Using Atomic Force Microscopy to Predict Tumor Specificity of ICAM1 Antibody-Directed Nanomedicines.

Atomic force microscopy (AFM) is a powerful tool to detect in vitro antibody-antigen interactions. To date, however, AFM-measured antibody-antigen interactions have yet to be exploited to predict in vivo tumor specificity of antibody-directed nanomedicines. In this study, we have utilized AFM to directly measure the biomechanical interaction between live triple negative breast cancer (TNBC) cells and an antibody against ICAM1, a recently identified TNBC target. For the first time, we provide proof-of-principle evidence that in vitro TNBC cell-ICAM1 antibody binding force measured by AFM on live cells more precisely correlates with in vivo tumor accumulation and therapeutic efficacy of ICAM1 antibody-directed liposomes than ICAM1 gene and surface protein overexpression levels. These studies demonstrate that live cell-antibody binding force measurements may be used as a novel in vitro metric for predicting the in vivo tumor recognition of antibody-directed nanomedicines.

Influence of the surface structure of a multiblock copolymer on the cellular behavior of primary cell cultures of the upper aerodigestive tract in vitro.

The influence of the surface topography of a biodegradable copolymer on adhesion, proliferation, and cellular activity of primary cell cultures of the upper aerodigestive tract (ADT) was investigated. On the basis of the important functions of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of MMPs (TIMPs) in regulating extracellular matrix remodeling, cellular adhesion and growth, the appearance and kinetics of these enzymes were investigated in primary cells of the upper ADT seeded on different surfaces of a polymeric biomaterial. Primary cell cultures of the upper ADT of Sprague-Dawley rats were seeded on different surfaces (smooth versus rough surface) of a biodegradable multiblock copolymer and on polystyrene surface as control. Conditioned media of the primary cells were analyzed for MMPs and TIMPs by both zymography and radiometric enzyme assay. Cell adhesion and proliferation as well as the kinetics of appearance and activity level of MMP-1, MMP-2, and TIMPs were significantly different depending on the cell type and the surface structure of the multiblock copolymer. In this study, the data obtained indicated that surface topography governed the biological response to biomaterials. Knowledge as to how cells interact with the interface of biomaterials will be necessary in order to eventually design the "ideal" surface of biomaterials, which will be both tissue and organ-optimized in order to best provide clinicians with specific and viable novel therapeutical options in medicine.

Expression of MMPs and TIMPs in primary epithelial cell cultures of the upper aerodigestive tract seeded on the surface of a novel polymeric biomaterial.

INTRODUCTION: Using standard cell biological and biochemical experimental approaches we were able to test the ability of a particular polymer construct to support the adhesion, proliferation, and the cellular acitivity of pharyngeal cells. The delicate balance between Matrix Metalloproteinases (MMPs) and their endogenous inhibitors (Tissue Inhibitor of MMPs, TIMPs) have a decisive function in the remodeling of the extracellular matrix during cellular ingrowth. Novel polymeric biomaterials may be useful to develop new therapeutic options in head and neck surgery. METHODS: Primary cell cultures of the pharynx of Sprague-Dawley rats were seeded on the surface of a thermoplastic multi-block copolymer and on a polystyrene surface as control. Conditioned media of the primary cells was analyzed for MMPs and TIMPs. The MMP and TIMP expression was analysed by zymography and a radiometric enzyme assay. RESULTS: No statistically significant differences in the levels of MMP-1, MMP-2, MMP-9 and TIMPs were detected between cells grown on the novel polymer surface versus control. CONCLUSION: An appropriate understanding of the molecular machinery that regulates gene expression and cellular growth in tissue engineered constructs is the requirement for an optimal adaptation of biodegradable biomaterials to develop new therapeutic options in otolaryngology and head and neck surgery.

[Cell proliferation and cellular activity of primary cell cultures of the oral cavity after cell seeding on the surface of a degradable, thermoplastic block copolymer]. / Zellproliferation und zelluläre aktivität von primärzellkulturen der mundhöhle nach oberflächenbesiedlung eines abbaubaren, thermoplastischen blockcopolymers.

Using standard cell biological and biochemical methods we were able to test the ability of a degradable, thermoplastic block copolymer to support the adhesion, proliferation, and the cellular activity of primary cell cultures of the oral cavity in vitro. The delicate balance between a group of endogenous enzymes, Matrix Metalloproteinases (MMPs), and their inhibitors (Tissue Inhibitor of MMPs, TIMPs) have a decisive function in the remodeling of the extracellular matrix during processes like wound healing or the integration of biomaterials in surrounding tissues after implantation. Recently developed, biodegradable thermoplastic elastomers with shape-memory properties may be the key to develop new therapeutical options in head and neck surgery. Primary cell cultures of the oral cavity of Sprague-Dawley rats were seeded on the surface of a thermoplastic block copolymer and on a polystyrene surface as control. Conditioned media of the primary cells were analyzed for MMPs and TIMPs after different periods of cell growth. The MMP and TIMP expression was analysed by zymography and a radiometric enzyme assay. No statistically significant differences in the appearance and the kinetic of MMP-1, MMP-2, MMP-9 and TIMPs were detected between cells grown on the polymer surface compared to the control. An appropriate understanding of the molecular processes that regulate cellular growth and integration of a biomaterial in surrounding tissue is the requirement for an optimal adaptation of biodegradable, polymeric biomaterials to the physiological, anatomical, and surgical conditions in vivo to develop new therapeutic options in otolaryngology and head and neck surgery.

Patch augmentation of the pulmonary artery with bioabsorbable polymers and autologous cell seeding.

OBJECTIVE: In recent years bioabsorbable synthetic or biologic materials have been used to augment the pulmonary artery or the right ventricular outflow tract. However, each of these polymers has one or more shortcomings. None of these patch materials has been seeded with cells. Thus, we have tested a fast-absorbing biopolymer, poly-4-hydroxybutyric acid, with autologous cell seeding for patch augmentation of the pulmonary artery in a juvenile sheep model. METHODS: Vascular cells were isolated from ovine peripheral veins (n = 6). Bioabsorbable porous poly-4-hydroxybutyric acid patches (porosity > 95%) were seeded on 3 consecutive days with a mixed vascular cell suspension (21.3 +/- 1.3 x 10(6) cells). Forty-five (+/- 2) days after the vessel harvest, 1 unseeded and 6 autologously seeded control patches were implanted into the proximal pulmonary artery. The animals received no postoperative anticoagulation. Follow-up was performed with echocardiography after 1 week and before explantation after 1, 7, and 24 weeks (2 animals each) for the seeded control patches and after 20 weeks for the nonseeded control patch. RESULTS: All animals survived the procedure. Postoperative echocardiography of the seeded patches demonstrated a smooth surface without dilatation or stenosis. Macroscopic appearance showed a smooth internal surface with increasing tissue formation. Histology at 169 days demonstrated a near-complete resorption of the polymer and formation of organized and functional tissue. Biochemical assays revealed increasing cellular and extracellular matrix contents. The control patch showed a slight bulging, indicating a beginning dilatation. CONCLUSION: This experiment showed that poly-4-hydroxybutyric acid is a feasible patch material in the pulmonary circulation.

Tissue engineering of autologous aorta using a new biodegradable polymer.

BACKGROUND: Ovine pulmonary valve leaflets and pulmonary arteries have been tissue-engineered (TE) from autologous cells and biodegradable polyglycolic acid (PGA)-polyglactin copolymers. Use of this cell-polymer construct in the systemic circulation resulted in aneurysm formation. This study evaluates a TE vascular graft in the systemic circulation which is based on a new copolymer of PGA and polyhydroxyalkanoate (PHA). METHODS: Ovine carotid arteries were harvested, expanded in vitro, and seeded onto 7-mm diameter PHA-PGA tubular scaffolds. The autologous cell-polymer vascular constructs were used to replace 3-4 cm abdominal aortic segments in lambs (group TE, n = 7). In a control group (n = 4), aortic segments were replaced with acellular polymer tubes. Vascular patency was evaluated with echography. All control animals were sacrificed when the grafts became occluded. Animals in TE group were sacrificed at 10 days (n = 1), 3 (n = 3), and 5 months (n = 3). Explanted TE conduits were evaluated for collagen content, deoxyribonucleic acid (DNA) content, structural and ultrastructural examination, mechanical strength, and matrix metalloproteinase (MMP) activity. RESULTS: The 4 control conduits became occluded at 1, 2, 55, and 101 days. All TE grafts remained patent, and no aneurysms developed by the time of sacrifice. There was one mild stenosis at the anastomotic site after 5 months postoperatively. The percent collagen and DNA contents approached the native aorta over time (% collagen = 25.7%+/-3.4 [3 months] vs 99.6%+/-11.7 [5 months], p < 0.05; and % DNA = 30.8%+/-6.0 [3 months] vs 150.5%+/-16.9 [5 months], p < 0.05). Histology demonstrated elastic fibers in the medial layer and endothelial specific von Willebrand factor on the luminal surface. The mechanical strain-stress curve of the TE aorta approached that of the native vessel. A 66 kDa MMP-2 was found in the TE and native aorta but not in control group. CONCLUSIONS: Autologous aortic grafts with biological characteristics resembling the native aorta can be created using TE approach. This may allow the development of "live" vascular grafts.

The importance of angiogenesis in the interaction between polymeric biomaterials and surrounding tissue.

The uncomplicated outcome of surgical interventions after biomedical application of biomaterials depends on successful wound healing. Wound healing is a highly complex process compossed of a number of overlapping phases, including inflammation, epithelialization, angiogenesis and matrix deposition. Inadequate angiogenesis limits the transport between the microvasculature and implanted biomaterials. The regulation of angiogenesis is based on numerous growth factors, proteolytic enzymes, extracellular matrix components, cell adhesion molecules, and vasoactive factors. Capillary endothelial cells were grown for different time periods (day 1, 3, 6, 9 and 12) on the surface of a recently developed biodegradable polymeric biomaterial. As control the cells were seeded on the gelatine coated polystyrene surface of commercially available cell cultures dishes. Endothelial cells became adherent and showed confluent cells layers during increasing time period on both surfaces. The total cell number of cells grown on the gelatine coated polystyrene surface was higher in comparison to the polymer surface. The chorioallantois membrane (CAM) assay was used as a sensitive assay to investigate the influence of angiogenesis in vivo. After 48 hours of exposure of the CAM to polymer samples no avascular zones, free of capillaries and/or thrombosis or hemorrhage were detectable. Considering the biofunctionality of our recently developed polymer in these experiments different surface modifications of the polymer are the topic of current investigation to support the biomaterial-microvasculature interactions in vivo.

Dynamics of extracellular matrix production and turnover in tissue engineered cardiovascular structures.

Appropriate matrix formation, turnover and remodeling in tissue-engineered small diameter vascular conduits are crucial requirements for their long-term patency and function. This complex process requires the deposition and accumulation of extracellular matrix molecules as well as the remodeling of this extracellular matrix (ECM) by matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). In this study, we have investigated the dynamics of ECM production and the activity of MMPs and TIMPs in long-term tissue-engineered vascular conduits using quantitative ECM analysis, substrate gel electrophoresis, radiometric enzyme assays and Western blot analyses. Over a time period of 169 days in vivo, levels of elastin and proteoglycans/glycosaminoglycans in tissue-engineered constructs came to approximate those of their native tissue counter parts. The kinetics of collagen deposition and remodeling, however, apparently require a much longer time period. Through the use of substrate gel electrophoresis, proteolytic bands whose molecular weight was consistent with their identification as the active form of MMP-2 (approximately 64--66 kDa) were detected in all native and tissue-engineered samples. Additional proteolytic bands migrating at approximately 72 kDa representing the latent form of MMP-2 were detected in tissue-engineered samples at time points from 5 throughout 55 days. Radiometric assays of MMP-1 activity demonstrated no significant differences between the native and tissue-engineered samples. This study determines the dynamics of ECM production and turnover in a long-term tissue-engineered vascular tissue and highlights the importance of ECM remodeling in the development of successful tissue-engineered vascular structures.