Sarah H. Cartmell

Effects of Medium Perfusion Rate on Cell-Seeded Three-Dimensional Bone Constructs in Vitro

Cellular activity at the center of tissue-engineered constructs in static culture is typically decreased relative to the construct periphery because of transport limitations. We have designed a tissue culture system that perfuses culture medium through three-dimensional (3D) porous cellular constructs to improve nutrient delivery and waste removal within the constructs. This study examined the effects of medium perfusion rate on cell viability, proliferation, and gene expression within cell-seeded 3D bone scaffolds. Human trabecular bone scaffolds were seeded with MC3T3-E1 osteoblast-like cells and perfused for 1 week at flow rates of 0.01, 0.1, 0.2, and 1.0 mL/min. Confocal microscopy after 1 week of culture indicated that a flow rate of 1.0 mL/min resulted in substantial cell death throughout the constructs whereas lowering the flow rate led to an increasing proportion of viable cells, particularly at the center of the constructs. DNA analysis showed increases in cell proliferation at a flow rate of 0.01 mL/min relative to 0.2 mL/min and static controls. Conversely, mRNA expressions of Runx2, osteocalcin, and alkaline phosphatase were upregulated at 0.2 mL/min compared with lower flow rates as quantified by real-time RT-PCR. These data suggest that medium perfusion may benefit the development of 3-D tissues in vitro by enhancing transport of nutrients and waste within the constructs and providing flow-mediated mechanical stimuli.

Microarchitectural and mechanical characterization of oriented porous polymer scaffolds.

Biodegradable porous polymer scaffolds are widely used in tissue engineering to provide a structural template for cell seeding and extracellular matrix formation. Scaffolds must often possess sufficient structural integrity to temporarily withstand functional loading in vivo or cell traction forces in vitro. Both the mechanical and biological properties of porous scaffolds are determined in part by the local microarchitecture. Quantification of scaffold structure-function relationships is therefore critical for optimizing mechanical and biological performance. In this study, porous poly(L-lactide-co-DL-lactide) scaffolds with axially oriented macroporosity and random microporosity were produced using a solution coating and porogen decomposition method. Microarchitectural parameters were quantified as a function of porogen concentration using microcomputed tomography (micro-CT) analysis and related to compressive mechanical properties. With increasing porogen concentration, volume fraction decreased consistently due to microarchitectural changes in average strut thickness, spacing, and density. The three-dimensional interconnectivity of the scaffold porosity was greater than 99% for all porogen concentration levels tested. Over a porosity range of 58-80%, the average compressive modulus and ultimate strength of the scaffolds ranged from 43.5-168.3 MPa and 2.7-11.0 MPa, respectively. Thus, biodegradable porous polymer scaffolds have been produced with oriented microarchitectural features designed to facilitate vascular invasion and cellular attachment and with initial mechanical properties comparable to those of trabecular bone.

Effect of fluid flow-induced shear stress on human mesenchymal stem cells: Differential gene expression of IL1B and MAP3K8 in MAPK signaling

Human bone marrow-derived mesenchymal stem cells (MSCs) can differentiate into numerous cell lineages, making them ideal for tissue engineering. Mechanical forces and mechanotransduction are important factors influencing cell responses, although such data are limited for MSCs. We investigated the effect of different profiles of fluid flow-induced shear stress on mitogen-activated protein kinase (MAPK) signaling pathway gene expression in MSCs using DNA microarray and quantitative real-time reverse transcription-PCR analysis. In response to different magnitudes and durations of fluid flow-induced shear stress, we observed significant differential gene expression for various genes in the MAPK signaling pathway. Independent of magnitude and duration, shear stress induced consistent and marked up-regulation of MAP kinase kinase kinase 8 (MAP3K8) and interleukin-1 beta (IL1B) [2-fold to >35-fold, and 4-fold to >50-fold, respectively]. We also observed consistent up-regulation of dual specificity phosphatase 5 and 6, growth arrest and DNA-damage-inducible alpha and beta, nuclear factor kappa-B subunit 1, Jun oncogene, fibroblast growth factor 1, and platelet-derived growth factor alpha. Our data support MAP3K8-induced activation of different MAPK signaling pathways in response to different profiles of shear stress, possibly as a consequence of shear-induced IL1B expression. Thus, MAP3K8 may be an important mediator of intracellular mechanotransduction in human MSCs.

Osteoblast: Osteoclast co-cultures on silk fibroin, chitosan and PLLA films

This study investigates the growth of a co-culture of osteoblasts and osteoclasts on four different types of degradable biomaterials with bone tissue engineering potential. Single or co-cultures of osteoblasts and osteoclasts (used at a ratio of 1:100 osteoblast:osteoclasts) were cultured on vapour stabilised silk fibroin, methanol stabilised silk fibroin, chitosan and poly (l lactic acid) (PLLA) films for 10 days. Osteoclast differentiation was determined by tartrate resistant acid phosphatase (TRAP) staining, total cell number by a picogreen DNA assay, cell morphology by scanning electron microscopy (SEM) and the material topography by atomic force microscopy (AFM). Samples were also monitored for degradation by differential scanning calorimetry (DSC) and fourier transform infrared (FTIR). Results demonstrated that vapour stabilised silk fibroin, methanol stabilised silk fibroin and chitosan all support the growth of osteoblasts and osteoclasts in both single and co-cultures. PLLA showed poor osteoclast differentiation in both single and co-cultures but supported osteoblast attachment and proliferation. Both silk fibroin materials showed sign of early degradation in the ten-day period, but very little change was seen in chitosan and PLLA samples. This study indicates that this novel co-culture approach for bone tissue engineering may be possible if scaffolds are created from silk fibroin or chitosan.

Principles and Design of a Novel Magnetic Force Mechanical Conditioning Bioreactor for Tissue Engineering, Stem Cell Conditioning, and Dynamic In Vitro Screening

Mechanical conditioning of cells and tissue constructs in bioreactors is an important factor in determining the properties of tissue being produced. Mechanical conditioning within a bioreactor environment, however, has proven difficult. This paper presents the theoretical basis, design, and initial results of a mechanical conditioning system for cell and tissue culture which is based on biocompatible magnetic micro- and nanoparticles acting as a remote stress mechanism without invasion of the sterile bioreactor environment

Development of magnetic particle techniques for long-term culture of bone cells with intermittent mechanical activation

Magnetic particles were coated with RGD and adhered to primary human osteoblasts. During a 21-day culture, the osteoblasts plus adhered magnetic particles underwent a daily exposure to a time-varying magnetic field via a permanent NdFeB magnet, thus applying a direct mechanical stress to the cells (Bmax approximately 60 mT). After 21 days, preliminary results show that the cells plus magnetic particles were viable and had proliferated. A von-kossa stain showed mineralized bone matrix produced at 21 days in the experimental group whereas the control groups showed no mineralized matrix production. Real-time reverse transcription-polymerase chain reaction at 21 days showed an upregulation of osteopontin from the experimental group in comparison to the control group of cells with adhered particles and no magnet applied. These preliminary results indicate that adherence of RGD-coated 4.5 microm ferromagnetic particles to primary human osteoblasts does not initiate cell necrosis up to 21 days in vitro. Also, mechanical stimulation of human osteoblasts by magnetic particle technology appears to have an influence on osteoblastic activity.

State of the art composites comprising electrospun fibres coupled with hydrogels: a review

UNLABELLED Research into scaffolds tailored for specific tissue engineering and biomaterial applications continues to develop as these structures are commonly impeded by their limitations. For example, electrospun fibres and hydrogels are commonly exploited because of their ability to mimic natural tissues; however, their clinical use remains restricted due to negligible cellular infiltration and poor mechanical properties, respectively. A small number of research groups are beginning to investigate composite scaffolds based on electrospun fibres and hydrogels in an attempt to overcome their individual shortcomings. This review paper discusses the various methodologies and approaches currently undertaken to create these novel composite structures and their intended applications. The combination of these two commonly used scaffold architectures to create synergistically superior structures is showing potential with regards to therapeutic use within the tissue engineering community. FROM THE CLINICAL EDITOR This review discusses methodologies to create novel electrospun nanofibers and hydrogels, and their intended applications. The combination of these two scaffold architectures has important future clinical applications, although their use is currently limited to the experimental tissue engineering community.

Controlled Release Scaffolds for Bone Tissue Engineering

Bone disease and trauma can sometimes create defects that require more than current clinical therapies can provide for effective healing. Scaffolds that release bioactive agents such as drugs offer additional benefits in this regard in comparison to those whose main requirements are space filling and load bearing. This review details a variety of recent research on scaffold carrier systems that release osteogenic drugs for orthopaedic treatment purposes. The controlled release scaffolds presented are created from a variety of materials (such as tricalcium phosphate, hydrogel systems and poly(D,L-lactide)) and release drugs such as statins, bisphosphonates and a variety of growth factors.

Biocompatibility of polyhydroxyalkanoate as a potential material for ligament and tendon scaffold material

There is a strong need for new biodegradable materials that are suitable for scaffolds in tissue engineering of tendons and ligaments. In many cases, quick degradation rates are favorable, however, with respect to ligament and tendon replacement, slowly degrading polymers are clearly favored. Prime candidates are members of the large class of polyhydroxyalkanoates (PHAs), which are thermoplastic/elastomeric biopolyesters that are slowly degraded by surface erosion. Moreover, their physico-mechanical properties can be tailored during biosynthesis in bacteria or by chemical modifications. They may be spun into fibers, coated on surfaces or be part of composites. This study has investigated the biocompatability of seven different thermoplastic or elastomeric PHAs using L929 murine fibroblast cells. Cell viability and proliferation over 7 days was analyzed with live/dead staining and a picogreen assay. In addition, extracellular matrix production was measured with the hydroxyproline assay after 14 days. It was found that cell attachment to the PHA film ranged from 85-99% after 7 days. Three PHA films (PHBV (92/8), PHOUE-POSS and PHUE-O3) supported similar cell viability in comparison to the controls performed on tissue culture plastic (polystyrene), whereas the biomaterials (PHUA, PHUE, PHB and PHOUE) showed fewer viable cells than in controls. PHB, PHUE-O3, and PHBV with a water contact angle below 85 degrees supported a similar amount of collagen production in comparison to the tissue culture plastic controls. PHUA, PHUE, PHOUE, and PHOUE-POSS showed a decrease in collagen production in comparison to the controls after 14 days. Overall, PHB, PHBV, and PHUE-O3 demonstrated good performance with regards to potential use as a tissue-engineering scaffold.

Polarization of hydroxyapatite:Influence on osteoblast cell proliferation

Hydroxyapatite (HA) has been used clinically to treat bone defects. However, modifications of the surface properties of HA could improve and control bone matrix deposition and localized host tissue integration. The aim of this study was to investigate the effect of developing a surface charge on HA discs with respect to osteoblast activity in vitro. HA discs (12 mm x 2 mm) were sintered in either air or water vapour. The HA discs were then electrically polarized (positive and negative surfaces) or non-polarized (controls) and seeded with MC3T3-E1 cells. Polarized HA sintered in water vapour was shown to retain six times more charge than polarized HA sintered in air. Picogreen analysis demonstrated that at 4h cell number was significantly higher on the negatively and positively charged HA surface (water sintered) in comparison to the non-charged water and air-sintered HA controls. At 7 days there was a significant increase in cell number on the negatively charged HA (air sintered) sample in comparison to the negatively charged water vapour sintered HA sample and the non-charged water vapour sintered control sample. Also at 7 days, the picogreen data showed a significant increase in cell number on the positively charged water-treated HA sample in comparison to both the air- and water-treated HA non-charged control HA samples. An alamarBlue assay at 7 days demonstrated significant cell metabolic activity on the charged surfaces (both positive and negative) in comparison to the non-charged HA and the tissue culture plastic controls. This study demonstrated that all of the HA discs tested supported cell viability/attachment. However, cell attachment/proliferation/metabolic activity was significantly increased as a result of developing a charge on the HA surface.