Enhanced osteoblastogenesis in three-dimensional collagen gels.

Growth and differentiation of osteoblasts are often studied in cell cultures. In vivo, however, osteoblasts are embedded within a complex three-dimensional (3D) microenvironment, which bears little relation to standard culture flasks. Our study characterizes osteoblast-like cells cultured in 3D collagen gels and compares them with cells in two-dimensional (2D) cultures. Primary rat osteoblasts and MC3T3-E1 cells were seeded within type I collagen gels, and differentiation was determined by mineral staining and gene expression analysis. Cells growing in 3D gels showed positive mineral staining and induction of osteoblast marker genes earlier than cells growing in 2D. A number of genes, including osteocalcin, bone sialoprotein, alkaline phosphatase and dentin matrix protein 1, were already highly upregulated in 3D cultures 24 h after seeding. The early expression of osteoblast genes was dependent on the 3D structure and was not induced in cells growing on collagen-coated dishes in 2D. Comparison of thymidine incorporation between cells in 3D and 2D cultures treated with agents that induce proliferation-transforming growth factor ß, platelet-derived growth factor and lactoferrin-showed a much greater response in 3D gels. Cells in 3D cultures were also much more sensitive to inhibition of proliferation by the protein kinase inhibitor imatinib mesylate. The 3D collagen gels better represent the physiological bone environment and offer a number of technical advantages for the study of osteoblasts in vitro. These studies have additional practical implications as 3D collagen gels are considered as a scaffold material in regenerative medicine for the repair of bone defects.

Fates and osteogenic differentiation potential of human mesenchymal stem cells in immunocompromised mice.

Human mesenchymal stem cells (hMSCs) from bone marrow were genetically marked by using a murine leukaemia virus construct encoding enhanced green fluorescent protein (eGFP). The marked cells were either directly implanted into the tibialis anterior muscle or introduced into a variety of other tissue sites in immunocompromised mice (NOD/SCID and C.B-17 SCID/beige) to investigate their fates and differentiation potentials. It was observed that the hMSCs survived for up to 12 weeks and showed site-specific morphological phenotypes. hMSCs delivered by intravenous injection were found mainly in the lungs and were detected rarely in other organs. Histomorphometry showed that, after implantation of hMSCs into the tibialis anterior muscle juxtaskeletally, the areas of reactive host callus formation at 1 and 2 weeks and of ectopic human bone formation at 1 week were significantly increased compared with the control group. Expression of eGFP and human RUNX2, alkaline phosphatase, osteocalcin, osteopontin, and collagen type I mRNAs were detected in mice implanted with the labelled hMSCs but not in sham-treated samples. Active clearance of the reactive callus and ectopic calcified tissue by osteoclast-like tartrate-resistant acid phosphatase-positive cells was observed. We conclude that the eGFP-labelled hMSCs can survive and retain the potential to differentiate morphologically into a variety of apparent mesenchymal phenotypes in vivo. Absolute confirmation of differentiation capacity requires further study and is complicated by known possibilities of fusion of donor and host cells or limited transfer of genetic material. Nevertheless, the genetically marked hMSCs are shown to participate extensively in bone formation and turnover. Control of the host osteoclast/macrophage responses resulting in clearance of formed osteogenic tissue warrants further investigation to promote prolonged human osteogenesis in immunocompromised mice. Furthermore, any proposed general cytotherapeutic strategy for enhanced osteogenesis is likely to require supplementation of local bone-forming biological signals.

Selective targeting of death receptor 5 circumvents resistance of MG-63 osteosarcoma cells to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a tumor necrosis factor superfamily member, targets death receptors and selectively kills malignant cells while leaving normal cells unaffected. However, unlike most cancers, many osteosarcomas are resistant to TRAIL. To investigate this resistance, we characterized the response of MG-63 osteosarcoma cells and hPOB-tert osteoblast-like cells to TRAIL and agonist antibodies to death receptor 4 (DR4) and death receptor 5 (DR5). We found that MG-63 osteosarcoma cells and hPOB-tert osteoblast-like cells show no or very little response to TRAIL or a DR4 agonist, but MG-63 cells undergo apoptosis in response to a DR5 agonist. Analysis of TRAIL receptor expression showed that normal osteoblastic and osteosarcoma cells express a variety of TRAIL receptors but this does not correlate to TRAIL responsiveness. Production of the soluble decoy receptor osteoprotegerin also could not explain TRAIL resistance. We show that TRAIL activates the canonical caspase-dependent pathway, whereas treatment with cycloheximide increases the sensitivity of MG-63 cells to TRAIL and anti-DR5 and can also sensitize hPOB-tert cells to both agents. Proapoptotic and antiapoptotic protein expression does not significantly differ between MG-63 and hPOB-tert cells or change following treatment with TRAIL or anti-DR5. However, sequencing the death domain of DR4 in several osteoblast-like cells showed that MG-63 osteosarcoma cells are heterozygous for a dominant-negative mutation, which can confer TRAIL resistance. These results suggest that although the dominant-negative form of the receptor may block TRAIL-induced death, an agonist antibody to the active death receptor can override cellular defenses and thus provide a tailored approach to treat resistant osteosarcomas.

Innate immune response to human bone marrow fibroblastic cell implantation in CB17 scid/beige mice.

Immunocompromised mouse models have been extensively used to assess human cell implantation for evaluation of cytotherapy, gene therapy and tissue engineering strategies, as these mice are deficient in T and B lymphoid cells. However, the innate immune response and its effect on human cell xenotransplantation in these mouse models are mainly unknown. The aim of this study is to characterise the myeloid populations in the spleen and blood of CB17 scid beige (CB17 sb) mice, and to study the inflammatory cell responses to xenogeneic implantation of enhanced green fluorescent protein (GFP)-labelled human bone marrow fibroblastic (HBMF) cells into CB17 sb mice. The results indicate that even though CB17 sb mice are deficient in B- and T-cells, they exhibit some increases in their monocyte (Mo), macrophage (Mphi) and neutrophil (Neu) populations. NK cell and eosinophil populations show no differences compared with wild-type Balb/C mice. An innate immune response, identified by CR3 (CD11b/CD18)-positive myeloid inflammatory cells and F4/80-positive macrophages, was evident in the tissues where HBMF cells were implanted. As a consequence, the majority of implanted HBMF cells were eliminated by 4 weeks after implantation. Interestingly, the mineralised matrix formed by osteogenic HBMF cells was also eroded by multinuclear Mphi-like giant cells. We conclude that CB17 sb mice retain active innate immune cells, which respond to HBMF cell xenotransplantation. This study highlights the importance of the innate immune cells in the anti-xenograft response and suggests that strategies to block the activities of these cells may ameliorate the progressive long-term elimination of xenotransplants.

Stimulation of osteoclast formation by inflammatory synovial fluid.

Peri-articular bone resorption is a feature of arthritis due to crystal deposition and rheumatoid disease. Under these conditions, the synovial fluid contains numerous inflammatory cells that produce cytokines and growth factors which promote osteoclast formation. The aim of this study was to determine whether inflammatory synovial fluid stimulates the formation of osteoclasts. Synovial fluid from rheumatoid arthritis (RA), pyrophosphate arthropathy (PPA) and osteoarthritis (OA) patients was added to cultures (n=8) of human peripheral blood mononuclear cells (PBMCs) in the presence and absence of macrophage colony-stimulating factor (M-CSF) and the receptor activator of NF-kappaB ligand (RANKL). Osteoclast formation was assessed by the formation of cells positive for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR) and the extent of lacunar resorption. The addition of 10% OA, RA and PPA synovial fluid to PBMC cultures resulted in the formation of numerous multinucleated or mononuclear TRAP(+) and VNR(+) cells which were capable of lacunar resorption. In contrast to PBMC cultures incubated with OA synovial fluid, there was marked stimulation of osteoclast formation and resorption in cultures containing inflammatory RA and PPA synovial fluid which contained high levels of tumour necrosis factor alpha, a factor which is known to stimulate RANKL-induced osteoclast formation.

Efficient characterisation of human cell-bioceramic interactions in vitro and in vivo by using enhanced GFP-labelled mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) were transfected using four retroviral pseudotypes, amphotropic murine leukemia viruses 4070 (MuLV-10A1), a modification of amphotropic pseudotype 4073 (A71G, Q74K, V139M), gibbon ape leukemia virus (GaLV), or feline endogenous virus (RD114) encoding the neomycin resistance (Neo(r)) gene and enhanced green fluorescent protein (eGFP) as genetic markers. It was observed that the MuLV4073 was the most efficient pseudotype for hMSC transfection. The proliferation and differentiation characteristics of eGFP-labelled hMSCs were not significantly different from control hMSCs. G418 selected eGFP-labelled cells were cultured for 3 weeks on two porous, commercially available calcium phosphate bioceramics, a "synthetic hydroxyapatite" and a "deproteinised bone", before implantation into NOD/SCID mice for up to 4 weeks. The eGFP-labelled hMSCs could be readily visualised by their intense green fluorescence both in vitro and in vivo. In "synthetic hydroxyapatite" implants the cells remained in a monolayer, whereas in "deproteinised bone" implants mineralised tissues were detected by histology, scanning electron microscopy and energy dispersive X-ray spectrometry. From the results, it is concluded that the use of eGFP-labelled hMSCs is an effective tool to trace the fate of hMSCs and evaluate the interactions between cells and ceramics both in vitro and in vivo. This is of great value in prospective assessments of these cell populations for use in tissue engineering applications.

A gain-of-function mutation of Fgfr2c demonstrates the roles of this receptor variant in osteogenesis.

The b and c variants of fibroblast growth factor receptor 2 (FGFR2) differ in sequence, binding specificity, and localization. Fgfr2b, expressed in epithelia, is required for limb outgrowth and branching morphogenesis, whereas the mesenchymal Fgfr2c variant is required by the osteocyte lineage for normal skeletogenesis. Gain-of-function mutations in human FGFR2c are associated with craniosynostosis syndromes. To confirm and extend this evidence, we introduced a Cys342Tyr replacement into Fgfr2c to create a gain-of-function mutation equivalent to a mutation in human Crouzon and Pfeiffer syndromes. Fgfr2c(C342Y/)(+) heterozygote mice are viable and fertile with shortened face, protruding eyes, premature fusion of cranial sutures, and enhanced Spp1 expression in the calvaria. Homozygous mutants display multiple joint fusions, cleft palate, and trachea and lung defects, and die shortly after birth. They show enhanced Cbfa1/Runx2 expression without significant change in chondrocyte-specific Ihh, PTHrP, Sox9, Col2a, or Col10a gene expression. Histomorphometric analysis and bone marrow stromal cell culture showed a significant increase of osteoblast progenitors with no change in osteoclastogenic cells. Chondrocyte proliferation was decreased in the skull base at embryonic day 14.5 but not later. These results suggest that long-term aspects of the mutant phenotype, including craniosynostosis, are related to the Fgfr2c regulation of the osteoblast lineage. The effect on early chondrocyte proliferation but not gene expression suggests cooperation of Fgfr2c with Fgfr3 in the formation of the cartilage model for endochondral bone.

Mediators of the biphasic responses of bone to intermittent and continuously administered parathyroid hormone.

Parathyroid hormone (PTH) has biphasic effects on bone: continuous treatment is catabolic whereas intermittent treatment is anabolic. The mechanism(s) responsible for these differing effects are still unclear, partly because of the previous non-availability of a model system in which effects on both formation and resorption indices could be studied concomitantly. In cultured marrow cells from 6-week old C57BL/6 mice, we demonstrated that 4 days of intermittent PTH treatment increased mRNA for osteoblast differentiation markers (Runx2, alkaline phosphatase (AP), and type I procollagen (COL1A1) whereas continuous treatment resulted in production of large numbers of TRAP-positive multinucleated osteoclasts. Although IGF-I mRNA did not increase after intermittent treatment, it was consistently higher than after continuous treatment, and the addition of an anti-IGF-I neutralizing antibody prevented the increase in bone formation indices observed with intermittent treatment. By contrast, after continuous treatment, gene expression of RANK ligand (RANKL) was increased and that of osteoprotegerin (OPG) was decreased, resulting in a 25-fold increase in the RANKL/OPG ratio. In this model system, the data suggest that intermittent PTH treatment enhances osteoblast differentiation through an IGF-I dependent mechanism and continuous PTH treatment enhances osteoclastogenesis through reciprocal increases in RANKL and decreases in OPG.