Electro-spinning of pure collagen nano-fibres – just an expensive way to make gelatin?

Scaffolds manufactured from biological materials promise better clinical functionality, providing that characteristic features are preserved. Collagen, a prominent biopolymer, is used extensively for tissue engineering applications, because its signature biological and physico-chemical properties are retained in vitro preparations. We show here for the first time that the very properties that have established collagen as the leading natural biomaterial are lost when it is electro-spun into nano-fibres out of fluoroalcohols such as 1,1,1,3,3,3-hexafluoro-2-propanol or 2,2,2-trifluoroethanol. We further identify the use of fluoroalcohols as the major culprit in the process. The resultant nano-scaffolds lack the unique ultra-structural axial periodicity that confirms quarter-staggered supramolecular assemblies and the capacity to generate second harmonic signals, representing the typical crystalline triple-helical structure. They were also characterised by low denaturation temperatures, similar to those obtained from gelatin preparations ( p > 0.05). Likewise, circular dichroism spectra revealed extensive denaturation of the electro-spun collagen. Using pepsin digestion in combination with quantitative SDS-PAGE, we corroborate great losses of up to 99% of triple-helical collagen. In conclusion, electro-spinning of collagen out of fluoroalcohols effectively denatures this biopolymer, and thus appears to defeat its purpose, namely to create biomimetic scaffolds emulating the collagen structure and function of the extracellular matrix.

Immunohistochemical analysis of structural changes in collagen for the assessment of osteoarthritis

Collagen fibrillation within articular cartilage (AC) plays a key role in joint osteoarthritis (OA) progression and, therefore, studying collagen synthesis changes could be an indicator for use in the assessment of OA. Various staining techniques have been developed and used to determine the collagen network transformation under microscopy. However, because collagen and proteoglycan coexist and have the same index of refraction, conventional methods for specific visualization of collagen tissue is difficult. This study aimed to develop an advanced staining technique to distinguish collagen from proteoglycan and to determine its evolution in relation to OA progression using optical and laser scanning confocal microscopy (LSCM). A number of AC samples were obtained from sheep joints, including both healthy and abnormal joints with OA grades 1 to 3. The samples were stained using two different trichrome methods and immunohistochemistry (IHC) to stain both colourimetrically and with fluorescence. Using optical microscopy and LSCM, the present authors demonstrated that the IHC technique stains collagens only, allowing the collagen network to be separated and directly investigated. Fluorescently-stained IHC samples were also subjected to LSCM to obtain three-dimensional images of the collagen fibres. Changes in the collagen fibres were then correlated with the grade of OA in tissue. This study is the first to successfully utilize the IHC staining technique in conjunction with laser scanning confocal microscopy. This is a valuable tool for assessing changes to articular cartilage in OA.

Fine tuning of elasticity via crosslinking collagen-based materials to mediate mechanotransduction and stability using corona treatment

The common goal of tissue engineering is to develop substitutes that can closely mimic the structure of extracellular matrix (ECM). However, similarly important is the intensive material properties which have often been overlooked, in particular, for soft tissues that are not to bear load assumingly. The mechanostructural properties determine not only the structural stability of biomaterials but also their physiological functionality by directing cellular activity and regulating cell fate decision. The aim here is to emphasize that cells could sense intensive material properties like elasticity and reside, proliferate, migrate and differentiate accordinglyno matter if the construct is from a natural source like cartilage, skin etc. or of synthetic one. Meanwhile, the very objective of this work is to provide a tunable scheme for manipulating the elasticity of collagen-based constructs to be used to demonstrate how to engineer cell behavior and regulate mechanotransduction. Articular cartilage was chosen as it represents one of the most complex hierarchical arrangements of collagen meshwork in both connective tissues and ECM-like biomaterials. Corona discharge treatment was used to produce constructs with varying density of crosslinked collagen and stiffness accordingly. The results demonstrated that elastic modulus increased up to 33% for samples treated up to one minute as crosslink density was found to increase with exposure time. According to the thermal analysis, longer exposure to corona increased crosslink density as the denaturation enthalpy increased. However the spectroscopy results suggested that despite the stabilization of the collagen structure the integrity of the triple helical structure remained intact. The in vitro superficial culture of heterologous chondrocytes also determined that the corona treatment can modulate migration with increased focal adhesion of cells due to enhanced stiffness, without cytotoxicity effects, and providing the basis for reinforcing three-dimensional collagen-based biomaterials in order to direct cell function and mediate mechanotransduction.

Collagen in the scarless fetal skin wound : detection with picrosirius-polarization

Our group has developed an ovine model of deep dermal, partial-thickness burn where the fetus heals scarlessly and the lamb heals with scar. The comparison of collagen structure between these two different mechanisms of healing may elucidate the process of scarless wound healing. Picrosirius staining followed by polarized light microscopy was used to visualize collagen fibers, with digital capture and analysis. Collagen deposition increased with fetal age and the fibers became thicker, changing from green (type III collagen) to yellow/red (type I collagen). The ratio of type III collagen to type I was high in the fetus (166), whereas the lamb had a much lower ratio (0.2). After burn, the ratios of type III to type I collagen did not differ from those in control skin for either fetus or lamb. The fetal tissue maintained normal tissue architecture after burn while the lamb tissue showed irregular collagen organization. In conclusion, the type or amount of collagen does not alter significantly after injury. Tissue architecture differed between fetal and lamb tissue, suggesting that scar development is related to collagen cross-linking or arrangement. This study indicates that healing in the scarless fetal wound is representative of the normal fetal growth pattern, rather than a "response" to burn injury.

Simultaneous magnetic resonance imaging and consolidation measurement of articular cartilage

Magnetic resonance imaging (MRI) offers the opportunity to study biological tissues and processes in a non-disruptive manner. The technique shows promise for the study of the load-bearing performance (consolidation) of articular cartilage and changes in articular cartilage accompanying osteoarthritis. Consolidation of articular cartilage involves the recording of two transient characteristics: the change over time of strain and the hydrostatic excess pore pressure (HEPP). MRI study of cartilage consolidation under mechanical load is limited by difficulties in measuring the HEPP in the presence of the strong magnetic fields associated with the MRI technique. Here we describe the use of MRI to image and characterize bovine articular cartilage deforming under load in an MRI compatible consolidometer while monitoring pressure with a Fabry-Perot interferometer-based fiber-optic pressure transducer.

A collagen network phase improves cell seeding of open-pore structure scaffolds under perfusion

Scaffolds with open-pore morphologies offer several advantages in cell-based tissue engineering, but their use is limited by a low cell seeding efficiency. We hypothesized that inclusion of a collagen network as filling material within the open-pore architecture of polycaprolactone-tricalcium phosphate (PCL-TCP) scaffolds increases human bone marrow stromal cells (hBMSC) seeding efficiency under perfusion and in vivo osteogenic capacity of the resulting constructs. PCL-TCP scaffolds, rapid prototyped with a honeycomb-like architecture, were filled with a collagen gel and subsequently lyophilized, with or without final crosslinking. Collagen-free scaffolds were used as controls. The seeding efficiency was assessed after overnight perfusion of expanded hBMSC directly through the scaffold pores using a bioreactor system. By seeding and culturing freshly harvested hBMSC under perfusion for 3 weeks, the osteogenic capacity of generated constructs was tested by ectopic implantation in nude mice. The presence of the collagen network, independently of the crosslinking process, significantly increased the cell seeding efficiency (2.5-fold), and reduced the loss of clonogenic cells in the supernatant. Although no implant generated frank bone tissue, possibly due to the mineral distribution within the scaffold polymer phase, the presence of a non crosslinked collagen phase led to in vivo formation of scattered structures of dense osteoids. Our findings verify that the inclusion of a collagen network within open morphology porous scaffolds improves cell retention under perfusion seeding. In the context of cell-based therapies, collagen-filled porous scaffolds are expected to yield superior cell utilization, and could be combined with perfusion-based bioreactor devices to streamline graft manufacture.

Physiological investigation of periosteum structure and its application in periosteum tissue engineering

Although many different materials, techniques and methods, including artificial or engineered bone substitutes, have been used to repair various bone defects, the restoration of critical-sized bone defects caused by trauma, surgery or congenital malformation is still a great challenge to orthopedic surgeons. One important fact that has been neglected in the pursuit of resolutions for large bone defect healing is that most physiological bone defect healing needs the periosteum and stripping off the periosteum may result in non-union or non-healed bone defects. Periosteum plays very important roles not only in bone development but also in bone defect healing. The purpose of this project was to construct a functional periosteum in vitro using a single stem cell source and then test its ability to aid the repair of critical-sized bone defect in animal models. This project was designed with three separate but closely-linked parts which in the end led to four independent papers. The first part of this study investigated the structural and cellular features in periostea from diaphyseal and metaphyseal bone surfaces in rats of different ages or with osteoporosis. Histological and immunohistological methods were used in this part of the study. Results revealed that the structure and cell populations in periosteum are both age-related and site-specific. The diaphyseal periosteum showed age-related degeneration, whereas the metaphyseal periosteum is more destructive in older aged rats. The periosteum from osteoporotic bones differs from normal bones both in terms of structure and cell populations. This is especially evident in the cambial layer of the metaphyseal area. Bone resorption appears to be more active in the periosteum from osteoporotic bones, whereas bone formation activity is comparable between the osteoporotic and normal bone. The dysregulation of bone resorption and formation in the periosteum may also be the effect of the interaction between various neural pathways and the cell populations residing within it. One of the most important aspects in periosteum engineering is how to introduce new blood vessels into the engineered periosteum to help form vascularized bone tissues in bone defect areas. The second part of this study was designed to investigate the possibility of differentiating bone marrow stromal cells (BMSCs) into the endothelial cells and using them to construct vascularized periosteum. The endothelial cell differentiation of BMSCs was induced in pro-angiogenic media under both normoxia and CoCl2 (hypoxia-mimicking agent)-induced hypoxia conditions. The VEGF/PEDF expression pattern, endothelial cell specific marker expression, in vitro and in vivo vascularization ability of BMSCs cultured in different situations were assessed. Results revealed that BMSCs most likely cannot be differentiated into endothelial cells through the application of pro-angiogenic growth factors or by culturing under CoCl2-induced hypoxic conditions. However, they may be involved in angiogenesis as regulators under both normoxia and hypoxia conditions. Two major angiogenesis-related growth factors, VEGF (pro-angiogenic) and PEDF (anti-angiogenic) were found to have altered their expressions in accordance with the extracellular environment. BMSCs treated with the hypoxia-mimicking agent CoCl2 expressed more VEGF and less PEDF and enhanced the vascularization of subcutaneous implants in vivo. Based on the findings of the second part, the CoCl2 pre-treated BMSCs were used to construct periosteum, and the in vivo vascularization and osteogenesis of the constructed periosteum were assessed in the third part of this project. The findings of the third part revealed that BMSCs pre-treated with CoCl2 could enhance both ectopic and orthotopic osteogenesis of BMSCs-derived osteoblasts and vascularization at the early osteogenic stage, and the endothelial cells (HUVECs), which were used as positive control, were only capable of promoting osteogenesis after four-weeks. The subcutaneous area of the mouse is most likely inappropriate for assessing new bone formation on collagen scaffolds. This study demonstrated the potential application of CoCl2 pre-treated BMSCs in the tissue engineering not only for periosteum but also bone or other vascularized tissues. In summary, the structure and cell populations in periosteum are age-related, site-specific and closely linked with bone health status. BMSCs as a stem cell source for periosteum engineering are not endothelial cell progenitors but regulators, and CoCl2-treated BMSCs expressed more VEGF and less PEDF. These CoCl2-treated BMSCs enhanced both vascularization and osteogenesis in constructed periosteum transplanted in vivo.

Feasibility of agent-based modelling of articular cartilage including a conceptual representation of its structure

Articular cartilage is a complex structure with an architecture in which fluid-swollen proteoglycans constrained within a 3D network of collagen fibrils. Because of the complexity of the cartilage structure, the relationship between its mechanical behaviours at the macroscale level and its components at the micro-scale level are not completely understood. The research objective in this thesis is to create a new model of articular cartilage that can be used to simulate and obtain insight into the micro-macro-interaction and mechanisms underlying its mechanical responses during physiological function. The new model of articular cartilage has two characteristics, namely: i) not use fibre-reinforced composite material idealization ii) Provide a framework for that it does probing the micro mechanism of the fluid-solid interaction underlying the deformation of articular cartilage using simple rules of repartition instead of constitutive / physical laws and intuitive curve-fitting. Even though there are various microstructural and mechanical behaviours that can be studied, the scope of this thesis is limited to osmotic pressure formation and distribution and their influence on cartilage fluid diffusion and percolation, which in turn governs the deformation of the compression-loaded tissue. The study can be divided into two stages. In the first stage, the distributions and concentrations of proteoglycans, collagen and water were investigated using histological protocols. Based on this, the structure of cartilage was conceptualised as microscopic osmotic units that consist of these constituents that were distributed according to histological results. These units were repeated three-dimensionally to form the structural model of articular cartilage. In the second stage, cellular automata were incorporated into the resulting matrix (lattice) to simulate the osmotic pressure of the fluid and the movement of water within and out of the matrix; following the osmotic pressure gradient in accordance with the chosen rule of repartition of the pressure. The outcome of this study is the new model of articular cartilage that can be used to simulate and study the micromechanical behaviours of cartilage under different conditions of health and loading. These behaviours are illuminated at the microscale level using the socalled neighbourhood rules developed in the thesis in accordance with the typical requirements of cellular automata modelling. Using these rules and relevant Boundary Conditions to simulate pressure distribution and related fluid motion produced significant results that provided the following insight into the relationships between osmotic pressure gradient and associated fluid micromovement, and the deformation of the matrix. For example, it could be concluded that: 1. It is possible to model articular cartilage with the agent-based model of cellular automata and the Margolus neighbourhood rule. 2. The concept of 3D inter connected osmotic units is a viable structural model for the extracellular matrix of articular cartilage. 3. Different rules of osmotic pressure advection lead to different patterns of deformation in the cartilage matrix, enabling an insight into how this micromechanism influences macromechanical deformation. 4. When features such as transition coefficient were changed, permeability (representing change) is altered due to the change in concentrations of collagen, proteoglycans (i.e. degenerative conditions), the deformation process is impacted. 5. The boundary conditions also influence the relationship between osmotic pressure gradient and fluid movement at the micro-scale level. The outcomes are important to cartilage research since we can use these to study the microscale damage in the cartilage matrix. From this, we are able to monitor related diseases and their progression leading to potential insight into drug-cartilage interaction for treatment. This innovative model is an incremental progress on attempts at creating further computational modelling approaches to cartilage research and other fluid-saturated tissues and material systems.

Aligned poly(L-lactic-co-ε-caprolactone) electrospun microfibers and knitted structure: a novel composite scaffold for ligament tissue engineering

We developed a novel technique involving knitting and electrospinning to fabricate a composite scaffold for ligament tissue engineering. Knitted structures were coated with poly(L-lactic-co-e-caprolactone) (PLCL) and then placed onto a rotating cylinder and a PLCL solution was electrospun onto the structure. Highly aligned 2-μm-diameter microfibers covered the space between the stitches and adhered to the knitted scaffolds. The stress–strain tensile curves exhibited an initial toe region similar to the tensile behavior of ligaments. Composite scaffolds had an elastic modulus (150 ± 14 MPa) similar to the modulus of human ligaments. Biological evaluation showed that cells proliferated on the composite scaffolds and they spontaneously orientated along the direction of microfiber alignment. The microfiber architecture also induced a high level of extracellular matrix secretion, which was characterized by immunostaining. We found that cells produced collagen type I and type III, two main components found in ligaments. After 14 days of culture, collagen type III started to form a fibrous network. We fabricated a composite scaffold having the mechanical properties of the knitted structure and the morphological properties of the aligned microfibers. It is difficult to seed a highly macroporous structure with cells, however the technique we developed enabled an easy cell seeding due to presence of the microfiber layer. Therefore, these scaffolds presented attractive properties for a future use in bioreactors for ligament tissue engineering.

Gelatinase A (MMP-2) activation by skin fibroblasts : dependence on MT1-MMP expression and fibrillar collagen form

The respective requirements of collagen and MT1-MMP in the activation of MMP-2 by primary fibroblast cultures were explored further. Three-dimensional gels enriched in human collagen types I and III or composed of recombinant human type II or III collagen, caused increased MT1-MMP production (mRNA and protein) and induced MMP-2 activation. Only marginal induction was seen with dried monomeric collagen confirming the need for collagen fibrillar organisation for activation. To our surprise, relatively low amounts (as low as 25 μg/ml) of acid soluble type I collagen added to fibroblast cultures also induced potent MMP-2 activation. However, the requirement for collagen fibril formation by the added collagen was indicated by the inhibition seen when the collagen was pre-incubated with a fibril-blocking peptide, and the reduced activation seen with alkali-treated collagen preparations known to have impaired fibrilisation. Pre-treatment of the collagen with sodium periodate also abrogated MMP-2 activation induction. Further evidence of the requirement for collagen fibril formation was provided by the lack of activation when type IV collagen, which does not form collagen fibrils, was added in the cultures. Fibroblasts derived from MT1-MMP-deficient mice were unable to activate MMP-2 in response to either three-dimensional collagen gel or added collagen solutions, compared to their littermate controls. Collectively, these data indicate that the fibrillar structure of collagen and MT1-MMP are essential for the MMP-2 activational response in fibroblasts.

Multimodal imaging of the collagen and elastic fibre networks in the bovine intervertebral disc

INTRODUCTION. The intervertebral disc is the largest avascular structure in the human body, withstanding transient loads of up to nine times body weight during rigorous physical activity. The key structural elements of the disc are a gel-like nucleus pulposus surrounded by concentric lamellar rings containing criss-crossed collagen fibres. The disc also contains an elastic fiber network which has been suggested to play a structural role, but to date the relationship between the collagen and elastic fiber networks is unclear. CONCLUSION. The multimodal transmitted and reflected polarized light microscopy technique developed here allows clear differentiation between the collagen and elastic fiber networks of the intervertebral disc. The ability to image unstained specimens avoids concerns with uneven stain penetration or specificity of staining. In bovine tail discs, the elastic fiber network is intimately associated with the collagen network.

Magnetic and morphological properties of ferrofluid-impregnated hydroxyapatite/collagen scaffolds

In this article we present the morphological and magnetic characterization of ferrofluid-impregnated biomimetic scaffolds made of hydroxyapatite and collagen used for bone reconstruction. We describe an innovative and simple impregnation process by which the ferrofluid is firmly adsorbed onto the hydroxyapatite/collagen scaffolds. The process confers sufficient magnetization to attract potential magnetic carriers, which may be used to transport bioactive agents that favour bone regeneration. The crystalline structure of the magnetite contained in the ferrofluid is preserved and its quantity, estimated from the weight gain due to the impregnation process, is consistent with that obtained from energy dispersive X-ray spectroscopy. The magnetization, measured with a superconducting quantum interference device, is uniform throughout the scaffolds, demonstrating the efficiency of the impregnation process. The field emission gun scanning electron microscopy characterization demonstrates that the process does not alter the morphology of the hydroxyapatite/collagen scaffolds, which is essential for the preservation of their bioactivity and consequently for their effectiveness in promoting bone formation.

New dimeric supramolecular structure of mixed (phthalocyaninato)(porphyrinato)-europium(III) sandwiches : preparation and spectroscopic characteristics

The mixed double-decker Eu\[Pc(15C5)4](TPP) (1) was obtained by base-catalysed tetramerisation of 4,5-dicyanobenzo-15-crown-5 using the half-sandwich complex Eu(TPP)(acac) (acac = acetylacetonate), generated in situ, as the template. For comparative studies, the mixed triple-decker complexes Eu2\[Pc(15C5)4](TPP)2 (2) and Eu2\[Pc(15C5)4]2(TPP) (3) were also synthesised by the raise-by-one-story method. These mixed ring sandwich complexes were characterised by various spectroscopic methods. Up to four one-electron oxidations and two one-electron reductions were revealed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). As shown by electronic absorption and infrared spectroscopy, supramolecular dimers (SM1 and SM3) were formed from the corresponding double-decker 1 and triple-decker 3 in the presence of potassium ions in MeOH/CHCl3.