Most of the lung's gas-exchange surface forms during alveologenesis and its disruption causes bronchopulmonary dysplasia (BPD) in infants, characterized by alveolar simplification and myofibroblast accumulation. BPD also increases the risk of adult emphysema, marked by alveolar loss. Despite this connection, mechanisms linking these conditions and effective treatments are still lacking. We identify hedgehog-interacting protein (HHIP), associated with both BPD and emphysema, as a critical regulator of alveologenesis. During this process, Hhip-expressing cells expanded, accompanied by hedgehog (Hh) signaling inhibition and myofibroblast transition. Stromal-specific Hhip deletion led to hyperactivation of Hh-IGF1 signaling axis, causing persistent SMA+ myofibroblasts and epithelial stem/progenitor cell senescence. Hyperactivation of this pathway was also observed in human BPD and hyperoxia-induced BPD models. Early Hhip deficiency resulted in adult emphysema with myofibroblast accumulation. We developed a therapeutic Fc-fused HHIP protein that mitigated BPD in neonatal mice and prevented adult emphysema. These findings establish HHIP as a critical regulator of alveologenesis and a therapeutic target for BPD and emphysema.

The role of cardiac CD (cluster of differentiation) 206+ macrophages in chronic heart failure (HF) is unknown. We examined whether CD206+ macrophages expressing IL (interleukin)-4Rα are key drivers of adverse left ventricular (LV) remodeling in HF.

Adult C57BL/6 mice underwent nonreperfused myocardial infarction to induce HF. Macrophages in murine and human hearts were profiled using flow cytometry and immunostaining. In vivo myeloid-specific IL-4Rα deletion and intramyocardial macrophage adoptive transfer defined the functional effects of M[IL-4] macrophages. Antisense oligonucleotides were used for in vivo IL-4Rα gene silencing in mice.

CD206+ macrophages steadily expanded in hearts after myocardial infarction, such that at 8 weeks after myocardial infarction, they comprised ≈85% of all macrophages. These macrophages were proliferative, predominantly CCR2- (C-C motif chemokine receptor) and MHC (major histocompatibility complex) IIhi, and correlated with LV dysfunction and fibrosis. Nearly half of CD206+ macrophages expressed IL-4Rα, and the majority of CD206+IL-4Rα+ macrophages coexpressed profibrotic FIZZ (found in inflammatory zone) 1. IL-4-polarized bone marrow-derived CD206+ macrophages also exhibited marked upregulation of FIZZ1 and induced FIZZ1-dependent myofibroblast differentiation of both cardiac mesenchymal stem cells and cardiac fibroblasts, in part related to DLL-4/Jagged1-Notch1 signaling in cardiac mesenchymal stem cells. Intramyocardial adoptive transfer of M[IL-4], but not M[IL-10], CD206+ macrophages to naïve mice induced progressive LV remodeling over 4 weeks, increasing fibrosis, cardiomyocyte hypertrophy, and apoptosis. Myeloid-specific IL-4Rα gene deletion in HF (initiated 4 weeks after myocardial infarction) in IL-4Rαf/fLysM-CreERT2 mice significantly reduced CD206+ macrophage proliferation and effectively depleted CD206+IL-4Rα+ cardiac macrophages. This was associated with abrogation of LV remodeling progression, reduction of cardiac fibrosis, and improved neovascularization. In vivo IL-4Rα gene silencing in mice with established HF effectively depleted cardiac CD206+IL-4Rα+ macrophages and reversed LV remodeling, improving fibrosis, neovascularization, and dysfunction, and suppressed both local and systemic inflammation. Last, alternatively activated CD206+ and CD163+ macrophages were significantly expanded in human failing hearts and correlated with fibrosis. The majority of CD163+ macrophages expressed IL-4Rα and FIZZ3, the human homolog of FIZZ1.

Cardiac CD206+IL-4Rα+ macrophages proliferate and expand in HF and are key mediators of pathological remodeling and fibrosis, in part through the secretion of FIZZ1. Inhibition of CD206+ macrophage IL-4Rα signaling alleviates LV remodeling in ischemic cardiomyopathy.

Rotator cuff tears are the most common upper extremity orthopaedic injury, causing degenerative changes within the bone, tendon, joint capsule, bursa and muscle. These degenerative changes are linked to poor rehabilitative and surgical outcomes, which has launched investigations into co-therapeutic biologics. Specifically, mesenchymal stem cells (MSCs) have shown promise in mitigating degenerative changes in animal models of rotator cuff tears, but reports of their impact on clinical outcomes remain mixed. Here we describe an alternative source of MSCs in the human shoulder, adipose stromal cells (ASCs) from the subacromial fat (SAF) pad. Compared to the gold-standard subcutaneous (SQ) fat, we show that SAF ASCs are less sensitive to chemical and mechanical fibrotic cues, (1) retaining smaller cell area with reduced actin stress fibre alignment across a range of physiological and pathological stiffnesses, (2) having reduced traction forces and extracellular matrix production, and (3) having reduced myofibroblastic conversion in response to cytokine challenge. Furthermore, we show that SAF ASCs enhance fusion of primary human myoblasts via paracrine signalling. Despite a fibrotic signature in SAF from rotator cuffs with tendon tears, SAF ASCs sourced from torn rotator cuffs were equally effective at resisting fibroblastic conversion and promoting myogenesis as those from intact rotator cuffs, further supporting autologous clinical use of these cells. In conclusion, this study describes human SAF ASCs as an alternative, and potentially superior, cell source for rotator cuff therapies. KEY POINTS: Adipose tissue within the rotator cuff is a novel and understudied source of therapeutic adipose stromal cells. Here, we detail the impact rotator cuff tears have on adipose tissue within the shoulder, its resident adipose stromal cells, and make a comparison of shoulder adipose stromal cells to subcutaneous adipose stromal cells. Rotator cuff tears cause fibrosis of rotator cuff adipose tissue; this fibrosis does not impact downstream adipose stromal cell morphology or pro-myogenic signaling. Rotator cuff adipose stromal cells resist fibrotic microenvironmental cues and have enhanced pro-myogenic paracrine signaling compared with traditional subcutaneous adipose stromal cells. Rotator cuff adipose stromal cells represent a new cell type that can be impactful in advancing rotator cuff therapies.

Recent studies have shown that mechanical properties such as extracellular matrix stiffness, fluid flow, weight loading, compression, and stretching can affect cellular functions. Some examples of cell responses to mechanical properties could be the migration of cancer cells from rigid to soft surfaces or the differentiation of fibroblasts into myofibroblasts. Cellular responses to mechanical changes can modify the insertion of proteins in the extracellular matrix (ECM), causing an increase in tissue stiffness with functional consequences. In general, mechanical and physical factors can affect any kind of cell phenotype in culture conditions and in vivo tissues. Cells sense mechanical stimuli by applying force and restructuring their shape and functions in response to the resistance of the stimuli. Furthermore, mechanical triggers can develop a "memory" for altering cellular plasticity and adaptation. This phenomenon is called cellular mechanical memory (CMM), a singular feature of mesenchymal stem cells (MSCs). Controlled targeting of CMM may resolve the scarcity of viable cells needed for cell based therapy (CBT) and implement studies concerning cancer research, fibrosis, and senescence. This review focusses on cells from the mesodermal lineage, such as MSCs, fibroblasts and chondrocytes, and the role of CMM as a potential target for CBT.

The delivery of adipose-derived stromal cells (ASCs) on cell-instructive decellularized adipose tissue (DAT) scaffolds is a promising strategy for stimulating host-derived soft tissue regeneration. However, a better understanding of the mechanisms through which ASCs modulate regeneration in vivo is needed to harness these cells more effectively. In this study, DAT scaffolds, both with and without seeded syngeneic DsRED+ mouse ASCs, were implanted into immunocompetent C57BL/6 mice. Downstream analyses focused on assessing donor ASC persistence and phenotype, as well as the effects of ASC seeding on host macrophage polarization and the perfused host vascular network. Notably, most donor ASCs were cleared from the scaffolds by 2 weeks. Mass spectrometry-based proteomics indicated that the transplanted ASCs maintained their pre-implantation phenotype up to 1 week in vivo, suggesting that the cells were not undergoing programmed cell death. A higher fraction of the infiltrating host macrophages expressed CD68 and Arginase-1 in the ASC-seeded implants up to 1-week post-implantation. Interestingly, a small population of phagocytic macrophages, identified by uptake of DsRED protein, was present in the DAT implants in the first 2 weeks and showed enhanced expression of CD68, Arginase-1, and CD163, along with reduced expression of iNOS. MicroCT angiography revealed a similar perfused vessel network in the seeded and unseeded groups at 4- and 8-weeks post-implantation. Overall, seeding with syngeneic ASCs modulated the host macrophage response to the DAT bioscaffolds at early timepoints, but did not impact long-term regenerative outcomes, potentially due to the rapid clearance of the donor cell population in this model. STATEMENT OF SIGNIFICANCE: Decellularized adipose tissue (DAT) is a promising biomaterial for treating soft tissue defects. Seeding with adipose-derived stromal cells (ASCs) can augment fat regeneration within DAT in pre-clinical models, but our understanding of how ASCs contribute to tissue regeneration in vivo remains limited. Furthermore, ASC clearance from implanted biomaterials is well described, but poorly understood. Here, ASC-seeded DAT was implanted subcutaneously in immunocompetent mice to assess how ASCs altered the host macrophage response, functional vascular regeneration, and long-term integration with the host tissues. Additionally, ASC phenotype and persistence were assessed to determine how these cells might be cleared from the implants. Such understanding is critical to design biomaterials that can better harness the therapeutic benefits of ASCs.

Wolff's Law and the Mechanostat Theory elucidate how bone tissues detect and convert mechanical stimuli into biological signals, crucial for maintaining bone equilibrium. Abnormal mechanics can lead to diseases such as osteoporosis, osteoarthritis, and nonunion fractures. However, the detailed molecular mechanisms by which mechanical cues are transformed into biological responses in bone remain underexplored. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), key regulators of bone homeostasis, are instrumental in this process. Emerging research highlights bone cells' ability to sense various mechanical stimuli and relay these signals intracellularly. YAP/TAZ are central in receiving these mechanical cues and converting them into signals that influence bone cell behavior. Abnormal YAP/TAZ activity is linked to several bone pathologies, positioning these proteins as promising targets for new treatments. Thus, this review aims to provide an in-depth examination of YAP/TAZ's critical role in the interpretation of mechanical stimuli to biological signals, with a special emphasis on their involvement in bone cell mechanosensing, mechanotransduction, and mechanoresponse. The translational potential of this article: Clinically, appropriate stress stimulation promotes fracture healing, while bed rest can lead to disuse osteoporosis and excessive stress can cause osteoarthritis or bone spurs. Recent advancements in the understanding of YAP/TAZ-mediated mechanobiological signal transduction in bone diseases have been significant, yet many aspects remain unknown. This systematic review summarizes current research progress, identifies unaddressed areas, and highlights potential future research directions. Advancements in this field facilitate a deeper understanding of the molecular mechanisms underlying bone mechanics regulation and underscore the potential of YAP/TAZ as therapeutic targets for bone diseases such as fractures, osteoporosis, and osteoarthritis.

Biofilms formed by several bacterial strains still pose a significant challenge to healthcare due to their resistance to conventional treatment approaches, including antibiotics. This study explores the potential of loading natural extracts with antimicrobial activities into β-cyclodextrin (βCD) nanoparticles, which are FDA-approved and have superior biocompatibility owing to their cyclic sugar structures, for biofilm eradication. An inclusion complex of βCD carrying Boswellia sacra essential oils (BOS) was prepared and characterized with regard to its physicochemical properties, antimicrobial efficacy, and antibiofilm activities. Encapsulation of BOS into βCD significantly enhanced the antimicrobial activity of BOS by 4-fold against Gram-positive (Staphylococcus aureus and Bacillus subtilis) and by 8-fold against Gram-negative (Escherichia coli and Pseudomonas putida) bacteria, with minimum inhibitory concentrations ranging from 2.5 to 5 mg mL-1. Furthermore, the BOS-βCD complex demonstrated a dual-action against bacterial biofilms where it prevented biofilm formation and disrupted established biofilms. This resulted in a significant reduction in biofilm biomass, with prevention and disruption rates reaching up to 93.78% and 82.17%, respectively. Additionally, the formula revealed an excellent biocompatibility profile with no induction of oxidative stress in human skin fibroblast cells. Our findings suggest that βCD nanoparticles loaded with BOS essential oils hold promise as an effective formula for preventing the formation of bacterial biofilms and combating preformed ones for use in relevant medical applications.

Adipose-derived mesenchymal stromal cells (AD-MSCs) and fibroblasts are both widely used in regenerative medicine, demonstrating significant potential for personalized cell therapy. A major challenge in their use lies in their high biological similarity, encompassing morphology, differentiation capabilities, and flow cytometric markers, making their distinction difficult.

In our study, we aimed to compare AD-MSCs obtained from two types of adipose tissue, subcutaneous and visceral, alongside skin fibroblasts. Notably, all tissue samples were sourced from the same donors. We analyzed the cells for surface antigens via flow cytometry and conducted single-cell RNA sequencing, followed by verification with quantitative PCR (qPCR).

Our results revealed phenotypic similarities between the isolated AD-MSCs and dermal fibroblasts, particularly in the expression of markers characteristic of AD-MSCs. However, through in-depth analyses, we identified distinct differences between these cell types. Specifically, we pinpointed 30 genes exhibiting the most significant variations in expression between AD-MSCs and fibroblasts. These genes are associated with biological processes such as tissue remodeling, cell movement, and activation in response to external stimuli. Among them, MMP1, MMP3, S100A4, CXCL1, PI16, IGFBP5, COMP were further validated using qPCR, clearly demonstrating their potential to differentiate between AD-MSCs and fibroblasts.

Our scRNA-seq analysis elucidates the transcriptional landscape of AD-MSCs and fibroblasts with unprecedented resolution, highlighting both the population-specific markers and the intrapopulation heterogeneity. Our findings underscore the importance of employing high-resolution techniques for cell identification.

Mutations in SOST can lead to various monogenic bone diseases. Its paralog, SOSTDC1, shares 55 % protein sequence homology and belongs to the BMP antagonist class. Sostdc1-/- mice exhibit distinct effects on cortical and trabecular bone. Genetic polymorphisms in SOSTDC1 impacting peak bone mass makes SOSTDC1 gene, a candidate for influencing BMD variation in humans. SOSTDC1 is upregulated in bone loss conditions, altering BMP-responsive genes and signaling modulators, suggesting its dual BMP/Wnt antagonist role may enhance both pathways. Overexpression of SOSTDC1 confirmed its role as an osteogenic antagonist. Glycine max (Soy)-derived miR4352b, identified for cross-kingdom applications, precisely targets SOSTDC1, a key regulator of bone. SOSTDC1 competitively binds to BMP2 receptor, BMPR1A. Gma-miR4352b suppresses SOSTDC1 expression, enhancing osteogenesis and countering SOSTDC1's inhibition of osteogenic potential. Modeling estrogen deficiency to mimic elevated SOSTDC1 levels, we observed an inverse correlation with SOSTDC1 expression, while serum BMP2 and PINP levels increased following gma-miR4352b supplementation. In fracture healing, SOSTDC1's crucial role becomes evident in conditions of delayed fracture healing. As healing progresses, SOSTDC1 expression decreases. Gma-miR4352b, compared to scrambled miRNA, remarkably promotes callus formation, achieving 68 % healing by day 10, surpassing the scrambled group at 44 %. By the day 13, the treatment group exhibits advanced healing, challenging to find the callus, while the scrambled group maintains a healing rate similar to day10. The accelerated healing in the treatment group underscores the importance of SOSTDC1 in influencing early fracture healing, potentially through the activation of both BMP2 and Wnt signaling pathways.

To enhance the treatment of patients' urethral defects, such as strictures and hypospadias, we investigated the potential of using artificial urethral tissue. Our study aimed to generate this tissue and assess its effectiveness in a rabbit model. Two types of bioprinted grafts, based on methacrylated gelatin-silk fibroin (GelMA-SF) hydrogels, were produced: acellular, as well as loaded with autologous rabbit stem cells. Rabbit adipose stem cells (RASC) were differentiated toward smooth muscle in the GelMA-SF hydrogel, while rabbit buccal mucosa stem cells (RBMC), differentiated toward the epithelium, were seeded on its surface, forming two layers of the cell-laden tissue. The constructs were then reinforced with polycaprolactone-polylactic acid meshes to create implantable multilayered artificial urethral grafts. In vivo experiments showed that the cell-laden tissue integrated into the urethra with less fibrosis and inflammation compared to its acellular counterpart. Staining to trace the implanted cells confirmed integration into the host organism 3 months postsurgery.

Collagenase digestion (d) and cellular outgrowth (og) are the current modalities of meniscus fibrochondrocytes (MFC) isolation for bioengineering and mechanobiology-related studies. However, the impact of these modalities on study outcomes is unknown. Here, we show that og- and d-isolated MFC have distinct proliferative capacities, transcriptomic profiles via RNA sequencing (RNAseq), extracellular matrix (ECM)-forming, and migratory capacities. Our data indicate that microtissue pellet models developed from og-isolated MFC display a contractile phenotype with higher expressions of alpha-smooth muscle actin (ACTA2) and transgelin (TAGLN) and are mechanically stiffer than their counterparts from d-MFC. Moreover, we introduce a novel method of MFC isolation designated digestion-after-outgrowth (dog). The transcriptomic profile of dog-MFC is distinct from d- and og-MFC, including a higher expression of mechanosensing caveolae-associated caveolin-1 (CAV1). Additionally, dog-MFC were superior chondrogenically and generated larger-size microtissue pellet models containing a higher frequency of smaller collagen fibre diameters. Thus, we demonstrate that the modalities of MFC isolation influence the downstream outcomes of bioengineering and mechanobiology-related studies.

To study the impact of myofibroblast differentiation (MD) on hyaluronan (HA) turnover in orbital fibroblasts (OFs) focusing on the expression of its key enzymes and their potential implications in the pathogenesis of thyroid eye disease (TED).

Primary cultures of OFs were established from tissue samples (TED OFs, n = 4; non-TED OFs, n = 5). MD was induced by TGF-β1 (5 ng/mL). Measurements were performed after 24- and 72-hour treatments. The proliferation rate was determined by 5-bromo-2'-deoxyuridine (BrdU) incorporation. HA level and size were measured using an aggrecan-based ELISA-like method and agarose gel electrophoresis, respectively. mRNA expressions of myofibroblast markers and enzymes with a role in HA metabolism were determined using real-time PCR.

Upregulation of type I collagen alpha1 chain, alpha-smooth muscle actin, and fibronectin indicated that OFs underwent MD after stimulation by TGF-β. After 72 hours, proliferation of untreated cultures declined, but it remained higher in myofibroblasts. Pericellular HA content, but not HA in the supernatant of myofibroblasts, increased compared to untreated cells. TGF-β was a potent stimulator of hyaluronan synthase 1 (HAS1) expression. The expression of hyaluronidase-1 and cell migration-inducing protein (CEMIP) diminished following MD, whereas the expression of transmembrane protein 2, the regulator of HA catabolism through CEMIP, was elevated. The size distribution of HA shifted toward a high-molecular-weight form following treatment with TGF-β.

OFs undergoing MD are characterized by decreased HA turnover as a consequence of the inhibition of hyaluronidases and HAS1 induction. Our results suggest that hyaluronidases could be potential targets in the treatment of TED.

Osteochondral injury is a prevalent condition for which no specific treatment is currently available. This study presents a piezoelectric-conductive scaffold composed of a piezoelectric cartilage-decellularized extracellular matrix (dECM) and piezoelectric-conductive modified gelatin (Gel-PC). The piezoelectricity of the scaffold is achieved through the modification of diphenylalanine (FF) assembly on the pore surface, while the conductive properties of scaffold are achieved by the incorporating poly(3,4-ethylenedioxythiophene). In vitro experiments demonstrate that bone marrow mesenchymal stem cells (BMSCs) undergo biphasic division during differentiation. In vivo studies using a Parma pig model of osteochondral defects demonstrate that the piezoelectric-conductive scaffold exhibits superior reparative efficacy. Notably, the generation of electrical stimulation is linked to joint movement. During joint activity, mechanical forces compress the scaffold, leading to deformation and the subsequent generation of an electric potential difference. The positive charges accumulated on the upper layer of the scaffold attract BMSCs, promoting their migration to the upper layer and chondrogenic differentiation. Meanwhile, the negative charges in the lower layer induce the osteogenic differentiation of BMSCs. Overall, this piezoelectric-conducive scaffold provides a promising platform for the effective repair of osteochondral defects.

Critical limb ischemia (CLI) is a condition characterized by insufficient blood flow to the lower limbs, resulting in severe ischemia and potentially leading to amputation. This study aims to identify novel vasculogenic precursor cells (VPCs) in human bone marrow and evaluate their efficacy in combination with bone marrow-derived mesenchymal stem cells (BM-MSCs) for the treatment of CLI.

Ex vivo cultured VPCs and BM-MSCs from bone marrow were characterized and their effects on neovascularization and long-term tissue regeneration were tested in a mouse CLI model.

VPCs, expressing high levels of hepatocyte growth factor and c-MET, were identified from human bone marrow aspirates. These cells exhibited strong vasculogenic capacity in vitro but possessed a cellular phenotype distinct from those of previously reported endothelial precursor cells in circulation or cord blood. They also expressed most surface markers of BM-MSCs and demonstrated multipotent differentiation ability. Screening of 376 surface markers revealed that VPCs uniquely display CD141 (thrombomodulin). CD141+VPCs are present in BM aspirates as a rare population and can be expanded ex vivo with a population doubling time of approximately 20 h, generating an elaborate vascular network even under angiogenic factor-deficient conditions and recruiting BM-MSCs to the network as pericyte-like cells. Intramuscular transplantation of a combination of human CD141+VPCs and BM-MSCs at a ratio of 2:1 resulted in limb salvage, blood flow recovery, and regeneration of large vessels in the femoral artery-removed CLI model, with an efficacy superior to that of singular transplantation. Importantly, large arteries and arterioles in dual cell transplantation expressed human CD31 in the intima and human α-smooth muscle actin in media layer at 4 and 12 weeks, likely indicating their lineage commitment to endothelial cells and vascular smooth muscle, respectively, in vivo.

Dual-cell therapy using BM-derived CD141+ VPCs and BM-MSCs holds potential for further development in clinical trials to treat peripheral artery disease and diabetic ulcers.

The effects of the hydrogen sulfide (H2S) slow-releasing donor, named GSGa, a glutathione-conjugate water-soluble garlic extract, on human mesenchymal stem cells (hMSCs) in both bidimensional (2D) and three-dimensional (3D) cultures were investigated, demonstrating increased expression of the antioxidant enzyme HO-1 and decreased expression of the pro-inflammatory cytokine interleukin-6 (IL-6). The administration of the H2S donor can therefore increase the expression of antioxidant enzymes, which may have potential therapeutic applications in osteoarthritis (OA). Moreover, GSGa was able to promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), but not of cardiac mesenchymal stem cells (cMSCs) in a 2D culture system. This result highlights the varying sensitivity of hMSCs to the H2S donor GSGa, suggesting that the induction of osteogenic differentiation in stem cells by chemical factors is dependent on the tissue of origin. Additionally, a 3D-printable mesenchymal stem cells-bone matrix array (MSCBM), designed to closely mimic the stiffness of bone tissue, was developed to serve as a versatile tool for evaluating the effects of drugs and stem cells on bone repair in chronic diseases, such as OA. We demonstrated that the osteogenic differentiation process in cMSCs can be induced just by simulating bone stiffness in a 3D system. The expression of osteocalcin, RUNX2, and antioxidant enzymes was also assessed after treating MSCs with GSGa and/or increasing the stiffness of the culture environment. The printability of the array may enable better customization of the cavities, enabling an accurate replication of real bone defects. This could optimize the BM array to mimic bone defects not only in terms of stiffness, but also in terms of shape. This culture system may enable a rapid screening of antioxidant and anti-inflammatory compounds, facilitating a more personalized approach to regenerative therapy.

To explore the role of fibrocytes in the recurrence and calcification of fibrous epulides.

Different subtypes of fibrous epulides and normal gingival tissue specimens were first collected for histological and immunofluorescence analyses to see if fibrocytes were present and whether they differentiated into myofibroblasts and osteoblasts upon stimulated by transforming growth factor-β1 (TGF-β1). Electron microscopy and elemental analysis were used to characterize the extracellular microenvironment in different subtypes of fibrous epulides. Human peripheral blood mononuclear cells (PBMCs) were subsequently isolated from in vitro models to mimic the microenvironment in fibrous epulides to identify whether TGF-β1 as well as the calcium and phosphorus ion concentration in the extracellular matrix (ECM) of a fibrous epulis trigger fibrocyte differentiation.

Fibrous epulides contain fibrocytes that accumulate in the local inflammatory environment and have the ability to differentiate into myofibroblasts or osteoblasts. TGF-β1 promotes fibrocytes differentiation into myofibroblasts in a concentration-dependent manner, while TGF-β1 stimulates the fibrocytes to differentiate into osteoblasts when combined with a high calcium and phosphorus environment.

Our study revealed fibrocytes play an important role in the fibrogenesis and osteogenesis in fibrous epulis, and might serve as a therapeutic target for the inhibition of recurrence of fibrous epulides.

Ovarian insufficiency is one of the common reproductive disorders affecting women with limited therapeutic aids. Mesenchymal stem cells have been investigated in such disorders before yet, the exact mechanism of MSCs in ovarian regeneration regarding their epigenetic regulation remains elusive. The current study is to investigate the role of the bone marrow-derived mesenchymal stem cells (BM-MSCs) lncRNA (Neat-1 and Hotair1) and miRNA (mir-21-5p, mir-144-5p, and mir-664-5p) in mitigating ovarian granulosa cell apoptosis as well as searching BM-MSCs in altering the expression of ovarian and hypothalamic IGF-1 - kisspeptin system in connection to HPG axis in a cyclophosphamide-induced ovarian failure rat model. Sixty mature female Sprague Dawley rats were divided into 3 equal groups; control group, premature ovarian insufficiency (POI) group, and POI + BM-MSCs. POI female rat model was established with cyclophosphamide. The result revealed that BM-MSCs and their conditioned media displayed a significant expression level of Neat-1, Hotair-1, mir-21-5p, mir-144-5p, and mir-664-5p. Moreover, BM-MSCs transplantation in POI rats improves; the ovarian and hypothalamic IGF-1 - kisspeptin, HPG axis, ovarian granulosa cell apoptosis, steroidogenesis, angiogenesis, energy balance, and oxidative stress. BM-MSCs expressed higher levels of antiapoptotic lncRNAs and microRNAs that mitigate ovarian insufficiency.

Epithelial-stromal interplay through chemomechanical cues from cells and matrix propels cancer progression. Elevated tissue stiffness in potentially malignant tissues suggests a link between matrix stiffness and enhanced tumor growth. In this study, employing chronic oral/esophageal injury and cancer models, it is demonstrated that epithelial-stromal interplay through matrix stiffness and Hedgehog (Hh) signaling is key in compounding cancer development. Epithelial cells actively interact with fibroblasts, exchanging mechanoresponsive signals during the precancerous stage. Specifically, epithelial cells release Sonic Hh, activating fibroblasts to produce matrix proteins and remodeling enzymes, resulting in tissue stiffening. Subsequently, basal epithelial cells adjacent to the stiffened tissue become proliferative and undergo epithelial-to-mesenchymal transition, acquiring migratory and invasive properties, thereby promoting invasive tumor growth. Notably, transcriptomic programs of oncogenic GLI2, mechano-activated by actin cytoskeletal tension, govern this process, elucidating the crucial role of non-canonical GLI2 activation in orchestrating the proliferation and mesenchymal transition of epithelial cells. Furthermore, pharmacological intervention targeting tissue stiffening proves highly effective in slowing cancer progression. These findings underscore the impact of epithelial-stromal interplay through chemo-mechanical (Hh-stiffness) signaling in cancer development, and suggest that targeting tissue stiffness holds promise as a strategy to disrupt chemo-mechanical feedback, enabling effective cancer treatment.

This study investigates the characteristics of cardiac mesenchymal stem cell-like cells (CMSCLCs) isolated from the right atrial appendage of human donors with ischemia and a young patient with endocarditis (NE-CMSCLCs). Typical CMSCLCs from ischemic heart patients were derived from coronary artery bypass grafting procedures and compared against bone marrow mesenchymal stromal cells (BM-MSCs). NE-CMSCLCs had a normal immunophenotype, but exhibited enhanced osteogenic differentiation potential, rapid proliferation, reduced senescence, reduced glycolysis, and lower reactive oxygen species generation after oxidative stress compared with typical ischemic CMSCLCs. These differences suggest a unique functional status of NE-CMSCLCs, influenced by the donor health condition. Despite large variances in their paracrine secretome, NE-CMSCLCs retained therapeutic potential, as indicated by their ability to protect hypoxia/reoxygenation-injured human cardiomyocytes, albeit less effectively than typical CMSCLCs. This research describes a unique cell phenotype and underscores the importance of donor health status in the therapeutic efficacy of autologous cardiac cell therapy.

Cells isolated from their native tissues and cultured in vitro face different selection pressures than those cultured in vivo. These pressures induce a profound transformation that reshapes the cell, alters its genome, and transforms the way it senses and generates forces. In this perspective, we focus on the evidence that cells cultured on conventional polystyrene substrates display a fundamentally different mechanobiology than their in vivo counterparts. We explore the role of adhesion reinforcement in this transformation and to what extent it is reversible. We argue that this mechanoadaptation is often understood as a mechanical memory. We propose some strategies to mitigate the effects of on-plastic culture on mechanobiology, such as organoid-inspired protocols or mechanical priming. While isolating cells from their native tissues and culturing them on artificial substrates has revolutionized biomedical research, it has also transformed cellular forces. Only by understanding and controlling them, we can improve their truthfulness and validity.

Mesenchymal stromal cells (MSCs) can be isolated from various tissues of healthy or patient donors to be retransplanted in cell therapies. Because the number of MSCs obtained from biopsies is typically too low for direct clinical application, MSC expansion in cell culture is required. However, ex vivo amplification often reduces the desired MSC regenerative potential and enhances undesired traits, such as activation into fibrogenic myofibroblasts. Transiently activated myofibroblasts restore tissue integrity after organ injury by producing and contracting extracellular matrix into scar tissue. In contrast, persistent myofibroblasts cause excessive scarring-called fibrosis-that destroys organ function. In this review, we focus on the relevance and molecular mechanisms of myofibroblast activation upon contact with stiff cell culture plastic or recipient scar tissue, such as hypertrophic scars of large skin burns. We discuss cell mechanoperception mechanisms such as integrins and stretch-activated channels, mechanotransduction through the contractile actin cytoskeleton, and conversion of mechanical signals into transcriptional programs via mechanosensitive co-transcription factors, such as YAP, TAZ, and MRTF. We further elaborate how prolonged mechanical stress can create persistent myofibroblast memory by direct mechanotransduction to the nucleus that can evoke lasting epigenetic modifications at the DNA level, such as histone methylation and acetylation. We conclude by projecting how cell culture mechanics can be modulated to generate MSCs, which epigenetically protected against myofibroblast activation and transport desired regeneration potential to the recipient tissue environment in clinical therapies.

The term 'fibroblast' often serves as a catch-all for a diverse array of mesenchymal cells, including perivascular cells, stromal progenitor cells and bona fide fibroblasts. Although phenotypically similar, these subpopulations are functionally distinct, maintaining tissue integrity and serving as local progenitor reservoirs. In response to tissue injury, these cells undergo a dynamic fibroblast-myofibroblast transition, marked by extracellular matrix secretion and contraction of actomyosin-based stress fibres. Importantly, whereas transient activation into myofibroblasts aids in tissue repair, persistent activation triggers pathological fibrosis. In this Review, we discuss the roles of mechanical cues, such as tissue stiffness and strain, alongside cell signalling pathways and extracellular matrix ligands in modulating myofibroblast activation and survival. We also highlight the role of epigenetic modifications and myofibroblast memory in physiological and pathological processes. Finally, we discuss potential strategies for therapeutically interfering with these factors and the associated signal transduction pathways to improve the outcome of dysregulated healing.

In the last few decades, mesenchymal stem cells (MSCs)-based regenerative therapies in clinical applications have gradually become a hot topic due to their long-term self-renewal and multilineage differentiation ability. In this scenario, placenta (p) has been considered as a good source of MSCs. As a tissue of fetal origin with abundant number of stem cells compared to other sources, their non-invasive acquisition, strong immunosuppression, and lack of ethical concerns make placenta an indispensable source of MSC in stem cell research and therapy. The mesenchymal stem cells were derived from human term placenta (p-MSCs) in xenofree condition using platelet lysate (PL) as a suitable alternative to fetal bovine serum (FBS). Upon isolation, p-MSCs showed plastic adherence with spindle-shaped, fibroblast-like morphology under microscope. p-MSCs flourished well in PL-containing media. Immunophenotyping showed classical MSC markers (> 90%) and lack expression of hematopoietic and HLA-DR (< 1%). Surprisingly, differentiation study showed differentiation of p-MSCs to mature adipocytes in both induced cells and control (spontaneous differentiation), as observed via oil red staining. This is in line with gene expression data where both control and induced cells were positive for visfatin and leptin. Thus, we propose that p-MSCs can be used for clinical applications in the treatment of various chronic and degenerative diseases.

Aging is associated with increased susceptibility to chronic inflammatory bone loss disorders, such as periodontitis, in large part due to the impaired regenerative potential of aging tissues. DEL-1 exerts osteogenic activity and promotes bone regeneration. However, DEL-1 expression declines with age. Here we show that systemically administered macrolide antibiotics and a non-antibiotic erythromycin derivative, EM-523, restore DEL-1 expression in 18-month-old ("aged") mice while promoting regeneration of bone lost due to naturally occurring age-related periodontitis. These compounds failed to induce bone regeneration in age-matched DEL-1-deficient mice. Consequently, these drugs promoted DEL-1-dependent functions, including alkaline phosphatase activity and osteogenic gene expression in the periodontal tissue while inhibiting osteoclastogenesis, leading to net bone growth. Macrolide-treated aged mice exhibited increased skeletal bone mass, suggesting that this treatment may be pertinent to systemic bone loss disorders. In conclusion, we identified a macrolide-DEL-1 axis that can regenerate bone lost due to aging-related disease.

Epithelial Mesenchymal Transition (EMT) is a dedifferentiation process implicated in many physio-pathological conditions including tumor transformation. EMT is regulated by several extracellular mediators and under certain conditions it can be reversible. Autophagy is a conserved catabolic process in which intracellular components such as protein/DNA aggregates and abnormal organelles are degraded in specific lysosomes. In cancer, autophagy plays a controversial role, acting in different conditions as both a tumor suppressor and a tumor-promoting mechanism. Experimental evidence shows that deep interrelations exist between EMT and autophagy-related pathways. Although this interplay has already been analyzed in previous studies, understanding mechanisms and the translational implications of autophagy/EMT need further study. The role of autophagy in EMT is not limited to morphological changes, but activation of autophagy could be important to DNA repair/damage system, cell adhesion molecules, and cell proliferation and differentiation processes. Based on this, both autophagy and EMT and related pathways are now considered as targets for cancer therapy. In this review article, the contribution of autophagy to EMT and progression of cancer is discussed. This article also describes the multiple connections between EMT and autophagy and their implication in cancer treatment.

Fibrosis is one of the most worrisome complications of chronic inflammatory diseases, leading to tissue damage, organ failure, and ultimately, death. The most notable pathological characteristic of fibrosis is the excessive accumulation of extracellular matrix (ECM) components such as collagen and fibronectin adjacent to foci of inflammation or damage. The human microfibrillar-associated protein 4 (MFAP4), an important member of the superfamily of fibrinogen-related proteins, is considered to have an extremely important role in ECM transformation of fibrogenesis. This review summarizes the structure, characteristics, and physiological functions of MFAP4 and the importance of MFAP4 in various fibrotic diseases. Meanwhile, we elaborated the underlying actions and mechanisms of MFAP4 in the development of fibrosis, suggesting that a better understand of MFAP4 broadens novel perspective for early screening, diagnosis, prognostic risk assessment, and treatment of fibrotic diseases.

Distal outflow bleb-forming procedures in ophthalmic surgery expose subconjunctival tissue to inflammatory cytokines present in the aqueous humor, resulting in impaired outflow and, consequently, increased intraocular pressure. Clinically, this manifests as an increased risk of surgical failure often necessitating revision. This study (1) introduces a novel high-throughput screening platform for testing potential anti-fibrotic compounds and (2) assesses the clinical viability of modulating the transforming growth factor beta-SMAD2/3 pathway as a key contributor to post-operative outflow reduction, using the signal transduction inhibitor verteporfin. Human Tenon's capsule fibroblasts (HTCFs) were cultured within a 3D collagen matrix in a microfluidic system modelling aqueous humor drainage. The perfusate was augmented with transforming growth factor beta 1 (TGFβ1), and afferent pressure to the tissue-mimetic was continuously monitored to detect treatment-related pressure elevations. Co-treatment with verteporfin was employed to evaluate its capacity to counteract TGFβ1 induced pressure changes. Immunofluorescent studies were conducted on the tissue-mimetic to corroborate the pressure data with cellular changes. Introduction of TGFβ1 induced treatment-related afferent pressure increase in the tissue-mimetic. HTCFs treated with TGFβ1 displayed visibly enlarged cytoskeletons and stress fiber formation, consistent with myofibroblast transformation. Importantly, verteporfin effectively mitigated these changes, reducing both afferent pressure increases and cytoskeletal alterations. In summary, this study models the pathological filtration bleb response to TGFβ1, while demonstrating verteporfin's effectiveness in ameliorating both functional and cellular changes caused by TGFβ1. These demonstrate modulation of the aforementioned pathway as a potential avenue for addressing post-operative changes and reductions in filtration bleb outflow capacity. Furthermore, the establishment of a high-throughput screening platform offers a valuable pre-animal testing tool for investigating potential compounds to facilitate surgical wound healing.

The regeneration of periodontal, periapical, and pulpal tissues is a complex process requiring the direct involvement of cells derived from pluripotent stem cells in the periodontal ligament and dental pulp. Dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) are spatially distinct with the potential to differentiate into similar functional and phenotypic cells. We aimed to identify the cell heterogeneity of DPSCs and PDLSCs and explore the differentiation potentials of their specialized organ-specific functions using single-cell transcriptomic analysis. Our results revealed 7 distinct clusters, with cluster 3 showing the highest potential for differentiation. Clusters 0 to 2 displayed features similar to fibroblasts. The trajectory route of the cell state transition from cluster 3 to clusters 0, 1, and 2 indicated the distinct nature of cell differentiation. PDLSCs had a higher proportion of cells (78.6%) at the G1 phase, while DPSCs had a higher proportion of cells at the S and G2/M phases (36.1%), mirroring the lower cell proliferation capacity of PDLSCs than DPSCs. Our study suggested the heterogeneity of stemness across PDLSCs and DPSCs, the similarities of these 2 stem cell compartments to be potentially integrated for regenerative strategies, and the distinct features between them potentially particularized for organ-specific functions of the dental pulp and periodontal ligament for a targeted regenerative dental tissue repair and other regeneration therapies.

Cellular heterogeneity and extracellular matrix (ECM) stiffening have been shown to be drivers of breast cancer invasiveness. Here, we examine how stiffness-dependent crosstalk between cancer cells and mesenchymal stem cells (MSCs) within an evolving tumor microenvironment regulates cancer invasion. By analyzing previously published single-cell RNA sequencing datasets, we establish the existence of a subpopulation of cells in primary tumors, secondary sites and circulatory tumor cell clusters of highly aggressive triple-negative breast cancer (TNBC) that co-express MSC and cancer-associated fibroblast (CAF) markers. By using hydrogels with stiffnesses of 0.5, 2 and 5 kPa to mimic different stages of ECM stiffening, we show that conditioned medium from MDA-MB-231 TNBC cells cultured on 2 kPa gels, which mimic the pre-metastatic stroma, drives efficient MSC chemotaxis and induces stable differentiation of MSC-derived CAFs in a TGFβ (TGFB1)- and contractility-dependent manner. In addition to enhancing cancer cell proliferation, MSC-derived CAFs on 2 kPa gels maximally boost local invasion and confer resistance to flow-induced shear stresses. Collectively, our results suggest that homing of MSCs at the pre-metastatic stage and their differentiation into CAFs actively drives breast cancer invasion and metastasis in TNBC.

Salivary gland (SG) extracellular matrix (ECM) has a major influence on tissue development, homeostasis, and tissue regeneration after injury. During aging, disease, and physical insult, normal remodeling of the SG microenvironment (i.e. ECM) becomes dysregulated, leading to alterations in matrix composition which disrupt tissue architecture/structure, alter cell activity, and negatively impact gland function. Matrix metalloproteinases (MMPs) are a large and diverse family of metalloendopeptidases which play a major role in matrix degradation and are intimately involved in regulating development and cell function; dysregulation of these enzymes leads to the production of a fibrotic matrix. In the SG this altered fibrotic ECM (or cell microenvironment) negatively impacts normal cell function and the effectiveness of gene and stem cell therapies which serve as a foundation for many SG regenerative therapies. For this reason, prospective regenerative strategies should prioritize the maintenance and/or restoration of a healthy SG ECM. Mesenchymal stem cells (MSCs) have great potential for mitigating damage to the SG microenvironment by ameliorating inflammation, reducing fibrosis, and repairing the damaged milieu of extracellular regulatory cues, including the matrix. This review addresses our current understanding of the impact of aging and disease on the SG microenvironment and suggests critical deficiencies and opportunities in ECM-targeted therapeutic interventions.

The ability of cells to sense and respond to changes in mechanical environment is vital in conditions of organ injury when the architecture of normal tissues is disturbed or lost. Among the various cellular players that respond to injury, fibroblasts take center stage in re-establishing tissue integrity by secreting and organizing extracellular matrix into stabilizing scar tissue. Activation, activity, survival, and death of scar-forming fibroblasts are tightly controlled by mechanical environment and proper mechanotransduction ensures that fibroblast activities cease after completion of the tissue repair process. Conversely, dysregulated mechanotransduction often results in fibroblast over-activation or persistence beyond the state of normal repair. The resulting pathological accumulation of extracellular matrix is called fibrosis, a condition that has been associated with over 40% of all deaths in the industrialized countries. Consequently, elements in fibroblast mechanotransduction are scrutinized for their suitability as anti-fibrotic therapeutic targets. We review the current knowledge on mechanically relevant factors in the fibroblast extracellular environment, cell-matrix and cell-cell adhesion structures, stretch-activated membrane channels, stress-regulated cytoskeletal structures, and co-transcription factors. We critically discuss the targetability of these elements in therapeutic approaches and their progress in pre-clinical and/or clinical trials to treat organ fibrosis.

The mechanism of action of MicroRNA-30a(miR-30a) and Snail, a transcription factor, in silica(SiO2) dust-induced pulmonary EMT and secondary pulmonary fibrosis remains elusive. In this study, the cellular EMT model induced by the stimulation of A549 cells with SiO2 was established. A549 cells were transfected with miR-30a mimic and miR-30a inhibitor and the SNAIL gene was silenced to examine the mechanism of miR-30a targeting Snail to regulate silica dust-induced EMT. The results showed that 50 μg/mL SiO2 stained A549 cells for 24 h could induce EMT in A549 cells. Exposure of A549 cells to SiO2 dust decreased miR-30a expression, as well as mRNA and protein expression levels of E-cad. Conversely, SiO2 exposure increased mRNA and protein expression levels of α-SMA, vimentin, and Snail. The miR-30a mimic upregulated mRNA and protein expression levels of E-cadherin in SiO2-induced A549 cells, while downregulating mRNA and protein expression levels of α-SMA, vimentin and Snail. MiR-30a inhibitors have the opposite effect. Silencing the SNAIL gene, followed by SiO2 dust-induced stimulation of A549 cells, could enhance mRNA and protein expression levels of E-cad, whereas those of α-SMA and vimentin were reduced. Altogether, we found that miR-30a directly targeted Snail and inhibited its expression, thereby delaying silica induced pulmonary EMT.

Hematologic malignancies comprise a diverse range of blood, bone marrow, and organ-related disorders that present significant challenges due to drug resistance, relapse, and treatment failure. Cancer-associated fibroblasts (CAFs) represent a critical component of the tumor microenvironment (TME) and have recently emerged as potential therapeutic targets. In this comprehensive review, we summarize the latest findings on the roles of CAFs in various hematologic malignancies, including acute leukemia, multiple myeloma, chronic lymphocytic leukemia, myeloproliferative neoplasms, and lymphoma. We also explore their involvement in tumor progression, drug resistance, and the various signaling pathways implicated in their activation and function. While the underlying mechanisms and the existence of multiple CAF subtypes pose challenges, targeting CAFs and their associated pathways offers a promising avenue for the development of innovative treatments to improve patient outcomes in hematologic malignancies.

The esophagus exhibits peristalsis via contraction of circularly and longitudinally aligned smooth muscles, and esophageal replacement is required if there is a critical-sized wound. In this study, we proposed to reconstruct esophageal tissues using cell electrospinning (CE), an advanced technique for encapsulating living cells into fibers that allows control of the direction of fiber deposition. After treatment with transforming growth factor-β, mesenchymal stem cell-derived smooth muscle cells (SMCs) were utilized for cell electrospinning or three-dimensional bioprinting to compare the effects of aligned micropatterns on cell morphology. CE resulted in SMCs with uniaxially arranged and elongated cell morphology with upregulated expression levels of SMC-specific markers, including connexin 43, smooth muscle protein 22 alpha (SM22α), desmin, and smoothelin. When SMC-laden nanofibrous patches were transplanted into a rat esophageal defect model, the SMC patch promoted regeneration of esophageal wounds with an increased number of newly formed blood vessels and enhanced the SMC-specific markers of SM22α and vimentin. Taken together, CE with its advantages, such as guidance of highly elongated, aligned cell morphology and accelerated SMC differentiation, can be an efficient strategy to reconstruct smooth muscle tissues and treat esophageal perforation.

Myofibroblasts are the construction workers of wound healing and repair damaged tissues by producing and organizing collagen/extracellular matrix (ECM) into scar tissue. Scar tissue effectively and quickly restores the mechanical integrity of lost tissue architecture but comes at the price of lost tissue functionality. Fibrotic diseases caused by excessive or persistent myofibroblast activity can lead to organ failure. This review defines myofibroblast terminology, phenotypic characteristics, and functions. We will focus on the central role of the cell, ECM, and tissue mechanics in regulating tissue repair by controlling myofibroblast action. Additionally, we will discuss how therapies based on mechanical intervention potentially ameliorate wound healing outcomes. Although myofibroblast physiology and pathology affect all organs, we will emphasize cutaneous wound healing and hypertrophic scarring as paradigms for normal tissue repair versus fibrosis. A central message of this review is that myofibroblasts can be activated from multiple cell sources, varying with local environment and type of injury, to either restore tissue integrity and organ function or create an inappropriate mechanical environment.

Jinshui Huanxian formula (JHF) ameliorates idiopathic pulmonary fibrosis patients. Active compounds, including icariin, isoliquiritigenin, nobiletin, peimine, and paeoniflorin, deriving from JHF were combined as effective-component compatibility ECC of JHF II (ECC-JHF II), which is an effective therapeutic strategy for pulmonary fibrosis (PF) induced by bleomycin (BLM) in rats.

This study aimed to explore the underlying mechanism of ECC-JHF II on pulmonary fibrosis.

A model of PF in rats was established through intratracheal instillation of BLM. Pulmonary function, pathological changes, and collagen deposition were examined. The gene and protein expressions in fibroblast activation were detected by quantitative real-time PCR and western blotting respectively.

ECC-JHF II significantly improved BLM-induced PF in rats, manifested as decreased collagen deposition, reduced pathological damage and improved pulmonary function. Furthermore, ECC-JHF II inhibited fibroblast activation by reducing the expression of α-smooth muscle actin (α-SMA) and fibronectin. We analyzed the targets of ECC-JHF II and differentially expressed genes (DEGs) of fibroblast activation induced by transforming growth factor-β1 (TGF-β1) and found that ECC-JHF II might regulate fibroblast activation by EGFR, PI3K-Akt or mTOR signaling pathway. In vitro experiments, we also found that ECC-JHF II suppressed the mTOR pathway, such as downregulating the phosphorylation levels of p70S6K in fibroblast activation induced by TGF-β1. After activating mTOR signaling, the inhibition of ECC-JHF II on fibroblast activation was blocked. These results suggested that ECC-JHF II potently ameliorated pulmonary fibrosis in rats and effectively suppressed fibroblast activation by interfering with mTOR signaling.

We combined transcriptomics with the network analysis to predict the mechanism underlying ECC-JHF II suppression of fibroblast activation. In summary, ECC-JHF II improved BLM-induced pulmonary fibrosis, which might be associated with the suppression of fibroblast activation by inhibiting the mTOR signaling.