NMR Metabolomics Assessment of Osteogenic Differentiation of Adipose-Tissue-Derived Mesenchymal Stem Cells.

This Article presents, for the first time to our knowledge, an untargeted nuclear magnetic resonance (NMR) metabolomic characterization of the polar intracellular metabolic adaptations of human adipose-derived mesenchymal stem cells during osteogenic differentiation. The use of mesenchymal stem cells (MSCs) for bone regeneration is a promising alternative to conventional bone grafts, and untargeted metabolomics may unveil novel metabolic information on the osteogenic differentiation of MSCs, allowing their behavior to be understood and monitored/guided toward effective therapies. Our results unveiled statistically relevant changes in the levels of just over 30 identified metabolites, illustrating a highly dynamic process with significant variations throughout the whole 21-day period of osteogenic differentiation, mainly involving amino acid metabolism and protein synthesis; energy metabolism and the roles of glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation; cell membrane metabolism; nucleotide metabolism (including the specific involvement of O-glycosylation intermediates and NAD+); and metabolic players in protective antioxidative mechanisms (such as glutathione and specific amino acids). Different metabolic stages are proposed and are supported by putative biochemical explanations for the metabolite changes observed. This work lays the groundwork for the use of untargeted NMR metabolomics to find potential metabolic markers of osteogenic differentiation efficacy.

Human Platelet Lysates-Based Hydrogels: A Novel Personalized 3D Platform for Spheroid Invasion Assessment.

Fundamental physiologic and pathologic phenomena such as wound healing and cancer metastasis are typically associated with the migration of cells through adjacent extracellular matrix. In recent years, advances in biomimetic materials have supported the progress in 3D cell culture and provided biomedical tools for the development of models to study spheroid invasiveness. Despite this, the exceptional biochemical and biomechanical properties of human-derived materials are poorly explored. Human methacryloyl platelet lysates (PLMA)-based hydrogels are herein proposed as reliable 3D platforms to sustain in vivo-like cell invasion mechanisms. A systematic analysis of spheroid viability, size, and invasiveness is performed in three biomimetic materials: PLMA hydrogels at three different concentrations, poly(ethylene glycol) diacrylate, and Matrigel. Results demonstrate that PLMA hydrogels perfectly support the recapitulation of the tumor invasion behavior of cancer cell lines (MG-63, SaOS-2, and A549) and human bone-marrow mesenchymal stem cell spheroids. The distinct invasiveness ability of each cell type is reflected in the PLMA hydrogels and, furthermore, different mechanical properties produce an altered invasive behavior. The herein presented human PLMA-based hydrogels could represent an opportunity to develop accurate cell invasiveness models and open up new possibilities for humanized and personalized high-throughput screening and validation of anticancer drugs.

Bilayered silk/silk-nanoCaP scaffolds for osteochondral tissue engineering: In vitro and in vivo assessment of biological performance.

Novel porous bilayered scaffolds, fully integrating a silk fibroin (SF) layer and a silk-nano calcium phosphate (silk-nanoCaP) layer for osteochondral defect (OCD) regeneration, were developed. Homogeneous porosity distribution was achieved in the scaffolds, with calcium phosphate phase only retained in the silk-nanoCaP layer. The scaffold presented compressive moduli of 0.4MPa in the wet state. Rabbit bone marrow mesenchymal stromal cells (RBMSCs) were cultured on the scaffolds, and good adhesion and proliferation were observed. The silk-nanoCaP layer showed a higher alkaline phosphatase level than the silk layer in osteogenic conditions. Subcutaneous implantation in rabbits demonstrated weak inflammation. In a rabbit knee critical size OCD model, the scaffolds firmly integrated into the host tissue. Histological and immunohistochemical analysis showed that collagen II positive cartilage and glycosaminoglycan regeneration presented in the silk layer, and de novo bone ingrowths and vessel formation were observed in the silk-nanoCaP layer. These bilayered scaffolds can therefore be promising candidates for OCD regeneration.

On-chip assessment of the protein-release profile from 3D hydrogel arrays.

As the formation of healthy tissue and the treatment of several diseases are often dependent on an effective and prolonged action of bioactive agents, the delivery of molecules for therapeutic or induction purposes in a tissue is a common procedure. The correct administration of those agents is often dependent on tailored delivery mechanisms from hydrogel or polymeric matrixes. To the best of our knowledge, methods for the high-throughput monitoring of bioactive agent delivery are nonexistent. The methods for the in vitro monitoring of molecule release are expensive and laborious. As a simple alternative to these methods, we propose the imprinting of superhydrophobic biomimetic surfaces with ring-shaped transparent spots with concentric superhydrophobic millimetric regions to be used as bioactive agent release study platforms. We designed an array where polymeric precursors mixed with a growth-factor model protein labeled with a fluorescent tag could be dispensed in the concentric highly repellent regions and cross-linked afterward, generating a polymeric protein-loaded sphere. The ring-shaped region was then filled with a physiological-like fluid that covered the polymeric sphere. The acquisition of sequential images of each spot over time using microscopy methods allowed one to easily monitor the protein release by image-based fluorescence quantification. As the platform is easily adaptable and amenable for future automation in order to mimic standardized organ dynamics, we concluded that the device shows applicability for rapid and efficient in vitro bioactive agent release studies.

Production methodologies of polymeric and hydrogel particles for drug delivery applications.

INTRODUCTION: Polymeric particles are ideal vehicles for controlled delivery applications due to their ability to encapsulate a variety of substances, namely low- and high-molecular mass therapeutics, antigens or DNA. Micro and nano scale spherical materials have been developed as carriers for therapies, using appropriated methodologies, in order to achieve a prolonged and controlled drug administration. AREAS COVERED: This paper reviews the methodologies used for the production of polymeric micro/nanoparticles. Emulsions, phase separation, spray drying, ionic gelation, polyelectrolyte complexation and supercritical fluids precipitation are all widely used processes for polymeric micro/nanoencapsulation. This paper also discusses the recent developments and patents reported in this field. Other less conventional methodologies are also described, such as the use of superhydrophobic substrates to produce hydrogel and polymeric particulate biomaterials. EXPERT OPINION: Polymeric drug delivery systems have gained increased importance due to the need for improving the efficiency and versatility of existing therapies. This allows the development of innovative concepts that could create more efficient systems, which in turn may address many healthcare needs worldwide. The existing methods to produce polymeric release systems have some critical drawbacks, which compromise the efficiency of these techniques. Improvements and development of new methodologies could be achieved by using multidisciplinary approaches and tools taken from other subjects, including nanotechnologies, biomimetics, tissue engineering, polymer science or microfluidics.

Macroporous hydroxyapatite scaffolds for bone tissue engineering applications: physicochemical characterization and assessment of rat bone marrow stromal cell viability.

In this work, a new methodology is reported for developing hydroxyapatite (HA) scaffolds using an organic sacrifice template. The novelty of work consists of possibility of obtaining porous and highly interconnected scaffolds mimicking the sacrificial component. Our purpose consisted of evaluating the physicochemical properties of the HA scaffolds by means of Fourier transform infra-red spectroscopy, X-ray diffraction analysis, and scanning electron microscopy (SEM) attached with an X-ray detector. The HA scaffolds obtained possess a porosity of approximately 70%, and macropores diameter in the range of 50-600 microm. In contrast, results regarding the microcomputed tomography analysis have demonstrated both high pore uniformity and interconnectivity across the scaffolds. The compressive strength of the HA scaffolds was found to be 30.2 +/- 6.0 MPa. Bioactivity of the HA scaffolds was assessed by immersion into a simulated body fluid solution, in vitro. SEM observations have showed a deposition of apatite on the surface of the HA scaffolds, with a "cauliflower-like" morphology after 1 day, and tend to be more pronounced with the immersion time. The changes in calcium and phosphorus concentration were monitored by inductively-coupled plasma optical emission spectrometry. Cytotoxicity of the HA scaffolds was preliminarily investigated by carrying direct observation of mouse fibroblasts cells (L929 cell-line) death in the inverted microscope, and then cell viability was determined by means of carrying out a MTS assay. Complementarily, a luminescent cell viability assay based on the quantification of adenosine triphosphate was performed using rat bone marrow stromal cells (RBMSCs). A LIVE/DEAD assay and SEM analysis allowed the visualization of the RBMSCs adhesion and proliferation on the surface of the HA scaffolds. According to the results obtained from 3D architecture, mechanical properties, biocompatibility, and adhesion tests, it is suggested that HA scaffolds has potential to find applications in bone tissue engineering scaffolding.