Influence of gamma and electron beam sterilization on the stability of a premixed injectable calcium phosphate cement for trauma indications.

Premixed calcium phosphate cements (CPC's) are becoming the material of choice for injectable cements as a result of their effective delivery to the target implantation site. For orthopaedic use, it is of vital importance that the attributes of these CPC's are not compromised by irradiation sterilization. Therefore, the aim of this study is to determine the influence of irradiation sterilization on a range of premixed CPC's, with an emphasis on improving product shelf life through the use of optimal packaging configurations and annealing steps. Electron spin resonance (ESR) confirmed the presence of free radicals in the inorganic phase of the CPC paste following irradiation. The inclusion of a 24-h annealing step was the only successful method in reducing the degree of free radical formation. Based on the results of injectability force testing, it was revealed that an annealing step greater than 24-h significantly altered the viscosity, however; at 24-h the key attributes of the CPC paste were minimally effected. Overall, it was established that vacuum packing the CPC paste, placing the contents into a foil pouch, gamma irradiating at the minimal dose required and using an annealing step of ≤ 24-h, has the potential to extend the shelf life of the cement.

Inflammatory cytokines induce a unique mineralizing phenotype in mesenchymal stem cells derived from human bone marrow.

Bone marrow contains mesenchymal stem cells (MSCs) that can differentiate along multiple mesenchymal lineages. In this capacity they are thought to be important in the intrinsic turnover and repair of connective tissues while also serving as a basis for tissue engineering and regenerative medicine. However, little is known of the biological responses of human MSCs to inflammatory conditions. When cultured with IL-1ß, marrow-derived MSCs from 8 of 10 human subjects deposited copious hydroxyapatite, in which authenticity was confirmed by Fourier transform infrared spectroscopy. Transmission electron microscopy revealed the production of fine needles of hydroxyapatite in conjunction with matrix vesicles. Alkaline phosphatase activity did not increase in response to inflammatory mediators, but PPi production fell, reflecting lower ectonucleotide pyrophosphatase activity in cells and matrix vesicles. Because PPi is the major physiological inhibitor of mineralization, its decline generated permissive conditions for hydroxyapatite formation. This is in contrast to MSCs treated with dexamethasone, where PPi levels did not fall and mineralization was fuelled by a large and rapid increase in alkaline phosphatase activity. Bone sialoprotein was the only osteoblast marker strongly induced by IL-1ß; thus these cells do not become osteoblasts despite depositing abundant mineral. RT-PCR did not detect transcripts indicative of alternative mesenchymal lineages, including chondrocytes, myoblasts, adipocytes, ligament, tendon, or vascular smooth muscle cells. IL-1ß phosphorylated multiple MAPKs and activated nuclear factor-κB (NF-κB). Certain inhibitors of MAPK and PI3K, but not NF-κB, prevented mineralization. The findings are of importance to soft tissue mineralization, tissue engineering, and regenerative medicine.

EWS-FLI-1-targeted cytotoxic T-cell killing of multiple tumor types belonging to the Ewing sarcoma family of tumors.

PURPOSE: The Ewing sarcoma family of tumors (ESFT) comprises a group of aggressive, malignant bone, and soft tissue tumors that predominantly affect children and young adults. These tumors frequently share expression of the EWS-FLI-1 translocation, which is central to tumor survival but not present in healthy cells. In this study, we examined EWS-FLI-1 antigens for their capacity to induce immunity against a range of ESFT types. DESIGN: Computer prediction analysis of peptide binding, HLA-A2.1 stabilization assays, and induction of cytotoxic T-lymphocytes (CTL) in immunized HLA-A2.1 transgenic mice were used to assess the immunogenicity of native and modified peptides derived from the fusion region of EWS-FLI-1 type 1. CTL-killing of multiple ESFT family members in vitro, and control of established xenografts in vivo, was assessed. We also examined whether these peptides could induce human CTLs in vitro. RESULTS: EWS-FLI-1 type 1 peptides were unable to stabilize cell surface HLA-A2.1 and induced weak CTL activity against Ewing sarcoma cells. In contrast, peptides with modified anchor residues induced potent CTL killing of Ewing sarcoma cells presenting endogenous (native) peptides. The adoptive transfer of CTL specific for the modified peptide YLNPSVDSV resulted in enhanced survival of mice with established Ewing sarcoma xenografts. YLNPSVDSV-specific CTL displayed potent killing of multiple ESFT types in vitro: Ewing sarcoma, pPNET, Askin's Tumor, and Biphenotypic sarcoma. Stimulation of human peripheral blood mononuclear cells with YLNPSVDSV peptide resulted in potent CTL-killing. CONCLUSIONS: These data show that YLNPSVDSV peptide is a promising antigen for ESFT immunotherapy and warrants further clinical development.

Dysregulated in vitro hematopoiesis, radiosensitivity, proliferation, and osteoblastogenesis with marrow from SAMP6 mice.

The senescence accelerated-prone mouse variant 6 (SAMP6) shows normal growth followed by rapid aging, development of osteopenia, and shortened lifespan, compared with control R1 mice. Because oxidative stress is a fundamental mechanism of tissue aging, we tested whether cellular parameters that are associated with oxidative stress are impaired with marrow from SAMP6 mice. We compared in vitro hematopoiesis, irradiation sensitivity, proliferative potential, and osteoblastogenesis with marrow cells from SAMP6 and R1 mice. Marrow cells from SAMP6 mice showed shortened in vitro hematopoiesis; their stromal cells showed greater radiation sensitivity and decreased proliferation. Consistent with those properties, there was constitutive upregulation of transforming growth factor-ß(1), an inhibitor of hematopoiesis, and of cell cycle inhibitory genes, p16(INK4A) and p19(ARF). Paradoxically, there was constitutive expression of osteoblast genes in stromal cells from SAMP6 mice, but in vitro matrix mineralization was impaired. These studies and data included in other reports indicate that impaired proliferation of osteoblast progenitors in SAMP6 marrow may be a major factor contributing to accelerated loss of bone mass. In sum, marrow from SAMP6 mice had diminished capacity for long-term hematopoiesis, increased radiosensitivity, and reduced proliferative capacity.

MicroRNA profiling reveals age-dependent differential expression of nuclear factor κB and mitogen-activated protein kinase in adipose and bone marrow-derived human mesenchymal stem cells.

INTRODUCTION: Mesenchymal stem cells (MSCs) play a central role in mediating endogenous repair of cell and tissue damage. Biologic aging is a universal process that results in changes at the cellular and molecular levels. In the present study, the role of microRNA (miRNA) in age-induced molecular changes in MSCs derived from adipose tissue (ASCs) and bone marrow (BMSCs) from young and old human donors were investigated by using an unbiased genome-wide approach. METHODS: Human ASCs and BMSCs from young and old donors were cultured, and total RNA was isolated. The miRNA fraction was enriched and used to determine the expression profile of miRNA in young and old donor MSCs. Based on miRNA expression, differences in donor MSCs were further investigated by using differentiation assays, Western blot, immunocytochemistry, and bioinformatics. RESULTS: Biologic aging demonstrated reduced osteogenic and adipogenic potential in ASCs isolated from older donors, whereas cell size, complexity, and cell-surface markers remained intact with aging. Analysis of miRNA profiles revealed that small subsets of active miRNAs changed secondary to aging. Evaluation of miRNA showed significantly decreased levels of gene expression of inhibitory kappa B kinase (IκB), interleukin-1α, inducible nitric oxide synthase (iNOS), mitogen-activated protein kinase/p38, ERK1/2, c-fos, and c-jun in MSCs from older donors by both bioinformatics and Western blot analysis. Nuclear factor kappa B (NF-κB), myc, and interleukin-4 receptor mRNA levels were significantly elevated in aged cells from both the adipose and bone marrow depots. Immunocytochemistry showed nuclear localization in young donors, but a cytosolic predominance of phosphorylated NF-κB in ASCs from older donors. Western blot demonstrated significantly elevated levels of NF-κB subunits, p65 and p50, and AKT. CONCLUSIONS: These findings suggest that differential expression of miRNA is an integral component of biologic aging in MSCs.

Bone matrix regulates osteoclast differentiation and annexin A8 gene expression.

While attachment to bone is required for optimal osteoclast function, the molecular events that underlie this fact are unclear, other than that the cell requires adhesion to mineralized matrix to assume a fully differentiated phenotype. To address this issue, we cultured murine bone marrow-derived osteoclasts on either cell culture plastic or devitalized mouse calvariae to identify the distinct genetic profile induced by interaction with bone. Among a number of genes previously unknown to be expressed in osteoclasts we found that Annexin A8 (AnxA8) mRNA was markedly up-regulated by bone. AnxA8 protein was present at high levels in osteoclasts present in human tissues recovered from sites of pathological bone loss. The presence of bone mineral was required for up-regulation of AnxA8 mRNA since osteoclasts plated on decalcified bone express AnxA8 at low levels as did osteoclasts plated on native or denatured type I collagen. Finally, AnxA8-regulated cytoskeletal reorganization in osteoclasts generated on a mineralized matrix. Thus, we used a novel approach to define a distinct bone-dependent genetic program associated with terminal osteoclast differentiation and identified Anxa8 as a gene strongly induced late in osteoclast differentiation and a protein that regulates formation of the cell's characteristic actin ring.