Timely reperfusion after a myocardial infarction is necessary to salvage the ischemic region; however, reperfusion itself is a major contributor to the final tissue damage. Currently, there is no clinically relevant therapy available to reduce ischemia-reperfusion injury. While many drugs have shown promise in reducing ischemia-reperfusion injury in preclinical studies, none of these drugs have demonstrated benefit in large clinical trials. Part of the failure to translate therapies can be attributed to the reliance on small animal models for preclinical studies. While animal models encapsulate the complexity of the systemic in vivo environment, they do not fully recapitulate human cardiac physiology. In this thesis, we utilized cardiac tissue engineering methods in conjunction with cardiomyocytes derived from human induced pluripotent stem cells, to establish a biomimetic human tissue-engineered model of ischemia-reperfusion injury. The resulting cardiac constructs were subjected to simulated ischemia or ischemia-reperfusion injury in vitro. We demonstrated that the presence of reperfusion injury can be detected and distinguished from ischemic injury. Furthermore, we demonstrated that we were able to detect changes in reperfusion injury in our model following ischemic preconditioning, modification of reperfusion conditions, and addition of cardioprotective therapeutics. This work establishes the utility of the human tissue model in studying ischemia-reperfusion injury and the potential of the human tissue platform to help translate therapeutic strategies into the clinical setting.

Forensic facial reconstruction may be the final option available to draw the public attention in cases where the identity of an individual cannot be established by standard identification methods. Two fundamental components of all forensic facial reconstruction techniques are cranial morphology and soft tissue depths databases. The purpose of this study was to extend such databases by providing a complete set of accurate facial soft tissue thickness measurements, acquired from a contemporary adult population from Germany, for use in forensic facial reconstruction. The aims were to measure the distance between well-defined landmarks on the skull and reference points on the face in a standardised manner, to analyse how sex, age and body mass index (BMI) influence facial soft tissue depths, to identify patterns of facial asymmetry, and to conduct a comparative analysis with other populations. The material for this study consisted of 320 (160 male, 160 female) anonymised multi-slice computerised tomography (MSCT) scans of individuals drawn from a German population. Individuals between the ages of 18 and 84 years were analysed. Their statures varied between 1.50 m and 1.96 m; their weights ranged between 40 kg and 145 kg. The BMI fluctuated between 16.6 kg/m2 and 45.8 kg/m2. Patients with severe trauma or pathologies that may compromise facial soft tissue depth were excluded from the study as were patients known to have been treated with specific medication (e.g. cortisone). In Amira®, 3D models of the surfaces of the skull and the facial skin were semi-automatically segmented using previously calculated thresholds and surface extraction algorithms. The parameters were adjusted to permit semi-transparent visualisation and examination of the structures of both the 3D skull and facial skin surface models simultaneously. Facial soft tissue depth was measured at 10 midline and 28 bilateral anatomical landmarks, according to the main orientations of the skull. Statistical analyses and tests were performed with SPSS® Version 22 and TDStats Version v2015.1. The analysis of facial soft tissue thickness versus BMI, sex and age, for each landmark separately, indicated that, at a number of the landmarks, facial soft tissue depth is significantly (p < 0.05) influenced by all three biometric variables. Facial soft tissue thickness increased with increasing BMI, but the correlations with age were insignificant. The differences between males and females were statistically significant (p < 0.05) for almost all anatomical landmarks with the exception of a few in the region of the nasal root and orbitals. Asymmetry was noted at over half of the bilateral landmarks. The differences between the results from this sample and those obtained from comparable databases contradict the hypothesis that population-specificity significantly influences facial soft tissue thickness. Nevertheless, this and future studies of craniofacial soft tissues will improve our knowledge of the complexity of the human face. The information gathered will be invaluable when considering forensic facial reconstruction methods for neighbouring populations.

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To correct, improve, and maintain tissues, and their functions, are common denominators in tissue engineering and reconstructive plastic surgery. This can be achieved by using autolo-gous tissues as in flaps or transplants. However, often autologous tissue is not useable. This is one of the reasons for the increasing interest among plastic surgeons for tissue engineering, and it has led to fruitful cross-fertilizations between the fields. Tissue engineering is defined as an interdisciplinary field that applies the principles of engineering and life sciences for development of biologic substitutes designed to maintain, restore, or improve tissue functions. These methods have already dramatically improved the possibilities to treat a number of medical conditions, and can arbitrarily be divided into two main principles: &gt; Methods where autologous cells are cultured in vitro and transplanted by means of a cell suspension, a graft, or in a 3-D biodegradable matrix as carrier. &gt; Methods where the tissue of interest is stimulated and given the right prerequisites to regenerate the tissue in vivo/situ with the assistance of implantation of specially designed materials, or application of substances that regulate cell functions - guided tissue regeneration. We have shown that human mammary epithelial cells and adipocytes could be isolated from tissue biopsies and that the cells kept their proliferative ability. When co-cultured in a 3-D matrix, patterns of ductal structures of epithelial cells embedded in clusters of adipocytes, mimicking the in vivo architecture of human breast tissue, were seen. This indicated that human autologous breast tissue can be regenerated in vitro. The adipose tissue is also generally used to correct soft tissue defects e.g. by autologous fat transplantation. Alas 30-70% of the transplanted fat is commonly resorbed. Preadipocytes are believed to be hardier and also able to replicate, and hence, are probably more useful for fat transplantation. We showed that by using cell culture techniques, significantly more pre-adipocytes could survive and proliferate in vitro compared to two clinically used techniques of fat graft handling. Theoretically, a biopsy of fat could generate enough preadipocytes to seed a biodegradable matrix that is implanted to correct a defect. The cells in the matrix will replicate at a rate that parallels the vascular development, the matrix subsequently degrades and the cell-matrix complex is replaced by regenerated, vascularized adipose tissue. We further evaluated different biodegradable scaffolds usable for tissue engineering of soft tissues. A macroporous gelatin sphere showed several appealing characteristics. A number of primary human ecto- and mesodermal cells were proven to thrive on the gelatin spheres when cultured in spinner flasks. As the spheres are biodegradable, it follows that the cells can be cultured and expanded on the same substrate that functions as a transplantation vehicle and scaffold for tissue engineering of soft tissues. To evaluate the in vivo behavior of cells and gelatin spheres, an animal study was performed where human fibroblasts and preadipocytes were cultured on the spheres and injected intra-dermally. Cell-seeded spheres were compared with injections of empty spheres and cell suspensions. The pre-seeded spheres showed a near complete regeneration of the soft tissues with neoangiogenesis. Some tissue regeneration was seen also in the ‘naked’ spheres but no effect was shown by cell injections. In a human pilot-study, intradermally injected spheres were compared with hyaluronan. Volume-stability was inferior to hyaluronan but a near complete regeneration of the dermis was proven, indicating that the volume-effect is permanent in contrast to hyaluronan which eventually will be resorbed. Further studies are needed to fully evaluate the effect of the macroporous gelatin spheres, with or without cellular pre-seeding, as a matrix for guided tissue regeneration. However, we believe that the prospect to use these spheres as an injectable, 3D, biodegradable matrix will greatly enhance our possibilities to regenerate tissues through guided tissue regeneration. / On the day of the defence date the status of article V was In Press.

The primary objective of this research was to investigate the feasibility of calibrating ultrasound scanners to measure surface impedance from reflection data. The method proposed uses calibration curves from known impedance interfaces. This plot, or calibration curve, may then be used, with interpolation, to relate measured grey level to impedance for the characterization of tissue specimens with unknown properties. This approach can be used independent of different medical ultrasound scanner systems to solve for reproducible tissue impedance values without offline data processing and complicated custom electronics. <p>Two medical ultrasound machines from different manufacturers were used in the experiment; a 30 MHz and a 7.5 MHz machine. The calibration curves for each machine were produced by imaging the interfaces of a vegetable oil floating over varying salt solutions. <p>To test the method, porcine liver, kidney, and spleen acoustical impedances were determined by relating measured grey levels to reflection coefficients using calibration curves and then inverting the reflection coefficients to obtain impedance values. The 30 MHz ultrasound machines calculated tissue impedances for liver, kidney, and spleen were 1.476 ± 0.020, 1.486 ± 0.020, 1.471 ± 0.020 MRayles respectively. The 7.5 MHz machines tissue impedances were 1.467 ± 0.088, 1.507 ± 0.088, and 1.457 ± 0.088 MRayles respectively for liver, kidney and spleen. The differences between the two machines are 0.61%, 1.41%, and 0.95% for the impedance of liver, kidney, and spleen tissue, respectively. If the grey level is solely used to characterize the tissue, then the differences are 45.9%, 40.3%, and 39.1% for liver, kidney, and spleen between the two machines. The results support the hypothesis that tissue impedance can be determined using calibration curves and be consistent between multiple machines.

Craniofacial defects resulting from trauma and resection present many challenges to reconstruction due to the complex structure, combinations of tissues, and environment, with exposure to the oral, skin and nasal mucosal pathogens. Tissue engineering seeks to regenerate the tissues lost in these defects; however, the composite nature and proximity to colonizing bacteria remain difficult to overcome. Additionally, many tissue engineering approaches have further hurdles to overcome in the regulatory process to clinical translation. As such these studies investigated a two stage strategy employing an antibiotic-releasing porous polymethylmethacrylate space maintainer fabricated with materials currently part of products approved or cleared by the United States Food and Drug Administration, expediting the translation to the clinic. This porous space maintainer holds the bone defect open allowing soft tissue to heal around the defect. The space maintainer can then be removed and one regenerated in the defect. These studies investigated the individual components of this strategy. The porous space maintainer showed similar soft tissue healing and response to non-porous space maintainers in a rabbit composite tissue defect. In humans, the porous space maintainers were well tolerated and maintained a soft tissue envelope for closure after implantation of a bone regeneration technology. The antibiotic-releasing space maintainers showed release of antibiotics from 1-5 weeks, which could be controlled by loading and fabrication parameters. In vivo, space maintainers releasing a high dose of antibiotics for an extended period of time increased soft tissue healing over burst release space maintainers in an infected composite tissue defect model in a rabbit mandible. Finally, stabilization of bone defects and regeneration could be improved through scaffold structures and delivery of a bone forming growth factor. These studies illustrate the possibility of the two stage strategy for repair of composite tissue defects of the craniofacial complex.

The primary objective of this research was to investigate the feasibility of calibrating ultrasound scanners to measure surface impedance from reflection data. The method proposed uses calibration curves from known impedance interfaces. This plot, or calibration curve, may then be used, with interpolation, to relate measured grey level to impedance for the characterization of tissue specimens with unknown properties. This approach can be used independent of different medical ultrasound scanner systems to solve for reproducible tissue impedance values without offline data processing and complicated custom electronics. <p>Two medical ultrasound machines from different manufacturers were used in the experiment; a 30 MHz and a 7.5 MHz machine. The calibration curves for each machine were produced by imaging the interfaces of a vegetable oil floating over varying salt solutions. <p>To test the method, porcine liver, kidney, and spleen acoustical impedances were determined by relating measured grey levels to reflection coefficients using calibration curves and then inverting the reflection coefficients to obtain impedance values. The 30 MHz ultrasound machines calculated tissue impedances for liver, kidney, and spleen were 1.476 ± 0.020, 1.486 ± 0.020, 1.471 ± 0.020 MRayles respectively. The 7.5 MHz machines tissue impedances were 1.467 ± 0.088, 1.507 ± 0.088, and 1.457 ± 0.088 MRayles respectively for liver, kidney and spleen. The differences between the two machines are 0.61%, 1.41%, and 0.95% for the impedance of liver, kidney, and spleen tissue, respectively. If the grey level is solely used to characterize the tissue, then the differences are 45.9%, 40.3%, and 39.1% for liver, kidney, and spleen between the two machines. The results support the hypothesis that tissue impedance can be determined using calibration curves and be consistent between multiple machines.

Our ability to analyze the microstructure of biological tissue in three dimensions (3D) has proven invaluable in modeling its functionality, and therefore providing a better understanding of the basic mechanisms of life. Volumetric imaging of tissue at the cellular level, using serial imaging of consecutive tissue sections, provides such ability to acquire microstructure in 3D. Three-dimensional light microscopy in biology can be broadly classified as using either optical sectioning or physical sectioning. Due to the inherent limitations on the depth resolution in optical sectioning, and the recent introduction of novel techniques, physical sectioning has become the sought-out method to obtain high-resolution volumetric tissue structure data. To meet this demand with increased processing speed in 3D biological imaging, this thesis provides an engineering study and formulation of the tissue sectioning process. The knife-edge scanning microscopy (KESM), a novel physical sectioning and imaging instrument developed in the Brain Networks Laboratory at Texas A&M University, has been used for the purpose of this study. However, the modes of characterizing chatter and its measurement are equally applicable to all current variants of 3D biological microscopy using physical sectioning. We focus on chatter in the physical sectioning process, principally characterizing it by its geometric and optical attributes. Some important nonlinear dynamical models of chatter in the sectioning process, drawn from the metal machining literature, are introduced and compared with observed measurements of chatter in the tissue cutting process. To understand the effects of the embedding polymer on tissue sectioning, we discuss methods to characterize the polymer material and present polymer measurements. Image processing techniques are introduced as a method to abate chatter artifacts in the volumetric data that has already been obtained. Ultra-precise machining techniques, using (1) free-form nanomachining and (2) an oscillating knife, are introduced as potential ways to acquire chatter-free higher-resolution volumetric data in less time. Finally, conclusions of our study and future work conclude the thesis. In this thesis, we conclude that to achieve ultrathin sectioning and high-resolution imaging, embedded plastic should be soft. To overcome the machining defects of soft plastics, we suggested free-form nanomachining and sectioning with an oscillating knife.