Assessment of the disruption effects of tetrabromobisphenol A and its analogues on lipid metabolism using multiple in vitro models.

Tetrabromobisphenol A (TBBPA), a widely-used brominated flame retardant, has been revealed to exert endocrine disrupting effects and induce adipogenesis. Given the high structural similarities of TBBPA analogues and their increasing exposure risks, their effects on lipid metabolism are necessary to be explored. Herein, 9 representative TBBPA analogues were screened for their interference on 3T3-L1 preadipocyte adipogenesis, differentiation of C3H10T1/2 mesenchymal stem cells (MSCs) to brown adipocytes, and lipid accumulation of HepG2 cells. TBBPA bis(2-hydroxyethyl ether) (TBBPA-BHEE), TBBPA mono(2-hydroxyethyl ether) (TBBPA-MHEE), TBBPA bis(glycidyl ether) (TBBPA-BGE), and TBBPA mono(glycidyl ether) (TBBPA-MGE) were found to induce adipogenesis in 3T3-L1 preadipocytes to different extends, as evidenced by the upregulated intracellular lipid generation and expressions of adipogenesis-related biomarkers. TBBPA-BHEE exhibited a stronger obesogenic effect than did TBBPA. In contrast, the test chemicals had a weak impact on the differentiation process of C3H10T1/2 MSCs to brown adipocytes. As for hepatic lipid formation test, only TBBPA mono(allyl ether) (TBBPA-MAE) was found to significantly promote triglyceride (TG) accumulation in HepG2 cells, and the effective exposure concentration of the chemical under oleic acid (OA) co-exposure was lower than that without OA co-exposure. Collectively, TBBPA analogues may perturb lipid metabolism in multiple tissues, which varies with the test tissues. The findings highlight the potential health risks of this kind of emerging chemicals in inducing obesity, non-alcoholic fatty liver disease (NAFLD) and other lipid metabolism disorders, especially under the conditions in conjunction with high-fat diets.

An in vitro assessment of teething gels' effects on human gingival mesenchymal stem cells.

BACKGROUND: The aim of this study is to examine the cytotoxic effects of dental gels with different contents, which are frequently used during teething, on gingival mesenchymal stem cells (G-MSCs). METHOD: The teething gels used in this study were Dentinox, Gengigel, Osanite, and Jack and Jill. The human gingival mesenchimal stem cells (hG-MSCs) were incubated with these teething gel solutions (0.1%, 50% and 80% concentrations). Reproductive behavior of G-MSCs was monitored in real time for 72 h using the xCELLigence real-time cell analyzer (RTCA) system. Two-way repeated Anova test and post hoc Bonferroni test were used to evaluate the effect of concentration and dental gel on 0-hour and 72-hour viability. Significance was evaluated at p < 0.05 level. RESULTS: Teething gels prepared at 50% concentration are added to the G-MSC culture, the "cell index" value of G-MSCs to which Dentinox brand gel is added is significantly lower than all other groups (p = 0.05). There is a statistically significant difference between the concentrations in terms of cell index values at the 72nd hour compared to the 0th hour (p = 0.001). CONCLUSIONS: The local anesthetic dental gels used in children have a more negative effect on cell viability as concentration increases.

Challenging applicability of ISO 10993-5 for calcium phosphate biomaterials evaluation: Towards more accurate in vitro cytotoxicity assessment.

Research on biomaterials typically starts with cytocompatibility evaluation, using the ISO 10993-5 standard as a reference that relies on extract tests to determine whether the material is safe (cell metabolic activity should exceed 70 %). However, the generalized approach within the standard may not accurately reflect the material's behavior in direct contact with cells, raising concerns about its effectiveness. Calcium phosphates (CaPs) are a group of materials that, despite being highly biocompatible and promoting bone formation, still exhibit inconsistencies in basic cytotoxicity evaluations. Hence, in order to test the cytocompatibility dependence on different experimental setups and material-cell interactions, we used amorphous calcium phosphate, α-tricalcium phosphate, hydroxyapatite, and octacalcium phosphate (0.1 mg/mL to 5 mg/mL) with core cell lines of bone microenvironment: mesenchymal stem cells, osteoblast-like and endothelial cells. All materials have been characterized for their physicochemical properties before and after cellular contact and once in vitro assays were finalized, groups identified as 'cytotoxic' were further analyzed using a modified Annexin V apoptosis assay to accurately determine cell death. The obtained results showed that indirect contact following ISO standards had no sensitivity of tested cells to the materials, but direct contact tests at physiological concentrations revealed decreased metabolic activity and viability. In summary, our findings offer valuable guidelines for handling biomaterials, especially in powder form, to better evaluate their biological properties and avoid false negatives commonly associated with the traditional standard approach.

Preclinical assessment of thrombin-preconditioned human Wharton's jelly-derived mesenchymal stem cells for neonatal hypoxic-ischaemic brain injury.

Hypoxic-ischaemic encephalopathy (HIE) is a type of brain injury affecting approximately 1 million newborn babies per year worldwide, the only treatment for which is therapeutic hypothermia. Thrombin-preconditioned mesenchymal stem cells (MSCs) exert neuroprotective effects by enriching cargo contents and boosting exosome biogenesis, thus showing promise as a new therapeutic strategy for HIE. This study was conducted to evaluate the tissue distribution and potential toxicity of thrombin-preconditioned human Wharton's jelly-derived mesenchymal stem cells (th-hWJMSCs) in animal models before the initiation of clinical trials. We investigated the biodistribution, tumorigenicity and general toxicity of th-hWJMSCs. MSCs were administered the maximum feasible dose (1 × 105 cells/10 µL/head) once, or at lower doses into the cerebral ventricle. To support the clinical use of th-hWJMSCs for treating brain injury, preclinical safety studies were conducted in newborn Sprague-Dawley rats and BALB/c nude mice. In addition, growth parameters were evaluated to assess the impact of th-hWJMSCs on the growth of newborn babies. Our results suggest that th-hWJMSCs are non-toxic and non-tumorigenic in rodent models, survive for up to 7 days in the brain and hold potential for HIE therapy.

Assessment of the structural and functional characteristics of human mesenchymal stem cells associated with a prolonged exposure of morphine.

The discovery of the expression of opioid receptors in the skin and their role in orchestrating the process of tissue repair gave rise to questions regarding the potential effects of clinical morphine treatment in wound healing. Although short term treatment was reported to improve tissue regeneration, in vivo chronic administration was associated to an impairment of the physiological healing process and systemic fibrosis. Human mesenchymal stem cells (hMSCs) play a fundamental role in tissue regeneration. In this regard, acute morphine exposition was recently reported to impact negatively on the functional characteristics of hMSCs, but little is currently known about its long-term effects. To determine how a prolonged treatment could impair their functional characteristics, we exposed hMSCs to increasing morphine concentrations respectively for nine and eighteen days, evaluating in particular the fibrogenic potential exerted by the long-term exposition. Our results showed a time dependent cell viability decline, and conditions compatible with a cellular senescent state. Ultrastructural and protein expression analysis were indicative of increased autophagy, suggesting a relation to a detoxification activity. In addition, the enhanced transcription observed for the genes involved in the synthesis and regulation of type I collagen suggested the possibility that a prolonged morphine treatment might exert its fibrotic potential risk, even involving the hMSCs.

Comparative assessment of proliferation and immunomodulatory potential of Hypericum perforatum plant and callus extracts on mesenchymal stem cells derived adipose tissue from multiple sclerosis patients.

BACKGROUND: Mesenchymal stem cells-derived adipose tissue (AT-MSCs) are recognized for the treatment of inflammatory diseases including multiple sclerosis (MS). Hypericum perforatum (HP) is an anti-inflammatory pharmaceutical plant with bioactive compounds. Plant tissue culture is a technique to improve desired pharmacological potential. The aim of this study was to compare the anti-inflammatory and proliferative effects of callus with field-growing plant extracts of HP on AT-MSCs derived from MS patients. MATERIALS AND METHODS: AT-MSCs were isolated and characterized. HP callus was prepared and exposure to light spectrum (blue, red, blue-red, and control). Total phenols, flavonoids, and hypericin of HP callus and plant extracts were measured. The effects of HP extracts concentrations on proliferation were evaluated by MTT assay. Co-culture of AT-MSCs: PBMCs were challenged by HP plant and callus extracts, and Tregs percentage was assessed by flow cytometry. RESULTS: Identification of MSCs was performed. Data showed that blue light could stimulate total phenols, flavonoids, and hypericin. MTT test demonstrated that plant extract in concentrations (0.03, 1.2, 2.5 and 10 µg/ml) and HP callus extract in 10 µg/ml significantly increased. Both HP extracts lead to an increase in Tregs percentage in all concentrations. In particular, a comparison between HP plant and callus extracts revealed that Tregs enhanced 3-fold more than control groups in the concentration of 10 µg/ml callus. CONCLUSIONS: High concentrations of HP extracts showed effectiveness on AT-MSCs proliferation and immunomodulatory properties with a certain consequence in callus extract. HP extracts may be considered as supplementary treatments for the patients who receiving MSCs transplantation.

The osteogenic differentiation of human bone marrow stromal cells induced by nanofiber scaffolds using bioinformatics.

This article aims to investigate the mechanism of behaviors of human bone marrow stromal cells (hBMSCs) affected by scaffold structure combining Monte Carlo feature selection (MFCS), incremental feature selection (IFS) and support vector machine (SVM). The specific differentially expressed genes (DEGs) of hBMSCs cultured on nanofiber (NF) scaffolds and freeform fabrication (FFF) scaffolds were obtained. Key genes were screened from common genes between osteogenic DEGs and NF specific DEGs with MFCS, IFS and SVM. The results demonstrated that NF scaffolds induced hBMSCs to express more genes related to osteogenic differentiation. Finally, 16 key genes were identified among the common genes. The common genes were significantly enriched in Rap1 signaling pathway, extracellular matrix and ossification. The results in this study suggested that the gene expression of hBMSCs was sensitive to NF scaffolds and FFF scaffolds, and the osteogenic differentiation of hBMSCs could be enhanced by NF scaffolds.

Assessment of novel surgical procedures using decellularised muscle and bioactive ceramic: a histological analysis.

Tissue regeneration and neovascularisation in cases of major bone loss is a challenge in maxillofacial surgery. The hypothesis of the present study is that the addition of resorbable bioactive ceramic Silica Calcium Phosphate Cement (SCPC) to Declluraized Muscle Scaffold (DSM) can expedite bone formation and maturation. Two surgical defect models were created in 18 nude transgenic mice. Group 1(n = 6), with a 2-mm decortication calvarial defect, was treated with a DSM/SCPC sheet over the corticated bone as an onlay then seeded with human Mesenchymal Stromal Cells hMSC in situ. In Group 2 (n = 6), a critical size (4 mm) calvarial defect was made and grafted with DSM/SCPC/in situ human bone marrow stromal cells (hMSCs). The control groups included Group 3 (n = 3) animals, with a 2-mm decortication defect treated with an onlay DSM sheet, and Group 4 (n = 3) animals, treated with critical size defect grafted with plain DSM. After 8 weeks, bone regeneration in various groups was evaluated using histology, immunohistochemistry and histomorphometry. New bone formation and maturation was superior in groups treated with DSM/SCPC/hMSC. The DMS/SCPC scaffold has the ability to augment and induce bone regeneration and neovascularisation in cases of major bone resorption and critical size defects.

Assessment of the Neuroprotective and Stemness Properties of Human Wharton's Jelly-Derived Mesenchymal Stem Cells under Variable (5% vs. 21%) Aerobic Conditions.

To optimise the culture conditions for human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) intended for clinical use, we investigated ten different properties of these cells cultured under 21% (atmospheric) and 5% (physiological normoxia) oxygen concentrations. The obtained results indicate that 5% O2 has beneficial effects on the proliferation rate, clonogenicity, and slowdown of senescence of hWJ-MSCs; however, the oxygen level did not have an influence on the cell morphology, immunophenotype, or neuroprotective effect of the hWJ-MSCs. Nonetheless, the potential to differentiate into adipocytes, osteocytes, and chondrocytes was comparable under both oxygen conditions. However, spontaneous differentiation of hWJ-MSCs into neuronal lineages was observed and enhanced under atmospheric oxygen conditions. The cells relied more on mitochondrial respiration than glycolysis, regardless of the oxygen conditions. Based on these results, we can conclude that hWJ-MSCs could be effectively cultured and prepared under both oxygen conditions for cell-based therapy. However, the 5% oxygen level seemed to create a more balanced and appropriate environment for hWJ-MSCs.

Comparative assessment of immunomodulatory, proliferative, and antioxidant activities of crocin and crocetin on mesenchymal stem cells.

Saffron (Crocus sativus L) is a well-known spice with active pharmacologic components including crocin, crocetin, safranal, and picrocrocin. Similar to crocin/crocetin, mesenchymal stem cells (MSCs) have been shown to display immunomodulatory and antioxidant properties, which could be beneficial in treatment of various diseases. In the current study, we have evaluated the effects of crocin and crocetin on the functions of MSCs. We used the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay to evaluate MSCs proliferation, and flow cytometry assay to measure the percentage of apoptotic MSCs and Tregs populations. Furthermore, we used the real-time polymerase chain reaction method to quantify messenger RNA (mRNA) expression of inflammatory and anti-inflammatory cytokines. Antioxidant assay was employed to quantify antioxidant parameters including nitric oxide and malondialdehyde levels besides superoxide dismutase activity. Our findings indicated that both crocin and crocetin at low concentrations (2.5 and 5 µM) exhibited significant effects on increasing MSCs viability and on protecting them against apoptosis-induced death. Furthermore, crocin and crocetin at low concentrations (2.5 and 5 µM) displayed a better antioxidant function. Moreover, increased Treg population was observed at lower doses. In addition, crocin/crocetin at low concentrations caused an elevation in mRNA expression of anti-inflammatory cytokines (transforming growth factor-ß, interleukin-10 [IL-10], and IL-4), while at higher doses (25 and 50 µM) they led to lowering inflammatory cytokines (IL-1ß, IL-6, IL-17, and interferon gamma). Altogether, both crocin and crocetin at lower concentrations exhibited more efficacies on MSCs with a better effect toward crocin. It seems that crocin and crocetin may be considered as complementary treatments for the patients who undergo MSCs transplantation.

Harnessing the Secretome of Mesenchymal Stromal Cells for Traumatic Spinal Cord Injury: Multicell Comparison and Assessment of In Vivo Efficacy.

Cell therapy offers significant promise for traumatic spinal cord injury (SCI), which despite many medical advances, has limited treatment strategies. Able to address the multifactorial and dynamic pathophysiology of SCI, cells present various advantages over standard pharmacological approaches. However, the use of live cells is also severely hampered by logistical and practical considerations. These include specialized equipment and expertise, standardization of cell stocks, sustained cell viability post-thawing, and cryopreservation-induced delayed-onset cell death. For this reason, we suggest a novel and clinically translatable alternative to live-cell systemic infusion, which retains the efficacy of the latter while overcoming many of its limitations. This strategy involves the administration of concentrated cell secretome and exploits the trophic mechanism by which stromal cells function. In this study, we compare the efficacy of intravenously delivered concentrated conditioned media (CM) from human umbilical cord matrix cells (HUCMCs), bone marrow mesenchymal stromal cells, as well as newborn and adult fibroblasts in a rat model of moderately severe cervical clip compression/contusion injury (C7--T1, 35 g). This is further paired with a thorough profile of the CM cytokines, chemokines, and angiogenic factors. The HUCMC-derived CM was most effective at limiting acute (48 h post-SCI) vascular pathology, specifically lesion volume, and functional vascularity. Principle component analysis (PCA), hierarchical clustering, and interaction analysis of proteins highly expressed in the HUCMC secretome suggest involvement of the MAPK/ERK, JAK/STAT, and immune cell migratory pathways. This "secretotherapeutic" strategy represents a novel and minimally invasive method to target multiple organ systems and several pathologies shortly after traumatic SCI.

Regulatory-compliant conditions during cell product manufacturing enhance in vitro immunomodulatory properties of infrapatellar fat pad-derived mesenchymal stem/stromal cells.

BACKGROUND AIMS: Mesenchymal stem/stromal cell (MSC)-based therapies have gained attention as potential alternatives for multiple musculoskeletal indications based on their trophic and immunomodulatory properties. The infrapatellar fat pad (IFP) serves as a reservoir of MSCs, which play crucial roles modulating inflammatory and fibrotic events at the IFP and its neighboring tissue, the synovium. In an effort to comply with the existing regulatory framework regarding cell-based product manufacturing, we interrogated the in vitro immunomodulatory capacity of human-derived IFP-MSCs processed under different conditions, including a regulatory-compliant protocol, in addition to their response to the inflammatory and fibrotic environments often present in joint disease. METHODS: Immunophenotype, telomere length, transcriptional and secretory immunomodulatory profiles and functional immunopotency assay were assessed in IFP-MSCs expanded in regular fetal bovine serum (FBS)-supplemented medium and side-by-side compared with same-donor cells processed with two media alternatives (i.e., regulatory-compliant pooled human platelet lysate [hPL] and a chemically reinforced/serum-reduced [Ch-R] formulation). Finally, to assess the effects of such formulations on the ability of the cells to respond to pro-inflammatory and pro-fibrotic conditions, all three groups were stimulated ex vivo (i.e., cell priming) with a cocktail containing TNFα, IFNγ and connective tissue growth factor (tumor-initiating cells) and compared with non-induced cohorts assessing the same outcomes. RESULTS: Non-induced and primed IFP-MSCs expanded in either hPL or Ch-R showed distinct morphology in vitro, similar telomere dynamics and distinct phenotypical and molecular profiles when compared with cohorts grown in FBS. Gene expression of IL-8, CD10 and granulocyte colony-stimulating factor was highly enriched in similarly processed IFP-MSCs. Cell surface markers related to the immunomodulatory capacity, including CD146 and CD10, were highly expressed, and secretion of immunomodulatory and pro-angiogenic factors was significantly enhanced with both hPL and Ch-R formulations. Upon priming, the immunomodulatory phenotype was enhanced, resulting in further increase in CD146 and CD10, significant CXCR4 presence and reduction in TLR3. Similarly, transcriptional and secretory profiles were enriched and more pronounced in IFP-MSCs expanded in either hPL or Ch-R, suggesting a synergistic effect between these formulations and inflammatory/fibrotic priming conditions. Collectively, increased indoleamine-2,3-dioxygenase activity and prostaglandin E2 secretion for hPL- and Ch-R-expanded IFP-MSCs were functionally reflected by their robust T-cell proliferation suppression capacity in vitro compared with IFP-MSCs expanded in FBS, even after priming. CONCLUSIONS: Compared with processing using an FBS-supplemented medium, processing IFP-MSCs with either hPL or Ch-R similarly enhances their immunomodulatory properties, which are further increased after exposure to an inflammatory/fibrotic priming environment. This evidence supports the adoption of regulatory-compliant practices during the manufacturing of a cell-based product based on IFP-MSCs and anticipates a further enhanced response once the cells face the pathological environment after intra-articular administration. Mechanistically, the resulting functionally enhanced cell-based product has potential utilization as a novel, minimally invasive cell therapy for joint disease through modulation of local immune and inflammatory events.

Assessment of cartilage regeneration on 3D collagen-polycaprolactone scaffolds: Evaluation of growth media in static and in perfusion bioreactor dynamic culture.

Efforts on bioengineering are directed towards the construction of biocompatible scaffolds and the determination of the most favorable microenvironment, which will better support cell proliferation and differentiation. Perfusion bioreactors are attracting growing attention as an effective, modern tool in tissue engineering. A natural biomaterial extensively used in regenerative medicine with outstanding biocompatibility, biodegradability and non-toxic characteristics, is collagen, a structural protein with undisputed beneficial characteristics. This is a study designed according to the above considerations. 3D printed polycaprolactone (PCL) scaffolds with rectangular pores were coated with collagen either as a coating on the scaffold's trabeculae, or as a gel-cell solution penetrating scaffolds' pores. We employed histological, molecular and imaging techniques to analyze colonization, proliferation and chondrogenic differentiation of Adipose Derived Mesenchymal Stem Cells (ADMSCs). Two different differentiation culture media were employed to test chondrogenic differentiation on gelated and non gelated PCL scaffolds in static and in perfusion bioreactors dynamic culture conditions. In dynamic culture, non gelated scaffolds combined with our in house TGF-ß2 based medium, augmented chondrogenic differentiation performance, which overall was significantly less favorable compared to StemPro™ propriety medium. The beneficial mechanical stimulus of dynamic culture, appears to outgrow the disadvantage of the "weaker" TGF-ß2 medium used for chondrogenic differentiation. Even though cells in static culture grew well on the scaffold, there was limited penetration inside the construct, so the purpose of the 3D culture was not fully served. In contrast dynamic culture achieved better penetration and uniform distribution of the cells within the scaffold.

Engineering a multifunctional 3D-printed PLA-collagen-minocycline-nanoHydroxyapatite scaffold with combined antimicrobial and osteogenic effects for bone regeneration.

3D-printing and additive manufacturing can be powerful techniques to design customized structures and produce synthetic bone grafts with multifunctional effects suitable for bone repair. In our work we aimed the development of novel multifunctionalized 3D printed poly(lactic acid) (PLA) scaffolds with bioinspired surface coatings able to reduce bacterial biofilm formation while favoring human bone marrow-derived mesenchymal stem cells (hMSCs) activity. For that purpose, 3D printing was used to prepare PLA scaffolds that were further multifunctionalized with collagen (Col), minocycline (MH) and bioinspired citrate- hydroxyapatite nanoparticles (cHA). PLA-Col-MH-cHA scaffolds provide a closer structural support approximation to native bone architecture with uniform macroporous, adequate wettability and an excellent compressive strength. The addition of MH resulted in an adequate antibiotic release profile that by being compatible with local drug delivery therapy was translated into antibacterial activities against Staphylococcus aureus, a main pathogen associated to bone-related infections. Subsequently, the hMSCs response to these scaffolds revealed that the incorporation of cHA significantly stimulated the adhesion, proliferation and osteogenesis-related gene expression (RUNX2, OCN and OPN) of hMSCs. Furthermore, the association of a bioinspired material (cHA) with the antibiotic MH resulted in a combined effect of an enhanced osteogenic activity. These findings, together with the antibiofilm activity depicted strengthen the appropriateness of this 3D-printed PLA-Col-MH-cHA scaffold for future use in bone repair. By targeting bone repair while mitigating the typical infections associated to bone implants, our 3D scaffolds deliver an integrated strategy with the combined effects further envisaging an increase in the success rate of bone-implanted devices.

Application of Hydroxycholesterols for Alveolar Cleft Osteoplasty in a Rodent Model.

BACKGROUND: Bone morphogenetic proteins (BMPs) have played a central role in the regenerative therapies for bone reconstruction, including alveolar cleft and craniofacial surgery. However, the high cost and significant adverse effect of BMPs limit their broad application. Hydroxycholesterols, naturally occurring products of cholesterol oxidation, are a promising alternative to BMPs. The authors studied the osteogenic capability of hydroxycholesterols on human mesenchymal stem cells and the impact of hydroxycholesterols on a rodent alveolar cleft model. METHODS: Human mesenchymal stem cells were treated with control medium or osteogenic medium with or without hydroxycholesterols. Evaluation of cellular osteogenic activity was performed. A critical-size alveolar cleft was created and one of the following treatment options was assigned randomly to each defect: collagen sponge incorporated with hydroxycholesterols, BMP-2, or no treatment. Bone regeneration was assessed by means of radiologic and histologic analyses and local inflammation in the cleft evaluated. Moreover, the role of the hedgehog signaling pathway in hydroxycholesterol-mediated osteogenesis was examined. RESULTS: All cellular osteogenic activities were significantly increased on human mesenchymal stem cells treated with hydroxycholesterols relative to others. The alveolar cleft treated with collagen sponge with hydroxycholesterols and BMP-2 demonstrated robust bone regeneration. The hydroxycholesterol group revealed histologically complete bridging of the alveolar defect with architecturally mature new bone. The inflammatory responses were less in the hydroxycholesterol group compared with the BMP-2 group. Induction of hydroxycholesterol-mediated in vitro osteogenesis and in vivo bone regeneration were attenuated by hedgehog signaling inhibitor, implicating involvement of the hedgehog signaling pathway. CONCLUSION: Hydroxycholesterols may represent a viable alternative to BMP-2 in bone tissue engineering for alveolar cleft.

Complete Assessment of Multilineage Differentiation Potential of Human Skeletal Muscle-Derived Mesenchymal Stem/Stromal Cells.

The minimal criteria for mesenchymal stem/stromal cell (MSC) identification set by the International Society for Cellular Therapy include plastic adherence, presence and absence of a set of surface antigens and in vitro multilineage differentiation. This differentiation is assessed through stimulation of MSCs with defined combination and concentration of growth factors towards specific lineages and histological confirmation of the presence of differentiated cells. Here we provide protocols for multilineage differentiation, namely, osteogenesis, adipogenesis, chondrogenesis and myogenesis. We also provide their respective histological analyses.

Fabrication and cytotoxicity assessment of cellulose nanofibrils using Bassia eriophora biomass.

Cellulose nanofibrils (CNs) are eco-friendly, biodegradable, biocompatible, renewable, cost-effective, and possess excellent mechanical properties. We fabricated CNs from Bassia eriophora biomass, and the structure and morphology were investigated by transmission electron microscopy that revealed 2-6 µm long fibrillated structures with diameters of 15-40 nm. CNs biocompatibility was assessed using in vitro based assays, including cell viability assay, AO/EB staining, Hoechst staining, JC-1 staining, and gene expression analysis. The assessment of cellular and nuclear morphologies of human mesenchymal stem cells (hMSCs) showed that CNs do not affect cell viability and morphology. JC-1 staining results revealed that CNs do not cause mitochondrial membrane potential of hMSCs. Cell-based in vitro assays revealed that CNs are biocompatible even at high concentrations. The CNs effect on cell cycle regulated gene expression was studied that results suggested that CCND1 and CCND3 gene expression levels increased slightly, when compared with control. But CCNG1, CYCS3, and CCNC1 genes has no significant difference was observed. Overall, our results suggested that CNs can be used for tissue engineering and regenerative medicine.

Assessment of sulforaphane-induced protective mechanisms against cadmium toxicity in human mesenchymal stem cells.

Cd is a hazardous substance and carcinogen that is present in the environment; it is known to cause toxic effects in living organisms. Sulforaphane is a naturally available phytochemical with antioxidant, anti-inflammatory, and anticarcinogenic properties. However, the effects of sulforaphane on Cd toxicity in human mesenchymal stem cells (hMSCs) are unknown. In the present study, we investigated the molecular mechanisms of the effects of sulforaphane on Cd toxicity in hMSCs by using MTT assays, acridine orange/ethidium bromide staining, Hoechst staining, LysoRed staining, assessment of mitochondrial membrane potential, and gene expression analysis. Cd decreased hMSC viability in a dose-dependent manner with an IC50 value of 56.5 µM. However, sulforaphane did not induce any significant reduction in cell viability. Nuclear morphological analysis revealed that Cd induced necrotic cell death. Additionally, Cd caused mitochondrial membrane potential loss in hMSCs. The treatment of Cd-exposed cells with sulforaphane (Cd-sulforaphane co-treatment) resulted in a significant recovery of the cell viability and nuclear morphological changes compared with that of cells treated with Cd only. The gene expression pattern of cells co-treated with Cd-sulforaphane was markedly different from that of Cd-treated cells, owing to the reduction in Cd toxicity. Our results clearly indicated that sulforaphane reduced Cd-induced toxic effects in hMSCs. Overall, the results of our study suggested that sulforaphane-rich vegetables and fruits can help to improve human health through amelioration of the molecular effects of Cd poisoning.

Design and development of low cost polyurethane biopolymer based on castor oil and glycerol for biomedical applications.

In the current study, we present the synthesis of novel low cost bio-polyurethane compositions with variable mechanical properties based on castor oil and glycerol for biomedical applications. A detailed investigation of the physicochemical properties of the polymer was carried out by using mechanical testing, ATR-FTIR, and X-ray photoelectron spectroscopy (XPS). Polymers were also tested in short term in-vitro cell culture with human mesenchymal stem cells to evaluate their biocompatibility for potential applications as biomaterial. FTIR analysis confirmed the synthesis of castor oil and glycerol based PU polymers. FTIR also showed that the addition of glycerol as co-polyol increases crosslinking within the polymer backbone hence enhancing the bulk mechanical properties of the polymer. XPS data showed that glycerol incorporation leads to an enrichment of oxidized organic species on the surface of the polymers. Preliminary investigation into in vitro biocompatibility showed that serum protein adsorption can be controlled by varying the glycerol content with polymer backbone. An alamar blue assay looking at the metabolic activity of the cells indicated that castor oil based PU and its variants containing glycerol are non-toxic to the cells. This study opens an avenue for using low cost bio-polyurethane based on castor oil and glycerol for biomedical applications.

Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects "stemness" properties.

BACKGROUND: Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term "stemness" of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion. METHODS: DPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (ß-galactosidase (SA-ß-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR. RESULTS: Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6-7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in "osteogenic pre-disposition", evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6-7 under all expansion conditions. CONCLUSIONS: These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.