Introduction Cutaneous scars represent a common surgical complication, yet no effective drug therapy for scar treatment currently exists despite huge patient and physician demand. A connexin 43 (Cx43) carboxyl terminus (CT) mimetic peptide, alpha Connexin Carboxy-Terminus 1 (αCT1), has demonstrated efficacy in improving long-term scar appearance in pre-clinical and clinical trials. However, current understanding of the mechanism-of-action by which αCT1 improves long-term scar appearance with early intervention treatment is not well understood. Methods In vivo: Scar biopsies from 1) human, 2) Sprague-Dawley rat, and 3) IAF Hairless guinea pig trials of αCT1 were examined for collagen matrix structure at 4 weeks (all models), and 2 and 6 weeks (rat and guinea pig models only). Collagen matrix variables examined included local disorganization of the fibers, a variable that is higher in unwounded skin compared to scar tissue, and density of the fibers, which is higher in scar tissue but can also be used as an early temporal marker of the rate of healing. In vitro: Primary murine dermal fibroblasts were isolated from the whole dermis of 3-4 week old transgenic mice expressing collagen 1(α2) GFP-tpz. Cells were sorted for expression via FACS and plated on prealigned collagen substrate for 7 days under conditions favorable to generating extracellular matrix. Results: All in vivo scar biopsies demonstrated some level of altered collagen matrix structure with αCT1 treatment. Treated scars had higher local disorganization of the collagen fibers within the wound, and an increase in collagen matrix density compared to control at certain earlier timepoints that tended to decrease or disappear at later timepoints. The IAF Hairless guinea pig, a novel splinted wound healing model presented herein, was found to closely replicate the human dermal collagen profile and changes in collagen profile spurred by αCT1, significantly outperforming the traditional rat model. Primary dermal murine fibroblasts treated in vitro with αCT1 significantly increased synthesis of procollagen 1, the precursor of collagen 1 necessary for constructing the extracellular matrix, suggesting that at least part of the reason for higher collagen density at early in vivo timepoints is due to increased collagen synthesis by fibroblasts. Conclusion: αCT1 treatment in the early stages of wound healing prompts individual fibroblasts to increase their output of collagen and create a more disorganized early collagen matrix. These early changes potentially spur the long-term scar appearance improvements seen in clinical trials, and provide a basis for future work to discover the cellular pathways to alter in order to improve wound healing and cutaneous scarring outcomes. / Doctor of Philosophy / Skin wounds frequently result in scars that can range from barely visible to enormous eyesores. Almost everyone will experience at least one skin wound in their lifetime leading to a scar that they wish were less visible, feeding the multi-billion dollar market for anti-scarring agents. However, many of the products on store shelves that claim to reduce scar appearance have not proven those claims. Most of the therapies that do have some degree of scientific evidence to support their claims are difficult to use properly, such as silicone sheeting, and often result in only minor improvements to scar appearance. Alpha Connexin Carboxy-Terminus 1 (αCT1), marketed in clinical trials as Granexin® gel, is a protein-based therapy that works on the cellular level to fundamentally alter the skin's initial reaction to wounding and improving long-term scar appearance. This dissertation explores the link between cellular processes altered by αCT1 and long-term clinical improvements in scar appearance by studying both the extracellular matrix present in the scar in human and animal models and the creation of that extracellular matrix by dermal fibroblasts. In both human and animal models, topical application of αCT1 had no effect on skin surface appearance at early timepoints of 2-6 weeks, correlating with previous research that found scar appearance only improved at 3+ months post-injury. However, deep within the newly constructed tissue of the scar, these studies show the collagen organizational structure of αCT1-treated scars is more similar to unwounded skin and slightly more dense at early timepoints, suggesting αCT1 marginally improved the speed of healing. These findings in humans and animals were also verified in part in cell culture experiments that found dermal fibroblasts increased collagen output in response to αCT1 treatment. A novel wound healing model in the hairless guinea pig, superior at replicating human skin than established models like the rat, is also presented and shown to have effects strongly similar to the human with αCT1 treatment. These results provide a fundamental insight into the mode-of-action by which αCT1 may improve long term scar appearance and identifies early collagen structure as a target for future therapeutics to modify, as well as a new animal model in which to test them.

<p>The mechanical behavior of tissues at the macroscale is tightly coupled to cellular activity at the microscale and tuned by microstructure at the mesoscale. Dermal wound healing is a prominent example of a complex system in which multiscale mechanics regulate restoration of tissue form and function. In cutaneous wound healing, a fibrin matrix is populated by fibroblasts migrating in from a surrounding tissue made mostly out of collagen. Fibroblasts both respond to mechanical cues such as fiber alignment and stiffness as well as exert active stresses needed for wound closure. </p> <p>To model wound healing mechanobiology, we first develop a multiscale model with a two-way coupling between a microscale cell adhesion model and a macroscale tissue mechanics model. Starting from the well-known model of adhesion kinetics proposed by Bell, we extend the formulation to account for nonlinear mechanics of fibrin and collagen and show how this nonlinear response naturally captures stretch-driven mechanosensing. We then embed the new nonlinear adhesion model into a custom finite element implementation of tissue mechanical equilibrium. Strains and stresses at the tissue level are coupled with the solution of the microscale adhesion model at each integration point of the finite element mesh. In addition, solution of the adhesion model is coupled with the active contractile stress of the cell population. The multiscale model successfully captures the mechanical response of biopolymer fibers and gels, contractile stresses generated by fibroblasts, and stress-strain contours observed during wound healing. We anticipate this framework will not only increase our understanding of how mechanical cues guide cellular behavior in cutaneous wound healing, but will also be helpful in the study of mechanobiology, growth, and remodeling in other tissues. </p> <p>Next, we develop another multiscale model with a bidirectional coupling between a microscale cell adhesion model and a mesoscale microstructure mechanics model. By mimicking the generation of fibrous network in experiment, we established a discrete fiber network model to simulate the microstructure of biopolymer gels. We then coupled the cell adhesion model to the discrete model to obtain the solution of microstructure equilibrium. This multiscale model was able to recover the volume loss of fibrous gels and the contraction from cells in the networks observed in experiment. We examined the influence of RVE size, stiffness of single fibers and stretch of the gels. We expect this work will help bridge the activity of cell to the microstructure and then to the tissue mechanics especially in wound healing. We hope this work will provide more rigorous understanding in the study of mechanobiology.</p> <p>At last, we established a computational model to accurately capture the mechanical response of fibrin gels which is a naturally occurring protein network that forms a temporary structure to enable remodeling during wound healing and a common tissue engineering scaffold due to the controllable structural properties. We formulated a strategy to quantify both the macroscale (1–10 mm) stress-strain response and the deformation of the mesoscale (10–1000 microns) network structure during unidirectional tensile tests. Based on the experimental data, we successfully predict the strain fields that were observed experimentally within heterogenous fibrin gels with spatial variations in material properties by developing a hyper-viscoelastic model with non-affined evolution under stretching. This model is also potential to predict the macroscale mechanics and mesoscale network organization of other heterogeneous biological tissues and matrices.</p>

Text in Afrikaans / Die kerklike diens van genesing bet deur die geskiedenis uitgestaan as een van die aktiwiteite van die kerk van Jesus Christus wat vanwee verskillende redes voortdurend in die brandpunt was. Die afgelope dekades is gekenmerk deur die toename van gelowiges uit verskillende denominasies en groepe wat by die nadenke oor en beoefening van die diens van genesing in die kerk betrokke geraak bet. Die toename bet meegebring dat die kerklike diens van genesing op verskillende wyses (praktyke) beoefen is en dat verskillende redes as regverdiging daarvoor aangebied is. Onderliggend hieraan le bepaalde teoriee waarvan die beoefenaars soms bewus was, en soms nie. Die huidige navorsing ondersoek die teoriee en praxes van verskillende genesingsbedienings binne 'n bepaalde akademiese en kerklike raamwerk. In die verband is 'n vergelykende studie deur middel van 'n kwalitatiewe ondersoek na die werk van twee uiteenlopende instansies gedoen, naamlik die London Healing Mission ( wat onder toesig van die Anglikaanse kerk funksioneer en daarom 'n vaste tradisie bet), en die Vineyard Christian Fellowship van Boise, in die VSA, wat 'n jong gemeente is, en by die sogenaamde 'Third Wave' beweging inpas. Die teoriee en praktyk van elke bediening word in die studie ge!dentifiseer en met mekaar vergelyk. Uit hierdie vergelyking word verskillende gevolgtrekkings gemaak, onder andere oor die bestaansreg van die kerklike diens van genesing en die formaat wat dit behoort aan te neem. Sekere opmerkinge word op grond van die navorsingsresultate gemaak wat in die Pinkster, Charismatiese en Gerefonneerde kerke asook vir die kerk in die algemeen, van waarde kan wees. Die navorsingsresultate word gebruik om 'n bedieningsmodel vir die genesingsbediening in die kerk te ontwerp. Die ondersoek word afgesluit met aanbevelings met die oog op moontlike toekomstige navorsing. / Throughout history the healing ministry has been one of the outstanding activities of the church of Jesus Christ which remained continuously and for various reasons the focal point of attention. The past decades were characterized by an escalation of believers from different denominations and groups who became involved in the meditation and practicing of the healing ministry in the church. This escalation has resulted in the healing ministry being practiced in multiple ways and various reasons being given as justification for doing so. This was done in the presence of underlying theories of which the practitioners were either consciously or subconsciously aware or unaware of. The present research explores the theories and practices of the healing ministry within a particular academic and ecclesiastical context. A comparative study was undertaken by means of a qualitative investigation covering the work of two divergent institutions namely: The London Healing Mission (which is supervised by the Anglican Church and therefore has a specific tradition) and the Vineyard Christian Fellowship, Boise, USA, which is a 'new' congregation and fits in with the so called Third Wave movement. The theories and practices of both these ministries are identified and compared with one another. Various conclusions are reached through this comparison, referring amongst others to the healing ministry's right of existence and the format in which it should be practiced. Remarks that are being·made with regard to the results of the research should be of value to the Pentecostal, Charismatic and Reformed churches in particular, but to the Church in general as well. These results are also used to design a ministering model for the healing ministry. The study concludes with certain recommendations for possible future research. / Philosophy, Practical & Systematic Theology / Th. D. (Praktiese Teologie)

Text in Afrikaans / Die kerklike diens van genesing bet deur die geskiedenis uitgestaan as een van die aktiwiteite van die kerk van Jesus Christus wat vanwee verskillende redes voortdurend in die brandpunt was. Die afgelope dekades is gekenmerk deur die toename van gelowiges uit verskillende denominasies en groepe wat by die nadenke oor en beoefening van die diens van genesing in die kerk betrokke geraak bet. Die toename bet meegebring dat die kerklike diens van genesing op verskillende wyses (praktyke) beoefen is en dat verskillende redes as regverdiging daarvoor aangebied is. Onderliggend hieraan le bepaalde teoriee waarvan die beoefenaars soms bewus was, en soms nie. Die huidige navorsing ondersoek die teoriee en praxes van verskillende genesingsbedienings binne 'n bepaalde akademiese en kerklike raamwerk. In die verband is 'n vergelykende studie deur middel van 'n kwalitatiewe ondersoek na die werk van twee uiteenlopende instansies gedoen, naamlik die London Healing Mission ( wat onder toesig van die Anglikaanse kerk funksioneer en daarom 'n vaste tradisie bet), en die Vineyard Christian Fellowship van Boise, in die VSA, wat 'n jong gemeente is, en by die sogenaamde 'Third Wave' beweging inpas. Die teoriee en praktyk van elke bediening word in die studie ge!dentifiseer en met mekaar vergelyk. Uit hierdie vergelyking word verskillende gevolgtrekkings gemaak, onder andere oor die bestaansreg van die kerklike diens van genesing en die formaat wat dit behoort aan te neem. Sekere opmerkinge word op grond van die navorsingsresultate gemaak wat in die Pinkster, Charismatiese en Gerefonneerde kerke asook vir die kerk in die algemeen, van waarde kan wees. Die navorsingsresultate word gebruik om 'n bedieningsmodel vir die genesingsbediening in die kerk te ontwerp. Die ondersoek word afgesluit met aanbevelings met die oog op moontlike toekomstige navorsing. / Throughout history the healing ministry has been one of the outstanding activities of the church of Jesus Christ which remained continuously and for various reasons the focal point of attention. The past decades were characterized by an escalation of believers from different denominations and groups who became involved in the meditation and practicing of the healing ministry in the church. This escalation has resulted in the healing ministry being practiced in multiple ways and various reasons being given as justification for doing so. This was done in the presence of underlying theories of which the practitioners were either consciously or subconsciously aware or unaware of. The present research explores the theories and practices of the healing ministry within a particular academic and ecclesiastical context. A comparative study was undertaken by means of a qualitative investigation covering the work of two divergent institutions namely: The London Healing Mission (which is supervised by the Anglican Church and therefore has a specific tradition) and the Vineyard Christian Fellowship, Boise, USA, which is a 'new' congregation and fits in with the so called Third Wave movement. The theories and practices of both these ministries are identified and compared with one another. Various conclusions are reached through this comparison, referring amongst others to the healing ministry's right of existence and the format in which it should be practiced. Remarks that are being·made with regard to the results of the research should be of value to the Pentecostal, Charismatic and Reformed churches in particular, but to the Church in general as well. These results are also used to design a ministering model for the healing ministry. The study concludes with certain recommendations for possible future research. / Philosophy, Practical and Systematic Theology / Th. D. (Praktiese Teologie)

No description available.

The purpose of this research was to study the synthesis and remodeling of extracellular matrix (ECM) by fibroblasts with special emphasis on the culture environment (media composition and initial ECM composition) and the resulting mechanical integrity of the ECM. This was investigated by culturing fibroblasts for 3 weeks in a variety of culture conditions consisting of collagen gels, fibrin gels, or media permissive to the self-production of ECM (Cell-Derived Matrix), and quantifying the mechanics of the resulting ECM. The mechanical characteristics were related to the biochemistry of the resulting ECM, notably in terms of collagen accumulation and collagen fibril diameters. The ultimate tensile strength (UTS) of the collagen gels and fibrin gels at the end of the 3-week period was 168.5 ± 43.1 kPa and 133.2 ± 10.6 kPa, respectively. The ultimate tensile strength of the cell-derived matrices was 223.2 ± 9 kPa, and up to 697.1 ± 36.1 kPa when cultured in a chemically-defined medium that was developed for the rapid growth of matrix in a more defined environment. Normalizing the strength to collagen density resulted in a UTS / Collagen Density in these groups of 6.4 ± 1.9 kPa/mg/cm3, 25.9 ± 2.4 kPa/mg/cm3, 14.5 ± 1.1 kPa/mg/cm3, and 40.0 ± 1.9 kPa/mg/cm3, respectively. Cells were synthetically more active when they produced their own matrix than when they were placed within gels. The resulting matrix was also significantly stronger when it was self-produced than when the cells rearranged the matrix within gels that corresponded to a significantly larger fraction of non-acid and pepsin extractable collagen. These studies indicate that cell-derived matrices have potential both as in vitro wound healing models and as soft connective tissue substitutes.