Eliane Antonioli

High OCT4 and Low p16 INK4A Expressions Determine In Vitro Lifespan of Mesenchymal Stem Cells

After long-term culture, mesenchymal stem cells alter their biological properties and enter into a state of replicative senescence. Although several classical biomarkers have been used for quantitative assessment of cellular senescence, no hallmark has been proven completely unique to the senescent state in cells. We used bone marrow-derived MSCs (BM-MSCs) from different healthy young donors and an in vitro model with well-defined senescence end points to identify a set of robust markers that could potentially predict the expansion capacity of MSCs preparations before reaching senescence. For each early passage BM-MSC sample (5th or 6th passages), the normalized protein expression levels of senescence-associated markers p16INK4A, p21WAF1, SOD2, and rpS6S240/244; the concentration of IL6 and IL8 in cell culture supernatants; and the normalized gene expression levels of pluripotency markers OCT4, NANOG, and SOX2 were correlated with final population doubling (PD) number. We revealed that the low expression of p16INK4A protein and a high OCT4 gene expression, rather than other evaluated markers, might be potential hallmarks and predictors of greater in vitro lifespan and growth potential, factors that can impact the successful therapeutic use of MSCs preparations.

An evaluation of chondrocyte morphology and gene expression on superhydrophilic vertically-aligned multi-walled carbon nanotube films

Cartilage serves as a low-friction and wear-resistant articulating surface in diarthrodial joints and is also important during early stages of bone remodeling. Recently, regenerative cartilage research has focused on combinations of cells paired with scaffolds. Superhydrophilic vertically aligned carbon nanotubes (VACNTs) are of particular interest in regenerative medicine. The aim of this study is to evaluate cell expansion of human articular chondrocytes on superhydrophilic VACNTs, as well as their morphology and gene expression. VACNT films were produced using a microwave plasma chamber on Ti substrates and submitted to an O2 plasma treatment to make them superhydrophilic. Human chondrocytes were cultivated on superhydrophilic VACNTs up to five days. Quantitative RT-PCR was performed to measure type I and type II Collagen, Sox9, and Aggrecan mRNA expression levels. The morphology was analyzed by scanning electron microscopy (SEM) and confocal microscopy. SEM images demonstrated that superhydrophilic VACNTs permit cell growth and adhesion of human chondrocytes. The chondrocytes had an elongated morphology with some prolongations. Chondrocytes cultivated on superhydrophilic VACNTs maintain the level expression of Aggrecan, Sox9, and Collagen II determined by qPCR. This study was the first to indicate that superhydrophilic VACNTs may be used as an efficient scaffold for cartilage or bone repair.

Update on biological therapies for knee injuries: osteoarthritis

Osteoarthritis (OA) is a progressive disease that causes functional impairment of the joints. Chronic forms of knee OA have been increasing worldwide, limiting patients’ quality of life. Recent studies have sought to increase effectiveness and quality in the development of new therapies, such as platelet-rich plasma, hyaluronic acid, stem cells, and nuclear factor κB, aimed principally at reducing proinflammatory activity, pain, and degeneration of the knee joints. The goal of this review is to present an update on biological therapies for knee OA and to provide guidance for future OA studies.

Effect of Human Adipose Tissue Mesenchymal Stem Cells on the Regeneration of Ovine Articular Cartilage

Cell therapy is a promising approach to improve cartilage healing. Adipose tissue is an abundant and readily accessible cell source. Previous studies have demonstrated good cartilage repair results with adipose tissue mesenchymal stem cells in small animal experiments. This study aimed to examine these cells in a large animal model. Thirty knees of adult sheep were randomly allocated to three treatment groups: CELLS (scaffold seeded with human adipose tissue mesenchymal stem cells), SCAFFOLD (scaffold without cells), or EMPTY (untreated lesions). A partial thickness defect was created in the medial femoral condyle. After six months, the knees were examined according to an adaptation of the International Cartilage Repair Society (ICRS 1) score, in addition to a new Partial Thickness Model scale and the ICRS macroscopic score. All of the animals completed the follow-up period. The CELLS group presented with the highest ICRS 1 score (8.3 ± 3.1), followed by the SCAFFOLD group (5.6 ± 2.2) and the EMPTY group (5.2 ± 2.4) (p = 0.033). Other scores were not significantly different. These results suggest that human adipose tissue mesenchymal stem cells promoted satisfactory cartilage repair in the ovine model.

Analysis of cytokine profile and growth factors in platelet-rich plasma obtained by open systems and commercial columns

ABSTRACT Objective: To evaluate growth factors and cytokines in samples of platelet-rich plasma obtained by three different centrifugation methods. Methods: Peripheral blood of six individuals with no hematological diseases, aged 18 to 68 years, was drawn to obtain platelet-rich plasma, using the open method and commercial columns by Medtronic and Biomet. The products obtained with the different types of centrifugation were submitted to laboratory analysis, including pro-inflammatory cytokines and chemokines by flow cytometry assays, the concentration of fibroblast growth factors-2 (FGF-2) and transforming growth factor-beta1 (TGF-β1). Results: The diverse separation methods generated systematically different profiles regarding number of platelets and leukocytes. The Medtronic system yielded a product with the highest concentration of platelets, and the open method, with the lowest concentration of platelets. The results of cytokine analysis showed that the different types of centrifugation yielded products with high concentrations of interleukin 8, interleukin 1β. The open system resulted in a product with high levels of interleukin 6. Other cytokines and chemokines measured were similar between systems. The product obtained with the open method showed higher levels of TGF-β1 in relation to other systems and low FGF-2 levels. Conclusion: The formed elements, growth factors and cytokines in samples of platelet-rich plasma varied according to the centrifugation technique used.

Modulation of Hyaluronan Synthesis by the Interaction between Mesenchymal Stem Cells and Osteoarthritic Chondrocytes

Bone marrow mesenchymal stem cells (BM-MSCs) are considered a good source for cellular therapy in cartilage repair. But, their potential to repair the extracellular matrix, in an osteoarthritic environment, is still controversial. In osteoarthritis (OA), anti-inflammatory action and extracellular matrix production are important steps for cartilage healing. This study examined the interaction of BM-MSC and OA-chondrocyte on the production of hyaluronan and inflammatory cytokines in a Transwell system. We compared cocultured BM-MSCs and OA-chondrocytes with the individually cultured controls (monocultures). There was a decrease in BM-MSCs cell count in coculture with OA-chondrocytes when compared to BM-MSCs alone. In monoculture, BM-MSCs produced higher amounts of hyaluronan than OA-chondrocytes and coculture of BM-MSCs with OA-chondrocytes increased hyaluronan production per cell. Hyaluronan synthase-1 mRNA expression was upregulated in BM-MSCs after coculture with OA-chondrocytes, whereas hyaluronidase-1 was downregulated. After coculture, lower IL-6 levels were detected in BM-MSCs compared with OA-chondrocytes. These results indicate that, in response to coculture with OA-chondrocytes, BM-MSCs change their behavior by increasing production of hyaluronan and decreasing inflammatory cytokines. Our results indicate that BM-MSCs per se could be a potential tool for OA regenerative therapy, exerting short-term effects on the local microenvironment even when cell:cell contact is not occurring.

Analysis of epidemiology, lifestyle, and psychosocial factors in patients with back pain admitted to an orthopedic emergency unit

ABSTRACT Objective: To correlate epidemiological data, lifestyle, and psychosocial factors as predictors for clinical manifestation of back pain in patients treated at the orthopedic emergency unit of a Brazilian tertiary care hospital, and to evaluate their interest in participating in a hypothetical program for physical rehabilitation. Methods: This is an observational cross-sectional study. We evaluated 210 patients from the emergency department of a tertiary hospital with a major complaint of back pain. We used: epidemiological multiple-choice questionnaires developed for this study; Oswestry questionnaire for physical disability; Hospital Anxiety and Depression Scale (HAD) scale. Data analyses were performed using SAS - Statistical Analysis System (SAS Institute, 2001). Measurements were performed with the SAS functions Proc MEANS and Proc Freq. Results: The mean age was 39.1 years and there was no predominance between genders. The usual work activity was administrative (65.2% of cases). The mean body mass index was 26.0, indicating overweight. The majority (83.3%) of patients had low physical disability (Oswestry 0 – 40%). The number of medical visits in the previous 6 months (p=0.04) and the scores of anxiety and depression (p=0.05), independently, were correlated with physical disability. Most patients (77%) would agree to participate in a hypothetical program of physical rehabilitation for prevention of back pain. Conclusion: Patients with back pain complaints were predominantly young adults, sedentary or hypoactive, overweight, and with recurrent complaints of symptoms. Most participants had low levels of physical disability and would accept participation in a hypothetical physical rehabilitation program for the prevention of back pain.

Individual response to mTOR inhibition in delaying replicative senescence of mesenchymal stromal cells

Background aims Delaying replicative senescence and extending lifespan of human mesenchymal stromal cells (MSCs) may enhance their potential for tissue engineering and cell based therapies. Accumulating evidence suggests that inhibitors of the mTOR signaling pathway, such as rapamycin, constitute promising pharmacological agents to retard senescence and extend stemness properties of various progenitor cell types. Here, we investigated whether the ability of rapamycin to postpone replicative senescence varies among bone marrow MSC samples (BM-MSCs) derived from different healthy donors, and explored the molecular mechanisms that drive rapamycin-mediated lifespan increment. Methods BM-MSCs at early passages were serially passaged either in absence or continuous presence of rapamycin and the number of cell population doublings until growth arrest was measured. The inhibition of mTOR signaling was assessed by the phosphorylation status of the downstream target RPS6. The expression levels of several senescence and pluripotency markers at early and late/senescent passages were analyzed by RT-qPCR, flow cytometry and western blot. Results We found that the lifespan extension in response to the continuous rapamycin treatment was highly variable among samples, but effective in most BM-MSCs. Despite all rapamycin-treated cells secreted significantly reduced levels of IL6, a major SASP cytokine, and expressed significantly higher levels of the pluripotency marker NANOG, the expression patterns of these markers were not correlated with the rapamycin-mediated increase in lifespan. Interestingly, rapamycin-mediated life-span extension was significantly associated only with repression of p16INK4A protein accumulation. Conclusions Taken together, our results suggest that some, but not all, BM-MSC samples would benefit from using rapamycin to postpone replicative arrest and reinforce a critical role of p16INK4A protein downregulation in this process.

Analysis of 500 anterior cruciate ligament reconstructions from a private institutional register

Purpose The aims of this study are to describe the epidemiological characteristics of anterior cruciate ligament reconstructions in a private hospital in Brazil and to determine trends in medical practice for comparison with previous studies. Methods We retrospectively reviewed the anterior cruciate ligament institutional register to obtain data from all patients who underwent primary anterior cruciate ligament reconstruction from July 2014 to June 2016. Descriptive statistics were used to summarize the sample. Specific statistical tests were used to assess associations between the meniscal lesion and other variables. Results During the study period, 72.6% out of 500 patients were male. The mean age at surgery was 35.1 years. The mean age was higher among females than among males (37.3 ± 12.1 vs 34.3 ± 10.8 years). The median time from injury to surgery was 44 days. The most common femoral and tibial fixations used were suspensory fixation (60.8%) and interference screw (96%), respectively. The most commonly used graft was hamstring tendon (70.2%), followed by bone-patellar tendon-bone (28.8%). A meniscal lesion was noted in 44.8% of cases. Partial meniscectomy was performed in 69.5% of meniscal lesions, and meniscal repair was performed in 14.1% of lesions. The mean length of hospital stay was 1.4 days. The proportion of men in the group of patients with an associated meniscal lesion was higher than that in the group of patients without a meniscal lesion (p = 0.007). Conclusions In this study, we identified that the vast majority of surgeries were performed in male patients in all age groups, and patients older than 30 years and with a short time from injury to surgery predominated. Concerning surgical technique, we noted a low rate of meniscal repair and a higher preference for the use of hamstring graft and suspensory fixation on the femoral side.