Rat Adipose-derived Mesenchymal Stem Cells Research Articles

Exploring the Potential Effects of Cryopreservation on the Biological Characteristics and Cardiomyogenic Differentiation of Rat Adipose-Derived Mesenchymal Stem Cells.

Cryopreservation is essential for the broad clinical application of mesenchymal stem cells (MSCs), yet its impact on their cellular characteristics and cardiomyogenic differentiation potential remains a critical concern in translational medicine. This study aimed to evaluate the effects of cryopreservation on the biological properties and cardiomyogenic capacity of rat adipose-derived MSCs (AD-MSCs). We examined their cellular morphology, surface marker expression (CD29, CD90, CD45), trilineage differentiation potential (adipogenic, osteogenic, chondrogenic), and gene expression profiles for the pluripotency marker REX1 and immunomodulatory markers TGFβ1 and IL-6. After inducing cardiomyocyte differentiation, we assessed cardiac-specific gene expressions (Troponin I, MEF2c, GSK-3β) using quantitative RT-qPCR, along with live/dead cell staining and immunofluorescence for cardiac-specific proteins (Troponin T, α-actinin, Myosin Heavy Chain). Cryopreserved AD-MSCs preserved their morphology, surface markers, and differentiation potential, but exhibited a reduced expression of REX1, TGFβ1, and IL-6. Additionally, cryopreservation diminished cardiomyogenic differentiation, as indicated by the lower levels of Troponin I, MEF2c, and GSK-3β seen compared to non-cryopreserved cells. Despite this, high cell viability (>90%) and maintained cardiac protein expression were observed post-cryopreservation. These findings highlight the necessity of optimizing cryopreservation protocols to ensure the full therapeutic potential of AD-MSCs, particularly in applications related to cardiac regenerative medicine.

PCL reinforced nano strontium hydroxyapatite-Butea monosperma-scaffold for bone regeneration

Biocomposite materials, composed of biodegradable polymers and supplements, are promising for bone regenerative matrices due to their mechanical strength and excellent osteoconductivity. This study focuses on fabricating electrospun composite scaffolds using the bark extract of Butea monosperma (BM), nano strontium hydroxyapatite (nSrHA), and polycaprolactone (PCL). Four types of scaffolds were prepared: PCL (control), BM-PCL, nSrHA-PCL, and BM-nSrHA-PCL. The scaffolds were evaluated through various techniques: bright field microscopy for standardization, SEM for structural evaluation, and SEM-EDX for elemental analysis. Additionally, mechanical strength, release study, degradation, and protein adsorption were assessed. Furthermore, cytotoxicity, proliferation, and cytocompatibility of the scaffolds were evaluated using rat adipose-derived mesenchymal stem cells (rADMSCs) through SEM, MTT assay, LDH assay, DAPI staining, and Direct contact assay. SEM analysis confirmed that all four fabricated scaffolds were nano-fibrous in nature. The incorporation of phytochemicals and nSrHA was confirmed via SEM-EDX. Mechanical testing revealed that the BM-nSrHA-PCL scaffolds had superior mechanical properties compared to the control PCL scaffolds. Protein adsorption studies indicated enhanced protein binding in BM-nSrHA-PCL scaffolds. Degradation studies showed that BM-nSrHA-PCL scaffolds had a controlled degradation rate suitable for tissue engineering applications. Cytotoxicity tests confirmed that all scaffolds were noncytotoxic. Proliferation and cytocompatibility assays demonstrated that BM-nSrHA-PCL scaffolds significantly promoted cell proliferation compared to other scaffold types. These results suggest that the BM-nSrHA-PCL scaffolds exhibit improved mechanical properties, protein adsorption, controlled degradation, and enhanced cell proliferation, making them promising candidates for bone tissue engineering applications.

Augmented reparative potential of adipose-derived stem cells with silver nanoparticles against CCl4-induced liver injury in rats

Cymbopogon citratus (lemongrass) is a widely used medicinal plant. The previous literature studies demonstrated its diverse pharmacological activities such as anti-inflammatory, anti-diabetic, antibacterial and antifungal properties. The present study involves the green synthesis of silver nanoparticles from lemongrass and their characterization by X-ray diffraction (XRD), scanning electron microscope (SEM) and Fourier transmission infrared (FTIR). The purpose of this study is to evaluate the hepatoprotective potential of silver nanoparticles derived from the aqueous extract of lemongrass (LG-NPs) in comparison with the rat adipose-derived mesenchymal stem cells (ASCs) treated with lemongrass-derived silver nanoparticles (LG-NPs + ASCs). To assess the hepatoprotective effect of LG-NPs + ASCs, CCl4-induced fibrosis model rats were employed and it was revealed that transplanting LG-NPs + ASCs decreased fibrosis development compared to transplanting naive ASCs and LG-NPs, as demonstrated by improved hepatic architecture and functions. Additionally, the liver parenchyma's expression of fibrosis-associated gene markers Interleukin 1 beta (IL-1β), Cytochrome P450 2E1 (CYP2E1), Transforming Growth Factor Beta 1 (TGF-β1), collagen type I alpha 1(Col1α1), α-smooth muscle actin (α-SMA) reduced after LG-NPs + ASCs transplantation. These results suggest that pretreating ASCs with LG-NPs increase their effectiveness.

Synergistic Hepatoprotective Effects of Mesenchymal Stem Cells and Platelet-Rich Plasma in a Rat Model of Bile Duct Ligation-Induced Liver Cirrhosis.

Liver cirrhosis poses a global health challenge marked by significant prevalence and mortality. Current therapeutic options are limited by high costs and immune-mediated rejection, necessitating the exploration of innovative strategies to enhance hepatic self-rehabilitation, and counteract the underlying pathological mechanisms. We evaluated the hepatoprotective activity of rat adipose-derived mesenchymal stem cells (ADMSCs) in combination with platelet-rich plasma (PRP) and recombinant human hepatocyte growth factor (rh-HGF) on a rat model of liver fibrosis/cirrhosis induced by bile duct ligation (BDL). Treatment with PRP or rh-HGF alone did not yield significant hepatoprotection in the BDL-induced liver cirrhosis model. However, ADMSC transplantation alone exhibited the potential to alleviate impaired liver conditions. The combination of PRP and rh-HGF demonstrated superior ameliorative effects compared to either treatment alone. Notably, the combination of ADMSC + PRP or ADMSC + rh-HGF significantly enhanced hepatoprotective capacity compared to individual or combined PRP and rh-HGF therapies. Injection of ADMSC via the tail vein reduced inflammation, hepatocyte damage, and collagen deposition, improving overall liver function. This improvement was more pronounced when ADMSC was administered with PRP and rh-HGF versus monotherapy. Our study concludes that ADMSCs exert antifibrotic effects by inhibiting hepatic stellate cell proliferation, collagen synthesis, and inducing apoptosis. ADMSCs also demonstrate immune-modulatory effects and transdifferentiate into hepatic progenitor cells, secreting trophic factors, cytokines, and chemokines that promote impaired liver regeneration. The observed arrest in liver fibrosis progression highlights the potential therapeutic impact of these interventions.

Adipose Stem Cell-Seeded Decellularized Porcine Pericardium: A Promising Functional Biomaterial to Synergistically Restore the Cardiac Functions Post-Myocardial Infarction.

Myocardial infarction (MI) is a serious cardiovascular disease as the leading cause of death globally. Hence, reconstruction of the cardiac tissue comes at the forefront of strategies adopted to restore heart functions following MI. In this investigation, we studied the capacity of rat adipose-derived mesenchymal stem cells (r-AdMSCs) and decellularized porcine pericardium (DPP) to restore heart functions in MI animals. MI was induced in four different groups, three of which were treated either using DPP (MI-DPP group), stem cells (MI-SC group), or both (MI-SC/DPP group). Cardiac functions of these groups and the Sham group were evaluated using echocardiography, the intraventricular pressure gradient (IVPG) on weeks 2 and 4, and intraventricular hemodynamics on week 4. On day 31, the animals were euthanized for histological analysis. Echocardiographic, IVPG and hemodynamic findings indicated that the three treatment strategies shared effectively in the regeneration process. However, the MI-SC/DPP group had a unique synergistic ability to restore heart functions superior to the other treatment protocols. Histology showed that the MI-SC/DPP group presented the lowest (p < 0.05) degeneration score and fibrosis % compared to the other groups. Conclusively, stem cell-seeded DPP is a promising platform for the delivery of stem cells and restoration of heart functions post-MI.

Cementum protein 1 gene-modified adipose-derived mesenchymal stem cell sheets enhance periodontal regeneration in osteoporosis rat.

Osteoporosis (OP) and periodontitis are both diseases with excessive bone resorption, and the number of patients who suffer from these diseases is expected to increase. OP has been identified as a risk factor that accelerates the pathological process of periodontitis. Achieving effective and safe periodontal regeneration in OP patients is a meaningful challenge. This study aimed to assess the efficacy and biosecurity of human cementum protein 1 (hCEMP1) gene-modified cell sheets for periodontal fenestration defect regeneration in an OP rat model. Rat adipose-derived mesenchymal stem cells (rADSCs) were isolated from Sprague-Dawley rats. After primary culture, rADSCs were subjected to cell surface analysis and multi-differentiation assay. And rADSCs were transduced with hCEMP1 by lentiviral vector, and hCEMP1 gene-modified cell sheets were generated. The expression of hCEMP1 was evaluated by reverse transcription polymerase chain reaction and immunocytochemistry staining, and transduced cell proliferation was evaluated by Cell Counting Kit-8. The hCEMP1 gene-modified cell sheet structure was detected by histological analysis and scanning electron microscopy. Osteogenic and cementogenic-associated gene expression was evaluated by real-time quantitative polymerase chain reaction. In addition, an OP rat periodontal fenestration defect model was used to evaluate the regeneration effect of hCEMP1 gene-modified rADSC sheets. The efficacy was assessed with microcomputed tomography and histology, and the biosecurity of gene-modified cell sheets was evaluated by histological analysis of the spleen, liver, kidney and lung. The rADSCs showed a phenotype of mesenchymal stem cells and possessed multi-differentiation capacity. The gene and protein expression of hCEMP1 through lentiviral transduction was confirmed, and there was no significant effect on rADSC proliferation. Overexpression of hCEMP1 upregulated osteogenic and cementogenic-related genes such as runt-related transcription factor 2, bone morphogenetic protein 2, secreted phosphoprotein 1 and cementum attachment protein in the gene-modified cell sheets. The fenestration lesions in OP rats treated with hCEMP1 gene-modified cell sheets exhibited complete bone bridging, cementum and periodontal ligament formation. Furthermore, histological sections of the spleen, liver, kidney and lung showed no evident pathological damage. This pilot study demonstrates that hCEMP1 gene-modified rADSC sheets have a marked ability to enhance periodontal regeneration in OP rats. Thus, this approach may represent an effective and safe strategy for periodontal disease patients with OP.

Neural Tissue Engineering with Rat Adipose-Derived Mesenchymal Stem Cells: The Role of an Injectable, Resorbable Hydrogel Scaffold Derived from Oxidized Alginate and Gelatin.

The central nervous system has limited regeneration potential. The multipotency of adipose-derived mesenchymal stem cells (ADMSC) makes them an ideal autologous cell source for the regeneration of neural tissues. However, the likelihood of their differentiation into unwanted cell lineages when transplanted into a hostile injury environment is a serious disadvantage. Transplanting predifferentiated cells via an injectable carrier may aid in site-specific delivery for better survival of cells. Here, we focus on identifying an appropriate injectable hydrogel system that favors stem/progenitor cell attachment and differentiation for neural tissue engineering. An injectable composition of the hydrogel, derived from alginate dialdehyde (ADA) and gelatin, was formulated for this purpose. This hydrogel promoted proliferation/differentiation of ADMSCs to neural progenitors, visualized from the generation of prominent neurospheres and stage-specific expression of a neural progenitor marker (nestin, day 4), an intermittent neuronal marker (β-III tub, day 5), and a mature neuronal marker (MAP-2, day 8) with neural branching and networking (>85%). The differentiated cells also expressed the functional marker synaptophysin. There was no negative impact on stem/progenitor cell survival (>95%) or differentiation (∼90%) as compared to two-dimensional (2D) culture. Addition of appropriate quantities of asiatic acid specific for neural niche supported cell growth and differentiation without affecting cell survival (>90%) and improved neural branching and elongation. Optimized interconnected porous hydrogel niche exhibited rapid gelation (3 min) and self-healing properties mimicking native neural tissue. Both ADA-gelatin hydrogel by itself and that incorporated with asiatic acid were found to support stem/neural progenitor cell growth and differentiation and have potential applications as antioxidants and growth promoters upon release at the cell transplantation site. In short, the matrix itself or incorporated with phytomoieties could serve as a potential minimally invasive injectable cell delivery vehicle for cell-based therapies of neural diseases.

Impact of Adipose Tissue Depot Harvesting Site on the Multilineage Induction Capacity of Male Rat Adipose-Derived Mesenchymal Stem Cells: An In Vitro Study.

Recently, substantial attention has been paid toward adipose-derived mesenchymal stem cells (AdMSCs) as a potential therapy in tissue engineering and regenerative medicine applications. Rat AdMSCs (r-AdMSCs) are frequently utilized. However, the influence of the adipose depot site on the multilineage differentiation potential of the r-AdMSCs is still ambiguous. Hence, the main objective of this study was to explore the influence of the adipose tissue harvesting location on the ability of r-AdMSCs to express the stem-cell-related markers and pluripotency genes, as well as their differentiation capacity, for the first time. Herein, we have isolated r-AdMSCs from the inguinal, epididymal, peri-renal, and back subcutaneous fats. Cells were compared in terms of their phenotype, immunophenotype, and expression of pluripotency genes using RT-PCR. Additionally, we investigated their potential for multilineage (adipogenic, osteogenic, and chondrogenic) induction using special stains confirmed by the expression of the related genes using RT-qPCR. All cells could positively express stem cell marker CD 90 and CD 105 with no significant in-between differences. However, they did not express the hematopoietic markers as CD 34 and CD 45. All cells could be induced successfully. However, epididymal and inguinal cells presented the highest capacity for adipogenic and osteogenic differentiation (21.36-fold and 11.63-fold for OPN, 29.69-fold and 26.68-fold for BMP2, and 37.67-fold and 22.35-fold for BSP, respectively, in epididymal and inguinal cells (p < 0.0001)). On the contrary, the subcutaneous cells exhibited a superior potential for chondrogenesis over the other sites (8.9-fold for CHM1 and 5.93-fold for ACAN, (p < 0.0001)). In conclusion, the adipose tissue harvesting site could influence the differentiation capacity of the isolated AdMSCs. To enhance the results of their employment in various regenerative cell-based therapies, it is thus vital to take the collection site selection into consideration.

Evaluation of the biocompatibility of fish skin collagen with the mesenchymal stem cells in in vitro cultures.

There are scarce published data suggesting, that collagen extracted from fish skin may be an attractive alternative to mammalian-derived collagen for the in vitro cell cultures. In this study, we investigated proliferation potential and differentiation capability into osteogenic and adipogenic lineages of rat adipose-derived mesenchymal stem cells (rASCs) and human adipose-derived mesenchymal stem cells (hASCs) cultured on collagen extracted from silver carp and African sharptooth catfish skins, compared with commercially available mammalian collagen and collagen-free culture dishes. Our results revealed no significant differences between fish collagen and mammalian collagen in supporting cell viability and proliferation capacity. Fish-derived collagen is a cheap material derived from production waste, does not contain transmissible pathogens of mammalian origin, supports human cell cultures at comparable level to conventional collagen sources, and may be considered as the product of choice for the in vitro cell cultures.

Combining chitin biological conduits with injectable adipose tissue-derived decellularised matrix hydrogels loaded with adipose-derived mesenchymal stem cells for the repair of peripheral nerve defects in rats

Mesenchymal stem cells (MSCs) have shown potential for the repair of defective tissues and peripheral nerve injuries. However, local treatment alone may lead to cell loss, low viability, and diminished paracrine action owing to the lack of effective biological scaffolds. To improve the therapeutic effects of MSCs, bioactive scaffolds that mimic the stem cell microenvironment in vivo should be constructed. We developed an injectable decellularised matrix hydrogel (DAM-gel) to simulate the stem cell microenvironment using combined physical, chemical, and enzymatic digestions of human adipose tissues. The DAM-gel was loaded with rat adipose-derived mesenchymal stem cells (ADSCs) to repair sciatic nerve defects. Compared with ADSCs alone, the ADSC-loaded DAM-gel promoted the proliferation of Schwann cells in vitro, which are important in sciatic nerve regeneration. Chitin biological conduits filled with ADSC-DAM-gel composites were designed to bridge the sciatic nerve defects. Axonal regeneration and the recovery of neurological function in the ADSC-DAM-gel group increased post-surgery compared with the control group (blank conduit, ADSCs, and DAM-gel group), as confirmed by a CatWalk gait analysis, electrophysiology, and nerve/muscle histology. The ADSC-loaded DAM-gel-treated rats retained the improved peripheral nerve regeneration. Therefore, DAM-gels have great potential for enhancing the repair capacity of ADSCs in peripheral nerve defects.

Zirconia substrate with periodic surface microstructures enhances osteogenic differentiation of rat adipose-derived stem cells

Zirconia substrates with periodic surface microstructures formed by irradiation with a femtosecond laser previously enhanced osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs), associated with a reduction in the cell attachment area. Adipose-derived mesenchymal stem cells (ADSCs) are clinically less difficult in harvesting the cells than BMSCs. In this study, we investigated the effects of zirconia substrates with the periodic surface microstructures on osteogenic differentiation of rat ADSCs. Zirconia substrates with the periodic surface microstructures enhanced not adipogenic differentiation but osteogenic differentiation of rat ADSCs, associated with a reduction in the cell attachment area. Therefore, zirconia substrates with surface periodic microstructures formed by irradiation with a femtosecond laser could be useful in bone tissue engineering using ADSCs when BMSCs are unavailable.

Macroporous Cell-Laden Gelatin/Hyaluronic Acid/Chondroitin Sulfate Cryogels for Engineered Tissue Constructs.

Cryogels are a unique macroporous material for tissue engineering. In this work, we study the effect of hyaluronic acid on the physicochemical properties of cryogel as well as on the proliferation of a 3D model of mesenchymal stem cells. The functional groups of the synthesized cryogels were identified using Fourier transform infrared spectroscopy. With an increase in the content of hyaluronic acid in the composition of the cryogel, an increase in porosity, gel content and swelling behavior was observed. As the hyaluronic acid content increased, the average pore size increased and more open pores were formed. Degradation studies have shown that all cryogels were resistant to PBS solution for 8 weeks. Cytotoxicity assays demonstrated no toxic effect on viability of rat adipose-derived mesenchymal stem cells (ADMSCs) cultured on cryogels. ADMSC spheroids were proliferated on scaffolds and showed the ability of the cryogels to orient cell differentiation into chondrogenic lineage even in the absence of inductive agents. Thus, our results demonstrate an effective resemblance to extracellular matrix structures specific to cartilage-like microenvironments by cryogels and their further perspective application as potential biomaterials.

Soybean protein isolate/chitosan composite microcarriers for expansion and osteogenic differentiation of stem cells

Bone tissue has limited ability of self-repair and regeneration. To repair complexly shaped bone tissue defects, chitosan microcarriers (CS microcarriers, being used as cell carriers) impregnated with soybean protein isolate were prepared for expansion and osteogenic differentiation of stem cells. Highly soluble 7S and 11S powders were obtained through purification and separation of soy protein by adopting Nagano. Pure CS microcarriers were prepared by using an anti-phase suspension combined with thermally induced phase separation technique. Thereafter, CS microcarriers was impregnated into 11S, 7S and SPI solution, forming 11S/CS, 7S/CS and SPI/CS composite microcarriers. The morphology, pore size, porosity and surface potential of the composite microcarriers were investigated. The microcarriers are all three-dimensional porous structures. Compared with CS and SPI/CS microcarriers, 11S/CS and 7S/CS composite microcarriers showed porous structures and high porosity with large pores. Thereafter, rat adipose-derived mesenchymal stem cells (rADSCs) were cultured on composite microcarriers for 14 days to evaluate the effect of composite microcarriers on stem cells proliferation and osteogenic differentiation ability. The outcomes show that composite microcarriers better-supported cell adhesion and proliferation than CS microcarriers. In addition, there were more cells inside 11S/CS and 7S/CS composite microcarriers than those of CS, SPI/CS microcarriers. Alkaline phosphatase (ALP), collagen type I (COLI) and Alizarin Red S staining showed that the addition of soybean protein isolate components could promote osteogenic differentiation of rADSCs.

Studying the Effect of Chondroitin Sulfate on the Physicochemical Properties of Novel Gelatin/Chitosan Biopolymer-Based Cryogels

The application of biopolymers in tissue engineering is of a great interest due to of their inherent properties such as cell adhesion, biodegradation, bioavailability, and viscoelasticity. In this study, we synthesized cryogels based on biopolymers of gelatin, chitosan, and chondroitin sulfate by cryopolymerization and studied the effect of chondroitin sulfate on changing the physicochemical properties of cryogels such as pore size, pore volume, density, gel fraction, and biodegradation. A macroporous surface of the synthesized polymers has been investigated by SEM. The glass transition temperatures of the crosslinked cryogels, determined by the DSC method, were higher compared to that of the non-crosslinked cryogel used as a reference. The results of the MTT test showed that aqueous extracts of the prepared cryogels had no toxic effect on rat adipose-derived mesenchymal stem cells. The research in this area is of great importance and provides new insights into novel, effective methods for obtaining biopolymers that can be used as carriers of cells.

Combined local delivery of tacrolimus and stem cells in hydrogel for enhancing peripheral nerve regeneration.

The application of scaffold-based stem cell transplantation to enhance peripheral nerve regeneration has great potential. Recently, the neuroregenerative potential of tacrolimus (a U.S. Food and Drug Administration-approved immunosuppressant) has been explored. In this study, a fibrin gel-based drug delivery system for sustained and localized tacrolimus release was combined with rat adipose-derived mesenchymal stem cells (MSC) to investigate cell viability in vitro. Tacrolimus was encapsulated in poly(lactic-co-glycolic) acid (PLGA) microspheres and suspended in fibrin hydrogel, using concentrations of 0.01and 100 ng/ml. Drug release over time was measured. MSCs were cultured in drug-released media collected at various days to mimic systemic exposure. MSCs were combined with (i) hydrogel only, (ii) empty PLGA microspheres in the hydrogel, (iii) 0.01, and (iv) 100 ng/ml of tacrolimus PLGA microspheres in the hydrogel. Stem cell presence and viability were evaluated. A sustained release of 100 ng/ml tacrolimus microspheres was observed for up to 35 days. Stem cell presence was confirmed and cell viability was observed up to 7 days, with no significant differences between groups. This study suggests that combined delivery of 100 ng/ml tacrolimus and MSCs in fibrin hydrogel does not result in cytotoxic effects and could be used to enhance peripheral nerve regeneration.

In-Vitro Differentiation Adipose-Derived Mesenchymal Stem Cells into Pancreatic Progenitor Cells

Background: Adult stem cells are currently reliable sources of mesenchymal stem cells for regenerativetherapy, include diabetes mellitus. The aim of this study is to develop endocrine pancreatic progenitor cellscharacterized by Pdx1 and insulin expression from rat adipose-derived mesenchymal stem cells using twosteps in-vitro differentiation.Methods: In this experimental study, ADMSCs were isolated from rat adipose tissue and exposed toinsulinogenic differentiation medium containing nicotinamide, activin A and glucagon-like peptide-1 (GLP1). After induction, the existence of pancreatic progenitor cells (PPCs) was confirmed by immune-stainingassay of Pdx1 and insulin.Results: After three weeks of in-vitro differentiation, expression of Pdx1 and insulin proteins showed upas green in the immunofluorescence assay. Immunofluorescence intensity of Pdx1 was higher in PPCs thanin ADMSCs control (p&lt;0.05). Immunofluorescence intensity of insulin was also higher in PPCs than inADMSCs control (p&lt;0.05). Therefore, in-vitro differentiation was successful to develop PPCs from ratADMSCsConclusion: This study has demonstrated the in-vitro differentiation of ADMSCs into PPCs that expressedPdx1 and insulin

Gelatin reduced Graphene Oxide Nanosheets as Kartogenin Nanocarrier Induces Rat ADSCs Chondrogenic Differentiation Combining with Autophagy Modification.

Biocompatible reduced graphene oxide (rGO) could deliver drugs for synergistically stimulating stem cells directed differentiation with influences on specific cellular activities. Here, we prepared a biodegradable gelatin reduced graphene oxide (rGO@Ge) to evaluate its functions in promoting rat adipose derived mesenchymal stem cells (ADSCs) chondrogenic differentiation through delivering kartogenin (KGN) into the stem cell efficiently. The optimum KGN concentration (approximately 1 μM) that promoted the proliferation and chondrogenic differentiation of ADSCs was clarified by a series of experiments, including immunofluorescent (IF) staining (Sox-9, Col II), alcian blue (Ab) staining, toluidine blue (Tb) staining and real-time quantitative PCR analysis of the chondrogenic markers. Meanwhile, the biocompatibility of rGO@Ge was evaluated to clearly define the nonhazardous concentration range, and the drug loading and releasing properties of rGO@Ge were tested with KGN for its further application in inducing ADSCs chondrogenic differentiation. Furthermore, the mechanism of rGO@Ge entering ADSCs was investigated by the different inhibitors that are involved in the endocytosis of the nanocarrier, and the degradation of the rGO@Ge in ADSCs was observed by transmission electron microscopy (TEM). The synergistic promoting effect of rGO@Ge nanocarrier on ADSCs chondrogenesis with KGN was also studied by the IF, Ab, Tb stainings and PCR analysis of the chondrogenic markers. Finally, the intracellular Reactive Oxygen Species (ROS) and autophagy induced by KGN/rGO@Ge complex composites were tested in details for clarification on the correlation between the autophagy and chondrogenesis in ADSCs induced by rGO@Ge. All the results show that rGO@Ge as a biocompatible nanocarrier can deliver KGN into ADSCs for exerting a pro-chondrogenic effect and assist the drug to promote ADSCs chondrogenesis synergistically through modification of the autophagy in vitro, which promised its further application in repairing cartilage defect in vivo.

RETRACTED ARTICLE: Effects of miR-672 on the angiogenesis of adipose-derived mesenchymal stem cells during bone regeneration

BackgroundSufficient vascular network plays an important role in the repair of bone defects. Bone morphogenetic protein 2 (BMP2) being a key regulator of angiogenesis has attracted the attention of researchers. In addition, evidence has suggested that BMP2 coordinates with microRNAs (miRNAs) to form intracellular networks regulating mesenchymal stem cells (MSCs) angiogenesis. Elucidating the underlying mechanisms that are regulating adipose-derived mesenchymal stem cells (ADSCs) angiogenesis might provide more effective method to enhance bone regeneration.MethodsWe identified the specific miRNA in rat ADSCs during BMP2-induced angiogenesis and chose the most significant differentially expressed miRNA, miR-672. Three lentiviral system named Lenti-miR-672, Lenti-as-miR-672, and Lenti-miR-NC were transduced into the ADSCs individually. Then, the quantitative real-time polymerase chain reaction (qPCR), western blotting, and blood vessel formation analysis were performed to investigate the effects of miR-672 on ADSCs angiogenesis. Bioinformation platforms were used to screen the potential target of miR-672. Small interfering RNA (siRNA) against TIMP2 (si-TIMP2) mRNA were obtained from GenePharma, and then si-TIMP2 miRNA and miR-672 were co-transfected into ADSCs to detect the effects of TIMP2 on angiogenesis. Calcium phosphate cement (CPC) scaffolds that seeded the lentiviral-modified ADSCs were constructed to test the vascularized bone regeneration in vivo.ResultsOur data showed that after the angiogenesis of ADSCs induced by BMP2, miR-672 was the most significantly upregulated miRNA. Overexpression of miR-672 promoted the angiogenesis of ADSCs, while knockdown of miR-672 repressed the angiogenesis of ADSCs. The bioinformation prediction showed that TIMP2 might be the one of miR-672′ potential targets. TIMP2 protein expression was gradually decreased in ADSCs with overexpressed miR-672. And the angiogenic factors were upregulated in the ADSCs which were transduced with si-TIMP2. Then, the CPC scaffolds coupled the miR-672-modified ADSCs and showed the good potential in vascularized bone regeneration. The overexpressed miR-672 could greatly enhance the blood vessel volume and Microfil-labeled blood vessel numbers in newly formed bone.ConclusionBMP2 could promote the angiogenesis of ADSCs through stimulating the expression of miR-672 in ADSCs. miR-672 acted as a positive regulator on the angiogenesis of ADSCs, and incorporating the miR-672-modified ADSCs in the CPC could significantly promote the vascularization and the bone regeneration.

Healing effects of a protein scaffold loaded with adipose-derived mesenchymal stem cells on radiation-induced vaginal injury in rats.

ObjectivesCervical cancer, the most common female cancer after breast cancer, is typically treated using radiotherapy. However, pelvic radiotherapy can cause irreversible damage to the vagina, seriously affecting patients’ quality of life. In this study, protein scaffolds loaded with rat adipose-derived mesenchymal stem cells (ADSCs) were implanted into irradiated tissue to assess their healing potential.MethodsWe established a rat model of radiation-induced vaginal injury. Complexes (consisting of protein scaffolds loaded with ADSCs) were implanted into injury sites. Histological analysis were used to assess regeneration of the vaginal epithelium. RNA sequencing was used to study the therapeutic mechanism of the complexes.ResultsThe complexes promoted vaginal epithelial cell regeneration, vaginal tissue repair and improved vaginal stenosis and contracture. Compared with rats transplanted with ADSCs, rats transplanted with complexes achieved better therapeutic effects.ConclusionsProtein scaffold-ADSC complexes had a beneficial therapeutic effect on radiation-induced vaginal injury in rats and may serve as the basis of a novel therapeutic approach for radiation dermatitis.

Effects of hypoxia-pretreated rat adipose-derived mesenchymal stem cells conditioned medium on wound healing of rats with full-thickness defects

Effects of hypoxia-pretreated rat adipose-derived mesenchymal stem cells conditioned medium on wound healing of rats with full-thickness defects