Bioscaffold Valve with and without Mechanically Conditioned Stem Cells for the Treatment of Critical Mitral Valve Diseases in the Young

Congenital heart disease, which includes heart valve defects, are the most common type of birth abnormality in the US. Infants with critical congenital valve disease have no established treatment-measure other than compassionate care options, owing to an absence of prosthetic valves in small sizes and their inability to support somatic growth. A regenerable valve would be appealing since these barriers could be overcome; it can potentially provide for growth, self-repair, infection resistance and be a permanent approach for replacing defective heart valves. Porcine small intestinal submucosa (PSIS) bioscaffold was used to create valvular constructs with the possibility to grow overtime. PSIS bio-scaffolds consisting of two different yarn-twist configurations (2ply and 4ply) were assessed for mechanical properties to determine which scaffold would withstand fatigue loading in a similar manner to the native heart valves. It was found that fatigued 2ply PSIS exhibited higher yield stress (p Next, a pilot study was investigated for implanting 2ply hand-made PSIS mitral valves into juvenile baboons (n=3) to assess their functionality and somatic growth longitudinally. Bioscaffold mitral heart valve function was assessed via echocardiography, while somatic growth was evaluated with a novel parameter, normalized aspect growth ratio (NAGR), where ideal growth is 1, and via histological analysis after the valves were explanted. Our results showed trivial to mild regurgitation up to 17-months post-implantation demonstrating proper functionality of the PSIS mitral valves. The NAGR was found to be roughly 1 within the first 2-4 months, showing ideal growth. The PSIS mitral valve explants were found to develop extracellular matrix (ECM) proteins of collagen, elastin, proteoglycans and fibrin at all explant time points (3-, 11- and 20- months). Overall, the PSIS mitral valves functioned well and regenerated the proper ECM components over their implantation durations. However, sudden valve failure (at 3-, 11- and 20-months post-bioscaffold mitral valve implantation) occurred in all 3 subjects. As a possible means to circumvent valve failure, PSIS tubular mitral bioscaffold valves were subsequently seeded in vitro with bone marrow stem cells and exposed to fluid-induced shear stress patterns in a perfusion bioreactor. The cells secreted a thin layer of ECM, which potentially could help mitigate chronic inflammatory responses, an underlying reason for the valve failure that was observed with the raw PSIS bioscaffolds. It was found that our flow-conditioned valve could produce ECM proteins significantly higher (pde novo ECM that was secreted and the valvular phenotype that resulted from the flow-based mechanical conditioning of allogeneic stem cells-seeded, bioscaffold mitral valves have the potential to accelerate in vivo valve tissue formation. We thus expect these flow-conditioned valves to have longer-term function post-implantation compared to what was possible with the bioscaffold valves-alone

The manufacture of bioscaffolds by printing.

Printing technology is mainly used for graphic arts and packaging applications, but also is a potential technology for the micro manufacture of electronic devices, biosensors and tissue engineering scaffolds. The main goal of this research is to print fine lines of biopolymer by means of volume printing processes. To assess the feasibility of printing fine features for bioscaffolds using conventional printing technology, an experimental investigation into the rheological behaviour of biopolymer inks was conducted. This is because the rheological characteristics of the biopolymers have a significant influence on the performance of different printing processes. The biopolymers undergo significant phase transition which affects the printed products. The rheological tests focus on ink viscosity, viscoelastic and gelation properties. Gelatine appeared to be more favourable than collagen for scaffolds fabrication by printing technologies. Thus this study was mainly focused on aqueous gelatine solution as printing ink. All inks display shear-thinning and thixotropic behaviours which are important for good printing. The temperature ramp and multi-frequency sweep tests yield information on gelation temperature and gel point. The rate of ordering and gel formation of biopolymer inks was found to be strongly concentration-, temperature- and time-dependent. An increase in gelatine concentrations caused a reduction in the dynamic surface tension of the inks. Printing conditions that are compatible with printing biopolymer inks were being optimised in terms of operation temperature interval to accommodate phase transition. Three printing processes inkjet printing, flexography and screen printing were evaluated for printing fine features of biopolymers. Surfactants were used to lower the ink surface tension to below 30 mN/m, so that the ink could be jetted onto substrate. These printing methods are aimed to produce cost effective bioscaffolds in mass production. The quality of printed lines was examined in terms of width and film thickness to establish the best printing method for gelatine printing. Fine lines printed by inkjet and flexographic printing processes were mostly broken. Screen printing process looks more promising because the printed gelatine fine lines showed the best quality. The line width and line film thickness measured were more consistent and closer to the desired dimensions. A laboratory screen printing trial was conducted using Lie orthogonal array technique to investigate the effect of process parameters on the reproduction of the screen printed fine gelatine lines in terms of line width and film thickness. Six parameters studied were ink type, mesh type, squeegee hardness, snap-off gap, squeegee speed and squeegee pressure. The most significant parameter was the ink type, followed by snap-off gap and mesh type. The effect of squeegee parameters (squeegee type, speed and pressure) was considered insignificant. The orientation has an effect on line width but insignificant effect on line film thickness. Most process parameters had interactions with one another which complicated the optimisation of the process parameters

Adipose-Derived Stromal/Stem Cells

Adipose tissue is a rich, ubiquitous, and easily accessible source for multipotent mesenchymal stromal/stem cells (MSCs), so-called adipose-derived stromal/stem cells (ASCs). Primary isolated ASCs are a heterogeneous preparation consisting of several subpopulations of stromal/stem and precursor cells. Donor-specific differences in ASC isolations and the lack of culture standardization hinder the comparison of results from different studies. Nevertheless, ASCs are already being used in different in vivo models and clinical trials to investigate their ability to improve tissue and organ regeneration. Many questions concerning their counterparts and biology in situ, their differentiation potential in vitro and in vivo, and the mechanisms of regeneration (paracrine effects, including regeneration-promoting factors and extracellular vesicles, differentiation, and immunomodulation) are not completely understood or remain unanswered. This Special Issue covers research articles investigating various adipose tissues as a source for ASC isolation, specific cultures methods to enhance proliferation or viability, and the differentiation capacity. Furthermore, other studies highlight aspects of various diseases, the immunosuppressive potential of ASCs and their derivates, or the in vivo tracking of transplanted ASCs. This edition is complemented by a review that summarizes the current knowledge of spheroid culture system methods and applications for mesenchymal stem cells

Toward personalized medicine: human adipose-derived stem cells (ASCs) and xenogenic-free media as enabling tools in tendon tissue engineering strategies.

L'abstract è presente nell'allegato / the abstract is in the attachmen

Implantable medical devices for drug and cell release

This work is focused on the research on how to leverage 3D printing technology in the field of cell transplantation. More specifically, the study of an artificial organ for hormone replacement therapies thanks to the close collaboration between the Methodist Hospital Research Institute, Houston, Texas and Politecnico di Torino, Turin, Italy. Cell transplantation offers an attractive therapeutic approach for many endocrine deficiencies. Transplanted endocrine cells or engineered cells encapsulated in the here presented 3D printed device, can act as biological sensors detecting changes in hormonal levels and secrete molecules in response to maintain homeostasis. The major advantage of this technology is that patients affected by endocrine disorder could potentially avoid the need of frequent hormone injections, such as insulin or testosterone, resulting in an improved quality of life and lower chronic side effects associated to external hormone supplementations. This implant was extensively tested both in vitro and in vivo condition, providing remarkable results that lead to several publications. The cell encapsulation system was fabricated via 3D printing technology adopting an FDA approved polymeric material. The structure, composed by an array of micro and macro channels, was specifically designed in order to allow vasculature formation within the device and for housing cells while avoiding cell clustering. Over the course of the Ph.D., the technology was designed, fabricated and tested for the encapsulation of several cell lines and for small and large animal models. According to the in vivo results, we demonstrated that our 3D printed device exemplifies a clinically translatable strategy for preserving viability and function of transplanted cells. Currently, is ongoing an experiment in Non-Human Primates (data not shown), last pre- clinical study before the possibility to move to the clinical development in humans. The pre-vascularization approach to achieve an ideal intra-device milieu prior to transplantation, transcutaneous cell loading and refilling capabilities, as well as the potential for rapid device retrievability, addresses current challenges in transplantation. This technology may offer exciting potential for clinical adoption in relevant medical areas of diabetes, hypogonadism, hypothyroidism, cancer, and neurological diseases among others

Development of a Tissue Engineered Mitral Valve

Heart valve diseases affect nearly 8 million people every year in the United States. Of these patients, 72% are affected by mitral valve diseases. Stenosis, regurgitation, and prolapse of the mitral valve are the primary pathologies affecting valve function resulting in atrial fibrillation, arterial thromboembolism, pulmonary edema, pulmonary hypertension, cardiac hypertrophy and heart failure. Surgical options to repair or replace the mitral valve are only palliative, especially for children with congenital defects, and do not exclude the need for reoperation. A tissue-engineered option is feasible and holds great potential through the combination of decellularized scaffolds, patient stem cells, and heart valve bioreactors. Development of living tissue engineered mitral valves have not been reported in the recent literature. The primary focus of my research was threefold: 1) develop an acellular ECM scaffold which is mechanically robust, and allows for sufficient bioactivity for cellular seeding and signaling by use of a non-toxic matrix-binding polyphenolic antioxidant, pentagalloyl glucose (PGG); 2) confirm this scaffold to be biologically compatible with future hosts and limiting inflammatory responses in vivo by virtue of PGG\u27s antioxidant properties; 3) achieve recellularization of the mitral valve scaffold and direct differentiation and maturation through bioreactor preconditioning. First, a complete decellularization of porcine mitral valves was established and optimized to remove all cellular and nuclear material from the scaffolds while still preserving ECM components and basal lamina proteins. Treatment with PGG recovered lost mechanical integrity due to the decellularization process. Seeded cells were able to grow and proliferate on and in the acellular scaffold confirming cytocompatibility. An in vivo rat study was conducted to evaluate the scaffolds\u27 biocompatibility. In comparing non-treated and PGG-treated groups, PGG –treatment regularly and significantly showed increased resistance to degradation, polarization of macrophages to the pro-healing M2 phenotype, discouragement of inflammatory markers, and no limitations towards cell infiltration. Lastly, PGG-treated acellular scaffolds were recellularized with pre-differentiated fibroblasts and endothelial cells and placed in a newly developed mitral valve bioreactor. Design of the bioreactor required a full understanding and appreciation for the four tissue types present in the mitral apparatus. Preconditioning of the seeded constructs yielded a mitral construct similar to a native valve. The overarching goal of this research was to develop a stable mitral valve construct. It is expected that the progress made by this project will have a positive impact on those that suffer from mitral valve pathologies. Our translatable approach towards this tissue engineered mitral valve should allow clinicians to readily adopt this regenerative replacement and contribute as a whole to the field of cardiovascular tissue engineering

Coalescence of ECM and chitosan biomaterials for an advanced sutureless technology

The ideal wound closure device should restore the original integrity of the tissue, offer easy application and seamless, fluid-tight seal. Commonly used wound closure devices including sutures, staples and tissue adhesives do not offer effective sealing of the wound and possess a range of associated disadvantages including dehiscence, infection, toxicity and iatrogenic related trauma. Laser tissue welding has been proposed as an alternative and is becoming increasingly popular. This technique uses laser irradiation to initiate chemical reactions in a target material, thereby activating chemical components within the material to form an immediate seal. However, laser tissue welding is reliant upon temperatures exceeding the collagen denaturation point and is associated with some tissue damage. SurgiLux is a chitosan-based thin film surgical adhesive that relies upon laser irradiation to increase the strength of chitosan binding to tissue without thermal damage to tissue and avoiding many of the disadvantages of current wound closure devices. Previous in vitro and in vivo studies of SurgiLux has demonstrated the potential of SurgiLux in sutureless repair of peripheral nerves. Recent approaches to regenerative medicine and tissue engineering involve the use of decellularised extracellular matrix as biological scaffolds to augment the formation of new functional tissue and facilitate successful tissue reconstruction. The aim of the work reported here was to combine SurgiLux with an extracellular matrix scaffold derived from porcine urinary bladder matrix to potentially improve the capacity of the SurgiLux technology to enhance wound healing by promoting functional tissue regeneration, for potential applications in peripheral nerve repair. The bio-scaffold was incorporated into the SurgiLux film in a variety of ways; bio-scaffold embedded into SurgiLux had a greater tensile strength (32.4 ± 5.2 MPa), crystallinity (12.1 ± 1.3 %) and hydrophilicity (75.0 ± 2.0)° than the chitosan adhesive alone (8.5 ± 3.1 MPa, 10.7 ± 1.2% crystallinity, Ө = 98.1 ± 2.03°). Tissue adhesion strengths using these hybrid biomaterials were maintained at ~15 kPa compared to 3 kPa for fibrin glue. Furthermore, histological analysis demonstrated that laser irradiation of the UBM-SurgiLux adhesive caused no thermal damage to tissue. In vitro biocompatibility of the composite films was assessed by examining their influence on the proliferation and health of olfactory ensheathing cells and human monocyte-derived macrophages. Incorporation of the bio-scaffold into the SurgiLux films increased the attachment and proliferation of olfactory ensheathing cells and decreased the cytotoxicity of the films. Similarly, while chitosan films induced a cell population to undergo early apoptotic activation, the composite films apparently increased biocompatibility, preventing the cells from undergoing necrosis. Similarly, while SurgiLux showed a significantly reduced macrophage response compared to chitosan film, introduction of the bio-scaffold into the SurgiLux reduced their response further. A quantitative real time PCR approach was undertaken to identify polarised macrophage phenotype, M1 (pro-inflammatory) and M2 (anti-inflammatory) through the detection of specific cytokines expressed by the macrophages. Reduced cell spread (6.2e3 ± 7.0e2 μm2) and lack of foreign body giant cell formation lead to significantly reduced expression of M1 markers, IL-23p19, IL-12p40 and IL-12p35; thereby suggesting the presence of an alternative anti-inflammatory, tissue remodelling pathway. A protein expression profile of the UBM scaffold was generated to identify novel proteins within the UBM via an advanced mass spectrometry methodology. A total of 129 proteins were identified with the majority of these revealing a role in maintaining cell structure (19%) and adhesion (13%), while the smallest groups (1 %) had remodelling and stimulatory roles. A number of growth promoting proteins including galectins 1 and 7, obscurin, fibulin and have been identified that may have enhanced cellular proliferation on the UBM-SurgiLux composite scaffolds compared to chitosan films alone. UBM also contains proteins that have neurotrophic, anti-angiogenic, tumour suppressor activity and proteins known to promote tissue remodelling and morphogenesis. Therefore, coalescence of the bio-scaffold within SurgiLux matrix resulted in a surgical adhesive with enhanced biocompatibility and reduced cytotoxicity compared to chitosan films. The results suggest that the unique combination of extracellular matrix bio-scaffold with SurgiLux technology has the potential to promote functional tissue regeneration leading to enhanced sutureless nerve repair