Comparison of adipose stem cells sources from various locations of rat body for their application for seeding on polymer scaffolds

Adipose tissue yields adult adipose stem cells (ASCs) in large quantities via less-invasive methods. These cells are of interest owing to their modulating properties and paracrine activities, which can be harnessed in regenerative medicine. Many studies on the use of rat fat tissue in an autologous animal model have been conducted; however, the different locations to obtain stromal vascular fraction of rat fat depots have not been fully characterized. The purpose of the current study was to identify optimal source of ASC from various locations of rat body. Animal experiments in vitro revealed that fat depots from cervical fat are an optimal ASC source. A high ASC yield facilitates subsequent studies on autologous transplantation in rats. The secondary objective was to compare the efficiency of osteoinductive media composition and evaluate of osteogenic potential of ASCs for seeding on scaffolds for bone repair. Scaffolds were assessed in vitro, using rat adipose stem cells and three-dimensional (3D) scaffolds comprising polycaprolactone (PCL) or polycaprolactone covered with tricalcium phosphate (PCL + 5%TCP). Seeded ASCs adhere to the surface and migrate to the scaffolds. Upon staining and determining alkaline phosphatase levels, PCL + 5%TCP scaffolds performed better than PCL scaffolds. Furthermore, growth factors such as BMP2 and FGF2 significantly increased ASC mineralization and induced osteogenesis (p?<?0.05). Our results may help select and develop pre-clinical animal model for confirming the use of ASC, alone or in association with appropriate biomaterials for bone repair.     

Functionalization of Polycaprolactone Electrospun Osteoplastic Scaffolds with Fluorapatite and Hydroxyapatite Nanoparticles: Biocompatibility Comparison of Human Versus Mouse Mesenchymal Stem Cells

A capability for effective tissue reparation is a living requirement for all multicellular organisms. Bone exits as a precisely orchestrated balance of bioactivities of bone forming osteoblasts and bone resorbing osteoclasts. The main feature of osteoblasts is their capability to produce massive extracellular matrix enriched with calcium phosphate minerals. Hydroxyapatite and its composites represent the most common form of bone mineral providing mechanical strength and significant osteoinductive properties. Herein, hydroxyapatite and fluorapatite functionalized composite scaffolds based on electrospun polycaprolactone have been successfully fabricated. Physicochemical properties, biocompatibility and osteoinductivity of generated matrices have been validated. Both the hydroxyapatite and fluorapatite containing polycaprolactone composite scaffolds demonstrated good biocompatibility towards mesenchymal stem cells. Moreover, the presence of both hydroxyapatite and fluorapatite nanoparticles increased scaffolds’ wettability. Furthermore, incorporation of fluorapatite nanoparticles enhanced the ability of the composite scaffolds to interact and support the mesenchymal stem cells attachment to their surfaces as compared to hydroxyapatite enriched composite scaffolds. The study of osteoinductive properties showed the capacity of fluorapatite and hydroxyapatite containing composite scaffolds to potentiate the stimulation of early stages of mesenchymal stem cells’ osteoblast differentiation. Therefore, polycaprolactone based composite scaffolds functionalized with fluorapatite nanoparticles generates a promising platform for future bone tissue engineering applications.     

Engineering calcium deposits on polycaprolactone scaffolds for intravascular applications using primary human osteoblasts

Total atherosclerotic occlusions often include significant calcium deposits. Current animal models do not mimic the pathology of gradual occlusion of arteries and lack cell‐mediated calcium. The primary goal of this project was to establish an animal model incorporating these features into chronic total occlusions, using biodegradable scaffolds. As the first step, this study sought to determine the optimal dosage of TGF‐β1 on polycaprolactone (PCL) scaffolds cultured with primary human osteoblasts (HOBs) to effectively induce in vitro calcification. HOBs were cultured in TGF‐β1 and dexamethsaone (Dex)‐supplemented medium in well plates. Calcium in the cultures was visualized using alizarin red. The highest calcification was observed in groups with both TGF‐β1 (0.02 ng/ml) and Dex (10^?10 M ) in the medium. Next, HOBs were cultured on PCL scaffolds with different loadings of TGF‐β1: 0 (control), 5, 10, 50 and 100 ng. These cultures were performed with or without Dex (10^?10 M ) in the medium. DNA content, ALP activity and the amount and distribution of calcium were examined at 7, 14, 21 and 28 days. TGF‐β1 appeared to have an inhibitory effect on scaffold calcification when grown in Dex‐supplemented medium. When cultured without Dex, the lower amount of TGF‐β1 loading (5 ng) showed the most calcification, high DNA synthesis and high ALP activity on scaffolds. This study demonstrates the potential of implanting a PCL–HOB construct in an animal artery to establish a model of atherosclerotic occlusion with calcification. Copyright © 2010 John Wiley & Sons, Ltd.     

A humanised tissue‐engineered bone model allows species‐specific breast cancer‐related bone metastasis in vivo

Bone metastases frequently occur in the advanced stages of breast cancer. At this stage, the disease is deemed incurable. To date, the mechanisms of breast cancer‐related metastasis to bone are poorly understood. This may be attributed to the lack of appropriate animal models to investigate the complex cancer cell–bone interactions. In this study, two established tissue‐engineered bone constructs (TEBCs) were applied to a breast cancer‐related metastasis model. A cylindrical medical‐grade polycaprolactone‐tricalcium phosphate scaffold produced by fused deposition modelling (scaffold 1) was compared with a tubular calcium phosphate‐coated polycaprolactone scaffold fabricated by solution electrospinning (scaffold 2) for their potential to generate ectopic humanised bone in NOD/SCID mice. While scaffold 1 was found not suitable to generate a sufficient amount of ectopic bone tissue due to poor ectopic integration, scaffold 2 showed excellent integration into the host tissue, leading to bone formation. To mimic breast cancer cell colonisation to the bone, MDA‐MB‐231, SUM1315, and MDA‐MB‐231BO breast cancer cells were cultured in polyethylene glycol‐based hydrogels and implanted adjacent to the TEBCs. Histological analysis indicated that the breast cancer cells induced an osteoclastic reaction in the TEBCs, demonstrating analogies to breast cancer‐related bone metastasis seen in patients.     

In vitro evaluation of random and aligned polycaprolactone/gelatin fibers via electrospinning for bone tissue engineering

Scaffold, as an essential element of tissue engineering, should provide proper chemical and structural cues to direct tissue regeneration. In this study, aligned and random polycaprolactone (PCL)/gelatin fibrous scaffolds with different mass ratio were electrospun. Chemical, structural, and mechanical properties of PCL/gelatin fibrous scaffolds were characterized by FTIR and tensile measurements. The average diameters of different groups were between 334.96?±?41.43?nm and 363.78?±?50.49?nm. Blending PCL with gelatin increased the mechanical properties of the scaffolds. The cell culture results demonstrated that the mass ratio of PCL and gelatin showed no obvious effects on cell behavior, whereas the cell growth behavior was affected by the fibers orientation. Higher elongation ratio, enhanced cell proliferation and elevated alkaline phosphatase activity were observed for cells cultured on aligned fibers. The findings in our research provide insightful information for the design and fabrication of scaffolds for bone tissue engineering.     

An Efficient and Cost‐Effective Technique to Construct an Intraoral Central Bearing Tracing Device

Intraoral central bearing tracing has been shown to be a predictable way of recording and verifying centric relation position for patients. Existing tracing devices are challenging to use due to several significant clinical limitations. In comparison to commercially available counterparts, this article presents a technique that simplifies instrumentation and clinical steps to make an intraoral tracer for making centric relation records, determining occlusal vertical dimension, and detecting deflective occlusal contacts in edentulous patients.     

In vitro and in vivo evaluation of porous PCL-PLLA 3D polymer scaffolds fabricated via salt leaching method for bone tissue engineering applications

Three dimensional porous scaffolds composed of various ratios of polycaprolactone and poly(L-lactic acid) (PLLA) were prepared using salt leaching method for bone regeneration applications. Surfaces of the scaffolds were visualized using scanning electron microscope (SEM) and the combination of the polymers was confirmed by FT-IR. Addition of PLLA increased the porosity and pore sizes of the scaffolds and also the scaffolds’ compressive strength initially. Osteoblast-like cells were used and it was found that the samples’ cell biocompatibility was further promoted with the increase in PLLA content as observed via cell proliferation assays using MTT, gene expression with RT-PCR, and micrographs from SEM and confocal microscopy. Samples were then implanted into male rabbits for 2 months, and histological staining and micro-CT histomorphometry show that new bone formations were detected in the site containing the implants of the scaffolds and that bone regeneration was further promoted with the increased concentration of PLLA in the scaffold.     

On the Production of Chitosan-Coated Polycaprolactone Nanoparticles in a Confined Impinging Jet Reactor

This work is focused on the synthesis of polycaprolactone nanoparticles, coated with chitosan, in a confined impinging jet reactor using the solvent displacement method. The role of the various reacting species was investigated, evidencing that a biocompatible polymer, for example, polycaprolactone, is required to support chitosan to obtain a monomodal particle size distribution, with low particle diameters. A surfactant is required to reduce the nanoparticle size (down to a mean diameter of about 260 nm) and obtain a positive zeta potential (about +31 mV), perfectly suitable for pharmaceutical applications. Different surfactants were tested, and Poloxamer 388 appeared to be preferable to polyvinyl alcohol. The effect of the concentration of Poloxamer 388 (in the range 0.5-5 mg mL^?1) and of chitosan (in the range 1.5-5 mg mL^?1) on both the mean particle size and zeta potential was also investigated, evidencing that chitosan concentration has the strongest effect on both parameters. Finally, the effect of solvent evaporation, quenching and feed flow rate was investigated, showing that the evaporation stage does not affect particle characteristics, quenching is required to avoid particle aggregation, and a minimum liquid flow rate of 80 mL min^?1 is required in the considered reactor to minimize the particle size.     

Nanofiber technology in the ex vivo expansion of cord blood-derived hematopoietic stem cells

Umbilical cord blood (CB) can be used as an alternative source of hematopoietic stem cells (HSCs) for transplantation in hematological and non-hematological disorders. Despite several recognized advantages the limited cell number in CB one unit still restricts its clinical use. The success of transplantation greatly depends on the levels of total nucleated cell and CD34+ cell counts. Thus, many ex vivo strategies have been developed within the last decade in order to solve this obstacle, with more or less success, mainly determined by the degree of difficulty related with maintaining HSCs self-renewal and stemness properties after long-term expansion. Different research groups have developed very promising and diverse CB-derived HSC expansion strategies using nanofiber scaffolds. Here we review the state-of-the-art of nanofiber technology-based CB-derived HSC expansion.