Tethered epidermal growth factor as a paradigm for growth factor-induced stimulation from the solid phase.

We have tethered epidermal growth factor (EGF) to a solid substrate in a manner permitting the factor to retain its biological activity as assessed by both mitogenic and morphological assays. Mouse EGF was covalently coupled to aminosilane-modified glass via star poly(ethylene oxide) (PEO), which allows the ligand to retain significant mobility and active conformation. Tethered EGF was as effective as soluble EGF in eliciting DNA synthesis and cell rounding responses of primary rat hepatocytes under different surface conditions. In contrast, physically adsorbed EGF at comparable surface concentrations showed no activity. Presentation of growth factors in this manner may help to expedite their clinical use by permitting greater control of temporal and spatial availability in the extracellular environment.

In vitro cell response to differences in poly-L-lactide crystallinity.

Many different processing techniques are currently being used to produce tissue regeneration devices from polyesters in the polylactide/polyglycolide family. While it is generally well recognized that processing techniques influence bulk mechanical and degradation properties of these materials, the effects on surface properties are relatively less well studied. We thus investigated the effects of processing conditions that are known to change bulk properties, but not composition, on the surface properties of poly-L-lactide (PLLA). Specifically, we investigated the role of bulk crystallinity of PLLA substrates on several physiochemical aspects of the surface and on the attachment, morphology, and differentiated function of cultured primary hepatocytes and growth of 3T3 fibroblasts. We fabricated smooth, clear PLLA films of 13-37% crystallinity. Glancing angle X-ray diffraction indicated that low crystallinity films lacked order in the first 50 A of the surface while relatively high crystallinity films had detectable order in this range. In other aspects, the surfaces of all PLLA substrates appeared identical with XPS, SEM, and advancing contact angle analysis, but contact angle hysteresis was slightly greater for more crystalline films. Although the physicochemical properties of the surfaces appeared almost identical, we observed differences in cell behavior on less crystalline versus more crystalline films. Hepatocytes formed spheroids on all PLLA substrates, but spheroid formation was faster (24-48 H) on crystalline substrates. quantitative image analysis was used to assess the average cell area as a function of time in culture, and our data confirm previous reports that retention of differentiated function is inversely related to cell spreading where function was assessed by P-450 enzyme activity. In addition, the growth rate of 3T3 fibroblasts was lower on crystalline substrates than on amorphous substrates. An important conclusion from this work is that processing techniques that lead to seemingly inconsequential changes in bulk and surface properties of these polymers may influence biological response.

De novo cartilage generation using calcium alginate-chondrocyte constructs.

These studies investigated the utility of calcium alginate as a biocompatible polymer matrix within which large numbers of chondrocytes could be held successfully in a three-dimensional structure and implanted. Further, the ability of chondrocyte-calcium alginate constructs to engraft and generate new cartilage was examined. Chondrocytes isolated from calf shoulders were mixed with a 1.5% sodium alginate solution to generate cell suspensions with densities of 0, 1.0, 5.0, and 10.0 x 10(6) chondrocytes/ml. The cell suspensions were gelled to create disks that were placed in subcutaneous pockets on the dorsums of nude mice. The alginate concentration and CaCl2 concentration used to make the disks also were varied. A total of 20 mice were implanted with 67 bovine chondrocyte-calcium alginate constructs. Samples with an initial cellular density of at least 5.0 x 10(6) chondrocytes/ml demonstrated gross cartilage formation 12 weeks after implantation. Cartilage formation was observed microscopically in specimens with a cellular density as low as 1.0 x 10(6) chondrocytes/ml. The histoarchitecture of the new cartilage closely resembled that of native cartilage. Cartilage formation was independent of CaCl2 concentration (15 to 100 mM) or alginate concentration (0.5% to 4.0%) used in gel polymerization.

Mechanical properties of dense polylactic acid structures fabricated by three dimensional printing.

Polylactic acid (PLA) is a bioresorbable polymer that is used in a number of clinical situations. Complex shapes of PLA are commonly machined for bone fixation and reconstruction. Solid free from fabrication methods, such as 3D printing, can produce complex-shaped articles directly from a CAD model. This study reports on the mechanical properties of 3D-printed PLLA parts. 3D printing is a solid free-form fabrication process which produces components by ink-jet printing a binder into sequential powder layers. Test bars were fabricated from low and high molecular weight PLA powders with chloroform used as a binder. The binder printed per unit line length of the powder was varied to analyze the effects of printing conditions on mechanical and physical properties of the PLA bars. Furthermore, cold isostatic pressing was performed after printing to improve the mechanical properties of the printed bars. The maximum measured tensile strength for the low molecular weight PLLA (53 000) is 17.40 +/- 0.71 MPa and for high molecular weight PLLA (312 000) is 15.94 +/- 1.50 MPa.

Injectable cartilage.

Slowly polymerizing calcium alginate gels were investigated as a means of delivering large numbers of isolated chondrocytes by means of injection to determine if these gels would promote engraftment and could provide three-dimensional templates for new cartilage growth. Chondrocytes isolated from the articular surface of calf forelimbs were added to a 1% sodium alginate dissolved in a 0.1 M potassium phosphate buffer solution (pH 7.4) to generate a final cellular density of 10 x 10(6)/ml (representing approximately 10 percent of the cellular density of human juvenile articular cartilage). The calcium alginate-chondrocyte mixture was injected through a 22-gauge needle in 100-microliters aliquots under the panniculus carnosus on the dorsum of nude mice and incubated for 6 (n = 4), 8 (n = 11), and 12 (n = 12) weeks in vivo. Time-zero specimens (n = 10) consisting of 100-microliters aliquots of the calcium alginate-chondrocyte mixture were used to calculate initial weight. At harvest, all calcium alginate-chondrocyte specimens exhibited a pearly opalescence and were firm to palpation as early as 6 weeks after injection. By 12 weeks of in vivo incubation, the specimens weighed 0.15 +/- 0.04 gm, significantly more than the initial weight of 0.11 +/- 0.01 gm (p < 0.05). Specimens stained with hematoxylin and eosin demonstrated lacunae within a basophilic ground-glass substance. Control specimens of calcium alginate without chondrocytes (n = 4) had a doughy consistency 12 weeks after injection and had no histologic evidence of cartilage formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Corneal epithelial wound healing on bilayer composite hydrogels.

We demonstrate that a bilayer composite hydrogel composed of corneal stroma crosslinked to poly(ethylene oxide) provides a substrate suitable for wound healing behavior of corneal epithelial cells and for formation and maintenance of a stable multilayered epithelium. Potential diffusion-limitation of nutrients or regulatory molecules across the hydrogel was investigated experimentally with a new in vitro ocular assay, using epithelial cell migration as an index of molecular diffusion limitations. Corneal epithelial cells explanted on the composite hydrogel in vitro exhibited morphology similar to those in vivo, and migrated effectively over the stromal surface. Importantly, our system yielded multilayered epithelium like that found in normal corneal tissue under conditions that closely simulate the in vivo physiologic arrangement. In addition, our results indicate that molecules of substantially greater molecular weight than glucose appear to control the cell migration rate. Thus, engineering design of this composite hydrogel system may allow it to be useful in corneal wound healing applications.

Polymer substrates for controlled biological interactions.

The identification of a myriad of small peptide and carbohydrate ligands recognized by cell surface receptors has generated enthusiasm for the use of these ligands of components of biomaterials for controlling cellular interactions. Achieving control of cell interactions via ligand modification of materials also requires that nonspecific interactions of cells with these materials due to surface adsorption of biological macromolecules is minimized. Polyethylene oxide (PEO) exhibits extraordinary inertness toward most biological macromolecules and is thus receiving increasing attention as a component of new materials for controlling cell behavior. Both surface and bulk modifications with PEO are being applied to develop a range of bland substrate materials as vehicles for ligand immobilization.

A hydrodynamic interpretation of crisis in sickle cell anemia.

The occurrence of crisis events in patients with sickle cell disease is associated with an increase in blood plasma viscosity, the hydrodynamic consequences of which are examined here. A mathematical model of the flow of sickle cells in capillaries predicts that for moderate increases in plasma viscosity, a regime of multivalued solutions for blood velocities is encountered, and the likely physical response is a precipitous drop to the lowest velocity solution. This behavior results from the coupling of the hydrodynamics with sickle erythrocyte rheology and oxygen transport to the surrounding tissue; no such catastrophe is predicted for normal erythrocytes. The type of velocity changes predicted by the model strongly suggest that plasma viscosity changes may play an important role in initiating or exacerbating crisis.

Injectable alginate seeded with chondrocytes as a potential treatment for vesicoureteral reflux.

Injection of polytetrafluoroethylene (Teflon) or collagen has been used in the endoscopic treatment of vesicoureteral reflux. Although the principle of an endoscopic treatment is valid, there are concerns regarding the long-term safety and effectiveness of these substances. The goal of several investigators has been to find alternate implant materials that would be safe for human use. Toward this goal we conducted a study to determine the effect of chondrocytes using a biodegradable polymer solution as a template. Hyaline cartilage was obtained from the articular surfaces of calf shoulders and chondrocytes were harvested. Chondrocyte suspensions were concentrated to 20, 30 and 40 x 10(6) cells per cc and mixed with dry alginate powder (a biodegradable polymer) to form a gel. Twelve athymic mice were injected subcutaneously with a chondrocyte-alginate solution. Each mouse had 4 injection sites, consisting of control, 10, 15 and 20 x 10(6) chondrocyte cells (48 injection sites). Mice were sacrificed at 2, 4, 6 and 12 weeks after injection. Histological examination of the injection sites demonstrated evidence of cartilage formation in 34 of the 36 experimental injection sites. Gross examination of the injection sites with increasing time showed that the polymer gels were progressively replaced by cartilage. The ultimate size of the cartilage formed was related to the initial chondrocyte concentration injected, and appeared to be uniform and stable within each category. There was no evidence of cartilage formation in the 12 controls. Histological analyses of distant organs showed no evidence of cartilage or alginate gel migration, or granuloma formation. In conclusion, chondrocyte-alginate gel suspensions are injectable, appear to be nonmigratory and are able to conserve their volume. In addition, the use of autologous cartilage cells would preclude an immunological reaction. These preliminary studies indicate that autologous cartilage-polymer gel solutions may be potentially useful in the endoscopic treatment of reflux.

Preparation of poly(glycolic acid) bonded fiber structures for cell attachment and transplantation.

A novel method was developed to prepare three-dimensional structures with desired shapes used as templates for cell transplantation. The produced biomaterials are highly porous with large surface/volume and provide the necessary space for attachment and proliferation of the transplanted cells. The processing technique calls for the formation of a composite material with nonbonded fibers embedded in a matrix followed by thermal treatment and the selective dissolution of the matrix. To evaluate the technique, poly(glycolic acid) (PGA) fiber meshes were bonded using poly(L-lactic acid) (PLLA) as a matrix. The bonded structures were highly porous with values of porosity up to 0.81 and area/volume ratios as high as 0.05 micron-1.

Design of synthetic polymeric structures for cell transplantation and tissue engineering.

Two approaches for cell transplantation and new tissue constructions are discussed. In one case, a novel synthetic polyphosphazene has been synthesized that can be gelled by simply adding ions to it at room temperature under aqueous conditions. This polymer has been shown to be compatible for several different cell types. Microcapsular membranes based on the complex of this polymer with poly (L-lysine) allow the inward diffusion of nutrients to nourish the encapsulated cells, but are impermeable to antibodies. In a second approach, biodegradable polyesters have been designed as scaffolds for liver cells and cartilage cells to aid in organ regeneration. Design of the polymer scaffold including the characterization of the surface chemistries for cell attachment, as well as in-vitro and in-vivo data on cell behavior are presented.

Hepatocyte culture on biodegradable polymeric substrates.

The interactions of primary rat liver cells with biodegradable polymeric substrates were investigated in vitro to assess the suitability of the polymer materials for use in cell transplantation devices. The kinetics of cell adhesion to, and the growth and biochemical function of cells maintained on, films formed from poly (D,L-lactic-co-glycolic acid, 88: 12) (PLGA) or from a 50/50 (w/w) blend of PLGA and poly (L-lactic acid) (PLLA) were evaluated in comparison to two control substrates, matrigel coated or collagen-coated polystyrene petri dishes. The rate of cell adhesion to both types of polymeric substrates was similar to the rate of adhesion to the collagen control substrate, but of the two polymers, only the blend was suitable for extended culture. Hepatocytes maintained on the polymer blend films showed retention of differentiated cell function as measured by the rate of albumin secretion-the rate of albumin secretion by cells on the films was the same as the rate for cells on matrigel and reached a level in the range of reported in vivo levels (140-160 microg/10(6) cells/24 h). In contrast, albumin secretion by hepatocytes maintained on collagen-coated polystyrene culture dishes declined over five days to a level one third that of the initial level and one fifth that of cells maintained on the polymer blend films on day five. Such retention of differentiated cell function by hepatocytes in culture has previously been observed only when hepatocytes were cultured in the presence of exogenous extracellular matrix proteins or were cocultured with another cell type. In addition to retention of differentiated function, the cells maintained on the polymer blend films also displayed rates of DNA synthesis similar to controls maintained on collagen-coated polystyrene, a substrate optimal for DNA synthesis.

Tissue engineering by cell transplantation using degradable polymer substrates.

This paper reviews our research in developing novel matrices for cell transplantation using bioresorbable polymers. We focus on applications to liver and cartilage as paradigms for regeneration of metabolic and structural tissue, but review the approach in the context of cell transplantation as a whole. Important engineering issues in the design of successful devices are the surface chemistry and surface microstructure, which influence the ability of the cells to attach, grow, and function normally; the porosity and macroscopic dimensions, which affect the transport of nutrients to the implanted cells; the shape, which may be necessary for proper function in tissues like cartilage; and the choice of implantation site, which may be dictated by the total mass of the implant and which may influence the dimensions of the device by the available vascularity. Studies show that both liver and cartilage cells can be transplanted in small animals using this approach.

Future directions in biomaterials.

Biomaterials have made a great impact on medicine. However, numerous challenges remain. This paper discusses three representative areas involving important medical problems. First, drug delivery systems; major considerations include drug-polymer interactions, drug transformation, diffusion properties of drugs and, if degradation occurs, of polymer degradation products through polymer matrices developing a more complete understanding of matrix degradation in the case of erodible polymers and developing new engineered polymers designed for specific purposes such as vaccination or pulsatile release. Second, cell-polymer interactions, including the fate of inert polymers, the use of polymers as templates for tissue regeneration and the study of polymers which aid cell transplantation. Third, orthopaedic biomaterials, including basic research in the behaviour of chondrocytes, osteocytes and connective tissue-free interfaces and applied research involving computer-aided design of biomaterials and the creation of orthopaedic biomaterials.