A theragenerative bio-nanocomposite consisting of black phosphorus quantum dots for bone cancer therapy and regeneration.

Recently, the term theragenerative has been proposed for biomaterials capable of inducing therapeutic approaches followed by repairing/regenerating the tissue/organ. This study is focused on the design of a new theragenerative nanocomposite composed of an amphiphilic non-ionic surfactant (Pluronic F127), bioactive glass (BG), and black phosphorus (BP). The nanocomposite was prepared through a two-step synthetic strategy, including a microwave treatment that turned BP nanosheets (BPNS) into quantum dots (BPQDs) with 5 ± 2 nm dimensions in situ. The effects of surfactant and microwave treatment were assessed in vitro: the surfactant distributes the ions homogenously throughout the composite and the microwave treatment chemically stabilizes the composite. The presence of BP enhanced bioactivity and promoted calcium phosphate formation in simulated body fluid. The inherent anticancer activity of BP-containing nanocomposites was tested against osteosarcoma cells in vitro, finding that 150 µg mL-1 was the lowest concentration which prevented the proliferation of SAOS-2 cells, while the counterpart without BP did not affect the cell growth rate. Moreover, the apoptosis pathways were evaluated and a mechanism of action was proposed. NIR irradiation was applied to induce further proliferation suppression on SAOS-2 cells through hyperthermia. The inhibitory effects of bare BP nanomaterials and nanocomposites on the migration and invasion of bone cancer, breast cancer, and prostate cancer cells were assessed in vitro to determine the anticancer potential of nanomaterials against primary and secondary bone cancers. The regenerative behavior of the nanocomposites was tested with healthy osteoblasts and human mesenchymal stem cells; the BPQDs-incorporated nanocomposite significantly promoted the proliferation of osteoblast cells and induced the osteogenic differentiation of stem cells. This study introduces a new multifunctional theragenerative platform with promising potential for simultaneous bone cancer therapy and regeneration.

Skin innervation across amyotrophic lateral sclerosis clinical stages: new prognostic biomarkers.

Over recent decades, peripheral sensory abnormalities, including the evidence of cutaneous denervation, have been reported among the non-motor manifestations in amyotrophic lateral sclerosis (ALS). However, a correlation between cutaneous innervation and clinical features has not been found. The aims of this study were to assess sensory involvement by applying a morpho-functional approach to a large population of ALS patients stratified according to King's stages and correlate these findings with the severity and prognosis of the disease. We recruited 149 ALS patients and 41 healthy controls. Patients undertook clinical questionnaires for small fibre neuropathy symptoms (Small Fiber Neuropathy Symptoms Inventory Questionnaire) and underwent nerve conductions studies (NCS) and 3-mm punch skin biopsies from leg, thigh and fingertip. We assessed intraepidermal nerve fibre (IENF) and Meissner corpuscle (MC) density by applying an indirect immunofluorescence technique. Moreover, a subset of 65 ALS patients underwent a longitudinal study with repeat biopsies from the thigh at 6- and 12-month follow-ups. Serum NfL levels were measured in 40 patients. Sensory symptoms and sensory NCS abnormalities were present in 32.2% and 24% of patients, respectively, and increased across clinical stages. Analogously, we observed a progressive reduction in amplitude of the sensory and motor ulnar nerve potential from stage 1 to stage 4. Skin biopsy showed a significant loss of IENFs and MCs in ALS compared with healthy controls (all P < 0.001). Across the clinical stages, we found a progressive reduction in MCs (P = 0.004) and an increase in IENFs (all P < 0.027). The increase in IENFs was confirmed by the longitudinal study. Interestingly, the MC density inversely correlated with NfL level (r = -0.424, P = 0.012), and survival analysis revealed that low MC density, higher NfL levels and increasing IENF density over time were associated with a poorer prognosis (all P < 0.024). To summarize, in patients with ALS, peripheral sensory involvement worsens in parallel with motor disability. Furthermore, the correlation between skin innervation and disease activity may suggest the use of skin innervation as a putative prognostic biomarker.

Eumelanin decorated poly(lactic acid) electrospun substrates as a new strategy for spinal cord injury treatment.

Spinal cord injury (SCI) is characterized by neuroinflammatory processes that are marked by an uncontrolled activation of microglia, which directly damages neurons. Natural and synthetic melanins represent an effective tool to treat neuroinflammation because they possess immunomodulatory properties. Here, the main objective was to evaluate the effect of eumelanin-coated poly(lactic acid) (EU@PLA) aligned microfibers on in vitro model of neuroinflammation related to spinal cord injury in terms of inflammatory mediators' modulation. Aligned fibers were chosen to provide physical cues to guide axonal growth in a specific direction thus restoring the synaptic connection. Eumelanin decorated PLA electrospun substrates were produced combining electrospinning, spin coating and solid-state polymerization processes (oxidative coupling under oxygen atmosphere). Biological response in terms of antioxidant and anti-inflammatory activity was analyzed on an in vitro model of neuroinflammation [microglial cells stimulated with lipopolysaccharide (LPS)]. Cell morphology and EU@PLA mechanism of action, in terms of toll-like receptor-4 (TLR-4) involvement were assessed. The results show that EU@PLA fibers were able to decrease reactive oxygen species, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кB) expression >50 % compared to PLA + LPS and interleukin 6 (IL-6) secretion about 20 %. Finally, the mechanism of action of EU@PLA in microglia was found to be dependent on the TLR-4 signaling. Protein expression analysis revealed a decreased in TLR-4 production induced by LPS stimulation in presence of EU@PLA. Overall, our results show that EU@PLA represents an innovative and effective strategy for the control of inflammatory response in central nervous system.

Label-Free Morpho-Molecular Imaging for Studying the Differential Interaction of Black Phosphorus with Tumor Cells.

Black phosphorus nanosheets (2D BP) are emerging as very promising, highly selective chemotherapeutic agents due to their fast degradation in the intracellular matrix of cancer cells. Here, optical diffraction tomography (ODT) and Raman spectroscopy were exploited as a powerful label-free approach to achieve integrated insights into the processes accompanying the administration of exfoliated 2D BP flakes in human prostatic adenocarcinoma and normal human prostate epithelial cells. Our ODT experiments provided unambiguous visualization of the 2D BP internalization in cancer cells and the morphological modifications of those cells in the apoptotic phase. The cellular internalization and damaging occurred, respectively, 18 h and 36-48 h after the 2D BP administration. Changes in the chemical properties of the internalized 2D BP flakes were monitored by Raman spectroscopy. Interestingly, a fast oxidation process of the 2D BP flakes was activated in the intracellular matrix of the cancer cells after 24 h of incubation. This was in sharp contrast to the low 2D BP uptake and minimal chemical changes observed in the normal cells. Along with the understanding of the 2D BP fate in the cancer cells, the proposed label-free morpho-molecular approach offers a powerful, rapid tool to study the pharmacokinetic properties of engineered nanomaterials in preclinical research.

Chitosan/hydroxyapatite nanocomposite scaffolds to modulate osteogenic and inflammatory response.

Considerable attention has been given to the use of chitosan (CS)-based materials reinforced with inorganic bioactive signals such as hydroxyapatite (HA) to treat bone defects and tissue loss. It is well known that CS/HA based materials possess minimal foreign body reactions, good biocompatibility, controlled biodegradability and antibacterial property. Herein, the bioactivity of these composite systems was analyzed on in vitro bone cell models for their applications in the field of bone tissue engineering (BTE). The combination of sol-gel approach and freeze-drying technology was used to obtain CS/HA scaffolds with three-dimensional (3D) porous structure suitable for cell in-growth. Specifically, our aim was to investigate the influence of bioactive composite scaffolds on cellular behavior in terms of osteoinductivity and anti-inflammatory effects for treating bone defects. The results obtained have demonstrated that by increasing inorganic component concentration, CS/HA (60 and 70% v/v) scaffolds induced a good biological response in terms of osteogenic differentiation of human mesenchymal stem cells (hMSC) towards osteoblast phenotype. Furthermore, the scaffolds with higher concentration of inorganic fillers are able to modulate the production of pro-inflammatory (TGF-ß) and anti-inflammatory (IL-4, IL-10) cytokines. Our results highlight the possibility of achieving smart CS/HA based composites able to promote a great osteogenic differentiation of hMSC by increasing the amount of HA nanoparticles used as bioactive inorganic signal. Contemporarily, these materials allow avoiding the induction of a pro-inflammatory response in bone implant site.

ROS-Generating Hyaluronic Acid-Modified Zirconium Dioxide-Acetylacetonate Nanoparticles as a Theranostic Platform for the Treatment of Osteosarcoma.

Materials that are able to produce free radicals have gained increasing attention for environmental and biomedical purposes. Free radicals, such as the superoxide anion (O2•-), act as secondary messengers in many physiological pathways, such as cell survival. Therefore, the production of free radicals over physiological levels has been exploited in the treatment of different types of cancer, including osteosarcoma (OS). In most cases, the production of reactive oxygen species (ROS) by materials is light-induced and requires the use of chemical photosensitisers, making it difficult and expensive. Here, for the first time, we propose photoluminescent hybrid ZrO2-acetylacetonate nanoparticles (ZrO2-acac NPs) that are capable of generating O2•- without light activation as an adjuvant for the treatment of OS. To increase the uptake and ROS generation in cancer cells, we modify the surface of ZrO2-acac NPs with hyaluronic acid (HA), which recognizes and binds to the surface antigen CD44 overexpressed on OS cells. Since these nanoparticles emit in the visible range, their uptake into cancer cells can be followed by a label-free approach. Overall, we show that the generation of O2•- is toxic to OS cells and can be used as an adjuvant treatment to increase the efficacy of conventional drugs.

2D exfoliated black phosphorus influences healthy and cancer prostate cell behaviors.

Nowadays, prostate cancer is the most widespread tumour in worldwide male population. Actually, brachytherapy is the most advanced radiotherapy strategy for the local treatment of prostate cancer. It consists in the placing of radioactive sources closed to the tumour side thus killing cancer cells. However, brachytherapy causes the same adverse effects of external-beam radiotherapy. Therefore, alternative treatment approaches are required for enhancing radiotherapy effectiveness and reducing toxic symptoms. Nanostructured exfoliated black phosphorus (2D BP) may represent a strategic tool for local cancer therapy because of its capability to induce singlet oxygen production and act as photosensitizer. Hence, we investigated 2D BP in vitro effect on healthy and cancer prostate cell behavior. 2D BP was obtained through liquid exfoliation. 2D BP effect on healthy and cancer prostate cell behaviors was analyzed by investigating cell viability, oxidative stress and inflammatory marker expression. 2D BP inhibited prostate cancer cell survival, meanwhile promoted healthy prostate cell survival in vitro by modulating oxidative stress and immune response with and without near-infrared light (NIR)-irradiation. Nanostructured 2D BP is able to inhibit in vitro prostate cancer cells survival and preserve healthy prostate cell vitality through the control of oxidative stress and immune response, respectively.

Chitosan/PEGDA based scaffolds as bioinspired materials to control in vitro angiogenesis.

In the current work, our purpose was based on the assessment of bioactive chitosan (CS)/Poly(ethylene glycol) diacrylate (PEGDA) based scaffolds ability to stimulate in vitro angiogenesis process. The bioactivation of the scaffolds was accomplished by using organic (BMP-2 peptide) and inorganic (hydroxyapatite nanoparticles) cues. In particular, the properties of the materials in terms of biological response promotion on human umbilical vein endothelial cells (HUVECs) were studied by using in vitro angiogenesis tests based on cell growth and proliferation. Furthermore, our interest was to examine the scaffolds capability to modulate two important steps involved in angiogenesis process: migration and tube formation of cells. Our data underlined that bioactive signals on CS/PEGDA scaffolds surface induce a desirable effect on angiogenic response concerning angiogenic marker expression (CD-31) and endothelial tissue formation (tube formation). Taken together, the results emphasized the concept that bioactive CS/PEGDA scaffolds may be novel implants for stimulating neovascularization of tissue-engineered constructs in regenerative medicine field.

Tricalcium phosphate cement supplemented with boron nitride nanotubes with enhanced biological properties.

A self-setting bone cement containing ß-tricalcium phosphate (TCP) supplemented with boron nitride nanotubes (BNNTs, 1 wt%) was synthesized and analyzed in situ for its kinetics of hardening and selected physicochemical and biological properties. Moderately delayed due to the presence of BNNTs, the hardening reaction involved the transformation of the TCP precursor to the dicalcium phosphate (DCPD) product. In spite of the short-lived chemical transformations in the cement upon its hardening, the structural changes in it were extended. As a result, the compressive strength increased from day 1 to day 7 of the hardening reaction and the presence of BNNTs further increased it by ~25%. Fitting of the time-resolved energy-dispersive diffractometric data to the Johnson-Mehl-Avrami-Kolmogorov crystallization kinetics model conformed to the one-dimensional nucleation at a variable rate during the growth of elongated DCPD crystals from round TCP grains. For the first seven days of growth of human mesenchymal stem cells (hMSCs) on the cement, no difference in their proliferation was observed compared to the control. However, between the 7th and the 21st day, the cell proliferation decreased compared to the control because of the ongoing stem cell differentiation toward the osteoblast phenotype. This differentiation was accompanied by the higher expression of alkaline phosphatase, an early marker of hMSC differentiation into a pre-osteoblast phenotype. The TCP cement supplemented with BNNTs was able to thwart the production of reactive oxygen species (ROS) in hMSCs treated with H2O2/Fe2+ and bring the ROS levels down to the concentrations detected in the control cells, indicating the good capability of the material to protect the cells against the ROS-associated damage. Simultaneously, the cement increased the expression of mediators of inflammation in a co-culture of osteoblasts and macrophages, thus attesting to the direct reciprocity between the degrees of inflammation and stimulated new bone production.

Osteogenic and Anti-Inflammatory Behavior of Injectable Calcium Phosphate Loaded with Therapeutic Drugs.

Bone fractures related to musculoskeletal disorders determine long-term disability in older people with a consequent significant economic burden. The recovery of pathologically impaired tissue architecture allows avoiding bone loss-derived consequences such as bone height reduction, deterioration of bone structure, inflamed bone pain, and high mortality for thighbone fractures. Actually, standard therapy for osteoporosis treatment is based on the systemic administration of biphosphonates and anti-inflammatory drugs, which entail several side effects including gastrointestinal (GI) diseases, fever, and articular pain. Hence, the demand of innovative therapeutic approaches for locally treating bone lesions has been increasing in the last few years. In this scenario, the development of injectable materials loaded with therapeutically active agents (i.e., anti-inflammatory drugs, antibiotics, and peptides mimicking growth factors) could be an effective tool to treat bone loss and inflammation related to musculoskeletal diseases, including osteoporosis and osteoarthritis. According to this challenge, here, we propose three different compositions of injectable calcium phosphates (CaP) as new carrier materials of therapeutic compounds such as bisphosphonates (i.e., alendronate), anti-inflammatory drugs (i.e., diclofenac sodium), and natural molecules (i.e., harpagoside) for the local bone disease treatment. Biological quantitative analyses were performed for screening osteoinductive and anti-inflammatory properties of injectable drug-loaded systems. Meanwhile, cell morphological features were analyzed through scanning electron microscopy and confocal investigations. The results exhibited that the three systems exerted an osteoinductive effect during later phases of osteogenesis. Simultaneously, all compositions showed an anti-inflammatory activity on inflammation in vitro models.

In-Situ Synthesis and Characterization of Chitosan/Hydroxyapatite Nanocomposite Coatings to Improve the Bioactive Properties of Ti6Al4V Substrates.

Ti6Al4V alloy is still attracting great interest because of its application as an implant material for hard tissue repair. This research aims to produce and investigate in-situ chitosan/hydroxyapatite (CS/HA) nanocomposite coatings based on different amounts of HA (10, 50 and 60 wt.%) on alkali-treated Ti6Al4V substrate through the sol-gel process to enhance in vitro bioactivity. The influence of different contents of HA on the morphology, contact angle, roughness, adhesion strength, and in vitro bioactivity of the CS/HA coatings was studied. Results confirmed that, with increasing the HA content, the surface morphology of crack-free CS/HA coatings changed for nucleation modification and HA nanocrystals growth, and consequently, the surface roughness of the coatings increased. Furthermore, the bioactivity of the CS/HA nanocomposite coatings enhanced bone-like apatite layer formation on the material surface with increasing HA content. Moreover, CS/HA nanocomposite coatings were biocompatible and, in particular, CS/10 wt.% HA composition significantly promoted human mesenchymal stem cells (hMSCs) proliferation. In particular, these results demonstrate that the treatment strategy used during the bioprocess was able to improve in vitro properties enough to meet the clinical performance. Indeed, it is predicted that the dense and crack-free CS/HA nanocomposite coatings suggest good potential application as dental implants.

Osteoinductive and anti-inflammatory properties of chitosan-based scaffolds for bone regeneration.

Current bone implants based on new biomaterials may cause a foreign body reaction (FBR) around the implant itself thus prolonging the healing time following bone fractures. In this paper, biomimetic chitosan-based scaffolds promoting bone tissue regeneration and controlling inflammatory response are proposed. First, the anti-inflammatory potential of scaffolds on hMSCs stimulated by lipopolysaccharide (LPS) was investigated by dosing the levels of some interleukins and oxidative stress metabolites (IL-1ß, IL-10 and nitrites) involved in immune response. Then, to mimic the inflammation process at osteoporotic site, the effect of scaffolds was evaluated on in vitro co-culture model based on osteoblasts and macrophages stimulated by LPS. Results demonstrated that bioactivated scaffolds are able to i) inhibit synthesis of inflammatory mediators such as IL-1ß; ii) reduce oxidative stress metabolites; and iii) promote anti-inflammatory markers generation (IL-10) in hMSCs. Finally, bioactivated scaffolds show an anti-inflammatory activity also on in vitro co-cultures, which better mimic in vivo damaged bone microenvironment.

Exfoliated Black Phosphorus Promotes in Vitro Bone Regeneration and Suppresses Osteosarcoma Progression through Cancer-Related Inflammation Inhibition.

Nowadays chemotherapy is the main treatment for osteosarcoma disease, even if limited by the lack of selectivity between healthy and cancer cells during the inhibition of cell division. Herein, we propose the use of few-layer two-dimensional black phosphorous (2D bP) as an alternative tool for osteosarcoma treatment and report how 2D bP can stimulate newly forming bone tissue generation after osteosarcoma resection. In our study, we have developed an in vitro model to evaluate the efficacy of 2D bP material with and without near-infrared light irradiation treatment, and we have demonstrated that the presence of 2D bP without treatment inhibits the metabolic activity of osteosarcoma cells (SAOS-2) while inducing both the proliferation and the osteogenic differentiation of human preosteoblast cells (HOb) and mesenchymal stem cells. Furthermore, we also propose an in vitro coculture model (SAOS-2 and HOb cell lines) in order to study the effect of 2D bP on inflammatory response related to cancer. On this coculture model, 2D bP may increase anti-inflammatory cytokine generation (i.e., interleukin-10) and inhibit proinflammatory mediators synthesis (i.e., interleukin-6), thus suggesting the opportunity to prevent cancer-related inflammation. Finally, we have demonstrated that 2D bP represents a promising candidate for future regenerative medicine and anticancer applications.

Cu-Releasing Bioactive Glass Coatings and Their in Vitro Properties.

Bioactive glasses are well-known materials suitable for bone-related applications thanks to their biocompatibility and osteoconductivity. In order to improve their in vivo performance, the modification of the glass composition by adding ions with specific biological functions is required. As copper (Cu) possesses antibacterial properties, in this study, 5 wt % of CuO has been added to the 45S5 bioactive glass composition. The investigation of the effect of the Cu-containing bioactive glass on cellular behavior has revealed that the presence of Cu induces an early differentiation of human mesenchymal stem cells through osteoblast phenotype, promotes the expression of anti-inflammatory interleukin, and reduces proinflammatory interleukin expression. With the aim to produce coatings with antibacterial properties, the Cu-containing bioactive glass was used as the target material for the pulsed laser deposition (PLD) of bioactive thin films. PLD experiments were carried out at different substrate temperatures to study the effect on the film's characteristics. All of the films are compact, crack-free, and characterized by a rough morphology and good wettability. The in vitro bioactivity was demonstrated by the apatite growth on the coating surface, after soaking in simulated body fluid, revealed by Raman spectroscopy and scanning electron microscopy-energy dispersive X-ray analyses. The antibacterial study proved that the material showed more effective activity against three Gram-negative bacteria ( Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica) rather than against Gram-positive bacteria ( Staphylococcus aureus).

Antimicrobial Imidazolium Ionic Liquids for the Development of Minimal Invasive Calcium Phosphate-Based Bionanocomposites.

Biofilm formation is one of the main obstacles that occur during in vivo implantation, which compromises the implant functionality and patients' health. This is the inspiration for the development of novel implant materials that contain broad-spectrum antimicrobial activity, including antibacterial and antifungal, and enable the local release of antimicrobial agents. Here, multifunctional calcium phosphate-ionic liquid (IL) materials, possessing antimicrobial and repair/regeneration features plus injectability, are proposed as implants in minimally invasive surgery. This approach was based on the loading of 1-alkyl-3-alkylimidazolium chloride ionic liquids (ILs) (C nMImCl ( n = 4, 10, 16) and (C10)2MImCl) during the in situ sol-gel synthesis of calcium phosphates (CaP) and study of their effects on CaP crystallization and biological properties. Physical, morphological, and biological investigations were performed to evaluate the bionanocomposites' properties. The IL N-alkyl chain length influenced the crystallization of CaP and, consequently, the biological properties, which afforded bionanocomposites (when loaded with C16MImCl or (C10)2MImCl) that, (i) inhibit both in vitro bacterial and fungal growth; (ii) reduce the in vitro inflammatory response; (iii) induce osteogenic differentiation in the basal medium of human mesenchymal stem cells; and (iv) are injectable. This will enable the design of multifunctional injectable implants with antimicrobial, anti-inflammatory, and regenerative properties to be used in minimally invasive surgery of bone and maxillofacial defects.

Macroporous alginate foams crosslinked with strontium for bone tissue engineering.

Nowadays, the need of novel strategies to repair and regenerate bone defects in the field of biomedical applications has increased. Novel approaches include the design of natural bioactive scaffolds mimicking bone tissue. These bioactive scaffolds have to possess biophysical properties suitable to address biological response towards newly bone tissue formation. In particular, scaffold porosity and pore size play a pivotal role in cell migration, adhesion and proliferation, thus increasing cell-material surface interaction and osteogenic signals transmission. Here we propose the development of macroporous alginate foams (MAFs) with porous and well interconnected structure, useful to enhance growth and osteogenic differentiation of human Mesenchymal Stem Cells (hMSCs). Moreover, in this study we report a new method for MAFs fabrication based on the combination of internal gelation technique with gas foaming. Strontium was employed in combination with calcium as cross-linking agent for the alginate chains and as enhancer of the osteogenic differentiation. The influence of strontium ions on the gelation kinetics, physical properties and degradation in physiological medium of MAFs was investigated. Our results suggest that the combination of internal gelation technique with gas foaming followed by freeze-drying is an easy and straightforward procedure to prepare alginate foams with high porosity and interconnectivity, able to support cell infiltration. Finally, biological assays showed how scaffolds with high strontium content are able to support cell growth and differentiation in long times by promoting osteogenic marker expression.

Effect of inorganic and organic bioactive signals decoration on the biological performance of chitosan scaffolds for bone tissue engineering.

The present work is focused on the design of a bioactive chitosan-based scaffold functionalized with organic and inorganic signals to provide the biochemical cues for promoting stem cell osteogenic commitment. The first approach is based on the use of a sequence of 20 amino acids corresponding to a 68-87 sequence in knuckle epitope of BMP-2 that was coupled covalently to the carboxyl group of chitosan scaffold. Meanwhile, the second approach is based on the biomimetic treatment, which allows the formation of hydroxyapatite nuclei on the scaffold surface. Both scaffolds bioactivated with organic and inorganic signals induce higher expression of an early marker of osteogenic differentiation (ALP) than the neat scaffolds after 3 days of cell culture. However, scaffolds decorated with BMP-mimicking peptide show higher values of ALP than the biomineralized one. Nevertheless, the biomineralized scaffolds showed better cellular behaviour than neat scaffolds, demonstrating the good effect of hydroxyapatite deposits on hMSC osteogenic differentiation. At long incubation time no significant difference among the biomineralized and BMP-activated scaffolds was observed. Furthermore, the highest level of Osteocalcin expression (OCN) was observed for scaffold with BMP2 mimic-peptide at day 21. The overall results showed that the presence of bioactive signals on the scaffold surface allows an osteoinductive effect on hMSC in a basal medium, making the modified chitosan scaffolds a promising candidate for bone tissue regeneration.

Gelatin/nano-hydroxyapatite hydrogel scaffold prepared by sol-gel technology as filler to repair bone defects.

This study reports on the development of a scaffold with a gradient of bioactive solid signal embedded in the biodegradable polymer matrix by combining a sol-gel approach and freeze-drying technology. The chemical approach based on the sol-gel transition of calcium phosphates ensures the particles dispersion into the gelatin matrix and a direct control of interaction among COOHgelatin /Ca2+ ions. Morphological analysis demonstrated that on the basis of the amount of inorganic component and by using specific process conditions, it is possible to control the spatial distribution of nanoparticles around the gelatin helix. In fact, methodology and formulations were able to discriminate between the different hydroxyapatite concentrations and their respective morphology. The good biological response represented by good cell attachment, proliferation and increased levels of alkaline phosphatase as an indicator of osteoblastic differentiation of human mesenchymal stem cells toward the osteogenic lineage, demonstrating the effect of bioactive solid signals on cellular behavior. Furthermore, the inhibition of reactive oxygen species production by composite materials predicted potential anti-inflammatory properties of scaffolds thus confirming their biocompatibility. Indeed, these interesting biological results suggest good potential application of this scaffold as filler to repair bone defects. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2007-2019, 2018.

Eumelanin Coated PLA Electrospun Micro Fibers as Bioinspired Cradle for SH-SY5Y Neuroblastoma Cells Growth and Maturation.

Within the framework of neurodegenerative disorder therapies, the fabrication of 3D eumelanin architectures represents a novel strategy to realize tissue-engineering scaffolds for neuronal cell growth and control by providing both mechanical support and biological signals. Here, an appropriate procedure combining electrospinning, spin coating and solid-state polymerization process is established to realize the scaffolds. For biological analysis, a human derived cell line SH-SY5Y from neuroblastoma is used. Cell maturation on eumelanin microfibers, random and aligned, is evaluated by using confocal analysis and specific markers of differentiating neurons (ßIII tubulin and GAP-43 expression). Cell morphology is tested by SEM analysis and immunofluorescence techniques. As results, eumelanin coated microfibers prove capable to support biological response in terms of cell survival, adhesion and spreading and to promote cell differentiation toward a more mature neuronal phenotype as confirmed by GAP-43 expression over the culture.

5-Azacytidine-mediated hMSC behavior on electrospun scaffolds for skeletal muscle regeneration.

Incomplete regeneration after trauma or muscular dysfunction is a common problem in muscle replacement therapies. Recent approaches in tissue engineering allow for the replication of skeletal muscle structure and function in vitro and in vivo by molecular therapies and implantable scaffolds which properly address muscle cells toward myotube differentiation and maturation. Here, we investigate the in vitro response of human mesenchymal stem cells (hMSC) on electrospun fibers made of polycaprolactone (PCL) in the presence of 5-azacytidine (5-AZA) to evaluate how fibrous network may influence the therapeutic effect of drug during in vitro myogenesis. Biological studies demonstrate the ability of hMSCs to differentiate in mature myofibers in supplemented (myogenic) and, preferentially, in 5-AZA-enriched culture. PCL electrospun fibers amplify the 5-AZA capability to induce a low proliferation rate in hMSC, thus promoting hMSC differentiation (MTT assay). Qualitative (Azan Mallory stain, immunofluorescence assay, SEM analyses) and quantitative (ELISA test) assays confirm the synergistic contribution of PCL electrospun fibers and 5-AZA on in vitro myotubes formation and maturation. This result is also confirmed by the expression of muscle-specific proteins related to the myogenic mechanisms in the presence of other muscle inductive signals (i.e., oxytocin, Tweak). Hence, we suggest the use of PCL electrospun fibers as interesting preclinical model to explore the effect of drugs and chemotherapeutics administration after damaged muscle resection. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2551-2561, 2017.