Irwin Olsen

Stem Cell Function, Self-Renewal, and Behavioral Heterogeneity of Cells from the Adult Muscle Satellite Cell Niche

Satellite cells are situated beneath the basal lamina that surrounds each myofiber and function as myogenic precursors for muscle growth and repair. The source of satellite cell renewal is controversial and has been suggested to be a separate circulating or interstitial stem cell population. Here, we transplant single intact myofibers into radiation-ablated muscles and demonstrate that satellite cells are self-sufficient as a source of regeneration. As few as seven satellite cells associated with one transplanted myofiber can generate over 100 new myofibers containing thousands of myonuclei. Moreover, the transplanted satellite cells vigorously self-renew, expanding in number and repopulating the host muscle with new satellite cells. Following experimental injury, these cells proliferate extensively and regenerate large compact clusters of myofibers. Thus, within a normally stable tissue, the satellite cell exhibits archetypal stem cell properties and is competent to form the basal origin of adult muscle regeneration.

The effect of MgO on the solubility behavior and cell proliferation in a quaternary soluble phosphate based glass system.

This paper presents a systematic study of the MgO–CaO–Na2O–P2O5 glass system, which has great potential to be used as temporary hard and soft tissue implant materials. An overall study of solubility behavior of ternary and quaternary-based phosphate glass system have been carried out in order to understand the out-leaching progress of different ions and to determine their effect on cell proliferation. Originally, soluble phosphate based glasses within the ternary glass system of Na2O–CaO–P2O5 have been developed to create a simple baseline system. This paper, however, presents the development of this system by introducing magnesium oxide as a partial calcium oxide substitute and solubility behaviors as well as cell studies have been carried out to check the effect on magnesium ions. Glasses have been prepared via standard glass melting techniques and their solubility behavior has been tested in distilled water via simple weight loss, pH and ion measurements. The way the glasses dissolve is an inverse exponential behavior which is mirrored by the calcium ion release. Other ions show a less exponential behavior. The MTT test has been used to check preliminary in vitro studies on a human MG63 cell line and the result indicates that cell proliferation is increased for glasses with minimal CaO substitution.

Effects of ultrasound on the growth and function of bone and periodontal ligament cells in vitro

The effects of therapeutic ultrasound (US) on tissue healing processes in vivo are likely to involve US-induced changes in key cellular functions. However, these have not yet been clearly delineated and the present study has, therefore, examined the effects of a single 5-min CW exposure of 3.00-MHz US on the growth and functional activity of a human osteoblast-like cell line (MG63 cells) and human periodontal ligament (PDL) cells in vitro. Although cell proliferation was found to be largely unaffected by spatial average intensity (I(SA)) values of between 140-990 mW/cm(2), flow cytometry (FCM) analysis showed that there were pronounced and differential effects on cell function. Thus, bone-associated proteins were down-regulated, whereas collagen type I (COL I) was unaffected and fibronectin (FN) was up-regulated at low intensities in MG63 cells. In contrast, bone protein expression was found to be dose-dependent, and FN and COL I were down-regulated in PDL cells. These results show that US has potentially important effects on the functional activities of connective tissue cells in vitro, which could markedly influence tissue repair and regeneration processes in vivo.

Expression of growth-factor receptors in normal and regenerating human periodontal cells

Growth factors are biologically active mediators that bind to specific receptors on target cells and regulate genes involved in cell growth, wound healing and regeneration. The expression of these receptors is thus of fundamental importance for the response of the cells to the factors. The aim here was to examine, using immunohistochemistry and flow cytometry, the expression of growth factor receptors in normal gingiva, periodontal ligament and in cells derived from these tissues, and also in regenerated tissues following guided tissue regeneration (GTR). By immunocytochemistry platelet-derived growth factor receptor-alpha (PDGF-Ralpha) was not detected in any of the tissues, whereas the PDGF-Rbeta and transforming growth factor-beta receptor types I and II (TGF-beta RI, RII) appeared to be upregulated in regenerated tissues compared with gingival and periodontal ligament tissues. Epidermal growth factor receptor (EGF-R) was also notably elevated in the regenerated tissue and was strongly expressed in the gingival epithelium but not in the periodontal ligament. Neither were fibroblast growth factor receptor-I (FGF-RI) or insulin-like growth factor receptor (IGF-R) detected in the periodontal ligament, nor in the gingiva, but they sometimes stained weakly in the regenerated tissues. Flow cytometry (FCM) showed that all the cells derived from the normal gingiva and the periodontal ligament expressed the PDGF-Rbeta, whereas the TGF-beta RI and RII, FGF-RI and IGF-R were detected in only a proportion of the total cells. In contrast, none of the cells expressed the PDGF-Ralpha or the EGF-R. These observations show that the growth factor receptors are differentially expressed by the periodontal tissues and cells and suggest that the corresponding factors may also be differentially involved in periodontal wound healing and regeneration.

Expression Profiling of Periodontal Ligament Cells Stimulated with Enamel Matrix Proteins in vitro: A Model for Tissue Regeneration

Several studies have examined the role of enamel matrix proteins in root formation and periodontal regeneration, although most of these have focused on a few specific genes which had previously been implicated. However, recent advances in expressional profiling have made it possible to examine the range of genetic responses involved in these processes. In the present experiments, we have therefore utilized this technique to determine the effects of enamel matrix proteins on the gene activities of periodontal ligament cells in vitro. Such cells were found to have an elevated level of RNA synthesis compared with control cells. Moreover, hybridization of the cDNA prepared from this RNA to gene array filters showed that there was differential expression of 121 genes, most of which had not previously been associated with periodontal regeneration. Some of these selective changes in gene activity might thus reflect the fundamental events that underlie periodontal development.

Up-regulation of bone morphogenetic protein receptor IB by growth factors enhances BMP-2-Induced human bone cell functions

Bone morphogenetic proteins (BMP) stimulate osteoblast differentiation by signal transduction via three BMP receptors (BMPR‐IA, ‐IB, and ‐II). Several growth factors, including transforming growth factor‐β1 (TGF‐β1), fibroblast growth factor‐2 (FGF‐2) and platelet‐derived growth factor‐AB (PDGF‐AB), have also been shown to play an important part in osteogenesis. The mechanism underlying these activities is unclear, but these growth factors could modulate the BMP/BMPR pathway by up‐regulating BMPR expression, thereby enhancing the osteogenic responses of bone cells to the BMP. In this study we have therefore examined the effects of TGF‐β1, FGF‐2, and PDGF‐AB on BMPR expression and BMP‐2‐mediated osteoblast functions in primary human bone cells. The results showed that although the ligand BMP‐2 and growth factors had little effect on BMPR‐IA and ‐II transcript expression, they significantly up‐regulated BMPR‐IB mRNA specifically. However, only the growth factors, but not the ligand BMP‐2, increased the surface expression of the BMPR‐IB antigen, which was found to be due to a differential effect of BMP‐2 and the growth factors on the Smurf1/Smad6‐induced breakdown process. Pre‐incubation of the cells with the growth factors significantly augmented BMP‐2‐induced Smad1/5/8 phosphorylation, and Dlx5 expression ALP activity, compared with that of cells treated with BMP‐2 alone. When cells were transfected with siRNA targeting BMPR‐IB, the growth factors neither up‐regulated BMPR‐IB transcript expression nor enhanced BMP‐2‐induced Smad1/5/8 phosphorylation, Dlx5 expression and ALP activity. The results indicate that increased BMPR‐IB by TGF‐β1, FGF‐2, and PDGF‐AB significantly enhances BMP‐2‐induced osteogenic functions in vitro, suggesting that they might positively modulate bone formation by up‐regulating BMPR‐IB in vivo. J. Cell. Physiol. 209: 912–922, 2006.

Glass reinforced hydroxyapatite for hard tissue surgery*Part II: in vitro evaluation of bone cell growth and function

Hydroxyapatite (HA)-based materials are considered to be potentially useful as bone implant materials, particularly those reinforced with glass to improve mechanical strength. However, the precise effects of glass-reinforced HA on the growth and functions of bone cells are still unclear. The present study has therefore examined the response of human osteoblast-like cells to HA and HA reinforced with two different proportions of glass, namely 2.5% and 5%. All materials enabled the cells to attach and proliferate during 7 days in culture and, although the growth was less than on control plastic surfaces, there was no deleterious effect of the 5% glass composite compared with HA alone. Flow cytometry analysis showed that there was no effect on cell size and granularity, but there were marked and highly selective changes in the expression of certain connective tissue proteins. Thus, while bone sialoprotein and osteonectin were down-regulated on HA alone, the expression of these antigens was relatively enhanced on the composite materials, and collagen type I was also up-regulated on the glass-reinforced HA. Thus, modulation of the glass composition of HA materials could be used to produce not only improved mechanical strength, but also enhanced biocompatibility.

Flow cytometry analysis of the effects of pre-immersion on the biocompatibility of glass-reinforced hydroxyapatite plasma-sprayed coatings.

Multilayered coatings composed of mixtures of HA and P2O5-based bioactive glasses are of potential clinical benefit in orthopaedic and dental surgery. Pre-immersion of these materials has been reported to further enhance their efficacy in vivo, although the precise biological effects of this treatment are not yet known. In this study we have therefore prepared double-layer plasma-sprayed coatings and evaluated the effects of pre-immersion on the growth and function of human osteosarcoma cells in vitro, using the MTT assay and flow cytometry analysis, respectively. The results showed that the increase in numbers of viable cells was the same or elevated following incubation on the pre-immersed HA and glass-reinforced HA coatings compared with the non-immersed materials. In addition, the expression of bone sialoprotein and fibronectin, two key connective tissue antigens, was up-regulated in cultures grown on the pre-immersed surfaces compared with the non-treated materials. Moreover, cell numbers and antigen expression both improved as the proportion of glass increased, particularly in the pre-immersed samples. Our findings thus suggest that the immersion treatment of these materials appeared to improve the response of these bone-like cells.

Direct and indirect effects of P2O5 glass reinforced-hydroxyapatite composites on the growth and function of osteoblast-like cells

Human osteoblast-like cells were plated on hydroxyapatite and P2O5-glass reinforced hydroxyapatite composite discs. They were also cultured in the presence of media obtained by incubating the discs in the absence of cells. The effects of these direct and indirect interactions were examined by measuring cell proliferation and the expression of certain key extracellular matrix antigens. One composite was found to initially delay cell growth, while the extract of a different composite appeared to down-regulate DNA synthesis. Flow cytometry analysis showed that growth directly on the discs had little effect on collagen type I, but reduced fibronectin and osteocalcin levels. The extracts of the materials generally had less effect, although one extract obtained from the glass-reinforced hydroxyapatite significantly down-regulated fibronectin. These in vitro studies thus suggest that there were only few differences overall in the growth of the cells directly on the glass-reinforced compared with the hydroxyapatite discs and also only relatively small effects of the extracts on the cells. However, the flow cytometry results suggest that both the materials and the extracts may have a potentially important influence on connective tissue production, and that these effects are both material- and antigen-specific.

Adhesion of bone cells to ion-implanted titanium

The use of ion-implantation to encourage osseointegration has been investigated using an in vitro model cell culture system and surface analysis. Polished titanium discs were implanted with calcium, potassium and argon ions. The adhesion of bone-derived cells was measured using radioactively labeled cells and the morphology examined using scanning electron microscopy. Similar numbers of cells were found to adhere to the potassium and argon-implanted titanium as to control (non-implanted) titanium. However, adhesion to the calcium-implanted titanium discs was significantly reduced. Moreover, although the cells were found to be well spread on the calcium and potassium-implanted titanium, a much greater proportion of cells appeared to remain rounded and poorly attached on the argon-implanted surface. These differences are discussed in relation to the observed surface roughness and chemistry, which were assessed using interferometry and X-ray photoelectron spectroscopy, respectively.