Low-density cultured cartilage cells expanded in platelet lysate present distinct features to develop an innovative clinical treatment for diffuse cartilage lesions.

PURPOSE: Chondrocyte-based cell therapies are effective for the treatment of chondral lesions, but remain poorly indicated for diffuse lesions in the context of early osteoarthritis (OA). The aim of this study was to develop a protocol to obtain chondroprogenitor cells suitable for the treatment of diffuse chondral lesions within early OA. METHODS: Cartilage cells were expanded at low density in human platelet lysate (hPL). A test was performed to exclude senescence. The expression of surface cluster of differentiation 146, cluster of differentiation 166, major histocompatibility complex (MHC)-I and MHC-II and of genes of interest were evaluated, as well as the trophic potential of these cells, by the assessment of lubricin and matrix production. The immunomodulatory potential was assessed through their co-culture with macrophages. RESULTS: Cartilage cells expanded at low density in hPL showed higher proliferation rate than standard-density cells, no replicative senescence, low immunogenicity and expression of lubricin. Moreover, they presented an increased expression of chondrogenic and antihypertrophic markers, as well as a superior matrix deposition if compared to cells cultured at standard density. Cartilage cells induced on macrophages an upregulation of CD206, although a higher increase of CD163 expression was observed in the presence of low-density cells. CONCLUSIONS: These findings lay the grounds to explore the clinical usefulness of low-density cultured cartilage cells to treat diffuse lesions in early OA joints for both autologous and allogenic use. LEVEL OF EVIDENCE: Not applicable.

Donor Sites and Harvesting Techniques Affect miRNA Cargos of Extracellular Vesicles Released by Human Adipose-Derived Mesenchymal Stromal Cells.

Osteoarthritis (OA) is a degenerative joint disorder characterized by the progressive deterioration of articular cartilage driven and sustained by catabolic and inflammatory processes that lead to pain and functional impairment. Adipose-derived stem cells (ASCs) have emerged as a promising therapeutic strategy for OA due to their regenerative potential, which mainly relies on the adaptive release of paracrine molecules that are soluble or encapsulated in extracellular vesicles (EVs). The biological effects of EVs specifically depend on their cargo; in particular, microRNAs (miRNAs) can specifically modulate target cell function through gene expression regulation. This study aimed to investigate the impact of collection site (abdominal vs. peri-trochanteric adipose tissue) and collection method (surgical excision vs. lipoaspiration) on the miRNAs profile in ASC-derived EVs and their potential implications for OA therapy. EV-miRNA cargo profiles from ASCs of different origins were compared. An extensive bioinformatics search through experimentally validated and OA-related targets, pathways, and tissues was conducted. Several miRNAs involved in the restoration of cartilage homeostasis and in immunomodulation were identified in all ASC types. However, EV-miRNA expression profiles were affected by both the tissue-harvesting site and procedure, leading to peculiar characteristics for each type. Our results suggest that adipose-tissue-harvesting techniques and the anatomical site of origin influence the therapeutic efficacy of ASC-EVs for tissue-specific regenerative therapies in OA, which warrants further investigation.

The total testing process harmonization: the case study of SARS-CoV-2 serological tests.

The total testing process harmonization is central to laboratory medicine, leading to the laboratory test's effectiveness. In this opinion paper the five phases of the TTP are analyzed, describing, and summarizing the critical issues that emerged in each phase of the TTP with the SARS-CoV-2 serological tests that have affected their effectiveness. Testing and screening the population was essential for defining seropositivity and, thus, driving public health policies in the management of the COVID-19 pandemic. However, the many differences in terminology, the unit of measurement, reference ranges and parameters for interpreting results make analytical results difficult to compare, leading to the general confusion that affects or completely precludes the comparability of data. Starting from these considerations related to SARS-CoV-2 serological tests, through interdisciplinary work, the authors have highlighted the most critical points and formulated proposals to make total testing process harmonization effective, positively impacting the diagnostic effectiveness of laboratory tests.

Treatment with CR500® improves algofunctional scores in patients with knee osteoarthritis: a post-market confirmatory interventional, single arm clinical investigation.

BACKGROUND: Knee osteoarthritis (OA) is a progressive and degenerative condition. Several pharmacological and non-pharmacological treatments are able to improve the OA symptoms and the structural characteristics of the affected joints. Among these, infiltrative therapy with hyaluronic acid (HA) is the most used and consolidated procedure for the pain management. The addition of skin conditioning peptides to HA promotes the cartilage remodeling processes and a better permeation of the HA-based gel containing a peptide mixture, CR500®. Furthermore, the topic route of administration is convenient over the routinely used intra-articular injective procedures. In this study, the effectiveness of CR500® was evaluated in terms of improvement of the algo-functional symptoms related to unilateral knee OA. METHODS: 38 mild and moderate OA patients were enrolled at a screening visit (V-1), treated at baseline visit (V1), and then continued the topical application of CR500® twice a week for 4 weeks, and followed-up for 3 visits (V2-V4) from week 2 to 4. Lequesne Knee Index (LKI) and Knee injury and Osteoarthritis Outcome Score (KOOS) were collected. Synovial fluid was collected and used for the quantification of neoepitope of type II collagen (C2C), C-terminal telopeptide of type II collagen (CTX-II), type II collagen propeptide (CPII), tumor necrosis factor alpha (TNFα) and HA. The expression of CD11c and CD206 was evaluated on cell pellets. RESULTS: Three patients were excluded, thus 35 patients were included in the analysis. The treatment with CR500® was safe and well tolerated, with 7.9% patients had mild adverse events, not related to the device. The LKI total score showed a significant decrease from V1 to V4. KOOS score also showed a significant improvement of patient condition at V2, V3 and V4 in comparison with V1 for all subscales, except for KOOS sport subscale which improved only from V3. At V1 a negative correlation among KOOS pain subscale values and C2C, CPII and TNFα levels was observed, as well as a positive correlation between KOOS pain subscale and CD11c/CD206 ratio. CONCLUSION: CR500® is safe and appear to be effective in improving pain and function in OA patients during the 4 weeks of treatment. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT05661162. This trial was registered on 22/12/2022.

Autologous chondrocyte implantation provides good long-term clinical results in the treatment of knee osteoarthritis: a systematic review.

PURPOSE: To evaluate the mid- and long-term efficacy of autologous chondrocyte implantation (ACI) and matrix-assisted chondrocyte implantation (MACI) to treat patients with knee cartilage defects in the presence of osteoarthritis (OA). METHODS: PubMed and Cochrane databases were systematically searched for studies describing the treatment of knee OA with ACI or MACI (Kellgren-Lawrence (KL) ≥ 1, minimum follow-up 36 months). Results were reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines and included Lysholm, Western Ontario McMaster University and International Knee Documentation Committee scores. RESULTS: Of the 127 full-text articles assessed for eligibility, only five studies were selected based on inclusion/exclusion criteria (2 on ACI and 3 on MACI). In both groups, the defects were mainly located at femoral level, size 2.2-15.1 cm2 in the ACI and 2.0-7.6 cm2 in the MACI group. ACI was mostly used for patients affected by KL I, whereas MACI for patients with KL II-IV. The data obtained from 235 patients (161 ACI, 74 MACI) showed that ACI and MACI sustained stable clinical improvements up to 11 and 15 years, respectively, with a failure rate of about 10% up to 11 years. Scarce biological details regarding chondrocyte implantation were reported. CONCLUSIONS: ACI and MACI procedures for the treatment of knee cartilage lesions associated to OA showed long-term success and allowed delaying arthroplasty. Additional trials reporting homogenous data and precise patient characterization are needed to conduct an effective literature meta-analysis and identify the clinical relevance of these procedures. LEVEL OF EVIDENCE: IV.

Micro-fragmented adipose tissue (mFAT) associated with arthroscopic debridement provides functional improvement in knee osteoarthritis: a randomized controlled trial.

PURPOSE: Current conservative treatments for knee OA provide limited benefits, with symptoms relief for a short amount of time. Regenerative medicine approaches such as the use of microfragmented adipose tissue (mFAT) showed promising results in terms of durable effects and the possibility to enhance tissue healing and counteract the progression of the pathology. Nevertheless, up to today, the large part of clinical data about mFAT use refers to uncontrolled studies, especially in the surgical setting. The purpose of this study was to evaluate the effectiveness of mFAT applied in association with arthroscopic debridement (AD) for the treatment of knee OA, in terms of symptoms relief and tissue healing. METHODS: This study is a prospective, randomized controlled clinical trial. 78 patients affected by knee OA grade 3-4 according to KL classification were randomly assigned to AD or AD + mFAT treatment groups. Clinical, radiological and serological assessments were performed at 6 months after treatment. Additional clinical evaluation was performed at the end of the study with an average follow-up of 26.1 ± 9.5 months. VAS, KOOS, WOMAC and SF-12 were also collected at both timepoints, KSS only at 6 months. RESULTS: Treatment with AD + mFAT improved functional scores at both 6 months (KOOS-PS: + 11.7 ± 20.2 vs + 24.4 ± 22.5, in AD and AD + mFAT, respectively, p = 0.024; KSS: + 14.9 ± 15.9 vs + 24.8 ± 23.5, in AD and AD + mFAT, respectively, p = 0.046) and 24-month follow-ups (KOOS-PS Functional subscale: - 2.0 ± 3.5 vs - 4.7 ± 4.2, in AD and AD + mFAT, respectively, p = 0.012). Lower T2-mapping scores were obtained in AD + mFAT-treated group in medial and lateral condyle compartments (p < 0.001). Slight increase was observed in the levels of a serum biomarker of cartilage deposition (PIIINP) in both groups at 6-month follow-up (p = 0.037). CONCLUSION: mFAT improves functional outcome and MRI appearance when used in association with AD, therefore supporting its use in the treatment of knee OA in an arthroscopic setting.

Exploratory assessment of serological tests to determine antibody titer against SARS-CoV-2: Appropriateness and limits.

BACKGROUND: Serological tests can be used to detect antibodies in the serum of subject's after SARS-CoV-2 infection and vaccination. Currently, variability in antibody titers and the availability of a multiplicity of serological tests have made it necessary to highlight their appropriateness and limitations in various diagnostic settings. METHODS: This study is part of Covidiagnostix, a multicenter project aimed at the assessment of the health technology used in SARS-CoV-2 serological tests. Based on data gained from the analysis of over 5000 subjects, a selected number of serum samples, representative of different diagnostic settings, were analyzed first by qualitative immunoassays (IgA, M, and G MILLIPLEX® SARS-CoV-2 tests based on Luminex® ) to define the immunoglobulins serum composition and subsequently by four serological diagnostic tests (Elecsys Anti-SARS-CoV-2 and Elecsys Anti-SARS-CoV-2 S by Roche, SARS-CoV-2 IgG by Siemens Healthcare, and CHORUS SARS-CoV-2 "NEUTRALIZING" Ab by DIESSE). The first WHO International Standard for SARS-CoV-2 was also analyzed using the same methods. RESULTS: This study evaluated the antibody content and titer of the WHO Standard and serum of subjects with/without previous infection and before/after vaccination for SARS-CoV-2. CONCLUSION: The definition of antibodies in the WHO standard and the analysis of serum samples allowed for the identification of the appropriateness of serological tests in each diagnostic setting, increasing the effectiveness of the resulting laboratory data. Furthermore, we found that it would be optimal to produce new international standards against the S1 domain and RBD of the SARS-CoV-2 spike protein for a more effective serological monitoring of vaccination.

A single step, centrifuge-free method to harvest bone marrow highly concentrated in mesenchymal stem cells: results of a pilot trial.

PURPOSE: The aims of the present study were: (1) to characterize the bone-marrow aspirate (BMA) obtained with a centrifuge-free process, employing a dedicated aspiration device; (2) to test the in vitro efficacy of BMA in a model of cartilage inflammation; and (3) to report the preliminary clinical results in a small cohort of patients affected by knee OA. METHODS: Ten patients (4 M, 6 W; mean age: 51.9 ± 9.2 yy) affected by mild to moderate unicompartmental knee OA (KL grade 2-3) were treated by intra-articular and subchondral injections of BMA obtained by a centrifuge-free process. To evaluate the effectiveness of the device in harvesting mesenchymal stem cells (MSCs), samples of the obtained BMA were tested by flow cytometry before and after subculture; BMA ability to counteract inflammation was also tested in an in vitro model of cartilage cell inflammation, evaluating the expression of MMP1, MMP3, TGFß and TIMP-1 by real-time PCR. Patients were also evaluated up to two years' follow-up by using: VAS for pain, IKDC-subjective and KOOS scores. RESULTS: The laboratory analysis showed that BMSCs accounted for 0.011% of BMA cells, similar to what had been expected in native bone marrow. The paracrine activity of BMA was able to reduce in vitro the catabolic response of human chondrocyte, as shown by the decrease in metalloproteases concentration and increase in anti-inflammatory mediators. Moreover, the clinical evaluation showed significant improvements in all scores adopted, with stable results up to two years. CONCLUSION: The present data showed the effectiveness of the study device to harvest pure bone marrow with minimal peripheral blood contamination. The relevant content of MSCs resulted in the ability to counteract the catabolic cascade through a paracrine action. The clinical outcomes in patients affected by unicompartmental knee OA were encouraging in terms of pain reduction and functional improvement up to mid-term evaluation.

Tendon Cells Derived From The Long Head Of The Biceps And The Supraspinatus Tendons Of Patients Affected By Rotator Cuff Tears Show Different Expression Of Inflammatory Markers.

AIM OF THE STUDY: Tendons are exposed to mechanical stress constantly during movements and thus they are frequently subjected to injuries. Rotator cuff tears are common musculoskeletal disorders, mainly involving the supraspinatus tendon. The characterization of the tenocytes derived from this tendon and the comparison to cells isolated from the long head of the biceps tendon obtained from donors affected by rotator cuff disease may improve the knowledge of the cellular mechanisms involved in the initiation and progression of the pathology. Thus, the aim of the present study was to characterize and compare donor-matched human tendon cells (TCs) isolated from the long head of the biceps (LHB-TCs) and the supraspinatus tendons (SSP-TCs) of patients affected by rotator cuff tears. METHODS: donor-matched LHB-TCs and SSP-TCs were isolated and cultured up to passage 3. Phenotypic appearance, metabolic activity, DNA content, production of soluble mediators (IL-1Ra, IL-1ß, IL-6, and VEGF) and gene expression of tendon markers (SCX, COL1A1, COL3A1), inflammatory (PTGS2), and catabolic enzymes (MMP-1, MMP-3) were evaluated. RESULTS: LHB-TCs showed an elongated fibroblast-like shape, while SSP-TCs appeared irregular with jagged membrane. SSP-TCs gene expression revealed an augmented production of PTGS2, a marker of inflammation, whereas they produced a reduced amount of IL-6, in respect to LHB-TCs. CONCLUSION: SSP-TCs showed higher cellular stress and expression of inflammatory markers with respect to donor-matched LHB-TCs, suggesting that addressing the physio-pathological state of supraspinatus tendon cells during treatment of rotator cuff tears could favor tissue healing and possibly prevent relapses.

Classification of endplate lesions in the lumbar spine and association with risk factors, biochemistry, and genetics.

PURPOSE: To detect the associations between the degree of the endplate (EP) lesions with the presence of risk factors, biochemical and genetic markers previously observed in low back pain (LBP) patients with EP defects in comparison with hernia/discopathy patients and healthy controls. METHODS: In this observational retrospective study, T2-weighted sagittal MRI images (n = 223 LBP patients) were scored for EP lesions by two independent observers. Total MRI score and number of affected levels (L1/L2-L5/S1) have been considered for the correlation with demographic, behavioral, clinical, biochemical (25(OH)D, CTx-I and CTx-II levels, n = 69 males) and VDR variables. RESULTS: Males showed higher BMI and total MRI score than females. Patients bearing TT compared to tt VDR genotypes showed significant higher total MRI scores. Among males (n = 125), TT, bb and aa genotypes showed increased total MRI scores. Higher total MRI score directly correlates with higher levels of CTx-I and CTx-II (n = 69 males). CONCLUSIONS: The markers previously identified as associated with the presence of EP lesions have been confirmed as related to their severity and could be used to follow the pathology progression.

Autologous microfragmented adipose tissue reduces inflammatory and catabolic markers in supraspinatus tendon cells derived from patients affected by rotator cuff tears.

PURPOSE: Rotator cuff tears are common musculoskeletal disorders, and surgical repair is characterized by a high rate of re-tear. Regenerative medicine strategies, in particular mesenchymal stem cell-based therapies, have been proposed to enhance tendon healing and reduce the re-tear rate. Autologous microfragmented adipose tissue (µFAT) allows for the clinical application of cell therapies and showed the ability to improve tenocyte proliferation and viability in previous in vitro assessments. The hypothesis of this study is that µFAT paracrine action would reduce the catabolic and inflammatory marker expression in tendon cells (TCs) derived from injured supraspinatus tendon (SST). METHODS: TCs derived from injured SST were co-cultured with autologous µFAT in transwell for 48 h. Metabolic activity, DNA content, the content of soluble mediators in the media, and the gene expression of tendon-specific, inflammatory, and catabolic markers were analyzed. RESULTS: µFAT-treated TCs showed a reduced expression of PTGS2 and MMP-3 with respect to untreated controls. Increased IL-1Ra, VEGF, and IL-6 content were observed in the media of µFAT-treated samples, in comparison with untreated TCs. CONCLUSION: µFAT exerted an anti-inflammatory action on supraspinatus tendon cells in vitro through paracrine action, resulting in the reduction of catabolic and inflammatory marker expression. These observations potentially support the use of µFAT as adjuvant therapy in the treatment of rotator cuff disease.

High-Throughput Gene and Protein Analysis Revealed the Response of Disc Cells to Vitamin D, Depending on the VDR FokI Variants.

Vitamin D showed a protective effect on intervertebral disc degeneration (IDD) although conflicting evidence is reported. An explanation could be due to the presence of the FokI functional variant in the vitamin D receptor (VDR), observed as associated with spine pathologies. The present study was aimed at investigating-through high-throughput gene and protein analysis-the response of human disc cells to vitamin D, depending on the VDR FokI variants. The presence of FokI VDR polymorphism was determined in disc cells from patients with discopathy. 1,25(OH)2D3 was administered to the cells with or without interleukin 1 beta (IL-1ß). Microarray, protein arrays, and multiplex protein analysis were performed. In both FokI genotypes (FF and Ff), vitamin D upregulated metabolic genes of collagen. In FF cells, the hormone promoted the matrix proteins synthesis and a downregulation of enzymes involved in matrix catabolism, whereas Ff cells behaved oppositely. In FF cells, inflammation seems to hamper the synthetic activity mediated by vitamin D. Angiogenic markers were upregulated in FF cells, along with hypertrophic markers, some of them upregulated also in Ff cells after vitamin D treatment. Higher inflammatory protein modulation after vitamin D treatment was observed in inflammatory condition. These findings would help to clarify the clinical potential of vitamin D supplementation in patients affected by IDD.

Development, evaluation, and validation of machine learning models for COVID-19 detection based on routine blood tests.

Objectives: The rRT-PCR test, the current gold standard for the detection of coronavirus disease (COVID-19), presents with known shortcomings, such as long turnaround time, potential shortage of reagents, false-negative rates around 15-20%, and expensive equipment. The hematochemical values of routine blood exams could represent a faster and less expensive alternative. Methods: Three different training data set of hematochemical values from 1,624 patients (52% COVID-19 positive), admitted at San Raphael Hospital (OSR) from February to May 2020, were used for developing machine learning (ML) models: the complete OSR dataset (72 features: complete blood count (CBC), biochemical, coagulation, hemogasanalysis and CO-Oxymetry values, age, sex and specific symptoms at triage) and two sub-datasets (COVID-specific and CBC dataset, 32 and 21 features respectively). 58 cases (50% COVID-19 positive) from another hospital, and 54 negative patients collected in 2018 at OSR, were used for internal-external and external validation. Results: We developed five ML models: for the complete OSR dataset, the area under the receiver operating characteristic curve (AUC) for the algorithms ranged from 0.83 to 0.90; for the COVID-specific dataset from 0.83 to 0.87; and for the CBC dataset from 0.74 to 0.86. The validations also achieved good results: respectively, AUC from 0.75 to 0.78; and specificity from 0.92 to 0.96. Conclusions: ML can be applied to blood tests as both an adjunct and alternative method to rRT-PCR for the fast and cost-effective identification of COVID-19-positive patients. This is especially useful in developing countries, or in countries facing an increase in contagions.

Correction to: Interplay between low plasma RANKL and VDR-FokI polymorphism in lumbar disc herniation independently from age, body mass, and environmental factors: a case-control study in the Italian population.

Under the headline "Correlation of RANKL concentrations and VDR-FokI polymorphism on disc herniation" in the description text for Table 2, the term "allelic frequency" was used erroneously for "genotypic frequency".

Secreted Factors and EV-miRNAs Orchestrate the Healing Capacity of Adipose Mesenchymal Stem Cells for the Treatment of Knee Osteoarthritis.

Mesenchymal stem cells (MSCs) derived from adipose tissue and used either as expanded cells or minimally manipulated cell preparations showed positive clinical outcomes in regenerative medicine approaches based on tissue restoration and inflammation control, like in osteoarthritis (OA). Recently, MSCs' healing capacity has been ascribed to the large array of soluble factors, including soluble cytokines/chemokines and miRNAs conveyed within extracellular vesicles (EVs). Therefore, in this study, 200 secreted cytokines, chemokines and growth factors via ELISA, together with EV-embedded miRNAs via high-throughput techniques, were scored in adipose-derived MSCs (ASCs) cultivated under inflammatory conditions, mimicking OA synovial fluid. Both factors (through most abundantly expressed TIMP1, TIMP2, PLG and CTSS) and miRNAs (miR-24-3p, miR-222-3p and miR-193b-3p) suggested a strong capacity for ASCs to reduce matrix degradation activities, as those activated in OA cartilage, and switch synovial macrophages, often characterized by an M1 inflammatory polarization, towards an M2 phenotype. Moreover, the crucial importance of selecting the target tissue is discussed, showing how a focused search may greatly improve potency prediction and explain clinical outcomes. In conclusion, herein presented data shed light about the way ASCs regulate cell homeostasis and regenerative pathways in an OA-resembling environment, therefore suggesting a rationale for the use of MSC-enriched clinical products, such as stromal vascular fraction and microfragmented adipose tissue, in joint pathologies.

Mesenchymal stem cells in the treatment of articular cartilage degeneration: New biological insights for an old-timer cell.

Osteoarthritis (OA) is a debilitating, degenerative joint disease characterized by progressive destruction of articular cartilage. Given the poor repair capacity of articular cartilage and the associated local destructive immune/inflammatory responses involving all joint structures, OA frequently ends up as a "whole joint failure" requiring prosthetic replacement. Current pharmacological efforts, belatedly started, mainly aim at symptomatic pain relief, underscoring the need for novel therapeutic schemes designed to modify the course of the disease. Mesenchymal stem cell (MSC)-based therapy has gained significant interest, sparking the design of multiple trials proving safety while providing promising preliminary efficacy results. MSCs possess 'medicinal signaling cell' properties related to their immunomodulatory and anti-inflammatory effects, which induce the establishment of a pro-regenerative microenvironment at the injured tissue. Those trophic effects are paralleled by the long-established chondroprogenitor capacity that can be harnessed to ex vivo fabricate engineered constructs to repair damaged articular cartilage. The present review focuses on these two aspects of the use of MSCs for articular cartilage damage, namely, cell therapy and tissue engineering, providing information on their use criteria, advancements, challenges and strategies to overcome them.

Injective mesenchymal stem cell-based treatments for knee osteoarthritis: from mechanisms of action to current clinical evidences.

PURPOSE: Osteoarthritis (OA) represents a relevant social and economic burden worldwide. "Mesenchymal stem cells" or, as recently proposed, "medicinal signaling cells" (MSCs) have been recently introduced as injective treatments for OA with the aim of restoring joint homeostasis. The aim of this review is to provide the reader with the tools necessary to interpret the currently available clinical data, focusing on the MSC mechanisms of action which might help to clarify what we should expect from this treatment. METHODS: Clinical studies reporting MSC injections for the treatment of knee OA, either freshly isolated or culture-expanded cells, have been included and commented in relation to the supposed therapeutic effect that MSCs might exert giving their supposed mode of actions. RESULTS: The majority of the studies reports significant improvements in terms of pain and knee function compared to baseline values, up to 24 months of follow-up. Although these data support the expected therapeutic effect of this therapy giving the features of these cells, only 14% of the studies present a control group and more than one-third of them report the results on less than ten patients. CONCLUSIONS: Despite the constant presence of positive and satisfactory results in the studies analyzed, the complexity of MSC metabolism and related therapeutic effects as well as the weakness of most of the studies do not allow withdrawing definitive conclusions about the superiority of one tissue source over another, as well as about the best cell dose and the long-term durability of the effects of these procedures. Given the high potential value of these therapies in the treatment of OA, further studies accurately designed, carefully defining the type of patients to be included and pursuing minimal standard requirements in terms of follow-up, number of patients, and types of measurements should be conducted to finally assess the efficacy of MSC-based injective treatments.

Identification of miRNA Reference Genes in Extracellular Vesicles from Adipose Derived Mesenchymal Stem Cells for Studying Osteoarthritis.

Osteoarthritis (OA) leads to chronic pain and disability, and traditional conservative treatments are not effective in the long term. The intra-articular injection of mesenchymal stem cells (MSCs) is considered a novel therapy for OA whose efficacy mainly relies on the adaptive release of paracrine molecules which are either soluble or extracellular vesicles (EVs) embedded. The correct quantification of EV-miRNAs using reliable reference genes (RGs) is a crucial step in optimizing this future therapeutic cell-free approach. The purpose of this study is to rate the stabilities of literature-selected proposed RGs for EV-miRNAs in adipose derived-MSCs (ASCs). EVs were isolated by ultracentrifugation from ASCs cultured with or without inflammatory priming mimicking OA synovial fluid condition. Expression of putative RGs (let-7a-5p, miR-16-5p, miR-23a-3p, miR-26a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p, U6 snRNA) was scored by using the algorithms geNorm, NormFinder, BestKeeper and ΔCt method. miR-16a-5p/miR-23a-3p yielded the most stable RGs, whereas let-7a-5p/miR-425-5p performed poorly. Outcomes were validated by qRT-PCR on miR-146a-5p, reported to be ASC-EVs enriched and involved in OA. Incorrect RG selection affected the evaluation of miR-146a-5p abundance and modulation by inflammation, with both values resulting strongly donor-dependent. Our findings demonstrated that an integrated approach of multiple algorithms is necessary to identify reliable, stable RGs for ASC-EVs miRNAs evaluation. A correct approach would increase the accuracy of embedded molecule assessments aimed to develop therapeutic strategies for the treatment of OA based on EVs.

In Vitro Induction of Tendon-Specific Markers in Tendon Cells, Adipose- and Bone Marrow-Derived Stem Cells is Dependent on TGFß3, BMP-12 and Ascorbic Acid Stimulation.

Mesenchymal Stem Cells (MSCs) and tissue-specific progenitors have been proposed as useful tools for regenerative medicine approaches in bone, cartilage and tendon-related pathologies. The differentiation of cells towards the desired, target tissue-specific lineage has demonstrated advantages in the application of cell therapies and tissue engineering. Unlike osteogenic and chondrogenic differentiation, there is no consensus on the best tenogenic induction protocol. Many growth factors have been proposed for this purpose, including BMP-12, b-FGF, TGF-ß3, CTGF, IGF-1 and ascorbic acid (AA). In this study, different combinations of these growth factors have been tested in the context of a two-step differentiation protocol, in order to define their contribution to the induction and maintenance of tendon marker expression in adipose tissue and bone marrow derived MSCs and tendon cells (TCs), respectively. Our results demonstrate that TGF-ß3 is the main inducer of scleraxis, an early expressed tendon marker, while at the same time inhibiting tendon markers normally expressed later, such as decorin. In contrast, we find that decorin is induced by BMP-12, b-FGF and AA. Our results provide new insights into the effect of different factors on the tenogenic induction of MSCs and TCs, highlighting the importance of differential timing in TGF-ß3 stimulation.

Vitamin D's Effect on the Proliferation and Inflammation of Human Intervertebral Disc Cells in Relation to the Functional Vitamin D Receptor Gene FokI Polymorphism.

Vitamin D is known to have immunomodulatory effects, is involved in osteo-cartilaginous metabolism, and may have a role in human intervertebral disc pathophysiology. Although a link between vitamin D receptor (VDR) gene variants and disc degeneration-related pathologies has been observed, its functional contribution to pathologic processes has not been assessed yet. The aim of this study was to investigate the response of disc cells to vitamin D in terms of the regulation of proliferation, metabolism, and inflammatory processes, with a particular focus on the FokI VDR genotype. However, although it was found that vitamin D had a pro-apoptotic effect regardless of genotype, an up-regulation of IL-1Ra and downregulation of IL-6 was found to be evident only in Ff cells. Regarding the metabolic effects, in Ff cells, vitamin D promoted an upregulation of the aggrecan in inflammatory conditions but did not have an effect on the expression of collagen-related markers. Moreover, cells bearing the Ff genotype were the most responsive to vitamin D in the upregulation of catabolic markers. In addition, in contrast to the FF genotype, vitamin D downregulated the vitamin D-dependent signaling pathway in inflamed Ff cells, counteracting the inflammation-mediated catabolic effects. In conclusion, Ff cells were found to be more responsive to the anti-inflammatory and catabolic effects of vitamin D, which is likely to be related to matrix remodeling.