PEGylated liposomes associate with Wnt3A protein and expand putative stem cells in human bone marrow populations

Aim: To fabricate PEGylated liposomes which preserve the activity of hydrophobic Wnt3A protein, and to demonstrate their efficacy in promoting expansion of osteoprogenitors from human bone marrow. Methods: PEGylated liposomes composed of several synthetic lipids were tested for their ability to preserve Wnt3A activity in reporter and differentiation assays. Single-molecule microspectroscopy was used to test for direct association of protein with liposomes. Results: Labeled Wnt3A protein directly associated with all tested liposome preparations. However, Wnt3A activity was preserved or enhanced in PEGylated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes but not in PEGylated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes. PEGylated Wnt3A liposomes associated with skeletal stem cell populations in human bone marrow and promoted osteogenesis. Conclusion: Active Wnt protein-containing PEGylated liposomes may have utility for systemic administration for bone repair.The authors acknowledge funding support from the Medical Research Council, UK (grant number MR/J004103/1), Wessex Medical Research (grant number SO2), UoS Research Management Committee and the Institute for Life Sciences, Southampton. The authors would like to thank the Royal Society for the University Research Fellowship of Steven F Lee (UF120277)

Prospects of micromass culture technology in tissue engineering

Tissue engineering of bone and cartilage tissue for subsequent implantation is of growing interest in cranio- and maxillofacial surgery. Commonly it is performed by using cells coaxed with scaffolds. Recently, there is a controversy concerning the use of artificial scaffolds compared to the use of a natural matrix. Therefore, new approaches called micromass technology have been invented to overcome these problems by avoiding the need for scaffolds. Technically, cells are dissociated and the dispersed cells are then reaggregated into cellular spheres. The micromass technology approach enables investigators to follow tissue formation from single cell sources to organised spheres in a controlled environment. Thus, the inherent fundamentals of tissue engineering are better revealed. Additionally, as the newly formed tissue is devoid of an artificial material, it resembles more closely the in vivo situation. The purpose of this review is to provide an insight into the fundamentals and the technique of micromass cell culture used to study bone tissue engineering

Three-dimensional culture of human meniscal cells: Extracellular matrix and proteoglycan production

<p>Abstract</p> <p>Background</p> <p>The meniscus is a complex tissue whose cell biology has only recently begun to be explored. Published models rely upon initial culture in the presence of added growth factors. The aim of this study was to test a three-dimensional (3D) collagen sponge microenvironment (without added growth factors) for its ability to provide a microenvironment supportive for meniscal cell extracellular matrix (ECM) production, and to test the responsiveness of cells cultured in this manner to transforming growth factor-β (TGF-β).</p> <p>Methods</p> <p>Experimental studies were approved prospectively by the authors' Human Subjects Institutional Review Board. Human meniscal cells were isolated from surgical specimens, established in monolayer culture, seeded into a 3D scaffold, and cell morphology and extracellular matrix components (ECM) evaluated either under control condition or with addition of TGF-β. Outcome variables were evaluation of cultured cell morphology, quantitative measurement of total sulfated proteoglycan production, and immunohistochemical study of the ECM components chondroitin sulfate, keratan sulfate, and types I and II collagen.</p> <p>Result and Conclusion</p> <p>Meniscal cells attached well within the 3D microenvironment and expanded with culture time. The 3D microenvironment was permissive for production of chondroitin sulfate, types I and II collagen, and to a lesser degree keratan sulfate. This microenvironment was also permissive for growth factor responsiveness, as indicated by a significant increase in proteoglycan production when cells were exposed to TGF-β (2.48 μg/ml ± 1.00, mean ± S.D., vs control levels of 1.58 ± 0.79, p < 0.0001). Knowledge of how culture microenvironments influence meniscal cell ECM production is important; the collagen sponge culture methodology provides a useful in vitro tool for study of meniscal cell biology.</p

Altered mRNA expression of genes related to nerve cell activity in the fracture callus of older rats: A randomized, controlled, microarray study

BACKGROUND: The time required for radiographic union following femoral fracture increases with age in both humans and rats for unknown reasons. Since abnormalities in fracture innervation will slow skeletal healing, we explored whether abnormal mRNA expression of genes related to nerve cell activity in the older rats was associated with the slowing of skeletal repair. METHODS: Simple, transverse, mid-shaft, femoral fractures with intramedullary rod fixation were induced in anaesthetized female Sprague-Dawley rats at 6, 26, and 52 weeks of age. At 0, 0.4, 1, 2, 4, and 6 weeks after fracture, a bony segment, one-third the length of the femur, centered on the fracture site, including the external callus, cortical bone, and marrow elements, was harvested. cRNA was prepared and hybridized to 54 Affymetrix U34A microarrays (3/age/time point). RESULTS: The mRNA levels of 62 genes related to neural function were affected by fracture. Of the total, 38 genes were altered by fracture to a similar extent at the three ages. In contrast, eight neural genes showed prolonged down-regulation in the older rats compared to the more rapid return to pre-fracture levels in younger rats. Seven genes were up-regulated by fracture more in the younger rats than in the older rats, while nine genes were up-regulated more in the older rats than in the younger. CONCLUSIONS: mRNA of 24 nerve-related genes responded differently to fracture in older rats compared to young rats. This differential expression may reflect altered cell function at the fracture site that may be causally related to the slowing of fracture healing with age or may be an effect of the delayed healing