Matrix Formation Research Articles

20(S)-Ginsenoside Rh2 overcomes gemcitabine resistance in pancreatic cancer by inhibiting LAMC2-Modulated ABC transporters

IntroductionGemcitabine (GEM) is the first-line drug for pancreatic ductal adenocarcinoma (PDAC), but drug resistance severely restricts its chemotherapeutic efficacy. Laminin subunit γ2 (LAMC2) plays a crucial role in extracellular matrix formation in the development of GEM-resistance. However, the biological function of LAMC2 in GEM resistance and its molecular mechanisms are still unclear. 20(S)-Ginsenoside Rh2 (Rh2), one of the principal active components isolated from Ginseng Radix et Rhizoma, possesses strong anti-tumor effects. However, the effects of Rh2 on overcoming GEM resistance and its action mechanisms remain to be elucidated. ObjectivesThis study aimed to determine the efficacy of Rh2 on overcoming GEM resistance and to explore its underlying molecular mechanisms. MethodsClinical study, Western blotting, publicly available databasesand bioinformatic analyses were performed to investigate the protein expression of LAMC2 in the GEM-resistant PDAC patients and the acquired GEM-resistant PDAC cells. Then, the effects of Rh2 on overcoming the GEM resistance in PDAC were evaluated both in vitro and in vivo. Stable silencing or overexpression of LAMC2 in the GEM-resistant PDAC cells were established for validating the role of LAMC2 on Rh2 overcoming the GEM resistance in PDAC. ResultsThe protein expression of LAMC2 was markedly increased in the GEM-resistant PDAC patient biopsies compared to the sensitive cases. The protein expression of LAMC2 was significantly higher in the acquired GEM-resistant PDAC cells than that in their parental cells. Rh2 enhanced the chemosensitivity of GEM in the GEM-resistant PDAC cells, and inhibited the tumor growth of Miapaca-2-GR cell-bearing mice and Krastm4TyjTrp53tm1BrnTg (Pdx1-cre/Esr1*) #Dam/J (KPC) mice. Rh2 effectively reversed the GEM resistance in Miapaca-2-GR and Capan-2-GR cells by inhibiting LAMC2 expression through regulating the ubiquitin–proteasome pathway. Knockdown of LAMC2 enhanced the chemosensitivity of GEM and the effects of Rh2 on overcoming the GEM resistance in PDAC cells and the orthotopic PDAC mouse model. Conversely, LAMC2 overexpression aggravated the chemoresistance of GEM and abolished the effects of Rh2 on overcoming GEM resistance via modulating ATP-binding cassette (ABC) transporters leading to the active GEM efflux. ConclusionsLAMC2 plays an important role in the GEM resistance in PDAC, and Rh2 is a potential adjuvant for overcoming the chemoresistance of GEM in PDAC.

Real-time acid production and extracellular matrix formation in mature biofilms of three Streptococcus mutans strains with special reference to xylitol

BackgroundAcidogenicity and production of an extracellular matrix (ECM) are important virulence factors for the dental caries-associated bacteria, such as Streptococcus mutans, that live in biofilms on tooth surface. The ECM protects the bacteria from the flushing and buffering effects of saliva resulting in highly acidic microenvironments inside the biofilm. Materials and methodsIn this in vitro study, we applied real-time assays to follow biofilm formation and pH decrease in a growth medium and saliva by three S. mutans strains, as well as acid neutralization inside the mature biofilm. Results were compared with the biofilm composition. Effects of a non-fermentable polyol, xylitol, on acid production and acid neutralization in mature biofilms were evaluated by real-time pH measurements and confocal microscopy. ResultsCombination of real-time pH measurements with biofilm accumulation assays revealed growth media dependent differences in the pH decrease and biofilm accumulation, as well as strain differences in acid production and biofilm formation but not in the buffer diffusion through ECM. The presence of xylitol reduced the pH drop during biofilm formation of all strains. In addition, with strain Ingbritt xylitol reduced the amount of ECM in biofilm, which increased the rate of acid neutralization inside the biofilm after buffer exposure. ConclusionOur results stress the importance of biofilm matrix in creating the acidic environment inside a S. mutans biofilm, especially in the presence of saliva. In addition, our results suggest a novel mechanism of xylitol action. The observed increase in the permeability of the S. mutans ECM after xylitol exposure may allow acid-neutralizing saliva to reach deeper layer of the biofilms and thus, in part, explain previous clinical observations of reduced plaque acidogenicity after frequent xylitol use.

Interleukin-4-Loaded Gelatin Methacryloyl Hydrogel Promotes Subcutaneous Chondrogenesis of Engineered Auricular Cartilage in a Rabbit Model.

Tissue engineering technology offers a promising solution for ear reconstruction; however, it faces the challenge of foreign body reaction and neocartilage malformation. This study investigates the impact of interleukin-4 (IL-4), an anti-inflammatory factor, on cartilage regeneration of hydrogel encapsulating autologous auricular chondrocytes in a rabbit subcutaneous environment. Initially, we assessed the influence of IL-4 on chondrocyte proliferation and determined the appropriate concentration using the CCK-8 test in vitro. Subsequently, we loaded IL-4 into gelatin methacryloyl (GelMA) hydrogel containing chondrocytes and measured its release profile through ELISA. The constructs were then implanted autologously into rabbits' subcutis, and after 3, 7, 14, and 28 days, cartilage matrix formation was evaluated by histological examinations, and gene expression levels were detected by qRT-PCR. Results demonstrated that IL-4 promotes chondrocyte proliferation in vitro, and maximum release from constructs occurred during the first week. In the rabbit subcutaneous implantation model, IL-4-loaded constructs (20 ng/mL) maintained a superior chondrocytic phenotype compared to controls with increased expression of anti-inflammatory factors. These findings highlight IL-4 as a potential strategy for promoting chondrogenesis in a subcutaneous environment and improving ear reconstruction.

A Concise Overview of Biomaterials and the Diverse Uses of Nano Hydroxyapatite across Different Fields

Bruck (1900) defined biomaterials as substances, whether synthetic or natural, designed for use in prosthetic, diagnostic, therapeutic, and storage applications involving contact with tissue, blood, and biological fluids. They must not harm living organisms or their components. Biomaterials interact with tissues and bodily fluids over extended periods. They fall into two main categories: natural biomaterials sourced from plants or animals, which can enhance, replace, or restore biological tissues with properties like in-vivo immunomodulation, detoxification, and biomimicry (e.g., proteins, gelatin, alginate, silk, fibrin, cellulose, chitin, chitosan); and synthetic biomaterials, manufactured in laboratories or industries by human effort (e.g., metals, ceramics, polymers). Natural and synthetic biomaterials notably differ in their ability to provoke host responses upon implantation, including inflammation, matrix formation, blood interactions, and the development of granulation or fibrous capsules

Piezo channels modulate human lung fibroblast function.

Bronchial airways and lung parenchyma undergo both static and dynamic stretch in response to normal breathing as well as in the context of insults such as mechanical ventilation (MV) or in diseases such as asthma and chronic obstructive pulmonary disease (COPD) which lead to airway remodeling involving increased extracellular matrix (ECM) production. Here, the role of fibroblasts is critical, but the relationship between stretch- and fibroblast-induced ECM remodeling under these conditions is not well-explored. Piezo (PZ) channels play a role in mechanotransduction in many cell and organ systems, but their role in mechanical stretch-induced airway remodeling is not known. To explore this, we exposed human lung fibroblasts to 10% static stretch on a background of 5% oscillations for 48 h, with no static stretch considered controls. Collagen I, fibronectin, alpha-smooth muscle actin (α-SMA), and Piezo 1 (PZ1) expression was determined in the presence or absence of Yoda1 (PZ1 agonist) or GsMTx4 (PZ1 inhibitor). Collagen I, fibronectin, and α-SMA expression was increased by stretch and Yoda1, whereas pretreatment with GsMTx4 or knockdown of PZ1 by siRNA blunted this effect. Acute stretch in the presence and absence of Yoda1 demonstrated activation of the ERK pathway but not Smad. Measurement of [Ca2+]i responses to histamine showed significantly greater responses following stretch, effects that were blunted by knockdown of PZ1. Our findings identify an essential role for PZ1 in mechanical stretch-induced production of ECM mediated by ERK phosphorylation and Ca2+ influx in lung fibroblasts. Targeting PZ channels in fibroblasts may constitute a novel approach to ameliorate airway remodeling by decreasing ECM deposition.NEW & NOTEWORTHY The lung is an inherently mechanosensitive organ that can respond to mechanical forces in adaptive or maladaptive ways, including via remodeling resulting in increased fibrosis. We explored the mechanisms that link mechanical forces to remodeling using human lung fibroblasts. We found that mechanosensitive Piezo channels increase with stretch and mediate extracellular matrix formation and the fibroblast-to-myofibroblast transition that occurs with stretch. Our data highlight the importance of Piezo channels in lung mechanotransduction toward remodeling.

Development of Edible Films Based on Nostoc and Modified Native Potato Starch and Their Physical, Mechanical, Thermal, and Microscopic Characterization.

Considering the potential of biopolymers from underutilized Andean sources in Peru to improve the characteristics of edible films, this work aimed to evaluate the formation of a polymeric matrix composed of Nostoc and modified potato starch for the formulation of edible films for food coating. The effects of polymer matrix ratio and drying temperature on films obtained by thermoforming were studied, determining the water vapor permeability and mechanical properties using a multifactorial design. Additionally, thermal properties were characterized by TGA and DSC, and structural properties by FT-IR and scanning electron microscopy. The results showed that the films exhibited lower solubility, lighter hues, better water vapor resistance, higher tensile strength, and improved thermal stability with increasing modified starch content. The formulation with higher Nostoc content exhibited a more homogeneous surface according to microscopy images, and no new chemical bonds were formed by adding modified starch and Nostoc to the polymer matrix, according to FT-IR spectra. These findings are promising and suggest using Nostoc for elaborating edible films composed of native and modified starch from native Andean potatoes as bio-based materials with potential application in the food industry.

Liquid Crystalline Hydroxyapatite Nanorods Orchestrate Hierarchical Bone-Like Mineralization.

Bone matrix exhibits exceptional mechanical properties due to its unique nanocomposite structure of type I collagen fibrils and hydroxyapatite (HAp) nanoparticles in hierarchical liquid crystalline (LC) order. However, the regeneration mechanism of this LC structure is elusive. This study investigates the role of the LC structure of HAp nanorods in guiding aligned mineralization and its underlying molecular mechanism. A unidirectionally oriented LC phase of HAp nanorods is developed through engineering-assisted self-assembling. This is used to study the growth direction of long-range aligned extracellular matrix (ECM) and calcium deposit formation during the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. It is found that 2 key regulatory genes, COL1A1 and COL4A6, lead to the formation of aligned ECM. Activation of the PI3K-Akt pathway enhances osteogenesis and promotes ordered calcium deposits. This study provides evidence for elucidating the mechanism of LC-induced ordered calcium deposition at hierarchical levels spanning from the molecular to macro-scale, as well as the switch from ordered to disordered mineralization. These findings illuminate bone regeneration, contribute to the development of biomimetic artificial bone with long-range ordered structures, and suggest a basis for therapeutic targeting of microstructure-affected bone disorders and the broader field of cell-ECM interactions.

Electrospun PLGA composite hydrogel scaffold loaded with 3D extracellular vesicles for nasal septal cartilage defect repair

The nasal septum plays an important role in the growth and support of the human nose, and defects can cause nasal deformities. Extracellular vesicles (EVs) have shown great potential in tissue repair, especially cartilage repair, and stem cell EVs are widely used in the repair of articular cartilage defects but have not been reported in the repair of nasal septal cartilage defects. At the same time, due to the low yield of EVs, which are easy to lose during in-situ injection, better preparation methods are needed to obtain EVs and more suitable carriers for the sustained release of EVs in wounds. Firstly, swelling and degradation experiments were conducted on the scaffold, as well as mechanical performance testing, including observation of the scaffold morphology under SEM. Subsequently, in vitro cell experiments were conducted to evaluate the ability of 3D EVs to promote chondrocyte proliferation, migration, and extracellular matrix formation. Finally, Gel-PLGA loaded with EVs was implanted into the nasal septum defect site of rabbits in vivo animal experiments to observe the repair effect on the defect. In vitro experiments showed that the biological scaffold exhibited good biocompatibility and could effectively promote the proliferation and migration of chondrocytes. In vivo, a composite biological scaffold loaded with EVs was implanted into the nasal septum defect of rabbits, and the tissues were tested at 6 and 12 weeks after surgery. The results indicate that composite scaffolds loaded with EVs can effectively promote the repair of defect sites. The results showed that 3D EVs could promote tissue repair and healing, which provided a new idea for the clinical treatment of nasal septal defects.

Role of Growth Factors in Nasal Cartilage Development and Molding: A Comprehensive Review.

This review aims to investigate the properties of growth factors concerning the morphogenesis and development of nasal cartilage, which is fundamentally important for facial form and appearance. Since cartilagelacks a blood supply, it is more difficult to regenerate, as cartilage tissue obtains sustenance by diffusion. Cytokines are signalling molecules that control chondrocyte metabolism and extracellular matrix formation, which is required for cartilage development, homeostasis, and healing. Some craniofacial illnesses alter the composition of the cartilage and the structural organization of growth factors, allowing for moulding. TGF-β (transforming growth factor-β) encourages chondrocyte differentiation, whereas IGF-1 (insulin-like growth factor-1) stimulates cartilage-forming collagen synthesis and chondrocyte multiplication. We used the scoping review approach to present current research on the role of growth factors in the creation and architecture of nasal cartilage. We generally observed this structure before conducting specific experiments to determine the impact of growth agents on the development of chondrocytes and cartilage. Prominent findings increase our understanding of how growth factors influence the extracellular matrix, cell activities and features, and cartilage growth rate; all are critical for cartilage tissue development and repair. Research into growth factors and their physiological interactions with cartilage may help improve treatment's functional and aesthetic outcomes and our understanding of the origins and consequences of nasal congenital anomalies. This study emphasizes the importance of expanding knowledge and experience, as well as the use of growth factors in clinical practice, to stimulate cartilage development.

Is Exon Skipping a Viable Therapeutic Approach for Vascular Ehlers-Danlos Syndrome with Mutations in COL3A1 Exon 10 or 15?

Vascular Ehlers-Danlos syndrome or Ehlers-Danlos syndrome type IV (vEDS) is a connective tissue disorder characterised by skin hyperextensibility, joint hypermobility and fatal vascular rupture caused by COL3A1 mutations that affect collagen III expression, homo-trimer assembly and secretion. Along with collagens I, II, V and XI, collagen III plays an important role in the extracellular matrix, particularly in the inner organs. To date, only symptomatic treatment for vEDS patients is available. Fibroblasts derived from vEDS patients carrying dominant negative and/or haploinsufficiency mutations in COL3A1 deposit reduced collagen III in the extracellular matrix. This study explored the potential of an antisense oligonucleotide (ASO)-mediated splice modulating strategy to bypass disease-causing COL3A1 mutations reported in the in-frame exons 10 and 15. Antisense oligonucleotides designed to redirect COL3A1 pre-mRNA processing and excise exons 10 or 15 were transfected into dermal fibroblasts derived from vEDS patients and a healthy control subject. Efficient exon 10 or 15 excision from the mature COL3A1 mRNA was achieved and intracellular collagen III expression was increased after treatment with ASOs; however, collagen III deposition into the extracellular matrix was reduced in patient cells. The region encoded by exon 10 includes a glycosylation site, and exon 15 encodes hydroxyproline and hydroxylysine-containing triplet repeats, predicted to be crucial for collagen III assembly. These results emphasize the importance of post-translational modification for collagen III homo-trimer assembly. In conclusion, while efficient skipping of target COL3A1 exons was achieved, the induced collagen III isoforms generated showed defects in extracellular matrix formation. While therapeutic ASO-mediated exon skipping is not indicated for the patients in this study, the observations are restricted to exons 10 and 15 and may not be applicable to other collagen III in-frame exons.

Study on Electrochemical Characteristics of Crystal Structure Changes Effects of Silicon Anode Materials for Lithium Ion Batteries

Graphite is widely used as a representative anode material in commercialized lithium-ion secondary batteries. However, with a theoretical capacity of 370 mAh/g, it is difficult to manufacture high-capacity/high-density lithium-ion secondary batteries. Therefore, research is actively underway to find high-capacity substances to replace graphite. Silicon anode materials are currently attracting attention as next-generation high-capacity cathode materials. Silicon forms Li4.4Si when fully charged through an alloying/dealloying process with lithium during the reaction. Graphite is intercalation/deintercalation in reaction with lithium and LiC6 is formed when fully charged. That's why silicon has a theoretical capacity of 3800 mAh/g, about 10 times that of graphite.However, silicon is a high-capacity electrode material that overcomes the theoretical capacity limit of graphite, and has the disadvantage of experiencing volume expansion of about 400% during repeated charge/discharge cycles. Problems such as material cracks due to volume expansion can affect the safety of the battery. Various studies are being conducted to solve silicon-related challenges to effectively utilize silicon materials. Previous studies have explored the use of binders or additives to mitigate volume expansion. In addition, various types of silicon materials such as Si alloy and SiOx are being developed to suppress volume expansion through matrix formation. Nevertheless, despite the development of silicon materials, structural changes in the charging and discharging process of silicon remain unclear.This study aims to analyze the structural changes that occur during the charging and discharging process of silicon. The experiment evaluates the electrochemical characteristics of crystalline silicon and amorphous silicon and compares the behavior and structural changes during charging/discharging. The volumetric expansion measurements are performed with electrochemical characterization, and ex-situ X-ray diffraction (XRD) and transmission electron microscope (TEM) analyses are performed for comparison with crystal structural changes.

Single Cells Polydopamine Coating of Rhodobacter Sphaeroides for Enhanced Electron Transfer

Photosynthetic bacteria are anoxygenic microorganisms with highly versatile metabolism, as they use sunlight to oxidize a broad variety of organic compounds in addition to heterotrophic and photoautotropic alternative metabolisms. Recent advances in coupling light-harvesting microorganisms with electronic components has led to a new generation of biohybrid devices based on microbial photocatalysts. These devices are limited by the poorly conductive interface between phototrophs and synthetic materials that inhibit charge transfer.Polydopamine (PDA), produced by self-assembly of dopamine, is a very versatile and bioinspired polymer with widespread applications1 mostly due its ability to adhere and cover surfaces of different chemical composition. The oxidative conditions employed for the formation of this dark insoluble polymer are mild and biocompatible and have inspired scientists to develop novel nanomaterials. Post-functionalization of PDA2 also enables fine tuning of properties. Furthermore, the ability of this monomer to self-assemble and polymerize in the bacterial growth medium was considered one of the requirements of the polymer to be used as coating material beside the tunable conductive properties and the flexible structure.Biocompatibility of dopamine was tested by in vivo addition in the growth media of the photosynthetic purple non sulphur Rhodobacter (R.) sphaeroides 3 in anoxygenic conditions. This study focuses on overcoming the bottleneck of biohybrid devices through the metabolically-driven encapsulation of photosynthetic cells with a bio-inspired conductive polymer. The treated cells show preserved light absorption of the photosynthetic pigments in the presence of dopamine concentrations ranging between 0.05 – 3.5 mM. The thickness and nanoparticle formation of the membrane-associated PDA matrix was further shown to vary with the dopamine concentrations in this range. Compared to uncoated cells, the encapsulated cells show up to a 20-fold enhancement in transient photocurrent measurements under mediatorless conditions. The biologically synthesized PDA can thus act as a matrix for electronically coupling the light-harvesting metabolisms of cells with conductive surfaces. 1Liu, Y.; Ai, K.; Lu, L. Polydopamine and its derivative materials: Synthesis and promising applications in energy, environmental, and biomedical fields. Chem. Rev. 2014, 114, 5057–5115. 2Buscemi, G., Vona, D., Ragni, R., Comparelli, R., Trotta, M., Milano, F., Farinola, G.M., Polydopamine/Ethylenediamine Nanoparticles Embedding a Photosynthetic Bacterial Reaction Center for Efficient Photocurrent Generation. Adv. Sustainable Syst. 2021, 2000303. 3 Labarile, R.; Varsalona, M.; Vona, D.; Stufano, P.; Grattieri, M.; Farinola, G. M.; Trotta, M., A novel route for anoxygenic polymerization of dopamine via purple photosynthetic bacteria metabolism. MRS Advances 2023

Fibrin-konjac glucomannan-black phosphorus hydrogel scaffolds loaded with nasal ectodermal mesenchymal stem cells accelerated alveolar bone regeneration

BackgroundEffective treatments for the alveolar bone defect remain a major concern in dental therapy. The objectives of this study were to develop a fibrin and konjac glucomannan (KGM) composite hydrogel as scaffolds for the osteogenesis of nasal mucosa-derived ectodermal mesenchymal stem cells (EMSCs) for the regeneration of alveolar bone defect, and to investigate the osteogenesis-accelerating effects of black phosphorus nanoparticles (BPNs) embedded in the hydrogels.MethodsPrimary EMSCs were isolated from rat nasal mucosa and used for the alveolar bone recovery. Fibrin and KGM were prepared in different ratios for osteomimetic hydrogel scaffolds, and the optimal ratio was determined by mechanical properties and biocompatibility analysis. Then, the optimal hydrogels were integrated with BPNs to obtain BPNs/fibrin-KGM hydrogels, and the effects on osteogenic EMSCs in vitro were evaluated. To explore the osteogenesis-enhancing effects of hydrogels in vivo, the BPNs/fibrin-KGM scaffolds combined with EMSCs were implanted to a rat model of alveolar bone defect. Micro-computed tomography (CT), histological examination, real-time quantitative polymerase chain reaction (RT-qPCR) and western blot were conducted to evaluate the bone morphology and expression of osteogenesis-related genes of the bone regeneration.ResultsThe addition of KGM improved the mechanical properties and biodegradation characteristics of the fibrin hydrogels. In vitro, the BPNs-containing compound hydrogel was proved to be biocompatible and capable of enhancing the osteogenesis of EMSCs by upregulating the mineralization and the activity of alkaline phosphatase. In vivo, the micro-CT analysis and histological evaluation demonstrated that rats implanted EMSCs-BPNs/fibrin-KGM hydrogels exhibited the best bone reconstruction. And compared to the model group, the expression of osteogenesis genes including osteopontin (Opn, p < 0.0001), osteocalcin (Ocn, p < 0.0001), type collagen (Col , p < 0.0001), bone morphogenetic protein-2 (Bmp2, p < 0.0001), Smad1 (p = 0.0006), and runt-related transcription factor 2 (Runx2, p < 0.0001) were all significantly upregulated.ConclusionsEMSCs/BPNs-containing fibrin-KGM hydrogels accelerated the recovery of the alveolar bone defect in rats by effectively up-regulating the expression of osteogenesis-related genes, promoting the formation and mineralisation of bone matrix.

Abstract We020: Myh9 plays a vital role in cardiac myofibroblast differentiation and is indispensable for cardiac repair after myocardial infarction

After myocardial infarction (MI), quiescent cardiac fibroblasts (CFs) are activated and differentiate into myofibroblasts featured by elevated expression of smooth muscle alpha-actin (SMαA, encoded by Acta2 ), which form contractile stress fibers. However, compared to the matrix formation function of cardiac myofibroblasts, much less is known about the role of their contraction in post-MI cardiac repair. In our previous study using mice lacking Acta2 in CFs, all other 5 actin isoforms were activated to compensate for the loss of Acta2 , suggesting the low feasibility of abolishing cardiac myofibroblast contraction through deleting actin genes. An alternative strategy through targeting myosin was explored. Our transcriptomic study identified Myh9 and Myh10 [encoding non-muscle myosin IIA (NM IIA) and NM IIB, respectively] as the dominant myosin isoforms in cardiac myofibroblasts. In vitro studies found that Myh9 knockout (KO) but not Myh10 KO significantly affected cardiac myofibroblast morphology, completely abolished the stress fiber formation and contractility of these cells, and reduced their proliferation and ability to stabilize the surrounding matrix. Mice lacking Myh9 in CFs have an increased post-MI cardiac rupture rate compared to WT mice regardless of sexes ( p =0.004 and 0.02 for male and female mice, respectively). In vitro and in vivo transcriptomic and proteomics analysis revealed disrupted expression of stress fiber and mesenchymal identity genes/proteins caused by Myh9 KO. Mechanistically, Myh9 KO led to an increased globular to filamentous (G/F) actin ratio. The altered actin dynamics likely trigger multiple epigenetic events, which together lead to significantly altered cardiac myofibroblast gene expression and activities beyond contraction.

A novel design magnesium alloy suture anchor promotes fibrocartilaginous enthesis regeneration in rabbit rotator cuff repair

Regarding the current materials used for suture anchors for rotator cuff repair, there are still limitations in terms of degradability, mechanical properties, and bioactivities in clinical applications. Magnesium alloys have preliminarily been shown to promote tendon-bone healing with good prospects for application as anchor materials. However, the design of anchor structures for the degradation characteristics of magnesium alloy materials has not been considered, which is critical for the practical application of magnesium alloy anchors. The mechanism by which magnesium promotes tendon bone healing remains to be clarified. Here, we proposed a novel split hollowed magnesium alloy suture anchors for the repair of rabbit rotator cuff injury. We found that novel split hollowed magnesium alloy anchors structure effectively solved the problem of failure due to degradation of traditional eyelet structure, providing reliable suture fixation. The open architecture facilitates the metabolic resorption of the degradation products of and promotes the ingrowth of bone tissue. Histological staining showed that magnesium anchors have better ability to promote regeneration at the fibrocartilage interface compared to PLLA anchors. The higher expression of fibrocartilage markers (Aggrecan, COL2A1, and Sox9) at the tendon-bone interface in magnesium anchors, which promotes chondrocyte differentiation at the tendon-bone interface and matrix formation, which is more conducive to achieving regeneration and maturation of fibrocartilage enthesis. Hence, this study provides a basis for further research on the clinical application of degradable magnesium alloy suture anchors.

GSK3β Inhibitors Inhibit TGFβ Signaling in the Human Trabecular Meshwork.

Primary open-angle glaucoma (POAG) is a leading cause of blindness, and its primary risk factor is elevated intraocular pressure (IOP) due to pathologic changes in the trabecular meshwork (TM). We previously showed that there is a cross-inhibition between TGFβ and Wnt signaling pathways in the TM. In this study, we determined if activation of the Wnt signaling pathway using small-molecule Wnt activators can inhibit TGFβ2-induced TM changes and ocular hypertension (OHT). Primary human TM (pHTM) cells and transduced SBE-GTM3 cells were treated with or without Wnt and/or TGFβ signaling activators and used for luciferase assays; for the extraction of whole-cell lysate, conditioned medium, cytosolic proteins, and nuclear proteins for Western immunoblotting (WB); or for immunofluorescent staining. Human donor eyes were perfusion cultured to study the effect of Wnt activators on IOP. We found that the small-molecule Wnt activators (GSK3β inhibitors) (BIO, SB216763, and CHIR99021) activated canonical Wnt signaling in pHTM cells without toxicity at tested concentrations. This activation inhibited TGFβ signaling as well as TGFβ2-induced extracellular matrix deposition and formation of cross-linked actin networks in pHTM cells or SBE-GTM3 cells. We also observed nuclear translocation of both Smad4 and β-catenin in pHTM cells, which suggested that the cross-inhibition between the TGFβ and Wnt signaling pathways may occur in the nucleus. Using our ex vivo model, we found that CHIR99021 inhibited TGFβ2-induced OHT in perfusion-cultured human eyes. Our results showed that small-molecule Wnt activators have the potential for treating TGFβ signaling-induced OHT in patients with POAG.

Antimicrobial impact of a propolis/PVA/chitosan composite and its prospective application against methicillin resistance bacterial infection

Seriously damaged skin could be infected by methicillin-resistant bacteria, which delays restoration. Propolis has bioactivity linked with its minor components, such as antimicrobials and antioxidants. Active sites in polyvinyl alcohol (PVA) and chitosan (CS) can enhance the nano-loading of natural extracts with activity amelioration. Korean propolis extract (KPE) loading to a nanocomposite possibly enhances its antimicrobial and anti-inflammatory potency. Composites were formed using two PVA/CS structures (1:1; 2:1), and their skin-application appropriateness was determined by mechanical properties, moisture content, water activity, and color. The composite of PVA/CS (1:1) was more practicable for KPE-loading. Increasing KPE concentrations (50, 100, 150, and 200 ng/mL) alters composite bioactivity measured by Fourier transmission infrared (FT-IR). Antibacterial potency of 200 ng KPE/mL was the most effective concentration, followed by 150 ng KPE/mL, against Staphylococcus aureus (MRSA) and Clostridium perfringens. The composite activity was measured as minimum inhibition (MIC) and minimum bacterial concentrations (MBC). At 200 ng KPE/mL, MIC and MBC against MRSA were 14.93 ± 1.21 and 20.21 ± 1.97 mg composite/mL, respectively. Significant inhibition was also recorded for antibiofilm formation, where MRSA growth was not detected after 4 hours of time intervals to the stainless-steel coupon. Compared to planktonic bacteria, the formed barrier of PVA/CS restrained the biofilm matrix formation and supported KPE antimicrobial. The impact of inhibition against biofilm formation depends on two parallel mechanisms (PVA barrier with hydrogen bonds, besides nano-KPE particle penetration into bacterial cells). The KPE-composite application to rats’ wounds shows significantly reduced MRSA infection. The results demonstrate the capability of KPE composite in reducing infection, healing correctly, and restoring hair. The wound swabbed test emphasizes this capacity, in which bacterial growth rate restriction was evaluated using a plate count assay. The results recommended 150 ng KPE/mL loading into CS/PVA (1:1) as an effective anti-pathogenic treatment, particularly against the MRSA infection of wounds.

Developing double-crosslinking 3D printed hydrogels for bone tissue engineering

Bone defects are one of the main causes of disability worldwide. Due to the disadvantages associated with autografts, the latest advances have been focused on tissue regeneration approaches that use injectable hydrogels or 3D printed hydrogel-based structures that could refill appropriately the bone gap area without the need for external fixatives, leading to bone formation in the long term. Injectable hydrogels could be applied in extrusion-based 3D printing as inks; in this sense, double-crosslinking hydrogels appear as ideal candidates. In this work, injectable and printable double crosslinkable hydrogels based on oxidized xanthan gum (XGox) and methacrylate polyaspartylhydrazide (PAHy-MA) were produced. The formation of dynamic hydrazone bonds, occurring between aldehyde groups on the polysaccharide backbone and hydrazine moieties of PAHy-MA, induced an instant gelation, conferring, also, injectability and self-healing properties to the hydrogels. The presence of methacrylic moieties on the synthetic polymer allowed further crosslinking upon UV irradiation that stabilized the hydrogel shape and mitigated its susceptibility to hydrolytic degradation. Obtained hydrogels showed pseudoplastic behaviour and good recovery of viscoelastic properties over time. The physicochemical and rheological characterization highlighted increased stability and higher viscoelastic moduli after photo-crosslinking. The hydrogels also showed good printability, cytocompatibility and the early formation of a bone-like matrix when osteosarcoma-derived cells (MG-63) were cultured in the scaffolds for 21 days, with an increased collagen I deposition, mineralization and the expression of characteristic osteogenic markers.

Insulin promotes the bone formation capability of human dental pulp stem cells through attenuating the IIS/PI3K/AKT/mTOR pathway axis

BackgroundInsulin has been known to regulate bone metabolism, yet its specific molecular mechanisms during the proliferation and osteogenic differentiation of dental pulp stem cells (DPSCs) remain poorly understood. This study aimed to explore the effects of insulin on the bone formation capability of human DPSCs and to elucidate the underlying mechanisms.MethodsCell proliferation was assessed using a CCK-8 assay. Cell phenotype was analyzed by flow cytometry. Colony-forming unit-fibroblast ability and multilineage differentiation potential were evaluated using Toluidine blue, Oil red O, Alizarin red, and Alcian blue staining. Gene and protein expressions were quantified by real-time quantitative polymerase chain reaction and Western blotting, respectively. Bone metabolism and biochemical markers were analyzed using electrochemical luminescence and chemical colorimetry. Cell adhesion and growth on nano-hydroxyapatite/collagen (nHAC) were observed with a scanning electron microscope. Bone regeneration was assessed using micro-CT, fluorescent labeling, immunohistochemical and hematoxylin and eosin staining.ResultsInsulin enhanced the proliferation of human DPSCs as well as promoted mineralized matrix formation in a concentration-dependent manner. 10− 6 M insulin significantly up-regulated osteogenic differentiation-related genes and proteins markedly increased the secretion of bone metabolism and biochemical markers, and obviously stimulated mineralized matrix formation. However, it also significantly inhibited the expression of genes and proteins of receptors and receptor substrates associated with insulin/insulin-like growth factor-1 signaling (IIS) pathway, obviously reduced the expression of the phosphorylated PI3K and the ratios of the phosphorylated PI3K/total PI3K, and notably increased the expression of the total PI3K, phosphorylated AKT, total AKT and mTOR. The inhibitor LY294002 attenuated the responsiveness of 10− 6 M insulin to IIS/PI3K/AKT/mTOR pathway axis, suppressing the promoting effect of insulin on cell proliferation, osteogenic differentiation and bone formation. Implantation of 10− 6 M insulin treated DPSCs into the backs of severe combined immunodeficient mice and the rabbit jawbone defects resulted in enhanced bone formation.ConclusionsInsulin induces insulin resistance in human DPSCs and effectively promotes their proliferation, osteogenic differentiation and bone formation capability through gradually inducing the down-regulation of IIS/PI3K/AKT/mTOR pathway axis under insulin resistant states.

Bioprocessing of camptothecin from Alternaria brassicicola, an endophyte of Catharanthus roseus, with a strong antiproliferative activity and inhibition to Topoisomerases

Suppression of fungal camptothecin (CPT) biosynthesis with the preservation and successive subculturing is the challenge that impedes fungi from the industrial application, so, screening for a novel fungal isolate with a conceivable stable producing potency of CPT was the main objective of this work. Catharanthus roseus with diverse contents of bioactive metabolites could have a plethora of novel endophytes with unique metabolic properties. Among the endophytes of C. roseus, Alternaria brassicicola EFBL-NV OR131587.1 was the highest CPT producer (96.5 μg/L). The structural identity of the putative CPT was verified by HPLC, FTIR, HNMR and LC–MS/MS, with a molecular mass 349 m/z, and molecular fragmentation patterns that typically identical to the authentic one. The purified A. brassicicola CPT has a strong antiproliferative activity towards UO-31 (0.75 μM) and MCF7 (3.2 μM), with selectivity index 30.8, and 7.1, respectively, in addition to resilient activity to inhibit Topo II (IC50 value 0.26 nM) than Topo 1 (IC50 value 3.2 nM). The purified CPT combat the wound healing of UO-31 cells by ~ 52%, stops their matrix formation, cell migration and metastasis. The purified CPT arrest the cellular division of the UO-31 at the S-phase, and inducing their cellular apoptosis by ~ 20.4 folds, compared to the control cells. Upon bioprocessing with the surface response methodology, the CPT yield by A. brassicicola was improved by ~ 3.3 folds, compared to control. The metabolic potency of synthesis of CPT by A. brassicicola was attenuated with the fungal storage and subculturing, losing ~ 50% of their CPT productivity by the 6th month of storage and 6th generation. Practically, the CPT productivity of the attenuated A. brassicicola was restored by addition of 1% surface sterilized leaves of C. roseus, ensuring the eliciting of cryptic gene cluster of A. brassicicola CPT via the plant microbiome-A. brassicicola interactions. So, for the first time, a novel endophytic isolate A. brassicicola, from C. roseus, was explored to have a relatively stable CPT biosynthetic machinery, with an affordable feasibility to restore their CPT productivity using C. roseus microbiome, in addition to the unique affinity of the extracted CPT to inhibit Topoisomerase I and II.