<p>Transplantation of a functional engineered skeletal muscle substitute is a promising therapeutic option to repair irreversible muscle damage, and, on the other hand, functional muscle tissue constructs can serve as in vitro 3D tissue models that complement the conventional 2D cell cultures and animal models to advance our limited understanding of intrinsic myogenesis and muscle regeneration process. However, the engineering of skeletal muscle constructs with comparable contractile function to the native muscle is hampered by the lack of 1) effective and reproducible methods to form relatively large muscle constructs composed of viable, dense, aligned and matured myofibers, and 2) beneficial microenvironmental cues as well as physiological stimulations that favor the growth, differentiation and maturation of myogenic cells. Thus, in this thesis, I have developed a mesoscopic hydrogel molding approach to fabricate relatively large engineered muscle tissue networks with controllable thickness, pore dimensions, overall myofiber alignment and regional myofiber orientation. I then investigated the effect of variation in pore length on the force generation and passive properties of engineered muscle networks and the potential to improve the contractile function of engineered muscle networks with the treatment of a soluble neurotrophic factor, agrin.</p><p>Specifically, high aspect-ratio soft lithography was utilized to precisely fabricate elastomeric molds containing an array of staggered hexagonal posts which created elliptical pores in muscle tissue sheets made from a mixture of primary skeletal myoblasts, fibrin and Matrigel. The improved oxygen and nutrient access through the pores increased the viability of the embedded muscle cells and prevented the formation of an acellular core. The differentiated myofibers were locally aligned in tissue bundles surrounding the elliptical pores. The length and direction of the microfabricate posts arbitrarily determined the length of elliptical pores and the mean orientation of myofibers formed around the pores, which enables the control of pore dimensions and regional myofiber orientation. Contractile force analysis revealed that engineered muscle networks with more elongated pores generated larger contractile force due to the increased myonuclear density and degree of overall myofiber alignment, despite the larger porosity and reduced tissue volume. Furthermore, the introduction of elliptical pores resulted in distinct deformational changes in tissue bundles and node regions that connect the ends of bundles with the applied unaxial macroscopic stretch, but such spatial alteration of local strain field resulted in no significant change in macroscopic length- tension relationships among engineered muscle networks with different pore length. </p><p>In addition, supplementing culture medium with soluble recombinant agrin significantly increased contractile force production of engineered muscle networks in the absence of nerve-muscle interaction, primarily or partially due to the agrin-induced upregulation of dystrophin. As expected, alteration in the levels endogenous ACh or ACh-like compound affected the agrin-induced AChR aggregation. Furthermore, increased autocrine AChR stimulation, a novel mechanism underlying survival and maturation of aneural myotubes, attenuated the agrin-induced force increase, while suppressed autocrine AChR stimulation severely comproised the overall force production of engineered muscle networks, of which the underlying mechanisms remains to be elucidated in the future studies. </p><p>In summary, a novel tissue engineering methodology that enables the fabrication of relative large muscle tissue constructs with controllable structure and function has been developed in this thesis. Future studies, such as optimizing cell-matrix interaction, incorporating beneficial regulatory proteins in the fibrin-based matrix, and applying specific patterns of electro-mechanical stimulations are expected to further augment the contractile function of engineered muscle networks. The potential application of this versatile tissue fabrication approach to engineer different types of soft tissue might further advance the development of tissue regeneration therapies as well as deepen our understanding of intrinsic tissue morphogenesis and regeneration process.</p> / Dissertation

Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass of the blastocyst that can give rise to cells of the ectoderm, endoderm and mesoderm lineages. Once isolated from the blastocyst, ESCs can be cultured indefinitely in vitro in an undifferentiated state or can be induced to differentiate. In the case of mouse ESCs (mESCs), the cytokine leukemia inhibitory factor (LIF) is added to culture media to maintain pluripotency and is removed to induce differentiation. Although it is known that extracellular matrix (ECM) components influence stem cell maintenance, proliferation and differentiation, the precise effects of ECM environments on embryonic stem cell behavior have not been systematically studied. The main purpose of this thesis project was to investigate the behavior of undifferentiated mESCs cultured in different 3D hydrogel matrices and to determine whether viscoelastic and biochemical variations in the matrices differentially affect the ability of stem cells to self-renew; that is, retain their pluripotency or undifferentiated phenotype. Their behavior in 3D environments was compared to mESC behavior in traditional 2D culture. In addition, a new method of casting hydrogels in polydimethylsiloxane (PDMS) molds was developed in order to efficiently cast multiple hydrogels of varying sizes and shapes. The findings of this thesis project will benefit both the scientific and engineering community as it encourages researchers to re-evaluate the quality of standard 2D embryonic stem cell culture methods versus potentially novel and advantageous 3D hydrogel culture methods.

No description available.

This study investigated the effect of bone marrow derived mesenchymal stem cells (MSCs) in a PEGylated fibrin gel (PEG) seeded into a decellularized extracellular matrix (ECM) on recovery of skeletal muscle following a volumetric muscle loss (VML) injury. Six to nine month old male Sprague-Dawley rats were used in this study. Approximately one-third of the skeletal muscle mass of the lateral gastrocnemius (LGAS) was removed from the LGAS, which was immediately replaced with an acellular ECM of the same dimensions. Seven days after injury, animals were injected with one of four solutions: saline (SAL), MSCs (MSC), PEGylated fibrin hydrogel (PEG), or MSCs in PEG (PEG+MSC). Maximal isometric tetanic tension (Po) of the LGAS was assessed fifty-six days after VML injury, followed by histological evaluation. VML injury resulted in a functional impairment of the LGAS capable of producing 76.1± 4.9% of the force generated in the non-injured contralateral LGAS. Tetanic tension of the PEG+MSC treated group was significantly higher compared to all other treatment groups (p < 0.05), although specific tension (N/cm2) in the PEG+MSC group (79.7±4.0%) was only significantly higher compared to SAL (58.2±3.0) and PEG (64.0±2.1%) treated groups (p < 0.05). However, LGAS mass was significantly higher in the PEG+MSC group compared to all other groups (p < 0.05). These findings suggest the combination of the PEG+MSC did not lead to a significant increase in muscle function compared to MSC treatment alone, and demonstrates the importance of MSCs in skeletal muscle regeneration in VML injury models. However, as evident by the significant increase in LGAS mass, PEG+MSC treatment may lead to histological differences not evaluated in this study. Gross morphology of the repaired gastrocnemius was indistinguishable from the contralateral control. / text

Ischemia/reperfusion (I/R) injury causes extensive damage to skeletal muscle, often resulting in prolonged functional deficits. This current study determines the efficacy of controlled release of SDF-1α and IGF-1 by conjugation to biodegradable, polyethylene glycol, (PEG)ylated fibrin gel matrix in skeletal muscle repair of an I/R injury. Male Sprague-Dawley rats underwent a 2-hour tourniquet induced I/R injury on their hind limbs. Twenty-four hours post injury the following treatments were administered: PEGylated fibrin gel (PEG-Fib), SDF-1 conjugated PEGylated fibrin gel (PEG-Fib/SDF-1), or dual protein IGF-1 and SDF-1 conjugated PEGylated fibrin gel (PEG-Fibrin/SDF-1/IGF-1. Following 14 days after injury, functional and histological evaluations were performed. There was no significant difference in maximum tetanic force production recovery between PEG-Fib and PEG-Fib/SDF-1 groups. However, PEG-Fib/SDF-1/IGF-1 group resulted in significant improvement of force production relative to the other treatment groups. The same results were found for specific tension. Histological analysis revealed a greater distribution of small myofibers in the PEG-Fib/SDF-1 group than the PEG-Fib group, while the PEG-Fib/SDF-1/IGF-1 group had the smallest distribution of small fibers and similar to controls (uninjured). There were also a greater number of centrally located nuclei in the PEG-Fib/SDF-1 group than the PEG-Fib group, while the PEG-Fib/SDF-1/IGF-1 group had similar values to controls. Although these results confirm the protective role of exogenous IGF-1, SDF-1 did not have an effect on skeletal muscle repair. / text

I developed a system to release multiple growth factors from PEGylated fibrin gels with varying profiles to induce vasculogenesis from embedded human MSCs. Zero-order release can be obtained by conjugating a growth factor with a homobifunctional, amine-reactive, PEG derivative. Growth factors can be entrapped during thrombin-mediated crosslinking and released rapidly. Growth factors with physical affinity for fibrinogen or fibrin can be sequestered within the matrix and released via degradation and/or disassociation. PDGF-BB was loaded via entrapment while TGF-β1 was sequestered through a combination of physical affinity and conjugation. The affinity of TGF-β1 and fibrinogen had never been previously examined or quantified. I aimed to determine the Ka and Kd between TGF-β1 and fibrinogen through a variety of assays. Binding ELISAs were developed for TGF-β1 and fibronectin, a protein associated with fibrin gels, and TGF-β1 and fibrinogen. However, background was high due to insufficient blocking agents. Other assays explored included western blots, surface plasmon resonance, and radiolabeled TGF-β1 with limited success. The affect of TGF-β1 on human MSC differentiation towards vascular cell phenotypes was examined both in 2D and fibrin gels embedded with MSCs. With exposure to TGF-β1, MSC proliferation was significantly inhibited in both 2D and within fibrin gels indicating that loaded TGF-β1 maintained bioactivity for at least 7 days. Gene expression of MSCs exposed to TGF-β1 demonstrated inhibited endothelial cell differentiation and stimulated smooth muscle cell differentiation. However, confocal and light microscopy indicated that endothelial cell differentiation is maintained with TGF-β1 loaded PEGylated fibrin gels. The system developed is highly modular and can be applied to other tissue engineering systems. Furthermore, other growth factors could be incorporated to promote vascular cell differentiation. / text

The healing of tympanic membrane (TM) perforations is a complex wound healing process including inflammation, migration of keratinocytes and tissue remodelling. Most TM perforations in human heal spontaneously, however some perforations become chronic, and the reason to why is still largely unknown. In cutaneous wound healing plasminogen (plg) has been shown to play an important role. Plg is converted into the protease plasmin regulated by two plasminogen activators (PA), urokinase type PA (uPA) and tissue-type PA (tPA). The aim of the present thesis was to evaluate the role of plg in healing of TM perforations, both in vivo and in vitro. The main objectives were to determine the healing capacity of the TM, the involvement of keratinocytes, fibrin(ogen) and inflammatory cells in the healing process. The studies were performed in plg deficient and uPA deficient mice, with littermate wild type (wt) mice as controls It was shown that myringotomies of the TMs in plg deficient mice still remained open 143 days following a perforation. The wound area was characterized by an abundant recruitment and accumulation of inflammatory cells; mainly macrophages and neutrophils, an arrested keratinocyte migration and a fibrin deposition covering the surface of the TM. The TM perforations in the wt mice all healed within 11 days. Interestingly, the myringotomies of the plg deficient mice could be closed by reconstitution with systemic injections of plg, whereas injections of PBS had no affect on the healing. To characterize mechanisms involved in the development of persistent TM perforations in plg deficient mice after a myringotomy the early inflammatory response during the first 48 hours was studied. The recruitment and accumulation of inflammatory cells in the perforated TMs was found to be similar between the plg deficient and the wt mice. Myringotomized TMs in uPA deficient mice healed similar to perforations of wt controls. Neither did the keratinocyte migration nor the occurrence of inflammatory cells differ between these genotypes. In the in vitro experiments TMs from plg deficient and wt mice, were dissected out, perforated and cultured in absence or surplus of plg. A decrease in perforation size was seen in all groups regardless of genotype or amount of plg in the medium. In conclusion, the present studies show: • Plg is essential for the healing of TM perforations in mice. • The altered healing process after a myringotomy in plg deficient mice involves a disturbed keratinocyte migration, a massive deposition of fibrin and an abundant accumulation of inflammatory cells in the wound area. • Plasminogen deficiency does not alter the early inflammatory response, following a myringotomy. • Deficiency of uPA does not influence the healing of TM perforations. • During in vitro conditions healing of TM perforations is initiated irrespectively of genotype of the explant (plg deficient or wt) or supply of plg. The increased knowledge of the involvement of plg in the healing of TM perforations may open therapeutical possibilities in the treatment of chronic TM perforations in humans.

Motivation: Cardiovascular disease (CVD) is one of the leading causes of mortality and morbidity in South Africa and worldwide. Dyslipidaemia and an increased coagulation state contribute to the development of CVD. The quality of fibrin network structure (FNS) may also contribute to the risk for CVD and thrombosis. Changes in fibrinogen concentration directly affect FNS. Management of these risk factors is important and dietary intervention forms an essential part of this management program. An increased intake of vitamin C can lead to a decreased susceptibility to infection and subsequently to decreased levels of haemostatic factors (that give rise to an anti-thrombotic state) and thus reduction in CVD and mortality. Furthermore, vitamin C may prove to be beneficial by increasing the pro-fibrinolytx activities of FNS (formation of thick fibrin fibers and more lysable clots) that could result in a reduction in atherosclerosis and subsequent CVD. Obiective: To investigate the effects of FoodState Vitamin C complex supplementation on haemostatic factors, FNS, serum lipids and lipoprotein (a) (Lp(a)) in hyperlipideamic adults. Methods: Thirty free-living hiperlipidaemic volunteers from the Lipid Clinic, Potchefstroom University for Christian Higher Education (CHE), participated in this randomised placebo controlled double blind crossover study. The subjects were randomly divided into two groups (A or B). After a run-in period of 4 weeks during which the subjects excluded all vitamin supplements, Group A received 2 tablets/day of FoodState Vitamin C complex (500mg vitamin C, 600mg magnesium food complex, 900mg vitamin B complex and 160mg bioflavonoids) and Group B received 2 tablets/day of placebo, for at least 8 weeks. A washout period of 8 weeks followed after which the treatments were crossed-over for a further 8 weeks. Fasting blood samples were drawn 8 times (two samples, one week apart at the beginning and end of each treatment). Results: FoodState Vitamin C complex supplementation did not significantly influence the levels of plasma fibrinogen, plasminogen activator inhibitor 1 activity (PAI-I act), tissue plasminogen activator antigen (tPA ag) or d-dimer. Serum lipids and Lp(a) were also not affected. Median plasmin-antiplasmin complex (PAP) and thrombin-antithrombin complex (TAT) levels, which are markers of plasmin (initiate fibrinolysis) and thrombin (initiate coagulation) generation respectively, were both significantly decreased compared to placebo (PAP: 4.05[-23.39, -0.231% vs 1.81[-8.95, 8.091%; TAT: -5.81[-18,47, 0.391% vs 0.12[-8.03, 13.51%). FoodState Vitamin C complex beneficially affected FNS by significantly increasing compaction (49.95[47.55,53.70]% to 51.85[48.55,56.65]%). Conclusion: The decreases in TAT and PAP are possibly an indication that the FoodState Vitamin C complex decreased the initiation of haemostasis, which in turn led to a compensatory reduction in fibrinolysis. FoodState Vitamin C complex may, therefore be protective of cardiovascular disease by causing a new reduced steady state of hemostatic balance and more lysable clots (increased compaction). / Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2004.

Motivation: Cardiovascular disease (CVD) is one of the leading causes of mortality and morbidity in South Africa and worldwide. Dyslipidaemia and an increased coagulation state contribute to the development of CVD. The quality of fibrin network structure (FNS) may also contribute to the risk for CVD and thrombosis. Changes in fibrinogen concentration directly affect FNS. Management of these risk factors is important and dietary intervention forms an essential part of this management program. An increased intake of vitamin C can lead to a decreased susceptibility to infection and subsequently to decreased levels of haemostatic factors (that give rise to an anti-thrombotic state) and thus reduction in CVD and mortality. Furthermore, vitamin C may prove to be beneficial by increasing the pro-fibrinolytx activities of FNS (formation of thick fibrin fibers and more lysable clots) that could result in a reduction in atherosclerosis and subsequent CVD. Obiective: To investigate the effects of FoodState Vitamin C complex supplementation on haemostatic factors, FNS, serum lipids and lipoprotein (a) (Lp(a)) in hyperlipideamic adults. Methods: Thirty free-living hiperlipidaemic volunteers from the Lipid Clinic, Potchefstroom University for Christian Higher Education (CHE), participated in this randomised placebo controlled double blind crossover study. The subjects were randomly divided into two groups (A or B). After a run-in period of 4 weeks during which the subjects excluded all vitamin supplements, Group A received 2 tablets/day of FoodState Vitamin C complex (500mg vitamin C, 600mg magnesium food complex, 900mg vitamin B complex and 160mg bioflavonoids) and Group B received 2 tablets/day of placebo, for at least 8 weeks. A washout period of 8 weeks followed after which the treatments were crossed-over for a further 8 weeks. Fasting blood samples were drawn 8 times (two samples, one week apart at the beginning and end of each treatment). Results: FoodState Vitamin C complex supplementation did not significantly influence the levels of plasma fibrinogen, plasminogen activator inhibitor 1 activity (PAI-I act), tissue plasminogen activator antigen (tPA ag) or d-dimer. Serum lipids and Lp(a) were also not affected. Median plasmin-antiplasmin complex (PAP) and thrombin-antithrombin complex (TAT) levels, which are markers of plasmin (initiate fibrinolysis) and thrombin (initiate coagulation) generation respectively, were both significantly decreased compared to placebo (PAP: 4.05[-23.39, -0.231% vs 1.81[-8.95, 8.091%; TAT: -5.81[-18,47, 0.391% vs 0.12[-8.03, 13.51%). FoodState Vitamin C complex beneficially affected FNS by significantly increasing compaction (49.95[47.55,53.70]% to 51.85[48.55,56.65]%). Conclusion: The decreases in TAT and PAP are possibly an indication that the FoodState Vitamin C complex decreased the initiation of haemostasis, which in turn led to a compensatory reduction in fibrinolysis. FoodState Vitamin C complex may, therefore be protective of cardiovascular disease by causing a new reduced steady state of hemostatic balance and more lysable clots (increased compaction). / Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2004.

The regulation of fluid (water) volume in the body is crucial for tissue homeostasis. The interstitial fluid, which comprises almost 20% of the body fluid, is stored in the loose connective tissue and its volume is actively regulated by components of this tissue. The loose connective tissue provides a path for fluid flow from capillaries to the tissue and lymphatics. This fluid is partially stored in the interstitium and the remainder is directed to the lymphatics. The fibroblasts in the loose connective tissue actively compact the fibrous extracellular matrix (ECM) through mechanotransduction via integrins. This in turn, maintains the interstitial fluid pressure and keeps the ground substance underhydrated. The interstitial fluid pressure is part of the forces that regulate the efflux of fluid from capillaries and keep the ground substance underhydrated. The underhydrated ground substance has a potential to take up fluid 3-fold the plasma volume. Therefore, the active contraction of the ECM via fibroblasts is crucial to prevent the risk of evacuation of fluid from capillaries. During pathologies, such as inflammation and carcinogenesis, the interstitial fluid pressure and hence the interstitial fluid volume is altered. The results presented in this thesis show that the signaling events downstream of αVβ3 integrin, collagen-binding β1 integrins, and platelet-derived growth factor receptor β, that induce cell-mediated matrix contraction, included paired function of PI3K and PLCγ, cofilin activation, actin turnover, and generation of actomyosin forces. Furthermore, the results highlight new potential roles for fibrin and αVβ3 integrins, for instance during clearance of edema. Notably, fibrin extravasation at inflammatory sites induced αVβ3 integrin-dependent matrix contraction, leading to normalization of the altered interstitial fluid volume. It also reprograms the expression of ECM-related genes and hence induces ECM turnover. Taken together, these results provide further insight into the regulatory mechanism through which the loose connective tissue actively regulates the interstitial fluid volume.