These findings reveal that there is a high incidence of apoptosis in the intervertebral disc. Surviving cells are not synthetically inactive but are, rather, producing inappropriate matrix products during aging and degeneration. In certain instances it appears that the matrix surrounding the cell may form an isolation barrier, which may influence individual cell activity and intercellular communication. These results point to the need to 1) more fully understand the cause of disc cell death via apoptosis and to determine whether this programmed cell death can be reversed or halted, and 2) more fully understand the dynamic relation between disc cells and the surrounding extracellular matrix, which they produce and remodel. The factors regulating extracellular matrix-disc cell homeostasis in the disc are unknown, as is the relation between extracellular matrix and disc cell functional modulation. The morphologic findings of this study suggest that these issues are important considerations in disc cell biology. The identification of tartrate-resistant acid phosphatase activity in disc cells allows for a new area of study of disc extracellular matrix remodelling. In summary, these new perspectives provide new parameters with which to assess disc cell health and function.

Magnesium (Mg) is the second most abundant intracellular cation where it plays an important role in enzyme function and trans-membrane ion transport. Mg deficiency has been associated with a number of clinical disorders including osteoporosis. Osteoporosis is common problem accounting for 2 million fractures per year in the United States at a cost of over $17 billion dollars. The average dietary Mg intake in women is 68% of the RDA, indicating that a large proportion of our population has substantial dietary Mg deficits. The objective of this paper is to review the evidence for Mg deficiency-induced osteoporosis and potential reasons why this occurs, including a cumulative review of work in our laboratories and well as a review of other published studies linking Mg deficiency to osteoporosis. Epidemiological studies have linked dietary Mg deficiency to osteoporosis. As diets deficient in Mg are also deficient in other nutrients that may affect bone, studies have been carried out with select dietary Mg depletion in animal models. Severe Mg deficiency in the rat (Mg at <0.0002% of total diet; normal = 0.05%) causes impaired bone growth, osteopenia and skeletal fragility. This degree of Mg deficiency probably does not commonly exist in the human population. We have therefore induced dietary Mg deprivation in the rat at 10%, 25% and 50% of recommended nutrient requirement. We observed bone loss, decrease in osteoblasts, and an increase in osteoclasts by histomorphometry. Such reduced Mg intake levels are present in our population. We also investigated potential mechanisms for bone loss in Mg deficiency. Studies in humans and and our rat model demonstrated low serum parathyroid hormone (PTH) and 1,25(OH)(2)-vitamin D levels, which may contribute to reduced bone formation. It is known that cytokines can increase osteoclastic bone resorption. Mg deficiency in the rat and/or mouse results in increased skeletal substance P, which in turn stimulates production of cytokines. With the use of immunohistocytochemistry, we found that Mg deficiency resulted in an increase in substance P, TNFalpha and IL1beta. Additional studies assessing the relative presence of receptor activator of nuclear factor kB ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), found a decrease in OPG and an increase in RANKL favoring an increase in bone resorption. These data support the notion at dietary Mg intake at levels not uncommon in humans may perturb bone and mineral metabolism and be a risk factor for osteoporosis.

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Insufficient dietary magnesium (Mg) intake has been associated in humans with low bone mass. Mg deficiency in the rat has suggested bone loss is due to increased bone resorption and/or inadequate bone formation during remodeling. The purpose of this study was to assess the effect of a low Mg diet on bone and mineral metabolism in the young and mature BALB/c mouse and explore the hypothesis that inflammatory cytokines may contribute to Mg deficiency-induced osteoporosis. Using an artificial diet, we induced targeted Mg depletion (0.002% Mg) with all other nutrients maintained at the normal level. In all Mg-depleted mice, hypomagnesemia developed and skeletal Mg content fell significantly. The serum Ca in Mg-deficient mice was higher than in control mice; however, serum PTH levels were not significantly different. Osteoprotegerin (OPG) in dosages that inhibit osteoclastic bone resorption did not prevent hypercalcemia in Mg-deficient animals. No significant difference in serum Ca was observed between groups when dietary Ca was reduced by 50%, suggesting that a compensatory increase in intestinal absorption might account for the hypercalcemia. Growth plate width decreased 33% in young Mg-deficient animals and chondrocyte columns decreased in number and length, suggesting that Mg deficiency reduced bone growth. Trabecular bone volume in the metaphysis of the tibia in these animals was decreased and osteoclast number was increased by 135%. Osteoblast number was significantly reduced. Immunohistochemistry revealed that substance P increased 230% and 200% in megakaryocytes and lymphocytes, respectively, after 1 day of Mg depletion. IL-1 increased by 140% in osteoclasts by day 3 and TNF alpha increased in osteoclasts by 120% and 500% in megakaryocytes on day 12. This study demonstrates a profound effect of Mg depletion on bone characterized by impaired bone growth, decreased osteoblast number, increased osteoclast number in young animals, and loss of trabecular bone with stimulation of cytokine activity in bone.

Orthopaedic tissues, such as bone, cartilage, intervertebral disc and tendon, contain cells that are difficult to culture and stimulate in vitro for repair of damaged tissue. Stem cells have the ability to self-renew and differentiate into many tissue types. Recent progress in stem cell research has led to an enthusiastic effort to utilize stem cells for orthopaedic tissue regeneration. Due to ease of harvest and abundance, adipose-derived mesenchymal cells (ASC) are an attractive, readily available adult stem cell that has become increasingly popular for use in many stem cell applications. Recent progress has been made in characterizing ASC and looking mechanistically at gene expression and cellular pathways involved in differentiation. This review focuses on (i) the characterization of ASC through expression of appropriate surface markers; (ii) modulation of in vitro differentiation of ASC through different scaffolds, growth factors, and media; and (iii) the use of ASC in orthopaedic tissue repair. Strategies for repair involve the use of differentiated or undifferentiated, fresh or passaged ASC, in conjunction with appropriate choice of media, growth factors and scaffolds. Recent in vivo studies utilizing ASC are discussed giving results on defect repair and potential for clinical orthopaedic tissue regeneration.

Osteoblasts arise from partially differentiated osteogenic progenitor cells (OPCs) which in turn arise from undifferentiated marrow stromal mesenchymal stem cells (MSCs). It has been postulated that age-related defects in osteoblast number and function may be due to quantitative and qualitative stem cell defects. To examine this possibility, we compared osteogenic stem cell number and in vitro function in marrow cells from 4-month-old and 24-month-old male BALB/c mice. Histologic studies demonstrated that these mice undergo age-related bone loss resembling that seen in humans. In primary MSC cultures grown in media supplemented with 10 nM dexamethasone, cultures from older animals yielded an average of 41% fewer OPC colonies per given number of marrow cells plated (p < 0.001). This implies that for a given number of marrow cells there are fewer stem cells with osteogenic potential in older animals than there are in younger animals. The basal proliferative rate in cultures from older animals, as measured by 3H-thymidine uptake, was more than three times that observed in cultures from young animals (p < 0.005). However, the increase in proliferative response to serum stimulation was 10-fold in the younger cultures (p <0.001) and insignificant (p <0.4) in the older cultures. Colonies in both age groups became alkaline phosphatase positive at the same rate, and virtually all colonies were positive after 12 days of culture. Cultures from both age groups produced abundant type I collagen. These studies suggest that defects in the number and proliferative potential of MSCs may underlie age-related defects in osteoblast number and function.

Mutations in fukutin-related protein (FKRP) cause a common subset of muscular dystrophies characterized by aberrant glycosylation of alpha-dystroglycan (α-DG), collectively known as dystroglycanopathies. The clinical variations associated with FKRP mutations range from mild limb-girdle muscular dystrophy type 2I with predominantly muscle phenotypes to severe Walker-Warburg syndrome and muscle-eye-brain disease with striking structural brain and eye defects. In the present study, we have generated animal models and demonstrated that ablation of FKRP functions is embryonic lethal and that the homozygous-null embryos die before reaching E12.5. The homozygous knock-in mouse carrying the missense P448L mutation almost completely lacks functional glycosylation of α-DG in muscles and brain, validating the essential role of FKRP in the functional glycosylation of α-DG. However, the knock-in mouse survives and develops a wide range of structural abnormalities in the central nervous system, characteristics of neuronal migration defects. The brain and eye defects are highly reminiscent of the phenotypes seen in severe dystroglycanopathy patients. In addition, skeletal muscles develop progressive muscular dystrophy. Our results confirm that post-translational modifications of α-DG are essential for normal development of the brain and eyes. In addition, both the mutation itself and the levels of FKRP expression are equally critical for the survival of the animals. The exceptionally wide clinical spectrums recapitulated in the P448L mice also suggest the involvement of other factors in the disease progression. The mutant mouse represents a valuable model to further elucidate the functions of FKRP and develop therapies for FKRP-related muscular dystrophies.

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.