Publications by authors named "Mark A Kirkland"

26 Publications

  • Page 1 of 1

Controlling the Effective Oxygen Tension Experienced by Cells Using a Dynamic Culture Technique for Hematopoietic Ex Vivo Expansion.

Curr Protoc Stem Cell Biol 2018 02 28;44:2A.11.1-2A.11.13. Epub 2018 Feb 28.

Institute for Frontier Materials, Deakin University, Geelong, Victoria, Australia.

Clinical hematopoietic stem/progenitor cell (HSPC) transplantation outcomes are strongly correlated with the number of cells infused. Hence, to generate sufficient HSPCs for transplantation, the best culture parameters for expansion are critical. It is generally assumed that the defined oxygen (O ) set for the incubator reflects the pericellular O to which cells are being exposed. Studies have shown that low O tension maintains an undifferentiated state, but the expansion rate may be constrained because of limited diffusion in a static culture system. A combination of low ambient O and dynamic culture conditions has been developed to increase the reconstituting capacity of human HSPCs. In this unit, the protocols for serum-free expansion of HSPCs at 5% and 20% O in static and dynamic nutrient flow mode are described. Finally, the impact of O tension on HSPC expansion in vitro by flow cytometry and colony forming assays and in vivo through engraftment using a murine model is assessed. © 2018 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpsc.42DOI Listing
February 2018

Thiol surface functionalization via continuous phase plasma polymerization of allyl mercaptan, with subsequent maleimide-linked conjugation of collagen.

J Biomed Mater Res A 2017 Jul 3;105(7):1940-1948. Epub 2017 Apr 3.

Institute for Frontier Materials, Deakin University, Victoria, Australia.

Thiol groups can undergo a large variety of chemical reactions and are used in solution phase to conjugate many bioactive molecules. Previous research on solid substrates with continuous phase glow discharge polymerization of thiol-containing monomers may have been compromised by oxidation. Thiol surface functionalization via glow discharge polymerization has been reported as requiring pulsing. Herein, continuous phase glow discharge polymerization of allyl mercaptan (2-propene-1-thiol) was used to generate significant densities of thiol groups on a mixed macrodiol polyurethane and tantalum. Three general classes of chemistry are used to conjugate proteins to thiol groups, with maleimide linkers being used most commonly. Here the pH specificity of maleimide reactions was used effectively to conjugate surface-bound thiol groups to amine groups in collagen. XPS demonstrated surface-bound thiol groups without evidence of oxidation, along with the subsequent presence of maleimide and collagen. Glow discharge reactor parameters were optimized by testing the resistance of bound collagen to degradation by 8 M urea. The nature of the chemical bonding of collagen to surface thiol groups was effectively assessed by colorimetric assay (ELISA) of residual collagen after incubation in 8 M urea over 8 days and after incubation with keratinocytes over 15 days. The facile creation of useable solid-supported thiol groups via continuous phase glow discharge polymerization of allyl mercaptan opens a route for attaching a vast array of bioactive molecules. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1940-1948, 2017.
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http://dx.doi.org/10.1002/jbm.a.36064DOI Listing
July 2017

Surface-bound collagen 4 is significantly more stable than collagen 1.

J Biomed Mater Res A 2017 05 24;105(5):1364-1373. Epub 2017 Feb 24.

Barwon Biomedical Research, University Hospital Geelong, Victoria, Australia.

Collagen 1 (C1) is commonly used to improve biological responses to implant surfaces. Here, the stability of C1 was compared with collagen 4 (C4) on a mixed macrodiol polyurethane, both adsorbed and covalently bound via acetaldehyde glow discharge polymerization and reductive amination. Substrate specimens were incubated in solutions of C1 and C4. The strength of conjugation was tested by incubation in 8 M urea followed by enzyme linked immunosorbent assays to measure residual C1 and C4. The basal lamina protein, laminin-332 (L332) was superimposed via adsorption on C4-treated specimens. Keratinocytes were grown on untreated, C1-treated, C4-treated, and C4 + L332-treated specimens, followed by measurement of cell area, proliferation, and focal adhesion density. Adsorbed C4 was shown to be significantly more stable than C1 and covalent conjugation conferred even greater stability, with no degradation of C4 over twenty days in 8 M urea. Cell growth was similar for C1 and C4, with no additional benefit conferred by superimposition of L332. The greater resistance of C4 to degradation may be consequent to cysteine residues and disulphide bonds in its non-collagenous domains. The use of C4 on implants, rather than C1, may improve their long-term stability in tissues. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1364-1373, 2017.
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http://dx.doi.org/10.1002/jbm.a.36019DOI Listing
May 2017

Impact of Oxygen Levels on Human Hematopoietic Stem and Progenitor Cell Expansion.

Stem Cells Dev 2016 10 18;25(20):1604-1613. Epub 2016 Aug 18.

2 Deakin University , Geelong, Australia .

Oxygen levels are an important variable during the in vitro culture of stem cells. There has been increasing interest in the use of low oxygen to maximize proliferation and, in some cases, effect differentiation of stem cell populations. It is generally assumed that the defined pO in the incubator reflects the pO to which the stem cells are being exposed. However, we demonstrate that the pO experienced by cells in static culture can change dramatically during the course of culture as cell numbers increase and as the oxygen utilization by cells exceeds the diffusion of oxygen through the media. Dynamic culture (whereby the cell culture plate is in constant motion) largely eliminates this effect, and a combination of low ambient oxygen and dynamic culture results in a fourfold increase in reconstituting capacity of human hematopoietic stem cells compared with those cultured in static culture at ambient oxygen tension. Cells cultured dynamically at 5% oxygen exhibited the best expansion: 30-fold increase by flow cytometry, 120-fold increase by colony assay, and 11% of human CD45 engraftment in the bone marrow of NOD/SCID mice. To our knowledge, this is the first study to compare individual and combined effects of oxygen and static or dynamic culture on hematopoietic ex vivo expansion. Understanding and controlling the effective oxygen tension experienced by cells may be important in clinical stem cell expansion systems, and these results may have relevance to the interpretation of low oxygen culture studies.
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http://dx.doi.org/10.1089/scd.2016.0153DOI Listing
October 2016

Enhanced Ex Vivo Expansion of Human Hematopoietic Progenitors on Native and Spin Coated Acellular Matrices Prepared from Bone Marrow Stromal Cells.

Stem Cells Int 2016 14;2016:7231567. Epub 2016 Feb 14.

CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.

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.
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http://dx.doi.org/10.1155/2016/7231567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4769778PMC
March 2016

Toward a skin-material interface with vacuum-integrated capped macroporous scaffolds.

J Biomed Mater Res B Appl Biomater 2017 07 10;105(5):1307-1318. Epub 2016 Mar 10.

Barwon Biomedical Research, University Hospital Geelong, Geelong, Victoria, Australia.

Avulsion, epidermal marsupialization, and infection cause failure at the skin-material interface. A robust interface would permit implantable robotics, prosthetics, and other medical devices; reconstruction of surgical defects, and long-term access to blood vessels and body cavities. Torus-shaped cap-scaffold structures were designed to work in conjunction with negative pressure to address the three causes of failure. Six wounds were made on the backs of each of four 3-month old pigs. Four unmodified (no caps) scaffolds were implanted along with 20 cap-scaffolds. Collagen type 4 was attached to 21 implants. Negative pressure then was applied. Structures were explanted and assessed histologically at day 7 and day 28. At day 28, there was close tissue apposition to scaffolds, without detectable reactions from defensive or interfering cells. Three cap-scaffolds explanted at day 28 showed likely attachment of epidermis to the cap or cap-scaffold junction, without deeper marsupialization. The combination of toric-shaped cap-scaffolds with negative pressure appears to be an intrinsically biocompatible system, enabling a robust skin-material interface. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1307-1318, 2017.
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http://dx.doi.org/10.1002/jbm.b.33649DOI Listing
July 2017

Expansion of Human Hematopoietic Stem/Progenitor Cells on Decellularized Matrix Scaffolds.

Curr Protoc Stem Cell Biol 2016 Feb 3;36:1C.15.1-1C.15.16. Epub 2016 Feb 3.

CSIR Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India.

Umbilical cord blood (UCB) is one of the richest sources for hematopoietic stem/progenitor cells (HSPCs), with more than 3000 transplantations performed each year for the treatment of leukemia and other bone marrow, immunological, and hereditary diseases. However, transplantation of single cord blood units is mostly restricted to children, due to the limited number of HSPC per unit. This unit develops a method to increase the number of HSPCs in laboratory conditions by using cell-free matrices from bone marrow cells that mimic 'human-body-niche-like' conditions as biological scaffolds to support the ex vivo expansion of HSPCs. In this unit, we describe protocols for the isolation and characterization of HSPCs from UCB and their serum-free expansion on decellularized matrices. This method may also help to provide understanding of the biochemical organization of hematopoietic niches and lead to suggestions regarding the design of tissue engineering-based biomimetic scaffolds for HSPC expansion for clinical applications.
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http://dx.doi.org/10.1002/9780470151808.sc01c15s36DOI Listing
February 2016

Expansion of human hematopoietic stem/progenitor cells on decellularized matrix scaffolds.

Curr Protoc Stem Cell Biol 2014 Feb 6;28:Unit 1C.15.. Epub 2014 Feb 6.

CSIR Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India; Geelong Technology Precinct, Deakin University, Geelong, Victoria, Australia; Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia.

Umbilical cord blood (UCB) is one of the richest sources for hematopoietic stem/progenitor cells (HSPCs), with more than 3000 transplantations performed each year for the treatment of leukemia and other bone marrow, immunological, and hereditary diseases. However, transplantation of single cord blood units is mostly restricted to children, due to the limited number of HSPC per unit. This unit develops a method to increase the number of HSPCs in laboratory conditions by using cell-free matrices from bone marrow cells that mimic 'human-body niche-like' conditions as biological scaffolds to support the ex vivo expansion of HSPCs. In this unit, we describe protocols for the isolation and characterization of HSPCs from UCB and their serum-free expansion on decellularized matrices. This method may also help to provide understanding of the biochemical organization of hematopoietic niches and lead to suggestions regarding the design of tissue engineering-based biomimetic scaffolds for HSPC expansion for clinical applications.
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http://dx.doi.org/10.1002/9780470151808.sc01c15s28DOI Listing
February 2014

Osteohematopoietic stem cell niches in bone marrow.

Int Rev Cell Mol Biol 2012 ;298:95-133

CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India.

In adult mammals, maturation of blood and bone cells from their respective progenitors occurs in the bone marrow. The marrow region contains many progenitor and stem cell types that are confined by their biochemical and cellular microenvironments, referred to as stem cell niches. The unique properties of each niche assist the survival, proliferation, migration, and differentiation of that particular stem or progenitor cell type. Among the different niches of the bone marrow, our understanding of the osteohematopoietic niche is the most complete. Its properties, described in this chapter, are a model for studying adult stem cell differentiation, but a lot remains unknown. Our improved understanding of hematopoietic stem cell biology and its relationship with the properties of these niches are critical in the effective and safe use of these cells in regenerative medicine. Here, we review the current knowledge on the properties of these niches and suggest how the potential of hematopoietic progenitors can be utilized in regenerative medicine.
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http://dx.doi.org/10.1016/B978-0-12-394309-5.00003-1DOI Listing
April 2013

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

J Tissue Eng Regen Med 2013 Nov 17;7(11):871-83. Epub 2012 Apr 17.

CSIR Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India; Deakin University, Waurn Ponds, Geelong, VIC, Australia.

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, bone marrow failure and immunodeficiencies. The current methods for HSPC 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 acellular matrices 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 it may also provide insights into the constitution of the niche in which these cells reside in the bone marrow.
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http://dx.doi.org/10.1002/term.1479DOI Listing
November 2013

Systematic investigation of oxygen and growth factors in clinically valid ex vivo expansion of cord blood CD34(+) hematopoietic progenitor cells.

Cytotherapy 2012 Jul 16;14(6):679-85. Epub 2012 Mar 16.

Barwon Biomedical Research, Barwon Health, Geelong, Australia.

Background Aims: Cord blood is considered to be a superior source of hematopoietic stem and progenitor cells for transplantation, but clinical use is limited primarily because of the low numbers of cells harvested. Ex vivo expansion has the potential to provide a safe, effective means of increasing cell numbers. However, an absence of consensus regarding optimum expansion conditions prevents standard implementation. Many studies lack clinical applicability, or have failed to investigate the combinational effects of different parameters.

Methods: This is the first study to characterize systematically the effect of growth factor combinations across multiple oxygen levels on the ex vivo expansion of cord blood CD34(+) hematopoietic cells utilizing clinically approvable reagents and methodologies throughout.

Results: Optimal fold expansion, as assessed both phenotypically and functionally, was greatest with thrombopoietin, stem cell factor, Flt-3 ligand and interleukin-6 at an oxygen level of 10%. With these conditions, serial expansion showed continual target population expansion and consistently higher expression levels of self-renewal associated genes.

Conclusions: This study has identified optimized fold expansion conditions, with the potential for direct clinical translation to increase transplantable cell dose and as a baseline methodology against which future factors can be tested.
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http://dx.doi.org/10.3109/14653249.2012.666851DOI Listing
July 2012

M-CSF potently augments RANKL-induced resorption activation in mature human osteoclasts.

PLoS One 2011 29;6(6):e21462. Epub 2011 Jun 29.

The Department of Clinical and Biomedical Sciences, Barwon Health, The University of Melbourne, Geelong, Victoria, Australia.

Macrophage-CSF (M-CSF) is critical for osteoclast (OC) differentiation and is reported to enhance mature OC survival and motility. However, its role in the regulation of bone resorption, the main function of OCs, has not been well characterised. To address this we analysed short-term cultures of fully differentiated OCs derived from human colony forming unit-granulocyte macrophages (CFU-GM). When cultured on dentine, OC survival was enhanced by M-CSF but more effectively by receptor activator of NFκB ligand (RANKL). Resorption was entirely dependent on the presence of RANKL. Co-treatment with M-CSF augmented RANKL-induced resorption in a concentration-dependent manner with a (200-300%) stimulation at 25 ng/mL, an effect observed within 4-6 h. M-CSF co-treatment also increased number of resorption pits and F-actin sealing zones, but not the number of OCs or pit size, indicating stimulation of the proportion of OCs activated. M-CSF facilitated RANKL-induced activation of c-fos and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, but not NFκB nor nuclear factor of activated T-cells, cytoplasmic-1 (NFATc1). The mitogen-activated protein kinase kinase (MEK) 1 inhibitor PD98059 partially blocked augmentation of resorption by M-CSF. Our results reveal a previously unidentified role of M-CSF as a potent stimulator of mature OC resorbing activity, possibly mediated via ERK upstream of c-fos.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021462PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3126821PMC
December 2011

T cells fail to develop in the human skin-cell explants system; an inconvenient truth.

BMC Immunol 2011 Feb 18;12:17. Epub 2011 Feb 18.

Department of Internal Medicine, Division of Hematology, Maastricht University Medical Center+, Maastricht, The Netherlands.

Background: Haplo-identical hematopoietic stem cell (HSC) transplantation is very successful in eradicating haematological tumours, but the long post-transplant T-lymphopenic phase is responsible for high morbidity and mortality rates. Clark et al. have described a skin-explant system capable of producing host-tolerant donor-HSC derived T-cells. Because this T-cell production platform has the potential to replenish the T-cell levels following transplantation, we set out to validate the skin-explant system.

Results: Following the published procedures, while using the same commercial components, it was impossible to reproduce the skin-explant conditions required for HSC differentiation towards mature T-cells. The keratinocyte maturation procedure resulted in fragile cells with minimum expression of delta-like ligand (DLL). In most experiments the generated cells failed to adhere to carriers or were quickly outcompeted by fibroblasts. Consequently it was not possible to reproduce cell-culture conditions required for HSC differentiation into functional T-cells. Using cell-lines over-expressing DLL, we showed that the antibodies used by Clark et al. were unable to detect native DLL, but instead stained 7AAD+ cells. Therefore, it is unlikely that the observed T-lineage commitment from HSC is mediated by DLL expressed on keratinocytes. In addition, we did confirm expression of the Notch-ligand Jagged-1 by keratinocytes.

Conclusions: Currently, and unfortunately, it remains difficult to explain the development or growth of T-cells described by Clark et al., but for the fate of patients suffering from lymphopenia it is essential to both reproduce and understand how these co-cultures really "work". Fortunately, alternative procedures to speed-up T-cell reconstitution are being established and validated and may become available for patients in the near future.
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http://dx.doi.org/10.1186/1471-2172-12-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056828PMC
February 2011

Enhanced proliferation of human skeletal muscle precursor cells derived from elderly donors cultured in estimated physiological (5%) oxygen.

Cytotechnology 2009 Dec 22;61(3):93-107. Epub 2010 Jan 22.

Institute for Technology, Research and Innovation, Deakin University, Pigdons Road, Waurn Ponds, VIC, 3217, Australia.

Human skeletal muscle precursor cells (myoblasts) have significant therapeutic potential and are a valuable research tool to study muscle cell biology. Oxygen is a critical factor in the successful culture of myoblasts with low (1-6%) oxygen culture conditions enhancing the proliferation, differentiation, and/or viability of mouse, rat, and bovine myoblasts. The specific effects of low oxygen depend on the myoblast source and oxygen concentration; however, variable oxygen conditions have not been tested in the culture of human myoblasts. In this study, muscle precursor cells were isolated from vastus lateralis muscle biopsies and myoblast cultures were established in 5% oxygen, before being divided into physiological (5%) or standard (20%) oxygen conditions for experimental analysis. Five percent oxygen increased proliferating myoblast numbers, and since low oxygen had no significant effect on myoblast viability, this increase in cell number was attributed to enhanced proliferation. The proportion of cells in the S (DNA synthesis) phase of the cell cycle was increased by 50%, and p21(Cip1) gene and protein expression was decreased in 5 versus 20% oxygen. Unlike in rodent and bovine myoblasts, the increase in myoD, myogenin, creatine kinase, and myosin heavy chain IIa gene expression during differentiation was similar in 5 and 20% oxygen; as was myotube hypertrophy. These data indicate for the first time that low oxygen culture conditions stimulate proliferation, whilst maintaining (but not enhancing) the viability and the differentiation potential of human primary myoblasts and should be considered as optimum conditions for ex-vivo expansion of these cells.
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http://dx.doi.org/10.1007/s10616-009-9247-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2825300PMC
December 2009

RTKN2 Induces NF-KappaB Dependent Resistance to Intrinsic Apoptosis in HEK Cells and Regulates BCL-2 Genes in Human CD4(+) Lymphocytes.

J Cell Death 2009 7;2:9-23. Epub 2009 Sep 7.

Barwon Biomedical Research, Geelong Hospital, Barwon Health, Ryrie St, Geelong, Victoria, 3227, Australia.

The gene for Rhotekin 2 (RTKN2) was originally identified in a promyelocytic cell line resistant to oxysterol-induced apoptosis. It is differentially expressed in freshly isolated CD4(+) T-cells compared with other hematopoietic cells and is down-regulated following activation of the T-cell receptor. However, very little is known about the function of RTKN2 other than its homology to Rho-GTPase effector, rhotekin, and the possibility that they may have similar roles. Here we show that stable expression of RTKN2 in HEK cells enhanced survival in response to intrinsic apoptotic agents; 25-hydroxy cholesterol and camptothecin, but not the extrinsic agent, TNFα. Inhibitors of NF-KappaB, but not MAPK, reversed the resistance and mitochondrial pro-apoptotic genes, Bax and Bim, were down regulated. In these cells, there was no evidence of RTKN2 binding to the GTPases, RhoA or Rac2. Consistent with the role of RTKN2 in HEK over-expressing cells, suppression of RTKN2 in primary human CD4(+) T-cells reduced viability and increased sensitivity to 25-OHC. The expression of the pro-apoptotic genes, Bax and Bim were increased while BCL-2 was decreased. In both cell models RTKN2 played a role in the process of intrinsic apoptosis and this was dependent on either NF-KappaB signaling or expression of downstream BCL-2 genes. As RTKN2 is a highly expressed in CD4(+) T-cells it may play a role as a key signaling switch for regulation of genes involved in T-cell survival.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4474337PMC
http://dx.doi.org/10.4137/jcd.s2891DOI Listing
June 2015

Tissue compatibility of biomaterials: benefits and problems of skin biointegration.

ANZ J Surg 2008 Aug;78(8):654-9

Barwon Biomedical Research, Department of Clinical and Biomedical Sciences, The Geelong Hospital, Geelong, Australia.

The integration of biomaterials with skin is necessary to enable infection-free access to vasculature and body cavities. Also, integrating plastics and metals with skin increases options for the reconstruction of surgical and traumatic defects and enables the permanent implantation of robotic and electronic devices. Until now, attempts to integrate biomaterials with skin permanently have failed because of epidermal marsupialization and infection. This article reviews the general properties required of biomaterials to optimize integration with body tissues, the modifications that increase biocompatibility, focusing particularly on surface functionalization and the specific requirements for biomaterial integration into skin. Critical pathophysiological processes relating to biocompatibility are discussed with particular emphasis on the skin-biomaterial interface. Future directions are speculated on, in particular, the specific utility of subatmospheric pressure dressings in facilitating tissue integration into biomaterials.
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http://dx.doi.org/10.1111/j.1445-2197.2008.04609.xDOI Listing
August 2008

Multiple roles of M-CSF in human osteoclastogenesis.

J Cell Biochem 2007 Oct;102(3):759-68

The Department of Clinical and Biomedical Sciences: Barwon Health, The University of Melbourne, Geelong, Victoria 3220, Australia.

Although the critical role of M-CSF in osteoclastogenesis is well documented, there has been no detailed analysis of how it regulates human osteoclast formation and function in vitro. We used a human osteoclastogenesis model employing CFU-GM osteoclast precursors cultured for 14 days on dentine with RANKL, with varying exposure to exogenous human M-CSF. Short-term treatment of precursors with M-CSF (10-100 ng/mL) resulted in increased proliferation with or without RANKL. Treatment with M-CSF (1-100 ng/mL) for 14 days caused a biphasic concentration-dependent stimulation of formation, fusion, and resorption peaking at 10-50 ng/mL and almost complete abolition of resorption at 100 ng/mL. Time-course studies using M-CSF (25 ng/mL) showed that osteoclast size, nuclei/cell, and resorption increased with longer duration of M-CSF treatment. When treatment was restricted to the first 4 days, M-CSF (25-100 ng/mL) stimulated formation of normal numbers of osteoclasts that resorbed less. Blockade of endogenous M-CSF signaling with neutralizing M-CSF antibody during the first week of culture extensively inhibited osteoclastogenesis, whereas blockade during the second week produced only a small reduction in resorption. Treatment with M-CSF during the second week of culture caused a small increase in osteoclast number and a concentration-dependent increase in cytoplasmic spreading with inhibition of resorption. We have shown that M-CSF modulates multiple steps of human osteoclastogenesis, including proliferation, differentiation and fusion of precursors. In the later stages of osteoclastogenesis, M-CSF modulates osteoclast-resorbing activity, but is not required for survival. Modulation of M-CSF signaling is a potential therapeutic target for conditions associated with excess bone resorption.
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http://dx.doi.org/10.1002/jcb.21331DOI Listing
October 2007

Discrete stem cells: subsets or a continuum?

Blood 2006 Dec;108(12):3949; author reply 3950

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http://dx.doi.org/10.1182/blood-2006-06-029470DOI Listing
December 2006

GM-CSF cannot substitute for M-CSF in human osteoclastogenesis.

Biochem Biophys Res Commun 2004 Aug;321(1):7-12

Department of Clinical and Biomedical Sciences, Barwon Health, University of Melbourne, Geelong, Victoria 3220, Australia.

Osteopetrotic mice lacking functional M-CSF recover with ageing, suggesting alternate osteoclastogenesis pathways exist. One alternative is GM-CSF, treatment with which improves the osteopetrosis. Our objective was to determine whether GM-CSF could replace M-CSF in human osteoclastogenesis in vitro. Human CFU-GM precursors cultured with RANKL differentiate into osteoclasts without added M-CSF, indicating constitutive production of M-CSF. Addition of M-CSF antibody completely inhibited differentiation, demonstrating M-CSF-dependence in vitro. Co-treatment with low concentrations (0.01 ng/mL) of GM-CSF for 14 days or higher concentrations (10 ng/mL) for the first 1-2 days enhanced osteoclastogenesis but this effect was blocked with M-CSF antibody. Treatment with GM-CSF transiently increased M-CSF mRNA expression at 3 h but suppressed expression at 7-14 days. Neither FLT3-ligand nor VEGF supported osteoclastogenesis in the absence of M-CSF. Thus, in vitro human osteoclastogenesis is dependent on M-CSF and the stimulatory effects of GM-CSF are mediated by M-CSF. Rescue by GM-CSF in M-CSF-deficiency is unlikely to be directly mediated by FLT3-ligand or VEGF.
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http://dx.doi.org/10.1016/j.bbrc.2004.06.097DOI Listing
August 2004

Mechanisms of resistance to the cytotoxic effects of oxysterols in human leukemic cells.

J Steroid Biochem Mol Biol 2004 Mar;88(3):311-20

Stem Cell Laboratory, The Douglas Hocking Research Institute, Barwon Health, The Geelong Hospital, Geelong 3220, Vic., Australia.

We have developed hematopoietic cells resistant to the cytotoxic effects of oxysterols. Oxysterol-resistant HL60 cells were generated by continuous exposure to three different oxysterols-25-hydroxycholesterol (25-OHC), 7-beta-hydroxycholesterol (7beta-OHC) and 7-keto-cholesterol (7kappa-C). We investigated the effects of 25-OHC, 7beta-OHC, 7kappa-C and the apoptotic agent staurosporine on these cells. The effect of the calcium channel blocker nifedipine on oxysterol cytotoxicity was also investigated. Differential display and real-time PCR were used to quantitate gene expression of oxysterol-sensitive and -resistant cells. Our results demonstrate that resistance to the cytotoxic effects of oxysterols is relatively specific to the type of oxysterol, and that the cytotoxicity of 25-OHC but not that of 7beta-OHC and 7kappa-C, appears to occur by a calcium dependent mechanism. Oxysterol-resistant cells demonstrated no significant difference in the expression of several genes previously implicated in oxysterol resistance, but expressed the bcl-2 gene at significantly lower levels than those observed in control cells. We identified three novel genes differentially expressed in resistant cells when compared to HL60 control cells. Taken together, the results of this study reveal potentially novel mechanisms of oxysterol cytotoxicity and resistance, and indicate that cytotoxicity of 25-OHC, 7beta-OHC and 7kappa-C occur by independent, yet overlapping mechanisms.
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http://dx.doi.org/10.1016/j.jsbmb.2003.12.007DOI Listing
March 2004

Osteoclastic potential of human CFU-GM: biphasic effect of GM-CSF.

J Bone Miner Res 2004 Feb 16;19(2):190-9. Epub 2003 Dec 16.

Department of Clinical and Biomedical Sciences, Barwon Health, The Geelong Hospital, University of Melbourne, Geelong, Victoria, Australia.

Unlabelled: Human osteoclasts can be efficiently generated in vitro from cord blood mononuclear cells and derived CFU-GM colonies. However, CFU-M colonies are poorly osteoclastogenic. Short-term (2-48 h) treatment with GM-CSF stimulates osteoclast formation by proliferating precursors, whereas longer exposure favors dendritic cell formation.

Introduction: Osteoclasts (OC) differentiate from cells of the myelomonocytic lineage under the influence of macrophage-colony stimulating factor (M-CSF) and RANKL. However, cells of this lineage can also differentiate to macrophages and dendritic cells (DC) depending on the cytokine environment. The aims of this study were to develop an efficient human osteoclastogenesis model and to investigate the roles of granulocyte macrophage-colony stimulating factor (GM-CSF) and M-CSF in human OC differentiation.

Materials And Methods: A human osteoclastogenesis model, using as precursors colony forming unit-granulocyte macrophage (CFU-GM) colonies generated from umbilical cord mononuclear cells cultured in methylcellulose with GM-CSF, interleukin (IL)-3 and stem cell factor (SCF), has been developed. CFU-GM, colony forming unit-macrophage (CFU-M), or mixed colonies were cultured on dentine with soluble RANKL (sRANKL) and human M-CSF with and without GM-CSF. Major endpoints were OC number, dentine resorption, and CD1a+ DC clusters.

Results: Osteoclast generation from CFU-GM and mixed colonies treated with M-CSF and sRANKL for 7-14 days was highly efficient, but CFU-M colonies were poorly osteoclastogenic under these conditions. Pretreatment of precursors with M-CSF for 7 or 14 days maintained the precursor pool, but OCs were smaller and resorption was reduced. The effect of GM-CSF treatment was biphasic, depending on the timing and duration of exposure. Short-term treatment (2-48 h) at the beginning of the culture stimulated cell proliferation and enhanced OC formation up to 100%, independent of sRANKL. Longer-term GM-CSF treatment in the presence of sRANKL, however, inhibited OC generation with the formation of extensive CD1a+ DC clusters, accompanied by downregulation of c-Fos mRNA. Delaying the addition of GM-CSF resulted in progressively less inhibition of osteoclastogenesis.

Conclusions: Human CFU-GM, but not CFU-M, progenitors have high osteoclastogenic potential. GM-CSF plays an important role in osteoclastogenesis and has a biphasic effect: Short-term treatment potentiates OC differentiation by proliferating precursors, but persistent exposure favors DC formation.
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http://dx.doi.org/10.1359/JBMR.0301232DOI Listing
February 2004

Hereditary hyperferritinemia-cataract syndrome: prevalence, lens morphology, spectrum of mutations, and clinical presentations.

Arch Ophthalmol 2003 Dec;121(12):1753-61

Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria.

Objectives: To provide a comprehensive description of the clinical presentations, cataract morphology, and molecular basis of hereditary hyperferritinemia-cataract syndrome (HHCS) in 4 Australian pedigrees and to estimate its prevalence.

Methods: All known cases of HHCS in southeastern Australia were ascertained. Family members provided a medical history and underwent physical examination, lens photography, and venipuncture for measurement of serum ferritin levels and DNA extraction. Sequence analysis of the iron-responsive element of the ferritin light chain on chromosome 19q13.3-qter was performed.

Results: We investigated 26 affected individuals from 5 Australian pedigrees. Two pedigrees with HHCS ascertained independently were subsequently found to form 1 large kindred carrying the mutation A40G. The minimum estimated prevalence of HHCS is 1/200000. One pedigree had the mutation G32C. Among 2 smaller pedigrees studied, one carried a novel mutation (C39A), and the other was identified through the 2-year-old propositus with cataract but no positive family history. The latter case was shown to be due to a de novo mutation (G32U). All cataracts were highly distinctive in morphology, consisting of slowly progressive flecks, vacuoles, and distinctive crystalline deposits scattered predominantly in the lens cortex but also in the nucleus. Eight of 18 affected individuals examined have required cataract extraction to date. No other identified clinical manifestations of HHCS were delineated.

Conclusions: Cataract morphology in HHCS is highly distinctive. Longitudinal observation demonstrated slow progression of the cataracts. This study highlights that, although HHCS is an autosomal dominant condition, the diagnosis should be considered even in sporadic cataract of typical morphology. Furthermore, individuals with unexplained hyperferritinemia should be referred for ophthalmological assessment, as the cataract may be asymptomatic but lead to a correct diagnosis of HHCS. Clinical Relevance Progressive cataracts of highly distinctive morphology are an important feature of HHCS. Evaluation for this type of cataract may be of diagnostic value in patients with unexplained hyperferritinemia. Hereditary hyperferritinemia-cataract syndrome can be a cause of cataracts in pediatric patients even in the absence of any positive family history.
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http://dx.doi.org/10.1001/archopht.121.12.1753DOI Listing
December 2003

Mutation spectrum in Australian pedigrees with hereditary hyperferritinaemia-cataract syndrome reveals novel and de novo mutations.

Br J Haematol 2002 Sep;118(4):1179-82

Douglas Hocking Research Institute, Geelong, Australia.

Hereditary hyperferritinaemia-cataract syndrome (HHCS) (OMIM #600886) is a rare autosomal dominant condition identified by high serum ferritin levels with normal iron saturation and distinctive bilateral cataract. It may be misdiagnosed as haemochromatosis and such patients become anaemic as a result of inappropriate venesection. The elevated serum ferritin is due to a mutation in the iron-responsive element (IRE) of the l-ferritin gene, resulting in excessive l-ferritin production. We report the identification of three Australian pedigrees; one with a previously described mutation at position 40, a pedigree with a novel mutation at position 39 and an individual with a de novo mutation at position 32 of the l-ferritin IRE.
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http://dx.doi.org/10.1046/j.1365-2141.2002.03690.xDOI Listing
September 2002

Effect of oxysterols on hematopoietic progenitor cells.

Exp Hematol 2002 Jul;30(7):670-8

Stem Cell Laboratory, The Douglas Hocking Research Institute, The Geelong Hospital, Victoria, Australia.

Objectives: Oxysterols are hydroxylated derivatives of cholesterol detected in blood, cells, and tissues. They exhibit a number of biologic activities, including inhibition of cellular proliferation and cytotoxicity associated with induction of apoptosis. Given the important regulatory role of apoptosis in hematopoiesis, we investigated the effects of oxysterols on human hematopoietic progenitor cells (HPCs).

Materials And Methods: Colony-forming unit granulocyte-macrophage (CFU-GM) from human bone marrow and umbilical cord blood (UCB) were grown in the presence of varying concentrations of three different oxysterols-7-keto-cholesterol, 7-beta-hydroxycholesterol, and 25-hydroxycholesterol (25-OHC). Similarly, the effect of oxysterols on HL60 and CD34+ cells was investigated using annexin V staining and flow cytometry to measure apoptosis. Reduction of nitroblue tetrazolium was used to assess differentiative status of HL60 cells.

Results: CFU-GM derived from human bone marrow were inhibited by all three oxysterols tested, with 25-OHC being the most potent. In comparison, CFU-GM derived from UCB were less sensitive to the effects of all the oxysterols tested, with statistically significant inhibition observed only in the presence of 25-OHC. Oxysterol treatment of HL60 cells inhibited cell growth and increased the number of annexin V+ and nitroblue tetrazolium+ cells. The percentage of viable, CD34+ annexin V+ cells also was increased with oxysterol treatment of purified HPCs in liquid culture.

Conclusions: These experiments indicate that oxysterol inhibition of CFU-GM and HL60 cell growth can be attributed to induction of apoptosis and/or differentiation. These investigations revealed that oxysterols are a new class of inhibitors of HPC proliferation of potential relevance in vivo and in vitro.
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http://dx.doi.org/10.1016/s0301-472x(02)00833-0DOI Listing
July 2002

MERP1: a mammalian ependymin-related protein gene differentially expressed in hematopoietic cells.

Gene 2002 Mar;286(2):249-57

Stem Cell Laboratory, The Douglas Hocking Research Institute, Barwon Health, The Geelong Hospital, Geelong, VIC, 3220, Australia.

We have utilized differential display polymerase chain reaction to investigate the gene expression of hematopoietic progenitor cells from adult bone marrow and umbilical cord blood. A differentially expressed gene was identified in CD34+ hematopoietic progenitor cells, with low expression in CD34- cells. We have obtained the full coding sequence of this gene which we designated human mammalian ependymin-related protein 1 (MERP1). Expression of MERP1 was found in a variety of normal human tissues, and is 4- and 10-fold higher in adult bone marrow and umbilical cord blood CD34+ cells, respectively, compared to CD34- cells. Additionally, MERP1 expression in a hematopoietic stem cell enriched population was down-regulated with proliferation and differentiation. Conceptual translation of the MERP1 open reading frame reveals significant homology to two families of glycoprotein calcium-dependant cell adhesion molecules: ependymins and protocadherins.
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http://dx.doi.org/10.1016/s0378-1119(02)00434-1DOI Listing
March 2002

Influence of mode of birth and collection on WBC yields of umbilical cord blood units.

Transfusion 2002 Feb;42(2):210-5

Development Unit, Australian Red Cross Blood Service, Melbourne, Victoria, Australia.

Background: The aim of this study was to determine the influence of mode of birth and umbilical cord blood (CB) collection before (in utero) or after delivery of the placenta (ex utero) on total number of WBCs and CD34+ cells in CB units.

Study Design And Methods: Consecutively donated, banked CB units were assessed for net volume, WBC concentration, total number of WBCs, proportion of CD34+ cells, and total number of CD34+ cells. These parameters were then correlated with the mode of birth and the mode of CB collection relative to the delivery of the placenta.

Results: A significantly higher CB volume was seen following cesarean section (n = 61) than following vaginal delivery (n = 157; median volume, 76 vs. 63 mL, respectively; p < 0.0001). In contrast, CB from vaginal delivery had a significantly higher WBC concentration compared with CB from cesarean section (medians, 17.1 x 10(9) and 13.6 x 10(9) WBCs/L, respectively; p < 0.0001). The mode of birth did not influence the proportion of CD34+ cells. A correlation was demonstrated between the total number of CD34+ cells and the total number of WBCs. As a consequence of the opposing effects on volume and WBC counts by cesarean section and vaginal delivery, there were no significant differences in the total number of WBCs or CD34+ cells for the CB units with mode of delivery in this study. No significant differences were found in CB with mode of CB collection (in utero [n = 58] or ex utero [n = 99]) following vaginal delivery.

Conclusions: The mode of birth influences the CB WBC concentration and volume collected and should be taken into consideration for establishing any acceptance limits for CB units to be banked. There were no differences in CB with in utero or ex utero collections.
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http://dx.doi.org/10.1046/j.1537-2995.2002.00028.xDOI Listing
February 2002