Ex vivo investigation of novel wound healing therapies and development of a 3-D human skin equivalent wound model

It has previously been found that complexes comprised of vitronectin and growth factors (VN:GF) enhance keratinocyte protein synthesis and migration. More specifically, these complexes have been shown to significantly enhance the migration of dermal keratinocytes derived from human skin. In view of this, it was thought that these complexes may hold potential as a novel therapy for healing chronic wounds. However, there was no evidence indicating that the VN:GF complexes would retain their effect on keratinocytes in the presence of chronic wound fluid. The studies in this thesis demonstrate for the first time that the VN:GF complexes not only stimulate proliferation and migration of keratinocytes, but also these effects are maintained in the presence of chronic wound fluid in a 2-dimensional (2-D) cell culture model. Whilst the 2-D culture system provided insights into how the cells might respond to the VN:GF complexes, this investigative approach is not ideal as skin is a 3-dimensional (3-D) tissue. In view of this, a 3-D human skin equivalent (HSE) model, which reflects more closely the in vivo environment, was used to test the VN:GF complexes on epidermopoiesis. These studies revealed that the VN:GF complexes enable keratinocytes to migrate, proliferate and differentiate on a de-epidermalised dermis (DED), ultimately forming a fully stratified epidermis. In addition, fibroblasts were seeded on DED and shown to migrate into the DED in the presence of the VN:GF complexes and hyaluronic acid, another important biological factor in the wound healing cascade. This HSE model was then further developed to enable studies examining the potential of the VN:GF complexes in epidermal wound healing. Specifically, a reproducible partial-thickness HSE wound model was created in fully-defined media and monitored as it healed. In this situation, the VN:GF complexes were shown to significantly enhance keratinocyte migration and proliferation, as well as differentiation. This model was also subsequently utilized to assess the wound healing potential of a synthetic fibrin-like gel that had previously been demonstrated to bind growth factors. Of note, keratinocyte re-epitheliasation was shown to be markedly improved in the presence of this 3-D matrix, highlighting its future potential for use as a delivery vehicle for the VN:GF complexes. Furthermore, this synthetic fibrin-like gel was injected into a 4 mm diameter full-thickness wound created in the HSE, both keratinocytes and fibroblasts were shown to migrate into this gel, as revealed by immunofluorescence. Interestingly, keratinocyte migration into this matrix was found to be dependent upon the presence of the fibroblasts. Taken together, these data indicate that reproducible wounds, as created in the HSEs, provide a relevant ex vivo tool to assess potential wound healing therapies. Moreover, the models will decrease our reliance on animals for scientific experimentation. Additionally, it is clear that these models will significantly assist in the development of novel treatments, such as the VN:GF complexes and the synthetic fibrin-like gel described herein, ultimately facilitating their clinical trial in the treatment of chronic wounds.

Processing of polycaprolactone and polycaprolactone-based copolymers into 3D scaffolds and their cellular responses

Synthetic polymers have attracted much attention in tissue engineering due to their ability to modulate biomechanical properties. This study investigated the feasibility of processing poly(varepsilon-caprolactone) (PCL) homopolymer, PCL-poly(ethylene glycol) (PEG) diblock, and PCL-PEG-PCL triblock copolymers into three-dimensional porous scaffolds. Properties of the various polymers were investigated by dynamic thermal analysis. The scaffolds were manufactured using the desktop robot-based rapid prototyping technique. Gross morphology and internal three-dimensional structure of scaffolds were identified by scanning electron microscopy and micro-computed tomography, which showed excellent fusion at the filament junctions, high uniformity, and complete interconnectivity of pore networks. The influences of process parameters on scaffolds' morphological and mechanical characteristics were studied. Data confirmed that the process parameters directly influenced the pore size, porosity, and, consequently, the mechanical properties of the scaffolds. The in vitro cell culture study was performed to investigate the influence of polymer nature and scaffold architecture on the adhesion of the cells onto the scaffolds using rabbit smooth muscle cells. Light, scanning electron, and confocal laser microscopy showed cell adhesion, proliferation, and extracellular matrix formation on the surface as well as inside the structure of both scaffold groups. The completely interconnected and highly regular honeycomb-like pore morphology supported bridging of the pores via cell-to-cell contact as well as production of extracellular matrix at later time points. The results indicated that the incorporation of hydrophilic PEG into hydrophobic PCL enhanced the overall hydrophilicity and cell culture performance of PCL-PEG copolymer. However, the scaffold architecture did not significantly influence the cell culture performance in this study.

Characterisation and culturing of adipose-derived precursor cells

This book focuses on practical applications for using adult and embryonic stem cells in the pharmaceutical development process. It emphasizes new technologies to help overcome the bottlenecks in developing stem cells as therapeutic agents. A key reference for professionals working in stem cell science, it presents the general principles and methodologies in stem cell research and covers topics such as derivitization and characterization of stem cells, stem cell culture and maintenance, stem cell engineering, applications of high-throughput screening, and stem cell genetic modification with their use for drug delivery.

Disruption of androgen regulation in the prostate by the environmental contaminant hexachlorobenzene

Hexachlorobenzene (HCB) is a persistent environmental contaminant that has the potential to interfere with steroid hormone regulation. The prostate requires precise control by androgens to regulate its growth and function. To determine if HCB impacts androgen action in the prostate, we used a number of methods. Our in vitro cell-culture-based assay used a firefly luciferase reporter gene driven by an androgen-responsive promoter. In the presence of dihydrotestosterone, low concentrations (0.5-5 nM) of HCB increased the androgen-responsive production of firefly luciferase and high concentrations of HCB (> 10 microM) suppressed this transcriptional activity. Results from a binding assay showed no evidence of affinity between HCB and the androgen receptor. We also tested HCB for in vivo effects using transgenic mice in which the transgene was a prostate-specific, androgen-responsive promoter upstream of a chloramphenicol acetyl transferase (CAT) reporter gene. In 4-week-old mice, the proportion of dilated prostate acini, a marker of sexual maturity, increased in the low HCB dose group and decreased in the high HCB dose mice. In the 8-week-old mice, there was a significant decrease in both CAT activity and prostate weight upon exposure to 20 mg/kg/day HCB. Therefore, in vitro and in vivo data suggest that HCB weakly agonizes androgen action, and consequently, low levels of HCB enhanced androgen action but high levels of HCB interfered. Environmental contaminants have been implicated in the rising incidence of prostate cancer, and insight into the mechanisms of endocrine disruption will help to clarify their role.

Response of cells on surface-induced nanopatterns: Fibroblasts and mesenchymal progenitor cells

Ultrathin films of a poly(styrene)-block-poly(2-vinylpyrindine) diblock copolymer (PS-b-P2VP) and poly(styrene)-block-poly(4-vinylpyrindine) diblock copolymer (PS-b-P4VP) were used to form surface-induced nanopattern (SINPAT) on mica. Surface interaction controlled microphase separation led to the formation of chemically heterogeneous surface nanopatterns on dry ultrathin films. Two distinct nanopatterned surfaces, namely, wormlike and dotlike patterns, were used to investigate the influence of topography in the nanometer range on cell adhesion, proliferation, and migration. Atomic force microscopy was used to confirm that SINPAT was stable under cell culture conditions. Fibroblasts and mesenchymal progenitor cells were cultured on the nanopatterned surfaces. Phase contrast and confocal laser microscopy showed that fibroblasts and mesenchymal progenitor cells preferred the densely spaced wormlike patterns. Atomic force microscopy showed that the cells remodelled the extracellular matrix differently as they migrate over the two distinctly different nanopatterns

Osteogenic induction of human bone marrow-derived mesenchymal progenitor cells in novel synthetic polymer-hydrogel matrices

The aim of this project was to investigate the in vitro osteogenic potential of human mesenchymal progenitor cells in novel matrix architectures built by means of a three-dimensional bioresorbable synthetic framework in combination with a hydrogel. Human mesenchymal progenitor cells (hMPCs) were isolated from a human bone marrow aspirate by gradient centrifugation. Before in vitro engineering of scaffold-hMPC constructs, the adipogenic and osteogenic differentiation potential was demonstrated by staining of neutral lipids and induction of bone-specific proteins, respectively. After expansion in monolayer cultures, the cells were enzymatically detached and then seeded in combination with a hydrogel into polycaprolactone (PCL) and polycaprolactone-hydroxyapatite (PCL-HA) frameworks. This scaffold design concept is characterized by novel matrix architecture, good mechanical properties, and slow degradation kinetics of the framework and a biomimetic milieu for cell delivery and proliferation. To induce osteogenic differentiation, the specimens were cultured in an osteogenic cell culture medium and were maintained in vitro for 6 weeks. Cellular distribution and viability within three-dimensional hMPC bone grafts were documented by scanning electron microscopy, cell metabolism assays, and confocal laser microscopy. Secretion of the osteogenic marker molecules type I procollagen and osteocalcin was analyzed by semiquantitative immunocytochemistry assays. Alkaline phosphatase activity was visualized by p-nitrophenyl phosphate substrate reaction. During osteogenic stimulation, hMPCs proliferated toward and onto the PCL and PCL-HA scaffold surfaces and metabolic activity increased, reaching a plateau by day 15. The temporal pattern of bone-related marker molecules produced by in vitro tissue-engineered scaffold-cell constructs revealed that hMPCs differentiated better within the biomimetic matrix architecture along the osteogenic lineage.

Resorbable composite scaffolds for bone tissue engineering

Bone loss associated with trauma osteo-degenerative diseases and tumors has tremendous socioeconomic impact related to personal and occupation disability and health care costs. Bone grafting is often critical to surgical therapies. Autogenous bone is presently the preferred grafting material; however, this holds several disadvantages such as donor site morbidity. In the present climate of increasing life expectancy with an ensuing increase in bone-related injuries, orthopaedic surgery is undergoing a paradigm shift from bone-grafting to bone engineering, where a scaffold is implanted to provide adequate load bearing and enhance tissue regeneration. Our group at Queensland University of Technology (QUT) have developed, characterised and tested polycaprolactone/ tricalcium phosphate (PCL/TCP) composite scaffolds for low load-bearing bone defects. These scaffolds are being further developed for application in higher load bearing sites. Our approach emphasizes the importance of the biomaterials’ structural design, the scaffold architecture and structural and nutritional requirements for cell culture. These first-generation scaffolds made from medical grade PCL (mPCL) have been studied for more than 5 years within a clinical setting 1. This paper describes the application of second-generation scaffolds in small and large animal bone defect models and the ensuing bone regeneration as shown by histology and µCT.

Engineered silk fibroin protein 3D matrices for in vitro tumor model

3D in vitro model systems that are able to mimic the in vivo microenvironment are now highly sought after in cancer research. Antheraea mylitta silk fibroin protein matrices were investigated as potential biomaterial for in vitro tumor modeling. We compared the characteristics of MDA-MB-231 cells on A. mylitta, Bombyx mori silk matrices, Matrigel, and tissue culture plates. The attachment and morphology of the MDA-MB-231 cell line on A. mylitta silk matrices was found to be better than on B. mori matrices and comparable to Matrigel and tissue culture plates. The cells grown in all 3D cultures showed more MMP-9 activity, indicating a more invasive potential. In comparison to B. mori fibroin, A. mylitta fibroin not only provided better cell adhesion, but also improved cell viability and proliferation. Yield coefficient of glucose consumed to lactate produced by cells on 3D A. mylitta fibroin was found to be similar to that of cancer cells in vivo. LNCaP prostate cancer cells were also cultured on 3D A. mylitta fibroin and they grew as clumps in long term culture. The results indicate that A. mylitta fibroin scaffold can provide an easily manipulated microenvironment system to investigate individual factors such as growth factors and signaling peptides, as well as evaluation of anticancer drugs.

Mathematical modelling of chronic wound healing

Chronicwounds fail to proceed through an orderly process to produce anatomic and functional integrity and are a significant socioeconomic problem. There is much debate about the best way to treat these wounds. In this thesis we review earlier mathematical models of angiogenesis and wound healing. Many of these models assume a chemotactic response of endothelial cells, the primary cell type involved in angiogenesis. Modelling this chemotactic response leads to a system of advection-dominated partial differential equations and we review numerical methods to solve these equations and argue that the finite volume method with flux limiting is best-suited to these problems. One treatment of chronic wounds that is shrouded with controversy is hyperbaric oxygen therapy (HBOT). There is currently no conclusive data showing that HBOT can assist chronic wound healing, but there has been some clinical success. In this thesis we use several mathematical models of wound healing to investigate the use of hyperbaric oxygen therapy to assist the healing process - a novel threespecies model and a more complex six-species model. The second model accounts formore of the biological phenomena but does not lend itself tomathematical analysis. Bothmodels are then used tomake predictions about the efficacy of hyperbaric oxygen therapy and the optimal treatment protocol. Based on our modelling, we are able to make several predictions including that intermittent HBOT will assist chronic wound healing while normobaric oxygen is ineffective in treating such wounds, treatment should continue until healing is complete and finding the right protocol for an individual patient is crucial if HBOT is to be effective. Analysis of the models allows us to derive constraints for the range of HBOT protocols that will stimulate healing, which enables us to predict which patients are more likely to have a positive response to HBOT and thus has the potential to assist in improving both the success rate and thus the cost-effectiveness of this therapy.

Defective interfering viral particles in acute dengue infections

While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. While most reports of DI particles have referred to studies in vitro, there is some evidence for the presence of DI particles in chronic viral infections in vivo. In this study, short fragments of dengue virus (DENV) RNA containing only key regulatory elements at the 3' and 5' ends of the genome were recovered from the sera of patients infected with any of the four DENV serotypes. Identical RNA fragments were detected in the supernatant from cultures of Aedes mosquito cells that were infected by the addition of sera from dengue patients, suggesting that the sub-genomic RNA might be transmitted between human and mosquito hosts in defective interfering (DI) viral particles. In vitro transcribed sub-genomic RNA corresponding to that detected in vivo could be packaged in virus like particles in the presence of wild type virus and transmitted for at least three passages in cell culture. DENV preparations enriched for these putative DI particles reduced the yield of wild type dengue virus following co-infections of C6-36 cells. This is the first report of DI particles in an acute arboviral infection in nature. The internal genomic deletions described here are the most extensive defects observed in DENV and may be part of a much broader disease attenuating process that is mediated by defective viruses.

Preparation and characterisation of tri-calcium phosphate scaffolds with tunnel-like macro-pores for bone tissue engineering

Calcium Phosphate ceramics have been widely used in tissue engineering due to their excellent biocompatibility and biodegradability. In the physiological environment, they are able to gradually degrade, absorbed and promote bone growth. Ultimately, they are capable of replacing damaged bone with new tissue. However, their low mechanical properties limit calcium phosphate ceramics in load-bearing applications. To obtain sufficient mechanical properties as well as high biocompatibility is one of the main focuses in biomaterials research. Therefore, the current project focuses on the preparation and characterization of porous tri-calcium phosphate (TCP) ceramic scaffolds. Hydroxapatite (HA) was used as the raw material, and normal calcium phosphate bioglass was added to adjust the ratio between calcium and phosphate. It was found that when 20% bioglass was added to HA and sintered at 1400oC for 3 hours, the TCP scaffold was obtained and this was confirmed by X-ray diffraction (XRD) analysis. Test results have shown that by applying this method, TCP scaffolds have significantly higher compressive strength (9.98MPa) than those made via TCP powder (<3MPa). Moreover, in order to further increase the compressive strength of TCP scaffolds, the samples were then coated with bioglass. For normal bioglass coated TCP scaffold, compressive strength was 16.69±0.5MPa; the compressive strength for single layer mesoporous bioglass coated scaffolds was 15.03±0.63MPa. In addition, this project has also concentrated on sizes and shapes effects; it was found that the cylinder scaffolds have more mechanical property than the club ones. In addition, this project performed cell culture within scaffold to assess biocompatibility. The cells were well distributed in the scaffold, and the cytotoxicity test was performed by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay. The Alkaline Phosphatase (Alp) activity of human bone marrow mesenchymal stem cell system (hBMSCs) seeded on scaffold expressed higher in vitro than that in the positive control groups in osteogenic medium, which indicated that the scaffolds were both osteoconductive and osteoinductive, showing potential value in bone tissue engineering.

A fibrocontractive mechanochemical model of dermal wound closure incorporating realistic growth factor kinetics

Fibroblasts and their activated phenotype, myofibroblasts, are the primary cell types involved in the contraction associated with dermal wound healing. Recent experimental evidence indicates that the transformation from fibroblasts to myofibroblasts involves two distinct processes: the cells are stimulated to change phenotype by the combined actions of transforming growth factor β (TGFβ) and mechanical tension. This observation indicates a need for a detailed exploration of the effect of the strong interactions between the mechanical changes and growth factors in dermal wound healing. We review the experimental findings in detail and develop a model of dermal wound healing that incorporates these phenomena. Our model includes the interactions between TGFβ and collagenase, providing a more biologically realistic form for the growth factor kinetics than those included in previous mechanochemical descriptions. A comparison is made between the model predictions and experimental data on human dermal wound healing and all the essential features are well matched.