62 research outputs found
Synergistic effect of scaffold composition and dynamic culturing environment in multi-layered systems for bone tissue engineering
Bone extracellular matrix (ECM) is composed of mineralized collagen fibrils which support biological
apatite nucleation that participates in bone outstanding properties. Understanding and mimicking
bone morphological and physiological parameters at a biological scale is a major challenge in tissue
engineering scaffolding. Using emergent (nano)technologies scaffold designing may be critically
improved, enabling highly functional tissue substitutes for bone applications. This study aims to develop
novel biodegradable composite scaffolds of tricalcium phosphate (TCPs) and electrospun nanofibers
of poly(e-caprolactone) (PCL), combining TCPs osteoconductivity with PCL biocompatibility
and elasticity, mimicking bone structure and composition. We hypothesized that scaffolds with such
structure/composition would stimulate the proliferation and differentiation of bone marrow stromal
cells (BMSCs) towards the osteogenic phenotype. Composite scaffolds, developed by electrospining
using consecutive stacked layers of PCL and TCPs, were characterized by FTIR spectroscopy, X-Ray diffraction
and scanning electronic microscopy. Cellular behavior was assessed in goat BMSCs seeded
onto composite scaffolds and cultured in static or dynamic conditions, using basal or osteogenic media
during 7, 14 or 21 days. Cellular proliferation was quantified and osteogenic differentiation confirmed
by alkaline phosphatase activity, alizarin red staining and immunocytochemistry for osteocalcin and
collagen I. Results suggest that PCL-TCP scaffolds provide a 3D support for gBMSCs proliferation
and osteogenic differentiation with production of ECM. TCPs positively stimulate the osteogenic
process, especially under dynamic conditions, where PCL-TCP scaffolds are sufficient to promote
osteogenic differentiation even in basal medium conditions. The enhancement of the osteogenic
potential in dynamic conditions evidences the synergistic effect of scaffold composition and dynamic
stimulation in gBMSCs osteogenic differentiation.Fundação para a Ciência e a Tecnologia (FCT)European NoE EXPERTISSUES (NMP3-CT- 2004-500283
Osteoinduction of Human Mesenchymal Stem Cells by Bioactive Composite Scaffolds without Supplemental Osteogenic Growth Factors
The development of a new family of implantable bioinspired materials is a focal point of bone tissue engineering. Implant surfaces that better mimic the natural bone extracellular matrix, a naturally nano-composite tissue, can stimulate stem cell differentiation towards osteogenic lineages in the absence of specific chemical treatments. Herein we describe a bioactive composite nanofibrous scaffold, composed of poly-caprolactone (PCL) and nano-sized hydroxyapatite (HA) or beta-tricalcium phosphate (TCP), which was able to support the growth of human bone marrow mesenchymal stem cells (hMSCs) and guide their osteogenic differentiation at the same time. Morphological and physical/chemical investigations were carried out by scanning, transmission electron microscopy, Fourier-transform infrared (FTIR) spectroscopy, mechanical and wettability analysis. Upon culturing hMSCs on composite nanofibers, we found that the incorporation of either HA or TCP into the PCL nanofibers did not affect cell viability, meanwhile the presence of the mineral phase increases the activity of alkaline phosphatase (ALP), an early marker of bone formation, and mRNA expression levels of osteoblast-related genes, such as the Runt-related transcription factor 2 (Runx-2) and bone sialoprotein (BSP), in total absence of osteogenic supplements. These results suggest that both the nanofibrous structure and the chemical composition of the scaffolds play a role in regulating the osteogenic differentiation of hMSCs
Engineering large cartilage tissues using dynamic bioreactor culture at defined oxygen conditions
Mesenchymal stem cells maintained in appropriate culture conditions are capable of producing robust cartilage tissue. However, gradients in nutrient availability that arise during three-dimensional culture can result in the development of spatially inhomogeneous cartilage tissues with core regions devoid of matrix. Previous attempts at developing dynamic culture systems to overcome these limitations have reported suppression of mesenchymal stem cell chondrogenesis compared to static conditions. We hypothesize that by modulating oxygen availability during bioreactor culture, it is possible to engineer cartilage tissues of scale. The objective of this study was to determine whether dynamic bioreactor culture, at defined oxygen conditions, could facilitate the development of large, spatially homogeneous cartilage tissues using mesenchymal stem cell laden hydrogels. A dynamic culture regime was directly compared to static conditions for its capacity to support chondrogenesis of mesenchymal stem cells in both small and large alginate hydrogels. The influence of external oxygen tension on the response to the dynamic culture conditions was explored by performing the experiment at 20% O 2 and 3% O 2 . At 20% O 2 , dynamic culture significantly suppressed chondrogenesis in engineered tissues of all sizes. In contrast, at 3% O 2 dynamic culture significantly enhanced the distribution and amount of cartilage matrix components (sulphated glycosaminoglycan and collagen II) in larger constructs compared to static conditions. Taken together, these results demonstrate that dynamic culture regimes that provide adequate nutrient availability and a low oxygen environment can be employed to engineer large homogeneous cartilage tissues. Such culture systems could facilitate the scaling up of cartilage tissue engineering strategies towards clinically relevant dimensions
Three-Dimensional Bioprinting of Polycaprolactone Reinforced Gene Activated Bioinks for Bone Tissue Engineering
Regeneration of complex bone defects remains a significant clinical challenge. Multi-tool biofabrication has permitted the combination of various biomaterials to create multifaceted composites with tailorable mechanical properties and spatially controlled biological function. In this study we sought to use bioprinting to engineer nonviral gene activated constructs reinforced by polymeric micro-filaments. A gene activated bioink was developed using RGD-?-irradiated alginate and nano-hydroxyapatite (nHA) complexed to plasmid DNA (pDNA). This ink was combined with bone marrow-derived mesenchymal stem cells (MSCs) and then co-printed with a polycaprolactone supporting mesh to provide mechanical stability to the construct. Reporter genes were first used to demonstrate successful cell transfection using this system, with sustained expression of the transgene detected over 14 days postbioprinting. Delivery of a combination of therapeutic genes encoding for bone morphogenic protein and transforming growth factor promoted robust osteogenesis of encapsulated MSCs in vitro, with enhanced levels of matrix deposition and mineralization observed following the incorporation of therapeutic pDNA. Gene activated MSC-laden constructs were then implanted subcutaneously, directly postfabrication, and were found to support superior levels of vascularization and mineralization compared to cell-free controls. These results validate the use of a gene activated bioink to impart biological functionality to three-dimensional bioprinted constructs
Biocompatible Magnetite/Gold Nanohybrid Contrast Agents via Green Chemistry for MRI and CT Bioimaging
Magnetite/gold (Fe<sub>3</sub>O<sub>4</sub>/Au) hybrid
nanoparticles
were synthesized from a single iron precursor (ferric chloride) through
a green chemistry route using grape seed proanthocyanidin as the reducing
agent. Structural and physicochemical characterization proved the
nanohybrid to be crystalline, with spherical morphology and size ∼35
nm. Magnetic resonance imaging and magnetization studies revealed
that the Fe<sub>3</sub>O<sub>4</sub> component of the hybrid provided
superparamagnetism, with dark T<sub>2</sub> contrast and high relaxivity
(124.2 ± 3.02 mM<sup>–1</sup> s<sup>–1</sup>).
Phantom computed tomographic imaging demonstrated good X-ray contrast,
which can be attributed to the presence of the nanogold component
in the hybrid. Considering the potential application of this bimodal
nanoconstruct for stem cell tracking and imaging, we have conducted
compatibility studies on human Mesenchymal Stem Cells (hMSCs), wherein
cell viability, apoptosis, and intracellular reactive oxygen species
(ROS) generation due to the particle–cell interaction were
asessed. It was noted that the material showed good biocompatibility
even for high concentrations of 500 μg/mL and up to 48 h incubation,
with no apoptotic signals or ROS generation. Cellular uptake of the
nanomaterial was visualized using confocal microscopy and prussian
blue staining. The presence of the nanohybrids were clearly visualized
in the intracytoplasmic region of the cell, which is desirable for
efficient imaging of stem cells in addition to the cytocompatible
nature of the hybrids. Our work is a good demonstrative example of
the use of green aqueous chemistry through the employment of phytochemicals
for the room temperature synthesis of complex hybrid nanomaterials
with multimodal functionalities
- …