The Other Side of Plastics: Bioplastic-Based Nanoparticles for Drug Delivery Systems in the Brain.

Plastics have changed human lives, finding a broad range of applications from packaging to medical devices. However, plastics can degrade into microscopic forms known as micro- and nanoplastics, which have raised concerns about their accumulation in the environment but mainly about the potential risk to human health. Recently, biodegradable plastic materials have been introduced on the market. These polymers are biodegradable but also bioresorbable and, indeed, are fundamental tools for drug formulations, thanks to their transient ability to pass through biological barriers and concentrate in specific tissues. However, this "other side" of bioplastics raises concerns about their toxic potential, in the form of micro- and nanoparticles, due to easier and faster tissue accumulation, with unknown long-term biological effects. This review aims to provide an update on bioplastic-based particles by analyzing the advantages and drawbacks of their potential use as components of innovative formulations for brain diseases. However, a critical analysis of the literature indicates the need for further studies to assess the safety of bioplastic micro- and nanoparticles despite they appear as promising tools for several nanomedicine applications.

Contribution of peripheral blood mononuclear cells isolated by advanced filtration system to myogenesis of human bone marrow mesenchymal stem cells co-cultured with myoblasts.

Background: Contribution of peripheral blood mononuclear cells (PBMCs) in myogenesis is still under debate, even though blood filtration systems are commonly used in clinical practice for successfully management of critic limb ischemia. Objectives: A commercial blood filter used for autologous human PBMC transplantation procedures is characterized and used to collect PBMCs, that are then added to well-established 2D in vitro myogenic models assembled with a co-culture of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and skeletal myoblasts (hSkMs) whit the aim of investigating their potential contribution to stem cell myogenic commitment. Methods: A commercial blood filter was physically and chemically studied to understand its morphological characteristics and composition. PBMCs were concentrated using this system, further isolated by Ficoll-Paque density gradient centrifugation, and then added in an upper transwell chamber to a 2D co-culture of hBM-MSCs and hSkMs. Myogenic commitment was investigated by RT-PCR, immunofluorescence, and flow cytometry immunophenotyping. Cytokine levels were monitored by ELISA assay in culture media. Results: The blood filtration system was disassembled and appeared to be formed by twelve membranes of poly-butylene terephthalate fibers (diameters, 0.9-4.0 µm) with pore size distribution of 1-20 µm. Filter functional characterization was achieved by characterizing collected cells by flow cytometry. Subsequently, collected PBMCs fraction was added to an in-vitro model of hBM-MSC myogenic commitment. In the presence of PBMCs, stem cells significantly upregulated myogenic genes, such as Desmin and MYH2, as confirmed by qRT-PCR and expressed related proteins by immunofluorescence (IF) assay, while downregulated pro-inflammatory cytokines (IL12A at day 14) along the 21 days of culture. Novelty: Our work highlights chemical-physical properties of commercial blood filter and suggests that blood filtrated fraction of PBMC might modulate cytokine expression in response to muscle injury and promote myogenic events, supporting their clinical use in autologous transplantation.

Activity and Selectivity of Novel Chemical Metallic Complexes with Potential Anticancer Effects on Melanoma Cells.

Human malignant melanoma cells from lymph node metastatic site (MeWo) were selected for testing several synthesized and purified silver(I) and gold(I) complexes stabilized by unsymmetrically substituted N-heterocyclic carbene (NHC) ligands, called L20 (N-methyl, N'-[2-hydroxy ethylphenyl]imidazol-2-ylide) and M1 (4,5-dichloro, N-methyl, N'-[2-hydroxy ethylphenyl]imidazol-2-ylide), having halogenide (Cl- or I-) or aminoacyl (Gly=N-(tert-Butoxycarbonyl)glycinate or Phe=(S)-N-(tert-Butoxycarbonyl)phenylalaninate) counterion. For AgL20, AuL20, AgM1 and AuM1, the Half-Maximal Inhibitory Concentration (IC50) values were measured, and all complexes seemed to reduce cell viability more effectively than Cisplatin, selected as control. The complex named AuM1 was the most active just after 8 h of treatment at 5 µM, identified as effective growth inhibition concentration. AuM1 also showed a linear dose and time-dependent effect. Moreover, AuM1 and AgM1 modified the phosphorylation levels of proteins associated with DNA lesions (H2AX) and cell cycle progression (ERK). Further screening of complex aminoacyl derivatives indicated that the most powerful were those indicated with the acronyms: GlyAg, PheAg, AgL20Gly, AgM1Gly, AuM1Gly, AgL20Phe, AgM1Phe, AuM1Phe. Indeed, the presence of Boc-Glycine (Gly) and Boc-L-Phenylalanine (Phe) showed an improved efficacy of Ag main complexes, as well as that of AuM1 derivatives. Selectivity was further checked on a non-cancerous cell line, a spontaneously transformed aneuploid immortal keratinocyte from adult human skin (HaCaT). In such a case, AuM1 and PheAg complexes resulted as the most selective allowing HaCaT viability at 70 and 40%, respectively, after 48 h of treatment at 5 µM. The same complexes tested on 3D MeWo static culture induced partial spheroid disaggregation after 24 h of culture, with almost half of the cells dead.

Bottom-Up Strategy to Forecast the Drug Location and Release Kinetics in Antitumoral Electrospun Drug Delivery Systems.

Electrospun systems are becoming promising devices usable for topical treatments. They are eligible to deliver different therapies, from anti-inflammatory to antitumoral. In the current research, polycaprolactone electrospun membranes loaded with synthetic and commercial antitumoral active substances were produced, underlining how the matrix-filler affinity is a crucial parameter for designing drug delivery devices. Nanofibrous membranes loaded with different percentages of Dacarbazine (the drug of choice for melanoma) and a synthetic derivative of Dacarbazine were produced and compared to membranes loaded with AuM1, a highly active Au-complex with low affinity to the matrix. AFM morphologies showed that the surface profile of nanofibers loaded with affine substances is similar to one of the unloaded systems, thanks to the nature of the matrix-filler interaction. FTIR analyses proved the efficacy of the interaction between the amidic group of the Dacarbazine and the polycaprolactone. In AuM1-loaded membranes, because of the weak matrix-filler interaction, the complex is mainly aggregated in nanometric domains on the nanofiber surface, which manifests a nanometric roughness. Consequently, the release profiles follow a Fickian behavior for the Dacarbazine-based systems, whereas a two-step with a highly prominent burst effect was observed for AuM1 systems. The performed antitumoral tests evidence the high-cytotoxic activity of the electrospun systems against melanoma cell lines, proving that the synthetic substances are more active than the commercial dacarbazine.

3D in-vitro cultures of human bone marrow and Wharton's jelly derived mesenchymal stromal cells show high chondrogenic potential.

In this study, chondrogenic potentials of 3D high-density cultures of Bone Marrow (BM) and Wharton's Jelly (WJ)-derived mesenchymal stromal cells (MSCs) was investigated by chondrogenesis- and cytokine-related gene expression over a 16-day culture period supplemented with human transforming growth factor (hTGF)-ß1 at 10 ng/ml. In BM-MSC 3D models, a marked upregulation of chondrogenesis-related genes, such as SOX9, COL2A1, and ACAN (all p < 0.05) and formation of spherical pellets with structured type II collagen fibers were observed. Similarly, WJ-based high-density culture appeared higher in size and more regular in shape, with a significant overexpression of COL2A1 and ACAN (all p < 0.05) at day 16. Moreover, a similar upregulation trend was documented for IL-6 and IL-10 expression in both BM and WJ 3D systems. In conclusion, MSC-based high-density cultures can be considered a promising in vitro model of cartilage regeneration and tissue engineering. Moreover, our data support the use of WJ-MSCs as a valid alternative for chondrogenic commitment of stem cells in regenerative medicine.

Stem Cells from Healthy and Tendinopathic Human Tendons: Morphology, Collagen and Cytokines Expression and Their Response to T3 Thyroid Hormone.

The aim of this study was to investigate the effect of triiodothyronine (T3) on tendon specific markers and cytokines expression of stem cells extracted from human tendons. Indeed, thyroid hormones have been reported to be protective factors, maintaining tendons' homeostasis, whereas tendinopathy is believed to be related to a failed healing response. Healthy and tendinopathic human tendons were harvested to isolate tendon stem/progenitor cells (TSPCs). TSPCs obtained from pathological samples showed gene expression and morphological modifications at baseline in comparison with cells harvested from healthy tissues. When cells were maintained in a medium supplemented with T3 (10-6 M), only pathological populations showed a significant upregulation of tenogenic markers (DCN, TNC, COL1A1, COL3A1). Immunostaining revealed that healthy cells constantly released type I collagen, typical of tendon matrix, whereas pathological ones overexpressed and secreted type III collagen, typical of scarred and impaired tissue. Pathological cells also overexpressed pro- and anti-inflammatory cytokines, suggesting an impaired balance in the presence of T3, without STAT3 activation. Moreover, DKK-1 was significantly high in the culture medium of pathological cell cultures and was reversed by T3. This study opens perspectives on the complex biochemical alteration of cells from pathological tendons, which may lead to the chronic disease context with an impaired extracellular matrix.

Electrospun Membranes Designed for Burst Release of New Gold-Complexes Inducing Apoptosis of Melanoma Cells.

Two non-commercial metallic Au-based complexes were tested against one of the most aggressive malignant melanomas of the skin (MeWo cells), through cell viability and time-lapse live-cell imaging system assays. The tests with the complexes were carried out both in the form of free metallic complexes, directly in contact with the MeWo cell line culture, and embedded in fibers of Polycaprolactone (PCL) membranes produced by the electrospinning technique. Membranes functionalized with complexes were prepared to evaluate the efficiency of the membranes against the melanoma cells and therefore their feasibility in the application as an antitumoral patch for topical use. Both series of tests highlighted a very effective antitumoral activity, manifesting a very relevant cell viability inhibition after both 24 h and 48 h. In the case of the AuM1 complex at the concentration of 20 mM, melanoma cells completely died in this short period of time. A mortality of around 70% was detected from the tests performed using the membranes functionalized with AuM1 complex at a very low concentration (3 wt.%), even after 24 h of the contact period. The synthesized complexes also manifest high selectivity with respect to the MeWo cells. The peculiar structural and morphological organization of the nanofibers constituting the membranes allows for a very effective antitumoral activity in the first 3 h of treatment. Experimental points of the release profiles were perfectly fitted with theoretical curves, which easily allow interpretation of the kinetic phenomena occurring in the release of the synthesized complexes in the chosen medium.

Peripheral blood mononuclear cells contribute to myogenesis in a 3D bioengineered system of bone marrow mesenchymal stem cells and myoblasts.

In this work, a 3D environment obtained using fibrin scaffold and two cell populations, such as bone marrow-derived mesenchymal stem cells (BM-MSCs), and primary skeletal muscle cells (SkMs), was assembled. Peripheral blood mononuclear cells (PBMCs) fraction obtained after blood filtration with HemaTrate® filter was then added to the 3D culture system to explore their influence on myogenesis. The best cell ratio into a 3D fibrin hydrogel was 1:1 (BM-MSCs plus SkMs:PBMCs) when cultured in a perfusion bioreactor; indeed, excellent viability and myogenic event induction were observed. Myogenic genes were significantly overexpressed when cultured with PBMCs, such as MyoD1 of 118-fold at day 14 and Desmin 6-fold at day 21. Desmin and Myosin Heavy Chain were also detected at protein level by immunostaining along the culture. Moreover, the presence of PBMCs in 3D culture induced a significant downregulation of pro-inflammatory cytokine gene expression, such as IL6. This smart biomimetic environment can be an excellent tool for investigation of cellular crosstalk and PBMC influence on myogenic processes.

3D Biomimetic Scaffold for Growth Factor Controlled Delivery: An In-Vitro Study of Tenogenic Events on Wharton's Jelly Mesenchymal Stem Cells.

The present work described a bio-functionalized 3D fibrous construct, as an interactive teno-inductive graft model to study tenogenic potential events of human mesenchymal stem cells collected from Wharton's Jelly (hWJ-MSCs). The 3D-biomimetic and bioresorbable scaffold was functionalized with nanocarriers for the local controlled delivery of a teno-inductive factor, i.e., the human Growth Differentiation factor 5 (hGDF-5). Significant results in terms of gene expression were obtained. Namely, the up-regulation of Scleraxis (350-fold, p ≤ 0.05), type I Collagen (8-fold), Decorin (2.5-fold), and Tenascin-C (1.3-fold) was detected at day 14; on the other hand, when hGDF-5 was supplemented in the external medium only (in absence of nanocarriers), a limited effect on gene expression was evident. Teno-inductive environment also induced pro-inflammatory, (IL-6 (1.6-fold), TNF (45-fold, p ≤ 0.001), and IL-12A (1.4-fold)), and anti-inflammatory (IL-10 (120-fold) and TGF-ß1 (1.8-fold)) cytokine expression upregulation at day 14. The presented 3D construct opens perspectives for the study of drug controlled delivery devices to promote teno-regenerative events.

Chondrogenic Commitment of Human Bone Marrow Mesenchymal Stem Cells in a Perfused Collagen Hydrogel Functionalized with hTGF-ß1-Releasing PLGA Microcarrier.

Tissue engineering strategies can be relevant for cartilage repair and regeneration. A collagen matrix was functionalized with the addition of poly-lactic-co-glycolic acid microcarriers (PLGA-MCs) carrying a human Transforming Growth Factor ß1 (hTFG-ß1) payload, to provide a 3D biomimetic environment with the capacity to direct stem cell commitment towards a chondrogenic phenotype. PLGA-MCs (mean size 3 ± 0.9 µm) were prepared via supercritical emulsion extraction technology and tailored to sustain delivery of payload into the collagen hydrogel for 21 days. PLGA-MCs were coseeded with human Bone Marrow Mesenchymal Stem Cells (hBM-MSCs) in the collagen matrix. Chondrogenic induction was suggested when dynamic perfusion was applied as indicated by transcriptional upregulation of COL2A1 gene (5-fold; p < 0.01) and downregulation of COL1A1 (0.07-fold; p < 0.05) and COL3A1 (0.11-fold; p < 0.05) genes, at day 16, as monitored by qRT-PCR. Histological and quantitative-immunofluorescence (qIF) analysis confirmed cell activity by remodeling the synthetic extracellular matrix when cultured in perfused conditions. Static constructs lacked evidence of chondrogenic specific gene overexpression, which was probably due to a reduced mass exchange, as determined by 3D system Finite Element Modelling (FEM) analysis. Proinflammatory (IL-6, TNF, IL-12A, IL-1ß) and anti-inflammatory (IL-10, TGF-ß1) cytokine gene expression by hBM-MSC was observed only in dynamic culture (TNF and IL-1ß 10-fold, p < 0.001; TGF-ß1 4-fold, p < 0.01 at Day 16) confirming the cells' immunomodulatory activity mainly in relation to their commitment and not due to the synthetic environment. This study supports the use of 3D hydrogel scaffolds, equipped for growth factor controlled delivery, as tissue engineered models for the study of in vitro chondrogenic differentiation and opens clinical perspectives for injectable collagen-based advanced therapy systems.

Amnion-Derived Teno-Inductive Secretomes: A Novel Approach to Foster Tendon Differentiation and Regeneration in an Ovine Model.

Regenerative medicine has greatly progressed, but tendon regeneration mechanisms and robust in vitro tendon differentiation protocols remain to be elucidated. Recently, tendon explant co-culture (CO) has been proposed as an in vitro model to recapitulate the microenvironment driving tendon development and regeneration. Here, we explored standardized protocols for production and storage of bioactive tendon-derived secretomes with an evaluation of their teno-inductive effects on ovine amniotic epithelial cells (AECs). Teno-inductive soluble factors were released in culture-conditioned media (CM) only in response to active communication between tendon explants and stem cells (CMCO). Unsuccessful tenogenic differentiation in AECs was noted when exposed to CM collected from tendon explants (CMFT) only, whereas CMCO upregulated SCXB, COL I and TNMD transcripts, in AECs, alongside stimulation of the development of mature 3D tendon-like structures enriched in TNMD and COL I extracellular matrix proteins. Furthermore, although the tenogenic effect on AECs was partially inhibited by freezing CMCO, this effect could be recovered by application of an in vivo-like physiological oxygen (2% O2) environment during AECs tenogenesis. Therefore, CMCO can be considered as a waste tissue product with the potential to be used for the development of regenerative bio-inspired devices to innovate tissue engineering application to tendon differentiation and healing.

Supercritical emulsion extraction fabricated PLA/PLGA micro/nano carriers for growth factor delivery: Release profiles and cytotoxicity.

Controlled delivery of human growth factors is still a challenge in tissue engineering protocols, and poly-lactic acid and poly-lactic-co-glycolic acid carriers have been recently proposed for this purpose. Here, the microencapsulation of two human growth factors, namely Growth Differentiation Factor -5 (hGDF-5) and Transforming Growth Factor ß1 (hTGF-ß1) was tested, by processing different emulsions with Supercritical Emulsion Extraction (SEE) technology. Polymer molecular weight, co-polymer ratio and surfactant amount in aqueous phases as well as phases mixing rate were varied to fabricate carriers with suitable size and loadings. Carriers with different mean sizes from 0.4 ± 0.09 µm up to 3 ± 0.9 µm were obtained by SEE technology when processing emulsions with different formulations; carriers were loaded with 3 µg/g and 7 µg/g for hGDF-5 and hTGF-ß1 with controlled growth factor release over 25 days. Carriers displayed extremely low cytotoxicity when evaluated in Chinese Hamster Ovary cells (CHO-K1). Further, they also exhibited reduced cytotoxicity with respect to carriers obtained by conventional evaporation techniques, and low reactivity on human peripheral blood mononuclear cells (hPBMCs), suggesting their safety and potential use in tissue engineering protocols.

In Vitro Innovation of Tendon Tissue Engineering Strategies.

Tendinopathy is the term used to refer to tendon disorders. Spontaneous adult tendon healing results in scar tissue formation and fibrosis with suboptimal biomechanical properties, often resulting in poor and painful mobility. The biomechanical properties of the tissue are negatively affected. Adult tendons have a limited natural healing capacity, and often respond poorly to current treatments that frequently are focused on exercise, drug delivery, and surgical procedures. Therefore, it is of great importance to identify key molecular and cellular processes involved in the progression of tendinopathies to develop effective therapeutic strategies and drive the tissue toward regeneration. To treat tendon diseases and support tendon regeneration, cell-based therapy as well as tissue engineering approaches are considered options, though none can yet be considered conclusive in their reproduction of a safe and successful long-term solution for full microarchitecture and biomechanical tissue recovery. In vitro differentiation techniques are not yet fully validated. This review aims to compare different available tendon in vitro differentiation strategies to clarify the state of art regarding the differentiation process.

Dose-Response Tendon-Specific Markers Induction by Growth Differentiation Factor-5 in Human Bone Marrow and Umbilical Cord Mesenchymal Stem Cells.

Mesenchymal stem cells derived from human bone marrow (hBM-MSCs) are utilized in tendon tissue-engineering protocols while extra-embryonic cord-derived, including from Wharton's Jelly (hWJ-MSCs), are emerging as useful alternatives. To explore the tenogenic responsiveness of hBM-MSCs and hWJ-MSCs to human Growth Differentiation Factor 5 (hGDF-5) we supplemented each at doses of 1, 10, and 100 ng/mL of hGDF-5 and determined proliferation, morphology and time-dependent expression of tenogenic markers. We evaluated the expression of collagen types 1 (COL1A1) and 3 (COL3A1), Decorin (DCN), Scleraxis-A (SCX-A), Tenascin-C (TNC) and Tenomodulin (TNMD) noting the earliest and largest increase with 100 ng/mL. With 100 ng/mL, hBM-MSCs showed up-regulation of SCX-A (1.7-fold) at Day 1, TNC (1.3-fold) and TNMD (12-fold) at Day 8. hWJ-MSCs, at the same dose, showed up-regulation of COL1A1 (3-fold), DCN (2.7-fold), SCX-A (3.8-fold) and TNC (2.3-fold) after three days of culture. hWJ-MSCs also showed larger proliferation rate and marked aggregation into a tubular-shaped system at Day 7 (with 100 ng/mL of hGDF-5). Simultaneous to this, we explored the expression of pro-inflammatory (IL-6, TNF, IL-12A, IL-1ß) and anti-inflammatory (IL-10, TGF-ß1) cytokines across for both cell types. hBM-MSCs exhibited a better balance of pro-inflammatory and anti-inflammatory cytokines up-regulating IL-1ß (11-fold) and IL-10 (10-fold) at Day 8; hWJ-MSCs, had a slight expression of IL-12A (1.5-fold), but a greater up-regulation of IL-10 (2.5-fold). Type 1 collagen and tenomodulin proteins, detected by immunofluorescence, confirming the greater protein expression when 100 ng/mL were supplemented. In the same conditions, both cell types showed specific alignment and shape modification with a length/width ratio increase, suggesting their response in activating tenogenic commitment events, and they both potential use in 3D in vitro tissue-engineering protocols.

Biomechanical issues of tissue-engineered constructs for articular cartilage regeneration: in vitro and in vivo approaches.

BACKGROUND: Given the limited regenerative capacity of injured articular cartilage, the absence of suitable therapeutic options has encouraged tissue-engineering approaches for its regeneration or replacement. SOURCES OF DATA: Published articles in any language identified in PubMed and Scopus electronic databases up to August 2019 about the in vitro and in vivo properties of cartilage engineered constructs. A total of 64 articles were included following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. AREAS OF AGREEMENT: Regenerated cartilage lacks the biomechanical and biological properties of native articular cartilage. AREAS OF CONTROVERSY: There are many different approaches about the development of the architecture and the composition of the scaffolds. GROWING POINTS: Novel tissue engineering strategies focus on the development of cartilaginous biomimetic materials able to repair cartilage lesions in association to cell, trophic factors and gene therapies. AREAS TIMELY FOR DEVELOPING RESEARCH: A multi-layer design and a zonal organization of the constructs may lead to achieve cartilage regeneration.

An αB-Crystallin Peptide Rescues Compartmentalization and Trafficking Response to Cu Overload of ATP7B-H1069Q, the Most Frequent Cause of Wilson Disease in the Caucasian Population.

The H1069Q substitution is the most frequent mutation of the Cu transporter ATP7B that causes Wilson disease in the Caucasian population. ATP7B localizes to the Golgi complex in hepatocytes, but, in the presence of excessive Cu, it relocates to the endo-lysosomal compartment to excrete Cu via bile canaliculi. In contrast, ATP7B-H1069Q is strongly retained in the ER, does not reach the Golgi complex and fails to move to the endo-lysosomal compartment in the presence of excessive Cu, thus causing toxic Cu accumulation. We have previously shown that, in transfected cells, the small heat-shock protein αB-crystallin is able to correct the mislocalization of ATP7B-H1069Q and its trafficking in the presence of Cu overload. Here, we first show that the α-crystallin domain of αB-crystallin mimics the effect of the full-length protein, whereas the N- and C-terminal domains have no such effect. Next, and most importantly, we demonstrate that a twenty-residue peptide derived from the α-crystallin domain of αB-crystallin fully rescues Golgi localization and the trafficking response of ATP7B-H1069Q in the presence of Cu overload. In addition, we show that this peptide interacts with the mutant transporter in the live cell. These results open the way to attempt developing a pharmacologically active peptide to specifically contrast the Wilson disease form caused by the ATP7B-H1069Q mutant.

Differential phosphorylation-based regulation of αB-crystallin chaperone activity for multipass transmembrane proteins.

We have previously shown that αB-crystallin (CRYAB), a small heat shock protein (sHsp) that prevents irreversible aggregation of unfolded protein by an ATP-independent chaperone activity, plays a pivotal role in the biogenesis of multipass transmembrane proteins (TMPs) assisting their folding from the cytosolic side of the endoplasmic reticulum (ER) (D'Agostino et al., 2013). Here we present evidence, based on phosphomimetic substitutions, that the three phosphorytable serine residues at position 19, 45 and 59 of CRYAB play a different regulatory role in this novel chaperone activity: S19 and S45 have a strong inhibitory effect, either alone or in combination, while S59 has not and counteracts the inhibition caused by single phosphomimetic substitutions at S19 and S45. Interestingly, all phosphomimetic substitutions determine the formation of smaller oligomeric complexes containing CRYAB, indicating that the inhibitory effect seen for S19 and S45 cannot be ascribed to the reduction of oligomerization frequently associated to a decreased chaperone activity. These results indicate that phosphorylation finely regulates the chaperone activity of CRYAB with multipass TMPs and suggest a pivotal role for S59 in this process.