Limbal Epithelial Crypts (LECs) are stem cell niches located in the limbus, the area between the cornea and the conjunctiva, and are responsible for cornea epithelium regeneration. Their anatomical structures were identified and described by Dua et al (2005). Stem cells migrate from this area toward the cornea where they differentiate into corneal epithelial cells. The lack of the LECs determines a pathology called Limbal Stem Cell Deficiency (LSCD) that can cause conjunctivalization, neovascularisation, corneal opacification, eventually resulting in vision loss. Currently, the main carriers to support limbal stem cell proliferation and differentiation used in clinics do not include enclosed structures able to act as cell reservoirs. In addition, the driving forces that determine the migration and the differentiation of limbal stem cells are not clarified yet. The hypotheses of this thesis were that the stiffness and the geometry of the scaffold influence the limbal stem cell differentiation process. Understanding these key factors could help to inform the design of scaffolds for future cornea regeneration strategies. The effect of the stiffness on cell differentiation is a well-known mechanism, but is less investigated in the limbus-corneal tissue. In this work, it was studied by producing macro-scaffolds (d=8 mm) obtained by UV-crosslinking. The stiffness was varied by increasing the concentration of Gelatin Methacrylate (GelMA), a biocompatible, photocrosslinkable material. Compression tests, rheology, AFM, and swelling analysis were performed on the developed hydrogels and compared to the ex vivo human cornea values. The gels with a concentration between 5% and 15% (w/v) exhibited mechanical properties in the same order of magnitude obtained for the cornea. The results indicated that the increased stiffness did not have a significant impact either in the expression of cytokeratin (CK3/CK14, stem cell marker, and CK14, differentiation marker), or in the gene expression (KRT3 and KRT19). However, the secondary key factors explored, differentiation media and oxygen concentration, supported cell differentiation. Inkjet printing was investigated as an additive manufacturing technique to produce 3D architectures. Although with this technique it was possible to develop a 3D structure with a stiffness gradient, its resolution was not sufficiently high to obtain a structure with dimensions comparable to the in vivo limbal stem cell niche. The second hypothesis was verified by developing a biocompatible scaffold mimicking the structure of the limbal stem cell niche and by evaluating the impact of its architecture on stem cell differentiation. The crypt geometry was modelled as U-shaped scaffolds with a diameter narrowing from 200 μm to 20 μm and was micro-fabricated from GelMA/PEGDA-based hydrogels using a Two-Photon Polymerization system (2PP). However, it was proven that the use of riboflavin as photoinitiator was inefficient at 780 nm, the wavelength used in the 2PP system. For this reason, P2CK was used as photoinitator to obtain stable hydrogels. The 2PP system allowed the precise recreation of the exact dimensions of the native crypts. Swelling, susceptibility to enzymatic degradation and stiffness were all evaluated. The biocompatibility of the printed scaffolds was assessed using immortalized human corneal epithelial cell proliferation up to 14 days. The ability of limbal stem cells to repopulate the crypts was demonstrated via two strategies. In the first strategy, human limbal stem cells were seeded directly inside the niche whilst in the second strategy, primary human limbal explants were placed adjacent to the printed structures and cells migrated towards the scaffold. Cell distribution and differentiation along the z-axis were evaluated using confocal microscopy. Cytokeratin 14 (CK14) with p63 and Cytokeratin CK3/12 (CK3/12) were used as limbal stem cell and differentiated corneal epithelial cell markers, respectively. Limbal epithelial stem cells were cultured in two conditions: xeno-free media, and with primary cells in serum containing media on a feeder layer. Both conditions showed the zonation of markers along the z-axis, which was not observed on flat scaffolds, demonstrating that the geometry alone influences cell phenotype. This suggests that the enclosed geometry results in the generation of a microenvironment inside the niche that influences cell phenotype. The presence of soluble factors, generated by cellular secretions, a specific oxygen concentration, and a more ‘stressful’ biomechanical milieu for the cells are some hypotheses that need further investigation and will be the basis of future work. In conclusion, the hypotheses of this thesis were partially confirmed. The variation in gel stiffness did not allow for the control of the hLESC differentiation process. However, the results demonstrated the influence of the geometry on stem cell differentiation without the use of signaling molecules. Further studies are necessary to have a description of the detailed spatial variability of the scaffold’s characteristics. Overall, the 2PP approach is flexible and could be applied to the generation of stem cell niches of other tissues, and could represent a significant advance in regenerative medicine.

Chronic liver diseases represent a primary worldwide health concern. Liver transplantation is the only practical cure for severe and chronic diseases, but due to the lack of human livers donor an insufficient number. The alternative is the cell-based transplantation of hepatocytes or hepatocyte-like cells derived from human stem cells. We aim to produce a physiologically relevant human liver model from human stem cells and to scale-up their production to be utilised in disease modelling and drug testing. Wnt/ β-catenin signalling pathway involved in hepatoblast proliferation and differentiation during liver development and regeneration. Here we showed that inhibition of GSK3β to stabilise β-catenin during hESC- derived hepatoblast differentiation resulted in high level of β-catenin with the early appearance of truncated form, improvement of HLCs morphology and upregulation of HNF4α and albumin expression. Although hESC can differentiate into almost any cell type including hepatocytes, their genetic instability in cultures limited their utilisation in regeneration medicine and cell-based transplantation therapy. Adult liver stem/ hepatic progenitor cells (HPCs) on the other hand upon activation following severe liver injury can differentiate into both hepatocytes and biliary epithelial cells. EpCAM+ progenitor cells isolated from human livers presented their expandability with the maintenance of progenitor characteristics and capacity to differentiate into hepatocytes in 3D culture (organoids). Differentiation of organoids- resulted in a loss of hepatic progenitor cell markers expression while the expression of differentiated hepatocyte-specific genes increased. Differentiated organoids showed low albumin expression compared to primary human hepatocytes; suggesting that the differentiation may have arisen from a limited population of cells or the organoids- derived hepatocytes were not fully mature. FACS sorting using a combination of specific markers (EPCAM, CD133 and CD24) produced a more homogeneous cell population that differentiated more effectively into hepatocytes compared to EpCAM+ cells. The acellular rat liver scaffolds obtained by decellularisation using detergents was naturally directed the cultured HPCs and NPCs to their in localisation and improved the albumin expression in the differentiated HPC providing an applicable model for drug testing and bio-engineered liver transplantation. Generating functional hepatocytes from human liver progenitor cells could provide an unlimited supply of hepatocytes; replacing the animal use in drug screening, testing new chemical entities and modelling human liver diseases.

Thesis (MTech (Medical Technology))--Cape Technikon, Cape Town, 2002 / Erythropoietic response is the first indication of bone marrow recovery following bone marrow or peripheral blood stem cell transplantation. Manual reticulocyte counting has not only proven to be outdated but an extremely crude method of analysis, particularly if accurate and reliable means of assessing erythroid response is required to assess bone marrow recovery. Automated methods allow for the quantification of maturation within each reticulocyte, by measuring the amount of RNA present. The method of choice for our reticulocyte analysis was the Reticulocyte Maturation Index (RMI). The RMI was obtained by dividing the number of immature reticulocytes counted by the total number of reticulocytes counted producing a reportable value of International Units (IU). A normal Reticulocyte Maturation Index is 0.20 to 0.50 IU. The aim of the study was multifold. We wanted to prove that the Reticulocyte Maturation Index (RMI) is indeed the fastest means to assess bone marrow recovery in various types of transplants, including Bone Marrow Transplant (BMT) and Peripheral Blood Stem Cell Transplant (PBSCT). We also wanted to draw comparisons between allogeneic and autologous transplants, as well as further assessing different disease types. This was done by measuring the Reticulocyte Maturation Index (RMI), Absolute Neutrophil Count (ANe) and the Platelet Count (PLT) within the various groups. We further wanted to assess the effect of preconditioning treatment, Mononuclear Counts (MNC) and Colony Forming Unit - Granulocyte and Monocyte Counts (CFU-GM) on the early RMI response. These comparisons resulted in a need to establish a working range to determine patients response therein, and final outcome of the transplants. Finally we wanted to establish whether the "day 14" marrow biopsy is necessary, particularly if the three peripheral blood parameters, RMI, ANC and PLT were used as routine procedure following transplantation. The Reticulocyte Maturation Index (RMI) was measured on the Coulter EPICS ProfIle II flow cytometer; the ANC and PLT were measured on the Technicon H2 Haematology System. All other results such as the Mononuclear Counts (MNC), Colony Forming Unit - Granulocyte and Monocyte counts (CFU-GM), "day 14" and "day 28" bone marrow biopsies were retrieved from laboratory records. Forty nine transplant patients were evaluated for RMI over a period of six months, at the Department of Haematology, Groote Schuur Hospital, Cape Town. Four patients failed to engraft; and were not used in the calculations; but were evaluated as an aspect of the study in the final analysis. Forty five patients were analysed to establish the values used in the study, these patients were divided into eleven groups.

Thesis (Masters Diploma (Medical Technology) -- Cape Technikon, Cape Town, 1992 / Gastric cancer, a fatal malignancy, is endemic in the Coloured population of the Western Cape region of South Africa. Diagnosis is based mainly on histologic investigation with patients of either sex being mainly between 40-60 years of age. The extent to which genetic and environmental influences play a role in the aetiology of the disease is unknown. This study is an attempt to biochemically characterise gastric mucins or mucus glycoproteins, (the main gel forming components of crude mucus scrapings off the mucosal surface), in carcinoma of the stomach (HCA), as compared to those in ulcer disease (HGU), post mortem specimens (PM) and samples obtained from organ transplant donor stomachs (HD). The aim of this study is the development of a diagnostic marker within mucus secretions for the detection of pre-malignant disease amongst the high risk population of the Western Cape region of South Africa. Mucins were extracted from crude mucus gel scrapings according to a carefully designed technique in which proteolytic inhibitors were used to minimise the possibility of endogenous proteolysis in the laboratory through possible contamination. Two density gradient ultra-centrifugation steps for 48 hours each at 105,000g in caesium chloride, a well established standard isolation procedure for mucins, gave a yield of pure mucins which fractionated at a density of approximately 1.41gjml in all groups. These mucins, from the HO, PH, HGU and HCA groups eluted mainly in the included volume of a Sepharose 2B column as broad, polydisperse peaks, suggesting that they were degraded and comprised mainly lower molecular weight PAS positive material in relation to large polymeric gel forming mucin.

Thesis (BTech (Biomedical Technology))--Cape Technikon, 1992. / The steady state in a tumour rapidly changes with its growth and the subsequent deteriorating blood and nutrient supply. This adaptation in the steady state of the tumour is shown in the increased lactate dehydrogenase and acid phosphatase activity in the tumour during it's growth. These alterations in the tumour metabolism places an increased burden on the body to supply nutrient and to discard the waste products of the tumour. This is demonstrated at the macroscopic level by the decreasing body weight and food intake when the tumour burden increases, and also at the metabolic levels by the responses of certain glycolytic and Cori cycle enzymes. Furthermore three distinct stages were observed in the Cori cycle response to the influence of the tumour namely, a silent or preclinical stage, a hypermetabolic stage and a hypo metabolic stage. Although the decreasing body weight cannot be directly linked to the process of gluconeogenesis, the onset of anorexia appeared to coincide with the end of the hypermetabolic stage and the beginning of the hypometabolic stage in gluconeogenesis. This clearly shows that the body's steady state is adversely affected by the presence of the tumour and that the conditions at the metabolic level seem to cause the anorexia. Furthermore, it is well known that the success of cancer therapies depends entirely on the effectiveness ofthe modality to kill the tumour cell and on the ability' of the host to absorb the damage caused by the modality without being destroyed in the process itself. The second part of this study demonstrates the radioprotective effects of ATP at all levels. It is clear from this work that ATP had a bigger influence in protecting the normal tissue than it had on the tumour tissue. This was demonstrated by the response of acid phosphatase (AP) and glucose-6-phosphate dehydrogenase (G-6-PDH) in the tumour and testis. Furthermore, it would seem that ATP has a multifactorial interaction with the cell, two possible mechanisms of protection are indicated by these results. The first of these interactions is through the receptors of the cell to stimulate enhanced glycolysis, for higher energy production and thus repair. The second possibility is the interaction of ATP with the receptor of the cell to inhibit the production of free radicals and thus damage, as demonstrated by the response of G-6-PDH and AP.

Given the recent concern from multiple healthcare stakeholders that the pipeline of medical innovation is slowing, this thesis provides insights on how to spur breakthrough medical innovation in present day. The findings and recommendations are derived from one of the largest collections of interview transcripts from biomedical innovators (n=143) responsible for developing critical devices, drugs and diagnostics used in medicine today. An exemplary case (coronary artery stent) was selected for an in-depth analysis, which included a detailed recounting of stent development and an exhaustive analysis of the patent literature. External factors either catalyzed (e.g., supportive institutions, strong underlying science and collaboration) or hindered (e.g., technology transfer challenges, lack of funding and onerous conflict of interest rules) the development process. Strategies aimed towards promoting transformative medical innovation should focus on institutional-level policies targeting early stages of innovation. This includes providing individuals with unique expertise with the capacity to pursue innovative work. Technology transfer processes should be simplified to enable collaboration for individuals between institutions with disparate expertise. Policymakers should continue to support basic science research, which underlies future innovations. In contrast, policies that increase reimbursement or reduce taxes for industry or extend patent terms are less likely to impact transformative innovation.

Magister Scientiae - MSc / Most rural areas of developing countries are faced with problems like shortage of doctors in hospitals, illiteracy and poor power supply. Because of these issues, Information and Communication Technology (ICT) is often sees as a useful solution for these areas. Unfortunately, the social environment is often ignored. This leads to inappropriate systems being developed for these areas. The aims of this thesis were firstly, to learn how a communication system can be built for a rural telehealth environment in a developing country, secondly to learn how users can be supported to use such a system. / South Africa

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This licentiate thesis uses descriptive, mainly official,Swedish statistics to analyse industrial structure, productionand foreign trade in an industry that is traditionallydifficult to describe in numbers, that of medical devices. Forthe purposes of the thesis, the Swedish Medical Device industryis defined as companies classified in the SE-SIC manufacturingcodes 33101 (medical equipment and instruments, etc), 33102(dental products) and 35430 (invalid vehicles). Also otherbranches contribute, notably parts of SIC 51460 (wholesale inmedical equipment and pharmaceutical goods) and 73103 (medicalresearch and development) although their medical device volumecannot be specified. Additional items have been identified interms of specific product groups rather than as "belonging" toa specific SIC industrial code. Taken together, this is considered to correspond reasonablywell to the scope of the field as defined by the Global MedicalDevice Nomenclature (GMDN), a new European standard forclassifying medical devices in a more generic way than do theEuropean Medical Device Directives (or other pieces oflegislation). No quantification according to GMDN can be madeas yet, however, as that requires changing reporting habits inindustry as well as in official statistical classification andnomenclature regimes. With the manufacturing code SE-SIC 33101 as main object, thestudy for the first time presents data on the regionaldistribution, size classes of employment, company starting timeand company dynamics, in the form of entries to and exits fromthe code, over a six-year period. The latter analysis includesa follow-up of the "exits", some firms reappearing in otherparts of industry and others disappearing–surprisinglyfew among them being limited companies. Although this industryis comparatively mature, considerable mobility among themid-sized companies is indicated for reasons of real changes or(to some degree) factors inherent in the industrialclassification system. Some structural changes in companies inthe ≥50 employees bracket are identified. It is notedthat American actors, directly or indirectly, are increasinglyinvolved with the medical device industry in Sweden, and that anumber of technology-based companies that were started mostlyin the early eighties have recently reached the 50+ employeelevel. The analysis of identifiable production and internationaltrade in medical devices spans a period of 17 years based onofficial statistics following the HS/CN nomenclatures. Adatabase has been built, bottom-up, from the 8-digit CN levelwith production, exports and imports values for close to 100items collected in 12 product groups, for presentation purposesgrouped under three main headings. Compound annual growth ratesfor the latter are presented for three five-year periods1985-2000, showing that Swedish production and exports have hadan overall growth of 10 per cent p.a. This has kept Swedenahead of the international overall growth of 6- 7 per cent p.a.in recent years, products in the main group "Aids&Implants" growing more than 20 per cent p.a. Growth rates inthe most recent five-year period are lower, however. Healthynet exports figures are presented, the figure for 2002nominally representing 40 per cent of the production value incurrent as well as constant prices. Production figures are given at industry (local unit) levelas well as at product group level. The product-based figuresidentified for Production 2001 are estimated to SEK 13,3billion, Exports to SEK 13,7 billion and Imports to SEK 9,7billion. Figures for the Apparent Domestic Market arecalculated for the corresponding entities. It is obvious,however, that the statistics do not capture the real productionvalue as exports exceed production both at overall level and inmajor product groups, particularly those on a high systemstechnology level. The situation is not uncommon for a number ofreasons; further, cases in the statistics methodologyliterature confirm that medical instrument-related codes areliable to this phenomenon. Corrections, including adjustmentsof both production and exports values, are possible butdemanding already at one individual 4-digit HS/CN level. This,therefore, must be considered outside the scope of an academicstudy. The basic tablework developed for this thesis will be madefreely available to external parties for their own use providedthe author, with contact details, is named as the source.(Processing for commercial purposes is not expected, however.)Any suggestions for improvements are welcomed. / NR 20140805

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