Stretch and force generation induce rapid hypertrophy and myosin isoform gene switching in adult skeletal muscle.

Using electrical stimulation to control force generation and limb immobilization to alter the degree of stretch, we have studied the role of mechanical activity in inducing hypertrophy and in determining fast and slow muscle fibre phenotype. Changes in gene expression were detected by analysing the RNA in hybridization studies employing cDNA probes specific for fast and slow myosin heavy chains and other genes. As a result of overload in the stretched position, the fast contracting tibialis anterior muscle in an adult rabbit is induced to synthesize much new protein and to grow by as much as 30% within a period as short as 4 days. This very rapid hypertrophy was found to be associated with an increase of up to 250% in the RNA content of the muscles and an abrupt change in the species of RNA produced. Both stretch alone and electrical stimulation alone caused repression of the fast-type genes and activation of the slow-type genes. it appears that the fast-type IIB genes are the default genes, but that the skeletal slow genes are expressed as a response to overload and stretch. These findings have implications as far as athletic training and rehabilitation are concerned.     

Cytocentrifugation: a convenient and efficient method for seeding tendon-derived cells into monolayer cultures or 3-D tissue engineering scaffolds

Tendon and ligament injuries are very common, requiring some 200,000 reconstructions per year in the USA. Autografting can be used to repair these but donor tissue is limited and harvesting leads to morbidity at the graft sites. Tissue engineering has been used to grow simple tissues such as skin, cartilage and bone and due to their low vascularity and simple structure, tendons should be ideal candidates for such an approach. Scaffolds are essential for tissue engineering as they provide structure and signals that regulate growth. However, they present a physical barrier to cell seeding with the majority of the cells congregating at the scaffold surface. To address this we used centrifugation to enhance penetration of tendon-derived cells to the centres of 3-D scaffolds. The process had no apparent deleterious effects on the cells and both plating efficiency and cell distribution improved. After attachment the cells continued to proliferate and deposit a collagenous matrix. Scaffold penetration was investigated using layers of Azowipes allowing the separation and examination of individual leaves. At relatively low g-forces, cells penetrated a stack of 6 Azowipes leaving cells attached to each leaf. These data suggest that cytocentrifugation improves the penetration and homogeneity of tendon derived cells in 3-D and monolayer cultures.     

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.     

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.     

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.     

Stimulation of CFU-f formation by prostaglandin E(2) is mediated in part by its degradation product, prostaglandin A(2)

Prostaglandins (PG) of the E series are known to rapidly undergo non-enzymatic dehydration in culture medium containing serum albumin to produce the cyclopentenone PGs of the A series. We investigated the actions of PGA(1) and A(2) in the in vitro calcifying fibroblastic-colony forming unit assay which can partially mimic the in vivo anabolic effects of PGE(2). It was found that PGA(1) and A(2) both stimulated colony formation in a dose-dependent manner with a maximum at 10(-6) M and to a similar degree to PGE(2). In contrast to PGE(2), PGA(1) and PGA(2) both caused an inhibition of cAMP accumulation. Furthermore, the addition of protein kinase A inhibitors, H8 and H89, had no significant effect on the stimulation of colony number by PGE(2). These data suggest that (a) the bone anabolic effects of PGE(1) and E(2) are, in part at least, mediated by their dehydration products PGA(1) and A(2) and (b) that they are mediated via pathways not necessarily involving the cAMP/protein kinase A cascade.