Adipose-derived mesenchymal stem cells cultured in tenogenic serum-free medium express tendon-specific markers

Tendon injuries are common and present a clinical challenge, as they often respond poorly to treatment and result in long-term functional impairment. Poor tendon healing responses are mainly attributed to insufficient or failed tenogenesis. For optimal treatment, enhanced understanding of tendon physiology is necessary. Among others, growth factors (GFs) and cytokines modulate the differentiation of tendons during embryogenesis and the healing process of injured tendons. Cell-based therapy using mesenchymal stem cells (MSCs) in combination with GFs and biomaterials seems to be the most promising approach to heal tendon injuries. Adipose- derived MSCs (ASCs) are multipotent and immunoprivileged, making them ideal candidates for therapeutic purposes. Moreover, providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices (GMP), and GMP guidelines should be adhered to throughout the process of isolating, expanding and differentiating MSCs. For these reasons, the aims of this study were: i) to investigate the effect of several GFs already known to be involved in tendon development/healing process on human ASCs proliferation and expression of tendon-related markers; ii) to develop a tenogenic GMP-compliant serum free medium. Subcutaneous fat was obtained from 5 healthy donors by lipoaspiration, after written consent. Primary cultures of the stromal vascular fraction were established and characterized by flow cytometry analysis to evaluate cell viability (7AAD(-) and SYTO 40(+) expression), and ASC surface marker expression (CD45(-), CD146(-) and CD34(+)) and then cryopreserved. After thawing, ASCs were expanded until P3 culturing in a commercial human platelet lysate- supplemented culture medium (hPL) or in a well-defined serum free medium (SF) developed in our laboratories. At P4, tenogenic induction was performed: ASCs were cultured in 6-well plates coated with the tendon matrix protein type-I collagen and in tenogenic medium (TENO) consisting in hPL or SF medium supplemented with 100ng/ml CTGF, 10ng/ml TGFβ3, 50ng/ml BMP12 and 50μg/ml Ascorbic acid (AA) for 1, 3, 7 and 14 days. Cells cultured without any supplementations at the same time points were used as control (CTRL). Morphological appearance (optical microscopy), cell proliferation (lactate assay), gene (RT-PCR) and protein (immunofluorescence, SIRIUS-RED staining) expression were performed in all groups at all time points. Both SF-TENO and hPL-TENO cells appeared more rounded and with more cytoplasmic content and proliferated faster than respective CTRL. Tendon-marker genes (SCX, COL1A1, COL3A1, TNC, MMP3, MMP13) were significantly upregulated already after 3 to 14 days of differentiation in respect to CTRL without any significant differences between hPL and SF groups. In the meantime, stem cell gene (KLF4, NANOG, OCT4) expression decreased in TENO cells vs CTRL. SCX protein expression and the increase of collagen-matrix deposition were also observed in all TENO cells vs CTRL. These results demonstrate that ASCs possess tenogenic differentiation ability when exposed to CTGF, BMP12, TGFb3 and AA in both hPL and SF medium providing insights of the earliest events of tendon development and move forward the GMP-compliant approaches needed for cell-therapy strategies

3D Bioprinting of Human Adipose-Derived Stem Cells and Their Tenogenic Differentiation in Clinical-Grade Medium

Defining the best combination of cells and biomaterials is a key challenge for the development of tendon tissue engineering (TE) strategies. Adipose-derived stem cells (ASCs) are ideal candidates for this purpose. In addition, controlled cell-based products adherent to good manufacturing practice (GMP) are required for their clinical scale-up. With this aim, in this study, ASC 3D bioprinting and GMP-compliant tenogenic differentiation were investigated. In detail, primary human ASCs were embedded within a nanofibrillar-cellulose/alginate bioink and 3D-bioprinted into multi-layered square-grid matrices. Bioink viscoelastic properties and scaffold ultrastructural morphology were analyzed by rheology and scanning electron microscopy (SEM). The optimal cell concentration for printing among 3, 6 and 9 × 106 ASC/mL was evaluated in terms of cell viability. ASC morphology was characterized by SEM and F-actin immunostaining. Tenogenic differentiation ability was then evaluated in terms of cell viability, morphology and expression of scleraxis and collagen type III by biochemical induction using BMP-12, TGF-β3, CTGF and ascorbic acid supplementation (TENO). Pro-inflammatory cytokine release was also assessed. Bioprinted ASCs showed high viability and survival and exhibited a tenocyte-like phenotype after biochemical induction, with no inflammatory response to the bioink. In conclusion, we report a first proof of concept for the clinical scale-up of ASC 3D bioprinting for tendon TE

Clinical features and outcomes of elderly hospitalised patients with chronic obstructive pulmonary disease, heart failure or both

Background and objective: Chronic obstructive pulmonary disease (COPD) and heart failure (HF) mutually increase the risk of being present in the same patient, especially if older. Whether or not this coexistence may be associated with a worse prognosis is debated. Therefore, employing data derived from the REPOSI register, we evaluated the clinical features and outcomes in a population of elderly patients admitted to internal medicine wards and having COPD, HF or COPD + HF. Methods: We measured socio-demographic and anthropometric characteristics, severity and prevalence of comorbidities, clinical and laboratory features during hospitalization, mood disorders, functional independence, drug prescriptions and discharge destination. The primary study outcome was the risk of death. Results: We considered 2,343 elderly hospitalized patients (median age 81 years), of whom 1,154 (49%) had COPD, 813 (35%) HF, and 376 (16%) COPD + HF. Patients with COPD + HF had different characteristics than those with COPD or HF, such as a higher prevalence of previous hospitalizations, comorbidities (especially chronic kidney disease), higher respiratory rate at admission and number of prescribed drugs. Patients with COPD + HF (hazard ratio HR 1.74, 95% confidence intervals CI 1.16-2.61) and patients with dementia (HR 1.75, 95% CI 1.06-2.90) had a higher risk of death at one year. The Kaplan-Meier curves showed a higher mortality risk in the group of patients with COPD + HF for all causes (p = 0.010), respiratory causes (p = 0.006), cardiovascular causes (p = 0.046) and respiratory plus cardiovascular causes (p = 0.009). Conclusion: In this real-life cohort of hospitalized elderly patients, the coexistence of COPD and HF significantly worsened prognosis at one year. This finding may help to better define the care needs of this population

The “Diabetes Comorbidome”: A Different Way for Health Professionals to Approach the Comorbidity Burden of Diabetes

(1) Background: The disease burden related to diabetes is increasing greatly, particularly in older subjects. A more comprehensive approach towards the assessment and management of diabetes’ comorbidities is necessary. The aim of this study was to implement our previous data identifying and representing the prevalence of the comorbidities, their association with mortality, and the strength of their relationship in hospitalized elderly patients with diabetes, developing, at the same time, a new graphic representation model of the comorbidome called “Diabetes Comorbidome”. (2) Methods: Data were collected from the RePoSi register. Comorbidities, socio-demographic data, severity and comorbidity indexes (Cumulative Illness rating Scale CIRS-SI and CIRS-CI), and functional status (Barthel Index), were recorded. Mortality rates were assessed in hospital and 3 and 12 months after discharge. (3) Results: Of the 4714 hospitalized elderly patients, 1378 had diabetes. The comorbidities distribution showed that arterial hypertension (57.1%), ischemic heart disease (31.4%), chronic renal failure (28.8%), atrial fibrillation (25.6%), and COPD (22.7%), were the more frequent in subjects with diabetes. The graphic comorbidome showed that the strongest predictors of death at in hospital and at the 3-month follow-up were dementia and cancer. At the 1-year follow-up, cancer was the first comorbidity independently associated with mortality. (4) Conclusions: The “Diabetes Comorbidome” represents the perfect instrument for determining the prevalence of comorbidities and the strength of their relationship with risk of death, as well as the need for an effective treatment for improving clinical outcomes

Clinical features and outcomes of elderly hospitalised patients with chronic obstructive pulmonary disease, heart failure or both

Background and objective: Chronic obstructive pulmonary disease (COPD) and heart failure (HF) mutually increase the risk of being present in the same patient, especially if older. Whether or not this coexistence may be associated with a worse prognosis is debated. Therefore, employing data derived from the REPOSI register, we evaluated the clinical features and outcomes in a population of elderly patients admitted to internal medicine wards and having COPD, HF or COPD + HF. Methods: We measured socio-demographic and anthropometric characteristics, severity and prevalence of comorbidities, clinical and laboratory features during hospitalization, mood disorders, functional independence, drug prescriptions and discharge destination. The primary study outcome was the risk of death. Results: We considered 2,343 elderly hospitalized patients (median age 81 years), of whom 1,154 (49%) had COPD, 813 (35%) HF, and 376 (16%) COPD + HF. Patients with COPD + HF had different characteristics than those with COPD or HF, such as a higher prevalence of previous hospitalizations, comorbidities (especially chronic kidney disease), higher respiratory rate at admission and number of prescribed drugs. Patients with COPD + HF (hazard ratio HR 1.74, 95% confidence intervals CI 1.16-2.61) and patients with dementia (HR 1.75, 95% CI 1.06-2.90) had a higher risk of death at one year. The Kaplan-Meier curves showed a higher mortality risk in the group of patients with COPD + HF for all causes (p = 0.010), respiratory causes (p = 0.006), cardiovascular causes (p = 0.046) and respiratory plus cardiovascular causes (p = 0.009). Conclusion: In this real-life cohort of hospitalized elderly patients, the coexistence of COPD and HF significantly worsened prognosis at one year. This finding may help to better define the care needs of this population