abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The pulp organ is retained in the pulp chamber of teeth and maintains the biological and physiological vitality of the surrounding dentin. It works as a biosensor and generates secondary dentine and tertiary dentine to resist tooth abrasion and pathogenic stimuli (Zhang and Yelick, 2010). However, dental pulp is vulnerable to injury (Smulson and Sieraski, 1989). Most people experience some irreversible pulpal diseases during their lifetime. Hence, pulp regeneration is one of the research tasks in dentistry that attracts much attention. Stem cell transplantation is a plausible strategy for the regeneration of dental pulp organ. Dental pulp stem cells (DPSCs)derived from heavy or inflamed dental pulps have the natural advantage in pulp regeneration due to its dentinogenic potentiality (Huang et al., 2009). DPSCs are delivered into prepared root canal, which then differentiate into odontoblasts, fibroblasts, and other kinds of cells. It was shown that these transplanted DPSCs were able to produce dentin and formed a dentin-pulp like tissue both in vitro and in vivo(Huang, 2009).However, low survival rate of the transplanted cells is a common problem in pulp regeneration. Overexpression of Bcl-2 could enhance cell anti-apoptotic ability. Studies of many kinds of cell transplantation showed that a large number of cells died upon grafting and a large proportion of cell death seemed to have occurred due to apoptosis (Liu et al., 2013; Zhang et al., 2001).The aim of this study was to improve cell survival through making DPSCs overexpress lymphoma 2 (Bcl-2) protein.Bcl-2 is a proto-oncogene which playsa significant role in (anti) apoptosis. Former studies in the literature have provided evidences that overexpressing Bcl-2 could reduce cell apoptosis. However, this strategy has not been studied in the modification of DPSCs. In this study, DPSCs were isolated from discarded third molars of adults and manipulated to overexpressing Bcl-2. Proliferation of modified DPSCs was analyzed by static batch culture, CCK-8 test and BrdU based proliferation test. Apoptosis of modified DPSCs was analyzed by measuring DNA fragments in the cells. Modified DPSCs generated a higher maximum cell population during static batch culture and showed higher viability (the ratio of live cells to total cells). CCK-8 test showed that the population of modified DPSCs increased faster than control group cells and wild type cells. Modified DPSCs were not better than the other cells in proliferative ability, but had lower apoptosis level when culturing in serum free medium. Hence, overexpressing Bcl-2 could increase cell population, the mechanism is to help DPSCs survival rather than promote the proliferative ability of cells. / published_or_final_version / Dentistry / Master / Master of Philosophy

M. Tech. / Stem cells are regarded as undifferentiated cells that are capable of selfrenewal, proliferation, production of a great number of differentiated progeny, and regeneration of tissues (Blau et al., 2001). The therapeutic potential of multilineage stem cells for tissue engineering (TE) applications is vast. Two general types of stem cells are potentially useful for this application: embryonic stem cells (ESCs) and adult (autologous) stem cells (Zuk et al., 2001). Traditionally, ESCs are isolated from the inner cell mass (ICM) of blastocysts, however harvesting of these cells results in the death of the embryo, which has led to ethical, religious and political issues (Moore, 2007). In contrast, adult stem cells, by virtue of their nature, are immunocompatible and have no ethical issues associated with their use (Zuk et al., 2001). Subcutaneous adipose tissue is an active and highly complex tissue composed of several different cell types, and is derived from the mesodermal germ layer and contains a supportive stromal vascular fraction (SVF) that can be easily isolated. This SVF contains a heterogeneous mixture of cells including preadipocytes (Raposio et al., 2007; Schäffler and Büchler, 2007; Jurgens et al., 2008). The preadipoctyes are considered as the multipotent stem cells termed adipose derived stem cells (ADSCs) that have similar properties to bone marrow mesenchymal stem cells (BM-MSCs) (Fraser et al., 2006). ADSCs are idyllic for cellular therapy applications due to various factors: they can be harvested, multiplied and handled easily, efficiently and noninvasively, they have a pluripotential and proliferative capacity comparable to BM-MSCs, and morbidity to donors is considerably less, requiring only local anaesthesia and a short wound healing time. Human ADSCs (hADSCs) can be expanded in an undifferentiated state and have multipotential differentiation capacity along the classical mesenchymal lineages of adipogenesis, osteogenesis, chondrogenesis and myogenesis (de Villiers et al., 2009).

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: HISTORICAL PERSPECTIVE Stem cell therapy was commenced after using rabbits as research models. Once this process was successful, the first human transplant was done in 1974. Certain prerequisites were necessary and these were achieved - a protected environment, an apheresis unit, protocols and accreditation with International Registries. Initially, unmanipulated bone marrow and peripheral blood stem cells were used together with immunosuppressive drugs followed by the use of Cyclosporin A then the addition of ex vivo Campath®. AUDIT OF ACUTE ASSOCIATIONS (468 subjects in initial cohort) NEPHROLOGY Creatinine was used as an indication of renal function. Of the 76 available for analysis, 47% had acute kidney injury. Dialysis had a poor outcome as reported in the literature. Renal complications occurred frequently mostly due to infection. CARDIOLOGY A total of 119 individuals were available for analysis. Echocardiograms and electrocardiograms were part of pre-transplant assessment. Left ventricular systolic dysfunction predicted for increasing post transplant problems. Cardiac complications occurred at a lower frequency than other post-transplants side-effects consistent with the published data. DERMATOLOGY Cases were evaluated on a daily basis and referred to a dermatologist when necessary. To confirm Graft-Versus-Host Disease (GVHD), a skin biopsy was done to differentiate it from drug hypersensitivity or viral infections. The exposure to ex vivo Campath® significantly improved outcome by reducing the incidence and severity of GVHD. Quality of life was enhanced with substantial cost saving. GASTROENTEROLOGY Foregut symptoms occurred in 90% of patients. Nutritional problems were encountered. Altered liver functions were relatively common attributable to drugs, sepsis and conditioning regimens. Liver biopsies were not performed in this series and endoscopy performed only when necessary. A STUDY ON LATE COMPLICATIONS (55 subjects) RESPIRATORY Spirometry and diffusing capacity were done in this cohort. All the lung function studies were within the predicted normal range apart from some marginal reduction in diffusing capacity. In none of these patients did late consequences such as Bronchiolitis Obliterans Organising Pneumonia and Late Onset Non-Infectious Pulmonary complications occur. Cytomegalovirus reactivation was common but early intervention prevented serious complications. IMMUNOLOGY An in vitro functional study was done. Both the innate and adaptive systems were evaluated. Taken into consideration were the type of transplant, age from transplant, diagnosis and conditioning. The granulocyte Burst-test was done for the innate profile. Reduced activity was shown in all the subgroups. It appears as if the innate response of the granulocytic cells never recovered due to reduced granulocytic function in vitro. The adaptive responses were evaluated in vitro and only the autografts showed better CD4+ and CD8+ cytokine production. No major differences were seen in other groups. Normal cytokine production by CD4+ and CD8+ T cells were present when these were activated in vitro to produce regulatory cytokines, implying that their lymphoid component was intact post-transplant. BONE DISEASE Here both the Dual energy X-ray Absorptiometry (DXA) and Quantitative Computed Tomography (QCT) were used to evaluate bone mineral density. There was a discrepancy present between the two modalities. DXA showed no osteoporosis but QCT 22%. Biomarkers were normal in all. There was no history of fracture and no objective evidence of vertebral fractures using vertebral fracture assessment. Although QCT was used for the study, DXA remains the gold standard in South Africa. CONCLUSION This doctoral provided information on the non-haematological consequences in South Africa with the use of Campath® ex vivo. / AFRIKAANSE OPSOMMING: HISTORIESE PERSPEKTIEF Stamsel terapie is voortgesit nadat konyne aanvanklik as navorsingsmodelle gebruik is. Na suksesvolle voltooiing van hierdie proses, is die eerste menslike oorplanting gedoen in 1974. Sekere voorvereistes was nodig en hierdie was bereik – ʼn beskermde omgewing, ʼn aferese eenheid, protokolle en akkreditasie by Internasionale Registers. Aanvanklik is ongemanipuleerde beenmurg- en perifere bloed stamselle gebruik, tesame met immuunonderdrukkende middels, gevolg deur die gebruik van Sikloporien A en daarna die toevoeging van ex vivo Campath®. OUDIT VAN AKUTE ASSOSIASIES (468 GEVALLE IN DIE OORSPRONKLIKE GROEP) NEFROLOGIE Kreatinien is gebruik as ʼn aanduiding van nierfunksie. Van die 76 gevalle beskikbaar vir ontleding, het 47% akute nierbeserings gehad. Dialise het ʼn swak uitkoms gehad soos gerapporteer in publikasies. Nier komplikasies het gereeld voorgekom, meestal as gevolg van infeksie. KARDIOLOGIE ʼn Totaal van 119 gevalle was beskikbaar vir ontleding. Eggokardiogramme en elektrokardiogramme was deel van die pre-oorplanting assessering. Linker ventrikulêre disfunksie was voorspelbaar van verhoogde postoorplanting probleme. Kardiale komplikasies het konstant volgens publikasies minder geredelik voorgekom as ander post-oorplantings newe-effekte. DERMATOLOGIE Gevalle is op ʼn daaglikse basis geëvalueer en verwys na ʼn dermatoloog wanneer nodig. ʼn Velbiopsie is gedoen om “Graft-Versus-Host” siekte (GVHD) te bevestig en dit te onderskei van middel hipersensitiwiteit of virale infeksies. Die blootstelling aan ex vivo Campath® het uitkomste aansienlik verbeter deur die voorkoms en erns van GVHD te verminder. Kwaliteit van lewe is verhoog met aansienlike koste besparing. GASTROENTEROLOGIE Boonste gastro-intestinale simptome het voorgekom in 90% van die pasiënte. Wanvoeding het voorgekom.. Abnormale lewerfunksies was relatief algemeen toeskryfbaar aan middels, sepsis en kondisionerings protokolle. Lewer biopsies is nie in hierdie reeks uitgevoer nie en endoskopie slegs wanneer dit noodsaaklik was. DIE STUDIE VAN LAAT KOMPLIKASIES (55 GEVALLE) RESPIRATORIES Spirometrie en diffusie kapasiteit is gedoen in hierdie groep. Al die longfunksie ondersoeke was binne die voorspelde normale waardes behalwe ʼn paar marginale afnames in die diffusie kapasitiet. In geen van hierdie pasiënte het laat nagevolge soos Bronchoilitis Obliterans Organiserende Pneumonie en Laat Aanvangs Nieinfektiewe Long komplikasies voorgekom nie. Sitomegaal virus heraktivering was algemeen maar vroeë intervensie het ernstige komplikasies voorkom. IMMUNOLOGIE ʼn In vitro funksionele studie is gedoen. Beide die spesifieke en nie-spesifieke immuun stelsels is geëvalueer. Die tipe oorplanting, tyd vanaf oorplanting, diagnose en kondisionering is in ag geneem. Die “Granulocyte Burst” toets is gedoen vir die nie-spesifieke profiel. Verminderde aktiwiteite is bewys in al die subgroepe. Dit wil voorkom asof die nie-spesifieke respons van die granulosiete nooit herstel nie as gevolg van die verlaagde in vitro granulosiet funksie. Die spesifieke immuun respons is in vitro geëvalueer en slegs die outotransplantaat het beter CD4+ en CD8+ sitokiene produksie getoon. Geen groot verskille is gesien in ander groepe nie. By CD4+ en CD8+ T selle was normale sitokiene produksie teenwoordig toe dit in vitro geaktiveer is om regulatoriese sitokiene produksie te produseer, wat beteken dat hul limfoïede komponent na oorplanting ongeskonde was. BEEN SIEKTE Beide die Dubbele energie x-straal absorpsiemetrie (DXA) en Kwantitatiewe rekenaar tomografie (QCT) is hier gebruik om been mineraal digtheid te evalueer. Daar was ʼn teenstrydigheid teenwoordig tussen die twee modaliteite. DXA het geen osteoporose getoon nie maar QCT het 22% getoon. Biomerkers was normaal in albei. Daar was geen geskiedenis van frakture en geen objektiewe bewyse van vertebrale frakture met Vertebrale Fraktuur Assessering nie. Alhoewel QCT gebruik is vir die studie bly DXA die goue standaard in Suid-Afrika. GEVOLGTREKKING Hierdie doktoraal verskaf inligting oor die nie-hematologiese gevolge in Suid-Afrika met die gebruik van Campath® ex vivo.

published_or_final_version / abstract / Medicine / Master / Doctor of Medicine

published_or_final_version / abstract / Medicine / Master / Master of Philosophy

No description available.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Stem cell-based therapies are promising in regenerative medicine. However, the success of cell therapy is greatly limited by the low engraftment rate to the target tissues. The present study demonstrated that human mesenchymal stem cells (hMSCs) were subjected to a self selection process via microencapsulation in collagen barrier when they were induced to migrate out from this barrier. While retaining the immuophenotype and self renewal capacity, the selected hMSCs showed a significantly better in vitro migratory response of than those cultured in traditional monolayer. The migratory response could be controlled by varying the fabrication parameters of the collagen barrier, including initial collagen concentration and cells seeding density. Affinity to adhere on endothelial cells layer is another engraftment related property. Significant difference was observed between these selected hMSCs and hMSCs in monolayer culture. In order to investigate the engraftment potential of the selected hMSCs, an animal model was performed. The selected hMSCs were transplanted intravenously into NOD/SCID mice under partial hepatectomy. Presence of human cells in the residual liver was determined by the presence of human HLA-ABC using flow cytometry after 48 hours, 1 week and 1 month. Engraftment of the selected hMSCs was significantly higher than that of monolayer cultured hMSCs in time point of 1 month. It demonstrated that the selected hMSCs favor the engraftment to the injured liver. Further investigation is required to determine the fate of the engrafted hMSCs in order to truly confirm their therapeutic potential. The current work demonstrated that collagen-hMSCs microsphere could act as a barrier to select hMSCs with enhanced in vitro migratory response and in vivo engraftment properties. These findings may contribute towards the development of better stem cell therapies. / published_or_final_version / Mechanical Engineering / Master / Master of Philosophy