Biomedical subjects

Anthony J Freemont

Publications and source records attributed to Anthony J Freemont.

16 recordsLinked to original sources

PubMed2006

The differentiation of bone marrow mesenchymal stem cells into chondrocyte-like cells on poly-L-lactic acid (PLLA) scaffolds.

While intervertebral disc (IVD) degeneration is associated with the majority of cases of low back pain, current treatments are symptomatic rather than curative. Tissue engineering offers a treatment that both cures the problem of disc degeneration and restores normal disc function. One of the major problems for any tissue engineering strategy, however, is ensuring that both the cells and matrices used are suitable for the target tissue. In this study, we have developed and studied a potential system for tissue engineering of the nucleus pulposus (NP) of the severely degenerate IVD. While cells from degenerate discs are not suitable for tissue engineering, bone-marrow-derived mesenchymal stem cells, which are capable of differentiating into chondrocyte-like cells such as those found within the NP of the disc, offer a potential source of cells. We have used transfection with adenoviral SOX-9, a transcription factor involved in differentiation of MSCs along the chondrogenic lineage, combined with culture in a specialised medium, to differentiate monolayer MSCs to NP-like (chondrocyte-like) cells, as shown by real-time quantitative polymerase chain reaction for NP-marker genes. We have also replicated these findings on porous, biodegradable three-dimensional (3D) poly-l-lactic acid scaffolds and shown expression and deposition of NP matrix markers such as type II collagen and aggrecan. We are therefore proposing pre-differentiation of human MSCs and seeding on porous, biodegradable 3D synthetic polymer scaffolds as a realistic tissue engineering strategy for regeneration of the degenerate human IVD.

Adenoviridae↗

PubMed2006

A preliminary in vitro study into the use of IL-1Ra gene therapy for the inhibition of intervertebral disc degeneration.

Conventional therapies for low back pain (LBP) are purely symptomatic and do not target the cause of LBP, which in approximately 40% of cases is caused by degeneration of the intervertebral disc (DIVD). Targeting therapies to inhibit the process of degeneration would be a potentially valuable treatment for LBP. There is increasing evidence for a role for IL-1 in DIVD. A natural inhibitor of IL-1 exists, IL-1Ra, which would be an ideal molecular target for inhibiting IL-1-mediated effects involved in DIVD and LBP. In this study, the feasibility of ex vivo gene transfer of IL-1Ra to the IVD was investigated. Monolayer and alginate cultures of normal and degenerate human intervertebral disc (IVD) cells were infected with an adenoviral vector carrying the IL-1Ra gene (Ad-IL-1Ra) and protein production measured using an enzyme-linked immunosorbent assay. The ability of these infected cells to inhibit the effects of IL-1 was also investigated. In addition, normal and degenerate IVD cells infected with Ad-IL-1Ra were injected into degenerate disc tissue explants and IL-1Ra production in these discs was assessed. This demonstrated that both nucleus pulposus and annulus fibrosus cells infected with Ad-IL-1Ra produced elevated levels of IL-1Ra for prolonged time periods, and these infected cells were resistant to IL-1. When the infected cells were injected into disc explants, IL-1Ra protein expression was increased which was maintained for 2 weeks of investigation. This in vitro study has shown that the use of ex vivo gene transfer to degenerate disc tissue is a feasible therapy for the inhibition of IL-1-mediated events during disc degeneration.

Adenoviridae↗

PubMed2006

A comparative study of neovascularisation in atherosclerotic plaques using CD31, CD105 and TGF beta 1.

OBJECTIVES: This study aims to identify plaque neovascularisation using antibodies to CD31, CD105 and TGFbeta1, and to compare their patterns of expression. METHODS: Tissue expression of CD31, CD105 and TGFbeta1 was examined immunohistologically in atherosclerotic plaques from 53 patients who had undergone carotid endarterectomy and in 10 controls. RESULTS: CD31 was observed in a proportion of the microvessels within atheroma. The expression of CD105 was barely visible in normal arteries, but was markedly enhanced in atherosclerotic plaques. The vast majority of the microvessels in atheroma were positive for CD105 with pronounced expression around the periphery of the lipid core. In consecutive sections, microvessels showing negative staining for CD31 were positive for CD105. Although TGFbeta1 was seen in the thickened intima, it was more strongly expressed in well-formed fibrous plaques. Consecutive sections showed that some microvessels were stained by both CD105 and TGFbeta1, but in certain areas microvessels were exclusively CD105 positive. CONCLUSIONS: These observations highlight the distinctive expression patterns of CD31, CD105 and TGFbeta1, suggesting their specific roles in the development of atherosclerotic plaques. CD105 is almost universally expressed in microvessels within the atheroma and is therefore a better vascular marker than CD31 and TGFbeta1for assessing neovascularisation in atherosclerotic plaques.

Antigens, CD↗

PubMed2005

The differential expression of VEGF, VEGFR-2, and GLUT-1 proteins in disease subtypes of systemic sclerosis.

Our aim was to evaluate (a) whether there is differential expression of the endothelial regulator vascular endothelial growth factor (VEGF), its receptor (VEGFR-2), and the hypoxia-associated glucose transporter molecule, GLUT-1, in skin biopsies from different disease subtypes of systemic sclerosis (SSc) and (b) whether they associate with dermal calcinosis, a significant complication of SSc. Skin punch biopsies were taken from the forearms of 66 SSc patients including 18 with limited cutaneous disease without calcinosis (lcSSc), 23 with calcinosis (lcSSc/cal), and 25 with diffuse cutaneous disease (dcSSc) and from 12 healthy control subjects. The histological appearance of the skin was graded as G0 (normal), G1 (dermal edema), or G2 or G3 (increasing fibrotic changes). Immunohistochemistry was performed with antibodies to VEGF, VEGFR-2, and GLUT-1. Staining was assessed in the epidermis, microvessels, and fibroblasts. The Kruskal-Wallis 1-way analysis of variance was used to compare the data between disease groups. VEGF protein was located in the epidermis and in dermal endothelial cells, pericytes, fibroblasts, and inflammatory cells. In dcSSc only, there was a significant increase in VEGF staining intensity in the keratinocytes and pericytes and the lowest percentage of microvessels with VEGF-positive endothelial cells. GLUT-1 protein was located in the epidermis, erythrocytes, and perineurium. In both lcSSc/cal and dcSSC, but not lcSSc, there were significant increases in GLUT-1 staining intensity of keratinocytes. We propose that in patients with dcSSc, there is a net increase in unbound VEGF in skin that may account for the raised levels of VEGF in serum reported by others. Increased GLUT-1 expression in lcSSc/cal and dcSSc indicates that hypoxia is an associated factor.

Adult↗

PubMed2005

Intervertebral disc cell-mediated mesenchymal stem cell differentiation.

Low back pain is one of the largest health problems in the Western world today, and intervertebral disc degeneration has been identified as a main cause. Currently, treatments are symptomatic, but cell-based tissue engineering methods are realistic alternatives for tissue regeneration. However, the major problem for these strategies is the generation of a suitable population of cells. Adult bone marrow-derived mesenchymal stem cells (MSCs) are undifferentiated, multipotent cells that have the ability to differentiate into a number of cell types, including the chondrocyte-like cells found within the nucleus pulposus (NP) of the intervertebral disc; however, no method exists to differentiate these cells in an accessible monolayer environment. We have conducted coculture experiments to determine whether cells from the human NP can initiate the differentiation of human MSCs with or without cell-cell contact. Fluorescent labeling of the stem cell population and high-speed cell sorting after coculture with cell-cell contact allowed examination of individual cell populations. Real-time quantitative polymerase chain reaction showed significant increases in NP marker genes in stem cells when cells were cocultured with contact for 7 days, and this change was regulated by cell ratio. No significant change in NP marker gene expression in either NP cells or stem cells was observed when cells were cultured without contact, regardless of cell ratio. Thus, we have shown that human NP and MSC coculture with contact is a viable method for generating a large population of differentiated cells that could be used in cell-based tissue engineering therapies for regeneration of the degenerate intervertebral disc.

Adolescent↗

PubMed2005

Lineage plasticity and cell biology of fibrocartilage and hyaline cartilage: its significance in cartilage repair and replacement.

Cartilage repair is a major goal of modern tissue engineering. To produce novel engineered implants requires a knowledge of the basic biology of the tissues that are to be replaced or reproduced. Hyaline articular cartilage and meniscal fibrocartilage are two tissues that have excited attention because of the frequency with which they are damaged. A basic strategy is to re-engineer these tissues ex vivo by stimulating stem cells to differentiate into the cells of the mature tissue capable of producing an intact functional matrix. In this brief review, the sources of cells for tissue engineering cartilage and the culture conditions that have promoted differentiation are discussed within the context of natural cartilage repair. In particular, the role of cell density, cytokines, load, matrices and oxygen tension are discussed.

Biomechanical Phenomena↗

PubMed2005

The role of interleukin-1 in the pathogenesis of human intervertebral disc degeneration.

In this study, we investigated the hypotheses that in human intervertebral disc (IVD) degeneration there is local production of the cytokine IL-1, and that this locally produced cytokine can induce the cellular and matrix changes of IVD degeneration. Immunohistochemistry was used to localize five members of the IL-1 family (IL-1alpha, IL-1beta, IL-1Ra (IL-1 receptor antagonist), IL-1RI (IL-1 receptor, type I), and ICE (IL-1beta-converting enzyme)) in non-degenerate and degenerate human IVDs. In addition, cells derived from non-degenerate and degenerate human IVDs were challenged with IL-1 agonists and the response was investigated using real-time PCR for a number of matrix-degrading enzymes, matrix proteins, and members of the IL-1 family. This study has shown that native disc cells from non-degenerate and degenerate discs produced the IL-1 agonists, antagonist, the active receptor, and IL-1beta-converting enzyme. In addition, immunopositivity for these proteins, with the exception of IL-1Ra, increased with severity of degeneration. We have also shown that IL-1 treatment of human IVD cells resulted in increased gene expression for the matrix-degrading enzymes (MMP 3 (matrix metalloproteinase 3), MMP 13 (matrix metalloproteinase 13), and ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs)) and a decrease in the gene expression for matrix genes (aggrecan, collagen II, collagen I, and SOX6). In conclusion we have shown that IL-1 is produced in the degenerate IVD. It is synthesized by native disc cells, and treatment of human disc cells with IL-1 induces an imbalance between catabolic and anabolic events, responses that represent the changes seen during disc degeneration. Therefore, inhibiting IL-1 could be an important therapeutic target for preventing and reversing disc degeneration.

Adult↗

PubMed2005

Expression of receptors for putative anabolic growth factors in human intervertebral disc: implications for repair and regeneration of the disc.

Low back pain (LBP) is a common, debilitating and economically important disorder. Current evidence implicates loss of intervertebral disc (IVD) matrix consequent upon 'degeneration' as a major cause of LBP. Degeneration of the IVD involves increases in degradative enzymes and decreases in the extracellular matrix (ECM) component in a process that is controlled by a range of cytokines and growth factors. Studies have suggested using anabolic growth factors to regenerate the normal matrix of the IVD, hence restoring disc height and reversing degenerative disc disease. However, for such therapies to be successful it is vital that the target cells (i.e. the disc cells) express the appropriate receptors. This immunohistochemical study has for the first time investigated the expression and localization of four potentially beneficial growth factor receptors (i.e. TGFbetaRII, BMPRII, FGFR3 and IGFRI) in non-degenerate and degenerate human IVDs. Receptor expression was quantified across regions of the normal and degenerate disc and showed that cells of the nucleus pulposus (NP) and inner annulus fibrosus (IAF) expressed significantly higher levels of the four growth factor receptors investigated. There were no significant differences between the four growth factor expression in non-degenerate and degenerate biopsies. However, expression of TGFbetaRII, FGFR3 and IGFRI, but not BMP RII, were observed in the ingrowing blood vessels that characterize part of the disease aetiology. In conclusion, this study has demonstrated the expression of the four growth factor receptors at similar levels in the chondrocyte-like cells of the NP and IAF in both non-degenerate and degenerate discs, implicating a role in normal disc homeostasis and suggesting that the application of these growth factors to the degenerate human IVD would stimulate matrix production. However, the expression of some of the growth factor receptors on ingrowing blood vessels might be problematic in a therapeutic approach.

Adult↗

PubMed2004

Studies of human intervertebral disc cell function in a constrained in vitro tissue culture system.

STUDY DESIGN: This is a laboratory-based study examining a novel in vitro culture system for intervertebral disc tissue. OBJECTIVES: Address the hypothesis that "the novel culture system will preserve intervertebral disc tissue matrix and cell function and prevent cellular apoptosis for periods up to 21 days." SUMMARY OF BACKGROUND DATA: Studies of cell function in human intervertebral disc tissue are scarce. In vivo study of human intervertebral disc cells remains impracticable; in situ molecular biology in histologic sections lacks a dynamic dimension; and as for in vitro studies, cell culture often lacks physiologic relevance and explant cultures are subject to loss of tissue integrity and altered cell behavior. There is a biologic and therapeutic need for a satisfactory explant culture system for studying human intervertebral disc tissue in a controlled environment. METHODS: Samples of human intervertebral disc tissue, obtained at surgery, were examined for a number of tissue and cell parameters immediately after excision (controls) and following culture of tissue samples either in a plastic ring or unconstrained in tissue culture medium for up to 3 weeks. Data were compared between cultured tissue and controls. RESULTS: By comparison with control tissue, unconstrained explants swelled, tissue structure was disturbed, and there were profound changes in cell function. By contrast, tissue cultured in plastic rings maintained tissue structure, and after 3 weeks, the cellular parameters were the same as in controls. CONCLUSIONS: This is the first reported system to preserve cell function of human discal explants for long periods in tissue culture. It will be a useful tool for a wide range of investigations of intervertebral disc biology that have not hitherto been possible.

Adult↗

PubMed2004

Localization of degradative enzymes and their inhibitors in the degenerate human intervertebral disc.

The histological and biochemical changes that occur in the extracellular matrix of the intervertebral disc (IVD) during ageing and degeneration have been investigated extensively. However, the mechanisms behind these changes are not fully understood. A number of studies have suggested the involvement of matrix metalloproteinases (MMPs) and ADAMTS in IVD degeneration, but few have localized the site of production of these enzymes to the cells of the degenerate disc. This study uses immunohistochemical techniques to localize and quantify the production of degrading enzymes (MMPs 1, 3, and 13, and ADAMTS 4) and their inhibitors (TIMPS 1, 2, and 3) within non-degenerate and degenerate discs of varying severity of degeneration. In all discs investigated, the cells that produced the enzymes and their inhibitors were the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus (AF), with little immunopositivity in the outer AF. Non-degenerate discs showed low numbers of cells expressing the degradative enzymes MMP 1 and ADAMTS 4, suggesting a role for these enzymes in normal homeostasis. No MMP 3 or MMP 13 immunopositivity was observed in non-degenerate discs. In degenerate discs, the number of cells immunopositive for MMPs 1, 3, 13 and ADAMTS 4 increased with the severity of degeneration. This increase in degrading enzymes was also accompanied by increases in the number of cells immunopositive for TIMPs 1 and 2 but not TIMP 3. This study highlights that although the expression of a number of MMPs increases with degeneration, this is accompanied by an increase in their inhibitors. However, the increase in the number of cells immunoreactive for ADAMTS 4 with increasing degeneration was not paralleled by a rise in its inhibitor TIMP 3. This finding indicates that the aggrecanases, rather then the MMPs, are a possible therapeutic target for the inhibition of disc degeneration.

ADAM Proteins↗

PubMed2004

Cathepsin L-deficient mice exhibit abnormal skin and bone development and show increased resistance to osteoporosis following ovariectomy.

The role of cathepsin L in normal physiological processes was assessed using cathepsin L homozygous knockout mice (B6;129-Ctsl(tm1Alpk)). These mice were generated using gene targeting in embryonic stem cells. Null mice fail to express mRNA and protein to cathepsin L. They developed normally and were fertile. The distinct phenotypic change exhibited was a progressive hair loss, culminating in extensive alopecia by 9 months of age. Histological analysis of the skin from homozygous mice revealed diffuse epithelial hyperplasia, hypotrichosis, hair shaft fragmentation and utricle formation. These findings provide evidence that cathepsin L is involved in the regulation of epithelial cell proliferation and differentiation in the skin. In addition, the role of cathepsin L in bone remodelling was evaluated. Using bone histomorphometric measurements, trabecular, but not cortical, bone volume was found to be significantly decreased in the cathepsin L heterozygote and homozygote mice compared to the wild-type mice. Following ovariectomy, it was observed that loss of trabecular bone, the most metabolically active component of bone, occurred to a lesser extent in homozygote, and heterozygote mice, than was seen in wild-type mice. These observations suggest that cathepsin L is likely to have a role in controlling bone turnover during normal development and in pathological states.

Animals↗

PubMed2003

Bone histopathology and densitometry comparison between cyclosporine a monotherapy and prednisolone plus azathioprine dual immunosuppression in renal transplant patients.

BACKGROUND: No study has compared the bone histopathologic findings in renal transplant patients receiving cyclosporine A (CsA) monotherapy with those in patients receiving a non-CsA regimen. The aim of this study was to compare bone densitometry and histomorphometry findings in patients receiving CsA monotherapy versus those receiving azathioprine + prednisolone dual therapy. METHODS: A bone biopsy and densitometry were performed in 13 patients receiving CsA monotherapy and 12 patients receiving azathioprine + prednisolone, who had been on these regimens since the time of transplantation. Fourteen men and 11 women, age 51+/-12 years, with 140+/-75 months since transplantation, were included. RESULTS: A low bone mineral density (BMD) was observed in patients on both immunosuppressive schemes-most notably at the distal radius and less significantly at the lumbar spine. No significant differences in BMD were observed between immunosuppressive groups. Histopathologic analysis of the group as a whole revealed mixed uremic bone disease in 42%, adynamic bone in 29%, hyperparathyroid disease in 17%, and normal bone in 12%. Patients showed a slight increase in osteoclast number and function, decreased osteoblast number and function, and retardation of dynamic parameters. No differences in histopathologic diagnosis or histomorphometric findings were observed between the immunosuppressive therapy groups. In addition to the immunosuppressive drugs, male gender and old age negatively affected bone mass. CONCLUSIONS: Both prednisolone and CsA were associated with slight osteoclast stimulation and osteoblast suppression and marked retardation of mineral apposition and bone formation rates. Both drugs were also associated with reduced BMD at the axial and appendicular skeleton, even though a nonsignificant trend to a better-preserved lumbar spine BMD was observed in the CsA group.

Absorptiometry, Photon↗

PubMed2003

Evidence for cell-specific changes with age in expression of oestrogen receptor (ER) alpha and beta in bone fractures from men and women.

Oestrogen is recognized as important for maintaining bone mass in men and women. Oestrogen receptor (ER) alpha and the recently described ER-beta are both expressed in bone cells, but have different affinities for oestrogen agonists and plant oestrogens, which could be important in developing treatments for bone loss in both men and women. It is unclear, however, which isoform predominates in bone; cell type and age may influence their relative expression. The present study has compared ER-alpha and ER-beta expression in serial sections of human fracture callus from males (n = 19, age range 5-72 years) and females (n = 15, age range 3-86 years) by indirect immunoperoxidase. Fracture callus was used as it can be readily obtained from individuals over a wide age range and contains a variety of bone cells. Antibody specificity was confirmed by western blotting and comparison of immunoreactivity in sections of breast tumour and benign prostate hyperplasia. No gender difference in ER expression was found in bone from individuals less than 40 years old. Proliferative chondrocytes were positive for both isoforms, but few larger hypertrophic cells were immunoreactive. ER-alpha and ER-beta were co-expressed in osteoclasts, suggesting that oestrogen may act directly on these cells. Osteoblasts, osteocytes, and mesenchymal cells also expressed both isoforms. In women over 40 years of age, however, relatively fewer biopsies contained osteocytes positive for ER-alpha and ER-beta. Likewise, the proportions of osteoblasts and mesenchymal cells expressing ER-beta were reduced but ER-alpha remained unaffected. In contrast, in men over 40 years, only the proportion of biopsies containing ER-beta-positive mesenchymal cells was lower. In these older men and women, ER-alpha and ER-beta expression was retained by the small proliferative chondrocytes. These results demonstrate that gender, age, and cell type are important determinants of ER isoform expression in skeletal cells.

Adolescent↗

PubMed2002

Mast cells in the pathogenesis of chronic back pain: a hypothesis.

The pathophysiology of chronic low back pain is poorly understood, mainly because it is difficult to study experimentally or objectively. Recently it has been found that there is a relationship between neovascularization and innervation of the usually avascular and aneural intervertebral disc at the sites of discogenic pain. These data, together with the recognized involvement of mast cells in tissue repair, in the induction of angiogenesis, and in the production of and response to neurotrophic stimuli such as nerve growth factor, has suggested the hypothesis that mast cells may have a causative role in chronic low back pain. If so, the mast cell may represent an attractive therapeutic target.

Connective Tissue↗

PubMed2002

Biochemical measurements in the prediction of histologic subtype of renal transplant bone disease in women.

Renal transplant osteodystrophy encompasses several histologic subtypes. Bone histomorphometric examination reliably distinguishes these groups but is invasive, is time-consuming, and delays diagnosis. Establishing a noninvasive method of correctly predicting histologic subtype in an individual to direct management is an attractive proposition. We identified 19 female renal transplant recipients with histologic evidence of hyperparathyroid bone disease (HPTH) and 14 with adynamic bone (ADB). We evaluated serum osteocalcin and bone-specific alkaline phosphatase as bone formation markers and urinary hydroxyproline (Hypro) and deoxypyridinoline cross-links as bone resorption markers. Mean concentrations for all markers were higher in the HPTH group, reaching significance for Hypro (HPTH, 24.8 +/- 4.2 micromol/mmol creatinine; ADB, 13.2 +/- 5.0 micromol/mmol creatinine; P = 0.01). A cutoff of 16.4 micromol/mmol creatinine for Hypro (Youden's index, 0.65) gave a sensitivity of 93% and specificity and positive predictive value (PPV) of 72% in predicting HPTH. In combination, Hypro greater than 16.4 micromol/mmol creatinine and parathyroid hormone greater than 80 pg/mL gave a specificity of 100%, sensitivity of 32%, and PPV of 100%. Conversely, for predicting ADB, Hypro less than 15.1 micromol/mmol creatinine (Youden's index, 0.45) gave a specificity of 93%, sensitivity of 53%, and PPV of 91%. Hypro less than 15.1 micromol/mmol creatinine plus osteocalcin less than 6.8 microg/L gave a specificity of 84.2%, sensitivity of 64.3%, and PPV of 75%. Significant associations between markers and histomorphometry were evident only for Hypro and osteocalcin (with osteoblast surface) and all markers (except deoxypyridinoline cross-links) with cortical volume. Markers have limited utility in identifying histologic subtype (Hypro was most effective) and, with the exception of Hypro and osteocalcin, showed little association with cell surface markers of bone cell activity.

Adult↗

PubMed2002

Annexin VIII is differentially expressed by chondrocytes in the mammalian growth plate during endochondral ossification and in osteoarthritic cartilage.

Endochondral ossification is the developmental process that leads to the formation and coordinated longitudinal growth of the majority of the vertebrate skeleton. Central to this process is chondrocyte differentiation occurring in the growth plate that lies at the junction between the epiphyseal cartilage and the bone. To identify novel factors involved in this differentiation process, suppression subtractive hybridization was performed to amplify preferentially cDNAs uniquely expressed in fetal bovine growth plate chondrocytes as opposed to epiphyseal chondrocytes. The subtracted product was used to screen a fetal bovine chondrocyte cDNA library. One of the cDNA clones identified encoded the bovine orthologue of annexin VIII, a protein not previously described in the growth plate. Northern and Western blotting confirmed that annexin VIII was expressed by growth plate chondrocytes and not by epiphyseal chondrocytes. Immunohistochemistry of the fetal bovine growth plate identified a gradient of increasing annexin VIII protein from the proliferative to the hypertrophic zone. Immunofluorescence localized annexin VIII largely to the chondrocyte cell membrane. In a preliminary study, we examined the distribution of annexin VIII in normal and osteoarthritic (OA) articular cartilage. In OA cartilage, the protein was located in a subset of mid- to deep zone chondrocytes and in the matrix surrounding these cells; no annexin VIII was detected in normal articular cartilage. Thus annexin VIII is a marker for chondrocyte differentiation during normal endochondral ossification and may act as a marker for cells undergoing inappropriate differentiation in OA.

Amino Acid Sequence↗