Michael V. Sefton

Biodegradable scaffold with built-in vasculature for organ-on-a-chip engineering and direct surgical anastomosis

We report the fabrication of a scaffold (hereafter referred to as AngioChip) that supports the assembly of parenchymal cells on a mechanically tunable matrix surrounding a perfusable, branched, three-dimensional microchannel network coated with endothelial cells. The design of AngioChip decouples the material choices for the engineered vessel network and for cell seeding in the parenchyma, enabling extensive remodelling while maintaining an open-vessel lumen. The incorporation of nanopores and micro-holes in the vessel walls enhances permeability, and permits intercellular crosstalk and extravasation of monocytes and endothelial cells on biomolecular stimulation. We also show that vascularized hepatic tissues and cardiac tissues engineered by using AngioChips process clinically relevant drugs delivered through the vasculature, and that millimeter-thick cardiac tissues can be engineered in a scalable manner. Moreover, we demonstrate that AngioChip cardiac tissues implanted via direct surgical anastomosis to the femoral vessels of rat hindlimbs establish immediate blood perfusion.

In vitro and in vivo release of insulin from poly(lactic acid) microbeads and pellets

Abstract A feasibility study was carried out on developing an alternative insulin delivery system, for the treatment of insulin-requiring adult-onset (Type II) diabetes, which would by-pass some of the unresolved problems associated with mechanical insulin pumps. In our system, insulin delivery was accomplished by the sustained release of the hormone from a biodegradable polymer matrix, poly(1-lactic acid) (PLA). Injectable insulin—PLA microbeads and implantable pellets were prepared using an emulsion/solvent evaporation technique and a solvent casting technique respectively. Insulin—PLA microbeads retained between one-tenth and three-quarters of the loaded insulin. S.E.M. analysis of the microbeads revealed surface insulin crystals and distinct channels in the PLA matrix. It was found that the 1% to 2% poly(vinyl alcohol) emulsifier assisted in the formation of these surface insulin crystals. In vitroabout 50% of the insulin eluted from the microbeads into tris buffer within the first hour. The duration of action of the microbeads could be varied from a few hours to several days. Compared with the microbeads, insulin—PLA pellets showed a relatively small in vitro insulin burst effect and an almost constant insulin release rate during the first 13 hours (7.3 U/h). A pore-release model was used to describe the mechanism of insulin release from the polymer matrix. In animal studies, insulin—PLA preparations, administered subcutaneously as a single injection of microbeads or by implantation of a pellet, lowered the blood glucose levels of chemically induced diabetic rats for more than two weeks.

Microencapsulation of CHO cells in a hydroxyethyl methacrylate-methyl methacrylate copolymer.

Chinese hamster ovary fibroblasts, as model cells, have been microencapsulated in a hydroxyethyl methacrylate-methyl methacrylate copolymer (HEMA-MMA) by interfacial precipitation. The polymer containing approximately equal to 75 mol% HEMA, dissolved in polyethylene glycol 200 (PEG 200) was coextruded with the cell suspension (4-6 X 10(5) cells/ml in the alpha-MEM with 10% foetal calf serum +/- Ficoll 400/PBS) through a concentric needle assembly. Polymer solution droplets, containing cells, were blown off the end of the needle assembly by a coaxial filtered air stream into a nonsolvent bath containing phosphate buffered saline (PBS) with 5 ppm Pluronic L101, overlaid with hexadecane. The nascent capsules hang at the hexadecane/PBS interface while the solvent is extracted into the aqueous nonsolvent, to precipitate the polymer around the cells. The resultant capsules were 500 microns-1 mm in diam. with a microporous sponge-like interior, and also very tough and flexible. The cells survived encapsulation based on subculture ability, retention of some fluorescein diacetate (FDA) activity over 5 d and direct light microscopic evidence of cell growth over 10 d after histological sectioning and staining. However, cell growth was not uniformly observed (especially in the FDA assay) and this was attributed to space limitations for growth within the microporous interior. Continued development of this process and adaptation to cells such as pancreatic islets is expected to lead to hybrid artificial organs which are capable of ameliorating metabolic disorders such as diabetes.

Inactivation of thrombin by antithrombin III on a heparinized biomaterial

Abstract Heparin covalently bonded to polyvinyl alcohol (PVA) is potentially useful as a nonthrombogenic coating in the preparation of small diameter vascular prostheses and blood sampling catheters. PVA-heparin is highly stable: the elution rate of 35 S-heparin from the polymer was determined to be negligible (approx. 2 × 10 −11 g/cm 2 min) when washed with either buffered saline (pH 7.4) or citrated human plasma. The inactivation of thrombin by antithrombin III was studied on PVA-heparin. Using small columns of PVA-heparin beads eluted by 0.14M NaCl buffered at pH 7.4 both thrombin and antithrombin III bound to the immobilized heparin. If thrombin was loaded before an excess of antithrombin III, significant inactivation of thrombin was observed; however, loading antithrombin III before thrombin did not measurably inactivate thrombin. The results suggest that the covalently-bound heparin effectively participates in the inactivation of thrombin through the formation of surface-bound heparin-thrombin, which then reacts with antithrombin III to yield a surface-bound thrombin-antithrombin III complex. The fate of this surface-bound complex has yet to be clarified.

What really is blood compatibility

The criteria for nonthrombogenicity are classically defined as long clotting times and minimal platelet deposition. The inability to point to unequivocal progress in the development of truly nonthrombogenic materials, highlights the inadequacy if not actually invalidity of these criteria. Our approach is to define nonthrombogenicity in terms of: (1) a thrombin production rate constant, kp < 10-4 cm s-1; (2) low platelet consumption and low degree of platelet activation (e.g., microparticle formation); (3) perhaps some platelet spreading; and (4) low complement and leukocyte activation. Only when the target becomes clear, will it be possible to identify clear strategies for producing the materials we need.

Flow cytometric study of in vitro neutrophil activation by biomaterials

Neutrophil activation for adherent and nonadherent cells, as measured by flow cytometry, was not strongly dependent on material surface chemistry. We had hypothesized that material-induced neutrophil activation was an important parameter associated with material failure. All materials tested [cellophane, an acrylonitrile copolymer (AN69), Pellethane, nylon, polyethylene terephthalate, low density polyethylene, and polydimethylsiloxane] activated isolated human neutrophils, which were resuspended in plasma or serum, to similar extents based on L-selectin shedding, CD11b upregulation, and stimulation of the oxidative burst after 30-min exposure. Inhibition of complement activation by sCR1 unexpectedly had little effect if any on nonadherent neutrophils. However, neutrophil adhesion, but not the level of activation of the adherent cells, was strongly dependent on complement activation. Pretreatment with albumin did not inhibit adhesion or reduce neutrophil activation, but plasma pretreatment resulted in increased activation for nonadherent and adherent cells. More adhesion and a higher level of activation of adherent cells was observed following pretreatment with fibrinogen, a ligand of CD11b. Taken together these results suggest that upon contact with a material, neutrophil activation may occur though mechanisms that are not mediated by complement. For example, the presence of plasma proteins such as fibrinogen at the interface may trigger activation and the release of other activating agents. Although the material differences are small, the extent of activation may be significant and warrant further study of the mechanism and consequences of that activation.

Colorimetric assay fop cellular activity in microcapsules

Cellular activity in microcapsules was determined by a simple colorimetric assay, based on the cellular transformation of a tetrazolium salt, 3-(4,5-dimethyl-thiazol-2-yl)-2,5- diphenyl-tetrazolium bromide, into an insoluble formazan which was quantified in a spectrophotometer. The results showed that when encapsulated Chinese hamster ovary fibroblasts were exposed to the tetrazolium salt containing tissue culture medium, the formazan crystals were formed inside the poly(hydroxyethyl methacrylate-methyl methacrylate) microcapsules; capsules containing no cells or dead cells formed no formazan. A detectable amount of formazan was readily obtained even from single capsules. Formazan production was dependent on the incubation time, but not on the amount of added reagent. Capsules from a high cell-density encapsulation (4 X 10(6) cells/ml) formed more formazan than capsules from a low cell-density (4 X 10(5) cells/ml) encapsulation, suggesting a positive correlation between the cell density and tetrazolium transformation in microcapsules. The tetrazolium assay indicated the maintenance of cellular activity but slow, if any, proliferation in microcapsules over a 2 wk testing period.

Does surface chemistry affect thrombogenicity of surface modified polymers

With some exceptions, surface chemistry had little effect on platelet and leukocyte activation, and cell deposition, by scanning electron microscopy after blood exposure and clotting times among a group of 12 unmodified and plasma modified tubings. All materials activated platelets and leukocytes to detectable levels, although some materials increased the value of one activation parameter but not another. Unmodified materials [polyethylene (PE), Pellethane (PEU), latex, nylon, and Silastic] and modified materials (H(2)O plasma treated PE and PEU, CF(4) plasma treated PE, fluorinated PEU, NH(4) plasma treated PEU, polyethylene imine treated PEU, and heparin treated PEU) were characterised by XPS and contact angle. The objective of this project was to define a series of assays for the evaluation of hemocompatibility of cardiovascular devices with a view to clarify the specific requirements of ISO-10993-4, and to define an appropriate screening program for new blood contacting biomaterials. PE, PE--CF(4), PE--H(2)0, PEU--F, latex, and PEU-heparin were the exceptions to the general observations, although each behaved differently. PE proved to be least reactive, whereas PE-CF(4) was most reactive by several assays. Platelet microparticle formation (determined by flow cytometry), PTT, postblood exposure SEM, total SC5b-9, C3a, and platelet and leukocyte loss (cell counts) were able to distinguish differences among these materials, and often, but not always, showed expected correlations.

Dopamine secretion by PC12 cells microencapsulated in a hydroxyethyl methacrylate-methyl methacrylate copolymer

A rat pheochromocytoma cell line (PC12) was encapsulated in a water-insoluble hydroxyethyl methacrylate-methyl methacrylate copolymer by interfacial precipitation from a polyethylene glycol 200 solution into phosphate-buffered saline. The resulting capsules (660 +/- 44 microns in diameter; 84 +/- 27 microns wall thickness) contained viable PC12 cells in a spheroidal arrangement, much like tumour spheroids, the latter grown on surfaces unsuitable for cell attachment. In these spheroids, the viable cells formed a band approximately 100 microns thick, surrounding an inner core of necrotic cells. A similar arrangement was seen 14, 28 and 42 days after encapsulation, with capsules maintained in an in vitro tissue culture environment; the annular ring was roughly constant in size, although the packing density appeared to increase over the 6 week observation period. During the first 4 weeks, when measurements were made the encapsulated cells converted a tetrazolium dye (MTT) into an insoluble formazan product, in a time-after-encapsulation-dependent manner. This indicated that PC12 cells retained viability despite encapsulation and an ability to increase (at least in part) their metabolic capacity, presumably by a combination of proliferation and altered cellular activity. The encapsulated PC12 cells also secreted dopamine when incubated in a high potassium release medium but not in a low potassium, conventional tissue culture medium (RPMI 1640). Consistent with the MTT results, the amount of dopamine released was also dependent on the time after encapsulation, as well as the cell density at the time of encapsulation.

Endothelialized biomaterials for tissue engineering applications in vivo.

Rebuilding tissues involves the creation of a vasculature to supply nutrients and this in turn means that the endothelial cells (ECs) of the resulting endothelium must be a quiescent non-thrombogenic blood contacting surface. Such ECs are deployed on biomaterials that are composed of natural materials such as extracellular matrix proteins or synthetic polymers in the form of vascular grafts or tissue-engineered constructs. Because EC function is influenced by their origin, biomaterial surface chemistry and hemodynamics, these issues must be considered to optimize implant performance. In this review, we examine the recent in vivo use of endothelialized biomaterials and discuss the fundamental issues that must be considered when engineering functional vasculature.