The influence of architecture on degradation and tissue ingrowth into three-dimensional poly(lactic-co-glycolic acid) scaffolds in vitro and in vivo

The in vitro and in vivo degradation properties of poly(lactic-co-glycolic acid) (PLGA) scaffolds produced by two different technologies - thermally induced phase separation (TIPS), and solvent casting and particulate leaching (SCPL) were compared. Over 6 weeks, in vitro degradation produced changes in SCPL scaffold dimension, mass, internal architecture and mechanical properties. TIPS scaffolds produced far less changes in these parameters providing significant advantages over SCPL. In vivo results were based on a microsurgically created arteriovenous (AV) loop sandwiched between two TIPS scaffolds placed in a polycarbonate chamber under rat groin skin. Histologically, a predominant foreign body giant cell response and reduced vascularity was evident in tissue ingrowth between 2 and 8 weeks in TIPS scaffolds. Tissue death occurred at 8 weeks in the smallest pores. Morphometric comparison of TIPS and SCPL scaffolds indicated slightly better tissue ingrowth but greater loss of scaffold structure in SCPL scaffolds. Although advantageous in vitro, large surface area:volume ratios and varying pore sizes in PLGA TIPS scaffolds mean that effective in vivo (AV loop) utilization will only be achieved if the foreign body response can be significantly reduced so as to allow successful vascularisation, and hence sustained tissue growth, in pores less than 300 μm. © 2005 Elsevier Ltd. All rights reserved.

Noninvasive in vivo assessment of soft contact lens type on tear film surface quality

To evaluate the effect of soft contact lens type on the in vivo tear film surface quality (TFSQ) on daily disposable lenses and to establish whether two recently developed techniques for noninvasive measurement of TFSQ can distinguish between different contact lens types.

3D-printing of highly uniform CaSiO3 ceramic scaffolds : preparation, characterization and in vivo osteogenesis

Calcium silicate (CaSiO3, CS) ceramics have received significant attention for application in bone regeneration due to their excellent in vitro apatite-mineralization ability; however, how to prepare porous CS scaffolds with a controllable pore structure for bone tissue engineering still remains a challenge. Conventional methods could not efficiently control the pore structure and mechanical strength of CS scaffolds, resulting in unstable in vivo osteogenesis. The aim of this study is to set out to solve these problems by applying a modified 3D-printing method to prepare highly uniform CS scaffolds with controllable pore structure and improved mechanical strength. The in vivo osteogenesis of the prepared 3D-printed CS scaffolds was further investigated by implanting them in the femur defects of rats. The results show that the CS scaffolds prepared by the modified 3D-printing method have uniform scaffold morphology. The pore size and pore structure of CS scaffolds can be efficiently adjusted. The compressive strength of 3D-printed CS scaffolds is around 120 times that of conventional polyurethane templated CS scaffolds. 3D-Printed CS scaffolds possess excellent apatite-mineralization ability in simulated body fluids. Micro-CT analysis has shown that 3D-printed CS scaffolds play an important role in assisting the regeneration of bone defects in vivo. The healing level of bone defects implanted by 3D-printed CS scaffolds is obviously higher than that of 3D-printed b-tricalcium phosphate (b-TCP) scaffolds at both 4 and 8 weeks. Hematoxylin and eosin (H&E) staining shows that 3D-printed CS scaffolds induce higher quality of the newly formed bone than 3D-printed b-TCP scaffolds. Immunohistochemical analyses have further shown that stronger expression of human type I collagen (COL1) and alkaline phosphate (ALP) in the bone matrix occurs in the 3D-printed CS scaffolds than in the 3D-printed b-TCP scaffolds. Considering these important advantages, such as controllable structure architecture, significant improvement in mechanical strength, excellent in vivo osteogenesis and since there is no need for second-time sintering, it is indicated that the prepared 3D-printed CS scaffolds are a promising material for application in bone regeneration.

Problems in the assessment of magnesium depletion in the rat by in vivo 31P NMR

Prior in vitro studies, utilizing 31Pn uclear magnetic resonance (31PN MR) to measure the chemical shift (CT) of 0-ATP and lengthening of the phosphocreatine spin-spin (7"') relaxation time, suggested an assessment of their efficacy in measuring magnesium depletion in vivo. Dietary magnesium depletion (Me$) produced markedly lower magnesium in plasma (0.44 vs 1. I3 mmol/liter) and bone (1 30 vs 190 pmol/g) but much smaller changes in muscle (41 vs 45 pmol/g, P < 0.01), heart (42.5 vs 44.6 prnol/g), and brain (30 vs 32 pmollg). NMR experiments in anesthetized rats in a Bruker 7-T vertical bore magnet showed that in M e $ rats there was a significant change in brain j3-ATP shift (16.15 vs 16.03 ppm, P < 0.05). These chemical shifts gave a calculated free [Mg"] of 0.71 mM (control) and 0.48 mM (MgZ+$). In muscle the change in j3-ATP shift was not significant (Me$ 15.99 ppm, controls 15.96 ppm), corresponding to a calculated free M P of 0.83 and 0.95 mM, respectively. Phosphccreatine Tz (Carr-Purcell, spin-echo pulse sequence) was no different with M e $ in muscle in vivo (surface coil) (M$+$ 136, control 142 ms) or in isolated perfused hearts (Helmholtz coil) (control 83, M e $ 92 ms). 3'P NMR is severely limited in its ability to detect dietary magnesium depletion in vivo. Measurement of j3-ATP shift in brain may allow studies of the effects of interaction in group studies but does not allow prediction of an individual magnesium status.

Preparation, characterisation and in vivo osteogenesis of mesoporous bioactive glasses

Bone defects, especially large bone defects, remain a major challenge in orthopaedic surgery. Autologous bone transplantation is considered the most effective treatment, but insufficient donor tissue, coupled with concerns about donor site morbidity, has hindered this approach in large-scale applications. Alternative approaches include implanting biomaterials such as bioactive glass (BG), which has been widely used for bone defect healing, due to having generally good biocompatibility, and can be gradually biodegraded during the process of new bone formation. Mesoporous bioactive glass (MBG) is a newly developed bioactive glass which has been proven to have enhanced in-vitro bioactivity; however the in-vivo osteogenesis has not been studied. A critical problem in using the bone tissue engineering approach to restore large bone defects is that the nutrient supply and cell viability at the centre of the scaffold is severely hampered since the diffusion distance of nutrients and oxygen for cell survival is limited to 150-200µm. Cobalt ions has been shown to mimic hypoxia, which plays a pivotal role in coupling angiogenesis with osteogenesis in-vivo by activating hypoxia inducing factor-1α (HIF-1α) transcription factor, subsequently initiating the expression of genes associated with tissue regeneration. Therefore, one aim of this study is to investigate the in-vivo osteogenesis of MBG by comparison with BG and β-TCP, which are widely used clinically. The other aim is to explore hypoxia-mimicking biomaterials by incorporating Cobalt into MBG and β-TCP. MBG and β-TCP incorporated with 5% cobalt (5Co-MBG and 5CCP) have also been studied in-vivo to determine whether the hypoxic effect has a beneficial effect on the bone formation. The composition and microstructure of synthesised materials (BG, MBG, 5Co-MBG, 5CCP) were characterised, along with the mesopore properties of the MBG materials. Dissolution and cytotoxicity of the Co-containing materials were also investigated. Femoral samples with defects harvested at 4 and 8 weeks were scanned using micro-CT followed by processing for histology (H&E staining) to determine bone formation. Histology of MBG showed a slower rate of bone formation at 4 weeks than BG, however at 8 weeks it could be clearly seen that MBG had more bone formation. The in-vivo results show that the osteogenesis of MBG reciprocates the enhanced performance shown in-vitro compared to BG. Dissolution study showed that Co ions can be efficiently released from MBG and β-TCP in a controllable way. Low amounts of Co incorporated into the MBG and β-TCP showed no significant cytotoxicity and the Co-MBG powders maintained a mesopore structure although not as highly ordered as pure MBG. Preliminary study has shown that Co incorporated samples showed little to no bone formation, instead incurring high lymphocyte activity. Further studies need to be done on Co incorporated materials to determine the cause for high lymphocyte activity in-vivo, which appear to hinder bone formation. In conclusion, this study demonstrated the osteogenic activity of MBG and provided some valuable information of tissue reaction to Co-incorporated MBG and TCP materials.

Application of a human bone engineering platform to an in vitro and in vivo breast cancer metastasis model

Breast cancer in its advanced stage has a high predilection to the skeleton. Currently, treatment options of breast cancer-related bone metastasis are restricted to only palliative therapeutic modalities. This is due to the fact that mechanisms regarding the breast cancer celI-bone colonisation as well as the interactions of breast cancer cells with the bone microenvironment are not fully understood, yet. This might be explained through a lack of appropriate in vitro and in vivo models that are currently addressing the above mentioned issue. Hence the hypothesis that the translation of a bone tissue engineering platform could lead to improved and more physiological in vitro and in vivo model systems in order to investigate breast cancer related bone colonisation was embraced in this PhD thesis. Therefore the first objective was to develop an in vitro model system that mimics human mineralised bone matrix to the highest possible extent to examine the specific biological question, how the human bone matrix influences breast cancer cell behaviour. Thus, primary human osteoblasts were isolated from human bone and cultured under osteogenic conditions. Upon ammonium hydroxide treatment, a cell-free intact mineralised human bone matrix was left behind. Analyses revealed a similar protein and mineral composition of the decellularised osteoblast matrix to human bone. Seeding of a panel of breast cancer cells onto the bone mimicking matrix as well as reference substrates like standard tissue culture plastic and collagen coated tissue culture plastic revealed substrate specific differences of cellular behaviour. Analyses of attachment, alignment, migration, proliferation, invasion, as well as downstream signalling pathways showed that these cellular properties were influenced through the osteoblast matrix. The second objective of this PhD project was the development of a human ectopic bone model in NOD/SCID mice using medical grade polycaprolactone tricalcium phosphate (mPCL-TCP) scaffold. Human osteoblasts and mesenchymal stem cells were seeded onto an mPCL-TCP scaffold, fabricated using a fused deposition modelling technique. After subcutaneous implantation in conjunction with the bone morphogenetic protein 7, limited bone formation was observed due to the mechanical properties of the applied scaffold and restricted integration into the soft tissue of flank of NOD/SCID mice. Thus, a different scaffold fabrication technique was chosen using the same polymer. Electrospun tubular scaffolds were seeded with human osteoblasts, as they showed previously the highest amount of bone formation and implanted into the flanks of NOD/SCID mice. Ectopic bone formation with sufficient vascularisation could be observed. After implantation of breast cancer cells using a polyethylene glycol hydrogel in close proximity to the newly formed bone, macroscopic communication between the newly formed bone and the tumour could be observed. Taken together, this PhD project showed that bone tissue engineering platforms could be used to develop an in vitro and in vivo model system to study cancer cell colonisation in the bone microenvironment.

The active site residue V266 of Chlamydial HtrA is critical for substrate binding during both in vitro and in vivo conditions

HtrA is a complex, multimeric chaperone and serine protease important for the virulence and survival of many bacteria. Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that is responsible for severe disease pathology. C. trachomatis HtrA (CtHtrA) has been shown to be highly expressed in laboratory models of disease. In this study, molecular modelling of CtHtrA protein active site structure identified putative S1-S3 subsite residues I242, I265, and V266. These residues were altered by site-directed mutagenesis, and these changes were shown to considerably reduce protease activity on known substrates and resulted in a narrower and distinct range of substrates compared to wild type. Bacterial two-hybrid analysis revealed that CtHtrA is able to interact in vivo with a broad range of protein sequences with high affinity. Notably, however, the interaction was significantly altered in 35 out of 69 clones when residue V266 was mutated, indicating that this residue has an important function during substrate binding.

Tracking new directions in the evaluation of postgraduate supervision

This article focuses on the evaluation of thesis supervision and highlights the vast range of problem areas presently documented as significant areas of concern for both graduate students and their supervisors. Additionally, the authors report on a study completed in 1995, which surveyed all Australian universities about current evaluative practices in postgraduate supervision. The conclusion of this study was that the conduct of such evaluations appears to be minimal and is primarily designed to obtain an indicator of the general “health” of a university's postgraduate supervision rather than to foster improved supervisory practices. As part of the same study, the authors conducted University faculty-based student and supervisor focus groups. Key issues emerging from these focus groups were: (1) the importance of relational aspects of supervision as the student communicates over the long term with one or more supervisors; (2) the importance of systematic feedback, monitoring, and evaluation to the supervisory process; and (3) the lack of strategies to facilitate this evaluative feedback process. On the basis of these findings, the authors designed evaluative strategies to facilitate regular ongoing feedback between students and supervisors.

Characterization of in vitro Chlamydia muridarum persistence and utilization in an in vivo mouse model of Chlamydia vaccine

Problem: Chlamydia trachomatis genital tract infections are easily treated with antibiotics, however the majority of infections are asymptomatic and therefore untreated, highlighting the need for a vaccine. Because most infections are asymptomatic, vaccination could potentially be administered to individuals who may have an acute infection at that time. In such individuals the effect of vaccination on the existing infection is unknown; however one potential outcome could be the development of a persistent infection. In vitro chlamydial persistence has been well characterized in various strains, however there have been no reported studies in C. muridarum. Method of Study: We performed ultrastructural characterization, and transcriptome analysis of selected genes. We then used the transcriptional profiles of the selected genes to examine whether intranasal immunization of mice during an active genital infection would induce persistence in the upper reproductive tract of female mice. Results and Conclusions: We found that persistence developed in the oviducts of mice as a result of immunization. This is a significant finding, not only because it is the first time that C. muridarum persistence has been characterized in vitro, but also due to the fact that there is minimal characterization of in vivo persistence of any chlamydial species. This highlights the importance of the timing of vaccination in individuals.

Scaffold percolative efficiency : in vitro evaluation of the structural criterion for electrospun mats

Fibrous scaffolds of engineered structures can be chosen as promising porous environments when an approved criterion validates their applicability for a specific medical purpose. For such biomaterials, this paper sought to investigate various structural characteristics in order to determine whether they are appropriate descriptors. A number of poly(3-hydroxybutyrate) scaffolds were electrospun; each of which possessed a distinguished architecture when their material and processing conditions were altered. Subsequent culture of mouse fibroblast cells (L929) was carried out to evaluate the cells viability on each scaffold after their attachment for 24 h and proliferation for 48 and 72 h. The scaffolds’ porosity, pores number, pores size and distribution were quantified and none could establish a relationship with the viability results. Virtual reconstruction of the mats introduced an authentic criterion, “Scaffold Percolative Efficiency” (SPE), with which the above descriptors were addressed collectively. It was hypothesized to be able to quantify the efficacy of fibrous scaffolds by considering the integration of porosity and interconnectivity of the pores. There was a correlation of 80% as a good agreement between the SPE values and the spectrophotometer absorbance of viable cells; a viability of more than 350% in comparison to that of the controls.

Methods to estimate the size and shape of the unaccommodated crystalline lens in vivo

Purpose. The purpose of this article was to present methods capable of estimating the size and shape of the human eye lens without resorting to phakometry or magnetic resonance imaging (MRI). Methods. Previously published biometry and phakometry data of 66 emmetropic eyes of 66 subjects (age range [18, 63] years, spherical equivalent range [−0.75, +0.75] D) were used to define multiple linear regressions for the radii of curvature and thickness of the lens, from which the lens refractive index could be derived. MRI biometry was also available for a subset of 30 subjects, from which regressions could be determined for the vertex radii of curvature, conic constants, equatorial diameter, volume, and surface area. All regressions were compared with the phakometry and MRI data; the radii of curvature regressions were also compared with a method proposed by Bennett and Royston et al. Results. The regressions were in good agreement with the original measurements. This was especially the case for the regressions of lens thickness, volume, and surface area, which each had an R2 > 0.6. The regression for the posterior radius of curvature had an R2 < 0.2, making this regression unreliable. For all other regressions we found 0.25 < R2 < 0.6. The Bennett-Royston method also produced a good estimation of the radii of curvature, provided its parameters were adjusted appropriately. Conclusions. The regressions presented in this article offer a valuable alternative in case no measured lens biometry values are available; however care must be taken for possible outliers.