Stratified tissue biofabrication by rotational internal flow layer engineering

The bioassembly of layered tissue that closely mimics human histology presents challenges for tissue engineering. Existing bioprinting technologies lack the resolution and cell densities necessary to form the microscale cell-width layers commonly observed in stratified tissue, particularly when using low-viscosity hydrogels, such as collagen. Here we present rotational internal flow layer engineering (RIFLE), a novel, low-cost biofabrication technology for assembling tuneable, multi-layered tissue-like structures. Using high-speed rotating tubular moulds, small volumes of cell-laden liquids added to the inner surface were transitioned into thin layers and gelled, progressively building macroscale tubes composed of discrete microscale strata with thicknesses a function of rotational speed. Cell encapsulation enabled the patterning of high-density layers (108 cells ml−1) into heterogenous constructs. RIFLE versatility was demonstrated through tunica media assembly, encapsulating human smooth muscle cells in cell-width (12.5 µm) collagen layers. Such deposition of discrete microscale layers, facilitates the biofabrication of composite structures mimicking the nature of native stratified tissue. This enabling technology has the potential to allow researchers to economically create a range of representative layered tissue

3D printing in medicine for preoperative surgical planning: a review

The aim of this paper is to review the recent evolution of additive manufacturing (AM) within the medical field of preoperative surgical planning. The discussion begins with an overview of the different techniques, pointing out their advantages and disadvantages as well as an in-depth comparison of different characteristics of the printed parts. Then, the state-of-the-art with respect to preoperative surgical planning is presented. On the one hand, different surgical planning prototypes manufactured by several AM technologies are described. On the other hand, materials used for mimicking different living tissues are explored by focusing on the material properties: elastic modulus, hardness, etc. As a result, doctors can practice before performing surgery and thereby reduce the time needed for the operation. The subject of patient education is also introduced. A thorough review of the process that is required to obtain 3D printed surgical planning prototypes, which is based on different stages, is then carried out. Finally, the ethical issues associated with 3D printing in medicine are discussed, along with its future perspectives. Overall, this is important for improving the outcome of the surgery, since doctors will be able to visualize the affected organs and even to practice surgery before performing it.Postprint (author's final draft

Online Semantic Labeling of Deformable Tissues for Medical Applications

University of Minnesota Ph.D. dissertation. May 2017. Major: Mechanical Engineering. Advisor: Timothy Kowalewski. 1 computer file (PDF); ix, 133 pages.Surgery remains dangerous, and accurate knowledge of what is presented to the surgeon can be of great importance. One technique to automate this problem is non-rigid tracking of time-of-flight camera scans. This requires accurate sensors and prior information as well as an accurate non-rigid tracking algorithm. This thesis presents an evaluation of four algorithms for tracking and semantic labeling of deformable tissues for medical applications, as well as additional studies on a stretchable flexible smart skin and dynamic 3D bioprinting. The algorithms were developed and tested for this study, and were evaluated in terms of speed and accuracy. The algorithms tested were affine iterative closest point, nested iterative closest point, affine fast point feature histograms, and nested fast point feature histograms. The algorithms were tested against simulated data as well as direct scans. The nested iterative closest point algorithm provided the best balance of speed and accuracy while providing semantic labeling in both simulation as well as using directly scanned data. This shows that fast point feature histograms are not suitable for nonrigid tracking of geometric feature poor human tissues. Secondary experiments were also performed to show that the graphics processing unit provides enough speed to perform iterative closest point algorithms in real-time and that time of flight depth sensing works through an endoscope. Additional research was conducted on related topics, leading to the development of a novel stretchable flexible smart skin sensor and an active 3D bioprinting system for moving human anatomy

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction.

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering

Application of Collagen I and IV in Bioengineering Transparent Ocular Tissues.

Collagens represent a major group of structural proteins expressed in different tissues and display distinct and variable properties. Whilst collagens are non-transparent in the skin, they confer transparency in the cornea and crystalline lens of the eye. There are 28 types of collagen that all share a common triple helix structure yet differ in the composition of their α-chains leading to their different properties. The different organization of collagen fibers also contributes to the variable tissue morphology. The important ability of collagen to form different tissues has led to the exploration and application of collagen as a biomaterial. Collagen type I (Col-I) and collagen type IV (Col-IV) are the two primary collagens found in corneal and lens tissues. Both collagens provide structure and transparency, essential for a clear vision. This review explores the application of these two collagen types as novel biomaterials in bioengineering unique tissue that could be used to treat a variety of ocular diseases leading to blindness

Towards an in vitro innervated model of the cornea

Visual impairment due to corneal disease is a global health concern with few FDA-approved pharmaceuticals being developed. In the cornea, the interactions between the various cell types present are essential for its functioning. In particular, innervation through sensory nerves is crucial for optimal functioning of this tissue. However, the mechanisms underlying these interactions are poorly understood, and representative 3D innervated in vitro cornea models could be used as systems to model the native situation. Therefore, an innervated model of the cornea is proposed and initiated. Electrocompacted collagen constructs serve as a basis for mimicking the cornea, and its mechanical, optical, and degradative properties are shown to be favorable. Furthermore, three dimensional extrusion- based printing has been employed to print methacrylated gelatin, and this scaffold was shown to support neuronal cell survival (83.4% viability 1 day after printing). A sustained release of neural growth factor to induce differentiation was established through incorporation of growth- factor loaded microparticles within the electrocompacted collagen. Additionally, the bioactivity was confirmed through an in vitro PC12 cell assay. The two biomaterials have been interfaced to fabricate a model to guide neuronal innervation. The current model shows potential in mimicking the complex structure of the cornea, but some optimization is required for neurite outgrowth. In the future, a viable in vitro corneal model could be used to provide fundamental insight into the process of corneal innervation and corneal diseases, as well as pre-clinical toxicity testing of new ocular drugs

3D human skin bioprinting: a view from the bio side

Based on the 3D printing technologies and the concepts developed in tissue engineering during the last decades, 3D bioprinting is emerging as the most innovative and promising technology for the generation of human tissues and organs. In the case of skin bioprinting, thanks to the research process carried out during the last years, interfollicular skin has been printed with a structural and functional quality that paves the way for clinical and industrial applications. This review analyzes the present achievements and the future improvements that this area must bring about if bioprinted skin is to become widely used. We have made an effort to integrate the technological and the biological/biomedical sides of the subject.We thank the Spanish Fundación Ramón Areces for its continuous support. This work was partially supported by grant DPI2014-61887-EXP from the Spanish Ministerio de Economía y Competitividad

Cartilage Tissue Engineering For Rhinoplasty

Nasal surgery (rhinoplasty) has evolved considerably since its origins in Egypt around 1600BCE, yet modern reconstruction still relies on grafts harvested from autologous rib cartilage. Rib cartilage is an excellent graft material, but chest donor site morbidity can be a significant problem. The aim of this thesis was to create a patient specific cartilage surgical product using autologous stem cells that would provide surgeons with an effective alternative to rib cartilage. Adipose-derived stem cells (ADSCs) and cartilage-derived stem/precursor cells (CSPCs) were used in this thesis as they can be harvested through minimally invasive procedures and their chondrogenic potential already widely established. Using a novel tissue clearing protocol for whole mount imaging, primary experiments confirmed the ability of both cell types to self-organize and generate cartilage-like extracellular matrix (ECM) in 3D spheroids. Three different methods of engineering cartilage in 3D were investigated. Firstly, a clinically approved collagen matrix was used as a scaffold and seeded with cells. Immunocytochemistry and histological staining demonstrated cartilage like ECM on the scaffold surface in preference to deeper regions. The collagen matrix proved too be tight and constrictive on cell expansion. Secondly, a 3D bioprinter was used to print cells mixed with cellulose/alginate “bioink” hydrogels. This bioink failed to demonstrate cartilage like ECM in static culture and in a chick embryo chorioallantoic membrane (CAM) model. Lastly, a cell laden fibrin hydrogel was “sandwiched” between 2 layers of polycaprolactone (PCL) sheets to provide mechanical support and grafted onto CAM. Histological analysis of cell laden fibrin confirmed regions of chondrogenesis by positive staining of collagen and glycosaminoglycans. In conclusion, the results provide further understanding of how these cells respond to different 3D environments and the effect on chondrogenesis. Combining 3D bioprinting with a sandwich design may be an effective future approach to product development

Development of a Tissue Engineered Cardiac Patch

Cardiovascular Disease(CVD) is the leading cause of mortality in the developed world. CVD is most commonly manifested as atherosclerosis of the coronary arteries leading to Myocardial Infarction(MI). After MI, fibrosis of the ventricular wall leads to heart failure(HF), a pandemic affecting 26 million people globally. While therapies are continuously developed to combat HF, the treatment of choice, whole heart transplant, is limited by the availability of donor hearts. It is clear that there is a need to develop a long-term solution to combat HF and its enormous economic burden. Tissue Engineering and Regenerative Medicine holds promise as a possible solution through the development of cardiac grafts capable of returning the function lost from MI. Current efforts in the development of cardiac tissues have been plagued by the inability to generate tissues of sufficient thickness(\u3e100μm) or contractility of mature tissue, resulting in low engraftment rates or lacking efficacy. This leads to the focus of this research being the generation of a thick vascularized cardiac patch capable of long-term survival. First, we decelled whole porcine hearts for the production of ventricular flap and myocardial scaffolds. Scaffolds showed no evidence of retained cellular content or DNA, but retained key characteristics of the extracellular matrix and maintained the same mechanical properties as native tissue. We then performed a combined cell seeding to reendothelialize the vasculature of the ventricle wall and repopulate the myocardium. Finally, we developed a custom perfusion electromechanical bioreactor for the purpose of conditioning and maintaining the viability of our patch long term. We expect that this research will result in the development of cardiac grafts with long-term implications for therapy to solve MI related HF