PubMed Health⌕ Search

Biomedical subjects

Andrew Scutt

Publications and source records attributed to Andrew Scutt.

11 recordsLinked to original sources
PubMed2006

Effect of reduced culture temperature on antioxidant defences of mesenchymal stem cells.

Mesenchymal stem cells (MSC) promise to be valuable therapeutic tools but, due to their low numbers, require considerable in vitro expansion before use. This leads to in vitro aging, the accumulation of intracellular oxidative damage, and subsequently a decreased potential for proliferation and differentiation. Optimised culture conditions might help to reduce oxidative damage in MSC in vitro, and therefore, as reduced temperature is known to reduce oxidative stress in other somatic cells, we have investigated the effect of reduced temperature on rat MSC viability, differentiation, and oxidative damage. Temperature reduction did not affect MSC viability but increased differentiation and reduced apoptosis. Oxidative-damage-related indices were improved; reactive oxide species, nitric oxide, thiobarbituric acid reactive substances, carbonyl, and lipofuscin levels were reduced and glutathione peroxidase and superoxide dimutase levels increased. Levels of antiapoptotic heat shock proteins (HSP-27, -70, and -90) were raised and levels of the proapoptotic HSP-60 reduced. These data demonstrate that culturing MSC at reduced temperature decreases the accumulation of oxidative damage and therefore would probably improve long-term viability and successful engraftment of MSC used for tissue engineering or cell therapeutic purposes.

Animals↗
PubMed2006

hPTH-fragments (53-84) and (28-48) antagonize the stimulation of calcium release and repression of alkaline phosphatase activity by hPTH-(1-34) in vitro.

Different C-terminal fragments of parathyroid hormone (PTH)-(1-84) in blood participate in the regulation of calcium homeostasis by PTH-(1-84), and an antagonizing effect for the large carboxyl-terminal parathyroid hormone (C-PTH)-fragment (7-84) on calcium release has been described in vivo and in vitro. In this study the smaller C-PTH-fragment (53-84) and mid-regional PTH fragment (28-48), which represent discrete areas of activity in the PTH-(7-84) molecule, were assayed for their effects on calcium release and alkaline phosphatase (ALP) activity in a chick bone organ culture system. Neither PTH-(28-48) nor PTH-(53-84) had any effect on calcium release into the medium and both fragments stimulated ALP activity in the bone tissue, suggesting that the cAMP/PKA signalling pathway was not affected by these fragments. However they suppressed the calcium release induced by PTH-(1-34) and attenuated the down regulation of ALP activity caused by PTH-(1-34), suggesting that the effect on the cAMP/PKA signalling pathway may be indirectly. In conclusion, the study shows that the PTH-fragments (53-84) and (28-48) antagonize the PTH-(1-34) induced effects on calcium release and inhibition of ALP activity in a chick bone organ culture system.

Alkaline Phosphatase↗
PubMed2006

Glucocorticoids inhibit tenocyte proliferation and Tendon progenitor cell recruitment.

Corticosteroid injection is commonly used to treat tendon injuries but is often associated with tendon rupture and impaired tendon healing. The effects of dexamethasone on tenocytes have been studied in vitro but only using high concentrations of dexamethasone in monolayer cultures of tenocytes over short periods of time. We have therefore investigated the effects of physiological and pharmacological concentrations of dexamethasone on monolayer cultures of tenocytes over extended time periods. We have also used fibroblastic-colony forming unit cultures to examine the effects of dexamethasone on a progenitor cell population located in tendons. Culturing tenocytes in the presence of dexamethasone for a period of 24 days resulted in a concentration-related decrease in cell number and collagen synthesis as compared to control cultures. This effect was time dependent with cell number in both dexamethasone-treated and control cultures leveling off after 14 days with the control cultures reaching higher cell densities. In contrast in control cultures, collagen accumulation continued to increase until week 4, whereas in the presence of dexamethasone, this tended to level off after 14 days. To study the role of progenitor cell recruitment, the effects of dexamethasone were investigated using the fibroblastic-colony forming unit assay. Treatment with dexamethasone at concentrations of 0.1 nM to 10 microM leads to a progressive reduction in mean colony size as compared to control cultures. Colony number remained constant at concentrations below 10 nM but fell progressively at concentrations above this. In conclusion, dexamethasone reduces both cell number and collagen synthesis in tenocyte cultures in a concentration-dependent manner by both direct effects on tenocyte proliferation and collagen accumulation, and also by modulating the recruitment of tendon progenitor cells.

Animals↗
PubMed2006

Glucose-induced replicative senescence in mesenchymal stem cells.

Mesenchymal stem cells (MSCs) show great promise for use in a variety of cell-based therapies. Because isolated primary mesenchymal stem cells are low in numbers, in vitro expansion is necessary. However, the expansion potential is limited and in vitro aging leads to loss of multipotency and replicative senescence. Stress induced by culture conditions is likely to be a major cause of replicative senescence and reduced multipotency of MSC and optimization of culture conditions might be able to reduce this. Caloric restriction (CR) is the only established method to delay aging and extend lifespan. In vitro caloric restriction experiments are rare, but have demonstrated beneficial effects. Therefore, we investigated the effect of culture medium glucose concentration on the proliferative and differentiation potential of mesenchymal stem cells. Reduction in glucose concentrations led to decreased apoptosis and an increased rate of MSC proliferation and increased the number and size of fibroblastic colonies in the colony-forming unit assay.

Animals↗
PubMed2006

Age-related impairment of mesenchymal progenitor cell function.

In most mesenchymal tissues a subcompartment of multipotent progenitor cells is responsible for the maintenance and repair of the tissue following trauma. With increasing age, the ability of tissues to repair themselves is diminished, which may be due to reduced functional capacity of the progenitor cells. The purpose of this study was to investigate the effect of aging on rat mesenchymal progenitor cells. Mesenchymal progenitor cells were isolated from Wistar rats aged 3, 7, 12 and 56 weeks. Viability, capacity for differentiation and cellular aging were examined. Cells from the oldest group accumulated raised levels of oxidized proteins and lipids and showed decreased levels of antioxidative enzyme activity. This was reflected in decreased fibroblast colony-forming unit (CFU-f) numbers, increased levels of apoptosis and reduced proliferation and potential for differentiation. These data suggest that the reduced ability to maintain mesenchymal tissue homeostasis in aged mammals is not purely due to a decline in progenitor cells numbers but also to a loss of progenitor functionality due to the accumulation of oxidative damage, which may in turn be a causative factor in a number of age-related pathologies such as arthritis, tendinosis and osteoporosis.

Aging↗
PubMed2005

Aging of mesenchymal stem cells.

The role of adult mesenchymal stem cells (MSC) in tissue maintenance and regeneration has received significant attention of late. Questions arise to what extent these cells are either subject to, or causes of aging; whether age-related changes in these cells are due to intrinsic factors or induced by the somatic environment. This review collates and examines recent data in support of these different theories. By means of introduction, a brief overview is given of current MSC definitions and their basic role in tissue regeneration followed by a comparative analysis of gerontological studies involving MSC. Evidence for extrinsic aging and various aging markers relating to morphology, proliferation, signalling, senescence markers, telomeres and telomerase, and other indicators is discussed. We observe that while the literature might often appear to conflict, many apparent discrepancies are attributable to inconsistent methods of extracting and isolating MSC which in fact contains various subsets of adult stem cells, varying not only in their differentiation potential but also in their vulnerability to senescence--ranging from quasi-somatic lifespan to perennial vigour. Thus, mesenchymal stem cells emerge as both subject to and key mediators of organismal aging.

Aging↗
PubMed2004

EMD273316 & EMD95833, type 4 phosphodiesterase inhibitors, stimulate fibroblastic-colony formation by bone marrow cells via direct inhibition of PDE4 and the induction of endogenous prostaglandin synthesis.

BACKGROUND: Type 4 phosphodiesterase (PDE4) inhibitors have been shown to stimulate bone formation in vivo and to stimulate osteoblastic differentiation in vitro. As one possible mechanism for the stimulation of bone formation is the recruitment of osteoprogenitor cells from the bone marrow, we have investigated the effect of the PDE4 inhibitors EMD273316, EMD95833, EMD249615 and EMD 219906 on fibroblastic colony formation by whole bone marrow cells and on the ability of these colonies to adopt an osteoblastic phenotype. RESULTS: All four agents stimulated colony formation in a concentration dependent manner, however, in the case of EMD273316 & EMD95833, the effect was evident at lower concentrations and the addition of prostaglandin E2 (PGE2) was not necessary for maximal stimulation. It was subsequently found that co-incubation with indomethacin reduced the stimulatory effects of EMD273316 & EMD95833 but had no effect on the actions of EMD249615 and EMD 219906 and that EMD273316 & EMD95833 stimulated the synthesis of endogenous PGE2 by whole bone marrow cells whereas EMD249615 and EMD 219906 had no significant effect. CONCLUSIONS: These data suggest that EMD249615, EMD 219906, EMD273316 & EMD95833 can promote the recruitment of bone marrow osteoprogenitor cells leading to a stimulation of bone formation via their direct inhibitory effects on PDE4. The actions of EMD273316 & EMD95833 however, are augmented by their ability to stimulate endogenous prostanoids synthesis which acts synergistically with their direct effects on PDE4.

Animals↗
PubMed2002

Recruitment, augmentation and apoptosis of rat osteoclasts in 1,25-(OH)2D3 response to short-term treatment with 1,25-dihydroxyvitamin D3 in vivo.

BACKGROUND: Although much is known about the regulation of osteoclast (OC) formation and activity, little is known about OC senescence. In particular, the fate of of OC seen after 1,25-(OH)2D3 administration in vivo is unclear. There is evidence that the normal fate of OC is to undergo apoptosis (programmed cell death). We have investigated the effect of short-term application of high dose 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on OC apoptosis in an experimental rat model. METHODS: OC recruitment, augmentation and apoptosis was visualised and quantitated by staining histochemically for tartrate resistant acid phosphatase (TRAP), double staining for TRAP/ED1 or TRAP/DAPI, in situ DNA fragmentation end labelling and histomorphometric analysis. RESULTS: Short-term treatment with high-dose 1,25-(OH)2D3 increased the recruitment of OC precursors in the bone marrow resulting in a short-lived increase in OC numbers. This was rapidly followed by an increase in the number of apoptotic OC and their subsequent removal. The response of OC to 1,25-(OH)2D3 treatment was dose and site dependent; higher doses producing stronger, more rapid responses and the response in the tibiae being consistently stronger and more rapid than in the vertebrae. CONCLUSIONS: This study demonstrates that (1) after recruitment, OC are removed from the resorption site by apoptosis (2) the combined use of TRAP and ED1 can be used to identify OC and their precursors in vivo (3) double staining for TRAP and DAPI or in situ DNA fragmentation end labelling can be used to identify apoptotic OC in vivo.

Journal Article↗
PubMed2002

Histochemical localization of alkaline phosphatase activity in decalcified bone and cartilage.

We have developed methodology that enables alkaline phosphatase (ALP) to be histochemically stained reproducibly in decalcified paraffin-embedded bone and cartilage of rodents. Proximal tibiae and fourth lumbar vertebrae were fixed in periodate-lysine-paraformaldehyde (PLP) fixative, decalcified in an EDTA-G solution, and embedded in paraffin. In the articular cartilage of the proximal tibia, ALP activity was localized to the hypertrophic chondrocytes and cartilage matrix of the deep zone and the maturing chondrocytes of the intermediate zone. The cells and matrix in the superficial zone did not exhibit any enzyme activity. In tibial and vertebral growth plates, a progressive increase in ALP expression was seen in chondrocytes and cartilage matrix, with activity being weakest in the proliferative zone, higher in the maturing zone, and highest in the hypertrophic zone. In bone tissue, ALP activity was detected widely in pre-osteoblasts, osteoblasts, lining cells on the surface of trabeculae, some newly embedded osteocytes, endosteal cells, and subperiosteal cells. In areas of new bone formation, ALP activity was detected in osteoid. In the bone marrow, about 20% of bone marrow cells expressed ALP activity. In adult rats, the thickness of the growth plates was less and ALP activity was enhanced in maturing and hypertrophic chondrocytes, cartilage matrix in the hypertrophic zone, and primary spongiosa. This is the first time that ALP activity has been successfully visualized histochemically in decalcified, paraffin-embedded mineralized tissues. This technique should prove to be a very convenient adjunct for studying the behavior of osteoblasts during osteogenesis.

Alkaline Phosphatase↗
PubMedDate not supplied

Megakaryocyte-bone marrow stromal cell aggregates demonstrate increased colony formation and alkaline phosphatase expression in vitro.

Bone marrow stromal cells (BMSCs) possess certain stem celllike properties and can differentiate to adopt a number of mesenchymal phenotypes. BMSCs are usually investigated in vitro as homogeneous single-cell suspensions; however, these preparations lose much of their osteogenic capacity. Using the fibroblastic colony-forming unit assay, we have compared the proliferation and capacity to express alkaline phosphatase of BMSC-containing aggregates of bone marrow cells with single-cell suspensions of bone marrow cells from the same source. Aggregates were separated from single cells by density gradient centrifugation or cell sieving. The aggregate and single-cell preparations gave rise to similar numbers of colonies; however, the colonies produced by the aggregates were larger and expressed higher levels of alkaline phosphatase. When the aggregates were dissociated, colonies still formed; however, they expressed negligible levels of alkaline phosphatase. Immunomagnetic selection and immunofluorescent staining for CD61, N-methyl-D-aspartate (NMDA) receptor subunit 1, and acetylcholinesterase showed that the majority of the aggregates giving rise to osteoblastic colonies contained megakaryocytes. These data demonstrate that removing BMSCs from their normal environment reduces their osteoblastic capacity and that to achieve their maximal differentiation, BMSCs require direct physical contact with accessory cells such as megakaryocytes. These findings may be of direct relevance to the use of BMSCs for tissue-engineering purposes.

Animals↗