Background: Primary cells derived from connective tissues contain mesenchymal stem/stromal cell (MSC)–like progenitors with chondrogenic potential relevant for cartilage repair. However, donor- and tissue-specific variability and the lack of robust, high-content analytical methods limit their translational use. Objectives: This study aimed to develop and optimize a high-content imaging workflow for quantitative evaluation of chondrogenesis in three-dimensional (3D) pellets derived from primary cells. Methods: Primary human cells isolated from cartilage were chondrogenically differentiated in vitro. A systematic optimization of immunofluorescence staining parameters was performed, including staining platform, enzymatic matrix digestion, non-specific site blocking, membrane permeabilization, and nuclear counterstaining. Type II collagen was detected using an Alexa Fluor 488–conjugated antibody, and pellets were analyzed using high-content non-confocal imaging. Fluorescence intensities were adjusted to the pellet area to account for size-dependent effects. Results: Staining directly in imaging plates enabled streamlined high-content analysis. Controlled pepsin-mediated matrix digestion markedly enhanced antibody penetration, while excessive digestion compromised pellet integrity. Extended bovine serum albumin blocking improved type II collagen signal intensity and homogeneity. Triton X-100 permeabilization increased detection sensitivity but occasionally induced structural disruption in weakly organized control pellets. The optimized protocol enabled clear discrimination between chondrogenic pellets and controls, with approximately threefold higher type II collagen signal in chondrogenic samples. Conclusions: This study establishes a high-content imaging–based workflow for quantitative assessment of 3D chondrogenesis from primary cells. The approach provides a rapid, scalable platform with direct relevance for in vitro screening, potency testing, and quality control in cartilage-oriented advanced therapy development.

Cell fusion is a complex phenomenon that is key in maintaining tissue homeostasis, particularly in aiding tissue regeneration processes. Studies show that mesenchymal stem/stromal cells (MSCs) are capable of restoring damaged tissue by adopting the phenotype of various cell types via cell fusion. As cell fusion of MSCs with cells of different origin remains poorly researched, we have developed a protocol that allows successful electrofusion between human synovium-derived MSCs and human chondrocytes. Building on from our protocol can help researchers study the cell fusion processes in the in vitro environment and could set basis for development of fusion cell-based advanced therapy medicinal products (ATMPs). In our protocol, we provide a detailed description on how to culture both of the fusion partner cells, how to carry out the modified adherence method (MAM) to achieve a high yield of successfully fused cells, and how to determine the yield of cell fusion using either methyl violet or fluorescent cell trackers.

Human skin is a complex organ essential for protection, hydration and regeneration, with keratinocytes and fibroblasts playing pivotal roles in wound healing and tissue renewal. This study investigates the effects of selected vegetable oils, fatty acids and unsaponifiable compounds on the proliferation and migration of human keratinocytes and fibroblasts in vitro. Oils were selected based on their specific fatty acid profiles, including coconut, olive, linden, poppy, pomegranate, marigold and linseed oils. Using cell proliferation and gap closure assays, the impact of these oils and their constituents on cell proliferation and migration was evaluated. Results showed that the tested biological activity of the oils depended primarily on the fatty acid composition of their triglycerides. Oils rich in essential fatty acids, particularly linoleic acid, significantly promoted cell proliferation. In contrast, unsaponifiable compounds showed no effect, while punicic acid and pomegranate seed oil had a significant negative impact. The oils did not exert notable effects on the closure rate of the artificial wound gap; however, sterculic and oleic acids inhibited gap closure and induced changes in cell morphology. These findings support the role of plant oils and their composition in enhancing skin cell regeneration, and provide in vitro evidence to support their application in cosmetic and dermatological formulations aimed at skin repair and evidence-based skincare.

This study explores how nanofiber chemical composition influences human immune cell responses, a key factor in the body's defense against foreign substances. Nanofiber mats composed of polycaprolactone (PCL), alginate (ALG), chitosan (CS), and zein were incubated with both unstimulated and phytohemagglutinin-L-stimulated peripheral blood mononuclear cells (PBMCs), as well as immature and mature monocyte-derived dendritic cells (MoDCs). We evaluated viability, metabolic activity, and cytokine release of PBMCs while in MoDCs, we assessed surface marker expression, phagocytic capacity, and allogeneic T-cell stimulatory function of MoDCs. Results demonstrate that nanofiber composition significantly influenced immune cell responses. While PCL and ALG nanofibers showed no significant effects on PBMCs, those made of CS, reduced their metabolic activity, warranting caution in their use. In contrast, zein nanofibers increased both metabolic activity and proinflammatory cytokine release of PBMCs, suggesting their potential immunogenicity. In MoDCs, PCL and CS nanofibers impaired both their phenotypic maturation and functional performance, including antigen uptake and presentation. Zein nanofibers induced changes in MoDC surface marker expression, however their overall functionality remained intact. In contrast, ALG nanofibers exhibited the highest biocompatibility, with minimal impact on both immature and mature MoDCs. This favorable profile of ALG nanofibers supports their suitability for biomedical applications, such as tissue engineering and drug delivery. Overall, our findings underscore the importance of thorough immunological safety assessment of nanofiber-based biomaterials prior to their clinical use.

Purpose: Atopic dermatitis (AD) is the most common chronic inflammatory skin disease that severely impairs patient's life quality and represents significant therapeutic challenge due to its pathophysiology arising from skin barrier dysfunction. Topical corticosteroids, the mainstay treatment for mild to moderate AD, are usually formulated into conventional dosage forms that are impeded by low drug permeation, resulting in high doses with consequent adverse effects, and also lack properties that would strengthen the skin barrier. Herein, we aimed to develop biomimetic lamellar lyotropic liquid crystals (LLCs), offering a novel alternative to Methods: In screening studies, pseudoternary phase diagrams alongside polarized light microscopy (PLM) and viscosity measurements were utilized. Next, the selected LCCs underwent comprehensive characterization via PLM, small-angle X-ray scattering, differential scanning calorimetry, and rheological analysis. Lastly, their performance was evaluated and compared with the commercially available reference medicine in chemical stability study, in vitro permeation testing, in vitro safety assessment using cell proliferation assay, inverted light microscopy, and Raman mapping of keratinocytes, besides gap closure assay performed by live-cell imaging. Results: Formulation (L/T)Ho30, containing the highest amount of lecithin/Tween 80 mixture (21%) and hempseed oil (28%), demonstrated lamellar microstructure with high skin hydration potential and favourable rheological features for skin administration. Moreover, in comparison with the reference medicine, it stood out by providing suitable chemical BD (betamethasone dipropionate) stability, improved 3-fold BD permeation, and excellent biocompatibility with over 85% cell proliferation at all tested concentrations, ensuring keratinocytes' integrity, as well as promoting skin healing with gap closure observed after 36 hours. Conclusion: Unique multi-target drug delivery strategy depicted in newly developed bioinspired lamellar LCCs structurally resembling stratum corneum intercellular lipids, with incorporated BD drug, and composed of multifunctional components that synergistically strengthen skin barrier, was presented here and shows a promising approach for improved AD treatment.

Nanofiber safety, especially immunogenicity, is important for their successful translation to clinical setting. This study provides a comprehensive evaluation of how nanofiber physical properties influence immune cells cultured on them, specifically peripheral blood mononuclear cells (PBMCs). We prepared nanofibers with a wide range of physical properties including various diameters, interfibrillar pore sizes and mat thicknesses, using four main polymers: polycaprolactone, alginate, chitosan, and zein. Our findings show that nanofiber diameters had only a marginal influence on the activity of immune cells, whereas interfibrillar nanofiber pore sizes had a significant effect, and mat thickness proved to have the greatest impact. Cells that penetrated deeper into the thick nanofiber mats ceased to proliferate but did not experience cytotoxicity. Moreover, we discovered that PBMCs penetrating the zein/PVP nanofiber mesh exhibited increased metabolic activity, indicating potential immunogenicity, whereas the other tested non-immunogenic nanofibers reduced it. To best of our knowledge, this study is the first to report on the impact of various nanofiber physical properties on in vitro immune cell behavior, thereby expanding the knowledge in the relatively unexplored field of nanofiber immunological safety. It underscores the need for rigorous preclinical nanofiber assessment and setting new standards for designing nanofiber-based biomedical products.

Immunological safety of nanofibers remains poorly reported within the scientific literature and lacks specific in vitro testing models distinct from those used to test nanoparticles. To address the challenges of currently used conventional setups being described in the literature, we developed a novel in vitro model for nanofiber mats immunogenicity testing, which enables standardization of tested surface area, excludes nanofiber mat edges, and ensures stable contacts of cells with nanofibers during the experiment. The effect of nanofibers was assessed on peripheral blood mononuclear cells (PBMCs) by measuring their metabolic activity using MTS cell proliferation assay, where key performance parameters, i.e. cell number, phytohemagglutinin-L (PHA-L) concentration, incubation time and cell lysis were optimized. Repeatability of results obtained with non-activated and PHA-L-activated PBMCs in contact with differently thick polycaprolactone nanofiber mats was compared using both models. Our model provided more reproducible results with lower variability, exhibiting its higher reliability and accuracy than the conventional one. Furthermore, results showed the presence of thicker mats resulted in reduced metabolic activity and PBMC proliferation without any observed cytotoxicity, providing additional insights into their non-immunogenic characteristics. The developed model enables more accurate biological assessment that can support new guidelines for in vitro nanofiber testing and formulation.

Biofilm-associated diseases such as periodontitis are widespread and challenging to treat which calls for new strategies for their effective management. Probiotics represent a promising approach for targeted treatment of dysbiosis in biofilm and modulation of host immune response. In this interdisciplinary study, nanofibers with two autochthonous Bacillus strains 27.3.Z and 25.2.M were developed. The strains were isolated from the oral microbiota of healthy individuals, and their genomes were sequenced and screened for genes associated with antimicrobial and immunomodulatory activities, virulence factors, and transferability of resistance to antibiotics. Spores of two Bacillus strains were incorporated individually or in combination into hydrophilic poly(ethylene oxide) (PEO) and composite PEO/alginate nanofibers. The nanofiber mats were characterised by a high loading of viable spores (> 7 log CFU/mg) and they maintained viability during electrospinning and 6 months of storage at room temperature. Spores were rapidly released from PEO nanofibers, while presence of alginate in the nanofibers prolonged their release. All formulations exhibited swelling, followed by transformation of the nanofiber mat into a hydrogel and polymer erosion mediating spore release kinetics. The investigated Bacillus strains released metabolites, which were not cytotoxic to peripheral blood mononuclear cells (PBMCs) in vitro. Moreover, their metabolites exhibited antibacterial activity against two periodontopathogens, an anti -proliferative effect on PBMCs, and inhibition of PBMC expression of proinflammatory cytokines. In summary, the developed nanofiber-based delivery system represents a promising therapeutic approach to combat biofilm-associated disease on two fronts, namely via modulation of the local microbiota with probiotic bacteria and host immune response with their metabolites.

Conventional treatments for chronic wounds are often ineffective, thus new therapeutic approaches are needed, such as the delivery of immunomodulatory drugs that can reduce inflammation, restore immune cell function, and facilitate tissue regeneration. A potential drug for such an approach is simvastatin, which has major drawbacks including poor solubility and chemical instability. With the aim of developing a dressing for wound healing, simvastatin and an antioxidant were incorporated into alginate/poly(ethylene oxide) nanofibers by green electrospinning without the use of organic solvents, thanks to their prior encapsulation into liposomes. The composite liposome–nanofiber formulations exhibited fibrillar morphology (160–312 nm) and unprecedentedly high phospholipid and drug content (76%). Transmission electron microscopy revealed dried liposomes as bright ellipsoidal spots homogeneously distributed over the nanofibers. After nanofiber hydration, the liposomes reconstituted in two size populations (~140 and ~435 nm), as revealed by cutting-edge MADLS® analysis. Lastly, in vitro assays demonstrated that composite liposome–nanofiber formulations are superior to liposomal formulations due to a better safety profile in keratinocytes and peripheral blood mononuclear cells. Furthermore, both formulations exhibited similarly advantageous immunomodulatory effects, measured as decreased inflammation in vitro. A synergistic combination of the two nanodelivery systems shows promise for the development of efficient dressings for chronic wound treatment.

Human skeletal stem cells (hSSCs) were recently identified as podoplanin (PDPN)/CD73/CD164-positive and CD146-negative cells that decline with age, and play a role in the pathogenesis of osteoarthritis (OA). The aim of this study was to identify the hSSC-like properties of bone-derived mesenchymal stem/stromal cells (MSCs) of patients with late and early OA. Methods: First, we performed gene expression profiling for the hSSC markers in 32 patients with late and early OA, and donors without OA. Having identified the low expression of hSSC markers in late OA patients, we further performed trilineage differentiation and immunophenotyping for hSSC makers in the selected subsets from each donor group. Results: Our results show no differences in osteogenesis, chondrogenesis, and adipogenesis between the MSCs from the three groups. However, the immunophenotyping shows lower CD164 in MSCs from early OA patients in comparison with late and no OA subjects (p = 0.002 and p = 0.017). Conclusions: Our study shows that the in vitro hSSC-like properties of bone-derived MSCs are similar in patients with early and late OA, and in donors without OA. However, the lower percentage of CD164-positive MSCs in early OA patients indicates the potential of CD164 as a marker of the onset of OA.

Dear Editor, We investigated the clinical outcome of treating castration-resistant prostate cancer (CRPC) patients with autologous immunohybridoma cell (aHyC) vaccine generated by electrofusing autologous dendritic (DC) and tumor cells (TC), and tested whether the immunological response, involving the CD56brightCD16− natural killer (NK), putative pro-metastatic cells,1, 2 correlates with survival of CRPC patients. The results demonstrated that aHyC treatment is safe and prolongs patient survival correlating with a decrease in peripheral blood CD56brightCD16− NK cells. Despite advances in cancer immunotherapy, the only approved CRPC immunotherapy to date is a cell-based vaccine (sipuleucel-T),3 with a single antigen-specific response induction mechanism, consisting of a small fraction of DC markers. DCs are able to activate both naive and memory T cells, ideally suited for augmenting antitumor immune responses.4 Consistent with this, vaccination with enriched blood-derived DCs loaded with three tumor-associated antigens resulted in more frequent detection of antigen-specific T cells in CRPC patients.5 Here, whole TCs were electrofused with DCs to produce aHyC vaccine.6 The advantage of such hybridomas is their capacity of presenting both known and yet unknown tumor-associated antigens to T-lymphocytes. We used aHyC vaccine to treat chemotherapy-naive CRPC patients in a phase 1/2 randomized, placebo-controlled crossover trial to test primary outcomes—feasibility, safety, and quality of life (QL)—and also to evaluate clinical and immunological outcomes with overall survival (OS). Twenty-two men with CRPC were included (Table S1, Figure S1); 19 of them were treated with all four doses of the aHyC vaccine, either in first (aHyC-first group, n = 12) or in the second (placebo-first group, n = 10) trial session. Both groups were balanced with respect to most of the other considered variables (Table S2). The treatment with aHyC revealed only a few and mild (grade 1) intervention-related adverse events (AEs; Figure 1A–C), and did not cause additional or more frequent AEs than placebo, indicating that recorded AEs were not directly related to the aHyC application. None of the patients required hospitalization. Renal and liver functions remained stable during and after the aHyC treatment. These results show that the treatment of CRPC patients with aHyC is feasible and safe. QL was unchanged with aHyC treatment (QL scored 64.0 ± 3.7 before vs. 65.5 ± 4.5 after the first aHyC treatment; P = 0.67). Different modes of functioning, all scoring above 80 (Figure 1D), and various symptoms (scoring below 40; Figure 1E) were also comparable before and after treatment, indicating that the aHyC treatment did not affect the patients’ overall wellbeing. The demonstrated safety/nontoxicity is consistent with the completely autologous nature of aHyC. The baseline median prostate-specific antigen (PSA) value was higher in the aHyC group (8.9 ng/ml; interquartile range [IQR] = 5.6–23.7 ng/ml) than in the placebo-first group (4.3 ng/ml; IQR = 3.9–7.7 ng/ml; Figures 2 and S3), as reported.7 The median PSA progression time (PSA-P) and median PSA doubling time (PSA-DT) from first aHyC/placebo application (Table S2) were not significantly different between the two groups. High-sensitivity CRP, an inflammatory marker, was higher in the aHyC-first group (Figure 2B), which correlates with the kinetics of the PSA values (Figure 2A). In trials with DC vaccines, as well as in this study, there was no correlation between survival and PSA levels measured at different time points (not shown), likely due to the relatively delayed clinical response after immunotherapy compared with cytotoxic therapy.8 The standardized uptake values (SUVs) of [18F]fluorocholine PET–CT scans showed improvements in individual patients after treatment with aHyC. In the aHyC group, a continuous decrease in SUV was observed in the prostate (two patients; Figure S4) and in the lymph nodes and skeleton (one patient), and a transient SUV decrease in the prostate (six patients) and in the skeleton (two patients). In the placebo-first group, the SUV decreased transiently in four patients. There were no significant differences in the average SUVs between the two groups (Figure 2D). The SUV appears to have stabilized in the prostate and skeleton 6 months after the first aHyC treatment, but not in the lymph nodes (Figure 2D). Peripheral blood leukocytes were monitored regularly during the trial (Table 1). At baseline, the levels of all cell populations were similar between the two groups. After the first trial session, the total CD3+ T cells increased in both groups. However, an increase in regulatory CD25++CD127low, activated helper CD4+CD69+, and cytotoxic T cells (CD8+) and a decrease in total NK cells compared to baseline were recorded only in aHyC-first group (Table 1). Between treatment groups, a significant change was observed only in CD56brightCD16− NK cells, the level of which was significantly lower in the aHyC- versus the placebo-treated patients (P = 0.04; Figure 3A,B). Human NK lymphocytes are involved in antitumor immunity, and CD56brightCD16− NK cells are considered immunoregulatory cytokine-producing cells, representing 5%–10% of all NK cells in peripheral blood.9 The levels of counterpart CD56dimCD16+ NK cells were unaltered compared to baseline in both groups. These results indicate that the application of aHyC affects the immune system through NK cell subpopulation, consistent with observations in other cancers.1, 2 Survival analysis included all patients who received all four doses of aHyC vaccine (n = 19) and was determined from the first application of aHyC to the cutoff date or the patient's death (any cause). The median OS was 58.5 months (95% confidence interval [CI], 38.8–78.2; Figure 3C). The incidence of any cause of death was 58% (11 patients). Cancer-specific survival was 75.7 months (95% CI, 41.1–110.4). Compared to previous publication,5 aHyC treatment demonstrated to be beneficial for patient survival, especially since seven patients (37 %) were initially diagnosed with a less responsive, metastatic disease. Negative correlation between the survival time and change in the CD56brightCD16− fraction of NK cells at the end of the trial (Figure 3D, r = –0.80, 95% CI, –0.95 to –0.34, P = 0.005) suggests that a relatively high increase in peripheral CD56brightCD16− NK cells shortens survival. Similarly, a negative correlation between the abundance of CD56brightCD16− NK cells and OS in melanoma patients was observed.10 In conclusion, these results indicate that aHyC treatment attenuates an increase in CD56brightCD16− NK cell subpopulation in peripheral blood, benefiting CRPC patient survival. The support of nurses who coordinated the patients, Urška Naglič and Barbara Rijavec, and technical assistance by Miha Pate, Jelena Velebit, and Primož Runovc are acknowledged. H.H.C. and R.Z. wish to thank the support by Interreg EU project INTERREG Italia-Slovenija Immuno-Cluster. This study was conducted in accordance with the provisions of the Declaration of Helsinki and was approved in June 2013 by the National Medical Ethics Committee and the Agency for Medicinal Products and Medical Devices of the Republic of Slovenia, part of European Medical Agency (EMA). Trial EMA registration: EUDRACT: 2012-005498-29. All participants signed written informed consent prior to inclusion in the study. The authors declare no conflict of interest. This work was supported by grants P3 310, J3 6790, J3 6789, and J3 9266 from the Slovenian Research Agency, by CipKeBip, COST Action BM1002, EU COST Action CM1207-GLISTEN, and EU COST Action CA 15214 EuroCellNet. The funding sources had no involvement in study design, collection, analysis and interpretation of data, the writing of the report, and the decision to submit the article for publication. H.H.C., S.H., M. Gabrijel, M.K., A.I., M.J., and R.Z. conceptualized the study. H.H.C., M. Gabrijel, S.T.B., M.C., and M.J. contributed in methodology. H.H.C., M. Gabrijel, and S.T.B. helped in validation. H.H.C., M.K., and N.K. helped in formal analysis. H.H.C., S.H., M. Gabrijel, S.T.B., A.N.K., L.L., and M. Grmek investigated the study. H.H.C., S.H., A.N.K., L.L., and M. Grmek contributed in data curation. H.H.C. and S.H. wrote the original draft. All the authors reviewed and edited the manuscript. H.H.C. and R.Z. directed the study. H.H.C., S.H., M. Gabrijel, A.I., A.K., and R.Z. supervised the project. S.H., G.L., A.I., and A.K. provided resources. S.H., A.I., and R.Z. acquired funding. N.K. provided software. Data generated and analyzed during the current study are available from the corresponding author on reasonable request. Clinical trial protocol is available at link: lnmcp.mf.uni-lj.si/Protocol.pdf. Contact Matjaž Jeras for the immunology part. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Treatments of various forms of cancer with in vitro prepared dendritic cells of various modalities have been shown to be safe but have not led to the desired efficacy. In this study, we examined the safety and efficacy of an autologous vaccine based on the fusion of patient tumor and dendritic cells (aHyC) in the treatment of chemotherapy-naive patients with castration-resistant prostate cancer (CRPC). A randomized placebo-controlled cross-over trial was conducted between June 2013 and November 2016 and followed up for survival until September 2020. Twenty-two adult men with CRPC, asymptomatic or minimally symptomatic were enrolled and consecutively allocated to aHyC-first (12 patients) and placebo-first (10 patients) group according to previous randomization. Two patients were excluded during the trial. Autologous monocyte-derived dendritic cells and prostate tumor cells were electrofused to yield hybridomas and injected subcutaneously four times. The primary endpoints were safety, feasibility and quality of life assessments; the secondary endpoints were patients’ clinical and immune responses and overall survival. Twenty patients were analyzed. There were no serious adverse events (AEs) with aHyC treatment; mild AEs were observed in five patients in the aHyC arm (42%) and in three patients in the placebo arm (38%; P=0.78). The aHyC treatment preserved quality of life in the observed period of 4 months after the first application of aHyC vaccine. An increase in CD4+ cell subpopulations, an increase in cytotoxic T cells (CD8+), and a decrease in total NK cells were detected only in the aHyC arm compared to baseline. Moreover, the natural killer cell subpopulation remained at basal level, but increased in the placebo arm (P=0.004). The median overall survival from the first aHyC application was 58.5 months (95% CI, 45.4 to 71.7; n=19) and 65.2 months from CRPC diagnosis. The median time to next-in-line therapy (docetaxel, enzalutamide, abiraterone acetate) in patients receiving aHyC (n=19) was 28.0 months. Treatment with aHyC is safe and effective and may represent a new personalized therapeutic option for patients with CRPC.

Human cell-based assays for in vitro testing of drugs in preclinical and research studies, as well as in clinical practice, are gaining greater importance especially in view of personalized medicine, which is tailored to the individual needs and benefits of a patient.This chapter begins with an overview of contemporary cell-based assays, routinely used for a comparative in vitro potency testing of anti-TNF-α innovator biologics and their biosimilars.In sequel, based on the results of our original work, we will further discuss the establishment and use of 2D normal and osteoarthritic primary chondrocyte monolayer cultures and 3D microspheroidal articular cartilage tissues, prepared in hanging drops from osteoarthritic chondrocytes and chondrogenically differentiated mesenchymal stem cells.Both 2D and 3D cultures will be presented as models for assessing the neutralizing potency of the three wellknown anti-TNF-α biological drugs: adalimumab, etanercept, and infliximab.

The musculoskeletal system includes tissues that have remarkable regenerative capabilities. Bone and muscle sustain micro-damage throughout the lifetime, yet they continue to provide the body with the support that is needed for everyday activities. Our current understanding is that the regenerative capacity of the musculoskeletal system can be attributed to the mesenchymal stem/ stromal cells (MSCs) that reside within its different anatomical compartments. These MSCs can replenish various tissues with progenitor cells to form functional cells, such as osteoblasts, chondrocytes, myocytes, and others. However, with aging and in certain disorders of the musculoskeletal system such as osteoarthritis or osteoporosis, this regenerative capacity of MSCs appears to be lost or diverted for the production of other non-functional cell types, such as adipocytes and fibroblasts. In this review, we shed light on the tissue sources and subpopulations of MSCs in the musculoskeletal system that have been identified in animal models, discuss the mechanisms of their anti-inflammatory action as a prerequisite for their tissue regeneration and their current applications in regenerative medicine. While providing up-to-date evidence of the role of MSCs in different musculoskeletal pathologies, in particular in osteoporosis and osteoarthritis, we share some thoughts on their potential as diagnostic markers in musculoskeletal health and disease.

Inflammation plays a major role in progression of rheumatoid arthritis, a disease treated with antagonists of tumor necrosis factor‐alpha (TNF‐α) and interleukin 1β (IL‐1β). New in vitro testing systems are needed to evaluate efficacies of new anti‐inflammatory biological drugs, ideally in a patient‐specific manner. To address this need, we studied microspheroids containing 10,000 human osteoarthritic primary chondrocytes (OACs) or chondrogenically differentiated mesenchymal stem cells (MSCs), obtained from three donors. Hypothesizing that this system can recapitulate clinically observed effects of anti‐inflammatory drugs, spheroids were exposed to TNF‐α, IL‐1β, or to supernatant containing secretome from activated macrophages (MCM). The anti‐inflammatory efficacies of anti‐TNF‐α biologicals adalimumab, infliximab, and etanercept, and the anti‐IL‐1β agent anakinra were assessed in short‐term microspheroid and long‐term macrospheroid cultures (100,000 OACs). While gene and protein expressions were evaluated in microspheroids, diameters, amounts of DNA, glycosaminoglycans, and hydroxiproline were measured in macrospheroids. The tested drugs significantly decreased the inflammation induced by TNF‐α or IL‐1β. The differences in potency of anti‐TNF‐α biologicals at 24 h and 3 weeks after their addition to inflamed spheroids were comparable, showing high predictability of short‐term cultures. Moreover, the data obtained with microspheroids grown from OACs and chondrogenically differentiated MSCs were comparable, suggesting that MSCs could be used for this type of in vitro testing. We propose that in vitro gene expression measured after the first 24 h in cultures of chondrogenically differentiated MSCs can be used to determine the functionality of anti‐TNF‐α drugs in personalized and preclinical studies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1045–1058, 2018

In vitro cell-based models are important tools for assessing efficacies of new leads in early phases of drug development. Human osteoarthritic chondrocytes (OACs), obtained from biomedical waste material, represent a valuable, relatively accessible cellular source that could be used for this purpose. By employing reverse transcription-polymerase chain reaction (qRT-PCR) we compared gene expression profiles of key anabolic, catabolic and inflammatory genes of freshly isolated vs. monolayer cultured OACs (passages P0-P2) and non-stimulated vs. tumor necrosis factor alpha (TNF-α) stimulated P2 OACs. After expansion of OACs in monolayer cultures, the expression of almost all analyzed genes significantly decreased. The subsequent addition of TNF-α to OACs at P2 significantly increased expressions of all catabolic and inflammatory genes, leaving the anabolic profile almost unchanged. TNF-α-treated OACs were later utilized for efficacy testing of anti-TNF-α drugs infliximab and etanercept and both significantly reduced the expressions of all catabolic and inflammatory genes tested.

Bisphenols (BPs) are widely spread pollutants that act as estrogen-like endocrine disruptors and are potentially affecting human health on a long run. We explored the effects of BPA, BPF and BPAF, on in vitro differentiation and maturation of MDDCs. Monocytes were treated with 17β-estradiol (E2) and each BP at the beginning of their differentiation into iMDDCs. We found that 10 and 50 μM of BPA and BPF, 10 and 30μM of BPAF and 10 and 50 nM of E2 did not affect cell viability. However, 50 μM of BPA and BPF, as well as 10 and 30 μM of BPAF, significantly decreased the endocytotic capacity of iMDDCs. Both, BPA (50 μM) and BPAF (30 μM) decreased the expression of CD1a and increased the amount of DC-SIGN molecules on iMDDCs. The E2 pre-treatment moderately decreased expression of CD80, CD86 and CD83 co-stimulatory molecules while increasing the numbers of HLA-DR on mMDDCs. Only BPAF significantly influenced the expression of CD80 and CD86 (both decreased), as well as CD83 and HLA-DR molecules (both increased) on mMDDCs. In addition, BPAF modulated DC maturation signaling pathways by lowering the phosphorylation of p65 NF-κB (nuclear factor-kappaB) and ERK (extracellular signal regulated kinase) 1/2 proteins. Consequently, the in vitro proliferation of allogeneic T cells, stimulated with differently pre-treated iMDDCs and mMDDCs, was significantly reduced only in case of BPAF.

Background Glioblastoma multiforme (GBM) is among the most aggressive cancers with a poor prognosis in spite of a plethora of established diagnostic and prognostic biomarkers and treatment modalities. Therefore, the current goal is the detection of novel biomarkers, possibly detectable in the blood of GBM patients that may enable an early diagnosis and are potential therapeutic targets, leading to more efficient interventions. Experimental Procedures MicroRNA profiling of 734 human and human-associated viral miRNAs was performed on blood plasma samples from 16 healthy individuals and 16 patients with GBM, using the nCounter miRNA Expression Assay Kits. Results We identified 19 miRNAs with significantly different plasma levels in GBM patients, compared to the healthy individuals group with the difference limited by a factor of 2. Additionally, 11 viral miRNAs were found differentially expressed in plasma of GBM patients and 24 miRNA levels significantly correlated with the patients’ survival. Moreover, the overlap between the group of candidate miRNAs for diagnostic biomarkers and the group of miRNAs associated with survival, consisted of ten miRNAs, showing both diagnostic and prognostic potential. Among them, hsa miR 592 and hsa miR 514a 3p have not been previously described in GBM and represent novel candidates for selective biomarkers. The possible signalling, induced by the revealed miRNAs is discussed, including those of viral origin, and in particular those related to the impaired immune response in the progression of GBM. Conclusion The GBM burden is reflected in the alteration of the plasma miRNAs pattern, including viral miRNAs, representing the potential for future clinical application. Therefore proposed biomarker candidate miRNAs should be validated in a larger study of an independent cohort of patients.

The aim of our work was to produce a modern nanomaterial with incorporated blood-derived growth factors, produced by electrospinning, applicable in treatment of chronic wounds. Platelet-rich plasma was chosen as a natural source of growth factors. Results showed that platelet-rich plasma stimulates keratinocyte and fibroblast cell growth in vitro. Its optimal concentration in growth medium was 2% (v/v) for both types of skin cells, while higher concentrations caused alterations in cell morphology, with reduced cell mobility and proliferation. In the next step hydrophilic nanofibers loaded with platelet-rich plasma were produced from chitosan and poly(ethylene oxide), using electrospinning. The morphology of nanofibers was stable in aqueous conditions for 72 h. It was shown that electrospinning does not adversely affect the biological activity of platelet-rich plasma. The effects of nanofibers with incorporated platelet-rich plasma on cell proliferation, survival, morphology and mobility were examined. Nanofibers limited cell mobility, changed morphology and stimulated cell proliferation. Despite of the small amount of blood-derived growth factors introduced in cell culture via platelet-rich plasma-loaded nanofibers, such nanofibrillar support significantly induced cell proliferation, indicating synergistic effect of nanotopography and incorporated growth factors. The overall results confirm favorable in vitro properties of produced nanofibers, indicating their high potential as a nanomaterial suitable for delivery of platelet-rich plasma in wound healing applications.