Osteonectin is a calcium-binding matrix protein thought to play a role in regulating calcium distribution in a variety of biologic processes. To examine its role in endochondral bone formation, we examined the distribution of the protein during mineralization of the chicken tibial growth cartilage, using immunohistochemistry and immunoelectron microscopy. The synthesis of osteonectin was also determined in chondrocyte populations isolated from premineralizing and mineralizing regions of growth cartilage and assayed in short-term culture. The results show that a very low level of osteonectin is detectable in the resting, proliferating, and early hypertrophic zones of growth cartilage; in these zones, osteonectin is largely cell-associated. In contrast, a large amount of osteonectin is present in the mineralizing zone where it is associated with the matrix. Biosynthetic data from short-term culture experiments indicate, however, that osteonectin is synthesized and secreted by chondrocytes from both premineralizing and mineralizing zones. As indicated by immunoprecipitation, Northern hybridization, in vitro translation of hybrid-selected messenger RNA (mRNA), and electrophoretic analysis, osteonectin synthesized by chondrocytes of the premineralizing zones is not obviously different in structure from that synthesized by chondrocytes of the mineralizing zone. We conclude that osteonectin is a product of chondrocytes in each zone of growth cartilage but accumulates only in the mineralizing zone. The high affinity of the protein for calcium could favor its retention in calcifying matrix.
Background: Somatosensory impairment is common in patients who have had a stroke and can affect their motor function and activities of daily living (ADL). Therefore, detecting and treating somatosensory impairments properly is considered to be very important, and various examinations have been developed. However, the reliability and validity of few of them have been verified due to differences in the procedure of each examiner or poor quantification by the examination itself.Objective: We hypothesized that, with fixed procedures two convenient clinical examinations, the Semmes-Weinstein Monofilament Test (SWMT) and the Thumb Localizing Test (TLT), could provide reliable assessments of light touch sensation and proprioception. The purpose of this study was to verify the reliability and validity of these two examinations as indices of somatosensory impairment of the upper extremity (UE) in patients with chronic post-stroke hemiparesis.Methods: Fifty patients with chronic stroke (median time after onset of stroke, 848 [474–1708] days, mean age 57 [standard deviation 14] years) were enrolled at Keio University Hospital from 2017 to 2018. Examiners learned the original method of the SWMT and the TLT rigorously and shared it with each other. The TLT procedure was partially modified by dividing the location of the patient's thumb into four spaces. Two examiners evaluated the SWMT and the TLT for 2 days, and intra-rater and inter-rater reliabilities were calculated using weighted kappa statistics. In addition to this, the evaluator size score of the SWMT was assessed with Bland-Altman analysis to evaluate systematic bias. The Stroke Impairment Assessment Set (SIAS) sensory items were used to assess validity, and Spearman's rank correlation coefficients were calculated.Results: Intra/inter-rater agreements of the SWMT grade score were 0.89 (thumb, 95%CI: 0.83–0.95)/ 0.75 (0.60–0.91) and 0.80 (index finger, 0.67–0.93)/0.79 (0.66–0.92), and of the TLT they were 0.83 (navel level proximal space, 0.71–0.95)/ 0.83 (0.73–0.92), 0.90 (navel level distal space, 0.85–0.96)/ 0.80 (0.69–0.90), 0.80 (shoulder level proximal space, 0.68–0.92)/ 0.77 (0.65–0.89), and 0.87 (shoulder level distal space, 0.80–0.93)/ 0.80 (0.68–0.92) (P < 0.001, each item). All of them showed substantial agreement, but the MDC of the SWMT evaluator size was 1.28 to 1.79 in the inter-rater test and 1.94–2.06 in the intra-rater test. The SWMT grade score showed a strong correlation with the SIAS light touch sensation item (r = 0.65, p < 0.001), as did the TLT with the SIAS position sense item (r = −0.70–0.62, p < 0.001 each space).Conclusions: The reliability and validity of the SWMT and the TLT were verified. These tests can be used as reliable sensory examinations of the UE in patients with chronic stroke, and especially for the SWMT, it is more reliable for screening.
We have used in situ hybridization to examine expression of collagen type I, II, and X mRNA and osteonectin mRNA in the chick epiphysis. Tissue samples from the proximal tibial growth cartilage were fixed in modified Carnoy's solution, dehydrated in ethanol, and embedded in paraffin. Longitudinal and transverse sections were demineralized with HCl and digested with hyaluronidase and proteinase K. In situ hybridization was carried out using biotinylated cDNA probes; the hybridized probe was detected using a streptavidin-biotinylated alkaline phosphatase conjugate. This procedure permitted detection of the corresponding mRNAs in cartilage with high sensitivity and low background. Osteonectin mRNA was detected in proliferating cartilage; lower levels of osteonectin mRNA were seen in the mid-hypertrophic region. This mRNA species was also expressed in cells that border the vascular canals in the premineralized region of the epiphysis. Collagen type X mRNA was detected throughout the hypertrophic zone. As localization of collagen type X mRNA corresponded to the site of maximal synthesis of the protein, reported in other studies, our results would further support the suggestion that this protein is associated with mineralization of cartilage. Collagen type II mRNA was seen in both the proliferating and the hypertrophic regions of the cartilage. Highest levels of expression were observed in the proliferative region. The results suggest that the transcriptional control of collagen type II and X by cells of the proliferating and hypertrophic regions of the growth cartilage may be related.
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