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.

Evaluation of the cost-effectiveness of strategies claiming to reduce the risk of surgical site infections following primary total hip arthroplasty

Background Total hip arthroplasty (THA) is a commonly performed procedure and numbers are increasing with ageing populations. One of the most serious complications in THA are surgical site infections (SSIs), caused by pathogens entering the wound during the procedure. SSIs are associated with a substantial burden for health services, increased mortality and reduced functional outcomes in patients. Numerous approaches to preventing these infections exist but there is no gold standard in practice and the cost-effectiveness of alternate strategies is largely unknown. Objectives The aim of this project was to evaluate the cost-effectiveness of strategies claiming to reduce deep surgical site infections following total hip arthroplasty in Australia. The objectives were: 1. Identification of competing strategies or combinations of strategies that are clinically relevant to the control of SSI related to hip arthroplasty 2. Evidence synthesis and pooling of results to assess the volume and quality of evidence claiming to reduce the risk of SSI following total hip arthroplasty 3. Construction of an economic decision model incorporating cost and health outcomes for each of the identified strategies 4. Quantification of the effect of uncertainty in the model 5. Assessment of the value of perfect information among model parameters to inform future data collection Methods The literature relating to SSI in THA was reviewed, in particular to establish definitions of these concepts, understand mechanisms of aetiology and microbiology, risk factors, diagnosis and consequences as well as to give an overview of existing infection prevention measures. Published economic evaluations on this topic were also reviewed and limitations for Australian decision-makers identified. A Markov state-transition model was developed for the Australian context and subsequently validated by clinicians. The model was designed to capture key events related to deep SSI occurring within the first 12 months following primary THA. Relevant infection prevention measures were selected by reviewing clinical guideline recommendations combined with expert elicitation. Strategies selected for evaluation were the routine use of pre-operative antibiotic prophylaxis (AP) versus no use of antibiotic prophylaxis (No AP) or in combination with antibiotic-impregnated cement (AP & ABC) or laminar air operating rooms (AP & LOR). The best available evidence for clinical effect size and utility parameters was harvested from the medical literature using reproducible methods. Queensland hospital data were extracted to inform patients’ transitions between model health states and related costs captured in assigned treatment codes. Costs related to infection prevention were derived from reliable hospital records and expert opinion. Uncertainty of model input parameters was explored in probabilistic sensitivity analyses and scenario analyses and the value of perfect information was estimated. Results The cost-effectiveness analysis was performed from a health services perspective using a hypothetical cohort of 30,000 THA patients aged 65 years. The baseline rate of deep SSI was 0.96% within one year of a primary THA. The routine use of antibiotic prophylaxis (AP) was highly cost-effective and resulted in cost savings of over $1.6m whilst generating an extra 163 QALYs (without consideration of uncertainty). Deterministic and probabilistic analysis (considering uncertainty) identified antibiotic prophylaxis combined with antibiotic-impregnated cement (AP & ABC) to be the most cost-effective strategy. Using AP & ABC generated the highest net monetary benefit (NMB) and an incremental $3.1m NMB compared to only using antibiotic prophylaxis. There was a very low error probability that this strategy might not have the largest NMB (<5%). Not using antibiotic prophylaxis (No AP) or using both antibiotic prophylaxis combined with laminar air operating rooms (AP & LOR) resulted in worse health outcomes and higher costs. Sensitivity analyses showed that the model was sensitive to the initial cohort starting age and the additional costs of ABC but the best strategy did not change, even for extreme values. The cost-effectiveness improved for a higher proportion of cemented primary THAs and higher baseline rates of deep SSI. The value of perfect information indicated that no additional research is required to support the model conclusions. Conclusions Preventing deep SSI with antibiotic prophylaxis and antibiotic-impregnated cement has shown to improve health outcomes among hospitalised patients, save lives and enhance resource allocation. By implementing a more beneficial infection control strategy, scarce health care resources can be used more efficiently to the benefit of all members of society. The results of this project provide Australian policy makers with key information about how to efficiently manage risks of infection in THA.

Risk factors to predict the incidence of surgical adverse events following open or laparoscopic surgery for apparent early stage endometrial cancer: results from a randomised controlled trial

Aims: To identify risk factors for major Adverse Events (AEs) and to develop a nomogram to predict the probability of such AEs in individual patients who have surgery for apparent early stage endometrial cancer. Methods: We used data from 753 patients who were randomized to either total laparoscopic hysterectomy or total abdominal hysterectomy in the LACE trial. Serious adverse events that prolonged hospital stay or postoperative adverse events (using common terminology criteria 3+, CTCAE V3) were considered major AEs. We analyzed pre-surgical characteristics that were associated with the risk of developing major AEs by multivariate logistic regression. We identified a parsimonious model by backward stepwise logistic regression. The six most significant or clinically important variables were included in the nomogram to predict the risk of major AEs within 6 weeks of surgery and the nomogram was internally validated. Results: Overall, 132 (17.5%) patients had at least one major AE. An open surgical approach (laparotomy), higher Charlson’s medical co-morbidities score, moderately differentiated tumours on curettings, higher baseline ECOG score, higher body mass index and low haemoglobin levels were associated with AE and were used in the nomogram. The bootstrap corrected concordance index of the nomogram was 0.63 and it showed good calibration. Conclusions: Six pre-surgical factors independently predicted the risk of major AEs. This research might form the basis to develop risk reduction strategies to minimize the risk of AEs among patients undergoing surgery for apparent early stage endometrial cancer.

Improved surgical safety after laparoscopic compared to open surgery for apparent early stage endometrial cancer : results from a randomised controlled trial

AIM: To compare Total Laparoscopic Hysterectomy (TLH) and Total Abdominal Hysterectomy (TAH) with regard to surgical safety. METHODS: Between October 2005 and June 2010, 760 patients with apparent early stage endometrial cancer were enroled in a multicentre, randomised clinical trial (LACE) comparing outcomes following TLH or TAH. The main study end points for this analysis were surgical adverse events (AE), hospital length of stay, conversion from laparoscopy to laparotomy, including 753 patients who completed at least 6 weeks of follow-up. Postoperative AEs were graded according to Common Toxicity Criteria (V3), and those immediately life-threatening, requiring inpatient hospitalisation or prolonged hospitalisation, or resulting in persistent or significant disability/incapacity were regarded as serious AEs. RESULTS: The incidence of intra-operative AEs was comparable in either group. The incidence of post-operative AE CTC grade 3+ (18.6% in TAH, 12.9% in TLH, p 0.03) and serious AE (14.3% in TAH, 8.2% in TLH, p 0.007) was significantly higher in the TAH group compared to the TLH group. Mean operating time was 132 and 107 min, and median length of hospital stay was 2 and 5 days in the TLH and TAH group, respectively (p<0.0001). The decline of haemoglobin from baseline to day 1 postoperatively was 2g/L less in the TLH group (p 0.006). CONCLUSIONS: Compared to TAH, TLH is associated with a significantly decreased risk of major surgical AEs. A laparoscopic surgical approach to early stage endometrial cancer is safe.

Realistic irrigation visualization in a surgical wound debridement simulator

Wound debridement refers to the removal of necrotic, devitalized, or contaminated tissue and/or foreign material to promote wound healing. Surgical debridement uses sharp instruments to cut dead tissue from a wound and it is the quickest and most efficient method of debridement. A wound debridement simulator [1,2] can ensure that a medical trainee is competent prior to performing a procedure on a genuine patient. Irrigation is performed at different stages of debridement in order to remove debris and reduce the bacteria count through rinsing the wound. This paper presents a novel approach for realistic irrigation visualization based on texture representations of debris. This approach applies image processing techniques to a series of images, which model the cleanliness of the wound. The active texture is generated and updated dynamically based on the irrigation state, location, and range. Presented results demonstrate that texture mapping and image processing techniques can provide effective and efficient solutions for irrigation visualization in the wound debridement simulator.

A simulation-based training system for surgical wound debridement

A simulation-based training system for surgical wound debridement was developed and comprises a multimedia introduction, a surgical simulator (tutorial component), and an assessment component. The simulator includes two PCs, a haptic device, and mirrored display. Debridement is performed on a virtual leg model with a shallow laceration wound superimposed. Trainees are instructed to remove debris with forceps, scrub with a brush, and rinse with saline solution to maintain sterility. Research and development issues currently under investigation include tissue deformation models using mass-spring system and finite element methods; tissue cutting using a high-resolution volumetric mesh and dynamic topology; and accurate collision detection, cutting, and soft-body haptic rendering for two devices within the same haptic space.

The impact of surgical ward nurses practicing respiratory assessment on positive patient outcomes

Objective: A literature review to examine the incorporation of respiratory assessment into everyday surgical nursing practice; possible barriers to this; and the relationship to patient outcomes. Primary argument: Escalating demands on intensive care beds have led to highly dependent patients being cared for in general surgical ward areas. This change in patient demographics has meant the knowledge and skills required of registered nurses in these areas has expanded exponentially. The literature supported the notion that postoperative monitoring of vital signs should include the fundamental assessment of respiratory rate; depth and rhythm; work of breathing; use of accessory muscles and symmetrical chest movement; as well as auscultation of lung fields using a stethoscope. Early intervention in response to changes in a patient's respiratory health status impacts positively on patient health outcomes. Substantial support exists for the contention that technologically adept nurses who also possess competent respiratory assessment skills make a difference to respiratory care. Conclusions: Sub-clinical respiratory problems have been demonstrated to contribute to adverse events. There is a paucity of research knowledge as to whether respiratory education programs and associated inservice make a difference to nursing clinical practice. Similarly, the implications for associated respiratory educational needs are not well documented, nor has a research base been sufficiently developed to guide nursing practice. Further research has the potential to influence the future role and function of the registered nurse by determining the importance of respiratory education programs on post-operative patient outcomes.

Post-discharge surveillance : can patients reliably diagnose surgical wound infections?

Post-discharge surgical wound infection surveillance is an important part of many infection control programs. It is frequently undertaken by patient self-assessment, prompted either by a telephone or postal questionnaire. To assess the reliability of this method, 290 patients were followed for six weeks postoperatively. Their wounds were photographed and also covertly assessed for signs of infection by two experienced infection control nurses (ICNs). Patients also responded to a postal questionnaire seeking evidence of infection at both week four and week six post-surgery. Correlation between the patient's assessment of their wound and the ICNs diagnosis was poor (r=0.37) with a low positive predictive value (28.7%), although negative predictive value was high (98.2%). Assessment of photos for signs of infection by two experienced clinicians also correlated poorly with the ICNs diagnosis of infection (r=0.54). The patient's recall of prescription of an antibiotic by their general practitioner (GP) for wound infection during the postoperative period correlated best with the ICNs diagnosis (r=0.76). This latter measure, particularly when confirmed by the GP in those patients reporting an infection, appears to provide the most valid and resource efficient marker of post-discharge surgical wound infection.

Mutual enlightenment : augmenting human factors research in surgical robotics

This discussion has outlined a theoretical and pragmatic framework to demonstrate that future research involving the analysis of human performance in surgical should encourage the use of phenomenology to enhance the knowledge base of this area of study. Merging experiential (first-person) and experimental (third-person) methods may possibly help improve research designs and analyses in the investigation of robotics in surgical performance. By relying solely on third-person techniques, the current methodology and interpretation used to analyze human performance in surgical robotics is limited. Recent advances in cognitive science and psychology have also recognized this limitation and have now begun to shift to neurophenomenology. Finally, discussion on recent robotics research presented here demonstrates the potential phenomenology holds for augmenting the methodological and analysis techniques currently used by researchers of human performance in surgical robotics.

Surgical site infection prevention following total hip arthroplasty in Australia : a cost-effectiveness analysis

Background: Surgical site infection (SSI) is associated with substantial costs for health services, reduced quality of life, and functional outcomes. The aim of this study was to evaluate the cost-effectiveness of strategies claiming to reduce the risk of SSI in hip arthroplasty in Australia. Methods: Baseline use of antibiotic prophylaxis (AP) was compared with no antibiotic prophylaxis (no AP), antibiotic-impregnated cement (AP þ ABC), and laminar air operating rooms (AP þ LOR). A Markov model was used to simulate long-term health and cost outcomes of a hypothetical cohort of 30,000 total hip arthroplasty patients from a health services perspective. Model parameters were informed by the best available evidence. Uncertainty was explored in probabilistic sensitivity and scenario analyses. Results: Stopping the routine use of AP resulted in over Australian dollars (AUD) $1.5 million extra costs and a loss of 163 quality-adjusted life years (QALYs). Using antibiotic cement in addition to AP (AP þ ABC)generated an extra 32 QALYs while saving over AUD $123,000. The use of laminar air operating rooms combined with routine AP (AP þ LOR) resulted in an AUD $4.59 million cost increase and 127 QALYs lost compared with the baseline comparator. Conclusion: Preventing deep SSI with antibiotic prophylaxis and antibiotic-impregnated cement has shown to improve health outcomes among hospitalized patients, save lives, and enhance resource allocation. Based on this evidence, the use of laminar air operating rooms is not recommended.