue integration of a biomedical material is mainly determined by the inflammatory response after implantation. Macrophage behavior toward implanted materials is pivotal to determine the extent of the inflammatory response. Hydrogels with different properties have been developed for various biomedical applications such as wound dressings or cell-loaded scaffolds. However, there is limited investigation available on the effects of hydrogel mechanical properties on macrophage behavior and the further host inflammatory response. To this end, methacrylate-gelatin (GelMA) hydrogels were selected as a model material to study the effect of hydrogel stiffness (2, 10, and 29 kPa) on macrophage phenotype in vitro and the further host inflammatory response in vivo. Our data showed that macrophages seeded on stiffer surfaces tended to induce macrophages toward a proinflammatory (M1) phenotype with increased macrophage spreading, more defined F-actin and focal adhesion staining, and more proinflammatory cytokine secretion and cluster of differentiation (CD) marker expression compared to those on surfaces with a lower stiffness. When these hydrogels were further subcutaneously implanted in mice to assess their inflammatory response, GelMA hydrogels with a lower stiffness showed more macrophage infiltration but thinner fibrotic capsule formation. The more severe inflammatory response can be attributed to the higher percentage of M1 macrophages induced by GelMA hydrogels with a higher stiffness. Collectively, our data demonstrated that macrophage behavior and the further inflammatory response are mechanically regulated by hydrogel stiffness. The macrophage phenotype rather than the macrophage number predominately determined the inflammatory response after the implantation, which can provide new insights into the future design and application of novel hydrogel-based biomaterials.

Entanglement-Driven Adhesion, Self-Healing, and High Stretchability of Double-Network PEG-Based Hydrogels.

Hydrogels that are capable of wet adhesion and self-healing can enable major advances in a variety of biomedical applications such as tissue regeneration, wound dressings, wearable/implantable devices, and drug delivery. We hereby developed an innovative but simple strategy to achieve adhesive, self-healing, and highly stretchable double-network hydrogels, which were composed of a primary covalent polyethylene glycol diacrylate (PEGDA) network in combination with a noncovalent network of highly diffusive, giant PEG chains. The adhesion to substrates including tissue matrices was instant and repeatable due to the diffusive PEG chains that can spontaneously penetrate and entangle with the substrate network. Combining the intrinsic biocompatibility of PEG and rational design for tuning the hydrogel network properties, we exemplarily demonstrated that this hydrogel can be used as a three-dimensional matrix for cell culture or as a tissue adhesive for wound healing. The in vivo study showed that the hydrogel is capable of effectively triggering skin wound healing with a significantly lower immune response in comparison to commercial tissue adhesives currently used in clinics. Therefore, our study provides new and critical insights into the design strategy to achieve adhesion and rehealability by taking advantages of the entanglement effect from double-network hydrogels and opens up a new avenue for the application of entanglement-driven hydrogels in regenerative medicine.

Gab2 Ablation Reverses the Stemness of HER2-Overexpressing Breast Cancer Cells.

BACKGROUND/AIMS: HER2 has been implicated in mammary tumorigenesis as well as aggressive tumor growth and metastasis. Its overexpression is related to a poor prognosis and chemoresistance in breast cancer patients. Although Grb2-associated binding protein 2 (Gab2) is important in the development and progression of human cancer, its effects and mechanisms in HER2-overexpressing breast cancer are unclear. METHODS: Clone formation and MTT assays were used to examine cell proliferation. To detect the effect of Gab2 on the stemness of breast cancer cells, we used flow cytometry, a sphere formation assay, real-time PCR, and western blot. An animal model was created to validate the effect of Gab2 on tumor growth in vivo. Tissue slides were analyzed by immunohistochemistry. RESULTS: Knockdown of Gab2 suppressed PI3K/AKT and MAPK/ERK pathway activity. Gab2 ablation also reduced the stemness of HER2-overexpressing breast cancer cells. In vivo, knockdown of Gab2 inhibited tumor growth. CONCLUSION: This study unveils a potential function of Gab2 in HER2-overexpressing breast cancer cells. Gab2 might be a potential target in the clinical therapy of HER2-overexpressing breast carcinoma.

Effect of arenobufagin on human pancreatic carcinoma cells.

Pancreatic carcinoma (PC) is a deadly form of cancer with poor overall survival. Currently, chemotherapy such as gemcitabine and 5-fluorouracil (5-FU) are the most popular medications that can improve survival, but rapid drug-resistance makes the search for more effective drugs urgent. Upon looking for natural components to treat PC, it was found that arenobufagin, a cardiac glycosides-like compound, showed significant effects on the gemcitabine-resistant pancreatic carcinoma cell line Panc-1 and the gemcitabine-sensitive cell line ASPC-1 at nanomolar concentrations. The present study used MTT and clonogenic survival assays to examine survival and proliferation, and western blotting to assess changes in the associated mitogen activated protein kinase and phosphoinositide 3-kinase pathways and expression of apoptosis-related proteins. The current study also detected the cell cycle by flow cytometry. Arenobufagin inhibited cell survival and proliferation, decreased the phosphorylation of key downstream proteins of K-Ras, including protein kinase B and extracellular signal related kinase, induced cell cycle G2/M phase arrest and apoptosis, and downregulated the level of phosphorylated epidermal growth factor receptor. Notably, the present data also showed that arenobufagin can enhance the sensitivity of PC cells to gemcitabine and 5-FU. In conclusion, arenobufagin could enhance the effect of gemcitabine and 5-FU on PC cells by targeting multiple key proteins. Therefore, arenobufagin has potential as anadjuvant therapy for the treatment of PC.