CONTROLLED DELIVERY SYSTEMS FOR NEURONAL TISSUE ENGINEERING

Complete transection of peripheral nerves can result from trauma, tumor removal, infection, or as adverse consequences of various surgeries. Current commercially available nerve guides cannot repair large nerve defects because these guides are engineered to provide mechanical support for the developing axon and do not actively promote axonal growth. For large nerve gaps, targeting axonal growth is particularly important because the length of the nerve that must be regrown is the distance from the lesion to the innervated muscle. Therefore, there is enormous clinical potential for a nerve guide capable of improving axonal outgrowth across large nerve defects. Our underlying hypothesis is that delivery of Glial Cell Line-Derived Neurotrophic Factor (GDNF) from a nerve guide will improve peripheral nerve regeneration across large defects. To test this hypothesis, biodegradable poly(caprolactone) (PCL) nerve guides were prepared with manufacturing parameters optimized for protein delivery and retention at the injury site. Quantitative changes in the diffusion of small molecular weight proteins and glucose through PCL conduit walls were measured to determine the independent and combinatorial effects of three fabrication variables: wall thickness, pore size and porosity percentage. Double-walled microspheres were then fabricated as a method of sustained protein delivery, and were incorporated within the luminal wall of PCL nerve guides using a novel solvent specific embedding technique. The overall efficacy of our nerve guide design was confirmed by encapsulating and delivering GDNF in the rat sciatic nerve injury model. Evaluation of sensory reinnervation following a long gap, 1.5cm nerve injury at 16 weeks showed a significant increase in animal response time to stimuli from animals treated with GDNF as opposed to negative control PCL guides. Furthermore, the measured gastrocnemius contraction force in animals treated with GDNF was significantly higher than negative controls and was not significantly different from the isograft positive control group. Histological assessment of explanted conduits after 16 weeks showed improved tissue integration within GDNF releasing nerve guides compared to negative controls. Nerve fibers were present across the entire length of GDNF releasing guides, while nerve fibers were not detectable beyond the middle region of negative control guides. Therefore, the results reported within this dissertation support our original hypothesis that; the long-term delivery of a neurotrophic factor from nerve guides results in improved functional recovery above negative controls following large axonal defects in the peripheral nervous system

Engineering a 3D Vascularized Adipose Tissue Construct Using a Decellularized Lung Matrix

Critically sized defects in subcutaneous white adipose tissue result in extensive disfigurement and dysfunction and remain a reconstructive challenge for surgeons; as larger defect sizes are correlated with higher rates of complications and failure due to insufficient vascularization following implantation. Our study demonstrates, for the first time, a method to engineer perfusable, pre-vascularized, high-density adipose grafts that combine patient-derived adipose cells with a decellularized lung matrix (DLM). The lung is one of the most vascularized organs with high flow, low resistance, and a large blood–alveolar interface separated by a thin basement membrane. For our work, the large volume capacity within the alveolar compartment was repurposed for high-density adipose cell filling, while the acellular vascular bed provided efficient graft perfusion throughout. Both adipocytes and hASCs were successfully delivered and remained in the alveolar space even after weeks of culture. While adipose-derived cells maintained their morphology and functionality in both static and perfusion DLM cultures, perfusion culture offered enhanced outcomes over static culture. Furthermore, we demonstrate that endothelial cells seamlessly integrate into the acellular vascular tree of the DLM with adipocytes. These results support that the DLM is a unique platform for creating vascularized adipose tissue grafts for large defect filling

Evaluation of Porcine Versus Human Mesenchymal Stromal Cells From Three Distinct Donor Locations for Cytotherapy

Background: Mesenchymal stromal cell (MSC)-based cytotherapies fuel the hope for reduction of chronic systemic immunosuppression in allotransplantation, and our group has previously shown this capability for both swine and human cells. MSCs harvested from distinct anatomical locations may have different behavior and lead to different outcomes in both preclinical research and human trials. To provide an effective reference for cell therapy studies, we compared human and porcine MSCs from omental fat (O-ASC), subcutaneous fat (SC-ASC) and bone marrow (BM-MSC) under rapid culture expansion with endothelial growth medium (EGM). Methods: MSCs isolated from pigs and deceased human organ donors were compared for yield, viability, cell size, population doubling times (PDT), surface marker expression and differentiation potential after rapid expansion with EGM. Immunosuppressant toxicity on MSCs was investigated in vitro for four different standard immunosuppressive drugs. Immunomodulatory function was compared in mixed lymphocyte reaction assays (MLR) with/without immunosuppressive drug influence. Results: Human and porcine omental fat yielded significantly higher cell numbers than subcutaneous fat. Initial PDT was significantly shorter in ASCs than BM-MSCs and similar thereafter. Viability was reduced in BM-MSCs. Porcine MSCs were positive for CD29, CD44, CD90, while human MSCs expressed CD73, CD90 and CD105. All demonstrated confirmed adipogenic differentiation capacity. Cell sizes were comparable between groups and were slightly larger in human cells. Rapamycin revealed slight, mycophenolic acid strong and significant dose-dependent toxicity on viability/proliferation of almost all MSCs at therapeutic concentrations. No relevant toxicity was found for Tacrolimus and Cyclosporin A. Immunomodulatory function was dose-dependent and similar between groups. Immunosuppressants had no significant adverse effect on MSC immunomodulatory function. Discussion: MSCs from different harvest locations and donor species differ in terms of isolation yields, viability, PDT, and size. We did not detect relevant differences in immunomodulatory function with or without the presence of immunosuppressants. Human and pig O-ASC, SC-ASC and BM-MSC share similar immunomodulatory function in vitro and warrant confirmation in large animal studies. These findings should be considered in preclinical and clinical MSC applications

A review of adipocyte lineage cells and dermal papilla cells in hair follicle regeneration

Alopecia is an exceedingly prevalent problem effecting men and women of all ages. The standard of care for alopecia involves either transplanting existing hair follicles to bald areas or attempting to stimulate existing follicles with topical and/or oral medication. Yet, these treatment options are fraught with problems of cost, side effects, and, most importantly, inadequate long-term hair coverage. Innovative cell-based therapies have focused on the dermal papilla cell as a way to grow new hair in previously bald areas. However, despite this attention, many obstacles exist, including retention of dermal papilla inducing ability and maintenance of dermal papilla productivity after several passages of culture. The use of adipocyte lineage cells, including adipose-derived stem cells, has shown promise as a cell-based solution to regulate hair regeneration and may help in maintaining or increasing dermal papilla cells inducing hair ability. In this review, we highlight recent advances in the understanding of the cellular contribution and regulation of dermal papilla cells and summarize adipocyte lineage cells in hair regeneration

Comparison of adipose particle size on autologous fat graft retention in a rodent model

Aim: Unpredictable retention outcomes remain a significant issue in autologous fat grafting procedures. Liposuction cannula variation leads to variability in fat particle size. Recent data suggest that the size of fat particles is closely related to graft healing outcomes; however, this remains a point of contention due to potential confounding variables such as tissue trauma with harvest. The aim of this study was to compare autologous fat grafting outcomes with variable fat particle sizes in an animal model which isolated fat particle size as the primary experimental variable. The overall goal of this work was to determine if reducing fat particle size is an effective method for enhancing graft retention in autologous fat grafting.Methods: The range of fat particle diameter harvested by four common liposuction cannulas was quantified to define relevant small and large particle target diameters. To determine if particle size impacted nutrient and oxygen permeability, small and large particles were incubated in vitro in a spinner flask with an abundance of culture media and vascular endothelial growth factor secretion was measured with enzyme-linked immunosorbent assay. Finally, small and large fat grafts were prepared from subcutaneous mouse fat pads and grafted in syngeneic Balb/CJ mice. Weight and volume retention were evaluated at 1, 4, 8, and 12 weeks. Histological analysis with Masson’s trichrome and perilipin immunofluorescent staining was performed. Real-time quantitative polymerase chain reaction was performed for adipogenic, inflammatory and apoptotic genes.Results: The range of fat particle diameters harvested with four commonly used cannulas was 2-7 mm. In vitro studies showed that 5-7-mm particles had significantly increased VEGF secretion normalized to weight, indicating increased tissue hypoxia in these particles compared to 2-4-mm particles. Surprisingly, in vivo comparison in two unique studies showed 2-4-mm and 5-7-mm fat particles had comparable graft retention (P = 0.5329). Masson’s trichrome staining revealed increased extracellular matrix and fibrosis in the 5-7-mm particle group (P = 0.0115). Adipocyte survival with perilipin demonstrated comparable viability. Gene expression showed large particles experienced increased inflammation and apoptosis at one week after grafting, but overall there were no significant differences between groups.Conclusion: The ideal fat particle size should be large enough to contain adequate mesenchyme while not so thick as to preclude imbibition. This study suggests that, despite changes in hypoxia and VEGF levels, differing fat particles (2-4-mm and 5-7-mm) can achieve similar graft retention