Linda G. Cima

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.

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 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.

Solid free-form fabrication of drug delivery devices

Three-dimensional printing (3DP) is used to create resorbable devices with complex concentration profiles within the device. 3DP is an example of a solid free-form fabrication method where both the macro- and microstructure of the device can be controlled since objects are built by addition of very small amounts of matter. Application of this novel technology for fabrication of polymeric drug delivery systems is described in this article. The drug concentration profile is first specified in a computer model of the device which is then built using the 3DP process. Complex drug delivery regimes can be created in this way, such as the release of multiple drugs or multiphasic release of a single drug. This study demonstrates several simple examples of such devices and several construction methods that can be used to control the release of the drugs. Two dyes are used as model drugs in a matrix of biocompatible polymers. The dye release rate and release time are controlled by either specifying the position of the dye within the device or by controlling the local composition and microstructure with the 3DP process. The mechanism of resorption can also be controlled by manipulating the composition and microstructure of the device during construction. Polyethylene oxide and polycaprolactone were selected as matrix materials and methylene blue and alizarin yellow were used as drug models. Devices with erosion and diffusion controls are described in this report. Spectrophotometric analysis of dye release yielded reproducible results.

De novo cartilage generation using calcium alginate-chondrocyte constructs.

&NA; 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 × 106 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 × 106 chondrocytes/ml demonstrated gross cartilage formation 12 weeks alter implantation. Cartilage formation was observed microscopically in specimens with a cellular density as low as 1.0 × 106 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. (Plast. Reamslr. Surg. 97: 168, 1996.)

Cartilage engineered in predetermined shapes employing cell transplantation on synthetic biodegradable polymers.

Cartilage is often used as structural support tissue for cosmetic repair in plastic and reconstructive surgery. We describe the efficacy of a new approach for the generation of cartilage in predetermined shapes using specially configured biodegradable synthetic polymer devices as delivery vehicles for transplanted cells. Synthetic biodegradable polymer scaffolds were configured in one of four specific shapes, i.e., a triangle, a rectangle, a cross, and a cylinder. The polymer matrices were seeded with freshly isolated bovine articular chondrocytes and then implanted subcutaneously into nude mice. Gross examination of excised specimens 12 weeks after implantation revealed the presence of new hyaline cartilage of approximately the same dimensions as the original construct. This cartilage showed no signs of resorption or overgrowth over the 12-week time course of the experiment. Histologie evaluation using hematoxylin and eosin stains confirmed the presence of normal mature hyaline cartilage in 46 of 48 specimens. These results suggest that cartilage can be created in predetermined shapes and dimensions using cell transplantation on appropriate polymer templates. This technology would be useful in cosmetic and reconstructive surgery.

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.

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.

Tissue Engineered Growth of New Cartilage in the Shape of a Human Ear Using Synthetic Polymers Seeded with Chondrocytes

This report concerns the tissue-engineered growth of new cartilage in the shape of a human ear. Using synthetic biodegradable polyesters, a porous, three dimensional device in the shape of a human ear was fabricated. The polymer matrices were seeded with living chondrocytes isolated from a freshly sacrificed calf shoulder and implanted subcutaneously on the dorsum of athymic rats. This resulted in the formation of new cartilage in the shape of a human ear of approximately the same dimensions as the original implants. Histological analysis revealed the presence of mature cartilage in all specimens.

Studies in rat liver perfusion for optimal harvest of hepatocytes

Pediatric liver transplantation is successful but donor scarcity is a major limitation. We are studying hepatocyte transplantation as an alternative to provide functional hepatic replacement. This report details the study of rat liver perfusion for optimal harvest of hepatocytes and cell implantation. We performed 128 rat liver perfusions using a technique modified from the two-step enzymatic perfusion described by Seglen. We examined variations in the perfusion, rate, time, antegrade versus retrograde, pulsatile versus continuous flow, temperature, collagenase type, and variables of buffer composition. We have found optimal cell yield and viability under the following conditions: in situ perfusion, continuous flow at 25 cc/min, retrograde perfusion via the inferior vena cava, water bath temperature 38 degrees C, Boerhinger-Mannheim collagenase using a nonoxygenated HEPES based perfusion buffer, pH 7.4, for the initial perfusion and the same buffer with 4.8 mmol/L CaCl2 for the collagenase perfusion. These conditions consistently generate cell harvests of 500 to 700 x 10(5) cells/g of liver tissue with cell viability between 85% and 95%.