Marco Antonio Alvarez-Perez

Influence of Gelatin Cues in PCL Electrospun Membranes on Nerve Outgrowth

The design of functionalized polymers that can elicit specific biological responses and the development of methods to fabricate new devices that incorporate biological cues are of great interest to the biomedical community. The realization of nanostructured matrices that exhibit biological properties and that comprise fibers with diameters of similar scale to those of the natural extracellular matrix (ECM) would enable the provision of tailored materials for tissue engineering. Accordingly, the goal of this work is to create a biologically active functionalized electrospun matrix capable of guiding neurite growth for the regeneration of nerve tissue. In this study, nanoscale electrospun membranes made of poly ε-caprolactone enhanced with gelatin from calf skin were investigated to validate their biological response under in vitro culture of PC-12 nerve cells. Preliminary observations from SEM studies supported by image analysis highlighted the nanoscale texture of the scaffold with fiber diameters equal to 0.548 ± 0.140 μm. In addition, contact angle measurements confirmed the hydrophilic behavior of the membranes, ascribable to the gelatin content. We demonstrate that the balance of morphological and biochemical properties improves all the fundamental biological events of nerve regeneration, enhancing cell adhesion, proliferation, and differentiation in comparison with PCL nanofibrous scaffolds, as well as supporting the neurite outgrowth.

Conductive PANi/PEGDA Macroporous Hydrogels For Nerve Regeneration

Only recently polymers with intrinsic conductive properties have been studied in relation to their incorporation into bioactive scaffolds for use in tissue engineering. The reason for this interest is that such scaffolds could electrically stimulate cells and thus regulate specific cellular activities, and by this means influence the process of regeneration of those tissues that respond to electrical impulses. In our work, macroporous hydrogels are developed with controlled pore morphology and conductive properties to enable sufficient cell signaling to supply events inherent to nerve regeneration. A hybrid material has been prepared by in situ precipitation of polyaniline (PANi) in polyethyleneglycol diacrylate (PEGDA) solution, followed by crosslinking via UV irradiation. A porous architecture, characterized by macropores from 136 μm to 158 μm in size, has been achieved by sodium chloride particle leaching. In this work, we demonstrate that PANi synthesis and hydrogel crosslinking combine to enable the design of materials with suitable conductive behaviour. The presence of PANi evidently increased the electrical conductivity of the hybrid material from (1.1 ± 0.5) × 10(-3) mS/cm with a PANi content of 3wt%. The hydrophilic nature of PANi also enhanced water retention/proton conductivity by more than one order of magnitude. In vitro studies confirmed that 3 wt% PANi also improve the biological response of PC12 and hMSC cells. Hybrid PANi/PEGDA macroporous hydrogels supplement new functionalities in terms of morphological and conductive properties, both of which are essential prerequisites to drive nerve cells in regenerative processes.

hMSC interaction with PCL and PCL/gelatin platforms: A comparative study on films and electrospun membranes:

Polycaprolactone (PCL) and PCL/gelatin membranes and films were fabricated by electrospinning and solvent casting. A systematic analysis of the morphology evolution, as degradation occurred, was made to separate the contribution of fiber nanotexture and gelatin biochemical signal on cell adhesion and proliferation. Field emission scanning electron microscope was used to assess the contribution of platform architecture on the gelatin degradation by the morphological changes that occurred at different times. The evaluation of human mesenchymal stem cells’ biocompatibility confirmed the role of architecture and chemical composition on cell response. The nanostructured surfaces positively affected the cell recognition by increasing the surface area. The gelatin embedded in the PCL matrix of the nanofibers improved the cell/material interaction and provided support to the proliferation. The PCL/gelatin electrospun membranes showed an increase in mineralization when conditioned in osteogenic medium; this system has promise for long-term in vitro investigations of bone regeneration.

Tuning Size Scale and Crystallinity of PCL Electrospun Fibres via Solvent Permittivity to Address hMSC Response

The effect of solvent permittivity on the fibre morphology of PCL electrospun membranes for tissue engineering applications is studied. Morphological results indicate that polar solvents with higher permittivity are able to promote the formation of sub-micrometric fibres, while apolar solvents yield microfibres with an average fibre diameter of 2.86 ± 0.31 µm. Polymer/solvent interactions and electrospinning process parameters influence the mechanism of fibre and bead formation. It is shown that the dielectric properties of solvents influence the fibre size scale and crystallinity and directly contribute to the biological response of stem cells. Solvent permittivity is a key factor in controlling the morphological and physical properties of electrospun fibre meshes.

Immunolocalization of a Human Cementoblastoma-conditioned Medium-derived Protein

Little is known about the molecular mechanisms that regulate the cementogenesis process, because specific cementum markers are not yet available. To investigate whether a cementoblastoma-conditioned medium-derived protein (CP) could be useful as a cementum biological marker, we studied its expression and distribution in human periodontal tissues, human periodontal ligament, alveolar bone, and cementoblastoma-derived cells. In human periodontal tissues, immunoreactivity to anti-CP was observed throughout the cementoid phase of acellular and cellular cementum, cementoblasts, cementocytes, cells located in the endosteal spaces of human alveolar bone, and in cells in the periodontal ligament located near the blood vessels. Immunopurified CP promoted cell attachment on human periodontal ligament, alveolar bone-derived cells, and gingival fibroblasts. A monoclonal antibody against bovine cementum attachment protein (CAP) cross-reacted with CP. These findings indicate that CP identifies potential cementoblast progenitor cells, is immunologically related to CAP species, and serves as a biological marker for cementum.

Correlation between oxidative stress and alteration of intracellular calcium handling in isoproterenol-induced myocardial infarction

Myocardial Ca2+ overload and oxidative stress are well documented effects associated to isoproterenol (ISO)-induced myocardial necrosis, but information correlating these two issues is scarce. Using an ISO-induced myocardial infarction model, 3 stages of myocardial damage were defined: pre-infarction (0–12 h), infarction (12–24 h) and post-infarction (24–96 h). Alterations in Ca2+ homeostasis and oxidative stress were studied in mitochondria, sarcoplasmic reticulum and plasmalemma by measuring the Ca2+ content, the activity of Ca2+ handling proteins, and by quantifying TBARs, nitric oxide (NO) and oxidative protein damage (changes in carbonyl and thiol groups). Free radicals generated system, antioxidant enzymes and oxidative stress (GSH/GSSG ratio) were also monitored at different times of ISO-induced cardiotoxicity. The Ca2+ overload induced by ISO was counterbalanced by a diminution in the ryanodine receptor activity and the Na+-Ca+2 exchanger as well as by the increase in both calcium ATPases activities (vanadate- and thapsigargine-sensitive) and mitochondrial Ca2+ uptake during pre-infarction and infarction stages. Pro-oxidative reactions and antioxidant defences during the 3 stages of cardiotoxicity were observed, with maximal oxidative stress during the infarction. Significant correlations were found among pro-oxidative reactions with plasmalemma and sarcoplasmic reticulum Ca2+ ATPases, and ryanodine receptor activities at the onset and development of ISO-induced infarction. These findings could be helpful in the design of antioxidant therapies in this pathology.

Trapping tetracycline-loaded nanoparticles into polycaprolactone fiber networks for periodontal regeneration therapy

The controlled delivery of antibiotics, anti-inflammatory agents, or chemotherapeutic agents to the periodontal site is a recognized strategy to improve the efficiency of regenerative processes of hard tissues. A novel approach based on the trapping of tetracycline hydrochloride–loaded particles in polycaprolactone nanofibers was used to guide the regeneration processes of periodontal tissue at the gum interface. Chitosan nanoparticles loaded with different levels of tetracycline hydrochloride (up to 5% wt) were prepared by solution nebulization induced by electrical forces (i.e. electrospraying). The fine tuning of process parameters allows to obtain nanoparticles with tailored sizes ranging from 0.485 ± 0.147 µm to 0.639 ± 0.154 µm. The tetracycline hydrochloride release profile had a predominant burst effect for the first 70% of release followed by a relatively slow release over 24 h, which is promising for oral drug delivery. We also demonstrated that trapping tetracycline hydrochloride–loaded particles with submicrometer diameters into a polycaprolactone fiber network contributed to slowing the release of tetracycline hydrochloride from the nanoparticles, thus providing a more prolonged release in the periodontal pocket during clinical therapy. Preliminary studies on human mesenchymal stem cells confirm the viability of cells up to 5 days after culture, and thereby, validate the use of nanoparticle-/nanofiber-integrated systems in periodontal therapies.

Effect of citric acid crosslinking cellulose-based hydrogels on osteogenic differentiation

Understanding the relationships between material surface properties and cellular responses is essential to designing optimal material surfaces for implantation and tissue engineering. In this study, cellulose hydrogels were crosslinked using a non-toxic and natural component namely citric acid. The chemical treatment induces COOH functional groups that improve the hydrophilicity, roughness, and materials rheological properties. The physiochemical, morphological, and mechanical analyses were performed to analyze the material surface before and after crosslinking. This approach would help determine if the effect of chemical treatment on cellulose hydrogel improves the hydrophilicity, roughness, and rheological properties of the scaffold. In this study, it was demonstrated that the biological responses of human mesenchymal stem cell with regard to cell adhesion, proliferation, and differentiation were influenced in vitro by changing the surface chemistry and roughness.

Proliferation and osteoblastic differentiation of hMSCs on cellulose-based hydrogels

Purpose The aim of this project was to study the proliferation and differentiation of human Mesenchymal Stem Cells (hMSCs) onto a cellulose-based hydrogel for bone tissue engineering. Methods Modified-cellulose hydrogel was prepared via double esterification crosslinking using citric acid. The response of hMSCs in terms of cell proliferation and differentiation into osteoblastic phenotype was evaluated by using Alamar blue™ assay and alkaline phosphatase (ALP) activity. Results The results showed that CMCNa and CMCNa_CA have no negative effect on hMSC, adhesion and proliferation. Moreover, the increase of the ALP expression for CMCNa_CA confirms the ability of the hydrogels to support the osteoblastic differentiation. Conclusions The cellulose-based hydrogels have a potential application as filler in bone tissue regeneration.

MgCHA particles dispersion in porous PCL scaffolds: in vitro mineralization and in vivo bone formation.

In this work, we focus on the in vitro and in vivo response of composite scaffolds obtained by incorporating Mg,CO3‐doped hydroxyapatite (HA) particles in poly(ε‐caprolactone) (PCL) porous matrices. After a complete analysis of chemical and physical properties of synthesized particles (i.e. SEM/EDS, DSC, XRD and FTIR), we demonstrate that the Mg,CO3 doping influences the surface wettability with implications upon cell–material interaction and new bone formation mechanisms. In particular, ion substitution in apatite crystals positively influences the early in vitro cellular response of human mesenchymal stem cells (hMSCs), i.e. adhesion and proliferation, and promotes an extensive mineralization of the scaffold in osteogenic medium, thus conforming to a more faithful reproduction of the native bone environment than undoped HA particles, used as control in PCL matrices. Furthermore, we demonstrate that Mg,CO3‐doped HA in PCL scaffolds support the in vivo cellular response by inducing neo‐bone formation as early as 2 months post‐implantation, and abundant mature bone tissue at the sixth month, with a lamellar structure and completely formed bone marrow. Together, these results indicate that Mg2+ and CO32– ion substitution in HA particles enhances the scaffold properties, providing the right chemical signals to combine with morphological requirements (i.e. pore size, shape and interconnectivity) to drive osteogenic response in scaffold‐aided bone regeneration. Copyright