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.

The molecular mechanisms utilised by mesenchymal stem cells in migration to acute myocardial infarction

Heart damage caused by acute myocardial infarction (AMI) is a leading cause of death and disability in Australia. Novel therapies are still required for the treatment of this condition due to the poor reparative ability of the heart. As such, cellular therapies that assist in the recovery of heart muscle are of great current interest. Culture expanded mesenchymal stem cells (MSC) represent a stem and progenitor cell population that has been shown to promote tissue recovery in pre-clinical studies of AMI. For MSC-based therapies in the clinic, an intravenous route of administration would ideally be used due to the low cost, ease of delivery and relative safety. The study of MSC migration is therefore clinically relevant for a minimally invasive cell therapy to promote regeneration of damaged tissue. C57BL/6, UBI-GFP-BL/6 and CD44-/-/GFP+/+ mice were utilised to investigate mMSC migration. To assist in murine models of MSC migration, a novel method was used for the isolation of murine MSC (mMSC). These mMSC were then expanded in culture and putative mMSC were positive for Sca-1, CD90.2, and CD44 and were negative for CD45 and CD11b. Furthermore, mMSC from C57BL/6 and UBI-GFP-BL/6 mice were shown to differentiate into cells of the mesodermal lineage. Cells from CD44-/-/GFP+/+ mice were positive for Sca-1 and CD90.2, and negative for CD44, CD45 and CD11b however, these cells were unable to differentiate into adipocytes and chondrocytes and express lineage specific genes, PLIN and ACAN. Analysis of mMSC chemokine receptor (CR) expression showed that although mMSC do express chemokine receptors, (including those specific for chemokines released after AMI), these were low or undetectable by mRNA. However, protein expression could be detected, which was predominantly cytoplasmic. It was further shown that in both healthy (unperturbed) and inflamed tissues, mMSC had very little specific migration and engraftment after intravenous injection. To determine if poor mMSC migration was due to the inability of mMSC to respond to chemotactic stimuli, chemokine expression in bone marrow, skin injury and hearts (healthy and after AMI) was analysed at various time points by quantitative real-time PCR (qRT PCR). Many chemokines were up-regulated after skin biopsy and AMI, but the highest acute levels were found for CXCL12 and CCL7. Due to their high expression in infarcted hearts, the chemokines CXCL12 and CCL7 were tested for their effect on mMSC migration. Despite CR expression at both protein and mRNA levels, migration in response to CXCL12 and CCL7 was low in mMSC cultured on Nunclon plastic. A novel tissue culture plastic technology (UpCellTM) was then used that allowed gentle non-enzymatic dissociation of mMSC, thus preserving surface expression of the CRs. Despite this the in vitro data indicated that CXCL12 fails to induce significant migration ability of mMSC, while CCL7 induces significant, but low-level migration. We speculated this may be because of low levels of surface expression of chemokine receptors. In a strategy to increase cell surface expression of mMSC chemokine receptors and enhance their in vitro and in vivo migration capacity, mMSC were pre-treated with pro-inflammatory cytokines. Increased levels of both mRNA and surface protein expression were found for CRs by pre-treating mMSC with pro-inflammatory cytokines including TNF-á, IFN-ã, IL-1á and IL-6. Furthermore, the chemotactic response of mMSC to CXCL12 and CCL7 was significantly higher with these pretreated cells. Finally, the effectiveness of this type of cell manipulation was demonstrated in vivo, where mMSC pre-treated with TNF-á and IFN-ã showed significantly increased migration in skin injury and AMI models. Therefore this thesis has demonstrated, using in vitro and in vivo models, the potential for prior manipulation of MSC as a possible means for increasing the utility of intravenously delivery for MSC-based cellular therapies.

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.

Comparative effectiveness of population interventions to improve access to reperfusion for ST-segment–elevation myocardial infarction in Australia

Background Improving timely access to reperfusion is a major goal of ST-segment–elevation myocardial infarction care. We sought to compare the population impact of interventions proposed to improve timely access to reperfusion therapy in Australia. Methods and Results Australian hospitals, population, and road network data were integrated using Geographical Information Systems. Hospitals were classified into those that provided primary percutaneous coronary intervention (PPCI) or fibrinolysis. Population impact of interventions proposed to improve timely access to reperfusion (PPCI, fibrinolysis, or both) were modeled and compared. Timely access to reperfusion was defined as the proportion of the population capable of reaching a fibrinolysis facility ≤60 minutes or a PPCI facility ≤120 minutes from emergency medical services activation. The majority (93.2%) of the Australian population has timely access to reperfusion, mainly (53%) through fibrinolysis. Only 40.2% of the population had timely access to PPCI, and access to PPCI services is particularly limited in regional and nonexistent in remote areas. Optimizing the emergency medical services’ response or increasing PPCI services resulted in marginal improvement in timely access (1.8% and 3.7%, respectively). Direct transport to PPCI facilities and interhospital transfer for PPCI improves timely access to PPCI for 19.4% and 23.5% of the population, respectively. Prehospital fibrinolysis markedly improved access to timely reperfusion in regional and remote Australia. Conclusions Significant gaps in timely provision of reperfusion remain in Australia. Systematic implementation of changes in service delivery has potential to improve timely access to PPCI for a majority of the population and improve access to fibrinolysis to those living in regional and remote areas.

Langevin dynamics modeling of the water diffusion tensor in partially aligned collagen networks

In this work, a Langevin dynamics model of the diffusion of water in articular cartilage was developed. Numerical simulations of the translational dynamics of water molecules and their interaction with collagen fibers were used to study the quantitative relationship between the organization of the collagen fiber network and the diffusion tensor of water in model cartilage. Langevin dynamics was used to simulate water diffusion in both ordered and partially disordered cartilage models. In addition, an analytical approach was developed to estimate the diffusion tensor for a network comprising a given distribution of fiber orientations. The key findings are that (1) an approximately linear relationship was observed between collagen volume fraction and the fractional anisotropy of the diffusion tensor in fiber networks of a given degree of alignment, (2) for any given fiber volume fraction, fractional anisotropy follows a fiber alignment dependency similar to the square of the second Legendre polynomial of cos(θ), with the minimum anisotropy occurring at approximately the magic angle (θMA), and (3) a decrease in the principal eigenvalue and an increase in the transverse eigenvalues is observed as the fiber orientation angle θ progresses from 0◦ to 90◦. The corresponding diffusion ellipsoids are prolate for θ < θMA, spherical for θ ≈ θMA, and oblate for θ > θMA. Expansion of the model to include discrimination between the combined effects of alignment disorder and collagen fiber volume fraction on the diffusion tensor is discussed.