Processing of collagen gels to create in vitro cell growth matrix without damage to the collagen native structure

This preliminary work explores a technique for processing collagen gels to provide a structured matrix support for cell growth and other tissue engineering applications without using cyto-toxic photo-initiators. Collagen gels can be structured by techniques similar to those of rapid manufacturing and retain the fibril structure of native collagen. Incorporation of alpha-modified minimal essential medium (MEM) in the collagen solution improved the rate of gelation in a cell-friendly way. Local gelation of a collagen solution formulated with alpha-modified MEM can be achieved by exposure to radiation from a remote incandescent lamp source indicating that it may be possible to prepare structured gels by lithographically based rapid manufacturing processes. Exposure of the alpha-modified MEM collagen solution to the radiation also increased the thickness of the collagen fibrils formed during the gelation process to create a more structured gel. Methyl blue staining, scanning electron microscope (SEM), and differential scanning calorimetry (DSC) experiments confirmed the collagen was not denatured, i.e. the native structure of collagen was retained

Design modification and optimisation of the perfusion system of a tri-axial bioreactor for tissue engineering

A systematic design of experiments (DOE) approach was used to optimize the perfusion process of a tri-axial bioreactor designed for translational tissue engineering exploiting mechanical stimuli and mechanotransduction. Four controllable design parameters affecting the perfusion process were identified in a cause–effect diagram as potential improvement opportunities. A screening process was used to separate out the factors that have the largest impact from the insignificant ones. DOE was employed to find the settings of the platen design, return tubing configuration and the elevation difference that minimise the load on the pump and variation in the perfusion process and improve the controllability of the perfusion pressures within the prescribed limits. DOE was very effective for gaining increased knowledge of the perfusion process and optimizing the process for improved functionality. It is hypothesized that the optimized perfusion system will result in improved biological performance and consistency

MgO implanted in rat tibia bone marrow is osteoinductive through the formation of a matrix, containing hydroxyapatite

Healing of rat tibia after intramedullary implantation of MgO was analysed by Environmental Scanning Electron Microscopy (ESEM) and Energy-Dispersive X-ray spectroscopy (EDX). The results indicated the formation of hydroxyl-apatite (HA) in the entire intramedullary space after 1 week of healing. Then, corroded Mg, MgO and MgCO3 were incubated with DMEM in vitro for 24 h and the surface of the material was analysed by EDX and Time-of-Flight Secondary Ion Mass (ToF-SIMS). The chemical analysis of the Mg corrosion products indicate that HA is formed at the material surface and that MgCO3 was an efficient catalyzer of HA formation

Numerical modelling of effects of biphasic layers of corrosion products to the degradation of magnesium metal in vitro

© 2017 by the authors. Magnesium (Mg) is becoming increasingly popular for orthopaedic implant materials. Its mechanical properties are closer to bone than other implant materials, allowing for more natural healing under stresses experienced during recovery. Being biodegradable, it also eliminates the requirement of further surgery to remove the hardware. However, Mg rapidly corrodes in clinically relevant aqueous environments, compromising its use. This problem can be addressed by alloying the Mg, but challenges remain at optimising the properties of the material for clinical use. In this paper, we present a mathematical model to provide a systematic means of quantitatively predicting Mg corrosion in aqueous environments, providing a means of informing standardisation of in vitro investigation of Mg alloy corrosion to determine implant design parameters. The model describes corrosion through reactions with water, to produce magnesium hydroxide Mg(OH) 2 , and subsequently with carbon dioxide to form magnesium carbonate MgCO 3 . The corrosion products produce distinct protective layers around the magnesium block that are modelled as porous media. The resulting model of advection-diffusion equations with multiple moving boundaries was solved numerically using asymptotic expansions to deal with singular cases. The model has few free parameters, and it is shown that these can be tuned to predict a full range of corrosion rates, reflecting differences between pure magnesium or magnesium alloys. Data from practicable in vitro experiments can be used to calibrate the model's free parameters, from which model simulations using in vivo relevant geometries provide a cheap first step in optimising Mg-based implant materials

Mechanistic evaluation of long-term in-stent restenosis based on models of tissue damage and growth

Development and application of advanced mechanical models of soft tissues and their growth represent one of the main directions in modern mechanics of solids. Such models are increasingly used to deal with complex biomedical problems. Prediction of in-stent restenosis for patients treated with coronary stents remains a highly challenging task. Using a finite element method, this paper presents a mechanistic approach to evaluate the development of in-stent restenosis in an artery following stent implantation. Hyperelastic models with damage, verified with experimental results, are used to describe the level of tissue damage in arterial layers and plaque caused by such intervention. A tissue-growth model, associated with vessel damage, is adopted to describe the growth behaviour of a media layer after stent implantation. Narrowing of lumen diameter with time is used to quantify the development of in-stent restenosis in the vessel after stenting. It is demonstrated that stent designs and materials strongly affect the stenting-induced damage in the media layer and the subsequent development of in-stent restenosis. The larger the artery expansion achieved during balloon inflation the higher the damage introduced to the media layer, leading to an increased level of in-stent restenosis. In addition, the development of in-stent restenosis is directly correlated with the artery expansion during the stent deployment. The correlation is further used to predict the effect of a complex clinical procedure, such as stent overlapping, on the level of in-stent restenosis developed after percutaneous coronary intervention.</div

PDGF is a potent initiator of bone formation in a tissue engineered model of pathological ossification

Heterotopic ossification (HO) is a debilitating condition defined by the rapid formation of bone in soft tissues. What makes HO fascinating is firstly the rate at which bone is deposited, and secondly the fact that this bone is structurally and compositionally similar to that of a healthy adult. If the mechanisms governing HO are understood, they have the potential to be exploited for the development of potent osteoinductive therapies. With this aim, we utilised a tissue engineered skeletal muscle model to better understand the role of inflammation on this debilitating phenomenon. We showed myoblasts could be divided into two distinct populations, myogenic cells and undifferentiated "reserve" cells. Gene expression analysis of myogenic and osteo-regulatory markers confirmed that "reserve" cells were primed for osteogenic differentiation, but had a reduced capacity for myogenesis. Osteogenic differentiation was significantly enhanced in the presence of PDGF-BB and BMP2, and correlated with conversion to a Sca-1(+) /CD73(+) phenotype. Alizarin red staining showed that PDGF-BB promoted significantly more mineral deposition than BMP2. Finally, we showed that PDGF-induced mineralisation was blocked in the presence of the pro-inflammatory cytokines TNFα and IL1. In conclusion, the present study identified that PDGF-BB is a potent osteoinductive factor in a model of tissue engineered skeletal muscle, and that the osteogenic capacity of this protein was modulated in the presence of pro-inflammatory cytokines. These findings reveal a possible mechanism by which HO develops following trauma. Importantly, these findings have implications for the induction and control of bone formation for regenerative medicine

Cellular response to cyclic compression of tissue engineered intervertebral disk constructs composed of electrospun polycaprolactone

There is lack of investigation capturing the complex mechanical interaction of tissue engineered IVD (intervertebral disc) constructs in physiologically-relevant environmental conditions. In this study, mechanical characterisation of anisotropic eletrospinning (ES) substrates made of polycaprolactone (PCL) was carried out in wet and dry conditions and viability of human bone marrow derived mesenchymal stem cells (hMSCs) seeded within double layers of ES PCL was also studied. Cyclic compression of IVD-like constructs composed of an agarose core confined by ES PCL double-layers was implemented using a bioreactor and the cellular response to the mechanical stimulation was evaluated. Tensile tests showed decrease of elastic modulus of ES PCL as the angle of stretching increased and at 60° stretching angle in wet, maximum ultimate tensile strength was observed. Based on the configuration of IVD-like constructs, the calculated circumferential stress experienced by the ES PCL double layers was 40 times of the vertical compressive stress. Confined compression of IVD-like constructs at 5% and 10% displacement dramatically reduced cell viability, particularly at 10%, although cell presence in small and isolated area can still be observed after mechanical conditioning. Hence, material mechanical properties of tissue-engineered scaffolds, composed of fibril structure of polymer with low melting point, are affected by the testing condition. Circumferential stress induced by axial compressive stimulation, conveyed to the ES PCL double-layer wrapped around an agarose core, can affect the viability of cells seeded at the interface, depending on the mechanical configuration and magnitude of the load

The sensitivity of human mesenchymal stem cells to vibration and cold storage conditions representative of cold transportation

In the current study, the mechanical and hypothermic damage induced by vibration and cold storage on human mesenchymal stem cells (hMSCs) stored at 2–8°C was quantified by measuring the total cell number and cell viability after exposure to vibration at 50 Hz (peak acceleration 140 m s−2 and peak displacement 1.4 mm), 25 Hz (peak acceleration 140 m s−2, peak displacement 5.7 mm), 10 Hz (peak acceleration 20 m s−2, peak displacement 5.1 mm) and cold storage for several durations. To quantify the viability of the cells, in addition to the trypan blue exclusion method, the combination of annexin V-FITC and propidium iodide was applied to understand the mode of cell death. Cell granularity and a panel of cell surface markers for stemness, including CD29, CD44, CD105 and CD166, were also evaluated for each condition. It was found that hMSCs were sensitive to vibration at 25 Hz, with moderate effects at 50 Hz and no effects at 10 Hz. Vibration at 25 Hz also increased CD29 and CD44 expression. The study further showed that cold storage alone caused a decrease in cell viability, especially after 48 h, and also increased CD29 and CD44 and attenuated CD105 expressions. Cell death would most likely be the consequence of membrane rupture, owing to necrosis induced by cold storage. The sensitivity of cells to different vibrations within the mechanical system is due to a combined effect of displacement and acceleration, and hMSCs with a longer cold storage duration were more susceptible to vibration damage, indicating a coupling between the effects of vibration and cold storage

Experimental and computational studies of poly-L-lactic acid for cardiovascular applications: recent progress

Stents are commonly used in medical procedures to alleviate the symptoms of coronary heart disease, a prevalent modern society disease. These structures are employed to maintain vessel patency and restore blood flow. Traditionally stents are made of metals such as stainless steel or cobalt chromium; however, these scaffolds have known disadvantages. An emergence of transient scaffolds is gaining popularity, with the structure engaged for a required period whilst healing of the diseased arterial wall occurs. Polymers dominate a medical device sector, with incorporation in sutures, scaffolds and screws. Thanks to their good mechanical and biological properties and their ability to degrade naturally. Polylactic acid is an extremely versatile polymer, with its properties easily tailored to applications. Its dominance in the stenting field increases continually, with the first polymer scaffold gaining FDA approval in 2016. Still some challenges with PLLA bioresorbable materials remain, especially with regard to understanding their mechanical response, assessment of its changes with degradation and comparison of their performance with that of metallic drug-eluting stent. Currently, there is still a lack of works on evaluating both the pre-degradation properties and degradation performance of these scaffolds. Additionally, there are no established material models incorporating non-linear viscoelastic behaviour of PLLA and its evolution with in-service degradation. Assessing these features through experimental analysis accompanied by analytical and numerical studies will provide powerful tools for design and optimisation of these structures endorsing their broader use in stenting. This overview assesses the recent studies investigating mechanical and computational performance of poly(l-lactic) acid and its use in stenting applications

Human cell culture process capability: a comparison of manual and automated production

Cell culture is one of the critical bioprocessing steps required to generate sufficient human-derived cellular material for most cell-based therapeutic applications in regenerative medicine. Automated cell expansion is fundamental to the development of scaled, robust and cost effective commercial production processes for cell-based therapeutic products. This paper describes the first application of process capability analysis to establish and compare the short-term process capability of manual and automated processes for the in vitro expansion of a selected anchorage-dependent cell line. Estimates of the process capability indices (Cp, Cpk) have been used to assess the ability of both processes to consistently meet the requirements for a selected productivity output and to direct process improvement activities. Point estimates of Cp and Cpk show that the manual process has poor capability (Cp = 0.55, Cpk = 0.26) compared to the automated process (Cp = 1.32, Cpk = 0.25), resulting from excess variability. Comparison of point estimates, which shows that Cpk < Cp, indicates that the automated process mean was off-centre and that intervention is required to adjust the location of the process mean. A process improvement strategy involving an adjustment to the automated process settings has demonstrated in principle that the process mean can be shifted closer to the centre of the specification to achieve an estimated seven-fold improvement in process performance. In practice, the 90% confidence bound estimate of Cp (Cp = 0.90) indicates that that once the process is centred within the specification, a further reduction of process variation is required to attain an automated process with the desired minimum capability requirement