Highlights Early/middle stages of differentiation are not affected by decel NR, decel RPE or RP... more Highlights Early/middle stages of differentiation are not affected by decel NR, decel RPE or RPE conditioned media (CM RPE) supplementation Supplementation with decel RPE enhances RPE generation CM RPE and decel RPE enhance the formation of rod photoreceptors Decel RPE and decel NR enhance the expression of synaptic markers and the light driven responses Our findings provide the first substantive evidence for the role of retinal decellularised matrices in enhancing the differentiation and functionality of hPSCderived retinal organoids
The extracellular matrix (ECM) plays an important role in numerous processes including cellular p... more The extracellular matrix (ECM) plays an important role in numerous processes including cellular proliferation, differentiation, migration, maturation, adhesion guidance and axonal growth. To date, there has been no detailed analysis of the ECM distribution during retinal ontogenesis in humans and the functional importance of many ECM components is poorly understood. In this study, the expression of key ECM components in adult mouse and monkey retina, developing and adult human retina and retinal organoids derived from human pluripotent stem cells was studied. Our data indicate that basement membrane ECMs (Fibronectin and Collagen IV) were expressed in Bruch's membrane and the inner limiting membrane of the developing human retina, whilst the hyalectins (Versican and Brevican), cluster of differentiation 44 (CD44), photoreceptor-specific ECMs Interphotoreceptor Matrix Proteoglycan 1 (IMPG1) and Interphotoreceptor Matrix Proteoglycan 2 (IMPG2) were detected in the developing inter...
No treatments exist to effectively treat many retinal diseases. Retinal pigmented epithelium (RPE... more No treatments exist to effectively treat many retinal diseases. Retinal pigmented epithelium (RPE) and neural retina can be generated from human embryonic stem cells/induced pluripotent stem cells (hESCs/hiPSCs). The efficacy of current protocols is, however, limited. It was hypothesised that generation of laminated neural retina and/or RPE from hiPSCs/hESCs could be enhanced by three dimensional (3D) culture in hydrogels. hiPSC- and hESC-derived embryoid bodies (EBs) were encapsulated in 0.5% RGD-alginate; 1% RGD-alginate; hyaluronic acid (HA) or HA/gelatin hydrogels and maintained until day 45. Compared with controls (no gel), 0.5% RGD-alginate increased: the percentage of EBs with pigmented RPE foci; the percentage EBs with optic vesicles (OVs) and pigmented RPE simultaneously; the area covered by RPE; frequency of RPE cells (CRALBP+); expression of RPE markers (TYR and RPE65) and the retinal ganglion cell marker, MATH5. Furthermore, 0.5% RGD-alginate hydrogel encapsulation did n...
Human pluripotent stem cells (hPSCs) offer huge potential within the field of tissue engineering ... more Human pluripotent stem cells (hPSCs) offer huge potential within the field of tissue engineering since they can theoretically be differentiated into any cell type and therefore be used to replace any degenerated or diseased tissue. Importantly, it offers the possibility to replace regenerative and nonregenerative tissue including cardiac, retinal and CNS tissue. The first human embryonic stem cell lines were reported in 1998 [1], but human embryonic stem cell line-derived cells are allogenic and therefore could potentially evoke an immune reaction, and are also associated with ethical problems. Technology to produce humaninduced pluripotent stem cells (hiPSCs) was first reported in 2007 [2], offering the exciting potential to generate autologous tissue. Key advancements have been made toward the clinical application of hPSC-derived cells/tissues including the development of feeder-free and defined culture systems, the use of defined extracellular matrix (ECM) substrates (e.g., laminin subtypes) and nonintegrative methods of hiPSC production. Protocols enabling the derivation of a huge range of cell types have been developed including corneal cells, cardiomyocytes, osteoblasts and neurons [3]. Furthermore, multicellular 3D organoids resembling developmental tissue have been achieved, including mini brains, guts and retina [4]. The ability to genetically modify hPSCs due to the
There is a clinical need for effective alternative skin replacements to autografts, allografts an... more There is a clinical need for effective alternative skin replacements to autografts, allografts and xenografts. In this thesis a bi-layer skin graft was developed by encapsulation of fibroblasts in calcium-alginate hydrogel and culture of keratinocytes on the surface. Initially, the use of 5% and 2% w/v alginate hydrogels were investigated. Both scaffolds maintained fibroblast viability for at least 150 days encapsulation and caused reversible mitotic and catabolic inhibition, as assessed by fluorescent staining, immunochemistry and the thiazolyl blue assay. Sustained expression of angiogenic factors such as vascular endothelial growth factor, interleukin 6 and nerve growth factor were seen by fibroblasts encapsulated in both scaffolds, by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay. Histological staining demonstrated that following degradation of the scaffolds, fibroblasts secreted ECM to facilitate dermal repair. Comparison of degradation of the scaffolds over time by measuring release of calcium, and changes in rheological properties, morphology and mass, indicated that 5% w/v alginate hydrogel degraded more slowly and was preferable to 2% w/v alginate hydrogel. Fibroblasts encapsulated in 5% w/v alginate hydrogel were shown to express keratinocyte growth factor by RT-PCR, to support keratinocyte proliferation and differentiation, and keratinocytes cultured on the 5% w/v alginate hydrogel surface were seen to form multi-layered epidermal structures by histology, immunostaining and RT-PCR.
Calcium-alginate hydrogel has been widely studied as a material for cell encapsulation for tissue... more Calcium-alginate hydrogel has been widely studied as a material for cell encapsulation for tissue engineering. At present, the effect that cells have on the degradation of alginate hydrogel is largely unknown. We have shown that fibroblasts encapsulated at a density of 7.5 Â 10 5 cells ml À1 in both 2% and 5% w/v alginate remain viable for at least 60 days. Rheological analysis was used to study how the mechanical properties exhibited by alginate hydrogel changed during 28 days in vitro culture. Alginate degradation was shown to occur throughout the study but was greatest within the first 7 days of culture for all samples, which correlated with a sharp release of calcium ions from the construct. Fibroblasts were shown to increase the rate of degradation during the first 7 days when compared with acellular samples in both 2% and 5% w/v gels, but after 28 days both acellular and cell-encapsulating samples retained disc-shaped morphologies and gel-like spectra. The results demonstrate that although at an early stage cells influence the mechanical properties of encapsulating alginate, over a longer period of culture, the hydrogels retain sufficient mechanical integrity to exhibit gel-like properties. This allows sustained immobilization of the cells at the desired location in vivo where they can produce extracellular matrix and growth factors to expedite the healing process.
Knee surgery, sports traumatology, arthroscopy : official journal of the ESSKA, 2015
The popliteus tendon is crucial to postero-lateral stability and prone to iatrogenic injury intra... more The popliteus tendon is crucial to postero-lateral stability and prone to iatrogenic injury intra-operatively. Its role in the stability of the replaced knee remains contentious. The aim of this study was to use computer navigation to quantify the effect of popliteus sectioning on the 'envelope of laxity' (EoL) offered by a posterior-stabilised (PS) total knee arthroplasty (TKA) and compare with that of the native knee. Loaded cadaveric legs were mounted on a purpose built rig. EoL was measured in 3 degrees of freedom using computer navigation. Knees were subjectively stressed in varus/valgus, internal/external rotation and anterior draw. This was performed preoperatively, during TKA and after sectioning of the popliteus tendon. Real-time data were recorded at 0°, 30°, 60° and 90° of flexion as the operating surgeon stressed the knee in 3 degrees of freedom to its subjective endpoint. Mixed-effect modelling was used to quantify the effects of intervention on degree of laxity...
The aim of this review was to identify a reliable sequential medial release protocol for restorat... more The aim of this review was to identify a reliable sequential medial release protocol for restoration of soft tissue balance in total knee arthroplasty of the varus osteoarthritic knee and to allow for improved intraoperative decision-making. Current medial release sequences and applicability based upon pre-operative deformity have been reviewed. Furthermore, risks associated with over release, and the necessity of medial release, are discussed. The different medial release sequences are discussed in relation to pre-operative deformity, along with potential complications associated with medial release. It was found that release sequences may include the deep and superficial components of the medial collateral ligament, the posteromedial capsule, the posterior oblique ligament, the pes anserinus (pes A), and tendons of the semimembranosus and medial gastrocnemius muscle. The sequences described were found to vary substantially between studies, and very few studies had systematically quantified the effect of each release on balance. While medial release is the standard intraoperative mode of balancing, there is a lack of evidence to support current methods. The correct method for defining intraoperatively the sequence, extent and magnitude of releases required remains ill-defined. It could be argued that the classic extensive medial release may be unnecessary and may be associated with iatrogenic injury to the pes A and saphenous nerve, instability and abnormal knee kinematics. Minimal medial release may allow for improved soft tissue balancing leading ultimately to improved functional outcome. V (expert opinion).
2010 4th International Conference on Bioinformatics and Biomedical Engineering, 2010
Tactile sensation is important for the performance of daily tasks and prevention of injury. Full ... more Tactile sensation is important for the performance of daily tasks and prevention of injury. Full thickness wounds of greater than 4cm diameter will not spontaneously heal. Tissue engineering can be used to replace the lost skin, but this skin will often fail to be reinnervated. This results in loss of tactile sensations. Here we have developed a novel, silicon based
Patients with total knee arthroplasties (TKAs) continue to report dissatisfaction in functional o... more Patients with total knee arthroplasties (TKAs) continue to report dissatisfaction in functional outcome. Stability is a major factor contributing to functionality of TKAs. Implants with single-radius (SR) femoral components are proposed to increase stability throughout the arc of flexion. Using computer navigation and loaded cadaveric legs, we characterized the "envelope of laxity" (EoL) offered by a SR cruciate retaining (CR)-TKA compared with that of the native knee through the arc of flexion in terms of anterior drawer, varus/valgus stress, and internal/external rotation. In both the native knee and the TKA laxity increased with increasing knee flexion. Laxities measured in the three planes of motion were generally comparable between the native knee and TKA from 0° to 110° of flexion. Our results indicate that the SR CR-TKA offers appropriate stability in the absence of soft tissue deficiency.
The potential of cell therapy for the regeneration of diseased and damaged tissues is now widely ... more The potential of cell therapy for the regeneration of diseased and damaged tissues is now widely -recognized. As a consequence there is a demand for the development of novel systems that can deliver cells to a particular location, maintaining viability, and then degrade at a predictable rate to release the cells into the surrounding tissues. Hydrogels have attracted much attention in this area, as the hydrogel structure provides an environment that is akin to that of the extracellular matrix. One widely investigated hydrogel is alginate, which has been used for cell encapsulation for more than 30 years. Alginate gels have the potential to be used as 3D cell culture systems and as prosthetic materials, both are applied to regeneration of the cornea. Here, we describe an alginate-based process that has been used for encapsulation of mammalian cells including corneal cells, with high levels of viability, and which allows subsequent retrieval of cell cultures for further characterization.
Vascularization of engineered or damaged tissues is essential to maintain cell viability and prop... more Vascularization of engineered or damaged tissues is essential to maintain cell viability and proper tissue function. Revascularization of the left ventricle (LV) of the heart after myocardial infarction is particularly important, since hypoxia can give rise to chronic heart failure due to inappropriate remodeling of the LV after death of cardiomyocytes (CMs). Fibroblasts can express vascular endothelial growth factor (VEGF), which plays a major role in angiogenesis and also acts as a chemoattractant and survival factor for CMs and cardiac progenitors. In this in vitro model study, mouse NIH 3T3 fibroblasts encapsulated in 2% w/v Ca-alginate were shown to remain viable for 150 days. Semiquantitative reverse transcription-polymerase chain reaction and immunohistochemistry demonstrated that over 21 days of encapsulation, fibroblasts continued to express VEGF, while enzyme-linked immunosorbent assay showed that there was sustained release of VEGF from the Ca-alginate during this period. The scaffold degraded gradually over the 21 days, without reduction in volume. Cells released from the Ca-alginate at 7 and 21 days as a result of scaffold degradation were shown to retain viability, to adhere to fibronectin in a normal manner, and continue to express VEGF, demonstrating their potential to further contribute to maintenance of cardiac function after scaffold degradation. This model in vitro study therefore demonstrates that fibroblasts encapsulated in Ca-alginate provide sustained release of VEGF.
Background: Tibiofemoral instability is a common reason for total knee arthroplasty failure, and ... more Background: Tibiofemoral instability is a common reason for total knee arthroplasty failure, and may be attributed to soft tissue deficiency and incorrect ligament balancing. There are many different designs of implant with varying levels of constraint to overcome this instability; however there is little advice for surgeons to assess which is suitable for a specific patient, and soft tissue balance testing during arthroplasty is very subjective. Method: The current theories on primary and secondary soft tissue restraints to anterior/ posterior, varus/ valgus, and internal/ external rotational motion of the knee are discussed. The paper reviews biomechanics literature to evaluate instability in the intact and implanted knee. Findings: The paper highlights important intra-and extra-capsular structures in the knee and describes the techniques used by clinicians to assess instability perioperatively. In vitro cadaveric studies were found to be a very useful tool in comparing different implants and contributions of different soft tissues. Interpretation: In vitro cadaveric studies can be utilised in helping less experienced surgeons with soft tissue releases and determining the correct implant. For this to happen, more biomechanical studies must be done to show the impact of release sequences on implanted cadavers, as well as determining if increasingly constrained implants restore the stability of the knee to pre-deficient conditions.
There has been a consistent increase in the mean life expectancy of the population of the develop... more There has been a consistent increase in the mean life expectancy of the population of the developed world over the past century. Healthy life expectancy, however, has not increased concurrently. As a result we are living a larger proportion of our lives in poor health and there is a growing demand for the replacement of diseased and damaged tissues. While traditionally tissue grafts have functioned well for this purpose, the demand for tissue grafts now exceeds the supply. For this reason, research in regenerative medicine is rapidly expanding to cope with this new demand. There is now a trend towards supplying cells with a material in order to expedite the tissue healing process. Hydrogel encapsulation provides cells with a three dimensional environment similar to that experienced in vivo and therefore may allow the maintenance of normal cellular function in order to produce tissues similar to those found in the body. In this review we discuss biopolymeric gels that have been used for the encapsulation of mammalian cells for tissue engineering applications as well as a brief overview of cell encapsulation for therapeutic protein production. This review focuses on agarose, alginate, collagen, fibrin, hyaluronic acid and gelatin since they are widely used for cell encapsulation. The literature on the regeneration of cartilage, bone, ligament, tendon, skin, blood vessels and neural tissues using these materials has been summarised.
There is a clinical need for effective alternative skin replacements to autografts, allografts an... more There is a clinical need for effective alternative skin replacements to autografts, allografts and xenografts. In this thesis a bi-layer skin graft was developed by encapsulation of fibroblasts in calcium-alginate hydrogel and culture of keratinocytes on the surface. Initially, the use of 5% and 2% w/v alginate hydrogels were investigated. Both scaffolds maintained fibroblast viability for at least 150 days encapsulation and caused reversible mitotic and catabolic inhibition, as assessed by fluorescent staining, immunochemistry and the thiazolyl blue assay. Sustained expression of angiogenic factors such as vascular endothelial growth factor, interleukin 6 and nerve growth factor were seen by fibroblasts encapsulated in both scaffolds, by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay. Histological staining demonstrated that following degradation of the scaffolds, fibroblasts secreted ECM to facilitate dermal repair. Comparison of degradation of the scaffolds over time by measuring release of calcium, and changes in rheological properties, morphology and mass, indicated that 5% w/v alginate hydrogel degraded more slowly and was preferable to 2% w/v alginate hydrogel. Fibroblasts encapsulated in 5% w/v alginate hydrogel were shown to express keratinocyte growth factor by RT-PCR, to support keratinocyte proliferation and differentiation, and keratinocytes cultured on the 5% w/v alginate hydrogel surface were seen to form multi-layered epidermal structures by histology, immunostaining and RT-PCR.
Limiting cell proliferation without reducing cell viability for in vivo tissue engineering applic... more Limiting cell proliferation without reducing cell viability for in vivo tissue engineering applications is important in co-culture applications where the growth of one cell type must be inhibited to prevent overgrowth of the scaffold at the expense of another cell type. Also, it is vital for maintaining viability of cells in large constructs before vascularisation occurs. In this study we have shown by means of the Thiazolyl blue (MTT) assay and immuno-staining for proliferating cell nuclear antigen (PCNA) that encapsulating fibroblasts in 2% and 5% w/v calcium-alginate at a density of 7.5 Â 10 5 cells/ml as uniformly dispersed entities, enabled cells to maintain viability and caused a reversible mitotic inhibition. Alginate encapsulation also caused reversible metabolic inhibition as demonstrated by the MTT assay and fluorescent staining for mitochondrial membrane potential. Histological evaluation of the alginate constructs containing fibroblasts showed that mitotic and metabolic inhibition was possibly due to cell isolation during the first five weeks of culture. The alginate scaffold degraded with time releasing encapsulated fibroblasts. Upon implantation to a wound site this should ensure that encapsulated cells are able to replace the damaged tissue after sufficient proliferation of the co-cultured cell type or sufficient vascularisation of the construct.
Highlights Early/middle stages of differentiation are not affected by decel NR, decel RPE or RP... more Highlights Early/middle stages of differentiation are not affected by decel NR, decel RPE or RPE conditioned media (CM RPE) supplementation Supplementation with decel RPE enhances RPE generation CM RPE and decel RPE enhance the formation of rod photoreceptors Decel RPE and decel NR enhance the expression of synaptic markers and the light driven responses Our findings provide the first substantive evidence for the role of retinal decellularised matrices in enhancing the differentiation and functionality of hPSCderived retinal organoids
The extracellular matrix (ECM) plays an important role in numerous processes including cellular p... more The extracellular matrix (ECM) plays an important role in numerous processes including cellular proliferation, differentiation, migration, maturation, adhesion guidance and axonal growth. To date, there has been no detailed analysis of the ECM distribution during retinal ontogenesis in humans and the functional importance of many ECM components is poorly understood. In this study, the expression of key ECM components in adult mouse and monkey retina, developing and adult human retina and retinal organoids derived from human pluripotent stem cells was studied. Our data indicate that basement membrane ECMs (Fibronectin and Collagen IV) were expressed in Bruch's membrane and the inner limiting membrane of the developing human retina, whilst the hyalectins (Versican and Brevican), cluster of differentiation 44 (CD44), photoreceptor-specific ECMs Interphotoreceptor Matrix Proteoglycan 1 (IMPG1) and Interphotoreceptor Matrix Proteoglycan 2 (IMPG2) were detected in the developing inter...
No treatments exist to effectively treat many retinal diseases. Retinal pigmented epithelium (RPE... more No treatments exist to effectively treat many retinal diseases. Retinal pigmented epithelium (RPE) and neural retina can be generated from human embryonic stem cells/induced pluripotent stem cells (hESCs/hiPSCs). The efficacy of current protocols is, however, limited. It was hypothesised that generation of laminated neural retina and/or RPE from hiPSCs/hESCs could be enhanced by three dimensional (3D) culture in hydrogels. hiPSC- and hESC-derived embryoid bodies (EBs) were encapsulated in 0.5% RGD-alginate; 1% RGD-alginate; hyaluronic acid (HA) or HA/gelatin hydrogels and maintained until day 45. Compared with controls (no gel), 0.5% RGD-alginate increased: the percentage of EBs with pigmented RPE foci; the percentage EBs with optic vesicles (OVs) and pigmented RPE simultaneously; the area covered by RPE; frequency of RPE cells (CRALBP+); expression of RPE markers (TYR and RPE65) and the retinal ganglion cell marker, MATH5. Furthermore, 0.5% RGD-alginate hydrogel encapsulation did n...
Human pluripotent stem cells (hPSCs) offer huge potential within the field of tissue engineering ... more Human pluripotent stem cells (hPSCs) offer huge potential within the field of tissue engineering since they can theoretically be differentiated into any cell type and therefore be used to replace any degenerated or diseased tissue. Importantly, it offers the possibility to replace regenerative and nonregenerative tissue including cardiac, retinal and CNS tissue. The first human embryonic stem cell lines were reported in 1998 [1], but human embryonic stem cell line-derived cells are allogenic and therefore could potentially evoke an immune reaction, and are also associated with ethical problems. Technology to produce humaninduced pluripotent stem cells (hiPSCs) was first reported in 2007 [2], offering the exciting potential to generate autologous tissue. Key advancements have been made toward the clinical application of hPSC-derived cells/tissues including the development of feeder-free and defined culture systems, the use of defined extracellular matrix (ECM) substrates (e.g., laminin subtypes) and nonintegrative methods of hiPSC production. Protocols enabling the derivation of a huge range of cell types have been developed including corneal cells, cardiomyocytes, osteoblasts and neurons [3]. Furthermore, multicellular 3D organoids resembling developmental tissue have been achieved, including mini brains, guts and retina [4]. The ability to genetically modify hPSCs due to the
There is a clinical need for effective alternative skin replacements to autografts, allografts an... more There is a clinical need for effective alternative skin replacements to autografts, allografts and xenografts. In this thesis a bi-layer skin graft was developed by encapsulation of fibroblasts in calcium-alginate hydrogel and culture of keratinocytes on the surface. Initially, the use of 5% and 2% w/v alginate hydrogels were investigated. Both scaffolds maintained fibroblast viability for at least 150 days encapsulation and caused reversible mitotic and catabolic inhibition, as assessed by fluorescent staining, immunochemistry and the thiazolyl blue assay. Sustained expression of angiogenic factors such as vascular endothelial growth factor, interleukin 6 and nerve growth factor were seen by fibroblasts encapsulated in both scaffolds, by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay. Histological staining demonstrated that following degradation of the scaffolds, fibroblasts secreted ECM to facilitate dermal repair. Comparison of degradation of the scaffolds over time by measuring release of calcium, and changes in rheological properties, morphology and mass, indicated that 5% w/v alginate hydrogel degraded more slowly and was preferable to 2% w/v alginate hydrogel. Fibroblasts encapsulated in 5% w/v alginate hydrogel were shown to express keratinocyte growth factor by RT-PCR, to support keratinocyte proliferation and differentiation, and keratinocytes cultured on the 5% w/v alginate hydrogel surface were seen to form multi-layered epidermal structures by histology, immunostaining and RT-PCR.
Calcium-alginate hydrogel has been widely studied as a material for cell encapsulation for tissue... more Calcium-alginate hydrogel has been widely studied as a material for cell encapsulation for tissue engineering. At present, the effect that cells have on the degradation of alginate hydrogel is largely unknown. We have shown that fibroblasts encapsulated at a density of 7.5 Â 10 5 cells ml À1 in both 2% and 5% w/v alginate remain viable for at least 60 days. Rheological analysis was used to study how the mechanical properties exhibited by alginate hydrogel changed during 28 days in vitro culture. Alginate degradation was shown to occur throughout the study but was greatest within the first 7 days of culture for all samples, which correlated with a sharp release of calcium ions from the construct. Fibroblasts were shown to increase the rate of degradation during the first 7 days when compared with acellular samples in both 2% and 5% w/v gels, but after 28 days both acellular and cell-encapsulating samples retained disc-shaped morphologies and gel-like spectra. The results demonstrate that although at an early stage cells influence the mechanical properties of encapsulating alginate, over a longer period of culture, the hydrogels retain sufficient mechanical integrity to exhibit gel-like properties. This allows sustained immobilization of the cells at the desired location in vivo where they can produce extracellular matrix and growth factors to expedite the healing process.
Knee surgery, sports traumatology, arthroscopy : official journal of the ESSKA, 2015
The popliteus tendon is crucial to postero-lateral stability and prone to iatrogenic injury intra... more The popliteus tendon is crucial to postero-lateral stability and prone to iatrogenic injury intra-operatively. Its role in the stability of the replaced knee remains contentious. The aim of this study was to use computer navigation to quantify the effect of popliteus sectioning on the 'envelope of laxity' (EoL) offered by a posterior-stabilised (PS) total knee arthroplasty (TKA) and compare with that of the native knee. Loaded cadaveric legs were mounted on a purpose built rig. EoL was measured in 3 degrees of freedom using computer navigation. Knees were subjectively stressed in varus/valgus, internal/external rotation and anterior draw. This was performed preoperatively, during TKA and after sectioning of the popliteus tendon. Real-time data were recorded at 0°, 30°, 60° and 90° of flexion as the operating surgeon stressed the knee in 3 degrees of freedom to its subjective endpoint. Mixed-effect modelling was used to quantify the effects of intervention on degree of laxity...
The aim of this review was to identify a reliable sequential medial release protocol for restorat... more The aim of this review was to identify a reliable sequential medial release protocol for restoration of soft tissue balance in total knee arthroplasty of the varus osteoarthritic knee and to allow for improved intraoperative decision-making. Current medial release sequences and applicability based upon pre-operative deformity have been reviewed. Furthermore, risks associated with over release, and the necessity of medial release, are discussed. The different medial release sequences are discussed in relation to pre-operative deformity, along with potential complications associated with medial release. It was found that release sequences may include the deep and superficial components of the medial collateral ligament, the posteromedial capsule, the posterior oblique ligament, the pes anserinus (pes A), and tendons of the semimembranosus and medial gastrocnemius muscle. The sequences described were found to vary substantially between studies, and very few studies had systematically quantified the effect of each release on balance. While medial release is the standard intraoperative mode of balancing, there is a lack of evidence to support current methods. The correct method for defining intraoperatively the sequence, extent and magnitude of releases required remains ill-defined. It could be argued that the classic extensive medial release may be unnecessary and may be associated with iatrogenic injury to the pes A and saphenous nerve, instability and abnormal knee kinematics. Minimal medial release may allow for improved soft tissue balancing leading ultimately to improved functional outcome. V (expert opinion).
2010 4th International Conference on Bioinformatics and Biomedical Engineering, 2010
Tactile sensation is important for the performance of daily tasks and prevention of injury. Full ... more Tactile sensation is important for the performance of daily tasks and prevention of injury. Full thickness wounds of greater than 4cm diameter will not spontaneously heal. Tissue engineering can be used to replace the lost skin, but this skin will often fail to be reinnervated. This results in loss of tactile sensations. Here we have developed a novel, silicon based
Patients with total knee arthroplasties (TKAs) continue to report dissatisfaction in functional o... more Patients with total knee arthroplasties (TKAs) continue to report dissatisfaction in functional outcome. Stability is a major factor contributing to functionality of TKAs. Implants with single-radius (SR) femoral components are proposed to increase stability throughout the arc of flexion. Using computer navigation and loaded cadaveric legs, we characterized the "envelope of laxity" (EoL) offered by a SR cruciate retaining (CR)-TKA compared with that of the native knee through the arc of flexion in terms of anterior drawer, varus/valgus stress, and internal/external rotation. In both the native knee and the TKA laxity increased with increasing knee flexion. Laxities measured in the three planes of motion were generally comparable between the native knee and TKA from 0° to 110° of flexion. Our results indicate that the SR CR-TKA offers appropriate stability in the absence of soft tissue deficiency.
The potential of cell therapy for the regeneration of diseased and damaged tissues is now widely ... more The potential of cell therapy for the regeneration of diseased and damaged tissues is now widely -recognized. As a consequence there is a demand for the development of novel systems that can deliver cells to a particular location, maintaining viability, and then degrade at a predictable rate to release the cells into the surrounding tissues. Hydrogels have attracted much attention in this area, as the hydrogel structure provides an environment that is akin to that of the extracellular matrix. One widely investigated hydrogel is alginate, which has been used for cell encapsulation for more than 30 years. Alginate gels have the potential to be used as 3D cell culture systems and as prosthetic materials, both are applied to regeneration of the cornea. Here, we describe an alginate-based process that has been used for encapsulation of mammalian cells including corneal cells, with high levels of viability, and which allows subsequent retrieval of cell cultures for further characterization.
Vascularization of engineered or damaged tissues is essential to maintain cell viability and prop... more Vascularization of engineered or damaged tissues is essential to maintain cell viability and proper tissue function. Revascularization of the left ventricle (LV) of the heart after myocardial infarction is particularly important, since hypoxia can give rise to chronic heart failure due to inappropriate remodeling of the LV after death of cardiomyocytes (CMs). Fibroblasts can express vascular endothelial growth factor (VEGF), which plays a major role in angiogenesis and also acts as a chemoattractant and survival factor for CMs and cardiac progenitors. In this in vitro model study, mouse NIH 3T3 fibroblasts encapsulated in 2% w/v Ca-alginate were shown to remain viable for 150 days. Semiquantitative reverse transcription-polymerase chain reaction and immunohistochemistry demonstrated that over 21 days of encapsulation, fibroblasts continued to express VEGF, while enzyme-linked immunosorbent assay showed that there was sustained release of VEGF from the Ca-alginate during this period. The scaffold degraded gradually over the 21 days, without reduction in volume. Cells released from the Ca-alginate at 7 and 21 days as a result of scaffold degradation were shown to retain viability, to adhere to fibronectin in a normal manner, and continue to express VEGF, demonstrating their potential to further contribute to maintenance of cardiac function after scaffold degradation. This model in vitro study therefore demonstrates that fibroblasts encapsulated in Ca-alginate provide sustained release of VEGF.
Background: Tibiofemoral instability is a common reason for total knee arthroplasty failure, and ... more Background: Tibiofemoral instability is a common reason for total knee arthroplasty failure, and may be attributed to soft tissue deficiency and incorrect ligament balancing. There are many different designs of implant with varying levels of constraint to overcome this instability; however there is little advice for surgeons to assess which is suitable for a specific patient, and soft tissue balance testing during arthroplasty is very subjective. Method: The current theories on primary and secondary soft tissue restraints to anterior/ posterior, varus/ valgus, and internal/ external rotational motion of the knee are discussed. The paper reviews biomechanics literature to evaluate instability in the intact and implanted knee. Findings: The paper highlights important intra-and extra-capsular structures in the knee and describes the techniques used by clinicians to assess instability perioperatively. In vitro cadaveric studies were found to be a very useful tool in comparing different implants and contributions of different soft tissues. Interpretation: In vitro cadaveric studies can be utilised in helping less experienced surgeons with soft tissue releases and determining the correct implant. For this to happen, more biomechanical studies must be done to show the impact of release sequences on implanted cadavers, as well as determining if increasingly constrained implants restore the stability of the knee to pre-deficient conditions.
There has been a consistent increase in the mean life expectancy of the population of the develop... more There has been a consistent increase in the mean life expectancy of the population of the developed world over the past century. Healthy life expectancy, however, has not increased concurrently. As a result we are living a larger proportion of our lives in poor health and there is a growing demand for the replacement of diseased and damaged tissues. While traditionally tissue grafts have functioned well for this purpose, the demand for tissue grafts now exceeds the supply. For this reason, research in regenerative medicine is rapidly expanding to cope with this new demand. There is now a trend towards supplying cells with a material in order to expedite the tissue healing process. Hydrogel encapsulation provides cells with a three dimensional environment similar to that experienced in vivo and therefore may allow the maintenance of normal cellular function in order to produce tissues similar to those found in the body. In this review we discuss biopolymeric gels that have been used for the encapsulation of mammalian cells for tissue engineering applications as well as a brief overview of cell encapsulation for therapeutic protein production. This review focuses on agarose, alginate, collagen, fibrin, hyaluronic acid and gelatin since they are widely used for cell encapsulation. The literature on the regeneration of cartilage, bone, ligament, tendon, skin, blood vessels and neural tissues using these materials has been summarised.
There is a clinical need for effective alternative skin replacements to autografts, allografts an... more There is a clinical need for effective alternative skin replacements to autografts, allografts and xenografts. In this thesis a bi-layer skin graft was developed by encapsulation of fibroblasts in calcium-alginate hydrogel and culture of keratinocytes on the surface. Initially, the use of 5% and 2% w/v alginate hydrogels were investigated. Both scaffolds maintained fibroblast viability for at least 150 days encapsulation and caused reversible mitotic and catabolic inhibition, as assessed by fluorescent staining, immunochemistry and the thiazolyl blue assay. Sustained expression of angiogenic factors such as vascular endothelial growth factor, interleukin 6 and nerve growth factor were seen by fibroblasts encapsulated in both scaffolds, by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay. Histological staining demonstrated that following degradation of the scaffolds, fibroblasts secreted ECM to facilitate dermal repair. Comparison of degradation of the scaffolds over time by measuring release of calcium, and changes in rheological properties, morphology and mass, indicated that 5% w/v alginate hydrogel degraded more slowly and was preferable to 2% w/v alginate hydrogel. Fibroblasts encapsulated in 5% w/v alginate hydrogel were shown to express keratinocyte growth factor by RT-PCR, to support keratinocyte proliferation and differentiation, and keratinocytes cultured on the 5% w/v alginate hydrogel surface were seen to form multi-layered epidermal structures by histology, immunostaining and RT-PCR.
Limiting cell proliferation without reducing cell viability for in vivo tissue engineering applic... more Limiting cell proliferation without reducing cell viability for in vivo tissue engineering applications is important in co-culture applications where the growth of one cell type must be inhibited to prevent overgrowth of the scaffold at the expense of another cell type. Also, it is vital for maintaining viability of cells in large constructs before vascularisation occurs. In this study we have shown by means of the Thiazolyl blue (MTT) assay and immuno-staining for proliferating cell nuclear antigen (PCNA) that encapsulating fibroblasts in 2% and 5% w/v calcium-alginate at a density of 7.5 Â 10 5 cells/ml as uniformly dispersed entities, enabled cells to maintain viability and caused a reversible mitotic inhibition. Alginate encapsulation also caused reversible metabolic inhibition as demonstrated by the MTT assay and fluorescent staining for mitochondrial membrane potential. Histological evaluation of the alginate constructs containing fibroblasts showed that mitotic and metabolic inhibition was possibly due to cell isolation during the first five weeks of culture. The alginate scaffold degraded with time releasing encapsulated fibroblasts. Upon implantation to a wound site this should ensure that encapsulated cells are able to replace the damaged tissue after sufficient proliferation of the co-cultured cell type or sufficient vascularisation of the construct.
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Papers by Nicola Hunt