29 research outputs found
Autologous Microfragmented Adipose Tissue Reduces the Catabolic and Fibrosis Response in an in Vitro Model of Tendon Cell Inflammation
Background. Mesenchymal stem cells (MSCs) emerged as a promising therapy for tendon pathologies. Microfragmented adipose tissue (\u3bcFAT) represents a convenient autologous product for the application of MSC-based therapies in the clinical setting. In the present study, the ability of \u3bcFAT to counteract inflammatory processes induced by IL-1\u3b2 on human tendon cells (TCs) was evaluated. Methods. Cell viability and proliferation were evaluated after 48 hours of transwell coculture of TCs and autologous \u3bcFAT in the presence or absence of IL-1\u3b2. Gene expression of scleraxis, collagen type I and type III, metalloproteinases-1 and -3, and cyclooxygenase-2 was evaluated by real-time RT-PCR. The content of VEGF, IL-1Ra, TNF\u3b1, and IL-6 was evaluated by ELISA. Results. IL-1\u3b2-treated TCs showed augmented collagen type III, metalloproteases, and cyclooxygenase-2 expression. \u3bcFAT was able to reduce the expression of collagen type III and metalloproteases-1 in a significant manner, and at the same time, it enhanced the production of VEGF, IL-1Ra, and IL-6. Conclusions. In this in vitro model of tendon cell inflammation, the paracrine action of \u3bcFAT, exerted by anti-inflammatory molecules and growth factors, was able to inhibit the expression of fibrosis and catabolic markers. Then, these results suggest that the application of \u3bcFAT may represent an effective conservative or adjuvant therapy for the treatment of tendon disorders
Mirna reference genes in extracellular vesicles released from amniotic membrane-derived mesenchymal stromal cells
Human amniotic membrane and amniotic membrane-derived mesenchymal stromal cells (hAMSCs) have produced promising results in regenerative medicine, especially for the treatment of inflammatory-based diseases and for different injuries including those in the orthopedic field such as tendon disorders. hAMSCs have been proposed to exert their anti-inflammatory and healing potential via secreted factors, both free and conveyed within extracellular vesicles (EVs). In particular, EV miRNAs are considered privileged players due to their impact on target cells and tissues, and their future use as therapeutic molecules is being intensely investigated. In this view, EV-miRNA quantification in either research or future clinical products has emerged as a crucial paradigm, although, to date, largely unsolved due to lack of reliable reference genes (RGs). In this study, a panel of thirteen putative miRNA RGs (let-7a-5p, miR-16-5p, miR-22-5p, miR-23a-3p, miR-26a-5p, miR-29a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p, miR-660-5p and U6 snRNA) that were identified in different EV types was assessed in hAMSC-EVs. A validated experimental pipeline was followed, sifting the output of four largely accepted algorithms for RG prediction (geNorm, NormFinder, BestKeeper and \u394Ct method). Out of nine RGs constitutively expressed across all EV isolates, miR-101-3p and miR-22-5p resulted in the most stable RGs, whereas miR-423-5p and U6 snRNA performed poorly. miR-22-5p was also previously reported to be a reliable RG in adipose-derived MSC-EVs, suggesting its suitability across samples isolated from different MSC types. Further, to shed light on the impact of incorrect RG choice, the level of five tendon-related miRNAs (miR-29a-3p, miR-135a-5p, miR-146a-5p, miR-337-3p, let-7d-5p) was compared among hAMSC-EVs isolates. The use of miR-423-5p and U6 snRNA did not allow a correct quantification of miRNA incorporation in EVs, leading to less accurate fingerprinting and, if used for potency prediction, misleading indication of the most appropriate clinical batch. These results emphasize the crucial importance of RG choice for EV-miRNAs in hAMSCs studies and contribute to the identification of reliable RGs such as miR-101-3p and miR-22-5p to be validated in other MSC-EVs related fields
High Levels of Circulating Type II Collagen Degradation Marker (CTx-II) Are Associated with Specific VDR Polymorphisms in Patients with Adult Vertebral Osteochondrosis
Both vitamin D and collagen have roles in osteocartilaginous homeostasis. We evaluated the association between the circulating 25-hydroxyvitamin D (25(OH)D) type I and II collagen degradation products (CTx-I, and CTx-II), and four vitamin D receptor gene (VDR) polymorphisms, in Italian males affected by low back pain (LBP) due to herniation/discopathy and/or vertebral osteochondrosis. FokI, BsmI, ApaI, and TaqI VDR-polymorphisms were detected through PCR-restriction fragment length polymorphism (RFLP), and circulating 25(OH)D, CTx-I and CTx-II were measured by immunoassays in 79 patients (of which 26 had osteochondrosis) and 79 age-, sex- and body mass index (BMI)-matched healthy controls. Among all 158 subjects, carriers of FF and Ff genotypes showed lower 25(OH)D than ff, which suggested a higher depletion of vitamin D in F allele carriers. Higher CTx-I concentrations were observed in TT versus Tt among controls, and Tt versus tt among LBP cases, which suggested a higher bone-cartilaginous catabolism in subjects bearing the T allele. Higher CTx-II concentrations were observed in patients with osteochondrosis bearing FF, bb, TT, or Aa genotypes in comparison with hernia/discopathy patients and healthy controls. Vertebral osteochondrosis shows peculiar genotypic and biochemical features related to vitamin D and the osteocartilaginous metabolism. Vitamin D has roles in the pathophysiology of osteochondrosis
Effects of the pulsed electromagnetic field PST® on human tendon stem cells : A controlled laboratory study
Background: Current clinical procedures for rotator cuff tears need to be improved, as a high rate of failure is still observed. Therefore, new approaches have been attempted to stimulate self-regeneration, including biophysical stimulation modalities, such as low-frequency pulsed electromagnetic fields, which are alternative and non-invasive methods that seem to produce satisfying therapeutic effects. While little is known about their mechanism of action, it has been speculated that they may act on resident stem cells. Thus, the purpose of this study was to evaluate the effects of a pulsed electromagnetic field (PST\uae) on human tendon stem cells (hTSCs) in order to elucidate the possible mechanism of the observed therapeutic effects. Methods: hTSCs from the rotator cuff were isolated from tendon biopsies and cultured in vitro. Then, cells were exposed to a 1-h PST\uae treatment and compared to control untreated cells in terms of cell morphology, proliferation, viability, migration, and stem cell marker expression. Results: Exposure of hTSCs to PST\uae did not cause any significant changes in proliferation, viability, migration, and morphology. Instead, while stem cell marker expression significantly decreased in control cells during cell culturing, PST\uae-treated cells did not have a significant reduction of the same markers. Conclusions: While PST\uae did not have significant effects on hTSCs proliferation, the treatment had beneficial effects on stem cell marker expression, as treated cells maintained a higher expression of these markers during culturing. These results support the notion that PST\uae treatment may increase the patient stem cell regenerative potential
Systematic review and meta-analysis on the use of human platelet lysate for mesenchymal stem cell cultures: comparison with fetal bovine serum and considerations on the production protocol
Mesenchymal stem cell (MSC) culturing for cell therapies needs a step forward to be routinely used in clinical settings. Main concerns regard the use of animal origin reagents, in particular supplementing the culture medium with FBS. Lately, Human Platelet Lysate (HPL) has been proposed as animal-free alternative, described as an excellent supplement for culturing MSCs. The aim of this systematic review was to analyze the current literature on the effect of HPL and FBS on ASCs and BMSCs. The primary outcome was the proliferation rate of cells cultured with FBS and HPL. Differences in terms of doubling time (DT) and population doubling (PD) were evaluated by meta-analysis, subgrouping data according to the cell type. A total of 35 articles were included. BMSCs and ASCs were used in 65.7% (23) and 28.6% (10) studies, respectively. Only two studies included both cell types. Overall, 22 studies were eligible for the meta-analysis. Among them, 9 articles described ASCs and 13 BMSCs. The results showed that BMSCs and ASCs cultured with 10% HPL and 5% HPL have lower DT and higher PD compared to cells cultured with 10% FBS. A possible correlation between the DT decrease and the application of at least 3 freeze/thaw cycles to induce platelet lysis was found. Additionally, HPL increased VEGF secretion and maintained the immuno-modulatory abilities for both cell types. The clarification reported here of the higher efficiency of HPL compared to FBS can help the transition of the scientific community towards clinical-related procedures
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Reliable Reference Genes for Gene Expression Assessment in Tendon-Derived Cells under Inflammatory and Pro-Fibrotic/Healing Stimuli
Tendon cells (TCs) are important for homeostatic maintenance in the healthy tendon and to promote tissue healing after injury. Further, resident and rare populations of tendon stem/progenitor cells, located at various sites within the tendon, contribute to tendon recovery by differentiating into repairing TCs. Gene expression analysis, through quantitative reverse-transcription polymerase chain reaction (qRT-PCR), constitutes a useful tool to study cellular responses, including the transition from initial inflammation to healing processes. A critical step required for data normalization is the choice of reliable reference genes (RGs), a process highly underestimated in tendon biology. In this study, the suitability of five commonly used RGs (
,
,
,
, and
) was evaluated using TCs samples cultured in both standard and progenitor-enriching conditions, as well as under either inflammatory (IFNγ + TNFα) or pro-fibrotic/healing (CTGF) stimulation. The stability of the candidate RGs was computationally determined using NormFinder, geNorm, BestKeeper, and DeltaCt applets. Overall,
resulted as the most stable RG on the basis of the integration of each gene weight, whereas
and
performed poorly. To further validate
's optimal performance, we evaluated the expression of
, coding for an immune-related cell surface glycoprotein, and
, encoding collagen type I that is the main component of the tendon extracellular matrix (ECM), both known to be modulated by inflammation. The expression of both genes was heavily affected by the RGs used. Consequently, when analyzing gene expression in tendon-derived cells subjected to various stimulatory protocols, the use of a suitable RG should be considered carefully. On the basis of our results,
can be reliably used when analyzing different TC types exposed to pathological conditions
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CD146+CD107a+ Mesenchymal Stem/Stromal Cells with Signature Attributes Correlate to Therapeutic Potency as “First Responders” to Injury and Inflammation
ABSTRACT CD146 + bone marrow–derived Mesenchymal Stem/Stromal Cells (BM-MSC) play key roles in the perivascular niche, skeletogenesis and hematopoietic support, however elucidation of therapeutic potency has yet to be determined. Here, inflammatory challenge to crude BM-MSC captured a baseline of signatures including enriched expression of CD146 + with CD107a + , CXCR4 + , and LepR + , transcriptional profile, enhanced secretory capacity, robust secretome and immunomodulatory function with stimulated target immune cells. These responses were significantly more pronounced in CD146 + (POS)-selected subpopulation than in the CD146 - (NEG). Mechanistically, POS uniquely mediated robust immunosuppression while inducing significant frequencies of Naïve and Regulatory T cells in vitro . Moreover, POS promoted a pivotal M1-to-M2 macrophage shift in vivo, ameliorating inflammation/fibrosis of joint synovium and fat pad of the knee, failed by NEG. This study provides high-content evidence of CD146 + CD107a + BM-MSC, herein deemed ‘first responders’ to inflammation, as the underrepresented subpopulation within crude BM-MSC with innately higher secretory capacity and therapeutic potency. HIGHLIGHTS Signature phenotypic, transcriptional, and secretome profiles were identified and enriched in human CD146 + (POS)-selected subpopulation in response to inflammation Inflammatory challenge consistently altered stemness ( LIF ) and differentiation master regulators ( SOX9, RUNX2, PPARγ ) in crude, POS, and NEG BM-MSC, and deduced unique expressions in POS compared to NEG POS BM-MSC mediated the strongest immunomodulation, e.g. target immune cell suppression, Treg induction, diminished T cell differentiation POS BM-MSC promoted the largest M1-to-M2 shift in vivo alleviating induced synovitis and infrapatellar fat pad fibrosis of the kne