Ioannis Manousakas

Preparation and characterization of hyaluronan/collagen II microspheres under an electrostatic field system with disc electrodes.

Collagen II and hyaluronan are the two major components of the native extracellular matrix (ECM). Both biopolymers are responsible for providing the associated tissues with tensile strength, and also serve as a structural scaffold for cell adhesion and growth. Over the years, many researchers have focused on the preparation and evaluation of man-made ECM comprising the two polymers in the form of a membrane for chondrocyte culture applications. Here, a simple and in situ method, involving the injection of the hyaluronan/collagen II (HA/Col II) mixture solution through a pair of hollow-centered parallel disc electrodes (HCPDEs) of a high-voltage electrostatic field system, was developed and employed to prepare HA/Col II microspheres in watery phase. The HA/Col II microspheres were firmed up by a two-step cross-linking treatment (first by FeCl(3) and then by 1-ethyl-3-(3-dimethyl aminopropyl) carbodimide, EDC) to secure the spherical structure shape. Then, at 37 degrees C, reconstitution treatment of the Col II molecules was conducted to further strengthen the microspheres. Depending on treatment conditions, the resulting series of HA/Col II microspheres all exhibited good sphericity in the range of 486+/-43 to 679+/-24microm in diameter. Furthermore, the ratio and amount of HA/Col II in the mixture solutions would affect the morphological structure and basic characteristics, including mechanical strength, thermal properties and water content. In the preliminary study, the HA/Col II microspheres have shown to provide favorable ECM characteristics, with appropriate mechanical strength, and exhibited a 3D inclination.

EVALUATION OF CHITOSAN/CaSO4/PLATELET-RICH PLASMA MICROSPHERE COMPOSITES AS ALVEOLUS OSTEOGENESIS MATERIAL

Periodontal disease is the manifestation of serious bacteria infection that may extend to the gingival, periodontal ligaments, and alveolus bone. One commonly administrated treatment is the debridement therapy with the removal of infected area including the soft and hard lesion tissues. In some critical case, osteogenetic materials are being filled into the defective voids to improve the regeneration of slow-growing bony tissues. In attempt to improve bone regeneration, chitosan microsphere composites embedded with two osteogenesis beneficial ingredients, CaSO4 and platelet-rich plasma (PRP), were fabricated by using a high voltage electrostatic field system. Three groups, chitosan/CaSO4 microspheres (Group A), chitosan/CaSO4 microspheres mixed with thrombin (Group B), and chitosan/CaSO4/PRP microspheres mixed with thrombin (Group C) were prepared. And, these chitosan-based composites were evaluated together with a control group in pig oral model for the bone regeneration study. The chitosan/CaSO4/PRP microsphere composites, exhibiting good sphericity, were in the range of 457.5 ± 59.3 μm in diameter. Defects filled with Group B and Group C showed increases in new bone formation along with fibrous tissue regeneration as compared to that filled with Group A. The Massons Trichrome stain observations suggested more abundant presence of fibrous collagen matrices around the defects after implanted with Group B over that of Group C microsphere composites. The preparation of chitosan/CaSO4-based microspheres was straight forward by using high voltage electrostatic field system. Furthermore, Chitosan/CaSO4-based microspheres with thrombin could be used successfully in regenerating new bone around the alveolus bone area.

Preparation of Nano-Sized Collagen I and Collagen II Particles

This pilot study describes a new method to prepare nano-sized collagen type I and collagen type II particles by using a high-voltage electrostatic field system. Observations from transmission electron microscopy showed that, the nano-sized collagen II particles with a range of 150 nm in diameter could be produced with a good spherical shape under the experimental setting of 2.7~4.5 kVcm-1, under 23degC and a moderate concentration of collagen II solution at 0.2 mg/ml. When the treatment temperature increased to 30degC or higher, collagen II began to lose the tendency to individually separated spherical shaped nano-sized particles, instead, the fibrillogenesis of collagen II occurred by the spontaneous reconstitution of collagen II and then formed collagen II fibrils. More strict experimental settings were necessary for preparing nano-sized collagen I particles, that are, lower temperature setting (under 7degC environment), lower collagen concentration (0.1 mg/ml) and lower applied voltage (2.5 kVcm-1). Moreover, these prepared nano-sized collagen I particles exhibited smaller size in diameter (100 nm) but poorer sphericity than collagen II particles. They often aggregated together and exhibited an irregular outer shape.

The production of volvox spheres and their potential application in multi-drugs encapsulation and release.

Volvox sphere is a bio-mimicking concept of an innovative biomaterial structure of a sphere that contains smaller microspheres which then encapsulate chemicals, drugs and/or cells. The volvox spheres were produced via a high-voltage electrostatic field system, using alginate as the primary material. Encapsulated materials tested in this study include staining dyes, nuclear fast red and trypan blue, and model drugs, bovine serum albumin (BSA) and cytochrome c (CytC). The external morphology of the volvox spheres was observed via electron microscopy whereas the internal structure of the volvox spheres was observed via an optical microscope with the aid of the staining dyes, since alginate is colorless and transparent. The diameter of the microspheres was about 200 to 300 μm, whereas the diameter of the volvox spheres was about 1500 μm. Volvox spheres were durable, retaining about 95% of their mass after 4 weeks. Factors affecting entrapment efficiency, such as temperature and concentration of the bivalent cross-linker, were compared followed by a 7-day in vitro release study. The encapsulation efficiency of CytC within the microspheres was higher at cold (~4°C) and warm (~50°C) temperatures whereas temperature has no obvious effect on the BSA encapsulation. High crosslinking concentration (25% w/v) of calcium chloride has resulted higher entrapment efficiency for BSA but not for CytC. Furthermore, volvox spheres showed a different release pattern of BSA and CytC when compared to microspheres encapsulating BSA and CytC. Despite the fact that the mechanisms behind remain unclear and further investigation is required, this study demonstrates the potential of the volvox spheres for drug delivery.

A 3D ultrasound renal calculi fragmentation image analysis system for extracorporeal shock wave lithotripsy

Extracorporeal shock wave lithotripsy (ESWL) is the most effective and common practice for the treatment of renal calculi. After the treatment X-ray or ultrasound images are used to visually estimate the fragmentation of the stone. Unfortunately, up to now, there is not any method for quantitative estimation of the fragmentation level from ultrasound images. Conventional ultrasound image systems acquire two dimensional images. In this study we propose that such two dimensional images can assemble three dimensional images acquired along the duration of the treatment and apply Grey-Level Co-occurrence Matrix (GLCM) texture measurements to produce a fragmentation level measurement figure. Measurements with phantoms and simulated stone models were used to demonstrate the effectiveness of the proposed method. The results showed that there are significant variations between different texture features but different fragmentation levels can be correlated to different values of the texture features.

Alternative approach of cell encapsulation by Volvox spheres

Volvox sphere is a bio-mimicking concept of a biomaterial structure design able to encapsulate chemicals, drugs and/or cells. The aim of this study was to prepare Volvox spheres encapsulating AML12 liver cells and mesenchymal stem cells (MSCs) via a high voltage electrostatic field system. The results demonstrated that AML12 liver cells and MSCs could be successfully encapsulated into the inner spheres and the outer sphere of the Volvox spheres. The improved cell viability of MSCs was achieved by the addition of collagen and polyethylene glycol into the preparation components of the Volvox spheres. Collagen material potentially provides extracellular matrix-like structure for cell adhesion while polyethylene glycol provides a void/loose space for permeability of metabolites. The encapsulated MSCs were able to differentiate into hepatocytes or hepatocyte-like cells and express liver cell markers including albumin, alpha feto-protein and cytokeratin 18. The encapsulated cells secreted albumin to about 140 ng on day 14. Based on these observations, we conclude that Volvox spheres can be used as an alternative approach to encapsulate multiple types of cells, here AML12 hepatocyte cell line and MSCs. Nevertheless, efforts are still needed to improve the viability of the encapsulated cells and increase the differentiation of MSCs into functional liver cells.

In Vitro Study of the Revised Ultrasound Based Real-Time Tracking of Renal Stones for Shock Wave Lithotripsy: Part 1

PURPOSE Extracorporeal shock wave lithotripsy has been popular since the 1980s. Only 30% to 50% of the shock waves of all conventional lithotripters are focused on stones. We developed an ultrasound based, real-time stone tracking system (version 1) to improve accuracy and treatment efficiency. However, some problems remained. We revised the existing system (version 2) and tested its reliability and performance. MATERIALS AND METHODS We revised the system by adding more algorithms to decrease renal stone misidentification. We verified the advanced system by 2 tests using no tracking and tracking with 13 stone trajectories for versions 1 and 2. We performed the coincidence test to evaluate the accuracy of targeting the stone within the effective focal area and the stone fragmentation efficiency test to clarify the decrease in the number of shocks needed for stone fragmentation. RESULTS In the coincidence test the mean ± SD results of the nontracking system, and tracking versions 1 and 2 were 68.8% ± 18.8%, 89.9% ± 5.2% and 94.3% ± 4.5%, respectively. Version 2 was statistically significantly better than version 1 (p = 1.5 × 10(-4)). In the stone fragmentation efficiency test the mean results of the nontracking system, and versions 1 and 2 were 49.5% ± 14.2%, 85.1% ± 4.5% and 89.5% ± 4.2%, respectively. Version 2 was statistically significantly better than version 1 (p = 1.9 × 10(-8)). CONCLUSIONS The revised tracking system is better than version 1. It improves treatment efficiency, decreases stone misidentification and can shorten treatment time.

Preparation of Biopolymers Nanoparticles by Ultrasound Atomization

Microsphere particles have been introduced in a wide range of bio-medical applications, such as controlled drug delivery system and tissue repair materials. Microspheres can be produced in many ways including emulsion and stirring. These traditional methods have many restrictions such as large microspheres, production of air bubbles in the microspheres, uneven size and uneven morphology. All these can affect their applications. Ultrasound vibrations have frequencies higher than 20 kHz. Ultrasound can atomize medicines into tiny molecules. Through breathing, drugs can get into the respiratory tract for treatment. In order to improve the traditional preparation methods, this study suggests preparation using ultrasound atomization. Because the ultrasound transducer moves the micro-sized apertures very fast, materials can be atomized into particles, and the production rate is faster than traditional methods. The prepared alginate particles were analyzed by Dynamic Light Scattering System and the data expressed that alginate particles diameter distribution have two distinct peaks at 10 nm and 1000 nm. It was also expressed that while the viscosity of alginate increases, the diameter distribution of alginate particles decreases. This studys results show that ultrasound atomization can prepare biopolymers particles as complete particles of the same size and morphology. Because these materials have excellent biocompatibility and are biodegradable, they can be applied to tissue engineering.

A NOVEL TGD® DEVICE TO GENERATE THERAPEUTIC PLATELET GLUE

The surgical applications of platelet glue (a mixture of platelet gel and fibrin glue) are so far particularly prominent in plastic and orthopedic surgeries. It is shown to accelerate bone fracture and wound healing in many clinical practices. Over the years, one of the components used to prepare platelet glue (PG), thrombin, could only be obtained from pooled human plasma or bovine blood after repeated centrifugation processes. The quantity from this thrombin source is limited and its process deemed time consuming and the risk of infection does exist. Here, we present a stand alone device (TGD®, Thrombin Generation Device) which could prepare large quantities of human thrombin from autologous or single donor allogeneic plasma donations under sterile conditions. With this specific thrombin product, we could easily further mix with platelet and fibrinogen to prepare the platelet glue. PG not only contains various growth factors such as PDGF, TGF-β which are believed to be beneficial to wound healing but al...

Effects of culturing media on hepatocytes differentiation using Volvox sphere as co-culturing vehicle.

Volvox sphere is a unique design to mimic natural volvox consists of a large outer-sphere that contains smaller inner-spheres, which provide three-dimensional (3D) environment to culture cells. The purpose of this study is to co-culture mesenchymal stem cells (MSCs) and AML12 liver cells in Volvox spheres and to evaluate the effects of two media, DMEM and DMEM/F12 on the cultured cells. The results of this study shows that the 3D Volvox sphere can successfully be applied for co-culture of MSCs and AML12 liver cells, and the MSCs are able to differentiate into hepatocyte-like cells expressing hepatocyte-specific markers including albumin (ALB), alpha feto-protein (AFP) and cytokeratin 18 (CK18) mRNA expressions and producing CK18 and ALB proteins. Interestingly, the MSCs expressed higher ALB, AFP and CK18 mRNA expression at the initial 7-day culture by using DMEM, whereas, the MSCs expressed more mRNA expressions from 7-day to 14-day by the usage of DMEM/F12. The result demonstrated that DMEM and DMEM/F12 media could affect MSCs behaviors during a 14-day culture.