Multilayer fibroin/chitosan oligosaccharide lactate and pullulan immunomodulatory patch for treatment of hernia and prevention of intraperitoneal adhesion.

This study aims to develop a novel intraperitoneal two- or three-layered patch with immunomodulatory property for treatment of hernia, regeneration of abdominal wall and prevention of intraperitoneal adhesions. Polypropylene (PP) mesh, middle layer, was intended to provide mechanical support whereas pullulan (PUL) hydrogel coating layer was designed to prevent intraperitoneal adhesions. Fibroin/chitosan oligosaccharide lactate (F/COS) layer electrospun on one side of pullulan was chosen for immunomodulation and abdominal wall regeneration. Physical and mechanical properties and regenerative capacity of intraperitoneal patches were determined. Immunomodulatory property of electrospun layer and whole patch was studied by determining nitric oxide amount produced by RAW 264.7 macrophages. 25 % (w/v) PUL hydrogel and F/COS with 90:10 (w/w) ratio yielded optimal results. Here, we report that fabricated intraperitoneal patches successfully prevented cell adhesion on one side and increased cell viability and proliferation on other side, along with immunomodulation, in vitro.

Double entrapment of growth factors by nanoparticles loaded into polyelectrolyte multilayer films.

Delivery of growth factors and control of vascularization are prominent problems in regenerative medicine. Vascular endothelial growth factor (VEGF) has been used both in vitro and in vivo to promote angiogenesis but due to its short half-life its controlled delivery is a sought after method. In this study we present a new concept of degradable drug loaded nanoparticles entrapped into exponentially growing multilayer films. Through hydrolysis of the nanoparticles, the drug can be delivered over long periods in a controlled manner. Poly(ε-caprolactone) nanoparticles were loaded with VEGF and in turn the release of VEGF from a surface is controlled by a thick layer-by-layer polyelectrolyte film. Direct loading of VEGF inside the film was not efficient for long-term applications. When VEGF loaded nanoparticles were introduced into the film, the particles were equally distributed inside and were stable after several washes. Moreover, the presence of the film sustained the release of VEGF for 7 days. Addition of the nanoparticles to the film promoted endothelial cell proliferation, mainly due to the presence of VEGF. Mechanical properties of the film (Young's moduli) were also improved by the presence of nanoparticles. However, in the presence of the film loaded with nanoparticles and without any direct contact with this film, endothelial cell growth was also enhanced on polystyrene and on Transwell insert surfaces which demonstrates the effectiveness of the nanoparticles not only to improve the mechanical properties of the film but also to deliver active VEGF. An increase in nitric oxide levels as an indicator of endothelial cell activity was monitored and was correlated with the release of VEGF from the nanoparticle/film platform. Finally, such a system can be used as an auxiliary delivery body within implants to finely control the release of bioactive agent containing nanoparticles.

Microstructure, microhardness, and biocompatibility characteristics of yttrium hydroxyapatite doped with fluoride.

The current study focused on doping of hydroxyapatite (HA) with constant yttrium (Y(3+) ) and varying fluoride (F(-) ) compositions to investigate its microstructure, microhardness, and biocompatibility. HA was synthesized by precipitation method and sintered at 1100°C for 1 h. Y(3+) and F(-) ion dopings resulted in changes in densities. In x-ray diffraction analysis, no secondary phase formation was observed. Lattice parameters decreased upon ion substitutions. Scanning electron microscopy (SEM) results showed that ion addition resulted in smaller grains. In Fourier transform infrared spectroscopy analysis, F(-) ion substitution was confirmed. HA doped with 2.5% Y(3+) and 1% F(-) exhibited the highest microhardness. Y(3+) and F(-) ions improved Saos-2 cell proliferation on discs in Methylthiazolyldiphenyl-tetrazolium (MTT) assay. In SEM analysis, cells attached and proliferated on all disc surfaces. Alkaline phosphatase (ALP) assay showed that cell differentiation on the discs was improved by doping HA with an optimum F(-) amount. Dissolution tests revealed that structural stability of HA was improved with F(-) ion incorporation. The dissolution behavior of fluoridated samples exhibited a parallel pattern with the cell proliferation and differentiation behavior on these samples. Overall, this work shows that fluoride and yttrium cosubstitution into HA HA2.5Y1F was the most promising material for biomedical applications.

In vitro and in vivo evaluation of the effects of demineralized bone matrix or calcium sulfate addition to polycaprolactone-bioglass composites.

The objective of this study was to improve the efficacy of polycaprolactone/bioglass (PCL/BG) bone substitute using demineralized bone matrix (DBM) or calcium sulfate (CS) as a third component. Composite discs involving either DBM or CS were prepared by compression moulding. Bioactivity of discs was evaluated by energy dispersive X-ray spectroscopy (ESCA) and scanning electron microscopy (SEM) following simulated body fluid incubation. The closest Calcium/Phosphate ratio to that of hydroxyl carbonate apatite crystals was observed for PCL/ BG/DBM group (1.53) after 15 day incubation. Addition of fillers increased microhardness and compressive modulus of discs. However, after 4 and 6-week PBS incubations, PCL/BG/DBM discs showed significant decrease in modulus (from 266.23 to 54.04 and 33.45 MPa, respectively) in parallel with its highest water uptakes (36.3 and 34.7%). Discs preserved their integrity with only considerable weight loss (7.5-14.5%) in PCL/BG/DBM group. In vitro cytotoxicity tests showed that all discs were biocompatible.

Adipocyte differentiation defect in mesenchymal stromal cells of patients with malignant infantile osteopetrosis.

BACKGROUND: Malignant infantile osteopetrosis (MIOP) is a disorder of osteoclasts characterized by defective bone resorption and death in infancy. The multipotent mesenchymal stromal cells (MSC) and their progeny (osteoblasts) are major components of the bone marrow (BM) microenvironment and are found in close contact with cells of hematopoietic origin, including osteoclasts. We hypothesized that MSC defects may be associated with osteoclast dysfunction and osteopetrosis phenotype. METHODS: BM MSC, obtained from six patients with MIOP, were expanded in vitro and characterized by morphology, plastic-adherence, immunophenotype and multilineage differentiation potential. RESULTS: Physical and immunophenotypic characteristics of patient MSC were similar to healthy age-matched controls. However, an isolated in vitro differentiation defect toward adipogenic lineage was demonstrated in patient MSC and confirmed by low or absent expression of adipogenic transcripts (peroxisome proliferator-activated receptor-gamma, adipophilin, stearoyl-CoA desaturase, leptin and adiponectin) upon induction of adipogenesis. Following BM transplantation, minimal improvement in adipogenic potency of MSC was demonstrated by Oil Red O staining. DISCUSSION: MIOP is associated in vitro with a failure of MSC to differentiate into an adipogenic lineage, suggesting a BM microenvironment defect. The defect may contribute to osteoclast dysfunction, or may be attributed to the effect of the osteopetrotic marrow environment. Further investigations should determine the pathophysiologic importance of this novel defect, and could perhaps contribute to consideration of MSC therapy in MIOP.

A novel construct as a cell carrier for tissue engineering.

A 3D scaffold, in the form of a foam, with the top surface carrying a micropattern, was constructed from biodegradable polyesters poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) and poly(L-lactide-co-D,L-lactide) (P(L/DL)LA) to serve as a substitute for the extracellular matrix (ECM) of tissues with more than one cell type. The construct was tested in vitro for engineering of such tissues using fibroblasts (3T3) and epithelial cells (retinal pigment epithelial cells, D407). The patterned surface was seeded with D407 cells and the foam was seeded with 3T3 cells to represent a tissue with two different cell types. To improve cell adhesion, the construct was treated with fibronectin. The cells were seeded on the construct in a sequence allowing each type time for adhesion. Cell proliferation, studied by MTS assay, was significantly higher than that of tissue culture polystyrene control by day 14. Scanning electron and fluorescence microscopy showed that the foam side of the construct was highly porous and the pores were interconnected and this allowed cell mobility and proliferation. Immunostaining showed collagen deposition, indicating the secretion of the new ECM by the cells. On the film side of the construct D407 cells formed piles in the grooves and covered the surface completely. It was concluded that the 3D P(L/DL)LA-PHBV construct with one micropatterned surface has a serious potential for use as a tissue engineering carrier in the reconstruction of complex tissues with layered organization and different types of cells in each region.

In vitro characterization of micropatterned PLGA-PHBV8 blend films as temporary scaffolds for photoreceptor cells.

In developed countries the aging population faces increasing risks of blinding retinal diseases, for which there are few effective treatments available. Photoreceptor transplantation represents one approach, but generally results have been disappointing. We hypothesize that micropatterned biodegradable poly(L-lactic acid-co-glycolic acid)/poly(hydroxybutyrate-co-hydroxyvaleric acid) (PLGA-PHBV8) blend films could deliver photoreceptor cells in a more organized manner than bolus injections. Blending of PLGA and PHBV8 was used to optimize the degradation rate of the temporary template. At the end of 8 weeks, for both thin and thick films of PLGA-PHBV8 a 50% decrease of their initial weight with increasing water uptake was observed. When photoreceptor cells were seeded onto micropatterned PLGA-PHBV8 films with parallel grooves (21- and 42-microm-wide grooves and 20 microm ridge width and depth), the cells preferred laminin-deposited grooves to ridges and expressed rod- and cone-specific markers such as rhodopsin and arrestin. A loss in photoreceptor viability of 50% was observed after 7 days in culture. The effects of either retinal pigment epithelium (RPE)-derived or Muller glial cell-derived conditioned media or bFGF on the survival of photoreceptor cells seeded on PLGA-PHBV8 films were investigated. Addition of either RPE- and Muller-conditioned media increased statistically (p < 0.01) the viability of photoreceptor cells after 7 days of incubation. Our results suggest that such biodegradable micropatterned PLGA-PHBV8 blend films have a potential to deliver photoreceptor cells to the subretinal space and ensure laminar organization and maintenance of differentiation, and that incorporation of intrinsic factors within the scaffold would enhance the survival rate of transplanted cells.

Cornea engineering on polyester carriers.

In this study, biodegradable polyester based carriers were designed for tissue engineering of the epithelial and the stromal layers of the cornea, and the final construct was tested in vitro. In the construction of the epithelial layer, micropatterned films were prepared from blends of biodegradable and biocompatible polyesters of natural (PHBV) and synthetic (P(L/DL)LA) origin, and these films were seeded with D407 (retinal pigment epithelial) cells. To improve cell adhesion and growth, the films were coated with fibronectin. To serve as the stromal layer of the cornea, highly porous foams of P(L/DL)LA-PHBV blends were seeded with 3T3 fibroblasts. Cell numbers on the polyester carriers were significantly higher than those on the tissue culture polystyrene control. The cells and the carriers were characterized scanning electron micrographs showed that the foam was highly porous and the pores were interconnected. 3T3 Fibroblasts were distributed quite homogeneously at the seeding site, but probably because of the high thickness of the carrier ( approximately 6 mm); they could not sufficiently populate the core (central parts of the foam) during the test duration. The D407 cells formed multilayers on the micropatterned polyester film. Immunohistochemical studies showed that the cells retained their phenotype during culturing; D407 cells formed tight junctions characteristic of epithelial cells, and 3T3 cells deposited collagen type I into the foams. On the basis of these results, we concluded that the micropatterned films and the foams made of P(L/DL)LA-PHBV blends have a serious potential as tissue engineering carriers for the reconstruction of the epithelial and stromal layers of the cornea.

Retinal pigment epithelium cell culture on surface modified poly(hydroxybutyrate-co-hydroxyvalerate) thin films.

There is currently no effective treatment for the retinal disorders caused by retinal pigment epithelium (RPE) degeneration. Transplantation of allografts is the main strategy towards correction of this malady. Tissue engineering could offer hope and involve the use of biodegradable polymeric templates to replace diseased or lost RPE. In this study PHBV8 film was chosen as a temporary substrate for growing retinal pigment epithelium cells as an organized monolayer before their subretinal transplantation. The surface of the PHBV8 film was rendered hydrophilic by oxygen plasma treatment to increase the reattachment of D407 cells on the film surface. Power and duration was changed, from 50 W, 10 min to 100 W, 20 min during plasma treatment. The effect of these two parameters on surface hydrophilicity, morphology, topography, surface composition of PHBV8 thin films was studied using AFM, SEM, and phase contrast microscopy. The effect of changes in surface characteristics on cell reattachment, spreading and cell growth rate was investigated. It was found that as the treatment level was increased the surface hydrophilicity increased and roughness was decreased probably due to ablation. The PHBV8 film treated with 100 W 10 min was found to be the most suitable for 24 h reattachment of D407 cells. The cells were also grown to confluency as an organized monolayer suggesting PHBV8 film as a potential temporary substrate for subretinal transplantation to replace diseased or damaged retinal pigment epithelium.