Notochordal cell matrix as a bioactive lubricant for the osteoarthritic joint.

Notochordal cell derived matrix (NCM) can induce regenerative effects on nucleus pulposus cells and may exert such effects on chondrocytes as well. Furthermore, when dissolved at low concentrations, NCM forms a viscous fluid with potential lubricating properties. Therefore, this study tests the feasibility of the use of NCM as a regenerative lubricant for the osteoarthritic joint. Chondrocyte-seeded alginate beads were cultured in base medium (BM), BM with NCM (NCM), or BM with TGF-ß1 (TGF), as well as BM and NCM treated with IL-1ß. NCM increased GAG deposition and cell proliferation (stronger than TGF), and GAG/DNA ratio and hydroxyproline content (similar to TGF). These effects were maintained in the presence of IL-1ß. Moreover, NCM mitigated expression of IL-1ß-induced IL-6, IL-8, ADAMTS-5 and MMP-13. Reciprocating sliding friction tests of cartilage on glass were performed to test NCM's lubricating properties relative to hyaluronic acid (HA), and showed a dose-dependent reduction in coefficient of friction with NCM, similar to HA. NCM has anabolic and anti-inflammatory effects on chondrocytes, as well as lubricating properties. Therefore, intra-articular NCM injection may have potential as a treatment to minimize pain while restoring the affected cartilage tissue in the osteoarthritic joint.

Soluble and pelletable factors in porcine, canine and human notochordal cell-conditioned medium: implications for IVD regeneration.

During intervertebral disc (IVD) maturation, notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) in the nucleus pulposus, suggesting that NCs play a role in maintaining tissue health. Affirmatively, NC-conditioned medium (NCCM) exerts regenerative effects on CLC proliferation and extracellular matrix (ECM) production. The aim of this study was to identify NC-secreted substances that stimulate IVD regeneration. By mass spectrometry of porcine, canine and human NCCM, 149, 170 and 217 proteins were identified, respectively, with 66 proteins in common. Mainly ECM-related proteins were identified, but also organelle-derived and membrane-bound vesicle proteins. To determine whether the effect of NCCM was mediated by soluble and/or pelletable factors, porcine and canine NCCM were separated into a soluble (NCCM-S; peptides and proteins) and pelletable (NCCM-P; protein aggregates and extracellular vesicles) fraction by ultracentrifugation, and tested on bovine and canine CLCs in vitro, respectively. In each model, NCCM-S exerted a more pronounced anabolic effect than NCCM-P. However, glycosaminoglycan (GAG) uptake from the medium into the carrier gel prevented more definite conclusions. While the effect of porcine NCCM-P on bovine CLCs was negligible, canine NCCM-P appeared to enhance GAG and collagen type II deposition by canine CLCs. In conclusion, porcine and canine NCCM exerted their anabolic effects mainly through soluble factors, but also the pelletable NCCM factors showed moderate regenerative potential. Although the regenerative potential of NCCM-P should not be overlooked, future studies should focus on unraveling the protein-based regenerative mechanism from NCCM produced from isolated NCs, e.g. by NCCM fractionation and pathway blocking studies.

The species-specific regenerative effects of notochordal cell-conditioned medium on chondrocyte-like cells derived from degenerated human intervertebral discs.

During intervertebral disc (IVD) maturation, the main cell type shifts from notochordal cells (NCs) to chondrocyte-like cells (CLCs). NCs secrete factors with regenerative potential, making them an interesting focus for regenerative treatments. During initial development, these strategies preferably employ non-human donors due to easy availability of their NC-rich nucleus pulposus (NP) tissue. To increase the success of translating these strategies for clinical application, this study aimed to delineate whether NC-secreted factors of different species have a regenerative effect on human CLCs. Human, canine and porcine NC-rich NP tissue and NC-conditioned medium (NCCM) were analysed biochemically and histologically. Human CLC micro-aggregates from degenerated IVDs were cultured in human, canine or porcine NCCM. Collagen, glycosaminoglycan (GAG) and DNA content was determined and histology was performed. Canine and porcine NPs were richer in NCs than human NPs. Human NPs contained the highest collagen content, whereas the DNA and GAG content of canine NPs was significantly higher than that of human or porcine NPs. NCCM from all species significantly increased the DNA and GAG content of the human CLC micro-aggregates. Porcine and canine NCCM were significantly more potent than human NCCM in inducing GAG deposition, whereas only human NCCM induced collagen type II production. Secreted factors from human, canine and porcine NC-rich NPs exerted regenerative effects on human CLCs, indicating a cross-species effect. Bioactive compound(s) are present in NCCM of different species that may reverse human IVD degeneration, supporting further research into strategies based on NC-technology employing canine or porcine models for their translation into humans.

Liquid order at the interface of KDP crystals with water: evidence for icelike layers.

We present a surface x-ray diffraction study on the KDP-water interface in which the structure of both the crystalline and liquid part of the interface has been measured. We have been able to determine the ordering components in the liquid in both the perpendicular and parallel directions. We find interface-induced ordering in the first four layers of water molecules. The first two layers behave icelike and are strongly bound to the surface. The next two layers are more diffuse and show only minor lateral and perpendicular ordering. Subsequent layers are found to behave similar to a bulk liquid.

Slits as adjustable pinholes for coherent X-ray scattering experiments.

The combination of accurate translation stages with carefully polished slit blades leads to slits that have many advantages as pinholes for coherent X-ray scattering experiments. The size is adjustable and can be made as small as 0.5 mum. Setting up is easy, while the blade thickness (1 mm tungsten) also makes the slits useful for hard X-rays. A relation between the slit-sample distance and the minimum beam size, together with the corresponding slit size, is derived. This shows that a micrometer-sized beam can be achieved with this type of slits.

Dihydropyrimidine dehydrogenase deficiency leading to thymine-uraciluria. An inborn error of pyrimidine metabolism.

Three unrelated patients with excessive thymine-uraciluria due to dihydropyrimidine dehydrogenase deficiency are described. Excretory values (mmol/g creatinine) were: uracil 2.0-10.5, thymine 2.3-7.5, 5-hydroxymethyluracil 0.2-0.9. Orally administered (index patient) uracil and thymine were excreted for the greater part whilst dihydrouracil and S-dihydrothymine were mainly metabolised. Dihydropyrimidine dehydrogenase activities (nmol X h-1 X mg-1 protein) in leucocytes were 0.04, 0.01 and less than 0.01 in the patients, 0.31-1.66 in their parents, and 1.01-4.46 in controls (n = 4). The patients presented with a non-specific clinical picture of cerebral dysfunction.

Deficiency of fumarylacetoacetase in a patient with hereditary tyrosinemia.

A patient is described with type I tyrosinemia characterized by urinary excretion of succinylacetone together with increased excretion of tyrosine, p-hydroxyphenyllactic, p-hydroxyphenylpyruvic and p-hydroxyphenylacetic acids. Fumarylacetoacetase was measured in a liver biopsy and found to be very low compared to control liver. Furthermore the mass spectra of succinylacetone and fumarylacetoacetate (methoxime-TMS derivatives) are reported. Control jejunal mucosa, leucocytes and fibroblasts showed no enzyme activity; hence the prenatal diagnosis of this disease by measuring the fumarylacetoacetase activity in cultured amniotic fluid cells is not possible at present.

Effect of cyclic nucleotides on weight of gastrocnemius and creatine kinase activity after denervation of muscle in young rats.

Denervation of the gastrocnemius muscle at various stages of development reduces muscle weight and creatine kinase activity. An inverse relationship between the muscle-specific enzyme, creatine kinase, and the lysosomal enzyme, acid phosphatase, was shown. An increased percentage of the BB isoenzyme of creatine kinase is observed after long-term denervation. Apparently the muscle tissue has the ability to regenerate and presumptive myoblasts are formed from satellite cells. When the denervated muscle is treated with dibutyryl-cyclic-GMP administration new muscle tissue has been formed. Similar effects could not be demonstrated with either cyclic AMP or succinylcholine. The higher percentage of the BB isoenzyme after dibutyryl-cyclic-GMP administration supports the theory that presumptive myoblasts are derived from satellite cells. Succinylcholine also causes an increase of the B-type of creatine kinase. It can be concluded that cyclic GMP, generated via the nerve, has an important role in maintaining muscle weight.

Quantitative analysis of morphological changes in skeletal muscle of the rat after hormone administration.

Thyroxine and cortisone acetate administration of rats of 4--7 days of age is not only accompanied by the induction of the muscle-specific enzyme, creatine kinase, but the hormones also induce morphological changes in the gastrocnemius during this period. Administration of thyroxine to these rats causes a splitting of myofibrils as shown by stereological measurements on electron micrographs. This splitting of myofibrils was not observed upon cortisone acetate administration and when both hormones were given simultaneously. It is suggested that cortisone acetate counteracts the effect of thyroxine. Both thyroxine and cortisone acetate increase the volume percentage taken by the mitochondria at 7 days of age. The effect of the simultaneous injection of both hormones is equal to the sum of the separate effects of these hormones. These changes in volume percentage of the mitochondria were compared with changes in a mitochondrial marker enzyme, i.e. citrate synthase. The difference between the morphological measurements and citrate synthase activity is due to a change in the specific activity of citrate synthase in the mitochondria.

Effect of hormones on the development of creatine kinase activity in rat skeletal muscle.

Various approaches have been used in order to determine whether or not a certain hormone is a stimulus for the development of the muscle-specific enzyme, creatine kinase. Both thyroxine and glucocorticoids can be considered as naturally occurring stimuli for the synthesis of creatine kinase. The maximum increase of creatine kinase activity after stimulation by glucocorticoids (about 25%) occurs between 5 and 7 days after birth. A single injection of thyroxine has virtually no effect during this period. However, when a pretreatment with thyroxine is given, cortisone acetate administration increases creatine kinase activity to about 155%. The effect of cortisone acetate is due to de novo synthesis of creatine kinase. The augmentation of the effect of cortisone acetate by thyroxine is dependent on DNA synthesis. Thyroxine administration apparently causes the formation of more competent muscle cells. The effects of both hormones are age-dependent. Thyroxine and cortisone acetate administration to fetuses can prematurely evoke to MM isoenzyme of creatine kinase. Both hormones probably play a role in the activation of the M gene during embryonic development. Sex hormones are able to influence neither creatine kinase activity nor muscle growth. However, castration of male rats immediately after birth causes an impairment of growth at older ages. The androgen production by the testes immediately after birth seems to be of main importance for body growth development. It can be concluded from these results that creatine kinase in muscle is under multiple hormonal control, just as is observed for a number of enzymes in other tissues.

The development of creatine kinase in rat skeletal muscle. Changes in isoenzyme ratio, protein, RNA and DNA during development.

The activity of cytosolic creatine kinase in rat skeletal muscle rises stepwise during development. The increases occur simultnaeously with transient increases in DNA content. The second increase is accompanied by a rise in total protein, soluble sarcoplasmic protein and RNA/DNA ratio. Such changes are not observed at 20 days after birth, when creatine kinase finally accumulates to the adult level. Transient higher amounts of the MB and BB isoenzymes are observed after the first and second stepwise increase. The increase in creatine kinase activity observed after birth is predominantly due to an activation of the M gene. The BB isoenzyme is still present in adult skeletal muscle, but contributes little to the total activity.