Biomimetic proteolipid vesicles for reverting GPI deficiency in paroxysmal nocturnal hemoglobinuria.

Nano-vesicular carriers are promising tissue-specific drug delivery platforms. Here, biomimetic proteolipid vesicles (BPLVs) were used for delivery of glycosylphosphatidylinositol (GPI)-anchored proteins to GPI deficient paroxysmal nocturnal hemoglobinuria (PNH) cells. BPLVs were assembled as single unilamellar monodispersed (polydispersity index, 0.1) negatively charged (ζ-potential, -28.6 ± 5.6 mV) system using microfluidic technique equipped with Y-shaped chip. GPI-anchored and not-GPI proteins on BPLV surface were detected by flow cytometry. Peripheral blood mononuclear cells (PBMCs) from healthy and PNH subjects were treated with BPLVs (final concentration, 0.5 mg/mL), and cells displayed an excellent protein uptake, documented by flow cytometry immunophenotyping and confocal microscopy. BPLV-treated cells stressed with complement components showed an increased resistance to complement-mediated lysis, both healthy and PNH PBMCs. In conclusion, BPLVs could be effective nanocarriers for protein transfer to targeted cells to revert protein deficiency, like in PNH disease. However, further in vivo studies are required to validate our preclinical in vitro results.

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.

c-Kit M541L variant is related to ineffective hemopoiesis predisposing to clonal evolution in 3D in vitro biomimetic co-culture model of bone marrow niche.

Hematopoietic stem cell (HSC) maintenance in vitro is challenging because stem cell survival relies on cell-to-cell contacts and paracrine signals from bone marrow (BM) microenvironment. Indeed, HSCs easily differentiate in conventional culture systems, and in vitro study of stem cell biology, leukemogenesis, and evolutionary trajectories is limited. 3D-culture systems can mimic tissue architecture and microenvironment thus preserving HSC phenotype. In this study, we developed a calcium alginate hydrogel-based 3D co-culture system of BM mononuclear cells (BMMCs) and BM-derived mesenchymal stem cells (BM-MSCs) to study hemopoiesis in health and disease, such as biological roles of c-Kit M541L somatic mutation of unknown significance. BMMCs and peripheral blood stem cells were obtained from an acute myeloid leukemia patient who experienced graft failure and his haploidentical donor, and from a healthy donor. Cells embedded in alginate scaffolds were cultured for up to 21 days, and flow cytometry immunophenotyping was performed at baseline and every seven days. Our results showed suitability of our 3D culture system in preserving HSC vitality and phenotype throughout the culture period, and also in maintaining composition and vitality of total BMMCs. Moreover, 3D in vitro culture results suggested that M541L c-Kit somatic mutation could be a loss-of-function alteration by reducing HSC maintenance ability thus quickly promoting differentiation, as documented by in vivo graft failure and in vitro absence of long-term culture stability. In conclusions, our 3D BM-like biomimetic culture system allowed long-term stemness maintenance, making it a valid and effective tool for in vitro study of physiological and pathological hemopoiesis.

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.

Myogenic commitment of human stem cells by myoblasts Co-culture: a static vs. a dynamic approach.

An in-vitro model of human bone marrow mesenchymal stem cells (hBM-MSCs) myogenic commitment by synergic effect of a differentiation media coupled with human primary skeletal myoblasts (hSkMs) co-culture was developed adopting both conventional static co-seeding and perfused culture systems. Static co-seeding provided a notable outcome in terms of gene expression with a significant increase of Desmin (141-fold) and Myosin heavy chain II (MYH2, 32-fold) at day 21, clearly detected also by semi-quantitative immunofluorescence. Under perfusion conditions, myogenic induction ability of hSkMs on hBM-MSCs was exerted by paracrine effect with an excellent gene overexpression and immunofluorescence detection of MYH2 protein; furthermore, due to the dynamic cell culture in separate wells, western blot data were acquired confirming a successful cell commitment at day 14. A significant increase of anti-inflammatory cytokine gene expression, including IL-10 and IL-4 (15-fold and 11-fold, respectively) at day 14, with respect to the pro-inflammatory cytokines IL-12A (7-fold at day 21) and IL-1ß (1.4-fold at day 7) was also detected during dynamic culture, confirming the immunomodulatory activity of hBM-MSCs along with commitment events. The present study opens interesting perspectives on the use of dynamic culture based on perfusion as a versatile tool to study myogenic events and paracrine cross-talk compared to the simple co-seeding static culture.

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.

Stem Cell and Macrophage Roles in Skeletal Muscle Regenerative Medicine.

In severe muscle injury, skeletal muscle tissue structure and functionality can be repaired through the involvement of several cell types, such as muscle stem cells, and innate immune responses. However, the exact mechanisms behind muscle tissue regeneration, homeostasis, and plasticity are still under investigation, and the discovery of pathways and cell types involved in muscle repair can open the way for novel therapeutic approaches, such as cell-based therapies involving stem cells and peripheral blood mononucleate cells. Indeed, peripheral cell infusions are a new therapy for muscle healing, likely because autologous peripheral blood infusion at the site of injury might enhance innate immune responses, especially those driven by macrophages. In this review, we summarize current knowledge on functions of stem cells and macrophages in skeletal muscle repairs and their roles as components of a promising cell-based therapies for muscle repair and regeneration.

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.

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.

Circulating BPIFB4 Levels Associate With and Influence the Abundance of Reparative Monocytes and Macrophages in Long Living Individuals.

Long-Living Individuals (LLIs) delay aging and are less prone to chronic inflammatory reactions. Whether a distinct monocytes and macrophages repertoire is involved in such a characteristic remains unknown. Previous studies from our group have shown high levels of the host defense BPI Fold Containing Family B Member 4 (BPIFB4) protein in the peripheral blood of LLIs. Moreover, a polymorphic variant of the BPIFB4 gene associated with exceptional longevity (LAV-BPIFB4) confers protection from cardiovascular diseases underpinned by low-grade chronic inflammation, such as atherosclerosis. We hypothesize that BPIFB4 may influence monocytes pool and macrophages skewing, shifting the balance toward an anti-inflammatory phenotype. We profiled circulating monocytes in 52 LLIs (median-age 97) and 52 healthy volunteers (median-age 55) using flow cytometry. If the frequency of total monocyte did not change, the intermediate CD14++CD16+ monocytes counts were lower in LLIs compared to control adults. Conversely, non-classical CD14+CD16++ monocyte counts, which are M2 macrophage precursors with an immunomodulatory function, were found significantly associated with the LLIs' state. In a differentiation assay, supplementation of the LLIs' plasma enhanced the capacity of monocytes, either from LLIs or controls, to acquire a paracrine M2 phenotype. A neutralizing antibody against the phosphorylation site (ser 75) of BPIFB4 blunted the M2 skewing effect of the LLIs' plasma. These data indicate that LLIs carry a peculiar anti-inflammatory myeloid profile, which is associated with and possibly sustained by high circulating levels of BPIFB4. Supplementation of recombinant BPIFB4 may represent a novel means to attenuate inflammation-related conditions typical of unhealthy aging.

Longevity-Associated Variant of BPIFB4 Mitigates Monocyte-Mediated Acquired Immune Response.

One of the basis of exceptional longevity is the maintaining of the balance between inflammatory and anti-inflammatory networks. The monocyte-macrophages activation plays a major role in tuning the immune responses, by oscillating between patrolling-protective to inflammatory status. Longevity-associated variant (LAV) of bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4) activates calcium, PKC-alpha, and eNOS, rescuing endothelial dysfunction in aged mice and inducing revascularization. The BPIFB4's increment in serum of healthy long-living individuals (LLIs) compared to nonhealthy ones, its therapeutic potential in improving vascular homeostasis, which depends on immune system, together with its expression in bone marrow myeloid cells, suggests that LAV-BPIFB4 may improve immune regulation. Here we show that human monocytes exposed to LAV-BPIFB4 protein increased co-stimulatory molecules in resting state and reduced pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) after activating stimuli. Accordingly, a low percentage of CD69+ activated lymphocytes are found among LAV-BPIFB4-treated peripheral blood mononuclear cells (PBMCs). Moreover, human monocyte-derived dendritic cells (DCs) generated in presence of LAV-BPIFB4 secreted higher anti-(IL-10 and TGF-ß) and lower pro-inflammatory (TNF-α and IL-1ß) cytokines. Accordingly, LLIs' plasma showed higher levels of circulating IL-10 and of neutralizing IL-1 receptor antagonist (IL-1RA) compared to controls. Thus, LAV-BPIFB4 effects on myeloid compartment could represent one example of a genetic predisposition carried by LLIs to protect from immunological dysfunctions.