Human Adipose-derived Stem Cells Research Articles

Sexual Dimorphism's impact on adipogenesis: A three-dimensional in vitro model treated with 17β-estradiol and testosterone

Using a three-dimensional (3-D) in vitro culture model, we report the dose dependent effect of 17β-estradiol and testosterone on the adipogenic differentiation and maturation of human adipose derived stem cells (hASCs) obtained from female and male patients. Considering sexual dimorphism, we expected male and female adipocytes to respond differently to the sex steroids. Both male and female hASC spheroids were exposed to 100 nM and 500 nM of 17β-estradiol and testosterone either at the beginning of the adipogenic maturation (Phase I) to discourage intracellular triglyceride accumulation or exposed after adipogenic maturation (Phase II) to reduce the intracellular triglyceride accumulation. The results show that 17β-estradiol leads to a dose dependent reduction in intracellular triglyceride accumulation in female hASC spheroids compared to the both untreated and testosterone-treated cells. Affirming our hypothesis, 17β-estradiol prevented intracellular triglyceride accumulation during Phase I, while it stimulated lipolysis during Phase II. PPAR-γ and adiponectin gene expression also reduced upon 17β-estradiol treatment in female cells. Interestingly, 17β-estradiol and testosterone had only a modest effect on the male hASC spheroids. Collectively, our findings suggest that 17β-estradiol can prevent fat accumulation in adipocytes during early and late stages of maturation in females.

Silk Fibroin Films with Microscopic Surface Pattern as Promising Cartilage Tissue Engineering Scaffold in Supporting MSCs Amplification and Chondrogenic Differentiation

ABSTRACT Mesenchymal stem cells (MSCs) with multipotential differentiation ability are main cell source for cartilage tissue engineering, but its application is restricted by the difficulty in regulating committed chondrogenic differentiation. Study revealed that certain substrate topographic structure can enhance the chondrogenic differentiation of MSCs. This study prepared silk fibroin (SF) films with gradient microscopic surface patterns based on P280-P7000 type specification sandpapers, and investigated the adhesion, proliferation and chondrogenic differentiation of human adipose-derived stem cells (hADSCs) on the SF films. The results showed that the SF films surface had convex dots with diameter of 3–50 μm, and hADSCs could adhere and proliferate well on the film. Transcriptomics analysis indicated that hADSCs tend to differentiate differently on the SF films, among which hADSCs on P1000 SF films had higher expression of chondrogenic markers Sox-9 and Col-II, with activated yes associated protein (YAP) and restricted F-actin. In vitro co-culture of human OA knee cartilage with hADSCs seeded SF films found satisfactory cartilage protection effect in P1000 group. Overall, this study preliminarily revealed the microscopic surface pattern of SF films may induce chondrogenic differentiation of hADSCs through extracellular matrix mediated Hippo/Yap pathway, and provided a new strategy for cartilage repair.

Mitochondrial DNA as a target for analyzing the biodistribution of cell therapy products.

Biodistribution tests are crucial for evaluating the safety of cell therapy (CT) products in order to prevent unwanted organ homing of these products in patients. Quantitative polymerase chain reaction (qPCR) using intronic Alu is a popular method for biodistribution testing owing to its ability to detect donor cells without modifying CT products and low detection limit. However, Alu-qPCR may generate inaccurate information owing to background signals caused by the mixing of human genomic DNA with that of experimental animals. The aim of this study was to develop a test method that is more specific and sensitive than Alu-qPCR, targeting the mitochondrial DNA (mtDNA) sequence that varies substantially between humans and experimental animals. We designed primers for 12S, 16S, and cytochrome B in mtDNA regions, assessed their specificity and sensitivity, and selected primers and probes for the 12S region. Human adipose-derived stem cells, used as CT products, were injected into the tail vein of athymic NCr-nu/nu mice and detected, 7 d after administration, in their lungs at an average concentration of 2.22 ± 0.69 pg/μg mouse DNA, whereas Alu was not detected. Therefore, mtDNA is more specific and sensitive than Alu and is a useful target for evaluating CT product biodistribution.

Negative Printing for the Reinforcement of In Situ Tissue-Engineered Cartilage.

In the realm of in situ cartilage engineering, the targeted delivery of both cells and hydrogel materials to the site of a defect serves to directly stimulate chondral repair. Although the in situ application of stem cell-laden soft hydrogels to tissue defects holds great promise for cartilage regeneration, a significant challenge lies in overcoming the inherent limitation of these soft hydrogels, which must attain mechanical properties akin to the native tissue to withstand physiological loading. We therefore developed a system where a gelatin methacryloyl hydrogel laden with human adipose-derived mesenchymal stem cells is combined with a secondary structure to provide bulk mechanical reinforcement. In this study, we used the negative embodied sacrificial template 3D printing technique to generate eight different lattice-based reinforcement structures made of polycaprolactone, which ranged in porosity from 80% to 90% with stiffnesses from 28 ± 5 kPa to 2853 ± 236 kPa. The most promising of these designs, the hex prism edge, was combined with the cellular hydrogel and retained a stable stiffness over 41 days of chondrogenic differentiation. There was no significant difference between the hydrogel-only and hydrogel scaffold group in the sulfated glycosaminoglycan production (340.46 ± 13.32 µg and 338.92 ± 47.33 µg, respectively) or Type II Collagen gene expression. As such, the use of negative printing represents a promising solution for the integration of bulk reinforcement without losing the ability to produce new chondrogenic matrix.

Adipose-derived stem cells derived decellularized extracellular matrix enabled skin regeneration and remodeling.

The tissues or organs derived decellularized extracellular matrix carry immunogenicity and the risk of pathogen transmission, resulting in limited therapeutic effects. The cell derived dECM cultured in vitro can address these potential risks, but its impact on wound remodeling is still unclear. This study aimed to explore the role of decellularized extracellular matrix (dECM) extracted from adipose derived stem cells (ADSCs) in skin regeneration. Methods: ADSCs were extracted from human adipose tissue. Then we cultivated adipose-derived stem cell cells and decellularized ADSC-dECM for freeze-drying. Western blot (WB), enzyme-linked immunosorbent assay (ELISA) and mass spectrometry (MS) were conducted to analyzed the main protein components in ADSC-dECM. The cell counting assay (CCK-8) and scratch assay were used to explore the effects of different concentrations of ADSC-dECM on the proliferation and migration of human keratinocytes cells (HaCaT), human umbilical vein endothelia cells (HUVEC) and human fibroblasts (HFB), respectively. Moreover, we designed a novel ADSC-dECM-CMC patch which used carboxymethylcellulose (CMC) to load with ADSC-dECM; and we further investigated its effect on a mouse full thickness skin wound model. Results: ADSC-dECM was obtained after decellularization of in vitro cultured human ADSCs. Western blot, ELISA and mass spectrometry results showed that ADSC-dECM contained various bioactive molecules, including collagen, elastin, laminin, and various growth factors. CCK-8 and scratch assay showed that ADSC-dECM treatment could significantly promote the proliferation and migration of HaCaT, human umbilical vein endothelia cells, and human fibroblasts, respectively. To evaluate the therapeutic effect on wound healing in vivo, we developed a novel ADSC-dECM-CMC patch and transplanted it into a mouse full-thickness skin wound model. And we found that ADSC-dECM-CMC patch treatment significantly accelerated the wound closure with time. Further histology and immunohistochemistry indicated that ADSC-dECM-CMC patch could promote tissue regeneration, as confirmed via enhanced angiogenesis and high cell proliferative activity. Conclusion: In this study, we developed a novel ADSC-dECM-CMC patch containing multiple bioactive molecules and exhibiting good biocompatibility for skin reconstruction and regeneration. This patch provides a new approach for the use of adipose stem cells in skin tissue engineering.

Melt Electrowriting Combined with Fused Deposition Modeling Printing for the Fabrication of Three-Dimensional Biomimetic Scaffolds for Osteotendinous Junction Regeneration.

This study aims to explore a novel scaffold for osteotendinous junction regeneration and to preliminarily verify its osteogenic and tenogenic abilities in vitro. A polycaprolactone (PCL) scaffold with aligned and orthogonal fibers was created using melt electrowriting (MEW) and fused deposition modeling (FDM). The scaffold was coated with Type I collagen, and hydroxyapatite was carefully added to separate the regions intended for bone and tendon regeneration, before being rolled into a cylindrical shape. Human adipose-derived stem cells (hADSCs) were seeded to evaluate viability and differentiation. Scaffold characterization was performed with Scanning Electron Microscope (SEM). Osteogenesis was assessed by alkaline phosphatase (ALP) and Alizarin red staining, while immunostaining and transcription-quantitative polymerase chain reaction (RT-qPCR) evaluated osteogenic and tendogenic markers. Scaffolds were developed in four variations: aligned (A), collagen-coated aligned (A+C), orthogonal (O), and mineral-coated orthogonal (O+M). SEM analysis confirmed surface morphology and energy-dispersive X-ray spectroscopy (EDS) verified mineral coating on O+M types. Hydrophilicity and mechanical properties were optimized in modified scaffolds, with A+C showing increased tensile strength and O+M improved in compression. hADSCs demonstrated good viability and morphology across scaffolds, withO+M scaffolds showing higher cell proliferation and osteogenic potential, and A and A+C scaffolds supporting tenogenic differentiation. This study confirms the potential of a novel PCL scaffold with distinct regions for osteogenic and tenogenic differentiation, supporting the regeneration of osteotendinous junctions in vitro.

Effect of hydroxyapatite and pulsed electromagnetic field on osteogenic differentiation of human stem cells seeded onto 3D‐printed polycaprolactone scaffolds for future bone graft transplantation

AbstractBone graft transplantation is a risky procedure mainly due to postoperative complications. Tissue engineering is an alternative to regenerating bone tissue by seeding mesenchymal stem cells into 3D scaffolds by osteoinductive signaling. Poly(ε‐caprolactone) (PCL) is one of the most used biopolymers for fabricating scaffolds for bone tissue engineering. PCL is enriched with hydroxyapatite (HA) to promote the production of the bone‐mineralized matrix. Moreover, the application of pulsed electromagnetic fields (PEMFs) on scaffolds loaded with HA can significantly benefit collagen and HA networks favoring the interaction between organic and mineral phases of bones. In this context, we investigated, for the first time, the effect exerted by a PEMF exposure on the osteogenic differentiation of human adipose stem cells (hASCs) seeded onto 3D printed PCL‐HA scaffolds and cultured in growth and osteogenic medium (OM). After 21 days of culture, the results showed excellent colonization of scaffolds and the deposition of extracellular matrix (ECM) secreted by hASCs. Immunofluorescence staining confirmed the deposition of collagen type I and osteopontin both on scaffolds. Gene expression analysis indicated that both HA crystal and PEMF treatment were able to trigger osteogenic differentiation, as documented by the increase of osteogenic markers. These results suggest that HA, osteogenic factors, and PEMFs converge on the same signaling pathways as there are no additive effects and no change in protein synthesis between scaffolds with HA or treated with PEMFs for cells cultured in OM. The proposed study lays the foundations for exploring new approaches in bone graft transplantation.

Evaluation of the Treatment Effects of Conditioned Medium from Human Orbital Adipose-Derived Stem Cells in a Corneal Alkali Burn Rabbit Model.

Purpose: This study aimed to evaluate the effects of a new treatment-conditioned medium from human orbital adipose-derived stem cells (OASC-CM)-on corneal recovery after alkali burns in a rabbit model. Methods: The corneal alkali burn rabbit model was established and treated with OASC-CM, conditioned medium from human abdominal subcutaneous adipose-derived stem cells (ABASC-CM), and fresh control culture medium (con-CM) three times a day for 7 days, respectively. Subsequently, the treatment effects were evaluated and compared through clinical, histological, immunohistochemical, and cytokine evaluations. Results: Clinically, OASC-CM alleviated corneal opacity and edema and promoted recovery of corneal epithelium defect. Histologically and immunohistochemically, OASC-CM inhibited neovascularization, conjunctivalization, and immuno-inflammatory reaction, while promoting corneal regeneration and rearrangement. Increased secretion of interleukin-10 and inhibited protein levels of cluster of differentiation 45, interferon-γ, and tumor necrosis factor-α were observed in the alkali-burned cornea after OASC-CM treatment, which might be the relevant molecular mechanism. Conclusions: OASC-CM showed significant effects on the recovery of rabbit corneal alkali burns and eliminated immunological and ethical limitations, representing a new option for corneal wound treatment.

Incorporating bioactive glass nanoparticles in silk fibroin/bacterial nanocellulose composite scaffolds improves their biological and osteogenic properties for bone tissue engineering applications

Blend polymers composed of natural polymers are a ubiquitous biomaterial class due to their suitable mechanical and biological characterization. In the present study, composite scaffolds based on bacterial cellulose (BC)/silk fibroin (SF) with bioactive glass nanoparticles (BGNPs) were developed to enhance osteogenesis in human adipose derived stem cells (hASCs). The scanning electron microscopy (SEM) results of BGNPs indicated a spherical morphology and size ranging from 15 to 30 nm. The presence of BC and BGNPs reduced the pore diameter of SF scaffolds to about 210 ± 10 μm and 205 ± 10 μm, respectively, while increasing their compressive strength and compressive modulus. FTIR analyses proved the presence of BGNPs, BC and SF in the scaffolds. Flow cytometry data confirmed the surface markers for hASCs. The results also showed that BC and BGNPs addition to BC/SF scaffolds decreased degradation and swelling rate. The gene expression (Runx2, alkaline phosphatase and osteocalcin) studies signified the osteogenic potential of BGNPs in BC/SF scaffolds on hASCs. Eventually, the increased cell adhesion, viability and differentiation in the BC/SF and BC/SF/BGNPs composite scaffolds drawn from MTT, SEM, Alizarin red staining and alkaline phosphatase activity confirmed that these scaffolds promise to serve as a therapeutic candidate for bone defects.

Oxaloacetate as new inducer for osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells in vitro.

Mitochondrial organelles play a crucial role in cellular metabolism so different cell types exhibit diverse metabolic and energy demands. Therefore, alternations in the intracellular distribution, quantity, function, and structure of mitochondria are required for stem cell differentiation. Finding an effective inducer capable of modulating mitochondrial activity is critical for the differentiation of specific stem cells into osteo-like cells for addressing issues related to osteogenic disorders. This study aimed to investigate the effect of oxaloacetate (OAA) on the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs) in vitro. First, the most favorable OAA concentration was measured through MTT assay and subsequently confirmed using acridine orange staining. Human ADSCs were cultured in osteogenic medium supplemented with OAA and analyzed on days 7 and 14 of differentiation. Various assays including alkaline phosphatase assay (ALP), cellular calcium content assay, mineralized matrix staining with alizarin red, catalase (CAT) and superoxide dismutase (SOD) activity, and real-time RT-PCR analysis of three bone-specific markers (ALP, osteocalcin, and collagen type I) were conducted to characterize the differentiated cells. Following viability assessment, OAA at a concentration of 1 µM was considered the optimal dosage for further studies. The results of osteogenic differentiation assays showed that OAA at a concentration of 1 × 10- 6 M significantly increased ALP enzyme activity, mineralization, CAT and SOD activity and the expression of bone-specific genes in differentiated cells compared to control groups in vitro. In conclusion, the fundings from this study suggest that OAA possesses favorable properties that make it a potential candidate for application in medical bone regeneration.

Synthesis and characterization of a magnetic bacterial cellulose-chitosan nanocomposite and evaluation of its applicability for osteogenesis.

Natural biopolymers are used for various purposes in healthcare, such as tissue engineering, drug delivery, and wound healing. Bacterial cellulose and chitosan were preferred in this study due to their non-cytotoxic, biodegradable, biocompatible, and non-inflammatory properties. The study reports the development of a magnetic bacterial cellulose-chitosan (BC-CS-Fe3O4) nanocomposite that can be used as a biocompatible scaffold for tissue engineering. Iron oxide nanoparticles were included in the composite to provide superparamagnetic properties that are useful in a variety of applications, including osteogenic differentiation, magnetic imaging, drug delivery, and thermal induction for cancer treatment. The magnetic nanocomposite was prepared by immersing Fe3O4 in a mixture of bacterial cellulose-chitosan scaffold and then freeze-drying it. The resulting nanocomposite was characterized using FE-SEM and FTIR techniques. The swelling ratio and mechanical strength of the scaffolds were evaluated experimentally. The biodegradability of the scaffolds was assessed using PBS for 8 weeks at 37°C. The cytotoxicity and osteogenic differentiation of the nanocomposite were studied using human adipose-derived mesenchymal stem cells (ADSCs) and alizarin red staining. One-way ANOVA with Tukey's multiple comparisons test was used for statistical analysis. The FTIR spectra demonstrated the formation of bonds between functional groups of nanoparticles. FE-SEM images showed the integrity of the fibrillar network. The magnetic nanocomposite has the highest swelling ratio (2445% ± 23.34) and tensile strength (5.08 MPa). After 8 weeks, the biodegradation ratios of BC, BC-CS, and BC-CS-Fe3O4 scaffolds were 0.75% ± 0.35, 2.5% ± 0.1, and 9.5% ± 0.7, respectively. Magnetic nanocomposites have low toxicity (P < 0.0001) and higher osteogenic potential compared to other scaffolds. Based on its high tensile strength, low water absorption, suitable degradability, low cytotoxicity, and high ability to induce an increase in calcium deposits by stem cells, the magnetic BC-CS-Fe3O4 nanocomposite scaffold can be a suitable candidate as a biomaterial for osteogenic differentiation.

Three-Dimensional-Printed GelMA-KerMA Composite Patches as an Innovative Platform for Potential Tissue Engineering of Tympanic Membrane Perforations.

Tympanic membrane (TM) perforations, primarily induced by middle ear infections, the introduction of foreign objects into the ear, and acoustic trauma, lead to hearing abnormalities and ear infections. We describe the design and fabrication of a novel composite patch containing photocrosslinkable gelatin methacryloyl (GelMA) and keratin methacryloyl (KerMA) hydrogels. GelMA-KerMA patches containing conical microneedles in their design were developed using the digital light processing (DLP) 3D printing approach. Following this, the patches were biofunctionalized by applying a coaxial coating with PVA nanoparticles loaded with gentamicin (GEN) and fibroblast growth factor (FGF-2) with the Electrohydrodynamic Atomization (EHDA) method. The developed nanoparticle-coated 3D-printed patches were evaluated in terms of their chemical, morphological, mechanical, swelling, and degradation behavior. In addition, the GEN and FGF-2 release profiles, antimicrobial properties, and biocompatibility of the patches were examined in vitro. The morphological assessment verified the successful fabrication and nanoparticle coating of the 3D-printed GelMA-KerMA patches. The outcomes of antibacterial tests demonstrated that GEN@PVA/GelMA-KerMA patches exhibited substantial antibacterial efficacy against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. Furthermore, cell culture studies revealed that GelMA-KerMA patches were biocompatible with human adipose-derived mesenchymal stem cells (hADMSC) and supported cell attachment and proliferation without any cytotoxicity. These findings indicated that biofunctional 3D-printed GelMA-KerMA patches have the potential to be a promising therapeutic approach for addressing TM perforations.

Generating human skeletal myoblast spheroids for vascular myogenic tissue engineering

Engineered myogenic microtissues derived from human skeletal myoblasts offer unique opportunities for varying skeletal muscle tissue engineering applications, such as in vitro drug-testing and disease modelling. However, more complex models require the incorporation of vascular structures, which remains to be challenging. In this study, myogenic spheroids were generated using a high-throughput, non-adhesive micropatterned surface. Since monoculture spheroids containing human skeletal myoblasts were unable to remain their integrity, co-culture spheroids combining human skeletal myoblasts and human adipose-derived stem cells were created. When using the optimal ratio, uniform and viable spheroids with enhanced myogenic properties were achieved. Applying a pre-vascularization strategy, through addition of endothelial cells, resulted in the formation of spheroids containing capillary-like networks, lumina and collagen in the extracellular matrix, whilst retaining myogenicity. Moreover, sprouting of endothelial cells from the spheroids when encapsulated in fibrin was allowed. The possibility of spheroids, from different maturation stages, to assemble into a more large construct was proven by doublet fusion experiments. The relevance of using three-dimensional microtissues with tissue-specific microarchitecture and increased complexity, together with the high-throughput generation approach, makes the generated spheroids a suitable tool for in vitro drug-testing and human disease modeling.

Pore graded borosilicate bioactive glass scaffolds: in vitro dissolution and cytocompatibility

3D borosilicate bioactive glass (1393B20 and B12.5MgSr) scaffolds were prepared by robocasting, with and without a dense layer at the top. Pore graded scaffolds are promising as they allow for membrane deposition and could limit the risk of soft tissue infiltration. In vitro dissolution was studied in tris(hydroxymethyl)aminomethane (TRIS) and Simulated Body Fluid (SBF). 1393B20 scaffolds dissolved faster than B12.5MgSr in TRIS whereas they dissolved slower in SBF. The difference in dissolution profiles, as a function of the medium used, is assigned to the different rates of precipitation of hydroxyapatite (HA). While the precipitation of calcium phosphate (CaP) in the form of HA, first sign of bioactivity, was confirmed by ICP, FTIR-ATR and SEM-EDX analysis for both compositions, 1393B20 was found to precipitate HA at a faster rate. The presence of a dense top layer did not significantly impact the dissolution rate and CaP precipitation. In vitro cell culture was performed using human adipose-derived stem cells (hADSCs). Prior to cell plating, a preincubation of 3 days was found optimum to prevent burst ion release. In direct contact, cells proliferate and spread on the scaffolds while maintaining characteristic spindle morphology. Cell plated on 1393B20 scaffolds showed increased viability when compared to cell plated on B12.5MgSr. The lower cell viability, when testing B12.5MgSr, was assigned to the depletion of Ca2+ ions from culture medium and higher pH. Static cell culture leads to believe that the scaffold produced from the 1393B20 glass composition are promising in bone regeneration applications.Graphical

Generation of Highly Functional Hepatocyte-like Organoids from Human Adipose-Derived Mesenchymal Stem Cells Cultured with Endothelial Cells.

Previously, we successfully established a highly functional, three-dimensional hepatocyte-like cell (3D-HLC) model from adipose-derived mesenchymal stem cells (ADSCs) via a three-step differentiation protocol. The aim of the present study was to investigate whether generating hepatocyte-like organoids (H-organoids) by adding endothelial cells further improved the liver-like functionality of 3D-HLCs and to assess H-organoids' immunogenicity properties. Genes representing liver maturation and function were detected by quantitative reverse transcription-PCR analysis. The expression of hepatic maturation proteins was measured using immunofluorescence staining. Cytochrome P (CYP)450 metabolism activity and ammonia metabolism tests were used to assess liver function. H-organoids were successfully established by adding human umbilical vein endothelial cells at the beginning of the definitive endoderm stage in our 3D differentiation protocol. The gene expression of alpha-1 antitrypsin, carbamoyl-phosphate synthase 1, and apolipoprotein E, which represent liver maturation state and function, was higher in H-organoids than non-organoid 3D-HLCs. H-organoids possessed higher CYP3A4 metabolism activity and comparable ammonia metabolism capacity than 3D-HLCs. Moreover, although H-organoids expressed human leukocyte antigen class I, they expressed little human leukocyte antigen class II, cluster of differentiation (CD)40, CD80, CD86, and programmed cell death ligand 1, suggesting their immunogenicity properties were not significantly upregulated during differentiation from ADSCs. In conclusion, we successfully established an H-organoid model with higher liver-like functionality than previously established 3D-HLCs and comparable immunogenicity to ADSCs.

Noggin promotes osteogenesis in human adipose-derived mesenchymal stem cells via FGFR2/Src/Akt and ERK signaling pathway

The balance between Noggin and bone morphogenetic proteins (BMPs) is important during early development and skeletal regenerative therapies. Noggin binds BMPs in the extracellular space, thereby preventing BMP signaling. However, Noggin may affect cell response not necessarily through the modulation of BMP signaling, raising the possibility of direct Noggin signaling through yet unspecified receptors. Here we show that in osteogenic cultures of adipose-derived stem cells (ASCs), Noggin activates fibroblast growth factor receptors (FGFRs), Src/Akt and ERK kinases, and it stabilizes TAZ proteins in the presence of dexamethasone. Overall, this leads ASCs to increased expression of osteogenic markers and robust mineral deposition. Our results also indicate that Noggin can induce osteogenic genes expression in normal human bone marrow stem cells and alkaline phosphatase activity in normal human dental pulp stem cells. Besides, Noggin can specifically activate FGFR2 in osteosarcoma cells. We believe our findings open new research avenues to further explore the involvement of Noggin in cell fate modulation by FGFR2/Src/Akt/ERK signaling and potential applications of Noggin in bone regenerative therapies.

Development of a Composite Hydrogel Containing Statistically Optimized PDGF-Loaded Polymeric Nanospheres for Skin Regeneration: In Vitro Evaluation and Stem Cell Differentiation Studies.

Platelet-derived growth factor-BB (PDGF-BB) is a polypeptide growth factor generated by platelet granules faced to cytokines. It plays a role in forming and remodeling various tissue types, including epithelial tissue, through interaction with cell-surface receptors on most mesenchymal origin cells. However, it breaks down quickly in biological fluids, emphasizing the importance of preserving them from biodegradation. To address this challenge, we formulated and evaluated PDGF-encapsulated nanospheres (PD@PCEC) using polycaprolactone-polyethylene glycol-polycaprolactone. PD@PCECs were fabricated through the triple emulsion methodology and optimized by using the Box-Behnken design. The encapsulation efficiency (EE) of nanoencapsulated PDGF-BB was investigated concerning four variables: stirring rate (X1), stirring duration (X2), poly(vinyl alcohol) concentration (X3), and PDGF-BB concentration (X4). The selected optimized nanospheres were integrated into a gelatin-collagen scaffold (PD@PCEC@GC) and assessed for morphology, biocompatibility, in vitro release, and differentiation-inducing activity in human adipose-derived stem cells (hADSCs). The optimized PD@PCEC nanospheres exhibited a particle size of 177.9 ± 91 nm, a zeta potential of 5.2 mV, and an EE of 87.7 ± 0.44%. The release profile demonstrated approximately 85% of loaded PDGF-BB released during the first 360 h, with a sustained release over the entire 504 h period, maintaining bioactivity of 87.3%. The study also included an evaluation of the physicochemical properties of the scaffolds and an assessment of hADSC adhesion to the scaffold's surface. Additionally, hADSCs cultivated within the scaffold effectively differentiated into keratinocyte-like cells (KLCs) over 21 days, evidenced by morphological changes and upregulation of keratinocyte-specific genes, including cytokeratin 18, cytokeratin 19, and involucrin, at both transcriptional and protein levels.

MicroRNA-155 mediates multiple gene regulations pertinent to the role of human adipose-derived mesenchymal stem cells in skin regeneration.

Introduction: The role of Adipose-derived mesenchymal stem cells (AD-MSCs) in skin wound healing remains to be fully characterized. This study aims to evaluate the regenerative potential of autologous AD-MSCs in a non-healing porcine wound model, in addition to elucidate key miRNA-mediated epigenetic regulations that underlie the regenerative potential of AD-MSCs in wounds. Methods: The regenerative potential of autologous AD-MSCs was evaluated in porcine model using histopathology and spatial frequency domain imaging. Then, the correlations between miRNAs and proteins of AD-MSCs were evaluated using an integration analysis in primary human AD-MSCs in comparison to primary human keratinocytes. Transfection study of AD-MSCs was conducted to validate the bioinformatics data. Results: Autologous porcine AD-MSCs improved wound epithelialization and skin properties in comparison to control wounds. We identified 26 proteins upregulated in human AD-MSCs, including growth and angiogenic factors, chemokines and inflammatory cytokines. Pathway enrichment analysis highlighted cell signalling-associated pathways and immunomodulatory pathways. miRNA-target modelling revealed regulations related to genes encoding for 16 upregulated proteins. miR-155-5p was predicted to regulate Fibroblast growth factor 2 and 7, C-C motif chemokine ligand 2 and Vascular cell adhesion molecule 1. Transfecting human AD-MSCs cell line with anti-miR-155 showed transient gene silencing of the four proteins at 24h post-transfection. Discussion: This study proposes a positive miR-155-mediated gene regulation of key factors involved in wound healing. The study represents a promising approach for miRNA-based and cell-free regenerative treatment for difficult-to-heal wounds. The therapeutic potential of miR-155 and its identified targets should be further explored in-vivo.

Adiponectin Inhibits the Progression of Obesity-Associated Papillary Thyroid Carcinoma Through Autophagy.

Obesity is a risk factor for the development of papillary thyroid cancer (PTC). However, the molecular mechanisms by which obesity promotes PTC are unclear. This study aims to identify adipokines that are linked to PTC progression. An adipokine antibody array was used to determine the serum levels of 40 adipokines in normal-weight and obese PTC patients. Enzyme-linked immunosorbent assay was used to determine the serum levels of adiponectin. Recombinant human adiponectin was produced by human adipose-derived stem cells and used to treat PTC cells. Cell proliferation and migration were evaluated using the CCK8 and Transwell assays. Bioinformatics analysis was used to predict mechanisms by which adiponectin affects PTC. Adipokines differentially expressed between normal-weight and obese patients showed a gender-dependent pattern. Obese PTC patients had a significantly lower serum adiponectin level than normal-weight patients, especially in female individuals. Adiponectin levels were negatively correlated with aggressive features of PTC, including tumor diameter > 1 cm, extrathyroidal extension, and lymph node metastasis. Recombinant human adiponectin inhibited the proliferation and migration of human PTC cells in vitro. Bioinformatics analysis identified adiponectin receptor 2 (ADIPOR2) and the autophagy pathway as possible mediators of adiponectin function in TC. In vitro experiments confirmed that adiponectin activated autophagy in PTC cells. These findings shed new lights into the role and mechanisms of adiponectin in TC pathogenesis. Adiponectin is involved in development of obesity-related PTC. Adiponectin can directly inhibit thyroid cancer growth and metastasis through the autophagy pathway.

Microglial uptake of hADSCs-Exo mitigates neuroinflammation in ICH

Intracerebral hemorrhage (ICH) is associated with secondary neuroinflammation, leading to severe central nervous system damage. Exosomes derived from human adipose-derived mesenchymal stem cells (hADSCs-Exo) have shown potential therapeutic effects in regulating inflammatory responses in ICH. This study aims to investigate the role of hADSCs-Exo in ICH and its underlying mechanism involving miRNA-mediated regulation of formyl peptide receptor 1 (FPR1). Flow cytometry was used to identify hADSCs and extract exosomes. Transmission electron microscopy and Western blot were performed to confirm the characteristics of the exosomes. In vitro experiments were conducted to explore the uptake of hADSCs-Exo by microglia cells and their impact on inflammatory responses. In vivo, an ICH mouse model was established, and the therapeutic effects of hADSCs-Exo were evaluated through neurological function scoring, histological staining, and immunofluorescence. Bioinformatics tools and experimental validation were employed to identify miRNAs targeting FPR1. hADSCs-Exo were efficiently taken up by microglia cells and exhibited anti-inflammatory effects by suppressing the release of inflammatory factors and promoting M1 to M2 transition. In the ICH mouse model, hADSCs-Exo significantly improved neurological function, reduced hemorrhage volume, decreased neuronal apoptosis, and regulated microglia polarization. miR-342-3p was identified as a potential regulator of FPR1 involved in the neuroprotective effects of hADSCs-Exo in ICH. hADSCs-Exo alleviate neuroinflammation in ICH through miR-342-3p-dependent targeting of FPR1, providing a new therapeutic strategy for ICH.