Evaluation of fibroin-based scaffolds for ocular tissue reconstruction

The epithelium of the corneolimbus contains stem cells for regenerating the corneal epithelium. Diseases and injuries affecting the limbus can lead to a condition known as limbal stem cell deficiency (LSCD), which results in loss of the corneal epithelium, and subsequent chronic inflammation and scarring of the ocular surface. Advances in the treatment of LSCD have been achieved through use of cultured human limbal epithelial (HLE) grafts to restore epithelial stem cells of the ocular surface. These epithelial grafts are usually produced by the ex vivo expansion of HLE cells on human donor amniotic membrane (AM), but this is not without limitations. Although AM is the most widely accepted substratum for HLE transplantation, donor variation, risk of disease transfer, and rising costs have led to the search for alternative biomaterials to improve the surgical outcome of LSCD. Recent studies have demonstrated that Bombyx mori silk fibroin (hereafter referred to as fibroin) membranes support the growth of primary HLE cells, and thus this thesis aims to explore the possibility of using fibroin as a biomaterial for ocular surface reconstruction. Optimistically, the grafted sheets of cultured epithelium would provide a replenishing source of epithelial progenitor cells for maintaining the corneal epithelium, however, the HLE cells lose their progenitor cell characteristics once removed from their niche. More severe ocular surface injuries, which result in stromal scarring, damage the epithelial stem cell niche, which subsequently leads to poor corneal re-epithelialisation post-grafting. An ideal solution to repairing the corneal limbus would therefore be to grow and transplant HLE cells on a biomaterial that also provides a means for replacing underlying stromal cells required to better simulate the normal stem cell niche. The recent discovery of limbal mesenchymal stromal cells (L-MSC) provides a possibility for stromal repair and regeneration, and therefore, this thesis presents the use of fibroin as a possible biomaterial to support a three dimensional tissue engineered corneolimbus with both an HLE and underlying L-MSC layer. Investigation into optimal scaffold design is necessary, including adequate separation of epithelial and stromal layers, as well as direct cell-cell contact. Firstly, the attachment, morphology and phenotype of HLE cells grown on fibroin were directly compared to that observed on donor AM, the current clinical standard substrate for HLE transplantation. The production, transparency, and permeability of fibroin membranes were also evaluated in this part of the study. Results revealed that fibroin membranes could be routinely produced using a custom-made film casting table and were found to be transparent and permeable. Attachment of HLE cells to fibroin after 4 hours in serum-free medium was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. While HLE cultured on AM displayed superior stratification, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (cytokeratin pair 3/12 expression; CK3/12) and displayed a comparable number and distribution of ÄNp63+ progenitor cells to that seen in cultures grown on AM. These results confirm the suitability of membranes constructed from silk fibroin as a possible substrate for HLE cultivation. One of the most important aspects in corneolimbal tissue engineering is to consider the reconstruction of the limbal stem cell niche to help form the natural limbus in situ. MSC with similar properties to bone marrow derived-MSC (BM-MSC) have recently been grown from the limbus of the human cornea. This thesis evaluated methods for culturing L-MSC and limbal keratocytes using various serum-free media. The phenotype of resulting cultures was examined using photography, flow cytometry for CD34 (keratocyte marker), CD45 (bone marrow-derived cell marker), CD73, CD90, CD105 (collectively MSC markers), CD141 (epithelial/vascular endothelial marker), and CD271 (neuronal marker), immunocytochemistry (alpha-smooth muscle actin; á-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis), and co-culture experiments with HLE cells. While all techniques supported to varying degrees establishment of keratocyte and L-MSC cultures, sustained growth and serial propagation was only achieved in serum-supplemented medium or the MesenCult-XF„¥ culture system (Stem Cell Technologies). Cultures established in MesenCult-XF„¥ grew faster than those grown in serum-supplemented medium and retained a more optimal MSC phenotype. L-MSC cultivated in MesenCult-XFR were also positive for CD141, rarely expressed £\-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of L-MSC established in MesenCult-XF„¥ medium. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker £GNp63, along with the corneal differentiation marker CK3/12. Our findings conclude that MesenCult-XFR is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells. Following on from the findings of the previous two parts, silk fibroin was tested as a novel dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. In this section, the growth and phenotype of HLE cells on non-porous versus porous fibroin membranes was compared. Furthermore, the growth of L-MSC in either serum-supplemented medium or the MesenCult-XFR culture system within fibroin fibrous mats was investigated. Lastly, the co-culture of HLE and L-MSC in serum-supplemented medium on and within fibroin dual-layer constructs was also examined. HLE on porous membranes displayed a flattened and squamous monolayer; in contrast, HLE on non-porous fibroin appeared cuboidal and stratified closer in appearance to a normal corneal epithelium. Both constructs maintained CK3/12 expression and distribution of £GNp63+ progenitor cells. Dual-layer fibroin scaffolds consisting of HLE cells and L-MSC maintained a similar phenotype as on the single layers alone. Overall, the present study proposed to create a three dimensional limbal tissue substitute of HLE cells and L-MSC together, ultimately for safe and beneficial transplantation back into the human eye. The results show that HLE and L-MSC can be cultivated separately and together whilst maintaining a clinically feasible phenotype containing a majority of progenitor cells. In addition, L-MSC were able to be cultivated routinely in the MesenCult-XF® culture system while maintaining a high purity for the MSC characteristic phenotype. However, as a serum-free culture medium was not found to sustain growth of both HLE and L-MSC, the combination scaffold was created in serum-supplemented medium, indicating that further refinement of this cultured limbal scaffold is required. This thesis has also demonstrated a potential novel marker for L-MSC, and has generated knowledge which may impact on the understanding of stromal-epithelial interactions. These results support the feasibility of a dual-layer tissue engineered corneolimbus constructed from silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration. Further refinement of this technology should explore the potential benefits of using epithelial-stromal co-cultures with MesenCult-XF® derived L-MSC. Subsequent investigations into the effects of long-term culture on the phenotype and behaviour of the cells in the dual-layer scaffolds are also required. While this project demonstrated the feasibility in vitro for the production of a dual-layer tissue engineered corneolimbus, further studies are required to test the efficacy of the limbal scaffold in vivo. Future in vivo studies are essential to fully understand the integration and degradation of silk fibroin biomaterials in the cornea over time. Subsequent experiments should also investigate the use of both AM and silk fibroin with epithelial and stromal cell co-cultures in an animal model of LSCD. The outcomes of this project have provided a foundation for research into corneolimbal reconstruction using biomaterials and offer a stepping stone for future studies into corneolimbal tissue engineering.

Exploring the information practices of people with end stage kidney disease

This small exploratory study sought to understand how people with End Stage Kidney Disease (ESKD) experience the information environment and what information practices they employ in order to inform the decisions they make in relation to treatment and care. Using a constructivist methodology, in-depth interviews were conducted with five people who were receiving haemodialysis in two small satellite dialysis units located in regional and rural communities in New South Wales, Australia. Thematic analysis revealed two types of patients. The first type appears to adopt a received view of information, who do not question their condition; and passively accept information. In the other type, patients were found to be engaged; they actively identified their information needs and quickly learned what that they needed to ask and who to ask. Knowing the information practices of people with ESKD is useful for nephrology nurses when providing patient education.

What information-related activities do people with ESKD use?

Background Information practice is an emerging area of research that seeks to reveal how people learn to connect with the complex multimodal information landscapes that informs their ability to make decisions. Previous research has identified that people with end stage kidney disease (ESKD) tend to adopt a ‘received’ or ‘engaged’ view of information but little is known about the activities of information practice. Objectives This research project sought to identify the: i) information-related activities; and ii) how information is used. Methods Using a constructivist qualitative methodology, ten people with ESKD living in a large metropolitan city were purposively selected and interviewed. Data was subject to thematic analysis by researchers from nursing and information science. Saturation of themes was achieved. Results Participants were between 38 and 72 years, had been receiving kidney replacement therapy from 2 weeks to 31 years. Eight participants reported having access to the internet but none participated in chat rooms. The activities were conceptualized into themes as listening, seeking, searching, sharing and observing. These activities enabled people to create, reflect on and evaluate the information needed to inform their decision-making Conclusion/Application to Clinical Practice The information practice research approach will enable a better understanding of the underlying relationship between information, knowledge and experience to be better understood. For renal nurses who are involved in patient education being able to recognise the way people use information will assist in individualizing educational sessions and tailoring teaching strategies to make it more meaningful.

Job satisfaction in haemodialysis nurses : results from a two country study

• Australian nurses report lower levels of job satisfaction than the broader working population and perceive they have limited influence over important workplace decisions. • Higher levels of nurse job satisfaction is positively linked to improved quality of care, patient outcomes and staff retention. • Identifying factors that contribute to job satisfaction can improve retention of highly skilled and specialised haemodialysis nurses. • Contributors to job satisfaction and current levels of job satisfaction are poorly understood in the Australia and New Zealand context.

Micromarrows—Three-dimensional coculture of hematopoietic stem cells and mesenchymal stromal cells

Hematopoietic stem cell (HSC) transplant is a well established curative therapy for some hematological malignancies. However, achieving adequate supply of HSC from some donor tissues can limit both its application and ultimate efficacy. The theory that this limitation could be overcome by expanding the HSC population before transplantation has motivated numerous laboratories to develop ex vivo expansion processes. Pioneering work in this field utilized stromal cells as support cells in cocultures with HSC to mimic the HSC niche. We hypothesized that through translation of this classic coculture system to a three-dimensional (3D) structure we could better replicate the niche environment and in turn enhance HSC expansion. Herein we describe a novel high-throughput 3D coculture system where murine-derived HSC can be cocultured with mesenchymal stem/stromal cells (MSC) in 3D microaggregates—which we term “micromarrows.” Micromarrows were formed using surface modified microwells and their ability to support HSC expansion was compared to classic two-dimensional (2D) cocultures. While both 2D and 3D systems provide only a modest total cell expansion in the minimally supplemented medium, the micromarrow system supported the expansion of approximately twice as many HSC candidates as the 2D controls. Histology revealed that at day 7, the majority of bound hematopoietic cells reside in the outer layers of the aggregate. Quantitative polymerase chain reaction demonstrates that MSC maintained in 3D aggregates express significantly higher levels of key hematopoietic niche factors relative to their 2D equivalents. Thus, we propose that the micromarrow platform represents a promising first step toward a high-throughput HSC 3D coculture system that may enable in vitro HSC niche recapitulation and subsequent extensive in vitro HSC self-renewal.

Why renal nurses cope so well with their workplace stressors

Background: Previous studies have found significant stressors experienced by nurses working in haemodialysis units yet renal nurses appear to report less burnout than other nurses. Objectives: This study aims to undertake an inductive process to better understand the stressors and the coping strategies used by renal nurses that may lead to resilience. Method: Sixteen haemodialysis nurses from a metropolitan Australian hospital and two satellite units participated in open-ended interviews. Data were analysed from a grounded theory methodology. Measures of burnout and resilience were also obtained. Results: Two major categories of stressors emerged. First, due to prolonged patient contact, family-like relationships developed that lead to the blurring of boundaries. Second, participants experienced discrimination from both patients and staff. Despite these stressors, the majority of participants reported low burnout and moderately high-to-high levels of resilience. The major coping strategy that appeared to promote resilience was emotional distancing, while emotional detachment appeared to promote burn-out. Conclusion: Assisting nurses to use emotional distancing, rather than emotional detachment strategies to engender a sense of personal achievement may promote resilience.

Re-strengthening 20th century architectural heritage : A case study of Brisbane City Hall restoration

Restoring old buildings to conform the current building policies and standards is a great challenge to engineers and architects. The restoration of the Brisbane City Hall, a heritage building listed by the State of Queensland in Australia, developed an innovative approach to upgrade the building using the method called ‘concrete overlay’ following the guidelines of both the International Council on Monuments and Sites and the Burra Charter of Australia. Concrete overlay is a new method of structural strengthening by drilling new reinforcement and placing new concrete on top of the existing structure, akin to a bone transplant or bone grafting in the case of a human being. This method is popularly used for newer bridges which have suffered load stresses. However, this method had never been used on any heritage buildings which were built on different conditions and standards. The compatibility of this method is currently being monitored. Most of the modern historic buildings are rapidly deteriorating and require immediate interventions in order to be saved. As most of these heritage buildings are on the stage of advanced deterioration, significant attempts are being made and several innovations are being applied to upgrade these structures to conform with the current building requirements. To date, the knowledge and literature in regarding ‘concrete cancer’ in relation to rehabilitating these reinforced concrete heritage structures is significantly lacking. It is hoped that the method of concrete overlay and the case study of Brisbane City Hall restoration will contribute to the development of restoration techniques and policies for Modern Heritage Buildings.

Inflow cannula design for biventricular assist devices

Cardiovascular diseases are a leading cause of death throughout the developed world. With the demand for donor hearts far exceeding the supply, a bridge-to-transplant or permanent solution is required. This is currently achieved with ventricular assist devices (VADs), which can be used to assist the left ventricle (LVAD), right ventricle (RVAD), or both ventricles simultaneously (BiVAD). Earlier generation VADs were large, volume-displacement devices designed for temporary support until a donor heart was found. The latest generation of VADs use rotary blood pump technology which improves device lifetime and the quality of life for end stage heart failure patients. VADs are connected to the heart and greater vessels of the patient through specially designed tubes called cannulae. The inflow cannulae, which supply blood to the VAD, are usually attached to the left atrium or ventricle for LVAD support, and the right atrium or ventricle for RVAD support. Few studies have characterized the haemodynamic difference between the two cannulation sites, particularly with respect to rotary RVAD support. Inflow cannulae are usually made of metal or a semi-rigid polymer to prevent collapse with negative pressures. However suction, and subsequent collapse, of the cannulated heart chamber can be a frequent occurrence, particularly with the relatively preload insensitive rotary blood pumps. Suction events may be associated with endocardial damage, pump flow stoppages and ventricular arrhythmias. While several VAD control strategies are under development, these usually rely on potentially inaccurate sensors or somewhat unreliable inferred data to estimate preload. Fixation of the inflow cannula is usually achieved through suturing the cannula, often via a felt sewing ring, to the cannulated chamber. This technique extends the time on cardiopulmonary bypass which is associated with several postoperative complications. The overall objective of this thesis was to improve the placement and design of rotary LVAD and RVAD inflow cannulae to achieve enhanced haemodynamic performance, reduced incidence of suction events, reduced levels of postoperative bleeding and a faster implantation procedure. Specific objectives were: * in-vitro evaluation of LVAD and RVAD inflow cannula placement, * design and in-vitro evaluation of a passive mechanism to reduce the potential for heart chamber suction, * design and in-vitro evaluation of a novel suture-less cannula fixation device. In order to complete in-vitro evaluation of VAD inflow cannulae, a mock circulation loop (MCL) was developed to accurately replicate the haemodynamics in the human systemic and pulmonary circulations. Validation of the MCL’s haemodynamic performance, including the form and magnitude of pressure, flow and volume traces was completed through comparisons of patient data and the literature. The MCL was capable of reproducing almost any healthy or pathological condition, and provided a useful tool to evaluate VAD cannulation and other cardiovascular devices. The MCL was used to evaluate inflow cannula placement for rotary VAD support. Left and right atrial and ventricular cannulation sites were evaluated under conditions of mild and severe heart failure. With a view to long term LVAD support in the severe left heart failure condition, left ventricular inflow cannulation was preferred due to improved LVAD efficiency and reduced potential for thrombus formation. In the mild left heart failure condition, left atrial cannulation was preferred to provide an improved platform for myocardial recovery. Similar trends were observed with RVAD support, however to a lesser degree due to a smaller difference in right atrial and ventricular pressures. A compliant inflow cannula to prevent suction events was then developed and evaluated in the MCL. As rotary LVAD or RVAD preload was reduced, suction events occurred in all instances with a rigid inflow cannula. Addition of the compliant segment eliminated suction events in all instances. This was due to passive restriction of the compliant segment as preload dropped, thus increasing the VAD circuit resistance and decreasing the VAD flow rate. Therefore, the compliant inflow cannula acted as a passive flow control / anti-suction system in LVAD and RVAD support. A novel suture-less inflow cannula fixation device was then developed to reduce implantation time and postoperative bleeding. The fixation device was evaluated for LVAD and RVAD support in cadaveric animal and human hearts attached to a MCL. LVAD inflow cannulation was achieved in under two minutes with the suture-less fixation device. No leakage through the suture-less fixation device – myocardial interface was noted. Continued development and in-vivo evaluation of this device may result in an improved inflow cannulation technique with the potential for off-bypass insertion. Continued development of this research, in particular the compliant inflow cannula and suture-less inflow cannulation device, will result in improved postoperative outcomes, life span and quality of life for end-stage heart failure patients.

Development of an ultra-thin fibroin membrane for RPE cell transplantation

Purpose: One of the challenges associated with cell-based therapies for repairing the retina is the development of suitable materials on which to grow and transplant retinal cells. Using the ARPE-19 cell line, we have previously demonstrated the feasibility of growing RPE-derived cells on membranes prepared from the silk protein fibroin. The present study was aimed at developing a porous, ultra-thin fibroin membrane that might better support development of apical-basal polarity in culture, and to extend this work to primary cultures of human RPE cells. Methods: Ultra-thin fibroin membranes were prepared using a highly polished casting table coated with Topas® (a cyclic olefin copolymer) and a 1:0.03 aqueous solution of fibroin and PEO (Mv 900 000 g/mol). Following drying, the membranes were water annealed to make them water-stable, washed in water to remove PEO, sterilised by treatment with 95% ethanol, and washed extensively in saline. Primary cultures containing human RPE cells were established from donor posterior eye cups and maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum and antibiotics. First passage cultures were seeded onto fibroin membranes pre-coated with vitronectin and grown for 6 weeks in medium supplemented with 1% serum. Comparative cultures were established on porous 1.0 µm pore PET membrane (Millipore) and using ARPE-19 cells. Results: The fibroin membranes displayed an average thickness of 3 µm and contained numerous dimples/pore-like structures of up to 3-5 µm in diameter. The primary cultures predominantly contained pigmented epithelial cells, but mesenchymal cells (presumed fibroblasts) were also often present. Passaged cultures appeared to attach equally well to either fibroin or PET membranes. Over time cells on either material adopted a more cobblestoned morphology. Conclusions: Progress has been made towards developing a porous ultra-thin fibroin membrane that supports cultivation of RPE cells. Further studies are required to determine the degree of membrane permeability and RPE polarity.

Immunodeficiency-associated lymphomas

This article covers lymphoproliferative disorders in patients with primary or acquired immunodeficiencies. Primary immunodeficiences include Ataxia Telangiectasia and X-linked disorders such as Wiskott-Aldrich syndrome. Acquired immunodeficiencies predominantly occur in the setting of infection with the Human Immunodeficiency Virus or arise following immunosuppressive therapy administered after organ transplantation. The rising incidence of HIV throughout the world and the dramatic increase in transplant surgery since the 1990's suggest that these lymphomas will remain an important health problem. Evidence for lymphoma developing as a result of treatment with methotrexate or Tumour Necrosis Factor Antagonists for autoimmune entities will also be reviewed. The lymphoproliferations that occur with immunodeficiency are extremely heterogenous. In part this reflects the diversity of the causal immune defect. The most striking clinical characteristic is the high frequency of extranodal disease. Frequently, these lymphomas are driven by viruses such as Epstein-Barr virus (EBV), although the lack of EBV in a proportion indicates that alternate pathways must also be involved in the pathogenesis. Lastly, discussion will centre on mechanisms utilized by lymphomas in the immunodeficient as these may have applications to lymphomas in the "immunocompetent", by serving as a paradigm for the altered immunoregulatory environment present in many lymphoma sub-types.

Evaluation of inflow cannulation site for implantation of right-sided rotary ventricular assist device

Right heart dysfunction is one of the most serious complications following implantation of a left ventricular assist device (LVAD), often leading to the requirement for short or long term right ventricular support (RVAD). The inflow cannulation site induces major haemodynamic changes and so there is a need to optimize the site used depending on the patient's condition. Therefore, this study evaluated and compared the haemodynamic influence of right atrial (RAC) and right ventricular (RVC) inflow cannulation sites. An in-vitro, variable heart failure, mock circulation loop was used to compare RAC and RVC in mild and severe biventricular heart failure (BHF) conditions. In the severe BHF condition, higher ventricular ejection fraction (RAC: 13.6%, RVC: 32.7%) and thus improved heart chamber and RVAD washout was observed with RVC, which suggested this strategy might be preferable for long term support (ie. bridge to transplant or destination therapy) to reduce the risk of thrombus formation. In the mild BHF condition, higher pulmonary valve flow (RAC: 3.33 L/min, RVC: 1.97 L/min) and lower right ventricular stroke work (RAC: 0.10 W, RVC: 0.13 W) and volumes were recorded with RAC. These results indicate an improved potential for myocardial recovery, thus RAC should be chosen in this condition. This in-vitro study suggests that RVAD inflow cannulation site should be chosen on a patient-specific basis with a view to the support strategy to promote myocardial recovery or reduce the risk of long-term complications.

Re-strengthening Brisbane City Hall : a case study of heritage engineering

Upgrading old buildings with the evolution of building requirements, this project investigates new approaches that can be applied to strengthen our own heritage buildings using historical and comparative analysis of heritage building restorations locally and abroad. Within the newly developing field of Heritage Engineering, it evaluates the innovative Concrete Overlay technique adapted to building restoration of the Brisbane City Hall. This study aims to extend the application of Concrete Overlay techniques and determine its compatibility specifically to heritage buildings. Concrete overlay involves drilling new reinforcement and placing concrete on top of the existing structure. It is akin to a bone transplant or bone grafting in the case of a human being and has been used by engineers to strengthen newer bridges.

Dying feet in ICU : why might extracorporeal membrane oxygenation machines cause necrotic feet?

Background Extracorporeal membrane oxygenation (ECMO) is used for severe lung and/or heart failure in intensive care units (ICU). The Prince Charles Hospital (TPCH) has one of the largest ECMO units in Australia. Its use rapidly increased during the H1N1 (“swine flu”) pandemic and an increase in pedal complications resulted. The relationship between ECMO and pedal complications has been described, particularly in children, though no strong data exists. This paper presents a case series of foot complications in patients having received ECMO treatment. Methods We present nine cases of severe foot complications resulting from patients receiving ECMO treatment at TPCH in 2009–2012. Results Case ages ranged from 16 - 58 years and three were male. Six cases had an unremarkable medical history prior to H1N1 or H1N2 infection, one had Cardiomyopathy, one had received a lung transplant, and one had multi-organ failure post-sepsis. Common medications prescribed included vasopressors, antibiotics, and sedatives. All cases showed signs of markedly impaired peripheral perfusion whilst on ECMO and seven developed increasing areas of foot necrosis. Outcomes include two bilateral below knee amputations, two multiple digital amputations, one Reflex Sympathetic Dystrophy Syndrome, three pressure injuries, and three deaths. Conclusion Necrosis of the feet appears to occur more readily in younger people requiring ECMO treatment than others in ICU. The authors are conducting further studies to investigate associations between particular infections, medical history, medications, or machine techniques and severe foot complications. Some of these early results will also be presented at this conference.

Crucial roles for olfactory ensheathing cells and olfactory mucosal cells in the repair of damaged neural tracts

Olfactory ensheathing cells, the glial cells of the olfactory nervous system, exhibit unique growth-promoting and migratory properties that make them interesting candidates for cell therapies targeting neuronal injuries such as spinal cord injury. Transplantation of olfactory cells is feasible and safe in humans; however, functional outcomes are highly variable with some studies showing dramatic improvements and some no improvements at all. We propose that the reason for this is that the identity and purity of the cells is different in each individual study. We have shown that olfactory ensheathing cells are not a uniform cell population and that individual subpopulations of OECs are present in different regions of the olfactory nervous system, with strikingly different behaviors. Furthermore, the presence of fibroblasts and other cell types in the transplant can dramatically alter the behavior of the transplanted glial cells. Thus, a thorough characterization of the differences between olfactory ensheathing cell subpopulations and how the behavior of these cells is affected by the presence of other cell types is highly warranted.

An adipoinductive role of inflammation in adipose tissue engineering : key factors in the early development of engineered soft tissues

Tissue engineering and cell implantation therapies are gaining popularity because of their potential to repair and regenerate tissues and organs. To investigate the role of inflammatory cytokines in new tissue development in engineered tissues, we have characterized the nature and timing of cell populations forming new adipose tissue in a mouse tissue engineering chamber (TEC) and characterized the gene and protein expression of cytokines in the newly developing tissues. EGFP-labeled bone marrow transplant mice and MacGreen mice were implanted with TEC for periods ranging from 0.5 days to 6 weeks. Tissues were collected at various time points and assessed for cytokine expression through ELISA and mRNA analysis or labeled for specific cell populations in the TEC. Macrophage-derived factors, such as monocyte chemotactic protein-1 (MCP-1), appear to induce adipogenesis by recruiting macrophages and bone marrow-derived precursor cells to the TEC at early time points, with a second wave of nonbone marrow-derived progenitors. Gene expression analysis suggests that TNFα, LCN-2, and Interleukin 1β are important in early stages of neo-adipogenesis. Increasing platelet-derived growth factor and vascular endothelial cell growth factor expression at early time points correlates with preadipocyte proliferation and induction of angiogenesis. This study provides new information about key elements that are involved in early development of new adipose tissue.