Development of Avian Tibial Dyschondroplasia: Gene Expression and Protein Synthesis

Age-dependent gene expression and protein synthesis associated with chondrocyte differentiation were evaluated in the epiphyseal growth plates of normal and tibial dyschondroplasia (TD)-afflicted chickens. In the normal growth plate, collagen type II gene is expressed mainly by chondrocytes at the upper zone of the growth plate and by the chondrocytes in the articular cartilage. Collagen type X and osteopontin (OPN) genes are expressed in the lower zone of the growth plate and in the zone of cartilage-to-bone transition. No age-dependent changes in the pattern of OPN and collagen type II or X gene expression were observed up to 20 days of age. In the TD-afflicted growth plates, the lesion is enlarged with age, and chondrocytes expressing the collagen type II gene were observed in the hypertrophic zone as early as 8 days posthatching. Abnormal expression of OPN and collagen type X genes was also observed starting at 13 days of age. At day 20, the entire TD lesion—which was significantly enlarged—was surrounded by collagen type II, collagen type X, and OPN expressing cells. The level of OPN in TD was reduced with increasing age, and at 20 days almost no OPN could be detected in either the upper or the lower hypertrophic zones. The level of bone sialoprotein (BSP) also diminished with increasing age in the TD growth plates. In contrast to OPN, the age-dependent reduction in BSP levels was mainly in the lower hypertrophic zone (LHZ), and at 20 days of age, BSP was barely detected in the LHZ, whereas in the upper hypertrophic zone, the levels of BSP were similar to those in normal growth plate. In summary, our results suggest that the primary event of the TD lesion occurs in cells of proliferative phenotype within the hypertrophic zone. These cells divide and form the TD lesion, which consists of cells that do not express the genes associated with hypertrophy. Received: 11 June 1997 / Accepted: 11 May 1998     

Calcification in human intervertebral disc degeneration and scoliosis

Calcification is a pathological process that may lead to impairment of nutrient supply and disc metabolism in degenerative and scoliotic intervertebral discs (IVDs). The purpose of this study was to assess the calcification potential of IVDs in degenerative disc disease (DDD) and adolescent idiopathic scoliosis (AIS). For this purpose, 34 IVDs from 16 adult patients with DDD and 25 IVDs from 9 adolescent patients with AIS were obtained at surgery. The concave and convex parts of the scoliotic discs were analyzed separately. Von Kossa staining was performed to visualize calcium deposits, while type X collagen (COL X) expression associated with endochondral ossification was measured by immunohistochemistry. Alkaline phosphatase activity and calcium and inorganic phosphate concentrations were used as indicators of calcification potential. Results showed the presence of calcium deposits and COL X in degenerative and scoliotic IVDs, but not in control discs, and the level of the indicators of calcification potential was consistently higher in degenerative and scoliotic discs than in control discs. The results suggest that disc degeneration in adults is associated with ongoing mineral deposition and that mineralization in AIS discs might reflect a premature degenerative process. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1888–1895, 2011     

Elevated synthetic activity in the convex side of scoliotic intervertebral discs and endplates compared with normal tissues.

STUDY DESIGN: We measured concentrations of specific molecules reflecting matrix synthesis and degradation in normal and scoliotic intervertebral discs and endplates. OBJECTIVES: The aim of this work was to quantitate markers of matrix turnover in normal versus adolescent idiopathic scoliotic intervertebral discs and cartilaginous endplates. SUMMARY OF BACKGROUND DATA: Changes in the intervertebral disc and endplate composition have been implicated as possible etiologic factors in the pathogenesis of adolescent idiopathic scoliosis. To better understand this process, it is important to compare the turnover of matrix components in scoliotic and normal intervertebral disc and endplate tissues. This comparison may help to improve our understanding of the role that disc and endplate tissues may play in the induction and/or progression of idiopathic scoliosis. METHODS: Fifteen scoliotic and 17 normal intervertebral discs and endplates were analyzed for their water, collagen, proteoglycan, and protein content. In addition, newly synthesized aggrecan and collagen Types I and II were measured. Percent total denatured collagen was also determined. RESULTS: The total collagen content was significantly lower in the scoliotic anulus and endplate regions, whereas glycosaminoglycan (GAG) content was significantly lower in the scoliotic endplates and nucleus regions. Conversely, total protein content was significantly higher in scoliotic endplates and elevated in scoliotic nucleus regions. Water content was significantly lower in the scoliotic anulus and endplate regions. When comparing the concave and convex regions of scoliotic endplates, there was no significant difference in concentration of any matrix component. The major difference in the synthetic marker levels relates to the synthesis of Type II collagen, which was higher in the nucleus, anulus, and endplate regions of scoliotic discs than in the corresponding regions of normal tissues. By contrast, the percent total denatured collagen was significantly elevated in the nucleus of normal tissues compared with the scoliotic ones. CONCLUSIONS: The higher collagen Type II synthetic levels and increased total protein content with no matrix turnover suggest that scoliotic changes are due to an altered and ineffective synthetic response to a pathologic mechanical environment.     

Chondrocyte Differentiation in Human Osteoarthritis: Expression of Osteocalcin in Normal and Osteoarthritic Cartilage and Bone

Osteocalcin (OC), which is a marker of the mature osteoblasts, can also be found in posthypertrophic chondrocytes of the epiphyseal growth plate, but not in chondrocytes of the resting zone or in adult cartilage. In human osteoarthritis (OA), chondrocytes can differentiate to a hypertrophic phenotype characterized by type X collagen. The protein- and mRNA-expression pattern of OC was systematically analyzed in decalcified cartilage and bone sections and nondecalcified cartilage sections of human osteoarthritic knee joints with different stages of OA to investigate the differentiation of chondrocytes in OA. In severe OA, we found an enhanced expression of the OC mRNA in the subchondral bone plate, demonstrating an increased osteoblast activity. Interestingly, the OC protein and OC mRNA were also detected in osteoarthritic chondrocytes, whereas in chondrocytes of normal adult cartilage, both the protein staining and the specific mRNA signal were negative. The OC mRNA signal increased with the severity of OA and chondrocytes from the deep cartilage layer, and proliferating chondrocytes from clusters showed the strongest signal for OC mRNA. In this late stage of OA, chondrocytes also stained for alkaline phosphatase and type X collagen. Our results clearly show that the expression of OC in chondrocytes correlates with chondrocyte hypertrophy in OA. Although the factors including this phenotypic shift in OA are still unknown, it can be assumed that the altered microenvironment around osteoarthritic chondrocytes and systemic mediators could be potential inducers of this differentiation. Received: 20 May 1999 / Accepted: 10 February 2000     

Expression of p21CIP1/WAF1 in Chondrocytes

During endochondral ossification, proliferative activity of chondrocytes is arrested and the cells undergo terminal hypertrophic differentiation. We examined the expression of the cyclin-dependent kinase inhibitor, p21^CIP1/WAF1 in permanent cartilage (xyphoid and articular cartilage) and in cartilage undergoing endochondral ossification (growth plate, epiphyseal ossification centers, and costochondral junctions) to determine if p21 is up-regulated in chondrocytes during hypertrophic differentiation. Northern blot analyses demonstrated expression of p21 in chondrocytes undergoing endochondral ossification and from sites of permanent cartilage. Quantitative analyses of Northern data showed an association between expression of the hypertrophic-specific marker, collagen type X, and the level of 21 expression. In situ hybridization of rodent femoropatellar joints and costochondral junctions localized p21 mRNA to chondrocytes within both the proliferative and hypertrophic zones of the growth plates, in chondrocytes involved in formation of the epiphyseal ossification centers, and in articular chondrocytes. Immunohistochemical analyses of p21 expression in the same tissues demonstrated comparatively higher levels of p21 protein in postmitotic chondrocytes. These data suggest that p21 is active in cell cycle regulation in chondrocytes, and that increased p21 expression is associated with hypertrophic differentiation. Received: 11 October 1996 / Accepted: 23 April 1997     

The Role of Chondrocytes in Intramembranous and Endochondral Ossification During Distraction Osteogenesis in the Rabbit

We have used a rabbit leg-lengthening model for detailed studies of the histology of distraction osteogenesis. Some unusual features of the endochondral ossification that occurs during the rapid transition of cartilage to bone in the regenerate were observed. Histological staining techniques together with immunohistochemistry and nonradioactive in situ mRNA hybridization for cartilage and bone-related molecules have been used to document the presence of an overlapping cartilage-bone phenotype in cells of the cartilage-bone transitional region. In those particular areas, some chondrocytes appeared to be directly transformed into newly formed bone trabeculae which are surrounded by bone matrix. Acid phosphatases were found within the cartilage matrix in some of the cartilage/bone transitional regions and type I collagen mRNA and type II collagen protein were found together in some of the marginal hypertrophic chondrocytes. This study indicates an unusual role of chondrocytes in the process of ossification at a distraction rate of 1.3 mm/day in the rabbit. Further direct evidence is required to prove the hypothesis that the hypertrophic chondrocytes may transdifferentiate into bone cells in this model. Received: 13 March 1997 / Accepted: 22 September 1998     

Localization of bone morphogenetic protein 13 in human intervertebral disc and its molecular and functional effects in vitro in 3D culture

Our laboratory has demonstrated that bone morphogenetic protein 13 prevented the effects of annular injury in an ovine model, maintaining intervertebral disc height, cell numbers and increasing extracellular matrix production compared to degenerated controls. The present study sought to examine the molecular effects of bone morphogenetic protein 13 on human degenerated disc cells and localize its expression in both human degenerate and scoliotic disc tissue. Effect of bone morphogenetic protein 13 on human derived nucleus pulposus, annulus fibrosus and endplate cells cultured in alginate beads was evaluated by changes in proteoglycan and collagen content. Migratory potential of disc cells towards bone morphogenetic protein 13 was also examined. Bone morphogenetic protein 13 induced significant proteoglycan accumulation in nucleus (18%), annulus (21%) and endplate (23%) cells cultured in alginate beads (p < 0.05) compared to controls. Further bone morphogenetic protein 13 increased collagen I and II protein expression in nucleus and endplate cells. Nucleus cells displayed a significant chemotactic response towards bone morphogenetic protein 13. The endogenous expression of bone morphogenetic protein 13 in degenerate disc tissue was not different to scoliotic disc. Bone morphogenetic protein 13 has the potential to enhance extracellular matrix accumulation and induce cell migration in certain disc cells. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1769–1775, 2015.     

Electrochemical measurement of transport into scoliotic intervertebral discs in vivo using nitrous oxide as a tracer

STUDY DESIGN: An in vivo study measuring nitrous oxide concentrations in scoliotic intervertebral discs during surgery. OBJECTIVES: To determine pathways for nutrient transport into scoliotic human discs in vivo. SUMMARY OF BACKGROUND DATA: The intervertebral disc is the largest avascular structure in the body. Disc cells in the nucleus rely on the blood supply from the vertebral bodies for supply of nutrients and removal of waste. Loss of nutrient supply is thought to lead to disc degeneration, but solute transport has not been measured in vivo in humans. METHODS: We measured solute transport into the disc using N2O as a tracer, in 19 human discs from five patients with neuromuscular scoliosis (6-19 years of age) during surgery for correction of scoliotic deformities. During anesthesia N2O diffuses into the disc at a rate governed by effective permeability of the vertebral body-disc interface. Intradiscal N2O concentrations were measured amperometrically using silver needle microelectrodes, which were inserted into the discs once they were exposed by an anterior approach. RESULTS: For all spines N2O concentrations were very low in the disc at the curve apex (6% those expected from unimpeded diffusion) and, although still low, were significantly higher 2 discs below or above the apex. CONCLUSIONS: Because flux into the apical disc is most restricted, the decrease in solute transport is possibly induced by changes in mechanical stress on the disc; microfocal radiographs of a scoliotic spine suggest that increased endplate calcification could be partly responsible for limiting solute diffusion.     

Analysis of cell death and vertebral end plate bone mineral density in the annulus of the aging sand rat.

BACKGROUND CONTEXT: The relationship between disc degeneration and end plate sclerosis is poorly understood. The sand rat is an excellent, economical small-animal model in which disc degeneration is age related, spontaneous, reliable, and well characterized. This model is used here to evaluate disc degeneration, disc cell viability, and vertebral end plate bone mineral density (BMD) in lumbar sites. PURPOSE: To determine the proportion of live and dead cells and end plate bone mineral density in the aging sand rat annulus. STUDY DESIGN: Young and old sand rats were used in work approved by the Institutional Animal Care and Use Committee. Outcome measures were the percentage of live/dead annulus cells in the disc and the BMD of cranial and caudal end plates of lumbar vertebrae. METHODS: Bone densitometry was used to obtain endplate BMD on lumbar spines of 16 young sand rats aged 2 to 6 months and 26 older animals aged 22 to 46 months. X-ray films were analyzed for wedging, end plate calcification, and disc-space narrowing. Additional discs were also harvested and incubated with fluorochromes, and the percentage of live or dead cells were determined for the outer, inner annulus, and entire annulus. RESULTS: Radiographically old animals had significantly greater incidence of lumbar wedging (p<0.004) and a significantly greater incidence of end plate calcification and disc-space narrowing (p<0.01). In the live-dead study, the mean percentage of dead annulus cells for the three age groups were significantly different for the outer annulus (p<0.001), inner annulus (p=0.005), and total annulus (p<0.0001). The percentages of dead cells for the entire annulus were 46.14%+/-7.99% (age 2-6 months), 48.13%+/-17.32% (age, 13-19 months), and 76.80%+/-7.27% (age 26-38 months). The percentage of dead disc cells correlated significantly with age for outer annulus, inner annulus, and total annulus (p<0.006). The percentage of dead cells in the entire annulus and the inner annulus correlated significantly with end plate BMD (p<0.02). CONCLUSIONS: Data are novel and show that in very aged sand rats, end plate BMD is significantly greater than that of young animals. Live/dead cell analyses showed increasing cell death in both outer and inner annulus, which correlated significantly with age and with end plate BMD.     

Ⅸ型胶原基因在特发性脊柱侧凸患者顶椎椎间盘内表达的初步研究

目的　研究Ⅸ型胶原基因在椎间盘内分布,探讨其在特发性脊柱侧凸发病中的作用。方法　收集特发性脊柱侧凸患者椎间盘标本14例,已知病因脊柱侧凸标本13例,采用3例突然死亡的正常青少年的6个椎间盘作为正常对照。采用免疫组化技术和RNA探针原位杂交技术研究Ⅸ型胶原基因mRNA在椎间盘内不同部位的分布,对其mRNA含量进行半定量分析,比较Ⅸ型胶原mRNA含量在不同部位的分布。结果　Ⅸ型胶原主要分布于内层纤维环、髓核及终板软骨内,以内层纤维环与软骨终板内较高。Ⅸ型胶原主要由小圆形类软骨细胞分泌,在肥大的软骨细胞内无表达。Ⅸ型胶原mRNA含量特发性脊柱侧凸顶椎凹侧与已知病因组及正常对照组间差异均有统计学意义(P<0. 05),已知病因组端椎两侧含量均低于正常对照组,其余各组间均无明显差异。结论　Ⅸ型胶原在特发性脊柱侧凸患者椎间盘内分布无异常。Ⅸ型胶原mRNA含量在特发性侧凸患者顶椎椎间盘凹侧内含量明显低于正常人,而已知病因的侧凸患者则无明显下降,提示Ⅸ型胶原可能与特发性侧凸发病有关。