Moustapha Kassem

Demonstration of the presence of independent pre-osteoblastic and pre-adipocytic cell populations in bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells (MSC) are defined as plastic-adherent, clonal cells that are common progenitors for osteoblasts and adipocytes. An inverse relationship between bone and fat has been observed in several clinical conditions and has been suggested to be caused by re-directing MSC differentiation into one particular lineage. However, this inverse relationship between bone and fat is not consistent and under certain in vivo conditions, bone and fat can change independently suggesting separate precursor cell populations. In order to test for this hypothesis, we extensively characterized two plastic-adherent clonal MSC lines (mMSC1 and mMSC2) derived from murine bone marrow. The two cell lines grew readily in culture and have undergone more than 100 population doublings with no apparent differences in their growth rates. Both cell lines were positive for the murine MSC marker Sca-1 and mMSC1 was also positive for CD13. Both cell lines were exposed to in vitro culture induction of osteogenesis and adipogenesis. mMSC1 and not mMSC2 were only able to differentiate to adipocytes evidenced by the expression of adipocyte markers (aP2, adiponectin, adipsin, PPARgamma2 and C/EBPa) and the presence of mature adipocytes visualized by Oil Red O staining. On the other hand, mMSC2 and not mMSC1 differentiated to osteoblast lineage as demonstrated by up-regulation of osteoblastic makers (CBFA1/RUNX2, Osterix, alkaline phosphatase, bone sialoprotein and osteopontin) and formation of alizarin red stained mineralized matrix in vitro. Consistent with the in vitro results, mMSC2 and not mMSC1, were able to form bone in vivo after subcutaneous implantation in immune-deficient (NOD/SCID) mice. Our data suggest that contrary to the current belief, bone marrow contains clonal subpopulations of cells that are committed to either osteoblast or adipocyte lineage. These cell populations may undergo independent changes during aging and in bone diseases and thus represent important targets for therapy.

Telomere shortening during aging of human osteoblasts in vitro and leukocytes in vivo: lack of excessive telomere loss in osteoporotic patients

We have compared the telomere length, as assessed by Southern analysis, of telomere restriction fragments (TRFs) generated by RsaI/HinfI digestion of genomic DNA in: (i) in vitro cultured human trabecular osteoblasts undergoing cellular aging; and (ii) peripheral blood leukocytes (PBL) obtained from three groups of women: young (aged 20-26 years, n = 15), elderly (aged 48-85 years, n = 15) and osteoporotic (aged 52-81 years, n = 14). The mean TRF length in human osteoblasts undergoing aging in vitro decreased from an average of 9.32 kilobasepairs (kb) in middle-aged cells to an average of 7.80 kb in old cells. The rate of TRF shortening was about 100 bp per population doubling, which is similar to what has been reported for other cell types, such as human fibroblasts. Furthermore, there was a 30% decline in the total amount of telomeric DNA in senescent osteoblasts as compared with young cells. In the case of PBL, TRF length in the DNA extracted from young women was slightly longer (6.76 +/- 0.64 kb) than that from a group of elderly women (6.42 +/- 0.71 kb). A comparison of TRFs in the DNA extracted from the PBL from osteoporotic patients and from age-matched controls did not show any significant differences (6.47 +/- 0.94 versus 6.42 +/- 0.71 kb, respectively). Therefore, using TRF length as a marker for cellular aging in vitro and in vivo, our data comparing TRFs from osteoporotic patients and age-matched controls do not support the notion of the occurrence of a generalized premature cellular aging in osteoporotic patients.

Changes in the insulin-like growth factor-system may contribute to in vitro age-related impaired osteoblast functions

Age-related bone loss is thought to be due to impaired osteoblast functions. Insulin-like growth factors (IGFs) have been shown to be important stimulators of bone formation and osteoblast activities in vitro and in vivo. We tested the hypothesis that in vitro osteoblast senescence is associated with changes in components of the IGF-system including IGF-I, IGF-II, IGF-binding proteins (IGFBPs) and IGFBP-specific proteases. We employed a human diploid osteoblast cell line obtained from trabecular bone explants and that exhibit typical characteristics of in vitro senescence during serial subculturing. Using a non-competitive reverse-transcriptase polymerase-chain reaction (RT-PCR) assay, we found that the constitutive level of IGF-I mRNA decreased progressively to 49.9 +/- 4.9% in old osteoblasts as compared to the levels found in the young cells. No age-related change was found in IGF-II steady-state mRNA levels. Changes in IGFBPs gene expression and protein production were assessed using Northern blot analysis and Western ligand blotting (WLB), respectively. IGFBP-3 mRNA levels decreased to 30% and protein production to 16% in aged osteoblasts as compared to levels found in young cells. We also found age-related decreases in mRNA levels of both IGFBP-4 and IGFBP-5 to 70% and 60% in aged osteoblasts, respectively, compared to young cells. While IGFBP-5 protein was not detected by WLB, IGFBP-4 protein production showed a biphasic change with 50% decrease in middle-aged cells and a subsequent increase in aged osteoblasts to levels similar to those in young osteoblasts. We found an age-related increase in the immunoreactive levels of IGFBP-4 protease, however, no detectable IGFBP-4 or IGFBP-3 protease activities in conditioned media from osteoblast cultures were observed. Our findings demonstrate that osteoblast aging is associated with impaired production of the stimulatory components of the IGF-system, that may be a mechanism contributing to age-related decline in osteoblast functions.

Treatment with 1,25‐dihydroxyvitamin D3 reduces impairment of human osteoblast functions during cellular aging in culture

Adequate responses to various hormones, such as 1,25‐dihydroxyvitamin D3 (calcitriol) are a prerequisite for optimal osteoblast functions. We have previously characterized several human diploid osteoblastic cell lines that exhibit typical in vitro aging characteristics during long‐term subculturing. In order to study in vitro age‐related changes in osteoblast functions, we compared constitutive mRNA levels of osteoblast‐specific genes in early‐passage (< 50% lifespan completed) with those of late‐passage cells (> 90% lifespan completed). We found a significant reduction in mRNA levels of alkaline phosphatase (AP: 68%), osteocalcin (OC: 67%), and collagen type I (ColI: 76%) in in vitro senescent late‐passage cells compared to early‐passage cells, suggesting an in vitro age‐related impairment of osteoblast functions. We hypothesized that decreased osteoblast functions with in vitro aging is due to impaired responsiveness to calcitriol known to be important for the regulation of biological activities of the osteoblasts. Thus, we examined changes in vitamin D receptor (VDR) system and the osteoblastic responses to calcitriol treatment during in vitro osteoblast aging. We found no change in the amount of VDR at either steady state mRNA level or protein level with increasing in vitro osteoblast age and examination of VDR localization, nuclear translocation and DNA binding activity revealed no in vitro age‐related changes. Furthermore, calcitriol (10−8M) treatment of early‐passage osteoblastic cells inhibited their proliferation by 57 ± 1% and stimulated steady state mRNA levels of AP (1.7 ± 0.1‐fold) and OC (1.8 ± 0.2‐fold). Similarly, calcitriol treatment increased mRNA levels of AP (1.7 ± 0.2‐fold) and OC (3.0 ± 0.3‐fold) in late‐passage osteoblastic cells. Thus, in vitro senescent osteoblastic cells maintain their responsiveness to calcitriol and some of the observed in vitro age‐related decreases in biological markers of osteoblast functions can be reverted by calcitriol treatment. J. Cell. Physiol. 186:298–306, 2001.

European and North American experience with HRT for the prevention of osteoporosis

Hormone replacement therapy (HRT) has been the method of choice for the prevention of postmenopausal osteoporosis since the early 1990s. Although a number of routes of administration are now available, HRT is still predominantly administered orally. In the United States, HRT formulations traditionally comprise conjugated equine estrogens. In Europe, however, HRT preparations tend to be based on 17 beta-estradiol, a natural human estrogen. Furthermore, distinct patterns of HRT use are apparent based on the age of the woman receiving it. Current recommendations are that early postmenopausal women (in their early 50s) receive sequential combined estrogen/progestogen therapy with continued monthly bleeds, while in women who are at least 1 year postmenopausal, continuous combined HRT, which leads to endometrial atrophy and cessation of monthly bleeding, is preferred. Clinical experience to date clearly demonstrates that long-term HRT unequivocally increases bone mass and reduces the risk of fractures in postmenopausal women, with no significant differences between sequential and continuous combined prescribing regimens. Data demonstrating that antiestrogens such as tamoxifen may preserve bone mass have led to the initiation of large-scale trials to determine the potential clinical utility of such agents for the prevention of osteoporosis in postmenopausal women. Nonhormonal therapeutic approaches are now also available, most notably bisphosphonates and vitamin D analogs. At present, however, traditional HRT remains the regimen of choice for the prevention of postmenopausal osteoporosis, given its additional beneficial effects on acute menopausal symptoms, as well as on the cardiovascular system and brain.

Aging of human trabecular osteoblasts in culture

Osteoblasts are the bone forming cells, which synthesize and secrete the components of the bone matrix. An imbalance between the breakdown and the build up of bone and a decline in osteoblast activity is considered to be the basis of age-related changes in bone structure and function, including the origin of osteoporosis. In order to determine whether the osteoblast activity decreases because of a decreased proliferative capacity or some other reason, we have examined several cellular and biochemical characteristics of human trabecular osteoblasts serially passaged in culture. We have studied growth and maximum lifespan in terms of cumulative population doubling level achieved in culture. We have also determined changes in morphology, protein content and the synthesis of proteins, DNA and RNA during aging of osteoblasts. Furthermore, changes in the cytoskeletal components actin and microtubuli have been observed along with a comparison of one dimensional and two dimensional gel electrophoretic protein pattern during aging.

Evidence of a normal mean telomere fragment length in patients with Ullrich-Turner syndrome

Clinical and epidemiological studies suggest that premature ageing and increased morbidity and mortality is present in Ullrich-Turner syndrome. We studied telomere restriction fragment length (TRFL) in 30 women with Ullrich-Turner syndrome and 30 age-matched control women. All Turner women had the 45,X karyotype verified by karyotyping. We found no difference in the mean TRFL in the young age group (TS: 7011±521 vs C: 7285±917 bp, P = 0.3), or in the older age group (TS: 7357±573 vs C: 7221±621 bp, P = 0.6). In conclusion, our data suggest that Ullrich-Turner syndrome is not associated with excessive telomere loss, at least when studied in peripheral blood leucocytes, and thus quite different from other premature ageing syndromes.

In Vitro Senescence of Human Osteoblasts

Human aging is associated with bone loss leading to bone fragility and increased risk for fractures, a disease known as osteoporosis. Osteoporosis is one of the most prevalent and serious diseases affecting the elderly population and constitutes a major public health problem. The cellular and molecular causes of age-related bone loss are current intensive topic of investigation with the aim of identifying new approaches to abolish its negative effects on the skeleton. The aim of this chapter is to give a review on the current understanding of the contribution of aging of the osteoblasts (the bone forming cells) to the phenomenon of age-related boneloss.