Timely Supplementation of Hydrogels Containing Sulfated or Unsulfated Chondroitin and Hyaluronic Acid Affects Mesenchymal Stromal Cells Commitment Toward Chondrogenic Differentiation

Mesenchymal stromal cells (MSCs) are currently used for cartilage cell therapy because of their well proven capacity to differentiate in chondrocytes. The advantage of MSC-based therapy is the possibility of producing a high number of chondrocytes for implants. The transplant procedure, however, has some limitations, since MSCs may produce non-functional chondrocytes. This limit has been challenged by cultivating MSC in media with hydrogels containing hyaluronic acid (HA), extractive chondroitin sulfate (CS), or bio-fermentative unsulphated chondroitin (BC) alone or in combination. Nevertheless, a clear study of the effect of glycosaminoglycans (GAGs) on chondrocyte differentiation is still lacking, especially for the newly obtained unsulfated chondroitin of biotechnological origin. Are these GAGs playing a role in the commitment of stem cells to chondrocyte progenitors and in the differentiation of progenitors to mature chondrocytes? Alternatively, do they have a role only in one of these biological processes? We evaluated the role of HA, CS, and – above all – BC in cell commitment and chondrocyte differentiation of MSCs by supplementing these GAGs in different phases of in vitro cultivation. Our data provided evidence that a combination of HA and CS or of HA and BC supplemented during the terminal in vitro differentiation and not during cell commitment of MSCs improved chondrocytes differentiation without the presence of fibrosis (reduced expression of Type I collagen). This result suggests that a careful evaluation of extracellular cues for chondrocyte differentiation is fundamental to obtaining a proper maturation process

Hyaluronan and Derivatives: An In Vitro Multilevel Assessment of Their Potential in Viscosupplementation

In this research work, viscosupplements based on linear, derivatized, crosslinked and complexed HA forms were extensively examined, providing data on the hydrodynamic parameters for the water-soluble-HA-fraction, rheology, sensitivity to enzymatic hydrolysis and capacity to modulate specific biomarkers’ expression in human pathological chondrocytes and synoviocytes. Soluble HA ranged from 0 to 32 mg/mL and from 150 to 1330 kDa MW. The rheological behavior spanned from purely elastic to viscoelastic, suggesting the diversity of the categories that are suitable for restoring specific/different features of the healthy synovial fluid. The rheological parameters were reduced in a diverse manner upon dilution and hyaluronidases action, indicating different durations of the viscosupplementation effect. Bioactivity was found for all the samples, increasing the expression of different matrix markers (e.g., hyaluronan-synthase); however, the hybrid cooperative complexes performed better in most of the experiments. Hybrid cooperative complexes improved COLII mRNA expression (~12-fold increase vs. CTR), proved the most effective at preserving cell phenotype. In addition, in these models, the HA samples reduced inflammation. IL-6 was down-regulated vs. CTR by linear and chemically modified HA, and especially by hybrid complexes. The results represent the first comprehensive panel of data directly comparing the diverse HA forms for intra-articular injections and provide valuable information for tailoring products’ clinical use as well as for designing new, highly performing HA-formulations that can address specific needs

Potential of Biofermentative Unsulfated Chondroitin and Hyaluronic Acid in Dermal Repair

Chondroitin obtained through biotechnological processes (BC) shares similarities with both chondroitin sulfate (CS), due to the dimeric repetitive unit, and hyaluronic acid (HA), as it is unsulfated. In the framework of this experimental research, formulations containing BC with an average molecular size of about 35 KDa and high molecular weight HA (HHA) were characterized with respect to their rheological behavior, stability to enzymatic hydrolysis and they were evaluated in different skin damage models. The rheological characterization of the HHA/BC formulation revealed a G' of 92 ± 3 Pa and a G″ of 116 ± 5 Pa and supported an easy injectability even at a concentration of 40 mg/mL. HA/BC preserved the HHA fraction better than HHA alone. BTH was active on BC alone only at high concentration. Assays on scratched keratinocytes (HaCaT) monolayers showed that all the glycosaminoglycan formulations accelerated cell migration, with HA/BC fastening healing 2-fold compared to the control. In addition, in 2D HaCaT cultures, as well as in a 3D skin tissue model HHA/BC efficiently modulated mRNA and protein levels of different types of collagens and elastin remarking a functional tissue physiology. Finally, immortalized human fibroblasts were challenged with TNF-α to obtain an in vitro model of inflammation. Upon HHA/BC addition, secreted IL-6 level was lower and efficient ECM biosynthesis was re-established. Finally, co-cultures of HaCaT and melanocytes were established, showing the ability of HHA/BC to modulate melanin release, suggesting a possible effect of this specific formulation on the reduction of stretch marks. Overall, besides demonstrating the safety of BC, the present study highlights the potential beneficial effect of HHA/BC formulation in different damage dermal models

Unsulfated biotechnological chondroitin by itself as well as in combination with high molecular weight hyaluronan improves the inflammation profile in osteoarthritis in vitro model

Several studies suggest that inflammation has a pivotal role during the progression of osteoarthritis (OA) and cytokines have been identified as the main process mediators. This study aimed to explore the ability to modulate the main OA pro-inflammatory biomarkers of novel gels (H-HA/BC) based on high molecular weight hyaluronan (H-HA) and unsulfated biotechnological chondroitin (BC). For the first time, BC was tested also in combination with H-HA on human primary cells isolated from pathological knee joints. Specifically, the experiments were performed using an OA in vitro model based on human chondrocytes and synoviocytes. To evaluate the anti-inflammatory effects of H-HA/BC in comparison with H-HA and BC single gels, NF-kB, COMP-2, MyD88, MMP-13 and a wide range of cytokines, known to be specific biomarkers in OA (e.g., IL-6, IL-8, and TNF-α), were evaluated. In addition, cell morphology and proliferation occurring in the presence of either H-HA/BC or single components were assessed using time-lapse video microscopy. It was shown that synovial fluids and cells isolated from OA suffering patients, presented a cytokine pattern respondent to an ongoing inflammation status. H-HA and BC significantly reduced the levels of 23 biomarkers associated with cartilage damage. However, H-HA/BC decreased significantly 24 biological mediators and downregulated 19 of them more efficiently than the single components. In synoviocytes cultures, cytokine analyses proved that H-HA/BC gels re-established an extracellular environment more similar to a healthy condition reducing considerably the concentration of 11 analytes. Instead, H-HA and BC significantly modulated 7 (5 only with a longer treatment) and 8 biological cytokines, respectively. Our results suggest that H-HA/BC beyond the viscosupplementation effect typical for HA-based gels, can improve the inflammation status in joints and thus could be introduced as a valid protective and anti-inflammatory intraarticular device in the field of Class III medical devices for OA treatments

Acellular dermal matrix used in diabetic foot ulcers: Clinical outcomes supported by biochemical and histological analyses

Diabetic foot ulcer (DFU) is a diabetes complication which greatly impacts the patient’s quality of life, often leading to amputation of the affected limb unless there is a timely and adequate management of the patient. DFUs have a high economic impact for the national health system. Data have indeed shown that DFUs are a major cause of hospitalization for patients with diabetes. Based on that, DFUs represent a very important challenge for the national health system. Especially in developed countries diabetic patients are increasing at a very high rate and as expected, also the incidence of DFUs is increasing due to longevity of diabetic patients in the western population. Herein, the surgical approach focused on the targeted use of the acellular dermal matrix has been integrated with biochemical and morphological/histological analyses to obtain evidence-based information on the mechanisms underlying tissue regeneration. In this research report, the clinical results indicated decreased postoperative wound infection levels and a short healing time, with a sound regeneration of tissues. Here we demonstrate that the key biomarkers of wound healing process are activated at gene expression level and also synthesis of collagen I, collagen III and elastin is prompted and modulated within the 28-day period of observation. These analyses were run on five patients treated with Integra® sheet and five treated with the injectable matrix Integra® Flowable, for cavitary lesions. In fact, clinical evaluation of improved healing was, for the first time, supported by biochemical and histological analyses. For these reasons, the present work opens a new scenario in DFUs treatment and follow-up, laying the foundation for a tailored protocol towards complete healing in severe pathological conditions

Life course factors associated with metabolically healthy obesity: a protocol for the systematic review of longitudinal studies

Abstract Background There is heterogeneity among obese individuals, as some appear to have healthier metabolic profiles and decreased health risks. These individuals are defined as metabolically healthy obese (MHO), whilst those with unhealthy metabolic profiles are defined as metabolically unhealthy obese (MUO). To date, most research on MHO has been cross-sectional or focused on disease prognosis. However, longitudinal studies are required to provide greater insight into the life course factors that contribute to the development of MHO. This study aims to systematically review longitudinal studies investigating the association between life course exposures and future MHO status. Methods Electronic databases (MEDLINE, SCOPUS, and Web of Science) will be searched using a trialled search strategy. Studies will be included following a double-screening process according to inclusion criteria to assess eligibility. Studies eligible for inclusion will include those that have a longitudinal observational design where a life course exposure occurred or was measured at least 1 year before the outcome, investigate a human study population, are published in English after 1956, and investigate the association between ≥ 1 life course exposure and ≥ 1 outcome that reflects a measure of cardiometabolic resilience to obesity. Accepted life course exposures will include body size, body composition, pubertal development, smoking, diet, physical activity, sedentary behaviour, and psychosocial stress. The primary measure of cardiometabolic resilience to obesity will be MHO as an outcome (at follow-up). Studies investigating the development of cardiometabolic risk factors in an obese group without specifying MHO will also be accepted, such as the development of the metabolic syndrome (MetS) in an obese group. Key results of included studies will be tabulated, and a narrative synthesis will be conducted. Discussion This will be the first systematic review to summarise the literature on the life course correlates of MHO. Importantly, it may highlight which modifiable lifestyle factors could be targeted to delay the onset of cardiometabolic complications among the obese. Systematic review registration PROSPERO CRD4201705799

Hyaluronan-Based Gel Promotes Human Dental Pulp Stem Cells Bone Differentiation by Activating YAP/TAZ Pathway.

Background: Hyaluronans exist in different forms, accordingly with molecular weight and degree of crosslinking. Here, we tested the capability to induce osteogenic differentiation in hDPSCs (human dental pulp stem cells) of three hyaluronans forms: linear pharmaceutical-grade hyaluronans at high and (HHA) low molecular weight (LHA) and hybrid cooperative complexes (HCC), containing both sizes. Methods: hDPSCs were treated with HHA, LHA, HCC for 7, 14 and 21 days. The effects of hyaluronans on osteogenic differentiation were evaluated by qRT-PCR and WB of osteogenic markers and by Alizarin Red S staining. To identify the involved pathway, CD44 was analyzed by immunofluorescence, and YAP/TAZ expression was measured by qRT-PCR. Moreover, YAP/TAZ inhibitor-1 was used, and the loss of function of YAP/TAZ was evaluated by qRT-PCR, WB and immunofluorescence. Results: We showed that all hyaluronans improves osteogenesis. Among these, HCC is the main inducer of osteogenesis, along with overexpression of bone related markers and upregulating CD44. We also found that this biological process is subordinate to the activation of YAP/TAZ pathway. Conclusions: We found that HA's molecular weight can have a relevant impact on HA performance for bone regeneration, and we unveil a new molecular mechanism by which HA acts on stem cells. Keywords: YAP/TAZ pathway; dental pulp stem cells; hyaluronic acid; osteogenic differentiation

The new normal of global mobility - flexibility, diversity & data mastery

<p>Blue: Nuclei (Hoescth), Green: ELS, red: Cytoskeleton (Phalloidin).</p

In vitro model based on human keratinocyte to evaluate relaxin activity on wound healing exploiting time-lapse video microscopy

Wound repair is a well-ordered but complex process involving many cellular activities including inflammation, growth factor and cytokine production, and thus cell migration and proliferation. In vitro wound healing assays have been used and are well accepted, for the discovery and validation of biomolecules that may influence cell migration and improve repair (Liu et al. 2009). The aim of our research was to exploit time-lapse video-microscopy (Okolab, Italy) to observe and analyse scratched monolayers of keratinocyte cell line (HaCat), with specific interest in characterizing different relaxin preparations

Prevalence of Children with Severe Fetal Alcohol Spectrum Disorders in Communities Near Rome, Italy: New Estimated Rates Are Higher than Previous Estimates

Objective: To determine the population-based epidemiology of fetal alcohol syndrome (FAS) and other fetal alcohol spectrum disorders (FASD) in towns representative of the general population of central Italy. Methods: Slightly revised U.S. Institute of Medicine diagnostic methods were used among children in randomly-selected schools near Rome. Consented first grade children (n = 976) were screened in Tier I for height, weight, or head circumference and all childre