Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells.

Human bone marrow stromal cells (hMSCs) were stably transduced by a retroviral vector containing the gene for the catalytic subunit of human telomerase (hTERT). Transduced cells (hMSC-TERTs) had telomerase activity, and the mean telomere length was increased as compared with that of control cells. The transduced cells have now undergone more than 260 population doublings (PD) and continue to proliferate, whereas control cells underwent senescence-associated proliferation arrest after 26 PD. The cells maintained production of osteoblastic markers and differentiation potential during continuous subculturing, did not form tumors, and had a normal karyotype. When implanted subcutaneously in immunodeficient mice, the transduced cells formed more bone than did normal cells. These results suggest that ectopic expression of telomerase in hMSCs prevents senescence-associated impairment of osteoblast functions.

QCM-D studies of attachment and differential spreading of pre-osteoblastic cells on Ta and Cr surfaces.

The quartz crystal microbalance with dissipation (QCM-D) technique was employed to characterize initial cell adhesion in terms of attachment and spreading of pre-osteoblastic MC3T3-E1 cells on Ta and Cr surfaces. Evaluation of initial cell adhesion established a correlation between input cell number and the shifts in frequency (f) and dissipation (D). The f-shift was found to be much larger in serum-free medium as compared to a medium including serum; hence, initial cell adhesion was subsequently evaluated in serum-free medium. During the first hour of adhesion, we found a positive correlation between the QCM-D f-shift and the average area of the spread cells, as measured by cryo-scanning electron microscopy (cryo-SEM). Finally, the QCM-D technique was used to study cell adhesion on different metal oxide surfaces. Initial cell adhesion on Ta was found to induce a larger f-shift as compared to Cr, indicating larger spreading of cells on Ta. Cryo-SEM data confirmed that spreading of cells on Cr was on average only two-thirds the spreading on Ta. Our results demonstrate that the QCM-D technique is a versatile technique to quickly distinguish initial cell-surface interactions on different biomaterials.

Mice deficient in 11beta-hydroxysteroid dehydrogenase type 1 lack bone marrow adipocytes, but maintain normal bone formation.

Glucocorticoids (GCs) exert potent, but poorly characterized, effects on the skeleton. The cellular activity of GCs is regulated at a prereceptor level by 11beta-hydroxysteroid dehydrogenases (11betaHSDs). The type 1 isoform, which predominates in bone, functions as a reductase in intact cells and regenerates active cortisol (corticosterone) from circulating inert 11-keto forms. The aim of the present study was to investigate the role of this intracrine activation of GCs on normal bone physiology in vivo using mice deficient in 11betaHSD1 (HSD1(-/-)). The HSD1(-/-) mice exhibited no significant changes in cortical or trabecular bone mass compared with wild-type (Wt) mice. Aged HSD1(-/-) mice showed age-related bone loss similar to that observed in Wt mice. Histomorphometric analysis showed similar bone formation and bone resorption parameters in HSD1(-/-) and Wt mice. However, examination of bone marrow composition revealed a total absence of marrow adipocytes in HSD1(-/-) mice. Cells from Wt and HSD1(-/-) mice exhibited similar growth rates as well as similar levels of production of osteoblastic markers. The adipocyte-forming capacity of in vitro cultured bone marrow stromal cells and trabecular osteoblasts was similar in HSD1(-/-) and Wt mice. In conclusion, our results suggest that 11betaHSD1 amplification of intracellular GC actions in mice may be required for bone marrow adipocyte formation, but not for bone formation. The clinical relevance of this observation remains to be determined.

Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells.

Age-related decrease in bone formation is well described. However, the cellular causes are not known. Thus, we have established cultures of bone marrow stromal cells (MSC) from young (aged 18-29 years, n = 6) and old (aged 68-81 years, n = 5) donors. MSC were serially passaged until reaching maximal life span. Cell growth, markers of cellular senescence, and osteogenic and adipogenic potential were determined in early-passage and late-passage cells established from young and old donors. MSC from old donors exhibited a decreased maximal life span compared with cells from young donors (24 +/- 11 population doublings [PD] vs 41 +/- 10 PD, P < 0.05) and mean PD rate was lower in old donor cells (0.05 +/- 0.02 PD/day) compared with young donor cells (0.09 +/- 0.02 PD/day) (P < 0.05). No differences were detected in number of senescence-associated beta-galactosidase positive (SA beta-gal+) cells and mean telomere length in early-passage cells obtained from young and old donors. However, MSC from old donors exhibited accelerated senescence evidenced by increased number of SA beta-gal+ cells per PD as compared with young (4% per PD vs 0.4% per PD, respectively). MSC from young and old donors were able to form similar amounts of mineralized matrix in vitro and of normal lamellar bone in vivo. In adipogenic medium similar numbers of adipocytes formed in cultures of young and old donors. In conclusion, aging is associated with decreased proliferative capacity of osteoprogenitor cells, suggesting that decreased osteoblastic cell number, and not function, leads to age-related decrease in bone formation.

Subcutaneous adipocytes can differentiate into bone-forming cells in vitro and in vivo.

Interconversion of bone marrow osteoblasts and adipocytes has been reported previously. However, the osteogenic potential of extramedullary adipocytes is not known. Thus, we incubated a pure culture of human subcutaneous adipocytes in control medium for 1-2 weeks. Afterward, the cells were incubated in either osteoblast medium (OB medium) containing various combinations of calcitriol, dexamethasone, ascorbic acid, and beta-glycerophosphate or in adipocyte medium (AD medium) containing HEPES, biotin, pantothenate, insulin, triiodothyronine, dexamethasone, and isobutylmethylxanthine for 4 weeks. Expression of osteoblastic and adipocytic phenotypes was examined by determination of lineage-specific mRNA markers and in vitro adipocyte and osteoblast formation. Cells were also implanted, mixed with hydroxyapatite-tricalcium phosphate powder, in the subcutaneous tissue of immunodeficient mice in order to assess in vivo bone formation potential. One week after incubation in control medium, cells formed fusiform elongated fibroblast-like cells. In OB medium, cells stained positive for alkaline phosphatase (AP) and expressed mRNAs encoding Cbfa1/Runx2, AP, and osteocalcin. In AD medium cells reacquired adipocyte morphology with multilocular lipid-filled cells. Also, the cells expressed adipocyte-specific mRNA markers: lipoprotein lipase and peroxisome proliferator-activated receptor gamma2. Bone was formed only in the in vivo implants of cells incubated in OB medium. In conclusion, extramedullary adipocytes can transdifferentiate to bone-forming cells. Because of their ease of isolation, adipocytes may be good candidates for tissue-engineering protocols aimed at creating bone tissue for the repair of nonunion fractures and large bone defects.

The use of combinatorial topographical libraries for the screening of enhanced osteogenic expression and mineralization.

Nano- and microstructured surfaces are known to impact on the binding and differentiation of cells, but the detailed basic understanding of the underlying regulatory mechanisms is still scarce, which impedes the rational design of smart biomaterials. Towards a comprehensive analysis of the interplay between topographical parameters such as feature design and lateral and vertical dimensions we here report on a combinatorial screening approach, BioSurface Structure Array (BSSA) of test squares each with a distinct topography. Using such BSSA libraries of 504 topographically distinct surface structures, we have identified combinations of size, gap and height of structures which enhance mineralization as well as the expression of osteogenic markers of a preosteoblastic murine cell line. This generic BSSA screening platform is a versatile technology for the systematic identification of surfaces with specific biological properties, and it may for example be useful for optimizing the design of biomaterials for regulating cellular behaviour.