Enhanced bone regeneration via endochondral ossification using Exendin-4-modified mesenchymal stem cells.

Nonunions and delayed unions pose significant challenges in orthopedic treatment, with current therapies often proving inadequate. Bone tissue engineering (BTE), particularly through endochondral ossification (ECO), emerges as a promising strategy for addressing critical bone defects. This study introduces mesenchymal stem cells overexpressing Exendin-4 (MSC-E4), designed to modulate bone remodeling via their autocrine and paracrine functions. We established a type I collagen (Col-I) sponge-based in vitro model that effectively recapitulates the ECO pathway. MSC-E4 demonstrated superior chondrogenic and hypertrophic differentiation and enhanced the ECO cell fate in single-cell sequencing analysis. Furthermore, MSC-E4 encapsulated in microscaffold, effectively facilitated bone regeneration in a rat calvarial defect model, underscoring its potential as a therapeutic agent for bone regeneration. Our findings advocate for MSC-E4 within a BTE framework as a novel and potent approach for treating significant bone defects, leveraging the intrinsic ECO process.

Gelatin-based 3D biomimetic scaffolds platform potentiates culture of cancer stem cells in esophageal squamous cell carcinoma.

Cancer stem cells (CSCs) are crucial for tumorigenesis, metastasis, and therapy resistance in esophageal squamous cell carcinoma (ESCC). To further elucidate the mechanism underlying characteristics of CSCs and develop CSCs-targeted therapy, an efficient culture system that could expand and maintain CSCs is needed. CSCs reside in a complex tumor microenvironment, and three-dimensional (3D) culture systems of biomimetic scaffolds are expected to better support the growth of CSCs by recapitulating the biophysical properties of the extracellular matrix (ECM). Here, we established gelatin-based 3D biomimetic scaffolds mimicking the stiffness and collagen content of ESCC, which could enrich ESCC CSCs efficiently. Biological changes of ESCC cells laden in scaffolds with three different viscoelasticity emulating physiological stiffness of esophageal tissues were thoroughly investigated in varied aspects such as cell morphology, viability, cell phenotype markers, and transcriptomic profiling. The results demonstrated the priming effects of viscoelasticity on the stemness of ESCC. The highly viscous scaffolds (G': 6-403 Pa; G'': 2-75 Pa) better supported the enrichment of ESCC CSCs, and the TGF-beta signaling pathway might be involved in regulating the stemness of ESCC cells. Compared to two-dimensional (2D) cultures, highly viscous scaffolds significantly promoted the clonal expansion of ESCC cells in vitro and tumor formation ability in vivo. Our findings highlight the crucial role of biomaterials' viscoelasticity for the 3D culture of ESCC CSCs in vitro, and this newly-established culture system represents a valuable platform to support their growth, which could facilitate the CSCs-targeted therapy in the future.

Cell mediated ECM-degradation as an emerging tool for anti-fibrotic strategy.

Investigation into the role of cells with respect to extracellular matrix (ECM) remodeling is still in its infancy. Particularly, ECM degradation is an indispensable process during the recovery from fibrosis. Cells with ECM degradation ability due to the secretion of various matrix metalloproteinases (MMPs) have emerged as novel contributors to the treatment of fibrotic diseases. In this review, we focus on the ECM degradation ability of cells associated with the repertoire of MMPs that facilitate the attenuation of fibrosis through the inhibition of ECM deposition. Besides, innovative approaches to engineering and characterizing cells with degradation ability, as well as elucidating the mechanism of the ECM degradation, are also illustrated. Studies conducted to date on the use of cell-based degradation for therapeutic purposes to combat fibrosis are summarized. Finally, we discuss the therapeutic potential of cells with high degradation ability, hoping to bridge the gap between benchside research and bedside applications in treating fibrotic diseases.

In situ self-assembled organoid for osteochondral tissue regeneration with dual functional units.

The regeneration of hierarchical osteochondral units is challenging due to difficulties in inducing spatial, directional and controllable differentiation of mesenchymal stem cells (MSCs) into cartilage and bone compartments. Emerging organoid technology offers new opportunities for osteochondral regeneration. In this study, we developed gelatin-based microcryogels customized using hyaluronic acid (HA) and hydroxyapatite (HYP), respectively for inducing cartilage and bone regeneration (denoted as CH-Microcryogels and OS-Microcryogels) through in vivo self-assembly into osteochondral organoids. The customized microcryogels showed good cytocompatibility and induced chondrogenic and osteogenic differentiation of MSCs, while also demonstrating the ability to self-assemble into osteochondral organoids with no delamination in the biphasic cartilage-bone structure. Analysis by mRNA-seq showed that CH-Microcryogels promoted chondrogenic differentiation and inhibited inflammation, while OS-Microcryogels facilitated osteogenic differentiation and suppressed the immune response, by regulating specific signaling pathways. Finally, the in vivo engraftment of pre-differentiated customized microcryogels into canine osteochondral defects resulted in the spontaneous assembly of an osteochondral unit, inducing simultaneous regeneration of both articular cartilage and subchondral bone. In conclusion, this novel approach for generating self-assembling osteochondral organoids utilizing tailor-made microcryogels presents a highly promising avenue for advancing the field of tissue engineering.

Advanced glycation end-products as mediators of the aberrant crosslinking of extracellular matrix in scarred liver tissue.

The extracellular matrix of cirrhotic liver tissue is highly crosslinked. Here we show that advanced glycation end-products (AGEs) mediate crosslinking in liver extracellular matrix and that high levels of crosslinking are a hallmark of cirrhosis. We used liquid chromatography-tandem mass spectrometry to quantify the degree of crosslinking of the matrix of decellularized cirrhotic liver samples from patients and from two mouse models of liver fibrosis and show that the structure, biomechanics and degree of AGE-mediated crosslinking of the matrices can be recapitulated in collagen matrix crosslinked by AGEs in vitro. Analyses via cryo-electron microscopy and optical tweezers revealed that crosslinked collagen fibrils form thick bundles with reduced stress relaxation rates; moreover, they resist remodelling by macrophages, leading to reductions in their levels of adhesion-associated proteins, altering HDAC3 expression and the organization of their cytoskeleton, and promoting a type II immune response of macrophages. We also show that rosmarinic acid inhibited AGE-mediated crosslinking and alleviated the progression of fibrosis in mice. Our findings support the development of therapeutics targeting crosslinked extracellular matrix in scarred liver tissue.

Injectable bone marrow microniches by co-culture of HSPCs with MSCs in 3D microscaffolds promote hematopoietic reconstitution from acute lethal radiation.

Hematopoietic syndrome of acute radiation syndrome (h-ARS) is an acute illness resulted from the damage of bone marrow (BM) microenvironment after exposure to radiation. Currently, the clinical management of h-ARS is limited to medication-assisted treatment, while there is still no specific therapy for the hematopoietic injury from high-dose radiation exposure. Our study aimed to assemble biomimetic three-dimensional (3D) BM microniches by co-culture of hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem cells (MSCs) in porous, injectable and viscoelastic microscaffolds in vitro. The biodegradable BM microniches were then transplanted in vivo into the BM cavity for the treatment of h-ARS. We demonstrated that the maintenance of HSPCs was prolonged by co-culture with MSCs in the porous 3D microscaffolds with 84 µm in pore diameter and 11.2 kPa in Young's modulus compared with 2D co-culture system. Besides, the minimal effective dose and therapeutic effects of the BM microniches were investigated on a murine model of h-ARS, which showed that the intramedullary cavity-injected BM microniches could adequately promote hematopoietic reconstitution and mitigate death from acute lethal radiation with a dose as low as 1000 HSPCs. Furthermore, the mRNA expression of Notch1 and its downstream target gene Hes1 of HSPCs were increased when co-cultured with MSCs, while the Jagged1 expression of the co-cultured MSCs was upregulated, indicating the significance of Notch signaling pathway in maintenance of HSPCs. Collectively, our findings provide evidence that biomimetic and injectable 3D BM microniches could maintain HSPCs, promote hematopoiesis regeneration and alleviate post-radiation injury, which provides a promising approach to renovate conventional HSPCs transplantation for clinical treatment of blood and immune disorders.

Scar-Degrading Endothelial Cells as a Treatment for Advanced Liver Fibrosis.

Deposition of extracellular matrix (ECM) in the liver is an important feature of liver cirrhosis. Recovery from liver cirrhosis is physiologically challenging, partially due to the ECM in scar tissue showing resistance to cell-mediated degradation by secreted matrix metalloproteinases (MMPs). Here, a cell-mediated ECM-degradation screening system (CEDSS) in vitro is constructed for high-throughput searching for cells with tremendous degradation ability. ECM-degrading liver sinusoidal endothelial cells (dLSECs) are screened using CEDSS, which exhibit 17 times the ability to degrade collagen when compared to other cells. The degradation ability of dLSECs is mediated by the upregulation of MMP9. In particular, mRNA expression of MMP9 shows an 833-fold increase in dLSECs compared to normal endothelial cells (nLSECs), and MMP9 is regulated by transcription factor c-Fos. In vivo, single intrasplenic injection of dLSECs alleviates advanced liver fibrosis in mice, while intraperitoneal administration of liver-targeting peptide-modified dLSECs shows enhanced fibrosis-targeting effects. Degradative human umbilical vein endothelial cells (dHUVECs) prove their enhanced potential of clinical translation. Together, these results highlight the potential of ECM-degrading endothelial cells in alleviating advanced liver fibrosis, thus providing remarkable insights in the development of ECM-targeting therapeutics.

Mesenchymal Stem Cells (MSCs): A Novel Therapy for Type 2 Diabetes.

Although plenty of drugs are currently available for type 2 diabetes mellitus (T2DM), a subset of patients still failed to restore normoglycemia. Recent studies proved that symptoms of T2DM patients who are unresponsive to conventional medications could be relieved with mesenchymal stem/stromal cell (MSC) therapy. However, the lack of systematic summary and analysis for animal and clinical studies of T2DM has limited the establishment of standard guidelines in anti-T2DM MSC therapy. Besides, the therapeutic mechanisms of MSCs to combat T2DM have not been thoroughly understood. In this review, we present an overview of the current status of MSC therapy in treating T2DM for both animal studies and clinical studies. Potential mechanisms of MSC-based intervention on multiple pathological processes of T2DM, such as ß-cell exhaustion, hepatic dysfunction, insulin resistance, and systemic inflammation, are also delineated. Moreover, we highlight the importance of understanding the pharmacokinetics (PK) of transplanted cells and discuss the hurdles in MSC-based T2DM therapy toward future clinical applications.

Engineered meatballs via scalable skeletal muscle cell expansion and modular micro-tissue assembly using porous gelatin micro-carriers.

The emerging field of cultured meat faces several technical hurdles, including the scale-up production of quality muscle and adipose progenitor cells, and the differentiation and bioengineering of these cellular materials into large, meat-like tissue. Here, we present edible, 3D porous gelatin micro-carriers (PoGelat-MCs), as efficient cell expansion scaffolds, as well as modular tissue-engineering building blocks for lab-grown meat. PoGelat-MC culture in spinner flasks, not only facilitated the scalable expansion of porcine skeletal muscle satellite cells and murine myoblasts, but also triggered their spontaneous myogenesis, in the absence of myogenic reagents. Using 3D-printed mold and transglutaminase, we bio-assembled pork muscle micro-tissues into centimeter-scale meatballs, which exhibited similar mechanical property and higher protein content compared to conventional ground pork meatballs. PoGelat-MCs also supported the expansion and differentiation of 3T3L1 murine pre-adipocytes into mature adipose micro-tissues, which could be used as modular assembly unit for engineered fat-containing meat products. Together, our results highlight PoGelat-MCs, in combination with dynamic bioreactors, as a scalable culture system to produce large quantity of highly-viable muscle and fat micro-tissues, which could be further bio-assembled into ground meat analogues.

Mechanical communication in fibrosis progression.

Mechanical hallmarks of fibrotic microenvironments are both outcomes and causes of fibrosis progression. Understanding how cells sense and transmit mechanical cues in the interplay with extracellular matrix (ECM) and hemodynamic forces is a significant challenge. Recent advances highlight the evolvement of intracellular mechanotransduction pathways responding to ECM remodeling and abnormal hemodynamics (i.e., low and disturbed shear stress, pathological stretch, and increased pressure), which are prevalent biomechanical characteristics of fibrosis in multiple organs (e.g., liver, lung, and heart). Here, we envisage the mechanical communication in cell-ECM, cell-hemodynamics and cell-ECM-cell crosstalk (namely paratensile signaling) during fibrosis expansion. We also provide a comprehensive overview of in vitro and in silico engineering systems for disease modeling that will aid the identification and prediction of mechano-based therapeutic targets to ameliorate fibrosis progression.

Exendin-4 gene modification and microscaffold encapsulation promote self-persistence and antidiabetic activity of MSCs.

Mesenchymal stem cell (MSC)-based therapy to combat diabetic-associated metabolic disorders is hindered by impoverished cell survival and limited therapeutic effects under high glucose stress. Here, we genetically engineered MSCs with Exendin-4 (MSC-Ex-4), a glucagon-like peptide-1 (GLP-1) analog, and demonstrated their boosted cellular functions and antidiabetic efficacy in the type 2 diabetes mellitus (T2DM) mouse model. Mechanistically, MSC-Ex-4 achieved self-augmentation and improved survival under high glucose stress via autocrine activation of the GLP-1R-mediated AMPK signaling pathway. Meanwhile, MSC-Ex-4-secreted Exendin-4 suppressed senescence and apoptosis of pancreatic ß cells through endocrine effects, while MSC-Ex-4-secreted bioactive factors (e.g., IGFBP2 and APOM) paracrinely augmented insulin sensitivity and decreased lipid accumulation in hepatocytes through PI3K-Akt activation. Furthermore, we encapsulated MSC-Ex-4 in 3D gelatin microscaffolds for single-dose administration to extend the therapeutic effect for 3 months. Together, our findings provide mechanistic insights into Exendin-4-mediated MSCs self-persistence and antidiabetic activity that offer more effective MSC-based therapy for T2DM.

Cell engineering techniques improve pharmacology of cellular therapeutics.

Despite the rapid growth of clinical trials for cellular therapy worldwide, their clinical success is still afflicted with formidable challenges demanding conceptual and technological overhaul. Pharmacology, which is conventionally divided into pharmacokinetics (PK) and pharmacodynamics (PD) in drug discovery have emerged as a prominent research direction to elucidate the cell fate and ensure the efficacy and safety of the therapeutic cells. Herein, we concisely present the dilemmas of cellular therapies, the concept of cell pharmacology, and the advances in cell engineering that leverage the cell formulation technologies to modulate cellular PK/PD for development of more cogent and versatile cell-based therapies.

Structure of the ligand-binding domain (LBD) of human androgen receptor in complex with a selective modulator LGD2226.

The androgen receptor (AR) is a ligand-inducible steroid hormone receptor that mediates androgen action, determining male sexual phenotypes and promoting spermatogenesis. As the androgens play a dominant role in male sexual development and function, steroidal androgen agonists have been used clinically for some years. However, there is a risk of potential side effects and most steroidal androgens cannot be dosed orally, which limits the use of these substances. 1,2-Dihydro-6-N,N-bis(2,2,2-trifluoroethyl)amino-4-trifluoromethyl-2-quinolinone (LGD2226) is a synthetic nonsteroidal ligand and a novel selective AR modulator. The crystal structure of the complex of LGD2226 with the androgen receptor ligand-binding domain (AR LBD) at 2.1 A was solved and compared with the structure of the AR LBD-R1881 complex. It is hoped that this will aid in further explaining the selectivity of LGD2226 observed in in vitro and in vivo assays and in developing more selective and effective therapeutic agents.

[Study on quality analysis of essential oil from twig and leaf of Baeckea frutescens].

OBJECTIVE: To provide scientific methods for quality criterion by studying the chemical components of essential oil from Baeckea frutescens. METHOD: The chemical components of essential oil from B. frutescens were identified by GC-MS-DS, TLC and capillary GC. The relative contents of main components were determined by area normalization. RESULT: More than 50 peaks were separated, and 38 components were identified, which accounted for over 94% of the total GC peaks areas of the essential oil. The methods for quality evaluation of essential oil from B. frutescens by TLC and capillary GC were established. CONCLUSION: The chemical components of essential oil from B. frutescens collected from different habitats and collecting periods have common characteristics as well as differences. Some components, such as linalool, can be used as a standard and chromatography fingerprint to analyze the quality of essential oil from B. frutescens.