Ti/Ta-based composite polysaccharide scaffolds for guided bone regeneration in total hip arthroplasty.

Guided bone regeneration can play an important role in orthopedic applications. This work presents the synthesis and characterization of composite scaffolds based on polysaccharides loaded with microparticles of titanium or tantalum as novel materials proposed for composite systems with promising characteristics for guided bone regeneration. Ti/Ta composite scaffolds were synthesized using chitosan and gellan gum as organic substrates and crosslinked with oxidized dextran resulting in stable inorganic-organic composites. Physico-chemical characterization revealed a uniform distribution of metal nanoparticles within the scaffolds that showed a release of metals lower than 5 %. In vitro biological assays demonstrated that Ta composites exhibit a 2 times higher ALP activity than Ti and a higher capacity to support the full differentiation of human mesenchymal stem cells into osteoblasts. These results highlight their potential for bone regeneration applications.

Development of Methotrexate Complexes Endowed with New Biological Properties Envisioned for Musculoskeletal Regeneration in Rheumatoid Arthritis Environments.

Methotrexate (MTX) administration is the gold standard treatment for rheumatoid arthritis (RA), but its effects are limited to preventing the progression of the disease. Therefore, effective regenerative therapies for damaged tissues are still to be developed. In this regard, MTX complexes of general molecular formula M(MTX)·xH2O, where M = Sr, Zn, or Mg, were synthesized and physicochemically characterized by TGA, XRD, NMR, ATR-FTIR, and EDAX spectroscopies. Characterization results demonstrated the coordination between the different cations and MTX via two monodentate bonds with the carboxylate groups of MTX. Cation complexation provided MTX with new bioactive properties such as increasing the deposition of glycosaminoglycans (GAGs) and alternative anti-inflammatory capacities, without compromising the immunosuppressant properties of MTX on macrophages. Lastly, these new complexes were loaded into spray-dried chitosan microparticles as a proof of concept that they can be encapsulated and further delivered in situ in RA-affected joints, envisioning them as a suitable alternative to oral MTX therapy.

Development of bioactive catechol functionalized nanoparticles applicable for 3D bioprinting.

Efficient wound treatments to target specific events in the healing process of chronic wounds constitute a significant aim in regenerative medicine. In this sense, nanomedicine can offer new opportunities to improve the effectiveness of existing wound therapies. The aim of this study was to develop catechol bearing polymeric nanoparticles (NPs) and to evaluate their potential in the field of wound healing. Thus, NPs wound healing promoting activities, potential for drug encapsulation and controlled release, and further incorporation in a hydrogel bioink formulation to fabricate cell-laden 3D scaffolds are studied. NPs with 2 and 29 M % catechol contents (named NP2 and NP29) were obtained by nanoprecipitation and presented hydrodynamic diameters of 100 and 75 nm respectively. These nanocarriers encapsulated the hydrophobic compound coumarin-6 with 70% encapsulation efficiency values. In cell culture studies, the NPs had a protective effect in RAW 264.7 macrophages against oxidative stress damage induced by radical oxygen species (ROS). They also presented a regulatory effect on the inflammatory response of stimulated macrophages and promoted upregulation of the vascular endothelial growth factor (VEGF) in fibroblasts and endothelial cells. In particular, NP29 were used in a hydrogel bioink formulation using carboxymethyl chitosan and hyaluronic acid as polymeric matrices. Using a reactive mixing bioprinting approach, NP-loaded hydrogel scaffolds with good structural integrity, shape fidelity and homogeneous NPs dispersion, were obtained. The in vitro catechol NPs release profile of the printed scaffolds revealed a sustained delivery. The bioprinted scaffolds supported viability and proliferation of encapsulated L929 fibroblasts over 14 days. We envision that the catechol functionalized NPs and resulting bioactive bioink presented in this work offer promising advantages for wound healing applications, as they: 1) support controlled release of bioactive catechol NPs to the wound site; 2) can incorporate additional therapeutic functions by co-encapsulating drugs; 3) can be printed into 3D scaffolds with tailored geometries based on patient requirements.

Antitumor Activity of Nanoparticles Loaded with PHT-427, a Novel AKT/PDK1 Inhibitor, for the Treatment of Head and Neck Squamous Cell Carcinoma.

Currently, new treatments are required to supplement the current standard of care for head and neck squamous cell carcinoma (HNSCC). The phosphatidylinositol3-kinase (PI3K) signaling pathway is commonly altered and activated in HNSCC. PHT-427 is a dual PI3K-mammalian target of the AKT/PDK1 inhibitor; however, to the best of our knowledge, the effect of the PHT-427 inhibitor on HNSCC has not been investigated. This study aims to evaluate the antitumoral effect of PHT-427-loaded polymeric nanoparticles based on α-tocopheryl succinate (α-TOS). The in vitro activity of PHT-427 was tested in hypopharynx carcinoma squamous cells (FaDu) to measure the cell viability, PI3KCA/AKT/PDK1 gene expression, and PI3KCA/AKT/PDK1 levels. Apoptosis, epidermal growth factor receptor (EGFR), and reactive oxygen species (ROS) were also measured. The presence of PHT-427 significantly enhances its antiproliferative and proapoptotic activity by inactivating the PI3K/AKT/PDK1 pathway. Nanoparticles (NPs) effectively suppress AKT/PDK1 expression. Additionally, NPs loaded with PHT-427 produce high oxidative stress levels that induce apoptosis. In conclusion, these results are promising in the use of this nanoformulation as a PHT-427 delivery system for effective HNSCC treatment.

Oregano Essential Oil Micro- and Nanoencapsulation With Bioactive Properties for Biotechnological and Biomedical Applications.

Due to the preservative, antioxidant, antimicrobial, and therapeutic properties of oregano essential oil (OEO), it has received an emerging interest for biotechnological and biomedical applications. However, stability and bioactivity can be compromised by its natural volatile and hydrophobic nature, and by external factors including light, heat, or oxygen. Therefore, micro- and nanoencapsulation are being employed to guarantee oregano oil protection from outside aggressions and to maximize its potential. Oregano oil encapsulation is an interesting strategy used to increase its stability, enhance its bioactivity, and decrease its volatility. At the same time, the versatility that micro- and nanocarriers offer, allows to prepare tailored systems that can provide a controlled and targeted release of the encapsulated principle, influence its bioactive activities, or even provide additional properties. Most common materials used to prepare these carriers are based on lipids and cyclodextrins, due to their hydrophobic nature, polymers due to their versatility in composition, and hybrid lipid-polymer systems. In this context, recently developed micro- and nanocarriers encapsulating oregano oil with applications in the biotechnological and biomedical fields will be discussed.

Microfluidics generation of chitosan microgels containing glycerylphytate crosslinker for in situ human mesenchymal stem cells encapsulation.

Human mesenchymal stem cells (hMSCs) are an attractive source for cell therapies because of their multiple beneficial properties, i.e. via immunomodulation and secretory factors. Microfluidics is particularly attractive for cell encapsulation since it provides a rapid and reproducible methodology for microgel generation of controlled size and simultaneous cell encapsulation. Here, we report the fabrication of hMSC-laden microcarriers based on in situ ionotropic gelation of water-soluble chitosan in a microfluidic device using a combination of an antioxidant glycerylphytate (G1Phy) compound and tripolyphosphate (TPP) as ionic crosslinkers (G1Phy:TPP-microgels). These microgels showed homogeneous size distributions providing an average diameter of 104 ± 12 µm, somewhat lower than that of control (127 ± 16 µm, TPP-microgels). The presence of G1Phy in microgels maintained cell viability over time and upregulated paracrine factor secretion under adverse conditions compared to control TPP-microgels. Encapsulated hMSCs in G1Phy:TPP-microgels were delivered to the subcutaneous space of immunocompromised mice via injection, and the delivery process was as simple as the injection of unencapsulated cells. Immediately post-injection, equivalent signal intensities were observed between luciferase-expressing microgel-encapsulated and unencapsulated hMSCs, demonstrating no adverse effects of the microcarrier on initial cell survival. Cell persistence, inferred by bioluminescence signal, decreased exponentially over time showing relatively higher half-life values for G1Phy:TPP-microgels compared to TPP-microgels and unencapsulated cells. In overall, results position the microfluidics generated G1Phy:TPP-microgels as a promising microcarrier for supporting hMSC survival and reparative activities.

Vitamin B9 derivatives as carriers of bioactive cations for musculoskeletal regeneration applications: Synthesis, characterization and biological evaluation.

The development of new drugs for musculoskeletal regeneration purposes has attracted much attention in the last decades. In this work, we present three novel vitamin B9 (folic acid)-derivatives bearing divalent cations (ZnFO, MgFO and MnFO), providing their synthesis mechanism and physicochemical characterization. In addition, a strong emphasis has been placed on evaluating their biological properties (along with our previously reported SrFO) using human mesenchymal stem cells (hMSC). In all the cases, pure folate derivatives (MFOs) with a bidentate coordination mode between the metal and the folate anion, and a 1:1 stoichiometry, were obtained in high yields. A non-cytotoxic dose of all the MFOs (50 µg/mL) was demonstrated to modulate by their own the mRNA profiles towards osteogenic-like or fibrocartilaginous-like phenotypes in basal conditions. Moreover, ZnFO increased the alkaline phosphatase activity in basal conditions, while both ZnFO and MnFO increased the matrix mineralization degree in osteoinductive conditions. Thus, we have demonstrated the bioactivity of these novel compounds and the suitability to further studied them in vivo for musculoskeletal regeneration applications.

Characterization of Novel Synthetic Polyphenols: Validation of Antioxidant and Vasculoprotective Activities.

Antioxidant compounds, including polyphenols, have therapeutic effects because of their anti-inflammatory, antihypertensive, antithrombotic and antiproliferative properties. They play important roles in protecting the cardiovascular and neurological systems, by having preventive or protective effects against free radicals produced by either normal or pathological metabolism in such systems. For instance, resveratrol, a well-known potent antioxidant, has a counteracting effect on the excess of reactive oxygen species (ROS) and has a number of therapeutic benefits, like anti-inflammatory, anti-cancer and cardioprotective activities. Based on previous work from our group, and on the most frequent OH substitutions of natural polyphenols, we designed two series of synthetically accessible bis-polyhydroxyphenyl derivatives, separated by amide or urea linkers. These compounds exhibit high antioxidant ability (oxygen radical absorbance capacity (ORAC) assay) and interesting radical scavenging activity (RSA) values (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and α,α-diphenyl-ß-picrylhydrazyl (DPPH) tests). Some of the best polyphenols were evaluated in two biological systems, endothelial cells (in vitro) and whole aorta (ex vivo), highly susceptible for the deleterious effects of prooxidants under different inflammatory conditions, showing protection against oxidative stress induced by inflammatory stimuli relevant in cardiovascular diseases, i.e., Angiotensin II and IL-1ß. Selected compounds also showed strong in vivo antioxidant properties when evaluated in the model organism Saccharomyces cerevisiae.

Development of Biocomposite Polymeric Systems Loaded with Antibacterial Nanoparticles for the Coating of Polypropylene Biomaterials.

The development of a biocomposite polymeric system for the antibacterial coating of polypropylene mesh materials for hernia repair is reported. Coatings were constituted by a film of chitosan containing randomly dispersed poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles loaded with chlorhexidine or rifampicin. The chlorhexidine-loaded system exhibited a burst release during the first day reaching the release of the loaded drug in three or four days, whereas rifampicin was gradually released for at least 11 days. Both antibacterial coated meshes were highly active against Staphylococcus aureus and Staphylococcus epidermidis (106 CFU/mL), displaying zones of inhibition that lasted for 7 days (chlorhexidine) or 14 days (rifampicin). Apparently, both systems inhibited bacterial growth in the surrounding environment, as well as avoided bacterial adhesion to the mesh surface. These polymeric coatings loaded with biodegradable nanoparticles containing antimicrobials effectively precluded bacterial colonization of the biomaterial. Both biocomposites showed adequate performance and thus could have potential application in the design of antimicrobial coatings for the prophylactic coating of polypropylene materials for hernia repair.

Glycerylphytate crosslinker as a potential osteoinductor of chitosan-based systems for guided bone regeneration.

Chitosan-based membranes are promising systems for guided bone regeneration. In this work, we used glycerylphytate as ionic crosslinker and osteinductor compound for the fabrication of chitosan membranes as supports for human mesenchymal stem cells. Three different glycerylphytate-crosslinked membranes were developed by changing the crosslinker concentration, from 2.5-10 wt-%, respect to chitosan. Physico-chemical characterization in terms of composition, morphology, and thermal behavior was further analyzed. Swelling degree, crosslinking density, and crosslinker release showed a glycerylphytate content-dependent behavior. Glycerylphytate suggested to improve osteointegration ability of chitosan surfaces by the formation of apatite-like aggregates after incubation in body simulated fluid. Stem cells cultured on the membranes increased their viability over time, and the incorporation of glycerylphytate improved osteogenic and osteoinductivity potential of chitosan by increasing calcium deposition and alkaline phosphatase (ALP) activity on cultured stem cells. These results demonstrated a potential application of glycerylphytate-crosslinked chitosan systems for promising bone tissue regeneration.

Bioadhesive functional hydrogels: Controlled release of catechol species with antioxidant and antiinflammatory behavior.

Chronic wounds are particularly difficult to heal and constitute an important global health care problem. Some key factors that make chronic wounds challenging to heal are attributed to the incessant release of free radicals, which activate the inflammatory system and impair the repair of the wound. Intrinsic characteristics of hydrogels are beneficial for wound healing, but the effective control of free radical levels in the wound and subsequent inflammation is still a challenge. Catechol, the key molecule responsible for the mechanism of adhesion of mussels, has been proven to be an excellent radical scavenger and anti-inflammatory agent. Our approach in this work lies in the preparation of a hybrid system combining the beneficial properties of hydrogels and catechol for its application as a bioactive wound dressing to assist in the treatment of chronic wounds. The hydrogel backbone is obtained through a self-covalent crosslinking between chitosan (Ch) and oxidized hyaluronic acid (HAox) in the presence of a synthetic catechol terpolymer, which is subsequently coordinated to Fe to obtain an interpenetrated polymer network (IPN). The structural analysis, catechol release profiles, in vitro biological behavior and in vivo performance of the IPN are analyzed and compared with the semi-IPN (without Fe) and the Ch/HAox crosslinked hydrogels as controls. Catechol-containing hydrogels present high tissue adhesion strength under wet conditions, support growth, migration and proliferation of hBMSCs, protect cells against oxidative stress damage induce by ROS, and promote down-regulation of the pro-inflammatory cytokine IL-1ß. Furthermore, in vivo experiments reveal their biocompatibility and stability, and histological studies indicate normal inflammatory responses and faster vascularization, highlighting the performance of the IPN system. The novel IPN design also allows for the in situ controlled and sustained delivery of catechol. Therefore, the developed IPN is a suitable ECM-mimic platform with high cell affinity and bioactive functionalities that, together with the controlled catechol release, will enhance the tissue regeneration process and has a great potential for its application as wound dressing.

Glycerylphytate compounds with tunable ion affinity and osteogenic properties.

Phytic acid (PA) is a natural-occurring antioxidant, which plays an important role in many biological processes. PA is recognized as a potent inhibitor of lipid peroxidation because of its high affinity to multivalent cations, and it can play a role in osteogenic processes. However, its powerful chelating capacity is controversial because it can lead to a severe reduction of mineral availability in the organism. For this reason, compounds with beneficial biological properties of PA, but a modular ion binding capacity, are of high interest. In this work, we report the synthesis and physicochemical characterization of two hydroxylic derivatives of PA, named glycerylphytates (GPhy), through a condensation reaction of PA with glycerol (G). Both derivatives present antioxidant properties, measured by ferrozine/FeCl2 method and chelating activity with calcium ions depending on the content of glyceryl groups incorporated. Besides, the hydroxylic modification not only modulates the ion binding affinity of derivatives but also improves their cytocompatibility in human bone marrow mesenchymal cells (MSCs). Furthermore, GPhy derivatives display osteogenic properties, confirmed by COL1A and ALPL expression depending on composition. These positive features convert GPhy compounds into potent alternatives for those skeletal diseases treatments where PA is tentatively applied.

Novel Bioactive and Antibacterial Acrylic Bone Cement Nanocomposites Modified with Graphene Oxide and Chitosan.

Acrylic bone cements (ABCs) have played a key role in orthopedic surgery mainly in arthroplasties, but their use is increasingly extending to other applications, such as remodeling of cancerous bones, cranioplasties, and vertebroplasties. However, these materials present some limitations related to their inert behavior and the risk of infection after implantation, which leads to a lack of attachment and makes necessary new surgical interventions. In this research, the physicochemical, thermal, mechanical, and biological properties of ABCs modified with chitosan (CS) and graphene oxide (GO) were studied. Fourier transform infrared (FTIR) spectroscopy, proton nuclear magnetic resonance (1H-NMR) scanning electron microscopy (SEM), Raman mapping, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), compression resistance, mechanical dynamic analysis (DMA), hydrolytic degradation, cell viability, alkaline phosphatase (ALP) activity with human osteoblasts (HOb), and antibacterial activity against Gram-negative bacteria Escherichia coli were used to characterize the ABCs. The results revealed good dispersion of GO nanosheets in the ABCs. GO provided an increase in antibacterial activity, roughness, and flexural behavior, while CS generated porosity, increased the rate of degradation, and decreased compression properties. All ABCs were not cytotoxic and support good cell viability of HOb. The novel formulation of ABCs containing GO and CS simultaneously, increased the thermal stability, flexural modulus, antibacterial behavior, and osteogenic activity, which gives it a high potential for its uses in orthopedic applications.

Bioactive and Bioadhesive Catechol Conjugated Polymers for Tissue Regeneration.

The effective treatment of chronic wounds constitutes one of the most common worldwide healthcare problem due to the presence of high levels of proteases, free radicals and exudates in the wound, which constantly activate the inflammatory system, avoiding tissue regeneration. In this study, we describe a multifunctional bioactive and resorbable membrane with in-built antioxidant agent catechol for the continuous quenching of free radicals as well as to control inflammatory response, helping to promote the wound-healing process. This natural polyphenol (catechol) is the key molecule responsible for the mechanism of adhesion of mussels providing also the functionalized polymer with bioadhesion in the moist environment of the human body. To reach that goal, synthesized statistical copolymers of N-vinylcaprolactam (V) and 2-hydroxyethyl methacrylate (H) have been conjugated with catechol bearing hydrocaffeic acid (HCA) molecules with high yields. The system has demonstrated good biocompatibility, a sustained antioxidant response, an anti-inflammatory effect, an ultraviolet (UV) screen, and bioadhesion to porcine skin, all of these been key features in the wound-healing process. Therefore, these novel mussel-inspired materials have an enormous potential for application and can act very positively, favoring and promoting the healing effect in chronic wounds.

Amphiphilic self-assembled "polymeric drugs": morphology, properties, and biological behavior of nanoparticles.

This article reports the fabrication and characterization of NPs based on the self-assembling of polymeric drugs with amphiphilic character synthetized from oleyl 2-acetamido-2-deoxy-α-d-glucopyranoside methacrylate and vinyl pyrrolidone (OAGMA-VP). NPs were spherical, with an apparent hydrodynamic diameter between 91 and 226 nm and with zeta potential values that ensure stability. Atomic concentrations of C, O, and N, determined by X-ray photoelectron spectroscopy (XPS) of NPs, compared well with the corresponding theoretical values. High resolution XPS C1s spectra suggest that the carbons bound to heteroatoms or carbonyl groups are preferentially situated on the surface of the NP samples. ToF-SIMS spectra analyzed by principal component analysis (PCA) indicated that ions coming from acetyl and oleyl groups of OAGMA play important roles in the outer structure of NPs. Water contact angle and surface tension values of NPs were characteristic of hydrophilic surfaces, confirming the location of VP sequences on the surface. Cell culture assays showed that copolymeric NPs did not compromise biocompatibility of human fibroblasts according to ISO standard, but they were cytotoxic on a human glioblastoma cell line (A-172).