Visualising feasible operating ranges within tissue engineering systems using a "windows of operation" approach: A perfusion-scaffold bioreactor case study.

Tissue engineering approaches to developing functional substitutes are often highly complex, multivariate systems where many aspects of the biomaterials, bio-regulatory factors or cell sources may be controlled in an effort to enhance tissue formation. Furthermore, success is based on multiple performance criteria reflecting both the quantity and quality of the tissue produced. Managing the trade-offs between different performance criteria is a challenge. A "windows of operation" tool that graphically represents feasible operating spaces to achieve user-defined levels of performance has previously been described by researchers in the bio-processing industry. This paper demonstrates the value of "windows of operation" to the tissue engineering field using a perfusion-scaffold bioreactor system as a case study. In our laboratory, perfusion bioreactor systems are utilised in the context of bone tissue engineering to enhance the osteogenic differentiation of cell-seeded scaffolds. A key challenge of such perfusion bioreactor systems is to maximise the induction of osteogenesis but minimise cell detachment from the scaffold. Two key operating variables that influence these performance criteria are the mean scaffold pore size and flow-rate. Using cyclooxygenase-2 and osteopontin gene expression levels as surrogate indicators of osteogenesis, we employed the "windows of operation" methodology to rapidly identify feasible operating ranges for the mean scaffold pore size and flow-rate that achieved user-defined levels of performance for cell detachment and differentiation. Incorporation of such tools into the tissue engineer's armoury will hopefully yield a greater understanding of the highly complex systems used and help aid decision making in future translation of products from the bench top to the market place. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.</p

Bone as a composite material: the role of osteons as barriers to crack growth in compact bone.

This article summarises a number of studies in the area of bone microdamage which were carried out in our laboratory over the past 5 years. A technique was developed to label microcracks during mechanical testing. Fluorescent chelating agents were applied at intervals to bone specimens fatigue tested in cyclic compression until failure occurred. Microcrack densities were measured and microcrack length at the time of encountering the cement line surrounding an osteon was also recorded. Microcracks were shown to develop rapidly during the first stage of testing but then further accumulation of cracks did not occur until the period just before failure. The majority of microcracks were found in interstitial bone and did not penetrate cement lines. Only microcracks greater than 300 μm in length were found to be capable of penetrating osteons. This work provides experimental data to support the hypothesis that secondary osteons act as barriers to crack propagation in compact bone

Microcrack accumulation at different intervals during fatigue testing of compact bone.

Fatigue damage in bone occurs in the form of microcracks. This microdamage contributes to the formation of stress fractures and acts as a stimulus for bone remodelling. A technique has been developed, which allows microcrack growth to be monitored during the course of a fatigue test by the application of a series of fluorescent chelating agents. Specimens were taken from bovine tibiae and fatigue tested in cyclic compression at a stress range of 80MPa. The specimens were stained before testing with alizarin and up to three other chelating agents were applied during testing to label microcracks formed at different times. Microcracks initiated in interstitial bone in the early part of a specimen's life. Further accumulation of microcracks is then suppressed until the period late in the specimen's life. Microcracks were found to be longer in the longitudinal than in the transverse direction. Only a small proportion of cracks are actively propagating; these are longer than non-propagating cracks. These results support the concept of a microstructural barrier effect existing in bone, whereby cracks initiate easily but slow down or stop at barriers such as cement lines

The nature of fatigue damage in bone

Bone is unusual among structural materials as it is alive and capable of self-repair. Fatigue-induced microdamage is repaired by bone remodelling, but if damage accumulates too quickly, or remodelling is deficient, fatigue failure may result. Fatigue is thought to contribute to both stress and fragility fractures which are of major clinical importance. Despite this, we do not fully understand the nature of fatigue damage in bone. Human rib sections, containing microcracks stained with basic fuchsin, were serially sectioned and microcracks identified and reconstructed in three dimensions using computer software. Microcracks were elliptical in shape, 400 μm long and 100 μm wide, typical of a transversely isotropic material. Chelating agents which bind Ca2+ were found to label microcracks in rib, as well as mineralising bone surfaces and resorption sites, suggesting that microcracks are Ca2+ ion-lined discontinuities in the hydroxyapatite matrix. Ca2+ ions were exposed by scratching the surface of bovine bone specimens and labelled with chelating agents in sequence. The optimal four agent sequence was: alizarin, xylenol orange, calcein and calcein blue. Two dye sequences were used to differentiate between pre-existing and test-induced microdamage in bovine samples fatigue tested in compression and longer sequences labelled microcrack growth. Microcrack dimensions can be used to calculate stress intensity values and, together with fatigue test data, can aid theoretical models to predict fatigue failure in bone

An improved labelling technique for monitoring microcrack growth in compact bone.

Fatigue-induced damage plays an important role in bone remodelling and in the formation of stress and fragility fractures. Recently, a technique has been developed (Lee, T.C. et al., Sequential labelling of microdamage in bone using chelating agents. Journal of Orthopedic Research, 18 (2000) 322-325) which allows microcrack growth in trabecular bone to be monitored by the application of a series of chelating fluorochromes, however, some limitations were identified with the process. The aims of this study were to refine the method of detection using these agents in order to determine the optimal sequence of application and the optimal concentrations which allowed all the agents to fluoresce equally brightly using UV epifluorescence. A chemical analysis process, ion chromatography, followed by validation tests on bone samples showed that the optimal sequence of application and concentration of each agent was alizarin complexone (0.0005 M) followed by xylenol orange (0.0005 M), calcein (0.0005 M) and calcein blue (0.0001 M). A fifth agent, oxytetracycline was excluded from the study after recurring problems were found with its ability to chelate exposed calcium when applied in sequence with the other agents. This work has developed a sequential labelling technique, which allows for microcrack propagation during fatigue testing of bone specimens to be monitored without the problem of chelating agent substitution occurring

A theoretical model for the simulation of microdamage, repair and adaptation in compact bone.

This paper describes a new theoretical approach to bone microdamage, in which a population of cracks is explicitly modelled. A given sample of bone is assumed to contain a certain number of cracks, whose growth characteristics are described with an equation containing stochastic variables to create statistical differences from one crack to another. This type of model allows us to predict a wide variety of data. The present paper illustrates the different types of prediction which can be made, including: (i) standard damage parameters such as the number and length of cracks and the reduction in stiffness; (ii) fatigue test data such as the number of cycles to failure as a function of stress level, including scatter; (iii) effects due to the living system, including repair, remodelling and adaptation. A useful feature of the model is our ability to examine the statistics of the crack population in detail to find, for example, the number of cracks which are potentially dangerous as opposed to those which are dormant, and to investigate the reasons for increased crack numbers in the bones of older people. The potential also exists to use the model to investigate different theories of bone remodelling and adaptation

The effect of bone microstructure on the initiation and growth of microcracks.

Osteonal bone is often compared to a composite material and to metals as discontinuities within the material may provide sites of stress concentration for crack initiation and serve as barriers to crack growth. However, little experimental data exist to back up these hypotheses. Fluorescent chelating agents were applied at specific intervals to bone specimens fatigue tested in cyclic compression at a stress range of 80 MPa. The failed specimens were sectioned and labelled microcracks identified using UV epifluorescence microscopy. Microcrack lengths were measured and their relationship to cement lines surrounding secondary osteons recorded. Microcrack length at the time of encountering a cement line was also measured. Microcracks of less than 100mum stopped growing when they encountered a cement line. Microcracks of greater than 100mum in length continued to grow after encountering a cement line surrounding an osteon. Only microcracks greater than 300mum in length were capable of penetrating osteons and these microcracks were the only ones which were observed to cause failure in the specimen. These experimental data support the hypothesis that secondary osteons act as barriers to crack propagation in compact bone. However, it shows that this microstructural barrier effect is dependent on the crack length at the time of encountering an osteon. For the vast majority of cracks, osteons act as barriers to growth but for the minority of cracks that are long enough and do break through the cement line, an osteon may actually act as a weakness in the bone and facilitate crack propagation

Articulation inspired by nature: a review of biomimetic and biologically active 3D printed scaffolds for cartilage tissue engineering

In the human body, articular cartilage facilitates the frictionless movement of synovial joints. However, due to its avascular and aneural nature, it has a limited ability to self-repair when damaged due to injury or wear and tear over time. Current surgical treatment options for cartilage defects often lead to the formation of fibrous, non-durable tissue and thus a new solution is required. Nature is the best innovator and so recent advances in the field of tissue engineering have aimed to recreate the microenvironment of native articular cartilage using biomaterial scaffolds. However, the inability to mirror the complexity of native tissue has hindered the clinical translation of many products thus far. Fortunately, the advent of 3D printing has provided a potential solution. 3D printed scaffolds, fabricated using biomimetic biomaterials, can be designed to mimic the complex zonal architecture and composition of articular cartilage. The bioinks used to fabricate these scaffolds can also be further functionalised with cells and/or bioactive factors or gene therapeutics to mirror the cellular composition of the native tissue. Thus, this review investigates how the architecture and composition of native articular cartilage is inspiring the design of biomimetic bioinks for 3D printing of scaffolds for cartilage repair. Subsequently, we discuss how these 3D printed scaffolds can be further functionalised with cells and bioactive factors, as well as looking at future prospects in this field

Editorial: Novel composites and multi-material assembly approaches for tissue regeneration

In this Research Topic entitled “Novel composites and multi-material assembly approaches for Tissue Regeneration,” we aimed to bring forth new insights and ideas from our peers who combine various materials to create complex tissue-regeneration solutions. As we wrap up this topic for now, we are happy to report that we were able to attract several interesting research studies, which generated significant new insight for our field. On composite materials, we received studies combining collagen composites with other biological factors, native extracellular matrix composites with hydrogels and cellulose-hydrogel composites among biologically sourced materials; and polymer-drug, polymer-polymer and polymer-hydrogel composites among (mostly) synthetic materials. On multi-material fabrication approaches, the papers received covered pressure based extrusion (bioprinting), composite material electrospnning, and an automated sheet rolling technique to obtain 3D constructs from electrospun films

Biodegradable materials for surgical management of stress urinary incontinence: a narrative review

Stress urinary incontinence (SUI) was managed with techniques such as colposuspension, autologous fascia sling and urethral bulking agents. The introduction of the mid-urethral polypropylene (PP) sling in the 1990s led to a significant and rapid global change in SUI surgery. The synthetic non-degradable PP sling had superior results to traditional SUI procedures but its use has now declined due to significant complications such as pain and mesh erosion. These complications are attributed to its poor biocompatibility and integration into vaginal tissues. The efficacy of PP was extrapolated from studies on abdominal wall repair and it is now clear that integration of implanted materials in the pelvic floor differs from the abdominal wall. With PP prohibited in some jurisdictions, female patients with SUI have few management options. In the present review we summarise recent advances in SUI surgery and evaluate potential alternatives to PP slings with a particular focus on degradable materials. Allograft and xenograft materials demonstrate good biocompatibility but have yielded suboptimal cure rates. Tissue engineered synthetic degradable materials outperform unmodified synthetic degradable materials in terms of biomechanics and cell support. Synthetic tissue engineered degradable materials show promising results from in vitro studies and future research should focus on animal and human trials in this field