Topographical and Compositional Gradient Tubular Scaffold for Bone to Tendon Interface Regeneration.

The enthesis is an extremely specific region, localized at the tendon-bone interface (TBI) and made of a hybrid connection of fibrocartilage with minerals. The direct type of enthesis tissue is commonly subjected to full laceration, due to the stiffness gradient between the soft tissues and hard bone, and this often reoccurs after surgical reconstruction. For this purpose, the present work aimed to design and develop a tubular scaffold based on pullulan (PU) and chitosan (CH) and intended to enhance enthesis repair. The scaffold was designed with a topographical gradient of nanofibers, from random to aligned, and hydroxyapatite (HAP) nanoparticles along the tubular length. In particular, one part of the tubular scaffold was characterized by a structure similar to bone hard tissue, with a random mineralized fiber arrangement; while the other part was characterized by aligned fibers, without HAP doping. The tubular shape of the scaffold was also designed to be extemporarily loaded with chondroitin sulfate (CS), a glycosaminoglycan effective in wound healing, before the surgery. Micro CT analysis revealed that the scaffold was characterized by a continuous gradient, without interruptions from one end to the other. The gradient of the fiber arrangement was observed using SEM analysis, and it was still possible to observe the gradient when the scaffold had been hydrated for 6 days. In vitro studies demonstrated that human adipose stem cells (hASC) were able to grow and differentiate onto the scaffold, expressing the typical ECM production for tendon in the aligned zone, or bone tissue in the random mineralized part. CS resulted in a synergistic effect, favoring cell adhesion/proliferation on the scaffold surface. These results suggest that this tubular scaffold loaded with CS could be a powerful tool to support enthesis repair upon surgery.

Hybrid Lipid/Clay Carrier Systems Containing Annatto Oil for Topical Formulations.

Nanocomposites formed by clay and lipid carriers (NLCs) show a high potential for providing controlled release and specific delivery of bioactive molecules and have recently gained attention in the pharmaceutical sector due to their ability to transport hydrophilic and hydrophobic drugs. Recent studies have recognized the biological activity of the oil of Bixa orellana L. (AO) with regards to its healing, antioxidant, antibacterial, and anti-leishmanial properties. Therefore, the purpose of this study is the preparation and characterization of hybrid systems based on lipid nanocarriers and laponite for the delivery of AO. NLCs were prepared by the fusion-emulsification method, using cetyl palmitate (CP) or myristyl myristate (MM), AO, and Poloxamer 188. The morphology, hydrodynamic diameters, zeta potential (ZP), polydispersity index (PDI), thermal analysis, X-ray diffraction analysis (XRD), viscosity behavior, and cytotoxicity testing of the hybrid systems were performed. The thermal study and X-ray diffraction analyses (XRD) revealed polymorphic structural changes compatible with the amorphization of the material. Rheological assays highlighted a typical pseudoplastic behavior in all systems (MM and CP with LAP). The hybrid systems' morphology, size diameters, and PDIs were similar, preset spherical and monodisperse structures (≈200 nm; <0.3), without significant change up to sixty days. The ZP values differed from each other, becoming higher with increasing AO concentration. XEDS spectra and elemental X-ray maps show peaks of lipids (organic components, C and O) and inorganic components O, Mg, and Si. All samples showed cell viability above 60%. The results indicated a stable, biocompatible hybrid system that can be an alternative for topical application.

Smart Device for Biologically Enhanced Functional Regeneration of Osteo-Tendon Interface.

The spontaneous healing of a tendon laceration results in the formation of scar tissue, which has lower functionality than the original tissue. Moreover, chronic non-healing tendon injuries frequently require surgical treatment. Several types of scaffolds have been developed using the tissue engineering approach, to complement surgical procedures and to enhance the healing process at the injured site. In this work, an electrospun hybrid tubular scaffold was designed to mimic tissue fibrous arrangement and extracellular matrix (ECM) composition, and to be extemporaneously loaded into the inner cavity with human platelet lysate (PL), with the aim of leading to complete post-surgery functional regeneration of the tissue for functional regeneration of the osteo-tendon interface. For this purpose, pullulan (P)/chitosan (CH) based polymer solutions were enriched with hydroxyapatite nanoparticles (HP) and electrospun. The nanofibers were collected vertically along the length of the scaffold to mimic the fascicle direction of the tendon tissue. The scaffold obtained showed tendon-like mechanical performance, depending on HP content and tube size. The PL proteins were able to cross the scaffold wall, and in vitro studies have demonstrated that tenocytes and osteoblasts are able to adhere to and proliferate onto the scaffold in the presence of PL; moreover, they were also able to produce either collagen or sialoproteins, respectively-important components of ECM. These results suggest that HP and PL have a synergic effect, endorsing PL-loaded HP-doped aligned tubular scaffolds as an effective strategy to support new tissue formation in tendon-to-bone interface regeneration.

Electrospun Scaffolds in Periodontal Wound Healing.

Periodontitis is a set of inflammatory conditions affecting the tissues surrounding the teeth predominantly sustained by bacterial infections. The aim of the work was the design and the development of scaffolds based on biopolymers to be inserted in the periodontal pocket to restore tissue integrity and to treat bacterial infections. Nanofibrous scaffolds were prepared by means of electrospinning. Gelatin was considered as base component and was associated to low and high molecular weight chitosans and alginate. The scaffolds were characterized by chemico-physical properties (morphology, solid state-FTIR and differential scanning calorimetry (DSC)-surface zeta potential and contact angle), and mechanical properties. Moreover, preclinical properties (cytocompatibility, fibroblast and osteoblast adhesion and proliferation and antimicrobial properties) were assessed. All the scaffolds were based on cylindrical and smooth nanofibers and preserved their nanofibrous structure upon hydration independently of their composition. They possessed a high degree of hydrophilicity and negative zeta potentials in a physiological environment, suitable surface properties to enhance cell adhesion and proliferation and to inhibit bacteria attachment. The scaffold based on gelatin and low molecular weight chitosan proved to be effective in vitro to support both fibroblasts and osteoblasts adhesion and proliferation and to impair the proliferation of Streptococcus mutans and Aggregatibacter actinomycetemcomitans, both pathogens involved in periodontitis.

Wound Healing Activity of Nanoclay/Spring Water Hydrogels.

BACKGROUND: hydrogels prepared with natural inorganic excipients and spring waters are commonly used in medical hydrology. Design of these clay-based formulations continues to be a field scarcely addressed. Safety and wound healing properties of different fibrous nanoclay/spring water hydrogels were addressed. METHODS: in vitro biocompatibility, by means of MTT assay, and wound healing properties were studied. Confocal Laser Scanning Microscopy was used to study the morphology of fibroblasts during the wound healing process. RESULTS: all the ingredients demonstrated to be biocompatible towards fibroblasts. Particularly, the formulation of nanoclays as hydrogels improved biocompatibility with respect to powder samples at the same concentration. Spring waters and hydrogels were even able to promote in vitro fibroblasts motility and, therefore, accelerate wound healing with respect to the control. CONCLUSION: fibrous nanoclay/spring water hydrogels proved to be skin-biocompatible and to possess a high potential as wound healing formulations. Moreover, these results open new prospects for these ingredients to be used in new therapeutic or cosmetic formulations.

Norfloxacin-Loaded Electrospun Scaffolds: Montmorillonite Nanocomposite vs. Free Drug.

Infections in nonhealing wounds remain one of the major challenges. Recently, nanomedicine approach seems a valid option to overcome the antibiotic resistance mechanisms. The aim of this study was the development of three types of polysaccharide-based scaffolds (chitosan-based (CH), chitosan/chondroitin sulfate-based (CH/CS), chitosan/hyaluronic acid-based (CH/HA)), as dermal substitutes, to be loaded with norfloxacin, intended for the treatment of infected wounds. The scaffolds have been loaded with norfloxacin as a free drug (N scaffolds) or in montmorillonite nanocomposite (H-hybrid-scaffolds). Chitosan/glycosaminoglycan (chondroitin sulfate or hyaluronic acid) scaffolds were prepared by means of electrospinning with a simple, one-step process. The scaffolds were characterized by 500 nm diameter fibers with homogeneous structures when norfloxacin was loaded as a free drug. On the contrary, the presence of nanocomposite caused a certain degree of surface roughness, with fibers having 1000 nm diameters. The presence of norfloxacin-montmorillonite nanocomposite (1%) caused higher deformability (90%-120%) and lower elasticity (5-10 mN/cm2), decreasing the mechanical resistance of the systems. All the scaffolds were proven to be degraded via lysozyme (this should ensure scaffold resorption) and this sustained the drug release (from 50% to 100% in 3 days, depending on system composition), especially when the drug was loaded in the scaffolds as a nanocomposite. Moreover, the scaffolds were able to decrease the bioburden at least 100-fold, proving that drug loading in the scaffolds did not impair the antimicrobial activity of norfloxacin. Chondroitin sulfate and montmorillonite in the scaffolds are proven to possess a synergic performance, enhancing the fibroblast proliferation without impairing norfloxacin's antimicrobial properties. The scaffold based on chondroitin sulfate, containing 1% norfloxacin in the nanocomposite, demonstrated adequate stiffness to sustain fibroblast proliferation and the capability to sustain antimicrobial properties to prevent/treat nonhealing wound infection during the healing process.

Halloysite- and Montmorillonite-Loaded Scaffolds as Enhancers of Chronic Wound Healing.

The increase in life expectancy and the increasing prevalence of diabetic disease and venous insufficiency lead to the increase of chronic wounds. The prevalence of ulcers ranges from 1% in the adult population to 3-5% in the over 65 years population, with 3-5.5% of the total healthcare expenditure, as recently estimated. The aim of this work was the design and the development of electrospun scaffolds, entirely based on biopolymers, loaded with montmorillonite (MMT) or halloysite (HNT) and intended for skin reparation and regeneration, as a 3D substrate mimicking the dermal ECM. The scaffolds were manufactured by means of electrospinning and were characterized for their chemico-physical and preclinical properties. The scaffolds proved to possess the capability to enhance fibroblast cells attachment and proliferation with negligible proinflammatory activity. The capability to facilitate the cell adhesion is probably due to their unique 3D structure which are assisting cell homing and would facilitate wound healing in vivo.

Development of a Mucoadhesive in Situ Gelling Formulation for the Delivery of Lactobacillus gasseri into Vaginal Cavity.

Local administration of vaginal probiotics, especially lactobacilli, has been recently proposed as an effective prevention strategy against candidosis recurrences, which affect 40-50% of women. In this context, the aim of the present work was the development of a mucoadhesive in situ gelling formulation for the vaginal administration of Lactobacillus gasseri. Mixtures of poloxamer 407 (P407) and methylcellulose (MC), two thermosensitive polymers, were prepared and subjected to rheological analyses for the assessment of their sol/gel transition temperature. The association of P407 (15% w/w) with MC (1.5% w/w) produced an increase in gelation extent at 37 °C even after dilution in simulated vaginal fluid (SVF). The presence of 0.5% w/w pectin (PEC) produced a reduction of vehicle pH and viscosity at 25 °C that is the vehicle resistance to flow during administration. The presence of a low concentration of xyloglucan (XYL) (0.25% w/w) increases the mucoadhesive properties and the capability to gelify at 37 °C of the formulation after dilution with SVF. A three-component (P407/MC/PEC; 3cM) and a four-component (P407/MC/PEC/XYL; 4cM) mixture were selected as promising candidates for the delivery of L. gasseri to the vaginal cavity. They were able to preserve L. gasseri viability and were cytocompatible towards the HeLa cell line.

Montmorillonite-norfloxacin nanocomposite intended for healing of infected wounds.

Background: Chronic cutaneous wounds represent a major issue in medical care and are often prone to infections. Purpose: The aim of this study was the design of a clay mineral-drug nanocomposite based on montmorillonite and norfloxacin (NF, antimicrobial drug) as a powder for cutaneous application, to enhance wound healing in infected skin lesions. Methods: The nanocomposite has been prepared by means of an intercalation solution procedure. Adsorption isotherm, solid-state characterization of the nanocomposite, drug loading capacity and its release have been performed. Moreover, cytocompatibility, in vitro fibroblast proliferation and antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus were assessed. Results: The clay drug adsorption isotherm demonstrates that the mechanism of NF intercalation into montmorillonite galleries is the adsorption as one single process, due to the charge-charge interaction between protonated NF and negatively charged montmorillonite edges in the interlayer space. Nanocomposite is biocompatible and it is characterized by antimicrobial activity greater than the free drug: this is due to its nanostructure and controlled drug release properties. Conclusion: Considering the results obtained, NF-montmorillonite nanocomposite seems a promising tool to treat infected skin lesions or skin wounds prone to infection, as chronic ulcers (diabetic foot, venous leg ulcers) and burns.

Chitosan/Glycosaminoglycan Scaffolds: The Role of Silver Nanoparticles to Control Microbial Infections in Wound Healing.

Cutaneous wounds represent a major issue in medical care, with approximately 300 million chronic and 100 million traumatic wound patients worldwide, and microbial infections slow the healing process. The aim of this work was to develop electrospun scaffolds loaded with silver nanoparticles (AgNPs) to enhance cutaneous healing, preventing wound infections. AgNPs were directly added to polymeric blends based on chitosan (CH) and pullulan (PUL) with hyaluronic acid (HA) or chondroitin sulfate (CS) to be electrospun obtaining nanofibrous scaffolds. Moreover, a scaffold based on CH and PUL and loaded with AgNPs was prepared as a comparison. The scaffolds were characterized by chemico-physical properties, enzymatic degradation, biocompatibility, and antimicrobial properties. All the scaffolds were based on nanofibers (diameters about 500 nm) and the presence of AgNPs was evidenced by TEM and did not modify their morphology. The scaffold degradation was proven by means of lysozyme. Moreover, the AgNPs loaded scaffolds were characterized by a good propensity to promote fibroblast proliferation, avoiding the toxic effect of silver. Furthermore, scaffolds preserved AgNP antimicrobial properties, although silver was entrapped into nanofibers. Chitosan/chondroitin sulfate scaffold loaded with AgNPs demonstrated promotion of fibroblast proliferation and to possess antimicrobial properties, thus representing an interesting tool for the treatment of chronic wounds.

Platelet lysate loaded electrospun scaffolds: Effect of nanofiber types on wound healing.

In healthy individuals, wound healing is a highly efficient process. However, interruptions of normal healing give rise to chronic wounds, characterized by inflammation with impaired angiogenesis and re-epithelialization. The aim of this work was the design and the development of electrospun nanofibrous scaffolds based on sodium alginate (SA) and pullulan (PUL) and loaded with human platelet lysate (PL) intended for skin reparation, to take the advantage of nanofibrous scaffolds (with improved physical structure) and of SA as biopolymer. Two preparation approaches have been used to load PL in the scaffolds: as component of the PUL/SA matrix, to be electrospun, or as coating component, to cover the previously prepared electrospun PUL based membranes. A preformulation study to assess pullulan entanglement concentration and alginate or citric acid critical concentration, to obtain electrospun nanofibers, has been performed. The preparation process allowed to obtain insoluble systems starting from aqueous solutions and these were able to act as scaffolds for tissue engineering with suitable mechanical properties and PL release. PL loading in PUL/SA matrix nanofibers did not substantially modify the nanofiber morphology before crosslinking, while the crosslinking process, in presence of PL, determined less sharp nanofibers probably due to an increase in hydrophilicity caused by PL proteins. On the contrary, the coated nanofibers showed an increase in diameters due to PL loading. The two different approaches affected the fiber dimension and scaffold elasticity, especially for PL loaded systems. Anyhow, these differences were not crucial for fibroblast adhesion and proliferation which were mainly influenced by PL loading. In particular, fibroblasts presented different conformation and orientation mainly due to the presence of PL. This caused a cell random orientation compatible to a fibroblast-to-myofibroblast transition that could enhance wound healing.

The role of chitosan as coating material for nanostructured lipid carriers for skin delivery of fucoxanthin.

Fucoxanthin (FUCO) is a marine carotenoid characterized by antiproliferative properties against hyperproliferative cells. The aim of this work was to design and develop nanostructured lipidic carriers (NLCs) based on bacuri butter and tucumã oil and loaded with FUCO, intended for skin application to prevent skin hyperproliferative diseases and in particular psoriasis. The presence of FUCO should control the hyperproliferation of skin diseased cells and the lipids forming the NLC core, rich in antioxidants and characterized by wound healing properties, should favor the restoring of skin integrity. NLCs were coated with chitosan (CS) to improve their biopharmaceutical properties (bio/mucoadhesion and wound healing) and to combine the advantages of lipidic nanoparticles with the biological properties of CS. Chitosan coated and non-coated NLC were prepared by means of high shear homogenization and characterized for chemico-physical and biopharmaceutical properties (in vitro biocompatibility and cell uptake towards normal dermal human fibroblasts). Moreover, the pharmacological activity of FUCO loaded in NLCs was assessed in psoriatic-like cellular model. NLCs were characterized by dimensions ranging from about 250 to 400 nm. Moreover, the CS coating and FUCO loading determined an increase of size. Moreover, TEM and zeta potential analysis confirmed the presence of CS coating on nanoparticle surface, thus conferring to nanoparticle good bioadhesion properties. NLCs uptake in fibroblasts was observed and NLC-FUCO-CS caused a reduction of cell viability with a less marked effect in fibroblasts rather than in psoriatic cells, highlighting the capability of this system to control skin hyperproliferation and inflammation. The loading of NLC-FUCO-CS in pullulan film should render NLCs application easy, without impair prompt interaction of the drug with the skin. Considering the overall results skin application of CS coated NLCs loaded with FUCO seems a promising approach to control skin hyperproliferation and to preserve skin integrity in psoriatic skin.

Chitosan/glycosaminoglycan scaffolds for skin reparation.

Burns and chronic wounds, often related to chronic diseases (as diabetes and cancer), are challenging lesions, difficult to heal. The prompt and full reconstitution of a functional skin is at the basis of the development of biopolymer-based scaffolds, representing a 3D substrate mimicking the dermal extracellular matrix. Aim of the work was to develop scaffolds intended for skin regeneration, according to: fabrication by electrospinning from aqueous polysaccharide solutions; prompt and easy treatment to obtain scaffolds insoluble in aqueous fluids; best performance in supporting wound healing. Three formulations were tested, based on chitosan (CH) and pullulan (P), associated with glycosaminoglycans (chondroitin sulfate - CS or hyaluronic acid - HA). A multidisciplinary approach has been used: chemico-physical characterization and preclinical evaluation allowed to obtain integrated information. This supports that CS gives distinctive properties and optimal features to the scaffold structure for promoting cell proliferation leading tissue reparation towards a complete skin restore.

Development of a Mucoadhesive and In Situ Gelling Formulation Based on κ-Carrageenan for Application on Oral Mucosa and Esophagus Walls. I. A Functional In Vitro Characterization.

Oral mucositis and esophagitis represent the most frequent and clinically significant complications of cytoreductive chemotherapy and radiotherapy, which severely compromise the patient quality of life. The local application of polymeric gels could protect the injured tissues, alleviating the most painful symptoms. The present work aims at developing in situ gelling formulations for the treatment of oral mucositis and esophagitis. To reach these targets, κ-carrageenan (κ-CG) was selected as a polymer having wound healing properties and able to gelify in the presence of saliva ions, while hydroxypropyl cellulose (HPC) was used to improve the mucoadhesive properties of the formulations. CaCl2 was identified as a salt able to enhance the interaction between κ-CG and saliva ions. Different salt and polymer concentrations were investigated in order to obtain a formulation having the following features: (i) low viscosity at room temperature to facilitate administration, (ii) marked elastic properties at 37 °C, functional to a protective action towards damaged tissues, and (iii) mucoadhesive properties. Prototypes characterized by different κ-CG, HPC, and CaCl2 concentrations were subjected to a thorough rheological characterization and to in vitro mucoadhesion and washability tests. The overall results pointed out the ability of the developed formulations to produce a gel able to interact with saliva ions and to adhere to the biological substrates.

Nanofiber Scaffolds as Drug Delivery Systems to Bridge Spinal Cord Injury.

The complex pathophysiology of spinal cord injury (SCI) may explain the current lack of an effective therapeutic approach for the regeneration of damaged neuronal cells and the recovery of motor functions. A primary mechanical injury in the spinal cord triggers a cascade of secondary events, which are involved in SCI instauration and progression. The aim of the present review is to provide an overview of the therapeutic neuro-protective and neuro-regenerative approaches, which involve the use of nanofibers as local drug delivery systems. Drugs released by nanofibers aim at preventing the cascade of secondary damage (neuro-protection), whereas nanofibrous structures are intended to re-establish neuronal connectivity through axonal sprouting (neuro-regeneration) promotion, in order to achieve a rapid functional recovery of spinal cord.

Comparison of poloxamer- and chitosan-based thermally sensitive gels for the treatment of vaginal mucositis.

CONTEXT: The local treatment of vaginal mucositis requires an intimate and prolonged contact of anti-infective drugs with the mucosa. This can be achieved by means of mucoadhesive and thermally sensitive vehicles, capable of gelifying at the physiological temperature. OBJECTIVE: The aim of the present work was to compare the potentiality of poloxamer 407 (PLX)/chitosan lactate (CS-L) and CS-L/glycerophosphate (GP) mixtures as mucoadhesive thermally sensitive vehicles for the treatment of vaginal mucositis. MATERIALS AND METHODS: PLX/CS-L and CS-L/GP mixtures were characterized for gelation and mucoadhesion properties as well as for bioactive (antimicrobial and wound healing) properties. Finally, the mixtures were loaded with amoxicillin trihydrate as model drug and characterized for drug release and washability properties. RESULTS AND DISCUSSION: The addition of CS-L to PLX causes an increase in PLX gelation temperature from 30 °C to the physiological temperature. The dilution with simulated vaginal fluid causes an increase in gelation time of PLX/CS-L mixture, while no variation of such parameter is observed for CS-L/GP mixture which is nevertheless characterized by poorer elastic properties. The stronger mucoadhesion properties of CS-L/GP mixture counterbalance the poorer elasticity of the gel and are responsible for a longer drug contact with the biological substrate. CS-L/GP mixture is moreover characterized by better bioactive properties than PLX-based mixture. CONCLUSION: CS-L/GP mixture represents a promising thermally sensitive vehicle.