Accelerated stem cell attachment to ultrafine grained titanium

Commercial purity titanium with an average grain size in the low sub-micron range was produced by equal channel angular pressing (ECAP). Attachment of human bone marrow-derived mesenchymal stem cells (hMSCs) to the surface of conventional coarse grained and ECAP-modified titanium was studied. It was demonstrated that the attachment and spreading of hMSCs in the initial stages (up to 24h) of culture was enhanced by grain refinement. Surface characterization by a range of techniques showed that the main factor responsible for the observed acceleration of hMSC attachment and spreading on titanium due to grain refinement in the bulk is the attendant changes in surface topography on the nanoscale. These results indicate that, in addition to its superior mechanical properties, ECAP-modified titanium possesses improved biocompatibility, which makes it to a potent candidate for applications in medical implants.

Differential effects of exercise on tibial shaft marrow density in young female athletes

Context:
Increased mechanical loading can promote the preferential differentiation of bone marrow mesenchymal stem cells to osteoblastogenesis, but it is not known whether long-term bone strength-enhancing exercise in humans can reduce marrow adiposity.

Objective:
Our objective was to examine whether bone marrow density (MaD), as an estimate of marrow adiposity 1) differs between young female athletes with contrasting loading histories and bone strengths and 2) is an independent predictor of bone strength at the weight-bearing tibia.

Design:
Mid-tibial MaD, cortical area (CoA), total area, medullary area, strength strain index (SSI), and cortical volumetric bone mineral density (vBMD) (total, endocortical, midcortical, and pericortical) was assessed using peripheral quantitative computed tomography in 179 female athletes involved in both impact and nonimpact loading sports and 41 controls aged 17–40 years.

Results:
As we have previously reported CoA, total area, and SSI were 16% to 24% greater in the impact group compared with the controls (all P < .001) and 12% to 18% greater than in the nonimpact group (all P < .001). The impact group also had 0.5% higher MaD than the nonimpact and control groups (both P < .05). Regression analysis further showed that midtibial MaD was significantly associated with SSI, CoA, endocortical vBMD, and pericortical vBMD (P < .05) in all women combined, after adjusting for age, bone length, loading groups, medullary area, muscle cross-sectional area, and percent fat.

Conclusion:
In young female athletes, tibial bone MaD was associated with loading history and was an independent predictor of tibial bone strength. These findings suggest that an exercise-induced increase in bone strength may be mediated via reduced bone marrow adiposity and consequently increased osteoblastogenesis.

Novel characterization of monocyte-derived cell populations in the meninges and choroid plexus and their rates of replenishment in bone marrow chimeric mice

The mouse dura mater, pia mater, and choroid plexus contain resident macrophages and dendritic cells (DCs). These cells participate in immune surveillance, phagocytosis of cellular debris, uptake of antigens from the surrounding cerebrospinal fluid and immune regulation in many pathologic processes. We used Cx3cr1 knock-in, CD11c-eYFP transgenic and bone marrow chimeric mice to characterize the phenotype, density and replenishment rate of monocyte-derived cells in the meninges and choroid plexus and to assess the role of the chemokine receptor CX3CR1 on their number and tissue distribution. Iba-1 major histocompatibility complex (MHC) Class II CD169 CD68 macrophages and CD11c putative DCs were identified in meningeal and choroid plexus whole mounts. Comparison of homozygous and heterozygous Cx3cr1 mice did not reveal CX3CR1-dependancy on density, distribution or phenotype of monocyte-derived cells. In turnover studies, wild type lethally irradiated mice were reconstituted with Cx3cr1/-positive bone marrow and were analyzed at 3 days, 1, 2, 4 and 8 weeks after transplantation. There was a rapid replenishment of CX3CR1-positive cells in the dura mater (at 4 weeks) and the choroid plexus was fully reconstituted by 8 weeks. These data provide the foundation for future studies on the role of resident macrophages and DCs in conditions such as meningitis, autoimmune inflammatory disease and in therapies involving irradiation and hematopoietic or stem cell transplantation.

Ex vivo expansion of haematopoietic stem/progenitor cells from human umbilical cord blood on acellular scaffolds prepared from MS-5 stromal cell line

Lineage-specific expansion of haematopoietic stem/progenitor cells (HSPCs) from human umbilical cord blood (UCB) is desirable because of their several applications in translational medicine, e.g. treatment of cancer, bonemarrowfailure and immunodeficiencies. The currentmethods forHSPC expansion use either cellular feeder layers and/or soluble growth factors and selected matrix components coated on different surfaces. The use of cell-free extracellular matrices from bone marrow cells for this purpose has not previously been reported. We have prepared insoluble, cell- free matrices from a murine bone marrow stromal cell line (MS-5) grown under four different conditions, i.e. in presence or absence of osteogenic medium, each incubated under 5% and 20% O2 tensions. These acellularmatrices were used as biological scaffolds for the lineage-specific expansion of magnetically sorted CD34+ cells and the results were evaluated by flow cytometry and colony-forming assays. We could get up to 80-fold expansion of some HSPCs on one of the matrices and our results indicated that oxygen tension played a significant role in determining the expansion capacity of the matrices. A comparative proteomic analysis of the matrices indicated differential expression of proteins, such as aldehyde dehydrogenase and gelsolin, which have previously been identified as playing a role in HSPC maintenance and expansion. Our approach may be of value in identifying factors relevant to tissue engineering-based ex vivo HSPC expansion, and itmay also provide insights into the constitution of the niche in which these cells reside in the bone marrow.

Bone marrow chimeric mice reveal a role for CX3CR1 in maintenance of the monocyte-derived cell population in the olfactory neuroepithelium

Macrophages in the olfactory neuroepithelium are thought to play major roles in tissue homeostasis and repair. However, little information is available at present about possible heterogeneity of these monocyte-derived cells, their turnover rates, and the role of chemokine receptors in this process. To start addressing these issues, this study used Cx3cr1gfp mice, in which the gene sequence for eGFP was knocked into the CX3CR1 gene locus in the mutant allele. Using neuroepithelial whole-mounts from Cx3cr1gfp/+ mice, we show that eGFP+ cells of monocytic origin are distributed in a loose network throughout this tissue and can be subdivided further into two immunophenotypically distinct subsets based on MHC-II glycoprotein expression. BM chimeric mice were created using Cx3cr1gfp/+ donors to investigate turnover of macrophages (and other monocyte-derived cells) in the olfactory neuroepithelium. Our data indicate that the monocyte-derived cell population in the olfactory neuroepithelium is actively replenished by circulating monocytes and under the experimental conditions, completely turned over within 6 months. Transplantation of Cx3cr1gfp/gfp (i.e., CX3CR1-deficient) BM partially impaired the replenishment process and resulted in an overall decline of the total monocyte-derived cell number in the olfactory epithelium. Interestingly, replenishment of the CD68lowMHC-II+ subset appeared minimally affected by CX3CR1 deficiency. Taken together, the established baseline data about heterogeneity of monocyte-derived cells, their replenishment rates, and the role of CX3CR1 provide a solid basis to further examine the importance of different monocyte subsets for neuroregeneration at this unique frontier with the external environment.

Enhanced Ex Vivo expansion of human hematopoietic progenitors on native and spin coated acellular matrices prepared from bone marrow stromal cells

The extracellular microenvironment in bone marrow (BM) is known to regulate the growth and differentiation of hematopoietic stem and progenitor cells (HSPC). We have developed cell-free matrices from a BM stromal cell line (HS-5), which can be used as substrates either in native form or as tissue engineered coatings, for the enhanced ex vivo expansion of umbilical cord blood (UCB) derived HSPC. The physicochemical properties (surface roughness, thickness, and uniformity) of native and spin coated acellular matrices (ACM) were studied using scanning and atomic force microscopy (SEM and AFM). Lineage-specific expansion of HSPC, grown on these substrates, was evaluated by immunophenotypic (flow cytometry) and functional (colony forming) assays. Our results show that the most efficient expansion of lineage-specific HSPC occurred on spin coated ACM. Our method provides an improved protocol for ex vivo HSPC expansion and it offers a system to study the in vivo roles of specific molecules in the hematopoietic niche that influence HSPC expansion.

Tyrosine residues of the granulocyte colony-stimulating factor transmit proliferation and differentiation signals in murine bone marrow cells

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of granulopoiesis and acts through binding to its specific receptor (G-CSF-R) on neutrophilic granulocytes. Previous studies of signaling from the 4 G-CSF-R cytoplasmic tyrosine residues used model cell lines that may have idiosyncratic, nonphysiological responses. This study aimed to identify specific signals transmitted by the receptor tyrosine residues in primary myeloid cells. To bypass the presence of endogenous G-CSF-R, a chimeric receptor containing the extracellular domain of the epidermal growth factor receptor in place of the entire extracellular domain of the G-CSF-R was used. A series of chimeric receptors containing tyrosine mutations to phenylalanine, either individually or collectively, was constructed and expressed in primary bone marrow cells from G-CSF-deficient mice. Proliferation and differentiation responses of receptor-expressing bone marrow cells stimulated by epidermal growth factor were measured. An increased 50% effective concentration to stimulus of the receptor Ynull mutant indicated that specific signals from tyrosine residues were required for cell proliferation, particularly at low concentrations of stimulus. Impaired responses by mutant receptors implicated G-CSF-R Y764 in cell proliferation and Y729 in granulocyte differentiation signaling. In addition, different sensitivities to ligand stimulation between mutant receptors indicated that G-CSF-R Y744 and possibly Y729 have an inhibitory role in cell proliferation. STAT activation was not affected by tyrosine mutations, whereas ERK activation appeared to depend, at least in part, on Y764. These observations have suggested novel roles for the G-CSF-R tyrosine residues in primary cells that were not observed previously in studies in cell lines.

Ingrowth of human mesenchymal stem cells into porous silk particle reinforced silk composite scaffolds : an in vitro study

Silk fibroin protein is biodegradable and biocompatible, exhibiting excellent mechanical properties for various biomedical applications. However, porous three-dimensional (3-D) silk fibroin scaffolds, or silk sponges, usually fall short in matching the initial mechanical requirements for bone tissue engineering. In the present study, silk sponge matrices were reinforced with silk microparticles to generate protein-protein composite scaffolds with desirable mechanical properties for in vitro osteogenic tissue formation. It was found that increasing the silk microparticle loading led to a substantial increase in the scaffold compressive modulus from 0.3 MPa (non-reinforced) to 1.9 MPa for 1:2 (matrix:particle) reinforcement loading by dry mass. Biochemical, gene expression, and histological assays were employed to study the possible effects of increasing composite scaffold stiffness, due to microparticle reinforcement, on in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs). Increasing silk microparticle loading increased the osteogenic capability of hMSCs in the presence of bone morphogenic protein-2 (BMP-2) and other osteogenic factors in static culture for up to 6 weeks. The calcium adsorption increased dramatically with increasing loading, as observed from biochemical assays, histological staining, and microcomputer tomography (μCT) analysis. Specifically, calcium content in the scaffolds increased by 0.57, 0.71, and 1.27 mg (per μg of DNA) from 3 to 6 weeks for matrix to particle dry mass loading ratios of 1:0, 1:1, and 1:2, respectively. In addition, μCT imaging revealed that at 6 weeks, bone volume fraction increased from 0.78% for non-reinforced to 7.1% and 6.7% for 1:1 and 1:2 loading, respectively. Our results support the hypothesis that scaffold stiffness may strongly influence the 3-D in vitro differentiation capabilities of hMSCs, providing a means to improve osteogenic outcomes.

Embryonic stem cell research: the principal ethical issue-when does life begin?

Embryonic stem cell research is perhaps the most controversial ethical issue of the new century. This is not surprising. It promises unprecedented potential benefits to human health but arguably comes at the expense of violating the most fundamental moral virtue - the right to life. The debate has become increasingly emotive. The Catholic Church has labelled stem cell research as cannibalism.1 This has led perhaps the world's most famous moral philosopher, Peter Singer, to label the Church, which has over a billion followers, as irrelevant.2 The principal purpose of this paper is not to  discuss all of the relevant moral issues in the embryonic stem cell debate. Considerations of space do not permit this and in any event there are  numerous reports which catalogue the relevant issues.3 Rather we attempt  to identify the crux of the issues in the debate. In our view, the main issue is the point at which life commences. We offer some preliminary observations on this matter. This discussion appears in section four. In the next section, we provide a brief  overview of nature and potential benefits of stem cell  research. This is followed by a discussion of the current legal position. In the final section, we offer some concluding remarks including some  suggestions for law reform.

Stem cell research and ethics : a socio-economic and marketing perspective

This paper addresses the ethical and moral issues surrounding stem cell research (SCR) and development within the socio-economic and macro marketing environments. A two dimensional conceptual framework is developed towards broadening the understanding of the complexities of these issues in an international context. The conceptual model captures the two dimensions of narrow moral/ethical to broader imperatives and the cost-benefits realm of SCR. Four quadrants emerge within these two dimensions which are likely to help identify the dominant views and the people associating themselves with these views in terms of their demographics and psychographic characteristics. These findings have important socio-economic and marketing implications which are highlighted in the paper.

Replication of hematopoietic stem cell niche

This thesis models the hematopoietic stem cell (HSC) niche by using decellularized extracellular matrix (ECM) scaffolds prepared by MS-5 cell line. The ECM replicates many of the properties of HSC niches in vivo, providing insights into expansion of HSCs that may have several applications in translational medicine.