Karina Stewart

Integrin Expression and Function on Human Osteoblast-like Cells

The integrin family of cell adhesion molecules are a series of cell surface glycoproteins that recognize a range of cell surface and extracellular matrix (ECM)‐associated ligands. To date, the precise role of individual integrin molecules in bone cell–ECM interactions remains unclear. Cell binding assays were performed to examine the ability of normal human bone cells (NHBCs) to adhere to different ECM proteins in vitro. NHBCs displayed preferential adhesion to fibronectin over collagen types I, IV, and vitronectin and showed low affinity binding to laminin and collagen type V. No binding was observed to collagen type III. The integrin heterodimers α1β1, α2β1, α3β1, α5β1, αvβ3, and αvβ5 were found to be constitutively expressed on the cell surface of NHBCs by flow cytometric analysis. The integrins α4β1 and α6β1 were not expressed by NHBCs. Subsequent binding studies showed that NHBC adhesion to collagen and laminin was mediated by multiple integrins where cell attachment was almost completely inhibited in the presence of a combination of function‐blocking monoclonal antibodies (Mabs) to α1β1, α2β1, α3β1, and β1. In contrast, the adhesion of NHBCs to fibronectin was only partially inhibited (50%) in the presence of blocking Mabs to α3β1, α5β1, and β1. The attachment of NHBCs to collagen, laminin, fibronectin, and vitronectin was also found to be unaffected in the presence of a function‐blocking Mab to αvβ3. The results of this study indicate that β1 integrins appear to be the predominant adhesion receptor subfamily utilized by human osteoblast‐like cells to adhere to collagen and laminin and in part to fibronectin.

Bioreactors for tissue engineering

Bioreactors are essential in tissue engineering, not only because they provide an in vitro environment mimicking in vivo conditions for the growth of tissue substitutes, but also because they enable systematic studies of the responses of living tissues to various mechanical and biochemical cues. The basic principles of bioreactor design are reviewed, the bioreactors commonly used for the tissue engineering of cartilage, bone and cardiovascular systems are assessed in terms of their performance and usefulness. Several novel bioreactor types are also reviewed.

Further Characterization of Cells Expressing STRO‐1 in Cultures of Adult Human Bone Marrow Stromal Cells

Primitive cells of the osteoblast lineage are not well characterized but are known to be present within the STRO‐1+ fraction of adult human bone and marrow. A survey of human osteosarcoma cell lines revealed that STRO‐1 is expressed by MG‐63 but not SaOS‐2. Among murine cell lines tested, expression of STRO‐1 was detected in the bipotential (adipocyte/osteoblast) line BMS‐2 but not the committed osteoblast precursor MC3T3‐E1. A proportion of cultured adult human bone marrow stromal cells (BMSCs) consistently expressed the STRO‐1 antigen. The expression of a range of cell surface antigens was studied in relation to STRO‐1 by flow cytometry and several, including the bone/liver/kidney isoform of alkaline phosphatase (ALP), were found to subtype the STRO‐1+ population of BMSCs. Further, BMSCs dual‐labeled with antibodies recognizing STRO‐1 and ALP could be assigned to one of four fractions: STRO‐1−/ALP−, STRO‐1+/ALP−, STRO‐1+/ALP+, and STRO‐1−/ALP+. Cells from each fraction could be isolated in high purity and, when recultured, remained viable and exhibited a limited degree of phenotypic stability. Using reverse transcriptase‐polymerase chain reaction, cells in the four fractions were found to express different levels of transcripts for the parathyroid hormone receptor (PTHr) and bone sialoprotein (BSP). The expression of transcripts for the nuclear transcription factor core‐binding factor alpha 1/osteoblast‐specific factor‐2 (CBFA1/OSF2) was restricted to those fractions expressing STRO‐1 and/or ALP. Treatment with 10 nM dexamethasone consistently increased the proportion of cells present in those fractions which expressed the highest levels of transcripts for PTHr and BSP (STRO‐1+/ALP+ and STRO‐1−/ALP+) while simultaneously decreasing the proportion present in the STRO‐1+/ALP− fraction. In conclusion, the expression of STRO‐1 in vitro remains a characteristic of less well differentiated cells of the osteoblast lineage; in cultures of BMSCs and in established human osteosarcoma cell lines, there is an inverse association between the expression of STRO‐1 and ALP; dual labeling of BMSCs with monoclonal antibodies recognizing STRO‐1 and ALP permits the identification and isolation of cells of the osteoblast lineage at different stages of differentiation.

Expression of the developmental markers STRO-1 and alkaline phosphatase in cultures of human marrow stromal cells: regulation by fibroblast growth factor (FGF)-2 and relationship to the expression of FGF receptors 1–4

Autologous marrow stromal cells have been proposed as an adjuvant in the treatment of bone defects and diseases. This will require the development of culture conditions that permit their rapid expansion ex vivo while retaining their potential for further differentiation. Fibroblast growth factor (FGF)-2 has been proposed as a candidate for the ex vivo expansion of cells with enhanced osteogenic potential, and we have explored this possibility further using cells obtained from a large cohort of adult human donors. Treatment with FGF-2 (0.001-2.5 ng/mL) had no detectable effect on colony formation, but markedly increased their proliferative potential and that of their immediate progeny, as shown by the increases in colony size and cell number. Based on the observed increase in the expression of the developmental markers STRO-1 and alkaline phosphatase (AP), a major target for the actions of FGF-2 appears to be the more primitive cells of the osteoblast lineage, and that, when added in combination with the synthetic glucocorticoid dexamethasone (Dx), it interacts positively to promote further cell maturation. The maintenance of adequate levels of ascorbate was shown to be a critical component in determining the nature of the effect of FGF-2 on AP expression. Variation in the response (predominantly in the magnitude and/or sensitivity) of the cultured cell populations to treatment with FGF-2 was apparent, but a preliminary analysis indicated that this was not due to differences in the age or gender of the donors used. The cultured cell populations were found to express multiple FGF receptors (FGFRs; 1-4) and the observed changes in the spectrum and abundance of FGFRs expressed in relation to that of STRO-1 and AP are consistent with their expression being developmentally regulated during the process of osteogenic differentiation. These results provide novel insights into the mechanism of action of FGF-2 on human cells of the osteoblast lineage and support the use of this factor, alone or in combination with Dx, for the rapid, ex vivo expansion of cell populations with enhanced osteogenic potential.

IGF-I does not affect the proliferation or early osteogenic differentiation of human marrow stromal cells

The ability of insulin-like growth factor-I (IGF-I) to regulate the proliferation and differentiation of primitive osteogenic precursors (CFU-F) has been investigated in cultures of bone marrow stromal cells (BMSC) derived from a large cohort of adult human donors. Treatment with IGF-I (0.1-20 ng/mL, days 0-28) had no consistent effect on the number or size of colonies that formed or the proportion of colonies that expressed the developmental marker alkaline phosphatase (AP). At the end of primary culture, similar numbers of cells were harvested from the control and IGF-I-treated groups and there was no detectable difference in the expression of AP (activity or percentage of positive cells) or the developmental marker STRO-1. This was found to be the case whether IGF-I was added alone or in combination with 10 nM dexamethasone (Dx), a known inducer of osteogenic differentiation in this cell culture system. In contrast, cells derived from the same cohort of donors responded to treatment with fibroblast growth factor-2 (FGF-2) with an increase in the number and size of the colonies that formed, in proliferation and in the number of cells recovered in STRO-1(+)/AP(+) (osteoprogenitor) fraction. Further analysis revealed that the majority of BMSC expressed the alpha and beta subunits of the type 1 receptor for IGF-I (IGF-IR), in the expected 1:1 ratio. Treatment with Dx did not affect the expression of these receptor subunits (percentage of positive cells or number of sites per cell) but did increase the proportion of cells present in the IGF-I(+)/AP(+) fraction. The results of this investigation suggest that the beneficial effects of IGF-I on the skeleton are not mediated primarily via an effect on osteoprogenitor fraction and are thus consistent with the hypothesis that the effects of IGF-I are differentiation dependent and restricted largely to the more mature cells of the osteoblast lineage.

A Comparison of Colorimetric and DNA Quantification Assays for the Assessment of Meniscal Fibrochondrocyte Proliferation in Microcarrier Culture

We have developed and refined a rapid, reliable method for the evaluation of attachment and proliferation of ovine meniscal chondrocytes in microcarrier culture. Assays measuring both mitochondrial activity, using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and MTS [3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium], and DNA synthesis with a PicoGreen assay were compared. The MTT assay was the most sensitive at lower cell concentrations and enabled accurate assessment of cell proliferation over 14 day culture.

Culture of Meniscal Chondrocytes on Alginate Hydrogel Matrices

Alginate hydrogels, crosslinked ionically (with calcium ions) and covalently (with 1-ethyl-3-dimethylaminopropyl-N-hydroxysuccinimide carbodiiamide) have been investigated for their suitability as scaffolds for meniscal chondrocyte cell culture. Ovine meniscal chondrocytes were encapsulated at 2.6 ± 0.6 × 106 cells/ml in 1–2% w/v alginate beads and were also seeded onto the surface of flat alginate hydrogels. Alginate beads were cultured in stirred or static environments on DMEM media with 10% serum. The alginate beads supported cell growth for over 50 days, however, cell numbers and viability in the alginate beads were found to decrease over time, with a greater retention of cell viability in the stirred flask culture. When cultured on flat alginate gels, it was found that a much greater cell count was achieved on gels which had been pre-conditioned with culture media containing chondrocytes as compared with soaking in media alone. Covalently crosslinked gels were found to disintegrate over a 3 day period, and were generally unsuitable for supporting meniscal chondrocyte growth.