Search Results (113)

The development of reusable and biocompatible biomaterial-based culture substrates is increasingly relevant for improving sustainability in biomedical research workflows. In this study, polyethylene terephthalate glycol-modified (PETG) was evaluated as a potential alternative to conventional polystyrene (PS) for in vitro cell culture applications. PETG substrates were fabricated through laser cutting and tested for their ability to support cell adhesion, viability, proliferation, and lineage-specific differentiation across multiple human cell models, including calvarial mesenchymal stromal cells (CMSCs), bone marrow-derived mesenchymal stromal cells (hBM-MSCs), dermal fibroblasts, LHCN-M2 myoblasts, and SH-SY5Y neuroblastoma cells. Morphological and immunofluorescence analyses demonstrated that PETG supported cell attachment and focal adhesion formation, comparable to standard PS surfaces. Cell viability and proliferation assays confirmed metabolic activity and growth over time. Furthermore, PETG substrates supported osteogenic, adipogenic, myogenic, and neuronal differentiation, as demonstrated by histological staining, myotube formation, neurite outgrowth, and lineage-specific gene expression analyses. Finally, PETG maintained CMSC morphology and metabolic activity after repeated recovery, ethanol/UV treatment, and gelatin re-coating, with comparable results between new substrates and those reused for up to three cycles. These findings support PETG as a biocompatible culture substrate with preliminary short-term reuse potential and possible sustainability benefits for laboratory workflows. Full article

The amniotic membrane is widely recognized in regenerative medicine due to its rich content of extracellular matrix proteins and growth factors that confer anti-inflammatory and pro-regenerative properties. However, its rapid degradation restricts its standalone clinical use. To overcome these limitations, we developed biofilms by incorporating decellularized human amniotic membrane matrix (dHAM) into polycaprolactone (PCL) and polyvinylpyrrolidone (PVP) matrices using spin-coating. Bone marrow-derived mesenchymal stem cells (BM-MSCs) were used to evaluate film biocompatibility through cell viability, proliferation, and wound healing migration assays. Surface characterization was performed using contact angle measurements, Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy, and scanning electron microscopy. Soluble dHAM extracts (4–6 mg/mL) significantly enhanced BM-MSC proliferation at 48 h compared to controls (p ≤ 0.01 and p ≤ 0.0001). Both PCL-dHAM and PVP-dHAM biofilms exhibited high cell viability (>90%) and improved initial adhesion. Notably, dHAM incorporation significantly increased wound closure rates at 24 h, reaching 98.47% for PCL-dHAM and 93.13% for PVP-dHAM, compared to 76.56% and 64.20% for pure polymers (p = 0.0001). All scaffolds maintained hydrophilic surfaces (<90°), favorable for cell interaction. The integration of dHAM into PCL and PVP by spin-coating produces biofilms biocompatible with enhanced regenerative potential, representing promising candidates for wound healing applications. In conclusion, these coatings support BM-MSC adhesion, proliferation, and migration, while significantly accelerating wound closure, underscoring their value as advanced bioactive coatings for regenerative medicine. Full article

Background: Postmenopausal osteoporosis is associated with cellular senescence and the accumulation of the senescence-associated secretory phenotype (SASP). While mesenchymal stem cell (MSC)-derived exosomes show tissue repair potential, the efficacy and mechanisms of MSC-derived apoptotic vesicles (apoVs) remain unclear. This study compared MSC-apoVs and exosomes in postmenopausal osteoporosis and investigated the underlying epigenetic mechanisms. Methods: Therapeutic efficacy was evaluated in an ovariectomized (OVX) mouse model and senescent human bone marrow mesenchymal stem cells (hBMMSCs). Small RNA sequencing identified differential microRNA (miRNA) cargos between vesicle types. SASP-related cytokine expression (IL-6, TNF-α, MCP-1) and pathway activation were assessed by RT-qPCR, ELISA, and Western blot. Results: MSC-apoV treatment attenuated bone loss in OVX mice and reduced SASP expression in senescent hBMMSCs to a greater extent than exosomes. Small RNA sequencing revealed that apoVs were enriched with a specific miRNA cluster, including hsa-let-7b-5p, hsa-miR-92a-3p, and hsa-miR-98-5p. Bioinformatic analyses identified BRAF and CRKL as downstream targets of this miRNA cluster, supported by reduced protein levels after apoV treatment. Subsequent molecular assays showed that apoV treatment inhibited the phosphorylation of both the MAPK (p38 and JNK) and NF-κB (p65) signaling pathways, which correlated with reduced local inflammation in the bone marrow microenvironment and preserved osteogenic differentiation capacity. Conclusions: MSC-apoVs attenuate postmenopausal osteoporosis more effectively than exosomes. This enhanced efficacy is associated with the delivery of an enriched miRNA cluster that inhibits MAPK and NF-κB signaling, together with suppression of BRAF and CRKL protein expression. ApoVs may represent a cell-free therapeutic strategy for age-related bone loss. Full article

Background: Advanced glycation end products (AGEs) and oxidative stress (OS) have been linked to bone complications related to type 2 diabetes mellitus (T2DM). However, the effects of AGEs and OS on bone marrow mesenchymal stromal cells (BMMSCs), which play a key role in bone homeostasis and repair, remain unclear. Objectives: This study aimed to investigate the effects of AGEs on BMMSCs function and the ability of N-acetylcysteine (NAC) to alleviate AGE-induced OS in a T2DM context. Methods: Bone marrow (BM) and BMMSCs were isolated from Zucker diabetic fatty (ZDF) rats, which serve as a T2DM model, and their lean littermates (ZL, controls) at 24 weeks of age. Results: The results show that long-standing T2DM leads to changes in the BM’s cellular composition and BMMSCs function that are distinct from age-related changes. In vitro, AGEs decreased BMMSCs viability, proliferation, and migration. The effects of AGEs were stronger in BMMSCs derived from a T2DM microenvironment. In both T2DM- and ZL-BMMSCs, AGEs induced cytoplasmic ROS, which was differentially reduced by NAC. The effect of NAC on T2DM-BMMSCs was greater when the cells were pre-treated with NAC 24 h before exposure to AGEs, whereas simultaneous exposure to both resulted in a smaller effect. Conclusions: These results show that AGEs impair BMMSCs expansion and functionality. AGE-induced ROS generation may be a critical factor in this impairment, while NAC was able to reduce OS in BMMSCs from a T2DM context. These findings highlight the vicious negative effects of the T2DM microenvironment on BMMSCs and underscore the need for further studies to better understand the underlying mechanisms and to explore strategies aimed at mitigating OS in the T2DM context. Full article

Gelatin–alginate composite hydrogels are some of the most prevalent bioinks used for extrusion-based three-dimensional (3D) bioprinting because of their combined bioactivity and ability to ionically crosslink. Ionically crosslinked gelatin–alginate constructs containing human bone marrow–derived mesenchymal stem cells (hBM-MSCs) were characterised over time under standardised in vitro conditions to assess physicochemical properties and resultant cell behaviour. Water uptake and degradation were quantified over time in phosphate-buffered saline (PBS) and collagenase type II media for up to 21 days. Cell viability and metabolic activity were quantified, and osteogenic gene expression (RUNX2, COL1A1, OCN) was assessed. Raman spectroscopy and compressive mechanical characterisation were performed. Collagen and glycosaminoglycan-related peaks were observed from extracellular matrix (ECM)-associated components, with an increased presence of protein-associated signatures later in culture. Hydrogels displayed nonlinear elastic behaviour with increased stress after longer incubation times, suggesting no degradation of mechanical integrity over the duration of the study. Hydrogels experienced rapid hydration followed by decreased swelling over time, with a maximum swelling ratio at 24 h. Degradation rates significantly increased over longer incubation times (p < 0.001) and in collagenase media compared to PBS (p < 0.001). Observed differences were likely due to both ion-exchange-mediated network disassembly and the dissolution of gelatin components. Cell metabolic activity decreased under osteogenic culture conditions, while changes in osteogenic marker expression were sequential, suggesting a transition from proliferation to early osteogenic commitment in this 3D system. This work provides both physicochemical and biological characterisation of a commonly utilised gelatin–alginate bioink system, to provide future optimisations within the field of extrusion-based bone tissue engineering, a reproducible baseline for future optimisation of bioink systems in extrusion-based bone tissue engineering. Full article

Background/Objectives: Pulmonary fibrosis is a chronic, progressive lung disease marked by scarring and inflammation, leading to impaired respiratory function. This study aimed to investigate the combined therapeutic effects of pirfenidone (PFD), metformin (MET), and bone marrow-derived mesenchymal stem cells (BM-MSCs) on bleomycin (BLM)-induced pulmonary fibrosis in rats. Methods: Forty-eight Western Albino rats were divided into six groups: normal control, BLM-positive control, and four treatment groups receiving PFD, MET, BM-MSCs, and their combination. Treatments were administered for four weeks starting on day 21 post-BLM instillation. Lung tissues were analyzed for oxidative stress markers, inflammatory cytokines, apoptotic markers, and fibrogenic gene expression. Histopathological changes were assessed using hematoxylin and eosin (H&E) and Masson’s trichrome staining. Results: The combination therapy significantly reduced oxidative stress and inflammatory markers while enhancing antioxidant capacity. It decreased pro-apoptotic Bcl-2-associated X protein (BAX) and increased anti-apoptotic B-cell lymphoma 2 (Bcl-2) levels. Additionally, anti-inflammatory interleukin-10 (IL-10) was elevated, while tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta 1 (TGF-β1) levels were markedly lowered. Gene expression analysis showed a significant downregulation of matrix metalloproteinase-9 (MMP-9) and collagen type 1 alpha 1 (Col1α1). Histologically, the combination treatment group exhibited minimal fibrosis and inflammation, closely resembling normal lung tissue. Conclusions: The combination of PFD, MET, and BM-MSCs offered superior therapeutic efficacy in treating BLM-induced pulmonary fibrosis compared to individual treatments. This multimodal approach effectively targets oxidative stress, inflammation, apoptosis, and fibrosis, suggesting strong potential for future clinical application. Full article

Corilagin is a hydrolyzable ellagitannin and naturally occurring polyphenolic compound widely distributed in medicinal plants. It is also present in longan (Dimocarpus longan), known as lumyai in Thailand, a subtropical fruit extensively cultivated across China and Southeast Asia. Corilagin has been reported to exhibit strong antioxidant, anti-inflammatory, hepatoprotective, and anticancer activities through modulation of multiple cellular signaling pathways. However, despite these well-established pharmacological properties, its potential role in regulating bone marrow mesenchymal stem cell (BM-MSC) differentiation has not been fully explored in biomedical applications. In this study, we investigated the effects of corilagin on BM-MSC viability, protein-binding interactions, and lineage-specific differentiation toward osteogenic and chondrogenic pathways. Cytotoxicity assessment using human synovial SW-982 cells demonstrated that corilagin maintained cell viability at concentrations ranging from 1.56 to 50 µg/mL within 48 h, whereas prolonged exposure resulted in a time-dependent reduction in viability. In BM-MSCs, corilagin significantly enhanced osteogenic and chondrogenic differentiation in a dose-dependent manner, as evidenced by increased mineral deposition and cartilage matrix formation, as revealed by Alizarin Red S, Toluidine Blue, and Alcian Blue staining. Quantitative analyses further showed the upregulation of key lineage-specific genes, including Runx2 and osteopontin (OPN) for osteogenesis and Sox9 and aggrecan for chondrogenesis. Protein-binding assays confirmed the molecular interaction capacity of corilagin, supporting its biological activity. Overall, these findings demonstrate that corilagin promotes MSC-mediated osteogenic and chondrogenic differentiation while maintaining acceptable cytocompatibility, highlighting its potential as a natural small-molecule candidate for bone and cartilage tissue engineering and other biomedical fields with regenerative medicine applications. Full article

Kynurenic acid (KYNA), a small molecule derived from the tryptophan–kynurenine pathway, can readily diffuse across biological membranes and act as an endogenous ligand for receptors such as the aryl hydrocarbon receptor (Ahr). While KYNA dysregulation is implicated in neurodegenerative disorders, the role of the KYNA–Ahr-IL-6 axis in MSC proliferation and differentiation remains poorly defined. We investigated the impact of KYNA on murine bone marrow-derived MSCs (BM-MSCs) at various concentrations (10–200 μM) and time points (8–48 h). The BM-MSC phenotype was assessed via flow cytometry; proliferation, via cell counting; and the gene expression of Ahr, Cyp1a1, Cyp1b1, and Il-6, via quantitative real-time PCR. Multipotency was evaluated through adipogenic, osteogenic, and chondrogenic differentiation assays with histochemical confirmation. KYNA significantly upregulated Ahr mRNA expression. Among the tested concentrations, 100 μM KYNA induced the highest Ahr expression (~19.1 ± 1.5-fold greater than that of the untreated controls, p < 0.005). Notably, 10 and 50 μM KYNA caused moderate induction, whereas compared with 100 μM KYNA, 200 μM did not further increase expression. In addition, KYN treatment increased Cyp1a1, Cyp1b1, and Il-6 expression, with increases of ~64.6 ± 4.5-fold, ~43.6 ± 2.3-fold, and ~41.6 ± 1.2-fold, respectively. Compared with no treatment, 100 µM KYNA enhanced BM-MSC proliferation by 1.210 ± 0.02, 1.189 ± 0.03, and 1.242 ± 0.02-fold across passages P3, P4, and P5, respectively (p < 0.05), without altering Sca-1, CD90, or CD45 expression or impairing trilineage differentiation potential. KYNA may activate the AHR–IL-6 signaling axis to promote BM-MSC expansion. This controlled proliferative effect, without loss of phenotypic or functional integrity, highlights the pharmacological potential of KYNA as a small-molecule modulator for stem cell-based therapies. Full article

Mesenchymal stromal cells (MSC) have been shown to mitigate IRI through their anti-inflammatory and immune-modulating capacities. This study aims to demonstrate the feasibility, safety, and effectiveness of hepatic administration of bone marrow-derived (BM)-MSCs in a large pig model relevant to human anatomy. After complete vascular exclusion for 45 min, 3 × 10⁶ human BM-MSCs/kg body weight were infused via the portal vein or hepatic artery. BM-MSC infusion did not cause obstruction of hepatic or pulmonary blood flow within 6 h after infusion. Cells were effectively retained in the liver, being undetectable in peripheral blood, lung, and spleen samples. Human B2M expression, as a marker for BM-MSC presence, was significantly higher for the left liver lobe in arterial infusion compared to portal infusion. In liver samples with high BM-MSC levels, we identified the prevention of up- or downregulation of some genes related to inflammation and energy metabolism that was present in non-treated control samples, indicating biological effects within 6 h of infusion. We conclude that hepatic BM-MSC infusion is feasible and safe, with the hepatic artery serving as the optimal administration route for homogenous distribution. These findings pave the way for clinical studies on MSC infusion in IRI, either in situ in liver conditions or ex situ during machine perfusion. Full article

Nanotechnology is transforming contemporary medicine by providing cutting-edge tools for the treatment and diagnosis of complex disorders. Advanced techniques such as bioimaging and photodynamic therapy (PDT) combine early diagnosis and targeted therapy, offering a more precise approach than conventional treatments. However, a significant obstacle for PDT is the need to selectively deliver photosensitizers to disease sites while minimizing systemic side effects. In this context, mesenchymal stem cells have emerged as promising biological carriers due to their natural tropism towards tumors, low immunogenicity, and their ability to overcome biological barriers. In this study, two push–pull compounds, NPI-PTZ and BTZ-PTZ, phenothiazine derivatives featuring aggregation-induced emission (AIE) abilities, were analyzed. These molecules proved to be excellent fluorescent probes and photosensitizing agents. When administered to human bone marrow-derived multipotent stromal cells (hBM-MSCs) and human adipose multipotent stem cells (hASCs), the compounds were efficiently internalized, maintained a stable fluorescent emission for several days, and showed phototoxicity after irradiation, without inducing major cytotoxic effects under normal conditions. These results highlight the potential of NPI-PTZ and BTZ-PTZ combined with mesenchymal stem cells as theranostic tools, bridging bioimaging and PDT, and suggest new possibilities for advanced therapeutic approaches in clinical applications. Full article

Implant-associated infections, particularly those linked to Staphylococcus aureus (S. aureus), continue to compromise the clinical success of β-tricalcium phosphate (β-TCP) implants despite their excellent biocompatibility and osteoconductivity. This investigation aims to tackle these challenges by integrating femtosecond (fs)-laser surface processing with two complementary strategies: ion doping and functionalization with green-synthesized silver nanoparticles (AgNPs). AgNPs were produced via fs-laser photoreduction using green tea leaf extract (GTLE), noted for its anti-inflammatory and antioxidant properties. Fs-laser processing was applied to modify β-TCP scaffolds by systematically varying scanning velocities, fluences, and patterns. Lower scanning velocities generated organized nanostructures with enhanced roughness and wettability, as confirmed by scanning electron microscopy (SEM), optical profilometry, and contact angle measurements, whereas higher laser energies induced significant phase transitions between hydroxyapatite (HA) and α-tricalcium phosphate (α-TCP), as revealed by X-ray diffraction (XRD). AgNP-functionalized scaffolds demonstrated markedly superior antibacterial activity against S. aureus compared to the ion-doped variants, attributed to the synergistic interplay of nanostructure-mediated surface disruption and AgNP-induced bactericidal mechanisms. Although ion-doped scaffolds exhibited limited direct antibacterial effects, they showed concentration-dependent activity in indirect assays, likely due to controlled ion release. Both strategies promoted osteogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) under defined conditions, albeit with transient cytotoxicity at higher fluences and excessive ion doping. Overall, this approach holds promise for markedly improving antibacterial efficacy and osteogenic compatibility, potentially transforming bone regeneration therapies. Full article

Bone tissue engineering (BTE) emerged as a practical approach to tackle prosthetic industry limitations. We merge aspects from developmental biology, engineering and medicine with the aim to produce fully functional bone tissue. Mesenchymal stem cells have the capability of self-renewal and specific lineage differentiation. Herein lies their potential for BTE. Among MSCs, human dental pulp stem cells have a higher proliferation rate, shorter doubling times, lower cellular senescence, and enhanced osteogenesis than hBM-SCs under specific conditions. In addition, these cells are readily accessible and can be extracted through a subtle extraction procedure. Thus, they garner fewer moral concerns than most MSCs available and embody a promising cell source for BTE therapies able to replace hBM-MSCs. Interestingly, their study has been limited. Conversely, there is a need for their further study to harness their true value in BTE, with special emphasis in the design of bioprocesses able to produce viable, homogenous bone constructs in a clinical scale. Here, we study the osteogenic differentiation of hDPSCs encapsulated in alginate hydrogels under suspended culture in a novel perfusion bioreactor. The system is compared with traditional 3D static and fed-batch culture methodologies. The novel system performed better, producing higher alkaline phosphatase activity, and more homogeneous, dense and functional bone constructs. Additionally, cell constructs produced by the in-house-designed system were richer in mature osteoblast-like and mineralizing osteocyte-like cells. In conclusion, this study reports the development of a novel bioprocess able to produce hDPSC-based bone-like constructs, providing new insights into hDPSCs’ therapeutic potential and a system able to be transferred from the laboratory bench into medical facilities. Full article

Background: Transforming growth factor-β (TGF-β) and interleukin 1β (IL-1β) are key regulators of the chondrogenic differentiation, physiology and pathology of cartilage tissue, with TGF-β promoting chondrogenesis and matrix formation, while IL-1β exerts catabolic effects, inhibiting chondrogenesis and contributing to cartilage degradation. Both cytokines alter the intracellular calcium ion (iCa²⁺) levels; however, the exact pathways are not known. Objectives: This study aimed to evaluate the impact of TGF-β3 and IL-1β on calcium homeostasis in human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and chondrocytes during chondrogenesis. Results: TGF-β3 increased iCa²⁺ levels in both hBM-MSCs and chondrocytes. Furthermore, TGF-β3 increased the functional activity of L-type voltage-operated calcium channels (L-VOCCs) in hBM-MSCs but not in chondrocytes. TGF-β3 and IL-1β reduced L-VOCCs subunit CaV1.2 (CACNA1C) gene expression in chondrocytes. In hBM-MSCs, TGF-β3 and IL-1β increased SERCA pump (ATP2A2) gene expression, while in chondrocytes, this effect was observed only with TGF-β3. Conclusions: TGF-β3 increases iCa²⁺ both in osteoarthritic chondrocytes and hBM-MSCs during chondrogenesis. In hBM-MSCs, TGF-β3-mediated elevation in iCa²⁺ is related to the increased functional activity of L-VOCCs. IL-1β does not change iCa²⁺ in osteoarthritic chondrocytes and hBM-MSCs; however, it initiates the mechanisms leading to further downregulation of iCa²⁺ in both types of cells. The differential and cell-specific roles of TGF-β3 and IL-1β in the calcium homeostasis of osteoarthritic chondrocytes and hBM-MSCs during chondrogenesis may provide a new insight into future strategies for cartilage repair and osteoarthritis treatment. Full article

Background/Objectives: Bone regeneration using nanomaterial-based approaches shows promise for treating critical bone defects. However, developing sustainable and cost-effective therapeutic materials remains challenging. This study investigates the osteogenic potential of clove-derived carbon dots (C-CDs) for bone regeneration applications. Methods: C-CDs were synthesized using a green hydrothermal method. The osteogenic potential was evaluated in human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and validated using ectopic bone formation and calvarial defect models. Results: C-CDs demonstrated uniform morphology (~10 nm) with efficient cellular uptake. In vitro studies showed successful osteogenic differentiation through the upregulation of RUNX2, ALP, COL1A1, and BMP-2 mediated by Wnt/β-catenin/GSK3β and BMP signaling pathways. In vivo models have also demonstrated that C-CDs are effective in promoting bone regeneration. Conclusions: These findings establish C-CDs as promising candidates for bone regeneration therapy, offering a sustainable alternative to current treatments. While optimization is needed, their demonstrated osteogenic properties warrant further development for regenerative medicine applications. Full article

This study aimed to evaluate the effect of a newly formulated Commiphora molmol (CM) nano-irrigant on the morphology, viability, proliferation, migration, and wound healing of human bone marrow-derived mesenchymal stem cells (hBMMSCs). Different concentrations of CM nano-irrigant were prepared. The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined to be 25 and 30 mg/mL, respectively. The solution was dispersed into liposomes, which were subsequently coated with chitosan-forming chitosomes. Three concentrations of CM chitosomes were evaluated (25, 30, and 35 mg/mL) along with positive (5.25% NaOCl) and negative (basal culture media) control groups. Cellular viability and proliferation were quantified using AlamarBlue, while wound-healing ability was determined using the scratch assay, and 3D cellular migration was evaluated using the transwell migration assay. All tested concentrations induced observable changes in cellular morphology without any detrimental effects. Viability was monitored at 1, 6, and 24 h, with only Group 1 (25 mg/mL) showing no significant effect on cellular viability. Cellular proliferation was observed over 14 days, with Group 3 (35 mg/mL) being the only group that showed a significantly slower proliferative rate. All tested concentrations resulted in significant differences in transwell migration compared to the negative control. Significant differences were observed within each group across different time points (24–48 h). The results confirm the biocompatibility of the newly formulated CM nano-irrigant in terms of hBMMSCs’ viability, proliferation, morphology, migration, and wound healing. Full article