Cell therapy using encapsulated cells producing endostatin.

Despite aggressive surgery and post-operative radiation and chemotherapy, the prognosis is poor for glioblastoma patients. Anti-angiogenic therapy with compounds such as endostatin could delay the onset of relapse. However, the short systemic half-life of this proteins as well as the blood-brain barrier makes the use of this therapy difficult for brain cancer patients. The aim of this project is to develop and implant genetically engineered producer cells secreting endostatin that are encapsulated in calcium cross-linked alginate gel beads. Encapsulation of cells within alginate gels has a potential as a sustained release system in addition to the fact that the encapsulation technology protects the cells from rejection by the immune system. Human embryonal kidney 293 cells have been transfected with the gene for endostatin. These cells have been encapsulated in calcium cross-linked alginate gels and optimized for the secretion of endostatin. Alginate gel beads implanted into rat brain have shown only a moderate loss in cell viability but extended endostatin release for periods of up to 12 months. Visualization of the anti-angiogenic effect on C6 rat glioma growth, tumor vasculature and microhemodynamics has been demonstrated by using intravital video microscopy. The data indicates that endostatin greatly affects tumor-associated microcirculation but does not appear to affect normal microcirculation. The local delivery of endostatin seems to specifically affect tumor-associated microvessels by reduction of the vessel density, diameter and functionality. Tumor cell migration and invasion was greatly reduced in the endostatin treated animals.

The effect of chitin and chitosan on the proliferation of human skin fibroblasts and keratinocytes in vitro.

The effects of chitin [(1 --> 4)-2-acetamido-2-deoxy-beta-D-glucan] and its partially deacetylated derivatives, chitosans, on the proliferation of human dermal fibroblasts and keratinocytes were examined in vitro. Chitosans with relatively high degrees of deacetylation strongly stimulated fibroblast proliferation while samples with lower levels of deacetylation showed less activity. Fraction, CL313A, a shorter chain length, 89% deacetylated chitosan chloride was further evaluated using cultures of fibroblasts derived from a range of human donors. Some fibroblast cultures produced a positive mitogenic response to CL313A treatment with proliferation rates being increased by approximately 50% over the control level at an initial concentration of 50 microg/ml, whilst others showed no stimulation of proliferation or even a slight inhibition (< 10%). The stimulatory effect on fibroblast proliferation required the presence of serum in the culture medium suggesting that the chitosan may be interacting with growth factors present in the serum and potentiating their effect. In contrast to the stimulatory effects on fibroblasts, fraction CL313A inhibited human keratinocyte mitogenesis with up to 40% inhibition of proliferation being observed at 50 microg/ml. In general highly deacetylated chitosans were more active than those with a lower degree of deacetylation. These data demonstrate that highly deacetylated chitosans can modulate human skin cell mitogenesis in vitro. Analysis of their effects on cells in culture may be useful as a screen for their potential activity in vivo as wound healing agents, although in the case of fibroblasts it is important to select appropriate strains of cells for use in the screen.

Standards and guidelines for biopolymers in tissue-engineered medical products: ASTM alginate and chitosan standard guides. American Society for Testing and Materials.

The American Society for Testing and Materials (ASTM) is making a concerted effort to establish standards and guidelines for the entire field of tissue-engineered medical products (TEMPS). Safety, consistency, and functionality of biomaterials used as matrices, scaffolds, and immobilizing agents in TEMPS are a concern. Therefore, the ASTM has established a number of task groups to produce standards and guidelines for such biomaterials. Alginate is a naturally occurring biomaterial used for immobilizing living cells to form an artificial organ, such as encapsulated pancreatic islets. In order to aid in successful clinical applications and to help expedite regulatory approval, the alginate used must be fully documented. The ASTM alginate guide gives information on selection of testing methodologies and safety criteria. Critical parameters such as monomer content, molecular weight, and viscosity, in addition to more general parameters, such as dry matter content, heavy metal content, bioburden, and endotoxin content are described in the ASTM document. In a like manner, the characterization parameters for chitosan, a bioadhesive polycationic polysaccharide, are described in a separate guide. For chitosan, the degree of deacetylation is of critical importance. Control of protein content and, hence, potential for hypersensitivity, endotoxin content, and total bioburden are important in chitosan preparations for TEMPS. Together these two guides represent part of the effort on behalf of the ASTM and other interested parties to ensure quality and standardization in TEMPS.