Midface Membranous Bone Lengthening: A One-Year Histological and Morphological Follow-Up of Distraction Osteogenesis

Midface bone lengthening was performed on three young, adult sheep using distraction osteogenesis following osteotomy of the maxilla and mounting of an extraoral fixation device. The midface was gradually distracted, 2 mm/day, for 21 days, up to approximately 40 mm. A marked midface advancement was noted. Following a further 6 weeks of retention, the device was removed and the animals were followed for 1 year. Biopsies specimens were taken from the distracted area at the end of the distraction period, after the additional 6 weeks of retention, and finally 1 year later. A nondistracted area of the maxillary bone served as control. The specimens were analyzed histologically, histochemically, and by scanning electron microscopy for the ultrastructural pattern, mineralization, mineral content, and approximate Ca²⁺ concentration. Clinically and radiographically, all sheep fully bridged the experimental gap. Histologically, at the completion of distraction, collagen bundles and slender bone trabeculae oriented in the direction of the distraction could be seen. At the end of the retention period, the trabeculae thickened noticeably and were partially replaced by mature lamellar bone. At the end of 1 year and after completion of the process of remodeling, the pattern of the distracted area resembled the control area. The mineralization, as reflected by quantitative calcium analysis, compared with the nondistracted area, demonstrated a low rate of mineralization after 3 weeks of lengthening, increased 6 weeks later, and after 1 year became nearly the same as in the nondistracted area. In conclusion, distraction osteogenesis provides satisfactory quantitative and structural new bone. Received: 23 September 1996 / Accepted: 17 June 1997     

The Domain of Hypertrophic Chondrocytes in Growth Plates Growing at Different Rates

In this study, we tested the hypotheses that (a) both the domain volume (volume of the cell and the matrix it has formed) and matrix volume of juxtametaphyseal hypertrophic chondrocytes in the growth plate is tightly controlled, and that (b) the domain volume of juxtametaphyseal hypertrophic chondrocytes is a strong determinant of the rate of bone length growth. We analyzed the rate of bone length growth (oxytetracycline labeling techniques) and nine stereologic and kinetic parameters related to the juxtametaphyseal chondrocytic domain in the proximal and distal radial and tibial growth plates of 21- and 35-day-old rats. The domain volume increased with increasing growth rates, independent of the location of the growth plate and the age of the animal. Within age groups, the matrix volume per cell increased with increasing growth rates, but an identical growth plate had the same matrix volume per cell in 21- and 35-day-old rats. The most suitable regression model (R ²= 0.992) to describe the rate of bone length growth included the mean volume of juxtametaphyseal hypertrophic chondrocytes and the mean rate of cell loss/cell proliferation. This relationship was independent of the location of the growth plate and the age of the animal. The data suggest that the domain volume of juxtametaphyseal hypertrophic chondrocytes, as well as the matrix volume produced per cell, may be tightly regulated. In addition, the volume of juxtametaphyseal hypertrophic chondrocytes and the rate of cell loss/rate of cell proliferation may play the most important role in the determination of the rate of bone length growth. Received: 2 December 1996 / Accepted: 24 March 1997     

Expression and Role of Interleukin-6 in Distraction Osteogenesis

Distraction osteogenesis is a special form of bone healing in which well-controlled distraction stresses and consequent tensile strains within callus tissue induce very efficient new bone formation. Proinflammatory cytokines are involved during the early phase of fracture healing and callus remodeling. Temporal expression patterns of proinflammatory cytokines were assessed in Sprague-Dawley rat tibial models of distraction osteogenesis and acute lengthening, and only interleukin-6 (IL-6) was found to be specifically induced during the distraction phase. IL-6 immunoreactivity was detected not only in hemopoietic cells and osteoblasts but also in the spindle-shaped cells of the fibrous interzone, where most of the tensile strains are concentrated. In vitro study revealed that IL-6 did not affect the proliferation of C3H10T1/2 cells, mouse bone marrow stromal cells (MSCs), or MC3T3-E1 cells; but its blocking antibody reduced the proliferation of C3H10T1/2 cells and MSCs. The mRNA expression of COL1A1 and osteopontin were not changed by IL-6 or its blocking antibody, but the alkaline phosphatase activities of MC3T3-E1 cells were increased by IL-6 and decreased by its blocking antibody. These findings indicate that IL-6 is a proinflammatory cytokine that responds to tensile strain during distraction osteogenesis. IL-6 negatively affects the proliferation of primitive mesenchymal cells, whereas the differentiation of more mature osteoblastic lineage cells is enhanced by IL-6 in vitro. IL-6 appears to be one of the cytokines involved in the complex network of signal cascades evoked during distraction osteogenesis and may differentially affect immature and mature osteoblastic lineage cells.     

Expression of Cathepsins B, H, K, L, and S and Matrix Metalloproteinases 9 and 13 During Chondrocyte Hypertrophy and Endochondral Ossification in Mouse Fracture Callus

Fracture repair provides an interesting model for chondrogenesis and osteogenesis as it recapitulates in an adult organism the same steps encountered during embryonic skeletal development and growth. The fracture callus is not only a site of rapid production of cartilage and bone, but also a site of extensive degradation of their extracellular matrices. The present study was initiated to increase our understanding of the roles of different proteolytic enzymes, cysteine cathepsins B, H, K, L, and S, and matrix metalloproteinases (MMPs) 9 and 13, during fracture repair, as this aspect of bone repair has previously received little attention. Northern analysis revealed marked upregulation of cathepsin K, MMP-9, and MMP-13 mRNAs during the first and second weeks of healing. The expression profiles of these mRNAs were similar with that of osteoclastic marker enzyme tartrate-resistant alkaline phosphatate (TRAP). The changes in the mRNA levels of cathepsins B, H, L, and S were smaller when compared with those of the other enzymes studied. Immunohistochemistry and in situ hybridization confirmed the predominant localization of cathepsin K and MMP-9 and their mRNA in osteoclasts and chondroclasts at the osteochondral junction. MMP-13 was present in osteoblasts and individual hypertrophic chondrocytes near the cartilage-bone interphase. In cartilaginous callus, the expression of cathepsins B, H, L, and S was mainly related to chondrocyte hypertrophy. During bone remodeling both osteoblasts and osteoclasts contained these cathepsins. The present data demonstrate that degradation and remodeling of extracellular matrices during fracture healing involves activation of MMP-13 production in hypertrophic chondrocytes and osteoblasts, and cathepsin K and MMP-9 production in osteoclasts and chondroclasts. Received: 2 February 2000 / Accepted: 25 May 2000 / Online publication: 2 November 2000     

Morphometric Analysis of Type I Collagen Fibrils in the Osteoid of Osteogenesis Imperfecta

Electron microscopy and morphometric measurements of bone osteoid collagen diameter from 42 osteogenesis imperfecta (OI) patients and 25 age- and site-matched controls were carried out. Although the mean diameter did not correlate well with the severity of the disease, it related well with the clinical types and revealed collagen fibrils of reduced diameter in the osteoid of all OI types. Thus, OI type II (the severest type) demonstrated the smallest diameter (45 nm), followed by OI type I (the mildest form) with a mean diameter of 57 nm. The diameter obtained for type III (67 nm) and type IV (64 nm) was lower than the normal control mean diameter (73 nm) but did not show a statistical difference. The thinner fibrils observed in OI bone may be unable to provide nucleating and scaffolding sites for mineral propagation and may play a role in the fragility of bone in this disease. Received: 1 June 1998 / Accepted: 23 April 1999     

Inhibition of Chondrocyte Terminal Differentiation and Matrix Calcification by Soluble Factors Released by Articular Chondrocytes

Chondrocytes do not undergo terminal differentiation in normal articular cartilage, whereas growth plate chondrocytes synthesize ALPase and induce matrix calcification terminally. Articular chondrocytes in osteoarthritic joints have been reported to express the terminal differentiation phenotypes, suggesting that terminal differentiation of articular chondrocytes is inhibited in normal joints. In the present study, we investigated the underlying inhibitory mechanism of the terminal differentiation in articular cartilage using a culture on type II collagen-coated dishes or a novel culture model on Millipore filters. ALPase activity increased from day 7 to day 8 in growth plate chondrocyte cultures on the collagen-coated dishes, but not in articular chondrocyte cultures. The ALPase expression of growth plate chondrocytes on the collagen-coated dish was completely inhibited when the same number of articular chondrocytes was mixed in the growth plate chondrocyte cultures. When articular chondrocytes or growth plate chondrocytes were maintained on Millipore filters held in 16-mm dishes, they started to synthesize ALPase. The ALPase expression of the chondrocytes on Millipore filters was inhibited by the presence of articular chondrocytes maintained on the bottom collagen-coated substratum in the same dishes. These results indicate that factors that diffused into the medium through the Millipore filters are involved in the inhibition of terminal differentiation. Since the conditioned medium from articular chondrocyte cultures did not affect the ALPase expression, it is considered that the soluble factors, which are continuously released from articular chondrocytes, are responsible for the inhibition of terminal differentiation. Received: 23 April 1998 / Accepted: 12 March 1999     

Characterization of Demineralized Bone Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: III. Gene and Protein Expression

Our previous studies of rat cranial defect repairs after the implantation of demineralized bone matrix (DBM) have demonstrated that healing occurs initially and principally by the direct induction and proliferation of osteoblasts derived principally from resident mesenchymal stem cells of the dura, and to a lesser extent by resident mesenchymal stem cells of the connective tissues beneath the skin flap. A small amount of cartilage is also synthesized after the direct process of ossification occurs. To further confirm the molecular phenotypes of the repair cells in rat cranial defects, the present study evaluated mRNA expression and synthesis of collagens I, II, and X and osteocalcin in the DBM-induced repair tissue by Northern blot analyses, autoradiography after in vivo ³H-proline labeling of collagen, and immunohistochemistry. The results demonstrated that osteocalcin mRNA appeared in small amounts by day 4 and continued to increase over the experimental period. Much lesser quantities of collagen types II and X mRNAs appeared by day 6 and day 8, respectively. Collagen type I mRNA was present at all times examined but its expression significantly increased by day 5. Autoradiographic and immunohistochemical studies showed that type II collagen was not detected whereas type I collagen was synthesized on days 3–5. The data provide definitive molecular evidence confirming that the initial and by far the major pathway of cranial defects repair induced by implantation of DBM is by the direct induction of resident mesenchymal stem cells to osteoblasts and the direct formation of bone, which is spatially and temporarily distinct from the later formation of cartilage. Received: 30 November 1999 / Accepted: 21 March 2000     

Differing Lumbar Vertebral Mineralization Rates in Ambulatory Pediatric Patients with Osteogenesis Imperfecta

The purpose of this study was to evaluate serial changes in bone mineral density (BMD) of the lumbar spine in individual children and adolescents with untreated osteogenesis imperfecta (OI) using dual X-ray absorptiometry (DXA). Twenty-seven pediatric patients with OI who had no historical or radiographic evidence of lumbar fracture, required no assistive device for mobility, and were taking no medications known to affect skeletal mineralization during the study period comprised the investigational group. Absolute BMD and age- and gender-matched BMD (Z-scores) were assessed relative to standard parameters of growth (height, weight, age, height adjusted for age and gender and body surface area) and severity of disease (lifetime fracture rate). The spinal mineralization rate (SMR) between examinations for 15 patients with more than one measurement (n= 20 intervals) was expressed as the magnitude of the change in BMD Z-score per year. Both BMD and BMD Z-score were closely correlated with height, height Z-score, weight and body surface area and were inversely related to fracture rate (P < 0.001 for all comparisons). BMD was also highly correlated with patient age (P < 0.001). Stepwise regression analysis showed that together height Z-score and lifetime fracture rate improved the prediction of BMD Z-score (r= 0.71; P < 0.001). SMRs ranged from −0.5 to 3.5. The average change in SMR between sequential measurements was 168% for the five children who had more than two DXA examinations. Linear regression showed a significant negative correlation between SMR and height Z-score (r=−0.79, P < 0.001). We conclude that vertebral body size is a critical determinant of BMD and BMD Z-score in OI because DXA results are expressed per unit area, not per unit volume. Pediatric patients with OI mineralize their lumbar vertebrae at rates similar to healthy children but tend to lag behind in overall mineralization. The rate of mineralization at any age appears to be related to the patient's height (adjusted for age- and gender-matched controls) and inversely related to the patient's lifetime rate of fractures. Our data suggest that vertebral mineralization in children with OI is related primarily to rapid increases in vertebral volume and only secondarily to increases in vertebral mineral density. Received: 2 March 1997 / Accepted: 5 June 1997     

Bone Metabolism During Exercise and Recovery: The Influence of Plasma Volume and Physical Fitness

Understanding the reaction of bone to physical exercise is important for the development of strategies to increase and maintain bone mass. In this study the aim was to investigate the relationship among exercise intensity, physical capacity, and the biochemical responses, estimated by measuring biochemical markers of bone metabolism in serum. As a complement to the circulating concentrations we also accounted for the plasma volume shifts during and after exercise. The study included 10 men and 10 women, mean age 29 years, with a wide range of physical capacity, who performed a standardized running exercise test on a motor-driven treadmill with loads corresponding to 47 and 76% of VO₂ max (maximal oxygen uptake) followed by a maximal effort until exhaustion. Total work time was about 35 minutes. Venous blood samples were drawn at rest, after each load, and after 30 minutes and 24 hours of recovery. The reductions in plasma volume during exercise were 4.3% (P < 0.05) and 15.1% (P < 0.001) whereas after 24 hours in recovery there was an expansion of 7.5% (P < 0.001). There were marked, intensity-related, increases of PICP and tALP concentrations (P < 0.001) during exercise. Since these were of the order of plasma volume reduction they did not correspond to a change in the calculated circulating amount (content). However, as the concentrations returned to basal during recovery, the total circulating amounts were increased at this point (P < 0.05). Osteocalcin was also increased during recovery (P < 0.01), although concentrations were unchanged during the entire study. The amount (P < 0.001) and concentration (P < 0.05) of ICTP were also increased during follow-up. Serum PTH concentrations rose (P < 0.05) in proportion to the intensity of exercise and remained elevated during recovery. The subjects' VO₂ max demonstrated positive relationships to the biochemical responses to exercise in bone and BMD of the legs, and a negative relationship to basal PTH levels. Bone turnover and PTH secretion was stimulated by exercise, and low basal levels of PTH and high BMD were induced by a high level of physical fitness. These observations correlate well with the favorable effects of exercise and training on bone mass. Received: 5 November 1996 / Accepted: 23 April 1997     

Bone Geometry and Strength Measurements in Aging Mice with the oim Mutation

Mice with the naturally occurring oim mutation allows investigation of bone pathobiology in the setting of one mutation: a G deletion in the murine Cola-2 gene (exon 52) encoding the proα2(I) C-propeptide. As a result, normal sized mRNA is transcribed, but no secreted protein has been identified in oim/oim fibroblasts or osteoblasts. Here we report longitudinal changes in body mass, bone geometry, and bone structural properties of femurs tested in torsion from wild type (+/+) mice and mice homozygous (oim/oim) and heterozygous (+/oim) for the oim mutation. Femurs from mice 3 months, 6 months, 12 months, and >18 months of age were dissected and X-ray films were taken in anterioposterior and mediolateral views to estimate the geometric properties. The metaphyseal ends of femurs were potted in polymethylemethacrylate and mounted on a torsional test fixture designed to convert axial tensile deformation to a torsional load using an INSTRON model 4204 materials tester. Compared with +/+ samples, peak torque at failure was reduced in oim/oim mice. Also, the geometric distribution of midshaft bone for oim/oim mice in terms of cortical area and polar moment was significantly reduced. However, the impact of the mutation on bone distribution was relatively minor for +/oim mice. Consistent with a type III classification in human OI patients, the presence of two nonfunctional alleles in homozygous oim mice significantly reduced body mass compared with age-matched wild type mice. However, no statistical difference in body mass was detected between +/oim and +/+ mice. The absence of a gross phenotypic difference between +/oim and +/+ mice demonstrates a milder phenotype in +/oim mice. Received: 27 January 1997 / Accepted: 21 May 1997     

Involvement of Insulin-Like Growth Factor I in Development of Ossification of the Posterior Longitudinal Ligament of the Spine

In order to investigate the pathogenesis of ossification of the posterior longitudinal ligament (OPLL) of the spine, we examined the distribution of insulin-like growth factor I (IGF-I) in the posterior longitudinal ligaments of OPLL patients, and analyzed the effects of IGF-I on the cultured spinal ligament cells. For that purpose we established eight varieties of OPLL and non-OPLL cell lines obtained from spinal ligaments of corresponding patients, respectively. In contrast to non-OPLL cases, all the OPLL cases were histologically shown to contain round-shaped cartilage-like cells in the transitional region from preossifying to ossifying ligaments, and these cells were strongly stained with an antibody for IGF-I. In the vicinity of preossifying cartilaginous tissues, ligament cells also had a rod-like appearance and were positive for IGF-I immunohistochemically. The effects of IGF-I on cultured spinal ligament cells were assayed by alkaline phosphatase (AP) activity, DNA synthesis, and the amounts of collagen produced. The number of OPLL cell lines that increased AP activity, responding to IGF-I irrespective of 1,25(OH)₂D₃, was significantly larger than that of non-OPLL cell lines, although IGF-I stimulated DNA and procollagen type I carboxyl-terminal peptide synthesis in most of both OPLL and non-OPLL cell lines. These data demonstrate the dominant expression of IGF-I in the posterior longitudinal ligaments of OPLL patients, and suggest that IGF-I preferentially induces osteogenic differentiation in OPLL cells rather than in non-OPLL cells. IGF-I, therefore, may be involved in the local ossification process of spinal ligaments observed in OPLL patients. Received: 26 December 1996 / Accepted: 21 May 1997     

Bone Morphogenetic Protein-2 Increases the Rate of Callus Formation after Fracture of the Rabbit Tibia

The effects of human recombinant bone morphogenetic protein-2 (rhBMP-2) on rabbit fractures healing under both stable and unstable mechanical conditions were investigated. rhBMP-2 was administered (1) on bioerodible particles, (2) in a collagen gel, and (3) by injection. rhBMP-2 on bioerodible particles has no effect as the particles prevent the migration of cells that produce the callus. The collagen gel is resorbed more rapidly; the development of the callus of mechanically unstable fractures is similar to controls at 14 days. When rhBMP-2 is injected, the callus of mechanically unstable fractures develops more rapidly so that cortical union occurs by 21 days, as compared with 28 days in control fractures. The effects on fractures healing under stable mechanical conditions are minimal. It is argued that mechanical factors influence the size of the callus of normally healing fractures and, although BMP-2 accelerates the rate of development of the callus and cortical union, it does not affect the amounts of bone and cartilage produced. Received: 9 February 1998 / Accepted: 9 December 1998     

The Role of Collagen Abnormalities in Ultrasound and Densitometry Assessment: In Vivo Evidence

There is little information concerning how the mutation of collagen affects bone mineralization and the assessment of bone properties. To estimate these influences, we performed ultrasonic assessments of the calcaneus and bone mineral density (BMD) measurements of the hip and lumbar spine. Females with diseases related to the mutation of collagen [Ehlers-Danlos syndrome (EDS) type III and systemic sclerosis (SSc)] participated in this study. We compared the broadband ultrasound attenuation (BUA and UBI-4), the average transit time through the heel (TTH), and a multiple factor index (UBI-4T) with control subjects matched on age, race, and menstrual status. Both groups of patients had BMD of the spine (L2–L4) within the normal range for their age and sex (for EDS: n = 23, 1.14 ± 0.14 g/cm² and z-score = 0.37; for SSc: n = 15, 0.98 ± 0.15 g/cm² and z-score = 0.20). EDS and SSc subjects had lower BMD of the femoral neck (FN) compared with controls (for EDS: 0.91 ± 0.13 g/cm², z-score =−0.41, P= 0.025; for SSc 0.67 ± 0.13 g/cm², z-score =−0.92, P= 0.006). Subjects with EDS and SSc also had lower BUA values (P= 0.051–0.001) compared with controls. After adjusting for body weight, height, and the level of physical activity, the difference in FN BMD between EDS or SSc and controls became marginal (EDS: P= 0.072; SSc: P= 0.086). However, the significant difference for BUA between subjects and controls remained for EDS (P= 0.008), and disappeared for SSc (0.70) after adjusting for weight, height, level of physical activity, and BMD. These results suggest that the abnormalities of collagen may impact on bone mass measurements differently depending on skeletal site, modality of the assessment, and the source and nature of collagen defects. To determine whether collagen properties influence QUS, proper models in vivo and in vitro should be used. Received: 1 June 1998 / Accepted: 1 November 1998     

Gene Expression of Noncollagenous Bone Matrix Proteins in the Limb Joints and Intervertebral Disks of the twy Mouse

The twy (tiptoe walking Yoshimura) mouse is an autosomal recessive mutant manifesting multiple osteochondral lesions characterized by pathologic calcium deposition. To elucidate the pathophysiology of the limb joint lesions and the intervertebral disk lesions of the twy mouse, we assessed the mRNA expression of noncollagenous bone matrix proteins such as osteocalcin, osteonectin, osteopontin, and matrix Gla protein (MGP) by in situ hybridization, but only expression of MGP was observed in association with the pathologic calcium deposits in twy mice. Mild degeneration and abnormal growth of the cartilage in contact with the joint capsule was observed at 5 weeks in the articular cartilage of the ankle joint of the twy mouse, and MGP gene expression was observed at the same time. Simultaneous growth of synovial membrane cells and relatively undifferentiated articular cartilage cells in the knee joint, and of cartilage-like cells near the insertion of the cruciate ligament was observed in the twy mouse, and MGP gene expression was found to be present at the same time. Hypertrophy of abnormally proliferated chondrocyte-like cells, which are different from fibrocartilaginous cells of the annulus fibrosus, was observed in the intervertebral disks of the twy mouse at 3 weeks of age, and MGP gene expression was noted at the same time. These findings suggest that abnormal expression of MGP plays a major role in the pathologic calcification of the twy mouse. Received: 14 May 1997 / Accepted: 24 December 1997     

Increased Turnover of Small Proteoglycans Synthesized by Human Osteoblasts During Cultivation with Ascorbate and β-Glycerophosphate

The small proteoglycan decorin had been localized previously at the d-band in the gap zone of collagen fibrils in nonmineralizing tissues. In bone matrix this zone is proteoglycan free and is at least in some species the place where mineralization along collagen fibrils starts. To study the metabolism of the small proteoglycans decorin and biglycan under mineralizing conditions, osteoblasts from human nasal bone were cultured for several weeks in the presence or absence of β-glycerophosphate and ascorbate. An immediate consequence of the treatment was a reduced expression of decorin, as judged by immune precipitation, whereas the biosynthesis of biglycan was not affected. Pulse-chase experiments were performed with osteoblasts embedded in floating type I collagen gels. In the presence of β-glycerophosphate and ascorbate, a more rapid turnover of both proteoglycans was noted; the one of biglycan reached statistical significance. Indirect evidence for an enhanced rate of proteoglycan endocytosis was obtained. This effect was not seen in cultured skin fibroblasts. Thus, osteoblasts respond rapidly to mineralizing conditions with alterations of small proteoglycan biosynthesis and turnover. Received: 30 October 1995 / Accepted: 3 January 1997     

The Effect of Fluoride Treatment on Bone Mineral in Rabbits

Fluoride therapy has been used clinically for many years, but its use remains controversial and many basic questions remain unanswered. Accordingly, this study returns to an animal model to study the effects of high doses of fluoride on bone mineral in rabbits. Twelve rabbits, aged 3(1/2) months at the start of the study, received drinking water fluoridated at 100 ppm while their 12 control counterparts drank distilled water. All rabbits were sacrificed after 6 months. Fluoride was readily incorporated into femoral cortical bone (7473 +/- 966 ppm F versus 1228 +/- 57 ppm in controls; P < 0.00005). Fluoride therapy led to increased mineralization, as measured by density fractionation (P < 0.0005 for the distributions). The bone mineral itself was altered, with a significant increase in the width of crystals (66.2 +/- 2.0 A versus 61.2 +/- 0.9 A; P < 0.01). The microhardness of both cortical and cancellous bone in the femoral head of fluoride-treated rabbits was greater than that in the controls (P < 0.05). The phosphate, calcium, and carbonate contents in the bone was the same in both groups. Finally, fluoride administration did not affect the architecture or connectivity of cancellous bone in the femoral head. Previously published data [1] indicated that the mechanical properties of bone were adversely affected; this suggests that the effect of high doses of fluoride on the strength and stiffness of bone may be mediated by its effect on bone mineral.     

Variation with Age in the Pattern of Type X Collagen Expression in Normal and Scoliotic Human Intervertebral Discs

The distribution and expression of type X collagen, a calcium-binding collagen, which is a marker of hypertrophic chondrocytes and thought to be involved in cartilage calcification, was examined in situ in nondegenerate (grade I or II) human discs taken at autopsy over a wide age range (fetal–>80 years) and also in scoliotic discs removed at surgery. In the fetal vertebral column, type X collagen was strongly expressed in the hypertrophic chondrocytes of the endplate, but was not seen in other areas. In the cartilaginous endplate of adults, it was found over the whole age range examined, with intensity increasing with age. In the disc matrix itself, type X collagen was demonstrated around individual cells from all individuals older than 50 years, but not in any fetal or autopsy disc from individuals younger than 40 years. In scoliotic discs, however, focal type X collagen expression was seen in 3/8 patients younger than 40 years including one 12-year-old. No type X collagen was found in the outer annulus in any autopsy or scoliotic disc, supporting the idea that cells of the outer annulus are phenotypically distinct from cells of the inner annulus and the nucleus. Our results demonstrate for the first time that type X collagen is a possible gene product of the intervertebral disc cells and a potential biochemical component of the disc matrix. They indicate that with age or in scoliosis, some cells from the inner annulus or nucleus of the disc differentiate to the hypertrophic chondrocyte phenotype. This might be the initiating event for the abnormal calcification described in aged and scoliotic discs in other studies. Received: 13 February 1997 / Accepted: 23 January 1998     

Gene and Protein Expression During Differentiation and Matrix Mineralization in a Chondrocyte Cell Culture System

Endochondral bone formation occurs through a series of developmentally regulated cellular stages, from initial formation of cartilage tissue to calcified cartilage, resorption, and replacement by bone tissue. Nasal cartilage cells isolated by enzymatic digestion from rat fetuses were seeded at a final density of 10⁵ cell/cm² and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum in the presence of ascorbic acid and β-glycerophosphate. First, cells lost their phenotype but in this condition they rapidly reexpressed the chondrocyte phenotype and were able to form calcified cartilaginous nodules with the morphological appearance of cartilage mineralization that occurs in vivo during endochondral ossification. In this mineralizing chondrocyte culture system, we investigated, between day 3 and day 15, the pattern expression of types II and X collagen, proteoglycan core protein, characteristic markers of chondrocyte differentiation, as well as alkaline phosphatase and osteocalcin associated with the mineralization process. Analysis of labeled collagen and immunoblotting revealed type I collagen synthesis associated with the loss of chondrocyte phenotype at the beginning of the culture. However, our culture conditions promoted extracellular matrix mineralization and cell differentiation towards the hypertrophic phenotype. This differentiation process was characterized by the induction of type X collagen mRNA, alkaline phosphatase, and diminished expression of type II collagen and core protein of large proteoglycan after an increase in their mRNA levels before the mineralizing process. These results revealed distinct switches of the specific molecular markers and indicated a similar temporal expression to that observed in vivo recapitulating all stages of the differentiation program in vitro. Received: 12 December 1996 / Accepted: 26 June 1997