Biomechanical disc culture system: feasibility study using rat intervertebral discs.

A small-scale biomechanical disc culture system was designed to stimulate intervertebral disc (IVD) 'motion segment' in culture environment with load-controlled compression and combined load (compression+shear). After 7 days of diurnal mechanical loading, cell viability of discs stimulated with static compression load (0.25 MPa) and static combined load (compression (0.25 MPa)+shear (1.5N)) were similar (>90 per cent) to unloaded controls. Mechanically stimulated discs showed decrease in static/dynamic moduli, early stress relaxation, and loss of disc height after 7 days of diurnal loading. Histological data of discs indicated load-induced transformations that were not apparent in controls. The feasibility of studying the mechanobiology of intact IVD as a motion segment was demonstrated. Media conditioning (improve tissue stability in long-term culture) and application of biochemical gene expression assays (differential tissue response to types of mechanical stimulation) are proposed as future improvements. The study suggests that the limitations in studying mechanobiology of IVD pathology in vitro can be overcome and it is possible to understand the physiologically relevant mechanism of IVD pathology.

CD44 expression in the developing and growing rat intervertebral disc.

CD44 has been identified at the time of extracellular matrix formation and expansion in several sites of the developing embryo (Wheatley et al. [1993] Development 119:295-306). The nucleus pulposus, consisting of a hydrated extracellular matrix tissue at birth, not previously closely analyzed, was examined for expression of CD44 in the developing and aging rat intervertebral disc. CD44 was identified solely on notochordal cells from the first onset of intervertebral disc formation (day 15 embryo) through the loss of notochordal cells from the nucleus pulposus (12-24 months of age). No CD44 expression was found in the notochordal cells prior to disc formation or in any cells other than the notochordal cells in the annulus fibrosus or nucleus pulposus of the intervertebral disc. Using reverse transcriptase-polymerase chain reaction methodology, the single 365 amino acid CD44 standard, CD44s, open reading frame was amplified from notochordal cells isolated from the nucleus pulposus. Western blot analysis of a cultured nucleus pulposus notochordal cells total protein extract identified a single CD44 species devoid of chondroitin sulfate with a mass of approximately 85 kDa, characteristic of CD44s. Cell surface detection for CD44 was co-localized with hyaluronan and proteoglycans at first appearance of disc formation in the nucleus pulposus.

Spatial and temporal expression of CD44 isoforms in the developing and growing joints of the rat limb.

Hyaluronan is an integral component of proteoglycan-rich extracellular matrices such as hyaline cartilage. Hyaluronan is commonly found in embryonic tissue and is important in the formation of hydrated matrices that allow cellular expansion and migration. Cell surface hyaluronan-binding proteins such as CD44 are presumed to be important in the cellular interactions with hyaluronan in both of these processes. The primary aim of this study was to document the spatial and temporal expressions of CD44 isoforms during the development and growth of the diarthrodial joints of rat limbs. With use of in situ hybridization and immunohistochemistry, the CD44s isoform is selectively identified as localized to a single cell layer on opposing sides of the joint at the first appearance of joint cavitation (on the 18th day of gestation). After joint formation in the neonate, the expression of the CD44s isoform in the cells at the joint surface is lost. These findings suggest that the CD44s isoform has a role in the development of the diarthrodial joint, presumably through interaction with hyaluronan.

Characterisation of the neurosensory elements of the feline cranial cruciate ligament.

The identification and distribution of mechanoreceptors in the cranial cruciate ligament of the cat (analogous to the anterior cruciate ligament in other species) was studied histologically using a modified celloidin embedding technique to achieve serial sectioning of bone-ligament-bone preparations with gold chloride staining. We identified distinctive large elongated structures situated between the collagen bundles of the ligament (resembling endings described as Freeman and Wyke type III; also termed Golgi tendon receptors). These endings were found near the middle of the ligament well away from the bone-ligament junction. Axons seen entering only one end of each type III ending helped to confirm its neural basis. While we saw structures resembling types I and II endings (i.e. Ruffini and pacinian endings, respectively) in individual sections, serial sections failed to reveal convincing evidence of their existence. Such structures almost always appeared to be vascular in nature on adjacent sections, with vessels entering and exiting. We conclude that serial sections are critical to interpreting the presence or absence of mechanoreceptors.

Diazepam binding inhibitor processing in the rhesus monkey brain: an immunocytochemical study.

Immunocytochemical methods were used to study the regional and cellular distribution of diazepam binding inhibitor (DBI)-, ODN (DBI 33-50)- and TTN (DBI 17-50)-like immunoreactivity (LI) in monkey brain. DBI-, ODN- and TTN-LI were found to have similar regional but different cellular distributions. The most intense staining was detected in limbic structures, especially the amygdala. The most notable staining discrepancy was the weak-to-moderate uniform DBI-LI and the very high patchy ODN-LI and TTN-LI in the ventral striatum. At the cellular level, DBI-LI was preferentially localized in glia-like cells, whereas ODN-LI and TTN-LI were mainly found in neurons.