The paper presents a transient, continuum, two-phase model of the tissue engineering in fibrous scaffolds, including transport equations for the flowing culture medium, nutrient and cell concentration with transverse and in-plane diffusion and cell migration, a novel feature of local in-plane transport across a phenomenological pore and innovative layer-by-layer cell filling approach. The model is successfully validated for the smooth muscle cell tissue engineering of a vascular graft using crosslinked, electrospun gelatin fiber scaffolds for both static and dynamic cell culture, the latter in a dynamic bioreactor with a rotating shaft on which the tubular scaffold is attached. Parametric studies evaluate the impact of the scaffold microstructure, cell dynamics, oxygen transport, and static or dynamic conditions on the rate and extent of cell proliferation and depth of oxygen accessibility. An optimized scaffold of 75% dry porosity is proposed that can be tissue engineered into a viable and still fully oxygenated graft of the tunica media of the coronary artery within 2 days in the dynamic bioreactor. Such scaffold also matches the mechanical properties of the tunica media of the human coronary artery and the suture retention strength of a saphenous vein, often used as a coronary artery graft.

Insulin (Humulin® R IU500) has been delivered from an implantable artificial pancreas in diabetic rats and pigs. The artificial pancreas which was implanted in the peritoneum was fabricated from several biocompatible materials such as polycarbonate, stainless steel, polyurethane, titanium and a polyurethane resin. The device also contains a glucose responsive smart gel which controls the diffusion of insulin dependent on the surrounding glucose environment. As the insulin reservoir is refillable and in contact with the device materials, assessing its biocompatibility with these various device component materials was conducted. Insulin can undergo chemical degradation mainly via a deamidation reaction on glutamine and asparagine residues rendering its biological hormone functionality. Two Reverse Phase High Performance Liquid Chromatography (RP-HPLC) methods were developed and validated for detection of insulin and degradant Asn A21 desamido insulin (method A) and insulin and degradant Asn B3 desamido insulin (method B). Material biocompatibility studies show that stainless steel and titanium are suitable for an implantable insulin delivery device design over a 31-day period. The use of polycarbonate and polyurethane could be considered if the insulin reservoir in the device was only to remain in the device for less than 11 days after which time there is a loss in cresol which acts in a protective capacity for insulin stability.

The relationship between cellular oxygen consumption and cell growth is important for modelling and simulating tissue engineering processes but there is a lack of comprehensive data for cell growth as a function of oxygen consumption in the literature for human smooth muscle cells and osteoblasts. Cell growth was monitored as a function of oxygen concentration in the culture medium for human umbilical vein smooth muscle cells (HUVSMCs) and human foetal osteoblasts (hFOBs). The yield coefficients, coefficients of oxygen consumption, oxygen consumption constants, cell growth constants and specific oxygen uptake rates were determined for the cells. Cell concentration and oxygen concentration data were fitted to models for cell growth and oxygen consumption. The Monod model for cell growth rate was fitted as a function of oxygen consumption rate and cell concentration for the HUVSMCs whereas two different models were used to best fit the oxygen consumption rate as a function of cell growth rate and cell concentration for the hFOBs. These models can be utilised to predict cell growth and oxygen consumption parameters for HUVSMC and hFOB cells which may be useful for simulating vascular and bone tissue engineering processes respectively.

Thermoresponsive gelling materials constructed from natural and synthetic polymers can be used to provide triggered action and therefore customised products such as drug delivery and regenerative medicine types as well as for other industries. Some materials give Arrhenius-type viscosity changes based on coil to globule transitions. Others produce more counterintuitive responses to temperature change because of agglomeration induced by enthalpic or entropic drivers. Extensive covalent crosslinking superimposes complexity of response and the upper and lower critical solution temperatures can translate to critical volume temperatures for these swellable but insoluble gels. Their structure and volume response confer advantages for actuation though they lack robustness. Dynamic covalent bonding has created an intermediate category where shape moulding and self-healing variants are useful for several platforms. Developing synthesis methodology—for example, Reversible Addition Fragmentation chain Transfer (RAFT) and Atomic Transfer Radical Polymerisation (ATRP)—provides an almost infinite range of materials that can be used for many of these gelling systems. For those that self-assemble into micelle systems that can gel, the upper and lower critical solution temperatures (UCST and LCST) are analogous to those for simpler dispersible polymers. However, the tuned hydrophobic-hydrophilic balance plus the introduction of additional pH-sensitivity and, for instance, thermochromic response, open the potential for coupled mechanisms to create complex drug targeting effects at the cellular level.

The performance of a completely implantable peritoneal artificial pancreas (AP) has been demonstrated in principle in a live diabetic domestic pig. The device consists of a smart glucose-sensitive gel that forms a gateway to an insulin reservoir and is designed to both sense glucose and deliver insulin in the peritoneal cavity. It can be refilled with insulin via subcutaneous ports and surgery was developed to insert the AP. Diabetes was induced with streptozotocin (STZ), the device filled with insulin (Humulin® R U-500) in situ and the animal observed for several weeks, during which time there was normal access to food and water and several oral glucose challenges. Blood glucose (BG) levels were brought down from >30mmol/L (540mg/dL) to non-fasted values between 7 and 13mmol/L (126–234mg/dL) about five days after filling the device. Glucose challenge responses improved ultimately so that, starting at 10mmol/L (180mg/dL), the BG peak was 18mmol/L (324mg/dL) and fell to 7mmol/L (126mg/dL) after 30min, contrasting with intravenous attempts. The reservoir solution was removed after 8days of blood glucose levels during which they had been increasingly better controlled. A rapid return to diabetic BG levels (30mmol/L) occurred only after a further 24days implying some insulin had remained in the device after removal of the reservoir solution. Thus, the closed loop system appeared to have particular influence on the basal and bolus needs for the 8days in which the reservoir solution was in place and substantial impact for a further 3 weeks. No additional insulin manual adjustment was given during this period.

Human insulin was fluorescently labelled with fluorescein isothiocyanate (FITC) and the conjugate species produced were identified using high performance liquid chromatography and electrospray mass spectroscopy. Mono-labelled FITC-insulin conjugate (A1 or B1) was successfully produced using human insulin at short reaction times (up to 5 h) however the product always contained some unlabelled native human insulin. As the reaction time was increased over 45 h, no unlabelled native human insulin was present and more di-labelled FITC-insulin conjugate (A1B1) was produced than mono-labelled conjugate with the appearance of tri-labelled conjugate (A1B1B29) after 20 h reaction time. The quantities switch from mono-labelled to di-labelled FITC-insulin conjugate between reaction times 9 and 20 h. In the presence of phenol or m-cresol, there appears to be a 10 % decrease in the amount of mono-labelled conjugate and an increase in di-labelled conjugate produced at lower reaction times. Clinically used insulin analogues present in commercially available preparations were successfully fluorescently labelled for future biomedical applications.

Virtually all publications in the field of tissue engineering contain some description of the materials used and the characteristics of the scaffolds that are produced from them. However, the practical value of this information is often limited due to lack of consistency in the test methods used and in the way in which the data are reported. This chapter describes the key characteristics and properties of scaffolds that are likely to be important for ensuring optimal functionality and consistency and identifies those features that should be considered in planning experimental test programmes. References are also made to relevant international standards that are in place and being developed.

It is increasingly recognised that biomimetic, natural polymers mimicking the extracellular matrix (ECM) have low thrombogenicity and functional motifs that regulate cell-matrix interactions, with these factors being critical for tissue engineered vascular grafts especially grafts of small diameter. Gelatin constitutes a low cost substitute of soluble collagen but gelatin scaffolds so far have shown generally low strength and suture retention strength. In this study, we have devised the fabrication of novel, electrospun, multilayer, gelatin fibre scaffolds, with controlled fibre layer orientation, and optimised gelatin crosslinking to achieve not only compliance equivalent to that of coronary artery but also for the first time strength of the wet tubular acellular scaffold (swollen with absorbed water) same as that of the tunica media of coronary artery in both circumferential and axial directions. Most importantly, for the first time for natural scaffolds and in particular gelatin, high suture retention strength was achieved in the range of 1.8-1.94 N for wet acellular scaffolds, same or better than that for fresh saphenous vein. The study presents the investigations to relate the electrospinning process parameters to the microstructural parameters of the scaffold, which are further related to the mechanical performance data of wet, crosslinked, electrospun scaffolds in both circumferential and axial tubular directions. The scaffolds exhibited excellent performance in human smooth muscle cell (SMC) proliferation, with SMCs seeded on the top surface adhering, elongating and aligning along the local fibres, migrating through the scaffold thickness and populating a transverse distance of 186 μm and 240 μm 9 days post-seeding for scaffolds of initial dry porosity of 74 and 83%, respectively.

Peripheral nerve injury continues to be a major global health problem that can result in debilitating neurological deficits and neuropathic pain. Current state-of-the-art treatment involves reforming the damaged nerve pathway using a nerve autograft. Engineered nerve repair conduits can provide an alternative to the nerve autograft avoiding the inevitable tissue damage caused at the graft donor site. Commercially available nerve repair conduits are currently only considered suitable for repairing small nerve lesions; the design and performance of engineered conduits requires significant improvements to enable their use for repairing larger nerve defects. Carbon nanotubes (CNTs) are an emerging novel material for biomedical applications currently being developed for a range of therapeutic technologies including scaffolds for engineering and interfacing with neurological tissues. CNTs possess a unique set of physicochemical properties that could be useful within nerve repair conduits. This progress report aims to evaluate and consolidate the current literature pertinent to CNTs as a biomaterial for supporting peripheral nerve regeneration. The report is presented in the context of the state-of-the-art in nerve repair conduit design; outlining how CNTs may enhance the performance of next generation peripheral nerve repair conduits.

The "Workshop on Standards & Measurements for Tissue Engineering Scaffolds" was held on May 21, 2013 in Indianapolis, IN, and was sponsored by the ASTM International (ASTM). The purpose of the workshop was to identify the highest priority items for future standards work for scaffolds used in the development and manufacture of tissue engineered medical products (TEMPs). Eighteen speakers and 78 attendees met to assess current scaffold standards and to prioritize needs for future standards. A key finding was that the ASTM TEMPs subcommittees (F04.41-46) have many active "guide" documents for educational purposes, but few standard "test methods" or "practices." Overwhelmingly, the most clearly identified need was standards for measuring the structure of scaffolds, followed by standards for biological characterization, including in vitro testing, animal models and cell-material interactions. The third most pressing need was to develop standards for assessing the mechanical properties of scaffolds. Additional needs included standards for assessing scaffold degradation, clinical outcomes with scaffolds, effects of sterilization on scaffolds, scaffold composition, and drug release from scaffolds. Discussions highlighted the need for additional scaffold reference materials and the need to use them for measurement traceability. Workshop participants emphasized the need to promote the use of standards in scaffold fabrication, characterization, and commercialization. Finally, participants noted that standards would be more broadly accepted if their impact in the TEMPs community could be quantified. Many scaffold standard needs have been identified and focus is turning to generating these standards to support the use of scaffolds in TEMPs.

Crosslinked, multi-layer electrospun gelatin fiber scaffolds with generally ±45 degree fiber orientation have been used to grow human umbilical vein smooth muscle cells (HUVSMCs) to create a vascular tunica media graft. Scaffolds of different fiber diameter (2-5 μm in wet state), pore size, and porosity (16-21% in wet state) were assessed in terms of cell adherence and viability, cell proliferation, and migration in both in-plane and transverse directions through the scaffold as a function of time under static cell culture conditions. HUVSMC cell viability reached between 80 and 92% for all scaffolds after 9 days in culture. HUVSMCs adhered, elongated, and orientated in the fiber direction, and migrated through a scaffold thickness of 200-235 μm 9 days post-seeding under static conditions. The best scaffold was then used to assess the tissue engineering of HUVSMCs under dynamic conditions for a rotating, cell seeded, tubular scaffold in the bioreactor containing the culture medium. Dynamic conditions almost doubled the rate of cell proliferation through the scaffold, forming full tissue throughout a scaffold of 250-300 μm thickness 6 days post-seeding.

A closed loop implantable insulin delivery device that delivers insulin to the peritoneum in an automated fashion linked to changing glucose levels has been developed and previously tested in diabetic rats and pigs. The device delivers insulin via a glucose-sensitive gel that comprises of photopolymerized acrylic derivatives of dextran and concanavalin A and acts as both a sensor and controller of the amount of insulin released. In this work the long-term stability of these acrylic polymerized gels and also dextran and concanavalin A mixtures has been shown at 20 degrees C and 37 degrees C by rheological characterization when stored with and without 0.1% w/w glucose. Acrylic gels were found to have a stable complex viscosity for over 730 days at these temperatures indicating that over time they do not undergo degradation. Mixtures and polymerized gels were also dialyzed in the presence of chymotrypsin, which is present in the peritoneum (device implant site) to assess gel integrity across a range of pore size dialysis membranes. Polymerized acrylic gels contained in dialysis membranes of 50kDa were found to be resistant to degradation over a long time (>500 days). These results show that these gels would be ideal candidates as part of an implantable insulin delivery device.

The aim of this survey was to establish the limitations of open loop continuous subcutaneous insulin infusion (CSII) as perceived by current users of the technology, and to ascertain their interest in and requirements for a non-electronic implantable closed loop insulin pump, INSmart, currently under development for the treatment of type 1 diabetes. INSmart has been surgically implanted in the peritoneum in animal models and continuously restored normoglycaemia.A bottom-up survey design was used to determine both positive and negative experiences of patients currently using CSII to define the performance characteristics they would require from a non-electronic, implantable closed loop insulin pump.A total of 360 insulin pump users completed the survey. All respondents had type 1 diabetes, were predominantly from English-speaking countries and had been diagnosed before age 34 years. Most had well controlled blood glucose (BG) according to their selfreported HbAic results. They reported a reduction in this value after transferring to CSII from multi-dose injections. However, 70% of pump users had more than three hypoglycaemic episodes per week. Eighty percent reported self-measured BG values > 10mmol/L three or more times per month; 94% of respondents considered a (non-electronic implantable) closed loop insulin pump would make their BG management easier and improve their quality of life.The majority of respondents felt there were still many disadvantages to current external insulin pumps such as their constant visible presence, rotation of insertion sites and skin inflammation. These shortfalls could be overcome by a device, such as INSmart, that provides a relatively instant feedback mechanism for controlling insulin release due to its proposed location in the peritoneal cavity. Copyright (C) 2014 John Wiley & Sons.

A non-invasive, luminescence quenching technique is developed for continuous monitoring of oxygen spatial-temporal concentration distribution in fully hydrated gelatine gels, intended for use as scaffolds in tissue engineering. Two mass transfer-diffusion models were used to simulate the unsteady-state oxygen mass transport in the system. Oxygen diffusion coefficient and mass transfer coefficient at the water-gel interface were determined for un-crosslinked gelatine, as well as gelatine crosslinked with 1 and 1.5% w/v glutaraldehyde. While crosslinking and increased concentration of the crosslinking agent reduced oxygen mass transfer across the gel surface, both factors increased the diffusion coefficient of oxygen in the bulk of the gel. Voids in the gelatine's microstructure, which were generated during the crosslinking process due to shrinkage and associated internal stresses, were associated with both increasing the diffusion coefficient within the gel, as well as inhomogeneous diffusion of oxygen within the gel.

Optimizing the structural of porous tissue scaffolds to maximize their performance in tissue engineering applications is difficult and time-consuming. The interdependence of different processing parameters means that it is difficult to optimize the properties of the scaffolds during manufacture. The authors report a simple method of changing the permeability of polycaprolactone scaffolds through controlled exposure to gamma radiation. 30kGy of gamma radiation increases the permeability of water and bovine serum albumin through the scaffolds by approximately 80%. The authors also demonstrate that performance measures can detect subtle changes in complex structures that are difficult to detect using SEM.

This chapter describes current best practice in aspects of tissue engineering ranging from characterization of starting materials to assessing cell behaviour. Technical challenges to developing standards in this area are due in part to the range of materials, cell types, manufacturing and delivery routes that are used to produce products that can be used in many different applications. Guidance documents describing measurements that can be made and techniques to obtain them are a valuable source of information for specialists wishing to gain knowledge beyond their expertise in this multi-disciplinary subject.

The viscoelastic properties of fluids are key to their performance in industries ranging from biotechnology to the automotive industry. Traditionally, fluid viscoelastic properties are monitored with rheometers but these are expensive, require a skilled operator, function over a relatively limited frequency range and are not suitable for in situ monitoring. Piezoelectric cantilevers capable of in situ assessment of the rheological properties of relatively small fluid volumes have the potential to overcome many of these limitations and can be fabricated into low cost probes. Rheological assessment of test fluids using piezoelectric cantilevers is typically made through analysis of the cantilever's resonant oscillation in the fluids. For accurate results, the damping of the cantilever should be low as quantified by a high quality factor Q. This can be difficult in fluids of high viscosity particularly for microscopic cantilevers. In this paper, a "mesoscale" piezoelectric bimorph cantilever was used. The mesoscale refers to a size regime intermediate between microscopic and macroscopic, in this work the cantilever used has dimensions of the order of millimeters. This mesoscale cantilever displayed a sufficiently high Q to probe the rheological properties of highly damping and elastic fluids in situ. The developed probe will be ideally suited to in-process monitoring of high value products such as those in the biotechnology industry. (C) 2012 The Society of Rheology. [DOI: 10.1122/1.3670732]