Promotion of hMDSC differentiation by combined action of scaffold material and TGF-ß superfamily growth factors.

Objective: Herein we propose a combined action of collagen type I (CA) or synthetic collagen-like-peptide functionalized with the cell adhesive RGD motif (PEG-CLP-RGD) hydrogels and selected growth factors to promote chondrogenic differentiation of human muscle-derived stem cells (hMDSCs) under normal and reduced oxygen conditions. Methods: hMDSCs were set for differentiation towards chondrogenic lineage using BMP-7 and TGF-ß3. Cells were seeded onto hydrogels loaded with growth factors (75ng/scaffold) and cultured for 28 days under normal (21%) and severe hypoxic (1%) conditions. Chondrogenesis was evaluated by monitoring collagen type II and GAG deposition, and quantification of ACAN expression by RT-PCR. Results: Sustained release of TGFß3 from the hydrogels was observed, 8.7 ± 0.5% of the initially loaded amount diffused out after 24 h from both substrates. For the BMP-7 growth factor, 14.8 ± 0.3% and 18.2 ± 0.6% of the initially loaded amount diffused out after 24 h from CA and CLP-RGD, respectively. The key findings of this study are: i) the self-supporting hydrogels themselves can stimulate hMDSC chondrogenesis by inducing gene expression of cartilage-specific proteoglycan aggrecan and ECM production; ii) the effect of dual BMP-7 and TGF-ß3 loading was more pronounced on CA hydrogel under normal oxygen conditions; iii) dual loading on PEG-CLP-RGD hydrogels did not have the synergistic effect, TGF-ß3 was more effective under both oxygen conditions; iv) BMP-7 can improve chondrogenic effect of TGF-ß3 on CA scaffolds, and hydrogels loaded with both growth factors can induce cartilage formation in hMDSC cultures. Conclusion: Our results support the potential strategy of combining implantable hydrogels functionalized with differentiation factors toward improving cartilaginous repair.

Evaluation of Cartilage Integrity Following Administration of Oral and Intraarticular Nifedipine in a Murine Model of Osteoarthritis.

Osteoarthritis (OA) ranks as the prevailing type of arthritis on a global scale, for which no effective treatments are currently available. Arterial hypertension is a common comorbidity in OA patients, and antihypertensive drugs, such as nifedipine (NIF), may affect the course of OA progression. The aim of this preclinical study was to determine the effect of nifedipine on healthy and OA cartilage, depending on its route of administration. In this study, we used the destabilization of medial meniscus to develop a mouse model of OA. Nifedipine was applied per os or intraarticularly (i.a.) for 8 weeks to both mice with OA and healthy animals. Serum biomarker concentrations were evaluated using the Luminex platform and alterations in the knee cartilage were graded according to OARSI histological scores and investigated immunohistochemically. Nifedipine treatment per os and i.a. exerted protective effects, as assessed by the OARSI histological scores. However, long-term nifedipine i.a. injections induced the deterioration of healthy cartilage. Lubricin, cartilage intermediate layer matrix protein (CILP), collagen type VI (COLVI), CILP, and Ki67 were upregulated by the nifedipine treatment. Serum biomarkers MMP-3, thrombospondin-4, and leptin were upregulated in the healthy groups treated with nifedipine, while only the levels of MMP-3 were significantly higher in the OA group treated with nifedipine per os compared to the untreated group. In conclusion, this study highlights the differential effects of nifedipine on cartilage integrity, depending on the route of administration and cartilage condition.

The Expression of Inflammasomes NLRP1 and NLRP3, Toll-Like Receptors, and Vitamin D Receptor in Synovial Fibroblasts From Patients With Different Types of Knee Arthritis.

Activated rheumatoid arthritis (RA) synovial fibroblasts (SFs) are among the most important cells promoting RA pathogenesis. They are considered active contributors to the initiation, progression, and perpetuation of the disease; therefore, early detection of RASF activation could advance contemporary diagnosis and adequate treatment of undifferentiated early inflammatory arthritis (EA). In this study, we investigated the expression of nucleotide-binding, oligomerization domain (NOD)-like receptor family, pyrin domain containing (NLRP)1, NLRP3 inflammasomes, Toll-like receptor (TLR)1, TLR2, TLR4, vitamin D receptor (VDR), and secretion of matrix metalloproteinases (MMPs) in SFs isolated from patients with RA, osteoarthritis (OA), EA, and control individuals (CN) after knee surgical intervention. C-reactive protein, general blood test, anticyclic citrullinated peptide (anti-CCP), rheumatoid factor (RF), and vitamin D (vitD) in patients' sera were performed. Cells were stimulated or not with 100 ng/ml tumor necrosis factor alpha (TNF-α) or/and 1 nM or/and 0.01 nM vitamin D3 for 72 h. The expression levels of NLRP1, NLRP3, TLR1, TLR2, TLR4, and VDR in all examined SFs were analyzed by quantitative real-time PCR (RT-qPCR). Additionally, the secretion of IL-1ß by SFs and MMPs were determined by ELISA and Luminex technology. The expression of NLRP3 was correlated with the levels of CRP, RF, and anti-CCP, suggesting its implication in SF inflammatory activation. In the TNF-α-stimulated SFs, a significantly lower expression of NLRP3 and TLR4 was observed in the RA group, compared with the other tested forms of arthritis. Moreover, upregulation of NLRP3 expression by TNF-α alone or in combination with vitD3 was observed, further indicating involvement of NLRP3 in the inflammatory responses of SFs. Secretion of IL-1ß was not detected in any sample, while TNF-α upregulated the levels of secreted MMP-1, MMP-7, MMP-8, MMP-12, and MMP-13 in all patient groups. Attenuating effects of vitD on the expression of NLRP3, TLR1, and TLR4 suggest potential protective effects of vitD on the inflammatory responses in SFs. However, longer studies may be needed to confirm or fully rule out the potential implication of vitD in SF activation in inflammatory arthritis. Both VDR and NLRP3 in the TNF-α-stimulated SFs negatively correlated with the age of patients, suggesting potential age-related changes in the local inflammatory responses.

Protocol for the Isolation of Intact Chondrons from Healthy and Osteoarthritic Human Articular Cartilage.

Chondrons are the main functional microanatomical units in cartilage, consisting of chondrocytes and the directly surrounding pericellular matrix (PCM). They have attracted attention as a more physiological and biomimetic in vitro model for evaluating chondrocyte function and metabolism as compared to single chondrocytes. Chondrons may be more suitable for in vitro studies than primary chondrocytes that have been isolated without PCM since their in situ and in vivo states remain intact: chondrocytes within their PCM do not undergo the rapid dedifferentiation that proliferating single chondrocytes undergo in culture. Therefore, chondrons may be a better model for studying chondrocyte biology and responses to pro-inflammatory and anti-inflammatory cytokines, growth factors and novel therapeutics. In this chapter, we present a concise and unified protocol for enzymatic isolation of intact chondrons from human articular cartilage and determination of their viability.

Nanotechnological Strategies for Osteoarthritis Diagnosis, Monitoring, Clinical Management, and Regenerative Medicine: Recent Advances and Future Opportunities.

PURPOSE OF REVIEW: In this review article, we discuss the potential for employing nanotechnological strategies for the diagnosis, monitoring, and clinical management of osteoarthritis (OA) and explore how nanotechnology is being integrated rapidly into regenerative medicine for OA and related osteoarticular disorders. RECENT FINDINGS: We review recent advances in this rapidly emerging field and discuss future opportunities for innovations in enhanced diagnosis, prognosis, and treatment of OA and other osteoarticular disorders, the smart delivery of drugs and biological agents, and the development of biomimetic regenerative platforms to support cell and gene therapies for arresting OA and promoting cartilage and bone repair. Nanotubes, magnetic nanoparticles, and other nanotechnology-based drug and gene delivery systems may be used for targeting molecular pathways and pathogenic mechanisms involved in OA development. Nanocomposites are also being explored as potential tools for promoting cartilage repair. Nanotechnology platforms may be combined with cell, gene, and biological therapies for the development of a new generation of future OA therapeutics. Graphical Abstract.

The Antihypertensive Drug Nifedipine Modulates the Metabolism of Chondrocytes and Human Bone Marrow-Derived Mesenchymal Stem Cells.

Aging is associated with the development of various chronic diseases, in which both cardiovascular disorders and osteoarthritis are dominant. Currently, there is no effective treatment for osteoarthritis, whereas hypertension is often treated with L-type voltage-operated calcium channel blocking drugs, nifedipine being among the most classical ones. Although nifedipine together with other L-type voltage-operated calcium channel inhibitors plays an important role in controlling hypertension, there are unresolved questions concerning its possible effect on cartilage tissue homeostasis and the development of osteoarthritis. The aim of this study was to analyse the effects of nifedipine on metabolic processes in human chondrocytes and bone marrow mesenchymal stem cells. To better understand whether the metabolic effects are mediated specifically through L-type voltage-operated calcium channel, effects of the agonist BayK8644 were analyzed in parallel. Nifedipine downregulated and mitochondrial respiration and ATP production in both cell types. Analysis of cartilage explants by electron microscopy also suggested that a small number of chondrocyte mitochondria's lose their activity in response to nifedipine. Conversely, nifedipine enhanced glycolytic capacity in chondrocytes, suggesting that these cells have the capacity to switch from oxidative phosphorylation to glycolysis and alter their metabolic activity in response to L-type voltage-operated calcium channel inhibition. Such a metabolic switch was not observed in bone marrow mesenchymal stem cells. Nitric oxide activity was upregulated by nifedipine in bone marrow mesenchymal stem cells and particularly in chondrocytes, implying its involvement in the effects of nifedipine on metabolism in both tested cell types. Furthermore, stimulation with nifedipine resulted in elevated production of collagen type II and glycosaminoglycans in micromass cultures under chondrogenic conditions. Taken together, we conclude that the antihypertensive drug nifedipine inhibits mitochondrial respiration in both chondrocytes and bone marrow mesenchymal stem cells and that these effects may be associated with the increased nitric oxide accumulation and pro-inflammatory activity. Nifedipine had positive effects on the production of collagen type II and proteoglycans in both cell types, implying potentially beneficial anabolic responses in articular cartilage. These results highlight a potential link between antihypertensive drugs and cartilage health.

Paracrine Potential of the Human Adipose Tissue-Derived Stem Cells to Modulate Balance between Matrix Metalloproteinases and Their Inhibitors in the Osteoarthritic Cartilage In Vitro.

Adipose tissue represents an abundant source of stem cells. Along with anti-inflammatory effects, ASC secrete various factors that may modulate metabolism of extracellular matrix in osteoarthritic (OA) cartilage, suggesting that the presence of ASC could be advantageous for OA cartilage due to the recovery of homeostasis between matrix metalloproteinases (MMPs) and their tissue inhibitors of metalloproteinases (TIMPs). To evaluate these effects, cartilage explants (CE) were cocultured with ASC for 3 and 7 days under stimulation with or without IL-1ß. The pattern of gene expression in CE was modified by ASC, including the upregulation of COL1A1 and COL3A1 and the downregulation of MMP13 and COL10A1. The production of MMP-1, MMP-3, and MMP-13 by ASC was not significant; moreover, cocultures with ASC reduced MMP-13 production in CE. In conclusion, active production of TIMP-1, TIMP-2, TIMP-3, IL-6, IL-8, and gelatinases MMP-2 and MMP-9 by ASC may be involved in the extracellular matrix remodelling, as indicated by the altered expression of collagens, the downregulated production of MMP-13, and the reduced chondrocyte apoptosis in the cocultured CE. These data suggest that ASC modulated homeostasis of MMPs/TIMPs in degenerated OA cartilage in vitro and might be favourable in case of the intra-articular application of ASC therapy for the treatment of OA.

Native matrix-based human lung alveolar tissue model in vitro: studies of the reparatory actions of mesenchymal stem cells.

Studies of lung diseases in vitro often rely on flat, plastic-based monocultures, due to short lifespan of primary cells, complicated anatomy, lack of explants, etc. We hereby present a native 3D model with cues for repopulating epithelial cells. Abilities of mesenchymal stem cells (MSC) to modulate bacterial lipopolysaccharide (LPS) and cigarette smoke-induced injury to pulmonary epithelium were tested in our model. Post-mortem human lung tissue was sliced, cut and decellularized. Resulting matrix pads were reseeded with pulmonary epithelium (A549 line). Markers of the layer integrity and certain secreted proteins in the presence of cigarette smoke extract (CSE) and LPS were assessed via Western blot, ELISA and RT-PCR assays. In parallel, the effects of MSC paracrine factors on exposed epithelial cells were also investigated at gene and protein levels. When cultured on native 3D matrix, A549 cells obtain dual, type I- and II-like morphology. Exposure to CSE and LPS leads to downregulation of several epithelial proteins and suppressed proliferation rate. MSC medium added to the model restores proliferation rate and some of the epithelial proteins, i.e. e-cadherin and beta-catenin. CSE also increases secretion of pro-inflammatory cytokines by epithelial cells and upregulates transcription factor NFκB. Some of these effects might be counteracted by MSC in our model. We introduce repopulated decellularized lung matrix that highly resembles in vivo situation and is convenient for studies of disease pathogenesis, cytotoxicology and for exploring therapeutic strategies in the human lung context in vitro. MSC paracrine products have produced protecting effects in our model.

Relevance of HCN2-expressing human mesenchymal stem cells for the generation of biological pacemakers.

BACKGROUND: The transfection of human mesenchymal stem cells (hMSCs) with the hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2) gene has been demonstrated to provide biological pacing in dogs with complete heart block. The mechanism appears to be the generation of the ion current (If) by the HCN2-expressing hMSCs. However, it is not clear how the transfection process and/or the HCN2 gene affect the growth functions of the hMSCs. Therefore, we investigated survival, proliferation, cell cycle, and growth on a Kapton® scaffold of HCN2-expressing hMSCs. METHODS: hMSCs were isolated from the bone marrow of healthy volunteers applying a selective cell adhesion procedure and were identified by their expression of specific surface markers. Cells from passages 2-3 were transfected by electroporation using commercial transfection kits and a pIRES2-EGFP vector carrying the pacemaker gene, mouse HCN2 (mHCN2). Transfection efficiency was confirmed by enhanced green fluorescent protein (EGFP) fluorescence, quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). After hMSCs were transfected, their viability, proliferation, If generation, apoptosis, cell cycle, and expression of transcription factors were measured and compared with non-transfected cells and cells transfected with pIRES2-EGFP vector alone. RESULTS: Intracellular mHCN2 expression after transfection increased from 22.14 to 62.66 ng/mg protein (p < 0.05). Transfection efficiency was 45 ± 5 %. The viability of mHCN2-transfected cells was 82 ± 5 %; they grew stably for more than 3 weeks and induced If current. mHCN2-transfected cells had low mitotic activity (10.4 ± 1.24 % in G2/M and 83.6 ± 2.5 % in G1 phases) as compared with non-transfected cells (52-53 % in G2/M and 31-35 % in G1 phases). Transfected cells showed increased activation of nine cell cycle-regulating transcription factors: the most prominent upregulation was of AMP-dependent transcription factor ATF3 (7.11-fold, p = 0.00056) which regulates the G1 phase. mHCN2-expressing hMSCs were attached and made anchorage-dependent connection with other cells without transmigration through a 12.7-µm thick Kapton® HN film with micromachined 1-3 µm diameter pores. CONCLUSIONS: mHCN2-expressing hMSCs preserved the major cell functions required for the generation of biological pacemakers: high viability, functional activity, but low proliferation rate through the arrest of cell cycle in the G1 phase. mHCN2-expressing hMSCs attached and grew on a Kapton® scaffold without transmigration, confirming the relevance of these cells for the generation of biological pacemakers.

Cell surface markers and exogenously induced PpIX in synovial mesenchymal stem cells.

The aim of present study was to assess the expression of surface markers and the accumulation of protoporphyrin IX in synovial mesenchymal stem cells (SMSCs). SMSC from patients with rheumatoid arthritis (RA, n = 5) and osteoarthritis (OA, n = 5-6) were characterized and their PpIX accumulation rates were evaluated by flow cytometry. The expression of the 21 out of 24 tested surface markers, related to stem-like features and aggressiveness of cells showed no statistically significant differences between RA and OA groups. However, the cells from RA group had the significantly lower levels of expression for the integrin-associated protein CD47 and the grow factor receptor CD271 (P = 0.018), while the higher levels of cell membrane zinc-dependent metalloproteinase CD10 (P = 0.006), as compared to the cells from OA group. Comparison of the mean intensities of PpIX fluorescence revealed no statistically significant differences between the RA and OA groups, as well as no relation to proliferation rates or cell size, although some conspicuous distinction in PpIX accumulation was observed in certain specimens within these groups, suggesting possibilities of this method application for characterization of individual SMSC populations. CD10, CD47, and CD271 were differently expressed in RA and OA SMSC, while had no direct association with the PpIX fluorescence intensity.

Induction of protoporphyrin IX in patient-derived synoviocytes, cartilage explants and chondrons after application of 5-aminolevulinic acid or its methyl ester.

OBJECTIVE: To compare the accumulation of protoporphyrin IX between synoviocytes of patients with rheumatoid arthritis (RA) or osteoarthritis (OA) and cartilage explants (CE) as well as chondrons of patients with OA after the application of 5-aminolevulinic acid (ALA) or its methyl ester (ALA-Me). MATERIALS AND METHODS: Samples of synovial and cartilage tissues were obtained from joint replacement surgeries. The accumulation of PpIX was determined by measuring fluorescence spectra from 2 × 10(5) synoviocytes or chondrons suspended in a glass tube or directly from CE surface after 2, 4, 8 and 24h of incubation with ALA or ALA-Me. RESULTS: No differences were found between the average fluorescence intensity values of PpIX in synoviocytes of patients with RA and OA. These values were non-significantly higher after incubation with ALA in comparison with ALA-Me at almost all time points. The average fluorescence intensity of PpIX in CE and chondrons was about ten times lower than in synoviocytes. The presence of preparation of hyaluronic acid (HA) significantly enhanced PpIX induction in chondrons versus treatment only with ALA. CONCLUSIONS: A potential for the selective synovial sensitization with endogenous PpIX in comparison with cartilage tissue has been demonstrated in vitro after application of ALA or ALA-Me.