Bioactive glass-induced osteoblast differentiation: a noninvasive spectroscopic study.

Here, we report on a rapid, noninvasive biophotonics system using Raman spectroscopy to detect real-time biochemical changes in foetal osteoblasts (FOBs) following exposure to 45S5 Bioglass (BG)-conditioned media. Bio-Raman spectroscopy, combined with multivariate statistical analysis techniques (principal component analysis and least squares analysis), was able to noninvasively identify biochemical differences in FOBs cultured for different time periods and between FOBs exposed/or not to BG-conditioned media. Gene and protein expression studies were also performed for known markers of osteoblastic differentiation, namely, alkaline phosphatase, bone sialoprotein, and collagen type I. Quantitative RT-PCR confirmed upregulation of genes associated with osteoblast differentiation after exposure to BG-conditioned media. These results suggest that Raman spectroscopy can noninvasively detect biochemical changes in FOBs associated with differentiation. This technique could have important applications in the field of regenerative medicine by enabling rapid characterization of cell or organoid behavior on novel bioactive scaffolds without damage to either cell or biomaterial.

Enhanced differentiation and mineralization of human fetal osteoblasts on PDLLA containing Bioglass composite films in the absence of osteogenic supplements.

This study investigates the cellular response of fetal osteoblasts to bioactive resorbable composite films consisting of a poly-D,L-lactide (PDLLA) matrix and bioactive glass 45S5 Bioglass (BG) particles at three different concentrations (0% (PDLLA), 5% (P/BG5), and 40% (P/BG40)). Using scanning electron microscopy (SEM) we observed that cells were less spread and elongated on PDLLA and P/BG5, whereas cells on P/BG40 were elongated but with multiple protrusions spreading over the BG particles. Vinculin immunostaining revealed similar distribution of focal adhesion contacts on all cells independent of substratum, indicating that all materials permitted cell adhesion. However, when differentiation and maturation of fetal osteoblasts was examined, incorporation of 45S5 BG within the PDLLA matrix was found to significantly (p < 0.05) enhance alkaline phosphatase enzymatic activity and osteocalcin protein synthesis compared to tissue culture polystyrene controls and PDLLA alone. Alizarin red staining indicated extracellular matrix mineralization on both P/BG5 and P/BG40, with significantly more bone nodules formed than on PDLLA. Real time RT-PCR revealed that expression of bone sialoprotein was also affected by the BG containing films compared to controls, whereas expression of Collagen Type I was not influenced. By performing these investigations in the absence of osteogenic factors it appears that the incorporation of BG stimulates osteoblast differentiation and mineralization of the extracellular matrix, demonstrating the osteoinductive capacity of the composite.

Biocompatibility of poly-DL-lactic acid (PDLLA) for lung tissue engineering.

This study explores the possibility of growing lung cells on poly-DL-lactic acid (PDLLA) scaffolds, with a view to in future engineer pulmonary tissue for human implantation. As a first step in this process, the ability of PDLLA to maintain the growth of lung epithelium is tested using a robust cell line. Poly-DL-lactic acid has been investigated in two forms, as planar discs and as 3-D foams, and it has been demonstrated that PDLLA is not only nontoxic to pneumocytes but it also actively supports their growth. The initial findings suggest that the material is an appropriate matrix for engineering of distal lung tissue.

Repopulation of human pulmonary epithelium by bone marrow cells: a potential means to promote repair.

After lung injury and damage to the alveolar epithelium, the underlying basement membranes become exposed. Proliferation of type II pneumocytes and their differentiation to the type I phenotype have been considered to be the mechanism by which repopulation of the alveolar epithelium occurs. A growing body of evidence has shown that tissues can be repaired by cells acquired via the circulation. For the lung, bone marrow stem cells have been shown in mice to regenerate epithelium as well as give rise to the expected mesodermal derivatives. We hypothesized that extrapulmonary cells, including those from the bone marrow, can contribute to the reepithelialization of human alveoli. To investigate this, we examined samples of peripheral lung from patients who had undergone cross-gender transplantation of lung or bone marrow. Thus, archival blocks of peripheral lung were analyzed from male patients (surgical samples, n = 8) who had received a lung transplant from a female donor and female patients (postmortem samples, n = 3) who had male bone marrow transplants. In both cases, male cells were identified in the female lungs by Y chromosome in situ hybridization. Male cells could be identified in the alveolar epithelium where, in the better preserved, transplanted lungs, it was possible to show that some had differentiated to type II pneumocytes. In addition, Y chromosomes were found to be widespread in cells of mesenchymal lineage, including macrophages and endothelial cells. Concomitant visualization of Y and X chromosomes, using fluorescence immunolabeling, yielded no evidence of cellular fusion, although the poor quality of the autopsy samples studied meant that the possibility could not be excluded. These observations suggest that, as occurs in rodents, the epithelium of the adult human lung has the capacity to renew itself, using cells recruited from extrapulmonary sources, including the bone marrow. This finding could provide new therapeutic opportunities for a range of pulmonary diseases by providing means to repair the lung and a novel route for gene therapy.

Coculture of embryonic stem cells with pulmonary mesenchyme: a microenvironment that promotes differentiation of pulmonary epithelium.

Coculture of stem/progenitor cells with mature cells or tissues can drive their differentiation toward required lineages. Thus, we hypothesized that coculture of murine embryonic stem (ES) cells with embryonic mesenchyme from distal lung promotes the differentiation of pneumocytes. Murine ES cells were differentiated to embryoid bodies (EBs) and cultured for 5 or 12 days with pulmonary mesenchyme from embryonic day 11.5 or 13.5 murine embryos, in direct contact or separated by a membrane. Controls included EBs cultured alone or with embryonic gut mesenchyme. Histology revealed epithelium-lined channels in directly cocultured EBs, whereas EBs grown alone showed little structural organization. The lining cells expressed cytokeratin and thyroid transcription factor 1, an early developmental marker in pulmonary epithelium. Differentiation of type II pneumocytes specifically was demonstrated by the presence of surfactant protein C (SP-C) in some of the epithelial cells. None of these markers was seen in EBs cultured alone or with embryonic gut mesenchyme. Indirect coculture of EBs with lung mesenchyme resulted in a 14-fold increase in SP-C gene expression. Thus, provision of an appropriate microenvironment, in the form of pulmonary mesenchyme, appears to promote the differentiation of ES cells toward lung epithelium. Our findings may have applications in regenerative medicine strategies and the engineering of lung tissue.

Stem cells.

Stem cells derived from adult and embryonic sources have great therapeutic potential, but much research is still needed before their clinical use becomes commonplace. There is debate about whether adult stem cells can be used instead of those derived from embryos. Rationalisation is needed but can be exercised only once the various cells have been carefully compared and contrasted under appropriate experimental conditions. Some characteristics that might help resolve the issue of cell source can already be applied to the debate. Accessibility is important; some adult cells, such as neural stem cells, are difficult to obtain, at least from living donors. Other factors include the frequency and abundance of adult stem cells and their numbers and potency, which might decline with age or be affected by disease. For embryonic stem cells, ethical concerns have been raised, and the proposed practice of therapeutic cloning tends to be misrepresented in the lay media. For both adult and embryonic stem cells, stability, potential to transmit harmful pathogens or genetic mutations, and risk of forming unwanted tissues or even teratocarcinomas have yet to be fully assessed.

Pantoprazole in severe acid-peptic disease: the effectiveness and safety of 5 years' continuous treatment.

OBJECTIVE: This is our final report on the clinical effectiveness and safety of long-term pantoprazole in patients with severe peptic ulcer or reflux disease during continuous treatment for up to 5 years. METHODS: Patients (n= 150) with peptic ulcer or reflux erosive oesophagitis running an aggressive course or with complications, and refractory to H2-receptor antagonists, were entered into this 5-year programme. Assessment was by serial endoscopy, clinical examination, serum gastrin estimation, gastric mucosal histology and mucosal endocrine cell quantification. RESULTS: Healing results were presented earlier. The estimated rates of remission on maintenance treatment with pantoprazole (n = 115) were 82% at 1 year, 75% at 2 years, 72% at 3 years, 70% at 4 years and 68% at 5 years. Helicobacter pylori infection appeared not to influence the outcome in reflux patients, with roughly two-thirds continuing in remission irrespective of infection. Only four patients had adverse events considered to be definitely related to pantoprazole. Median gastrin levels rose by 1.5-2-fold and were higher in those with H. pylori infection; 13 patients had levels >500 ng/L on at least one occasion, but these high levels were not sustained. Histological changes were more marked in patients infected with H. pylori: chronic gastritis decreased in the antrum and increased in the corpus, which also showed atrophic changes. The total number of endocrine cells in the antrum showed little variation over 60 months but fell by around one-third in the corpus. CONCLUSION: Long-term treatment with pantoprazole is effective and safe.

Apoptosis--a significant cause of bone cell death in osteonecrosis of the femoral head.

Osteonecrosis of the femoral head usually affects young individuals and is responsible for up to 12% of total hip arthroplasties. The underlying pathophysiology of the death of the bone cells remains uncertain. We have investigated nitric oxide mediated apoptosis as a potential mechanism and found that steroid- and alcohol-induced osteonecrosis is accompanied by widespread apoptosis of osteoblasts and osteocytes. Certain drugs or their metabolites may have a direct cytotoxic effect on cancellous bone of the femoral head leading to apoptosis rather than purely necrosis.

Initial observations on the effect of medium composition on the differentiation of murine embryonic stem cells to alveolar type II cells.

The pluripotency and high proliferative index of embryonic stem (ES) cells make them a good potential source of cells for tissue engineering purposes. We have shown that ES cells can be induced to differentiate in vitro into pulmonary epithelial cells (type II pneumocytes) using a serum-free medium designed for the maintenance of mature distal lung epithelial cells in culture (SAGM). However, the resulting cell cultures were heterogeneous. Our aim in this study was to attempt to increase pneumocyte yield and differentiation state by determining which medium components enhance the differentiation of pneumocytes and modifying the medium accordingly. Quantitative RT-PCR was used to measure changes in the expression of a type II pneumocyte-specific gene, surfactant protein C (SPC), in response to alterations in the cell culture medium. Results suggested that most individual SAGM growth factors were inhibitory for type II pneumocyte differentiation, with the largest increases in SPC expression (approximately threefold) being observed upon removal of retinoic acid and triiodothryonine. However, large standard deviations occurred between replicates, illustrating the highly variable nature of ES cell differentiation. Nevertheless, these observations represent an initial step towards achieving directed differentiation of pneumocytes from stem cells that could lead to their purification for tissue engineering purposes.

Osteogenic differentiation of mouse embryonic stem cells: differential gene expression analysis by cDNA microarray and purification of osteoblasts by cadherin-11 magnetically activated cell sorting.

We have previously shown osteogenic differentiation of mouse embryonic stem (ES) cells and temporal enrichment with osteoblastic cells, by stimulation with serum-containing culture medium supplemented with beta-glycerophosphate, ascorbate, and dexamethasone. In our present study we have used similar culture conditions to further investigate osteogenic differentiation of mouse ES cells. Using reverse transcription-polymerase chain reaction (RT-PCR) we demonstrated the expression of genes associated with osteoblast differentiation including the bone matrix protein osteocalcin and the transcription factor Cbfa-1/runx2. Furthermore, results of cDNA microarray analysis, and subsequent RT-PCR analysis of differentiating ES cells after exposure to osteogenic stimuli, revealed a combination of upregulation of genes involved in osteoblast differentiation including osteopontin, HSP-47, and IGF-II coupled with downregulation of genes involved in differentiation of other phenotypes such as the neuroectoderm factor Stra-13. Finally, we have applied magnetically activated cell-sorting methods to ES cell cultures treated with osteogenic stimuli and, using an antibody to cadherin-11, have purified a subpopulation of cells with osteoblastic characteristics.

A histological study of the hph-1 mouse mutant: an animal model of phenylketonuria and infantile hypertrophic pyloric stenosis.

AIM: To quantify the chronological sequence of changes in the morphology and immunoreactivity for neurotransmitters in the pylorus of an animal model of infantile hypertrophic pyloric stenosis and phenylketonuria. METHOD: Thirty specimens of pylorus from hph-1 mice and age/sex matched controls (age range: 10-180 days) were examined using conventional histology and immunohistochemistry for a variety of antigens: protein gene product 9.5, a pan neuronal marker; vasoactive intestinal polypeptide; nitric oxide synthase two antigens coalesced to the same inhibitory neurons in humans; substance P, a potent excitatory neurotransmitter; and calcitonin gene related peptide, a neurotransmitter implicated in the somatic afferent innervation of the stomach. The changes in the morphology of the muscle layers were quantified and statistically analysed for each age group (10, 20, 40, 90 and 180 days). RESULTS: Between 10 and 90 days of age, all muscle layers of the hph-1 mice were hypertrophied, for example, 10 days, hph-1 longitudinal muscle mean diameter = 3.4, control = 1.8; hph-1 circular muscle width = 11.5, control = 4.7. The hph-1 mice were significantly smaller during this period (40 days, hph-1 weight = 10 g, control = 25 g). There was no change in the pattern of expression of the antigens examined within the hph-1 mice compared with the controls. CONCLUSION: Hph-1 mice develop a transient smooth muscle hypertrophy of the pylorus attended by gastric distension and failure to gain weight. These changes resolve as the pyloric muscle hypertrophy resolves.

In situ monitoring of cell death using Raman microspectroscopy.

We investigated the use of Raman microspectroscopy to monitor the molecular changes in human lung carcinoma epithelial cells (A549) when cell death was induced by a toxic chemical. We treated A549 cells with 100 microM Triton X-100 and carried out Raman microspectroscopy measurements in parallel with cell viability and DNA integrity assays at time points of 0, 24, 48, and 72 hours. We found that the important biochemical changes taking place during cell death, such as the degradation of proteins, DNA breakdown, and the formation of lipid vesicles, can be detected with Raman microspectroscopy. A decrease in the intensity of the O-P-O stretching Raman peak corresponding to the DNA molecule phosphate-sugar backbone at 788 cm(-1) indicated DNA disintegration, an observation which was confirmed by DNA integrity analysis. We also found a decrease in the intensity of the Raman peaks corresponding to proteins (1005 cm(-1), 1342 cm(-1)) and an increase in the concentration of lipids (1660 cm(-1), 1303 cm(-1)). These changes are the effects of the complex molecular mechanisms during the induction of cell death, such as protein cleavage due to the activation of caspases, followed by DNA fragmentation.

Spectroscopic study of human lung epithelial cells (A549) in culture: living cells versus dead cells.

The noninvasive analysis of living cells grown on 3-dimensional scaffold materials is a key point in tissue engineering. In this work we show the capability of Raman spectroscopy for use as a noninvasive method to distinguish cells at different stages of the cell cycle and living cells from dead cells. The spectral differences between cells in different stages of the cell cycle are characterized mainly by variations in DNA vibrations at 782, 788, and 1095 cm(-1). The Raman spectrum of dead human lung derived (A549 line) cells indicates the breakdown of both phosphodiester bonds and DNA bases. The most sensitive peak for identifying dead cells is the 788 cm(-1) peak corresponding to DNA Obond;Pbond;O backbone stretching. The magnitude of this peak is reduced by 80% in the spectrum of dead cells. Changes in protein peaks suggest significant conformational changes; for example, the magnitude of the 1231 cm(-1) peak assigned to random coils is reduced by 63% for dead cells. The sharp peak of phenylalanine at 1005 cm(-1) drops to half, indicating a decrease of stable proteins associated with cell death. The differences in the 1190-1385 cm(-1) spectral region also suggest a decrease in the amount of nucleic acids and proteins. Using curve fitting, we quantify these spectral differences that can be used as markers of cell death.

Scaffolds and biomaterials for tissue engineering: a review of clinical applications.

Tissue engineering is a multidisciplinary area of research aimed at regeneration of tissues and restoration of organ function. This is achieved through implantation of cells/tissues grown outside the body or by stimulating cells to grow into an implanted matrix. In this short review, we discuss the use of biomaterials, in the form of scaffolds, for tissue engineering and review clinical applications to otorhinolaryngology-head and neck surgery.

A quantitative study of the neural changes underlying pyloric stenosis in dogs.

This study aimed to quantify the neural changes in congenital pyloric stenosis in dogs and to study the comparative anatomy between this condition in dogs and that in infantile hypertrophic pyloric stenosis. Eight specimens from the pylorus of dogs with pyloric stenosis and six control specimens were examined using conventional histology and immunohistochemistry for a range of neural antigens. The changes in the proportion of nerves immunoreactive for each antigen were quantified and analysed statistically. The morphology of the nerves in the diseased dogs was similar to that in controls. Only vasoactive intestinal peptide was reduced in expression in dogs (median proportion in control dogs 0.57, in diseased dogs 0.17; P = 0.065). This study demonstrates both morphological similarities and significant differences between closely related conditions in dogs, humans and other species.

Gene expression: a review of clinical applications in otorhinolaryngology-head and neck surgery.

Tissue engineering is a multidisciplinary area of research aimed at regeneration of tissues and restoration of function of organs through implantation of cells/tissues grown outside the body or stimulating cells to grow into implanted matrix. In this short review, we aim to examine current techniques in gene expression analysis and their relevant clinical applications to the field of otorhinolaryngology-head and neck surgery.

Stem cells: sources and applications.

Tissue engineering is a multidisciplinary area of research aimed at regeneration of tissues and restoration of function of organs through implantation of cells/tissues grown outside the body, or stimulating cells to grow into implanted matrix. In this short review, some of the most recent developments in the use of stem cells for tissue repair and regeneration will be discussed. There is no doubt that stem cells derived from adult and embryonic sources hold great therapeutic potential but it is clear that there is still much research required before their use is commonplace. There is much debate over adult versus embryonic stem cells and whether both are required. It is probably too early to disregard one or other of these cell sources. With regard to embryonic stem cells, the major concern relates to the ethics of their creation and the proposed practice of therapeutic cloning.

Modulation of hydrogen peroxide induced injury to corneal endothelium by virus mediated catalase gene transfer.

AIM: To examine the effect of catalase gene transfer on survival of corneal endothelial cells (EC) following challenge with hydrogen peroxide (H(2)O(2)) in an ex vivo model of oxidative stress. METHODS: A recombinant adenovirus vector (AdCL) was used to transfer human catalase cDNA into EC of whole thickness rabbit corneas ex vivo. The resulting catalase protein concentration was measured in corneal lysates by ELISA; catalase functional activity in lysates was determined using a H(2)O(2) activity assay. To examine the morphological effects of catalase gene transfer in modulation of H(2)O(2) induced injury, transduced corneas were maintained in ex vivo culture and challenged with H(2)O(2). Laser scanning confocal microscopy was used to image EC injury. Cell density, cell morphology, and ratios of viable to necrotic cells were determined. RESULTS: Following incubation with AdCL, catalase expression reached maximum at 5-7 days. Corneas transduced with AdCL showed increased EC cell survival following challenge with H(2)O(2) on day 3 when compared to null vector control or mock infected corneas. CONCLUSIONS: Ex vivo catalase gene transfer can protect EC from death mediated by H(2)O(2). This gene based approach to the protection of corneal endothelium from oxidative stress may have application in prevention of EC loss in pathological conditions in which H(2)O(2) is involved and in ex vivo donor corneal storage before transplantation.