Effect of nanofiber-coated surfaces on the proliferation and differentiation of osteoprogenitors in vitro

The osteoconductive property of titanium (Ti) surfaces is important in orthopedic and dental implant devices. Surface modifications of Ti have been proposed to further improve osseointegration. In this study, three different materials, silicon (Si), silicon oxide (SiO(2)), and titanium oxide (TiO(2)), were used to construct nanofibers for surface coating of Ti alloy Ti-6Al-4 V (Ti alloy). MC3T3-E1 osteoprogenitor cells were seeded on nanofiber-coated discs and cultured for 42 days. DNA, alkaline phosphatase, osteocalcin, and mineralization nodules were measured using PicoGreen, enzyme-linked immunosorbent assay, and calcein blue staining to detect the attachment, proliferation, differentiation, and mineralization of MC3T3-E1 cells, respectively. The results demonstrated that the initial cell attachments on nanofiber-coated discs were significantly lower, although cell proliferation on Si and SiO(2) nanofiber-coated discs was better than on Ti alloy surfaces. TiO(2) nanofibers facilitated a higher cellular differentiation capacity than Ti alloy and tissue culture-treated polystyrene surfaces. Thus, surface modification using nanofibers of various materials can alter the attachment, proliferation, and differentiation of osteoprogenitor cells in vitro.     

Nanostructured surfaces for bone biotemplating applications.

A major goal of orthopedic biomaterials research is to design better surface chemistries and configurations to control behavior of bone cells such as osteoblasts. Nanostructured architecture significantly affects the response of several cell lines. In this work, nanostructured surfaces were prepared by vapor liquid solid growth of silicon nanowires from size-controlled gold colloid catalysts deposited on fused silica substrates. The lengths and surface densities of the nanowires were varied to assess the effect of these parameters on bone cell response. Osteoblasts were seeded on nanowire surfaces to investigate both short-term adhesion and proliferation and long-term functionality and matrix production. Cell adhesion and proliferation were characterized using a standard 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay and cell counting for up to 4 days of culture. The total protein content, alkaline phosphatase activity, and matrix production were quantified using standard colorimetric assays for up to 4 weeks of culture. Matrix production was also characterized by measuring surface concentrations of calcium and phosphorus using X-ray photoelectron spectroscopy. Further, scanning electron microscopy was used to investigate osteoblast morphology on nanostructured surfaces. Over the 4-week study, the nanostructured surfaces demonstrated improved osteoblast adhesion and proliferation and increased alkaline phosphatase activity and matrix production compared to non-nanostructured control surfaces.     

Oncolytic viruses: a novel form of immunotherapy

Oncolytic viruses are novel anticancer agents, currently under investigation in Phase I-III clinical trials. Until recently, most studies have focused on the direct antitumor properties of these viruses, although there is now an increasing body of evidence that the host immune response may be critical to the efficacy of oncolytic virotherapy. This may be mediated via innate immune effectors, adaptive antiviral immune responses eliminating infected cells or adaptive antitumor immune responses. This report summarizes preclinical and clinical evidence for the importance of immune interactions, which may be finely balanced between viral and tumor elimination. On this basis, oncolytic viruses represent a promising novel immunotherapy strategy, which may be optimally combined with existing therapeutic modalities.     

Antitumor effects of aminobisphosphonates on renal cell carcinoma cell lines

PURPOSE: Bisphosphonates are established as a supportive therapy for a number of malignancies that metastasize to bone. Previous reports have also suggested potent antitumor and anti-angiogenic properties. We investigated the in vitro activity of the 2 aminobisphosphonates pamidronate (Faulding Pharmaceuticals, Paramus, New Jersey) and zoledronic acid (Novartis, Basel, Switzerland) on the growth and survival of the 3 renal cell carcinoma cell lines Caki-2, 769-P (American Type Culture Collection, Manassas, Virginia) and D69581. MATERIALS AND METHODS: Cell lines were exposed to bisphosphonates in vitro and evaluated by MTS (3-(4,5-dimethylahiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and cell cycle analysis. Mechanisms of apoptotic cell death were investigated by ApoDIRECT assay (BioVision, Mountain View, California) and Kinetworks analysis. RESULTS: Zoledronic acid was consistently more potent than pamidronate for inducing apoptotic cell death. Zoledronic acid was capable of overcoming resistance to pamidronate in 1 cell line. Although it was ultimately less potent, the inhibitory effects of pamidronate appeared earlier than those of zoledronic acid. The pro-apoptotic effect of zoledronic acid was achieved through nonmitochondrial pathways and it was associated with the activation of caspase 6 and 3, and poly adenosine diphosphate-ribosyltransferase polymerase cleavage. Furthermore, we observed a marked decrease in and intracellular distribution of MSH2, a protein involved in DNA mismatch repair, as well as evidence of a greater cellular response to zoledronic acid as increased expression of superoxide dismutase. CONCLUSIONS: These findings add further support to the clinical use of aminobisphosphonates, particularly zoledronic acid, in patients with renal cell carcinoma with disease metastatic to bone.     

Immunotherapy of murine prostate cancer using whole tumour cells killed ex vivo by cytosine deaminase/5-fluorocytosine suicide-gene therapy

OBJECTIVE: To evaluate the efficacy of antitumour vaccines comprising irradiated allogeneic or autologous whole cells expressing cytosine deaminase (CD) which are first killed ex vivo by prodrug activation using 5-fluorocytosine (5-FC), as the immunogenicity of tumour cells used as irradiated vaccines depends both on antigen expression and on the mode of their death. MATERIALS AND METHODS: The PA3 rat prostate cell line and MATLyLu, an androgen-insensitive subline, were grown and transfected with CD (designated PCD and MCD). In vitro drug-sensitivity was assessed in the cell lines using a viability assay, and the mode of cell death quantified by assessing apoptosis. Bax and bcl-2 expression were assessed by Western blot analysis. For in vivo experiments, male 8-10-week-old Lobund-Wistar rats were vaccinated (using vehicle in control groups) with 5 x 10(6) cells, all cells being irradiated before injection, to give groups with PA3, PCD, PCD killed with 5-FC, MatLyLu, MCD, and MCD killed with 5-FC. After 7 days all animals were given a subcutaneous tumour challenge of PA3 cells, and tumour volume measured subsequently. Immune responses were assessed in splenocytes. RESULTS: The efficiency of cell kill varied between the cell lines assessed, but cell death was by induced apoptosis. Single doses of vaccine were most effective in the allogeneic setting, causing significantly slower growth of syngeneic tumour challenge (P < 0.01), and 25% better survival at 50 days (P < 0.02) than irradiated untransfected cells. This was consistent with the greater proliferative response after allogeneic than autologous vaccination. CONCLUSION: The immunogenicity of irradiated tumour cells is enhanced when they are killed ex-vivo using suicide-gene therapy. This approach would be clinically applicable in terms of ease of vaccine production, safety, storage and avoidance of potential toxicities of in vivo gene transfer.     

Unified approach to linear quadratic regulator with time‐scale property

A method for decomposition of unified (continuous/discrete) linear quadratic regulator with two‐time‐scale property is presented. The composite and the reduced‐order controls are formulated in standard unified domain. By appropriate transformations, it is shown that the results of continuous‐time (CT) and discrete‐ time (DT) obtained previously using two separate techniques, can be obtained by this single unified technique. With this method, there is no need to consider two‐time scale systems in continuous and the discrete domains separately. This paper also presents some numerical examples to illustrate the theoretical results. Copyright © 2001 John Wiley & Sons, Ltd.     

Cyclophosphamide facilitates antitumor efficacy against subcutaneous tumors following intravenous delivery of reovirus.

PURPOSE: The purpose of the present study was to investigate whether it is possible to achieve truly systemic delivery of oncolytic reovirus, in immunocompetent hosts, using cyclophosphamide to overcome some of the barriers to effective intratumoral delivery and replication of i.v. injected virus. EXPERIMENTAL DESIGN: I.v. delivery of reovirus was combined with different regimens of i.p. administered cyclophosphamide in C57Bl/6 mice bearing established s.c. B16 tumors. Intratumoral viral replication, tumor size, and survival were measured along with levels of neutralizing antibody (NAb) in the blood. Finally, differential toxicities of the virus/cyclophosphamide regimens were monitored through viral replication in systemic organs, survival, and cardiac damage. RESULTS: Repeated i.v. injection of reovirus was poorly effective at seeding intratumoral viral replication/oncolysis. However, by combining i.v. virus with cyclophosphamide, viral titers of between 10(7) and 10(8) plaque-forming units per milligram were recovered from regressing tumors. Doses of cyclophosphamide that ablated NAb were associated with severe toxicities, characterized by viral replication in systemic organs--toxicities that are mirrored by repeated reovirus injections into B-cell knockout mice. Next, we restructured the dosing of cyclophosphamide and i.v. virus such that a dose of 3 mg cyclophosphamide was administered 24 h before reovirus injection, and this schedule was repeated every 6 days. Using this protocol, high levels of intratumoral viral access and replication ( approximately 10(7) plaque-forming units per milligram tumor) were maintained along with systemically protective levels of NAb and only very mild, non-life-threatening toxicity. CONCLUSION: NAb to oncolytic viruses play a dual role in the context of systemic viral delivery; on one hand, they hinder repeated administration of virus but on the other, they provide an important safety mechanism by which virus released from vigorous intratumoral replication is neutralized before it can disseminate and cause toxicity. These data support the use of cyclophosphamide to modulate, but not ablate, patient NAb, in development of carefully controlled clinical trials of the systemic administration of oncolytic viruses.     

Immunotherapy of murine prostate cancer using whole tumor cells killed ex vivo by herpes simplex viral thymidine kinase/ganciclovir suicide gene therapy

Whole cell cancer vaccines are currently under clinical evaluation. Their immunogenicity may depend on the mode of death of the vaccine cells prior to uptake by professional antigen-presenting cells and crosspriming of T cells. Destruction of tumor in vivo by genetic prodrug activation therapy leads to a marked local and systemic immune response, local T-cell infiltration and the establishment of T-cell memory. We postulated that this immunostimulation may be due to induction of danger signals and the inherent immunogenicity of products of HSVtk/ganciclovir kill. Using established models of murine prostate cancer, we have evaluated the efficacy of anti-tumor vaccines comprising irradiated allogeneic or autologous whole cells expressing HSVtK, which are first killed in vitro by prodrug activation using ganciclovir. HSVtk/ganciclovir-induced cell kill was through the induction of apoptosis. The vaccine was found to be effective in both models and superior to traditional irradiated whole tumor cells even after single doses. Protection against tumor challenge was associated with marked proliferative and Th1 cytokine responses. This approach would be applicable clinically in terms of ease of vaccine production, safety, storage and avoidance of potential toxicities of in vivo gene transfer.