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"Engineered muscle tissue offers a promising solution for the treatment of large muscle defects. Three-dimensional tissue engineered matrices, such as microthreads, can be used to grow new myofibers that will reduce scar formation and integrate easily into native myofibers. We hypothesize that adsorbing growth factors to the surface of braided collagen scaffolds using crosslinking strategies will promote muscle derived fibroblastic cell (MDFC) attachment and growth, which will serve as a platform for delivering cells to large muscle defects for muscle regeneration. To test this hypothesis, self-assembled type I collagen threads were braided and crosslinked using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) with and without heparin and 5 ng/mL, 10 ng/mL, or 50 ng/mL fibroblast growth factor (FGF-2) bound to the surface. Using immunhistochemistry, braided collagen scaffolds showed the presence of FGF-2 on the surface, and braiding the microthreads increased the mechanical properties compared to single threads. To determine the effect of FGF-2 on MDFC attachment, growth, and alignment, scaffolds were seeded with a MDFC cell suspension for 4 hours using a PDMS mold with a sealed 1 mm by 12 mm channel and cultured for 1, 5, or 7 days. After 1 day of culture, the results show a significant increase in cell attachment on braids crosslinked with EDC/NHS with heparin and no significant difference in attachment between the different concentrations of FGF-2 and EDC/NHS crosslinked scaffolds. After 7 days in culture, the MDFCs responded to FGF-2 with a positive linear correlation between growth rate and concentration of FGF-2 on the surface. Additionally, all control scaffolds showed cellular alignment after 7 days, while MDFCs on FGF-2 modified scaffolds showed limited alignment. These results show braided collagen scaffolds crosslinked with EDC/NHS with heparin delivering a controlled quantity of FGF-2 can support MDFC attachment and growth, which may serve as an exciting new approach to facilitate the growth and ultimately the delivery of cells to large defects in muscle regeneration."

Digitized by Kansas Correctional Industries

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Extracellular matrix (ECM) microfibrils are critical components of connective tissues with a wide range of mechanical and cellular signalling functions. The focus of this PhD thesis is the study of two microfibrillar assemblies, fibrillin-1 and collagen VI. Fibrillin is a large ECM glycoprotein which facilitates the deposition of elastin in elastic tissues such aorta, skin and lung and sequesters growth factors in the matrix. Collagen VI is a heteromeric network-forming collagen which is expressed in tissues such as skin, lung, blood vessels and articular cartilage where it anchors cells into the ECM allowing for the transduction of biochemical and mechanical signals. The structures of some individual domains and short fragments of both fibrillin and collagen VI have been solved, but it is not fully understood how they are arranged into microfibrils and how these microfibrils are arranged into tissues. Therefore the aim of this project has been to determine the hierarchical organisation of fibrillin and collagen VI across multiple length scales. The nanoscale structure of the fibrillin microfibril was determined using negative stain TEM and single particle reconstruction. Microfibrils had a hollow tube-like structure with well-defined bead, arm, interbead and shoulder regions. To overcome flexibility observed in the microfibril, separate sub-models of the different fibrillin regions were modelled. The bead region had a complex double layered structure with an interwoven core and ring structures. The arm region had four separate densities which are potentially formed from dimers of fibrillin molecules. Serial block face scanning electron microscopy (SBF-SEM) and electron tomography allowed for the in situ 3D imaging of individual fibrillin microfibrils in ciliary zonule tissue. Microfibrils in ciliary zonule fibres were held together by cross bridges between microfibrils. These ciliary zonule fibres were then organised into larger fascicle-like structures which were stabilised by circumferentially arranged ciliary zonule fibres. The frozen hydrated structure of the collagen VI half-bead was reconstructed using cryo-TEM. The half-bead region had a compact hollow head, and flexible tail regions, the tail regions were linked together by the collagenous interbead region. SBF-SEM and electron tomography of the pericellular matrix (PCM) of murine articular cartilage revealed that the PCM had a meshwork-like organisation formed from globular densities ~30 nm in diameter. Together a combinatorial approach to image ECM microfibrils from the sub-molecular level to intact tissue structures spanning nanometre to millimetre length scales is presented. This provides a better understanding of how fibrillin and collagen VI microfibrils are organised in tissues.

Purpose: Corneal transparency depends upon the precise organization of corneal stromal extracellular matrix and corneal endothelial function. Stromal structure and extracellular matrix organization is responsible for proper refraction of light into the eye. The corneal endothelium is responsible for pumping excess fluid out of the cornea, effectively maintaining corneal hydration and thickness. Corneal transplantation is the current form of treatment for corneal endothelial and stromal dystrophies. The mechanisms controlling stromal collagen fibril packing and organization into orthogonal layers as well as maturation of the endothelium into a fully functioning cellular layer are unknown. Collagens XII and XIV, fibril associated collagens with interrupted triple helices (FACIT), have been implicated in matrix-matrix interactions regulating structure, cell behavior, and cell-matrix interactions. The overall aim is to determine the role of collagens XII and XIV in fibril assembly, fibril packing, lamellar assembly, stromal organization, corneal thickness, and endothelial maturation. The general hypothesis is that collagens XII and XIV regulate cornea stromal matrix development and structure, endothelial development, and corneal function. This dissertation assesses three specific hypotheses: 1) Collagen XIV regulates lateral fibril growth and fibril packing through fibrillar surface interactions; 2) Collagen XII regulates fibril packing, lamellar assembly, stromal organization, corneal thickness, and therefore, corneal function; and 3) FACIT collagens in the specialized posterior stroma regulate the acquisition of function in the corneal endothelium. Materials and Methods: The temporal and spatial expression patterns of collagens XII and XIV were determined in the murine cornea using quantitative PCR, semi-quantitative immuno-blots and immuno-localization approaches. To determine the regulatory roles of collagens XII and XIV in stromal and endothelial development, mouse models null for collagens XII or XIV were. This was coupled with ultrastructural and morphometric analyses of fibril assembly, fibril packing, lamellar organization, and endothelial maturation. The roles of collagens XII and XIV in corneal structure were determined using measurements of corneal thickness at postnatal day (P) 30 and P60. Results: Collagen XIV had a dynamic expression pattern in wild type (WT) corneal development. Corneas at P4 expressed the highest amount of collagen XIV with a sharp reduction by P10. Collagen XIV localized in the full thickness of the stroma at P4 and P14. At P30 and P90 there was less immuno-reactivity for collagen XIV in the WT stroma. The collagen XIV null stromas contained larger diameter fibrils when compared to P30 WT stromas. The null stromas also exhibited irregular spacing of fibrils. In the absence of collagen XIV there was an abnormal increase in corneal thickness. Unlike collagen XIV, collagen XII localized homogenously throughout the WT corneal stroma from P4 to P90. Collagen XII content was relatively constant in the cornea from P4 to P90. The collagen XII P30 null stromas contained areas of increased fibril density and disruption of lamellar organization. Corneal thickness increased in the absence of collagen XII at P60. Corneas deficient in Col12a1-/- and/or Col14a1-/- exhibited a delay in maturation. The null corneal endothelia retained vacuoles seen only in the immature WT P4 cornea. The P30 Col12a1-/- and Col14a1-/- endothelia had patchy localization of ZO-1 similar to that of an immature endothelium. There was an abnormal increase in thickness at P30 in the absence of collagens XII and XIV suggesting an increase in stromal hydration. Conclusions: Collagen XIV regulates fibril assembly, and regular fibril packing in early stromal development. Collagen XII regulates fibril packing, lamellar assembly, stromal organization, and influences the keratocyte network. Both collagens XII and XIV regulate endothelial maturation and acquisition of function through interactions between the stroma and underlying endothelium. Understanding the mechanisms behind stromal organization and endothelial maturation will improve treatment of stromal and endothelial dystrophies, as well as other diseases that involve extracellular matrix-cell interactions mediated by FACIT collagens.

Breast cancer is the most common cause of cancer death in Australian women. Current pathological analysis examines a small section of tissue for cellular and plasma abnormalities using a light microscope. However, this method of diagnosis, despite being the current gold standard, has its limitations, where human error and professional experience can influence a patient’s diagnosis. A potential alternative or adjunct to conventional histopathology for classifying tissue disease status is offered by Small Angle X-ray Scattering (SAXS). At the time of commencement of this work, there had been several small scale studies which examined the potential of SAXS to classify the disease status of breast tissue. These tended to focus on the supramolecular structure of collagen fibrils found in the breast, where it is known that the degradation of these fibres is related to the spread of disease. Most previous studies also used a synchrotron as an X-ray source, due to the intense and highly collimated flux available. This study used a synchrotron source, but also evaluated the use of a laboratory X-ray source, as a more convenient and relatively inexpensive alternative that could one day find application in the clinic. The work presented in this thesis analyses the largest cohort of patients and breast tissue samples studied to date using SAXS: 130 patients with 543 tissue samples. Tissues were sourced from surgical waste and classified into four groups: invasive carcinoma, benign, normal, and mammoplasty. Mammoplasty tissue samples were harvested from patients undergoing breast reduction and/or reconstruction, where no history or presence of disease was indicated. Normal tissue was sampled from patients with known disease, but pathological analysis of the tissue core diagnosed it as normal. A comprehensive analysis of the scattering patterns was carried out, analysing features arising from the collagen structure and orientation, the total scattered intensity, and adipose tissue in the breast. Features related to the axial D-spacing of the collagen fibrils within the breast tissue as well as the integrated scattering intensity (called amorphous scatter) demonstrated the highest ability to discriminate tissue types, in SAXS images acquired from both the synchrotron source and the laboratory X-ray source. The amorphous scatter intensities obtained using a synchrotron source showed highly significant differences (p < 0.01) for almost all of the tissue pair comparisons: invasive carcinoma vs. benign, invasive carcinoma vs. normal, invasive carcinoma vs. mammoplasty, benign vs. mammoplasty, and normal vs. mammoplasty. However, no significant difference was seen in the amorphous scatter between benign versus normal tissues (p = 0.30). The amorphous scatter values increased with severity of disease, i.e. it was the highest for invaded tissues and decreased progressively from benign to normal to mammoplasty. There was a significant difference between normal and mammoplasty tissue types using the amorphous scatter as a discriminator (p = 0.0025). Pathological assessment cannot differentiate between these two tissue types, which suggests that there may be changes occurring in these tissue structures at the supramolecular level that can be characterised using SAXS. The ability of SAXS to reveal structural differences between normal and mammoplasty tissue types is highly significant, for both disease diagnosis and treatment, as well as for understanding disease progression. For example, these differences might aid in determining surgical margin clearance of excised breast lesions as well as potentially provide a means of pre-screening or perhaps improve false-negative rates of diagnosis. The potential of SAXS to reveal macroscopic extent and directional spread of disease was explored using two-dimensional mapping of the amorphous scatter. These maps showed broad agreement with histopathological diagnosis, but further investigation regarding their reliability and interpretation for clinical utility is still needed. Changes in both the amorphous scatter and the axial D- spacing were seen in tissue samples up to 6 cm away from the primary site of disease. In particular, a significant decrease in both parameters was seen between the centre of the tumour (at 0 cm) and 2 cm away, suggesting that closer examination of the tissue structures over the disease/healthy tissue border may provide information regarding the mechanisms of metastasis and growth of cancerous tumours. The combination of the amorphous scattering results from the two X-ray sources indicates that the size of the scatterers may be the key in classifying tissue types. The synchrotron source was able to access a lower q-range (q = 0.1-0.6 nm-1) and the laboratory source covered a larger q-range (q = 0.25-2.3 nm-1). Mammoplasty tissues appear to be characterised by large scattering components (d > 25.13 nm), whereas normal tissues are characterised by slightly smaller scattering components (10.47 nm < d < 25.13 nm) and benign tissues by even smaller scattering components (4.83 < d < 10.47 nm). It appears that the size of the scatterers contributing to the total scattering intensity decreases with severity of disease, which was seen independently with both X-ray sources. Further investigation is warranted to determine the biological origin of these differences. These results also suggest that the optimum SAXS instrument may need to cover a scattering vector range of q < 0.25 nm-1 to identify differences in healthy tissue types, and q > 2.3 nm-1 to possibly investigate invasive carcinoma tissue types. A SAXS apparatus that can examine a large q-range may provide all of the necessary information from the amorphous scatter to differentiate between tissue groups. The periodic structure of collagen fibrils along their longitudinal axis can be characterised by the axial D-spacing, where this spacing was found to change with the presence of disease. The axial D-spacing for healthy breast tissues was found to be significantly lower in normal and mammoplasty tissues compared to invaded tissues (p = 0.0050 and p = 0.0093, respectively). However, no significant differences between the other tissue group pairs were seen (p > 0.05). These differences were evident in classification modelling of the four tissue groups, where the amorphous scatter and the amplitude of a collagen axial peak were used to build a probability model for disease status. The model showed high sensitivities (> 70%) and widely variable specificities (ranged from 18-97%) for the data examined with the synchrotron source. This means that the model was a good indicator of disease, but poor at indentifying healthy tissue types. The work presented in this thesis shows that SAXS is capable of distinguishing breast tissue types with high sensitivity and has the potential to become a significant tool for the investigation of cancer progression or even diagnosis. Further investigation into the amorphous scatter and axial D-spacing in particular may provide insight into the biological mechanisms related to tissue degradation associated with invasive disease.

Type IV collagens are components of basement membranes throughout the body and are involved in maintenance of the structural integrity of tissues as well as cellular differentiation, growth, and adhesion. Members of this collagen family are uniquely arranged in pairs in a head-to-head orientation and share a proximal promoter region. The COL4A5-COL4A6 gene pair is involved in numerous human diseases and cancer metastasis. For these reasons, defining the mechanisms that regulate collagen gene expression is of specific interest. To study type IV collagens, an in vitro model system was characterized. Comparative genomics was utilized to identify conserved, non-coding DNA in COL4A5 and COL4A6. These sequences were transfected into cell lines differing in type IV collagen expression and tested for the ability to regulate transcription of a reporter gene. Each cell line was also treated with the epigenetic modifying agents, 5-Aza and TSA. The effects on type IV collagen expression were determined. The COL4A5-COL4A6 promoter region was extensively characterized using ChIP analysis; antibodies against RNAPII, acetylated histone H3, and H3K9me2 were used. Additionally, bisulfite sequencing was carried out on each cell line to determine the methylation status of CpG dinucleotides in the promoter. Cell lines differing in expression of COL4A5 and COL4A6 were identified: 1) SCC-25 keratinocytes and HEK-293 cells transcribed both COL4A5 and COL4A6, 2) HT-1080 cells selectively activated COL4A5, and 3) SK-N-SH neuroblastoma cells did not express either gene. In SK-N-SH cells, histone modifications were shown to facilitate formation of condensed chromatin to prevent transcription initiation; repression was independent of DNA methylation. Activation of COL4A5 and COL4A6 in SCC-25 and HEK-293 cells involved acetylation of histones, although differences between the two cell types were seen. In addition, conserved, non-coding sequences were shown to affect transcription of a reporter gene; these sequences may be interacting with the transcription machinery to modulate collagen expression. Finally, repression of COL4A6 in HT-1080 cells appeared to be mediated through DNA methylation of the promoter; selective activation of COL4A5 may involve conserved, non-coding DNA. In summary, epigenetic modifications as well as conserved sequences are intimately involved in regulation of type IV collagen gene expression.

The commensal Streptococcus gordonii expresses numerous surface adhesins with which it interacts with other microorganisms, host cells and salivary proteins to initiate dental plaque formation. However, this Gram-positive bacterium can also spread to non-oral sites such as the heart valves and cause infective endocarditis. One of its surface adhesins, Sgo0707, is a large protein composed of a non-repetitive N-terminal region followed by several C-terminal repeat domains and a cell wall sorting motif. Here we present the crystal structure of the Sgo0707 N-terminal domains, refined to 2.1 Å resolution. The model consists of two domains, N1 and N2. The largest domain, N1, comprises a putative binding cleft with a single cysteine located in its centre and exhibits an unexpected structural similarity to the variable domains of the streptococcal Antigen I/II adhesins. The N2-domain has an IgG-like fold commonly found among Gram-positive surface adhesins. Binding studies performed on S. gordonii wild-type and a Sgo0707 deficient mutant show that the Sgo0707 adhesin is involved in binding to type-1 collagen and to oral keratinocytes.