Fibroblast expression of CD248 may contribute to exacerbation of microvascular damage during systemic sclerosis.

OBJECTIVES: CD248 is a glycoprotein, highly expressed on pericytes and fibroblasts (FBs), that is implicated in the fibrotic process. During angiogenesis, CD248 can promote vessel regression, binding multimerin-2 (MMRN-2). Thus, we investigated the expression of MMRN-2 in systemic sclerosis (SSc)-skin and of CD248 in isolated SSc-FBs. The anti-angiogenic property of CD248+ SSc-FBs was evaluated by co-culturing these cells with healthy control endothelial cells (HC-ECs). The apoptotic effect of CD248 on HC-ECs was evaluated. Finally, the ability of CD248 to prevent activation of VEGF receptor 2 (VEGFR2) was assessed. METHODS: By IF, MMRN-2 was investigated in SSc-skin and CD248 in SSc FBs. The anti-angiogenic property of CD248+ SSc-FBs was evaluated by HC-ECs/SSc-FBs co-cultures. Lentiviral-induced CD248 short-hairpin RNA delivery was employed for loss-of-function studies in SSc-FBs. HC-ECs were cultured in the presence of CD248 to assess apoptosis by IF and VEGFR2 phosphorylation by western blot. RESULTS: MMRN-2 expression was increased in skin SSc-ECs, whereas CD248 expression was increased in SSc-FBs. Functionally, CD248+-SSc-FBs suppressed angiogenesis in the organotypic model, as assessed by the reduction in total tube length of HC-ECs. This anti-angiogenetic behaviour was reversed by CD248 silencing. Furthermore, the presence of CD248 promoted the apoptosis of HC-ECs. Finally, CD248 prevented activation of VEGFR2 by reducing its phosphorylation after VEGF stimulation. CONCLUSION: CD248 was anti-angiogenic in vitro due to a reduction in tube formation and to induction of apoptosis of ECs. Increased expression of CD248 in SSc could contribute to the microvascular rarefaction observed at the tissue level in SSc. Our results suggest a pathogenic role for CD248-MMRN-2 in SSc.

Characterization of age-related changes of tendon stem cells from adult human tendons.

PURPOSE: The present study evaluated the presence of stem cells in hamstring tendons from adult subjects of different ages. The aim was to isolate, characterize and expand these cells in vitro, and to evaluate whether cell activities are influenced by age. METHODS: Tendon stem cells (TSCs) were isolated through magnetic sorting from the hamstring tendons of six patients. TSC percentage, morphology and clonogenic potential were evaluated, as well as the expression of specific surface markers. TSC multi-potency was also investigated as a function of age, and quantitative polimerase chain reaction was used to evaluate gene expression of TSCs cultured in suitable differentiating media. RESULTS: The presence of easily harvestable stem cell population within adult human hamstring tendons was demonstrated. These cells exhibit features such as clonogenicity, multi-potency and mesenchymal stem cells markers expression. The age-related variations in human TSCs affect the number of isolated cells and their self-renewal potential, while multi-potency assays are not influenced by tendon ageing, even though cells from younger individuals expressed higher levels of osteogenic and adipogenic genes, while chondrogenic genes were highly expressed in cells from older individuals. CONCLUSIONS: These results may open new opportunities to study TSCs to better understand tendon physiology, healing and pathological processes such as tendinopathy and degenerative age-related changes opening new frontiers in the management of tendinopathy and tendon ruptures.

On the compatibility of single-cell microcarriers (nanovials) with microfluidic impedance cytometry.

We investigate for the first time the compatibility of nanovials with microfluidic impedance cytometry (MIC). Nanovials are suspendable crescent-shaped single-cell microcarriers that enable specific cell adhesion, the creation of compartments for undisturbed cell growth and secretion, as well as protection against wall shear stress. MIC is a label-free single-cell technique that characterizes flowing cells based on their electrical fingerprints and it is especially targeted to cells that are naturally in suspension. Combining nanovial technology with MIC is intriguing as it would represent a robust framework for the electrical analysis of single adherent cells at high throughput. Here, as a proof-of-concept, we report the MIC analysis of mesenchymal stromal cells loaded in nanovials. The electrical analysis is supported by numerical simulations and validated by means of optical analysis. We demonstrate that the electrical diameter can discriminate among free cells, empty nanovials, cell-loaded nanovials, and clusters, thus grounding the foundation for the use of nanovials in MIC. Furthermore, we investigate the potentiality of MIC to assess the electrical phenotype of cells loaded in nanovials and we draw directions for future studies.

Modeling and Validation of an Ultra-Compact Regenerative Liver Dialysis Device.

The availability of a wearable artificial liver that facilitates extracorporeal dialysis outside of medical facilities would represent a significant advancement for patients requiring dialysis. The objective of this preliminary investigation is to explore, using validated mathematical models based on in vitro data, the feasibility of developing a novel, cost-effective, and highly compact extracorporeal liver support device that can be employed as a transitional therapy to transplantation outside of clinical settings. Such an innovation would offer substantial cost savings to the national healthcare system while significantly improving the patient's quality of life. The experimental components consisted of replacing traditional adsorbent materials with albumin-functionalized silica microspheres due to their capacity to adsorb bilirubin, one of the toxins responsible for liver failure. Two configurations of the dialysis module were tested: one involved dispersing the adsorbent particles in dialysis fluid, while the other did not require dialysis fluid. The results demonstrate the superior performance of the first configuration compared to the second. Although the clinical applicability of these models remains distant from the current stage, further studies will focus on optimizing these models to develop a more compact and wearable device.

Droplet-based microfluidic synthesis of nanogels for controlled drug delivery: tailoring nanomaterial properties via pneumatically actuated flow-focusing junction.

Conventional batch syntheses of polymer-based nanoparticles show considerable shortcomings in terms of scarce control over nanomaterials morphology and limited lot-to-lot reproducibility. Droplet-based microfluidics represents a valuable strategy to overcome these constraints, exploiting the formation of nanoparticles within discrete microdroplets. In this work, we synthesized nanogels (NGs) composed of hyaluronic acid and polyethyleneimine using a microfluidic flow-focusing device endowed with a pressure-driven micro-actuator. The actuator achieves real-time modulation of the junction orifice width, thereby regulating the microdroplet diameter and, as a result, the NG size. Acting on process parameters, NG hydrodynamic diameter could be tuned in the range 92-190 nm while preserving an extremely low polydispersity (0.015); those values are hardly achievable in batch syntheses and underline the strength of our toolbox for the continuous in-flow synthesis of nanocarriers. Furthermore, NGs were validated in vitro as a drug delivery system in a representative case study still lacking an effective therapeutic treatment: ovarian cancer. Using doxorubicin as a chemotherapeutic agent, we show that NG-mediated release of the drug results in an enhanced antiblastic effect vs. the non-encapsulated administration route even at sublethal dosages, highlighting the wide applicability of our microfluidics-enabled nanomaterials in healthcare scenarios.

Graphene-laden hydrogels: A strategy for thermally triggered drug delivery.

The synthesis of graphene-based materials has attracted considerable attention in drug delivery strategies. Indeed, the conductivity and mechanical stability of graphene have been investigated for controlled and tunable drug release via electric or mechanical stimuli. However, the design of a thermo-sensitive scaffold using pristine graphene (without distortions related to the oxidation processes) has not been deeply investigated yet, although it may represent a promising approach for several therapeutic treatments. Here, few-layer graphene was used as a nanofiller in a hydrogel system with a thermally tunable drug release profile. In particular, varying the temperature (25 °C, 37 °C and 44 °C), responsive drug releases were noticed and hypothesized depending on the formation and perturbation of π-π interactions involving graphene, the polymeric matrix and the model drug (diclofenac). As a result, these hybrid hydrogels show a potential application as thermally triggered drug release systems in several healthcare scenarios.

Nano-encapsulation of hydroxytyrosol into formulated nanogels improves therapeutic effects against hepatic steatosis: An in vitro study.

Nanomaterials hold promise as a straightforward approach for enhancing the performance of bioactive compounds in several healthcare scenarios. Indeed, nanoencapsulation represents a valuable strategy to preserve the bioactives, maximizing their bioavailability. Here, a nanoencapsulation strategy for the treatment of nonalcoholic fatty liver disease (NAFLD) is presented. NAFLD represents the most common chronic liver disease in Western societies, and still lacks an effective therapy. Hydroxytyrosol (HT), a naturally occurring polyphenol, has been shown to protect against hepatic steatosis through its lipid-lowering, antioxidant and anti-inflammatory activities. However, the efficient delivery of HT to hepatocytes remains a crucial aspect: the design of smart nanogels appears as a promising tool to promote its intracellular uptake. In this paper, we disclose the synthesis of nanogel systems based on polyethylene glycol and polyethyleneimine which have been tested in an in vitro model of hepatic steatosis at two different concentrations (0.1 mg/mL and 0.5 mg/mL), taking advantage of high-content analysis tools. The proposed HT-loaded nanoscaffolds are non-toxic to cells, and their administration showed a significant decrease in the intracellular triglyceride levels, restoring cell viability and outperforming the results achievable with HT in its non-encapsulated form. Moreover, nanogels do not induce oxidative stress, thus demonstrating their biosafety. Overall, the formulated nanogel system achieves superior performance compared to conventional drug administration routes and hence represents a promising strategy for the management of NAFLD.

Biofabrication of Hepatic Constructs by 3D Bioprinting of a Cell-Laden Thermogel: An Effective Tool to Assess Drug-Induced Hepatotoxic Response.

A thermoresponsive Pluronic/alginate semisynthetic hydrogel is used to bioprint 3D hepatic constructs, with the aim to investigate liver-specific metabolic activity of the 3D constructs compared to traditional 2D adherent cultures. The bioprinting method relies on a bioinert hydrogel and is characterized by high-shape fidelity, mild depositing conditions and easily controllable gelation mechanism. Furthermore, the dissolution of the sacrificial Pluronic templating agent significantly ameliorates the diffusive properties of the printed hydrogel. The present findings demonstrate high viability and liver-specific metabolic activity, as assessed by synthesis of urea, albumin, and expression levels of the detoxifying CYP1A2 enzyme of cells embedded in the 3D hydrogel system. A markedly increased sensitivity to a well-known hepatotoxic drug (acetaminophen) is observed for cells in 3D constructs compared to 2D cultures. Therefore, the 3D model developed herein may represent an in vitro alternative to animal models for investigating drug-induced hepatotoxicity.

Textured and smooth breast implants: is there a difference in the chemical structure of silicone?: an analysis with fourier transformation infrared and attenuated total reflectance spectroscopy.

Scientific controversy concerning silicone and its biocompatibility has been ongoing for the last 10 years. This study on textured and smooth silicone breast implant shells using fourier transformation infrared spectroscopy associated with attenuated total reflectance cells aimed to identify eventual chemical modifications of silicone induced by texturization. The surfaces of 8 new implants produced by 2 well-known manufactures have been taken into consideration. A sample 1 cm2 has been harvested from the anterior and posterior sides of textured and smooth shells. Infrared spectra were then recorded, evaluated, and compared with the reference spectrum of pure silicone. Potentially reactive groups, known as silanols, were identified, in all shells, intensity increasing in textured implants (P < 0.05), whereas no silanols were detected in the spectrum of pure silicone. These results suggest that polar groups, present in manipulated silicone might influence capsula formation.

Fabrication of bioactive glass-ceramic foams mimicking human bone portions for regenerative medicine.

A technique for the preparation of bioglass foams for bone tissue engineering is presented. The process is based on the in situ foaming of a bioglass-loaded polyurethane foam as the intermediate step for obtaining a bioglass porous monolith, starting from sol-gel synthesized bioglass powders. The obtained foams were characterized using X-ray diffraction analysis, Fourier transform infrared spectroscopy, and field emission scanning electron microscopy observations. The material was assessed by soaking samples in simulated body fluid and observing apatite layer formation. Diagnostic imaging taken from human patients was used to reconstruct a human bone portion, which was used to mould a tailored scaffold fabricated using the in situ foaming technique. The results confirmed that the obtained bioactive materials prepared with three-dimensional processing are promising for applications in reconstructive surgery tailored to each single patient.

Dermal filler complications from unknown biomaterials: identification by attenuated total reflectance spectroscopy.

BACKGROUND: In the last decade, an increase in complications related to dermal filler injections has been reported, especially in patients who underwent multiple treatments with different products. Imaging or histological examinations may suggest what kind of substance was used, but none can precisely identify the biomaterial. The aim of this study was to evaluate the use of Fourier transform infrared spectroscopy, using an attenuated total reflectance cell, in the identification of unknown dermal fillers. METHODS: In the preclinical study, samples from different manufacturers were analyzed according to attenuated total reflectance spectroscopy using the Nicolet 8700 FT-IR spectrophotometer (resolution, 0.125 cm; Thermo Fisher Scientific, Inc., Madison, Wis.). Spectra of each biomaterial were collected and included in a reference database. In the clinical study, seven patients affected by severe complications due to multiple injections with unknown fillers provided a sample of the pathological tissue for the analysis. RESULTS: Two granulomas, two infiltrated tissues, and three abscesses were studied. Attenuated total reflectance/Fourier transform infrared analysis of pathological tissues revealed the presence of absorption bands absent in the healthy tissue. Comparison of these bands to the filler database made it possible to identify the dermal fillers injected. CONCLUSIONS: This pilot study has demonstrated the absolute validity of the application of infrared spectroscopy in attenuated total reflectance for the determination of infiltrated biomaterial. The knowledge of the previously injected fillers may be crucial to selecting the appropriate medical or surgical treatment as well as to solving medical-legal issues.

Scaffold-based delivery of a clinically relevant anti-angiogenic drug promotes the formation of in vivo stable cartilage.

Standard cartilage tissue engineering approaches, for example, matrix-induced autologous chondrocyte implantation (MACI), consist of the implantation of cell-based constructs whose survival and further development first depend on the degree of graft maturity at the time of surgery (e.g., matrix production) and, subsequently, on initial host reaction. Indeed, blood vessel ingrowth and macrophage migration within the implant may endanger graft stability of immature constructs; so, control of angiogenesis was proposed as an adjuvant of cellular therapy for the treatment of cartilage defects. In this study, we hypothesized that engineered constructs with no in vitro precultivation, but functionalized to block angiogenesis right on implantation, might result in better survival, as well as superior long-term cartilaginous quality. Here, we propose a clinically compatible fibrin/hyaluronan scaffold seeded with nasal chondrocytes (NC) and functionalized with an FDA-approved anti-angiogenic drug (bevacizumab; Avastin(®)), which sequestrates vascular endothelial growth factor from the surrounding environment. Our results show that the sustained bevacizumab release from NC-loaded scaffolds after subcutaneous implantation in nude mice efficiently blocked host vessels ingrowth (five times lower CD31(+) cells infiltration vs. control group, at 3 weeks after implant), and enhanced constructs survival rate (75% vs. 18% for the control, at 6 weeks after implant). In vitro assays, developed to elucidate the role of specific construct components in the in vivo remodeling, allowed to determine that fibrin degradation products enhanced the in vitro endothelial cell proliferation, as well as the macrophage migration; whereas the presence of bevacizumab was capable of counteracting these effects. The proposed pharmacological control of angiogenesis by a therapeutic drug released from a scaffold might enhance cartilage regeneration by MACI approaches, possibly allowing it to bypass the complex and costly phase of graft preculture to gain increased functionality.

The differentiation of humane adult mesenchimal stem cells of bone marrow (hMSC) into urothelial cells on bio-engineering support (scaffold): preliminary experience of tissue engineering.

OBJECTIVES: Tissue Engineering can develop scaffolds of Poly-L-Lactic Acid (PLLA) for tissue regeneration. The purpose of the present job is to test the possibility to seed human adult mesenchymal stem cells on a scaffold supplemented with specific grow factors to differentiate them into urothelium. METHODS: The Electrospinning technique was used to realize three scaffolds. The first one was seeded with urothelial cells, of a primary culture, and Keratinocyte serum free medium (KSFM); the second one was seeded with human mesenchymal stem cells (hMSC) and a minimum essential medium (aMEM); the third one was seeded with hMSC and conditioned medium. RESULTS: Electron microscopy showed scaffolds with cellular vitality (>90%) and their cellular proliferation. Moreover, the differentiation of hMSC, seeded in conditioned medium, into urothelial cells was demonstrated through immunofluorescence assays. CONCLUSIONS: Tissue Engineering can develop PLLA scaffolds thanks to the Electrospinning technique. The scaffold is a perfect environment for cellular culture and proliferation; a protocol for the differentiation of hMSC into urothelial cells is now available. Immunofluorescence assays can demonstrate the hMSC differentiation into urothelial cells.

A biomimetic three-layered compartmented scaffold for vascular tissue engineering.

Tissue engineering of vascular grafts still presents several shortcomings. Aiming to vascular regeneration, we developed a biomimetic multilayered scaffold with a middle pivotal collagen lamina between two functionalized layers of poly-L-lactide by means of electrospinning technique, with oriented drug-delivery capacity for the differentiation of human mesenchymal stem cells seeded therein. Applying appropriate cytokines, the inner layer is able to act as a drug delivery system in order to generate a pro-angiogenic and anti-thrombotic environment and the outer one is used to induce the media and adventitia generation. Our findings are consistent with an adequate cell engrafting and a double type of differentiation in each side of the scaffold, in particular cells exhibited morphostructural changes resulting in the achievement of an endothelial-like phenotype in cells populating the inner side of the scaffold and SMA positivity with cell elongation resembling muscular phenotype in the cells of the outer layer. The proposed "smart" vascular bio-prosthesis will recapitulate the structure and microenvironment of native cardiovascular tissues. It could surmount many hurdles to clinical use and would be relevant for therapeutic applications in a variety of medical fields.