The objective of the present study was to assess proteoglycan production by human intervertebral disc cells cultured in vitro in selected cell carriers. Based on previous studies which evaluated disc cells seeded into collagen sponge, collagen gel, agarose, alginate or fibrin gel three-dimensional (3D) cell carriers, collagen sponge and agarose were found to provide superior microenvironments for formation of extracellular matrix (ECM). A standardized test design was used to evaluate ECM formed after 14 days of culture using the 1,9-dimethylmethylene blue (DMB) assay to assess sulfated glycosaminoglycan (S-GAG) production. Although agarose culture showed higher S-GAG levels compared to collagen sponge (2.94±2.20 (19) μg/ml S-GAG (mean±S.D. (n)) vs. 0.94±0.77 (22), respectively, p=0.0003), this is off-set by the significantly lower proliferation rate associated with culture of disc cells in agarose.

SPARC (secreted protein, acidic, and rich in cysteine) is a matricellular protein that is present in the intervertebral disc; in man, levels of SPARC decrease with aging and degeneration. In this study, we asked whether targeted deletion of SPARC in the mouse influenced disc morphology. SPARC-null and wild-type (WT) mice were studied at 0.3-21 months of age. Radiologic examination of spines from 2-month-old SPARC-null mice revealed wedging, endplate calcification, and sclerosis, features absent in age-matched WT spines. Discs from 3-month-old SPARC-null mice had a greater number of annulus cells than those of WT animals (1884.6 +/- 397.9 [mean +/- SD] vs 1500.2 +/- 188.2, p=0.031). By 19 months discs from SPARC-null mice contained fewer cells than WT counterparts (1383.6 +/- 363.3 vs 1466.8 +/- 148.0, p=0.033). Histology of midsagittal spines showed herniations of lower lumbar discs of SPARC-null mice ages 14-19 months; in contrast, no herniations were seen in WT age-matched animals. Ultrastructural studies showed uniform collagen fibril diameters in the WT annulus, whereas in SPARC-null disc fibrils were of variable size with irregular margins. Consistent with the connective tissue deficits observed in other tissues of SPARC-null mice, our findings support a fundamental role for SPARC in the production, assembly, or maintenance of the disc extracellular matrix.

Low dietary magnesium (Mg) may be a risk factor for osteoporosis. In animals, severe Mg deficiency (0.04% of nutrient requirement [NR]) results in bone loss. We have also found that a more moderate dietary Mg restriction (10% of NR) also resulted in loss of bone. We now report the effect of Mg intake of 25% NR on bone and mineral metabolism in the rat. Serum Mg, Ca, PTH, 1,25(OH)2-vitamin D, alkaline phosphatase, osteocalcin, and pyridinoline were measured at 2,4, and 6 months in control and Mg-deficient animals. Femurs and tibias were collected for mineral content, micro-computerized tomography, histomorphometry, and immunocytochemical localization. Profound Mg deficiency developed as assessed by marked hypomagnesemia and 27% reduction in bone Mg content. Serum calcium was not significantly different between groups. Mg depletion resulted in a significantly lower serum PTH concentrations. Serum 1,25(OH)2-vitamin D was also significantly lower. No difference was noted in markers of bone turnover. Histomorphometry and micro-computerized tomography demonstrated decreased bone volume and trabecular thickness. No difference was observed for osteoclast or osteoblast number. Inflammatory cytokines may contribute to bone loss. We found that immunocytochemical localization of TNFα in osteoclasts was increased 138–150%. This increase in TNFα may be due to increased substance P as it was found to be elevated from 179% to 432%. These data demonstrate that Mg intake of 25% NR in the rat causes lower bone mass which may be related to increased release of substance P and TNFα.

Matrix metalloproteinases (MMPs) degrade components of the extracellular matrix of the disc, but the presence of MMP-19 has not been explored. In other tissues, MMP-19 is known to act in proteolysis of the insulin-like growth factor (IGF) binding protein-3, thereby exposing this protein to make it available to influence cell behavior. MMP-19 also has been shown to inhibit capillary-like formation and thus play a role in the avascular nature of the disc. Using immunohistochemistry, normal discs from six subjects aged newborn through 10 years and 20 disc specimens from control donors or surgical patients aged 15-76 (mean age 40.2 years) were examined for immunolocalization of MMP-19; six Thompson grade I discs, five Thompson grade II, eight Thompson grade III, five Thompson grade IV, and one Thompson grade V discs were analyzed. The results indicate that in discs from young subjects, MMP-19 was uniformly localized in the outer annulus. In discs from adult donors and surgical patients, outer and inner annulus cells only occasionally showed MMP-19 localization. The greatest expression of MMP-19 was observed in young discs, and little expression was seen in older or degenerating discs. Because MMP-19 has been shown to regulate IGF-mediated proliferation in other tissues, its decline in the aging/degenerating disc may contribute to the age-related decrease in disc cell numbers.

Insufficient dietary magnesium (Mg) intake has been associated with low bone mass in humans,and recent basic science studies have indicated that this bone loss may be secondary to increased release of substance P and TNFc Much less is known about the effects of low Mg intake on cartilage. We have evaluated growth plate and articular cartilage in rats following a 6 month dietary Mg restriction. Histomorphometry demonstrated significantly decreased distal femur articular cartilage chondrocyte density and decreased tibial growth plate width in experimental animals compared to controls. Growth plates of Mg-restricted animals showed reduced chondrocyte column formation. Extracellular matrix of both articular cartilage and growth plates in experimental animals contained reduced amounts of proteoglycans. Immunolocalization of Sox9 was decreased in both articular and growth plate cartilage in experimental animals compared to controls, suggesting that reduced Mg intake causes cartilage changes that may be secondary to reduced levels of the SOX9 transcription factor.

Study Design. Human intervertebral disc cells from the anulus were tested in a study of colony formation and extracellular matrix (ECM) production during long-term three-dimensional culture with exposure to selected cytokines. Experimental studies were approved by the authors’ Human Subjects Institutional Review Board. Objectives. To quantitatively evaluate colony formation and qualitatively assess ECM production (using immunohistochemistry and in situ hybridization) in cells derived from Thompson Grades I to V discs and tested in culture with cytokines and nutrient supplementation. Summary of the Background Data. Human intervertebral disc cells offer special in vitro challenges because of the slow-growing nature of these cells and their need for specialized three-dimensional in vitro conditions, which permit the expression and production of proteoglycans and Type II collagen, two ECM products that are important for disc cell biology. Methods. Discs from 9 human subjects (2 control donors and 7 surgical patients, Thompson Grades I–V), mean age 35.8 years, were used to obtain anulus cells to be tested in three-dimensional agarose culture. Tests of specialized growth conditions included treatment with ITS (insulin-transferrin-sodium selenite supplement), insulin-like growth factor I (IGF-I), and transforming growth factor-&bgr;1 (TGF-&bgr;1). Cultures were evaluated after 14 to 36 days of culture for % colony formation and cell numbers/colony; immunocytochemistry, in situ hybridization, and quantitative histology were used to evaluate colony formation and ECM production. Results. Data showed that compared with the average 17.5% colony formation observed in controls, ITS, TGF-&bgr;1 and ITS with IGF-I significantly increased colony formation (28.4%, 30.4%, and 30.4%, respectively, P ≤ 0.04). Even cells derived from Thompson Grade V disc showed responsiveness to cytokines and improved production of ECM in vitro. Conclusions. Findings indicated that cells derived from discs with advanced degeneration were still responsive to cytokines and could be modulated to produce Type II collagen and proteoglycans in three-dimensional culture by the addition of enriched media and selected cytokines. Such findings are important since they advance our understanding of how to modulate disc cell behavior in vitro, and may have application to potential future biologic therapies for disc degeneration.

Although osteoporosis is a major health concern for our growing population of the elderly, there continues to be a need for well-designed clinical and animal studies on the link between dietary magnesium (Mg) intake and osteoporosis. Relatively few animal studies have assessed the skeletal and hormonal impact of long-term low Mg intake; however, these studies have demonstrated that Mg deficiency results in bone loss. Potential mechanisms include a substance P-induced release of inflammatory cytokines as well as impaired production of parathyroid hormone and 1,25-dihydroxyvitamin D. Abnormal mineralization of bones may also contribute to skeletal fragility. Clinical studies have often varied greatly in study design, subject age, menopausal status and outcome variables that were assessed. Most studies focused on female subjects, thus pointing to the great need for studies on aging males. According to the U.S. Department of Agriculture, the mean Mg intake for males and females is 323 and 228 mg/day, respectively. These intake levels suggest that a substantial number of people may be at risk for Mg deficiency, especially if concomitant disorders and/or medications place the individual at further risk for Mg depletion. In this paper, we will review animal and human evidence of the association of Mg deficiency with osteoporosis and explore possible mechanisms by which this may occur.

Objective: To determine the optimal approach to prevent adhesions comparing leuprolide acetate (GnRH-a), Interceed (oxidized regenerated cellulose; Johnson & Johnson Medical, Me., New Brunswick, NJ), and a combination of leuprolide with Interceed in a rabbit uterine horn adhesion model.Design: Prospective, randomized, blinded study.Setting: Certified animal care facility.Animal(s): Twenty-eight sexually mature, female New Zealand White rabbits.Intervention(s): Animals were prospectively randomized (by number generator) to receive GnRH-a or saline. After 6 weeks, standard surgical manipulations were performed at three sites in each uterine horn by [1] suture, [2] unipolar cautery, and [3] superficial abrasion. Interceed was applied over one randomly assigned uterine horn only. Six weeks after surgery, uterine adhesions were assessed visually, and tissue fibrosis was assessed by histology.Main Outcome Measure(s): Presence or absence of adhesions and microscopic tissue fibrosis.Result(s): Gonadotropin-releasing hormone agonist significantly decreased adhesions, whereas Interceed alone did not reduce adhesions. However, GnRH agonist plus Interceed was the most effective measure to reduce tissue fibrosis.Conclusion(s): Preoperative GnRH-a is more effective than Interceed in preventing surgical adhesions in the rabbit uterine horn. However, preoperative GnRH-a plus Interceed may provide optimal results in this animal model, because microscopic tissue fibrosis is minimized with this combination. (C) 2004 by American Society for Reproductive Medicine.)

Dietary Mg intake has been linked to osteoporosis. Previous studies have demonstrated that severe Mg deficiency [0.04% of nutrient requirement (NR)] results in,osteoporosis in rodent models. We assessed the effects of more moderate dietary Mg restriction (10% of NR) on bone and mineral metabolism over a 6-mo experimental period in rats. At 2, 4 and 6 mo, serum Mg, Ca, parathyroid hormone (PTH), 1,25-dihydroxy-vitamin D, alkaline phosphatase, osteocalcin and urine pyridinoline were measured. Femurs and tibiae were collected for measurement of mineral content, microcomputerized tomography, histomorphometry, and immunocytochemical localization. By 2 mo, profound Mg deficiency had developed as assessed by marked hypomagnesemia and up to a 51% reduction in bone Mg content. These features continued through 6 mo of study. Serum Ca was slightly but significantly higher in Mg-deficient rats than in controls at all time points. At 2 mo, serum PTH was elevated in Mg-deficient rats but was significantly decreased at 6 mo in contrast to control rats in which PTH rose. Serum 1,25-dihydroxy-vitamin D was significantly lower than in controls at 4 and 6 mo. A significant fall in both serum alkaline phosphatase and osteocalcin suggested decreased osteoblast activity. Histomorphometry demonstrated decreased bone volume and trabecular thickness. This was confirmed by microcomputerized tomography analysis, which also showed that trabecular volume, thickness and number were significantly lower in Mg-deficient rats. Increased bone resorption was suggested by an increase in osteoclast number over time compared with controls as well as surface of bone covered by osteoclasts and eroded surface, but there was no difference in osteoblast numbers. The increased bone resorption may be due to an increase in TNF-alpha because immunocytochemical localization of TNF-alpha in osteoclasts was 199% greater than in controls at 2 mo, 75% at 4 mo and 194% at 6 mo. The difference in TNF-alpha may be due to substance P, which was 250% greater than in controls in mononuclear cells at 2 mo and 266% at 4 mo. These data demonstrated that a Mg intake of 10% of NR in rats causes bone loss that may be secondary to the increased release of substance P and TNF-alpha.

The objective of the present study was to examine the relation between gene expression and the shape of human intervertebral disc cells cultured in vitro in three-dimensional (3D) scaffolds. Disc cells from 19 subjects were seeded into either a collagen sponge or collagen gel and cultured for 10 days. In situ hybridization was performed on serial sections of paraffin embedded specimens and assessed for expression of selected genes important for extracellular matrix formation: Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase. Rounded cells grown in collagen gel showed expression of Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase; expression of these genes was absent in spindle shaped cells. Cells in the collagen sponge that lay on the sponge margin were frequently spindle shaped; these cells expressed type I collagen, but not type II collagen, aggrecan or chondroitin-6 sulfotransferase. Results presented here provide novel data concerning disc cell gene expression with collagen 3D constructs. This information is useful for future tissue engineering studies that have the challenging goal of selectively modulating gene expression.

In the present study, we used osteoprotegerin (OPG), which blocks osteoclastogenesis, to correct and thus explain the hypercalcemia that is seen during dietary Mg deficiency in the mouse. Control and Mg-deficient mice received injections for 12 days of either OPG or vehicle only. Serum Ca was similar in Mg-deficient mice treated with OPG and in control mice receiving OPG (9.2 +/- 0.3 mg/dl vs. 9.2 +/- 0.5). Both groups had significantly higher serum Ca than controls or Mg-deficient animals receiving vehicle alone. Surprisingly, Mg-depleted mice that received OPG in doses that inhibit osteoclastic bone resorption remained hypercalcemic. Because mature osteoclasts still present in the marrow might be hyperactive, we examined osteoclast morphology at the light microscopic and ultrastructural level. Light microscopic examination of trabecular bone showed few osteoclasts in OPG-treated mice. Ultrastructural examination revealed that osteoclasts in OPG-treated mice have decreased contact with the endosteal bone surface and absence of a ruffled border. Because the morphology of the existing pool of mature osteoclasts did not enhance resorption, another mechanism, such as increased intestinal absorption of Ca in Mg-deficient mice, likely contributes to the hypercalcemia observed during Mg deficiency.

STUDY DESIGN:Human intervertebral disc tissue from the annulus was obtained in a prospective study investigating the ultrastructural features of disc cells and extracellular matrix. Experimental studies were approved by the authors' Human Subjects Institutional Review Board. Discs were obtained from surgical specimens and control donors.OBJECTIVE:To compare the cellular and extracellular matrix characteristics of the annulus from control and surgical disc specimens using electron microscopy and specialized fixation that visualizes proteoglycans.SUMMARY OF THE BACKGROUND DATA:The ultrastructural features of disc cells and the disc matrix have received little attention, as compared with the literature on age- and disease-related changes in bone and cartilage.METHODS:Ultrastructural studies investigated disc tissue obtained from control and surgical disc specimens using transmission electron microscopy. Specialized fixation with ruthenium red was used to highlight matrix proteoglycans.RESULTS:Cellular and extracellular matrix fine structure was assessed in disc specimens from 29 control donors (newborns to 79-year-olds) and surgical disc specimens from 49 patients (16- to 77-year-olds). Control and surgical tissue showed similar ultrastructural features. Unusual matrix surrounding and encircling single cells or clusters of cells was common (48% of control and 63% of surgical specimens) and often contained fibrous long-spacing collagen (41.3% of control and 36.7% of surgical specimens). Ruthenium red greatly aided visualization of proteoglycans pooled in lacunar spaces. Variable cross-sectional diameters of collagen fibrils was present in 34% of control and 59% of surgical specimens. Regions with sparse interterritorial matrix were common. Cell morphology showed both cells with apoptotic nuclei and synthetically active cells that appeared healthy.CONCLUSIONS:Control and surgical specimens of the annulus showed similar ultrastructural features. Heterogeneity of collagen fibril diameter is an important observation because it is believed that fibril size relates to biomechanical disc function. Fibrous long-spacing collagen may reflect extracellular matrix remodeling or the presence of previous fibril depolymerization followed by repolymerization and reassociation with proteoglycans. Synthetic activity of disc cells is reflected in active rough endoplasmic reticulum, Golgi, and pools of proteoglycans in lacunar spaces and unusual extracellular matrix components that encircle cells and cell clusters. Such components may influence biomechanical quality. Departures from normal extracellular matrix organization of the aging or degenerating disc undoubtedly contribute to decreased biomechanical function of the annulus because they disrupt the normal annulus architecture. This study underscores the need for a fuller understanding of the dynamic relation between disc cells and the surrounding extracellular matrix, which they continually produce and remodel.

Study Design. Work presented here used a small animal model to illustrate the feasibility of autologous disc cell implantation. Objectives. To develop a small animal model for autologous disc cell implantation. Summary of the Background Data. The use of autologous disc cells in the potential treatment of disc degeneration offers attractive possibilities for novel therapies. Results are presented on the use of the sand rat (Psammomys obesus), a small rodent that spontaneously develops disc degeneration during aging, in experimental studies in which cells were harvested from a lumbar intervertebral disc, expanded in monolayer tissue culture, labeled with agents that allow subsequent immunolocalization of these cells, and implanted in a second disc site of the donor animal. Methods. Tissue culture, disc surgery, histology, and immunocytochemistry were used. Cells were either engrafted in a bioresorbable carrier tested for cell compatibility or injected into the recipient disc. Results were assessed with radiographic examination of the implantation site and with histology and immunocytochemistry. Conclusion. Data from 15 animals were obtained with engraftment resident in the animal for up to 33 weeks. Immunocytologic identification of engrafted cells showed that they integrated into the disc and were surrounded by normal matrix at time points up to 8 months postengraftment. Engrafted cells exhibited either a spindle-shaped morphology in the annulus or a rounded chondrocyte-like morphology in the nucleus. Although technically challenging, the authors' experience showed that autologous disc cell implantation can be successful and that the sand rat is a valuable model for autologous disc cell studies.

Study Design. This report is composed of two studies, one cross-sectional and one prospective cohort study, that analyze the radiologic features of disc degeneration in the sand rat (Psammomys obesus). Objectives. To statistically assess progressive disc degeneration in this useful animal model in terms of a cross-sectional study and a prospective monthly evaluation of individual animals. Summary of Background Data. P. obesus is an attractive small rodent model for spontaneous age-related disc degeneration. Because disc degeneration is spontaneous, the model avoids use of chemonucleolysis or surgical injury to cause disc degeneration. Little is understood, however, about specific details of the progressive disc deterioration. Methods. This study statistically assessed 158 animals in a cross-sectional study and 22 animals in a longitudinal study, which followed individual animals to 12 months of age. Radiologic features involving irregular disc margins, disc wedging, disc narrowing, endplate calcification, subchondral sclerosis, ligament calcification, and osteophyte formation were studied. Results. Significant age-related cross-sectional changes were present for all features (P ≤ 0.005). Males showed a statistically greater incidence of wedging at 6 and 12 months, wedging at 2 and 6 months, and endplate calcification at 2 months than did females. By 6 months of age, however, endplate calcification had a higher incidence in females than in males. Prospective analysis showed that wedging, narrowing, endplate calcification, and irregular disc margins were more common at 12 months of age than at 2 months (P = 0.0001). By the age of 12 months, all lumbar sites of both males and females showed endplate calcification and the majority of animals showed narrowing and wedging. Conclusions. Radiographic signs of degeneration were evident by age 2 months; wedging, narrowing, irregular disc margins, and endplate calcification were the most common degenerative changes in older animals. These data show that the sand rat provides a reliable, useful model of spontaneous disc degeneration.

Our objective for this study was to determine the presence and distribution of tenascin in the human intervertebral disc. The tenascins are a family of extracellular matrix proteins with repeated structural domains homologous to epidermal growth factor, fibronectin type III and the fibrinogens. Little is known about the presence of this protein in the disc. Ten normal human discs donated from subjects newborn to 15 years old, 10 control discs from adult donors aged 2441 years, and 11 surgical disc specimens from patients aged 26-76 years were examined for immunolocalization of tenascin. In young discs, tenascin was localized throughout the annulus; in the nucleus, localization was confined to pericellular matrix. In adult control and degenerating disc specimens, tenascin in the annulus was localized primarily in pericellular matrix regions encircling either single cells or clusters of disc cells; in rare instances localization was more diffuse in the intraterritorial matrix. In young, healthy disc, tenascin was abundant throughout the annulus. In contrast, degenerating discs in adults showed a localization restricted to the pericellular, and rarely, more restricted intraterritorial matrix. These observations indicate that changes in the amount and distribution of tenascin may have a role in disc aging and degeneration, possibly by modulating fibronectin-disc-cell interactions, and causing alterations in the shape of disc cells.

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.

STUDY DESIGN:Human anulus cells were cultured under control and experimental conditions to study associations between proliferation and clinical-demographic features of subjects from which cells were obtained. Statistical multiple regression analyses were applied to develop mathematic models relating proliferation to age, gender, Thompson score (denoting stage of disc degeneration), and status (control donor [postmortem]; surgical patient). OBJECTIVES:To identify the effect of donor characteristics on proliferative capacities of human disc cells. SUMMARY OF BACKGROUND DATA:As therapeutic options for disc degeneration increase, novel biologic options are important future considerations. Little is known about the influence of clinical-demographic features on cell proliferation. METHODS:Anulus cells were studied in two designs: 1) Cells from 12 individuals were grown in monolayer with 50 ng/mL interleukin growth factor-1 (IGF-I), 100 ng/mL insulin, or control conditions. 2) Cells from nine individuals were grown in three-dimensional culture with 10 ng/mL IGF-I or control conditions. Cell proliferation data and data on age, gender, Thompson score, and status were collected. Standard statistical analyses were used to develop correlation models. RESULTS:Data from monolayer experiments produced significant models fitting proliferation in the presence of low serum, 50 ng/mL IGF-I, or insulin, with age, gender, Thompson score, and status (respective R2: 0.827, 0.680, 0.850). Three-dimensional cultures exposed to 10 ng/mL IGF-I resulted in proliferation that correlated in a significant negative manner with Thompson score (r = -0.798). CONCLUSIONS:Clinical-demographic prognostic indicators may help predict levels of proliferation. Greater age, greater disc degeneration, female gender, and surgical derivation had deleterious effects on proliferation potential in this model.

The identification of a tapeworm (Rodentolepis nana, formerly named Hymenolepis nana) infection in a research breeding colony of sand rats (Psammomys obesus obesus) was complicated because of the unexpected long length (< 150 mm) of the worms. Other morphologic features that were consistent with this identification included the number (24), size (16 mm), and shape of the hooks on the rostellum. No evidence of intermediate hosts was found in the colony. Previous surveys of natural populations of sand rats had not identified this tapeworm. However, a detailed search of the literature revealed that variation in the size of R. nana had been reported, thus supporting the final identification of the tapeworm. R. nana is important and interesting because of its zoonotic potential and because it is the only tapeworm that is able to infect its definitive host without use of an intermediate host. This report is presented to help clarify the ambiguity found in the laboratory animal literature about the differences in the size of R. nana among rodent species used in research.

Data are presented which provide evidence for gap junction formation and connexin (Cx) 43 and 45 gene expression in human intervertebral disc cells in vivo and in vitro. These findings in cells from the annulus are important in conjunction with the well-recognized loss of disc cells during aging and disc degeneration. As a result of this loss of cells, cell-cell communication, which we propose is an important, but as yet poorly understood, mechanism which links and coordinates cellular function throughout the entire population of disc cells, is also disrupted. These studies provide additional information on the fundamental cell biology of the disc cell and provide an additional framework for understanding aging, degeneration and potential repair of the human disc.