Showing papers on "Tissue engineering published in 1993"

Cell origin and differentiation in the repair of full-thickness defects of articular cartilage.

Abstract: The origin and differentiation of cells in the repair of three-millimeter-diameter, cylindrical, full-thickness drilled defects of articular cartilage were studied histologically in New Zealand White rabbits. The animals were allowed to move freely after the operation. Three hundred and sixty-four individual defects from 122 animals were examined as long as forty-eight weeks postoperatively. In the first few days, fibrinous arcades were established across the defect, from surface edge to surface edge, and this served to orient mesenchymal cell ingrowth along the long axes. The first evidence of synthesis of a cartilage extracellular matrix, as defined by safranin-O staining, appeared at ten days. At two weeks, cartilage was present immediately beneath the surface of collagenous tissue that was rich in flattened fibrocartilaginous cells in virtually all specimens. At three weeks, the sites of almost all of the defects had a well demarcated layer of cartilage containing chondrocytes. An essentially complete repopulation of the defects occurred at six, eight, ten, and twelve weeks, with progressive differentiation of cells to chondroblasts, chondrocytes, and osteoblasts and synthesis of cartilage and bone matrices in their appropriate locations. At twenty-four weeks, both the tidemark and the compact lamellar subchondral bone plate had been re-established. The cancellous woven bone that had formed initially in the depths of the defect was replaced by lamellar, coarse cancellous bone. Autoradiography after labeling with 3H-thymidine and 3H-cytidine demonstrated that chondrocytes from the residual adjacent articular cartilage did not participate in the repopulation of the defect. The repair was mediated wholly by the proliferation and differentiation of mesenchymal cells of the marrow. Intra-articular injections of 3H-thymidine seven days after the operation clearly labeled this mesenchymal cell pool. The label, initially taken up by undifferentiated mesenchymal cells, progressively appeared in fibroblasts, osteoblasts, articular chondroblasts, and chondrocytes, indicating their origin from the primitive mesenchymal cells of the marrow. Early traces of degeneration of the cartilage matrix were seen in many defects at twelve to twenty weeks, with the prevalence and intensity of the degeneration increasing at twenty-four, thirty-six, and forty-eight weeks. Polarized light microscopy demonstrated failure of the newly synthesized repair matrix to become adherent to, and integrated with, the cartilage immediately adjacent to the drill-hole, even when light microscopy had shown apparent continuity of the tissue. In many instances, a clear gap was seen between repair and residual cartilage.(ABSTRACT TRUNCATED AT 400 WORDS)

Prevascularization of porous biodegradable polymers.

Abstract: Highly porous biocompatible and biodegradable polymers in the form of cylindrical disks of 13.5 mm diameter were implanted in the mesentery of male syngeneic Fischer rats for a period of 35 days to study the dynamics of tissue ingrowth and the extent of tissue vascularity, and to explore their potential use as substrates for cell transplantation. The advancing fibrovascular tissue was characterized from histological sections of harvested devices by image analysis techniques. The rate of tissue ingrowth increased as the porosity and/or the pore size of the implanted devices increased. The time required for the tissue to fill the device depended on the polymer crystallinity and was smaller for amorphous polymers. The vascularity of the advancing tissue was consistent with time and independent of the biomaterial composition and morphology. Poly(L-lactic acid) (PLLA) devices of 5 mm thickness, 24.5% crystallinity, 83% porosity, and 166 μm median pore diameter were filled by tissue after 25 days. However, the void volume of prevascularized devices (4%) was minimal and not practical for cell transplantation. In contrast, for amporphous PLLA devices of the same dimensions, and the similar porosity of 87% and median pore diameter of 179 μm, the tissue did not fill completely prevascularized devices, and an appreciable percentage (21%) of device volume was still available for cell engraftment after 25 days of implantation. These studies demonstrate the feasibility of creating vascularized templates of amorphous biodegradable polymers for the transplantation of isolated or encapsulated cell populations to regenerate metabolic organs and tissues. © 1993 John Wiley & Sons, Inc.

Cultivation of cell-polymer cartilage implants in bioreactors.

Abstract: Cartilage implants for potential use in reconstructive or orthopedic surgery can be created by growing isolated cartilage cells (chondrocytes) in vitro on synthetic, biodegradable polymer scaffolds. The scaffolds provide specific three-dimensional structures which support cell proliferation and biodegrade in a controlled fashion in parallel to cellular regeneration of cartilaginous tissue. Cartilage implants based on chondrocytes and fibrous polyglycolic acid scaffolds were recently shown to closely resemble normal cartilage histologically as well as with respect to cell density and matrix composition (collagen, glycosaminoglycan) [Freed et al., J Biomed Mater Res 27:11-23, 1993a]. These findings form the basis for developing straightforward procedures to obtain implants for clinical use from small, autologous cartilage specimens without any limitations in terms of availability of donor tissue or implant dimensions. Chondrocyte growth and cartilage matrix regeneration on polymer scaffolds are interdependent and also depend on in vitro tissue culture conditions. Under static culture conditions, cell growth rates are diffusionally limited due to increasing cell mass and decreasing effective implant porosity resulting from cartilage matrix regeneration. Optimization of the in vitro culture environment is thus essential for the cultivation of large, clinically useful cartilage implants. Preliminary studies indicate that major improvements can be achieved using bioreactors that provide efficient mass transfer and controlled shear rates at the cell and implant surfaces.

Tissue engineering of biomaterials for composite reconstruction: an experimental model.

Abstract: The fibrovascular integration of a biomaterial scaffold was examined in a rabbit model. Eight disks of expanded polytetrafluoroethylene measuring 2 x 2 x 0.2 cm were placed adjacent to dissected vascular pedicles in the ears of four rabbits and sealed between leaves of silicone sheeting. Fibrovascular outgrowth from the vascular pedicles completely integrated the biomaterial scaffolds by 6 weeks and was sufficient to sustain split-thickness skin grafts. Division of the distal vascular pedicle allowed isolation of the blood flow circuit to the construct. Successful microsurgical transfer was achieved in one construct. This model has proved useful in the study of tissue engineering of biomaterials for reconstruction and may well have use in the study of neovascularization as an isolated event.

Cultivation of recombinant, insulin-secreting AtT-20 cells as free and entrapped spheroids

Abstract: Animal cells from endocrine glands have potential applications in bioprocessing, for the production of hormones, enzymes, possibly also recombinant proteins, and in tissue engineering, for the development of immunoisolated, implantable devices for long-term treatment of endocrine disorders. Immunoisolation can be achieved by surrounding cells with a biocompatible polymer which allows diffusion of nutrients and metabolites, including hormones, but excludes higher molecular weight antibodies and cytotoxic cells. Primary hormone-secreting cells cannot be effectively amplified in culture, so the large-scale application of implantable systems based on such cells is limited by cell availability. In this study, we conducted an initial assessment of the feasibility of using transformed, continuous cell lines in immunoisolated devices. The model system employed consisted of mouse pituitary tumor AtT-20 cells which secrete recombinant proinsulin and an insulin-like peptide and exhibit a high growth potential. Cells were cultivated as spheroids in spinner flasks and entrapped as such in alginate/polylysine/alginate beads. Free and entrapped spheroids were propagated in fed-batch, suspension cultures. Entrapment did not significantly affect spheroid metabolism or basal secretion. Entrapped spheroids did not increase in size or number and maintained roughly constant metabolic and basal secretory activities over a 15-day period. Free spheroids in suspension increased in size during the sami period, but also maintained constant metabolism and basal secretion, apparently because of a concomitant increase in hypoxic and/or necrotic cells. The potential of using continuous cell lines in the development of bioartificial endocrine organs is discussed.

Extracellular matrix, cellular mechanics and tissue engineering

Abstract: One of the major recurring themes in this symposium on Tissue Engineering is that to design effective artificial tissues, we must first understand the critical chemical and structural determinants that control tissue development. Current tissue engineering approaches commonly use cell attachment scaffolds that are complex composites of naturally occuring extracellular matrix (ECM) molecules (e.g., collagens, glycosaminoglycans). Unfortunately, these “artificial ECMs” are restricted from an engineering standpoint: they exhibit a limited range of structural and chemical properties and are not easily chemically modified. Also, their large-scale fabrication can be limited by “batch to batch” variability during purification of the individual ECM molecules. An alternative approach for cell transplantation is to develop a completely synthetic attachment foundation that can support a high degree of cell function and yet be highly biocompatible (Vacanti et al., 1988; Cima et al., 1991). To accomplish this objective, recent advances in ECM biology must be merged with new developments in bioengineering and polymer chemistry.

The Tissue Engineering Approach to Ligament Reconstruction

Abstract: Previous studies in our laboratory showed that acellular collagen scaffold implants induce tissue ingrowth and perform similar to autografts following reconstruction of rabbit Achilles tendon or anterior cruciate ligament (ACL). We chronologically review these and related studies, and report preliminary development of fibroblast-seeded collagen scaffolds potentially useful for ACL reconstruction. The ‘healing potential’ of fibroblasts was measured within collagen scaffolds in vitro, as a function of fibroblast source. Aligned collagen scaffolds were seeded with fibroblasts from rabbit ACL, synovium, patellar tendon, or skin. Fibroblast viability, adherence, spreading, proliferation, and protein and collagen deposition were measured on collagen scaffolds. The fibroblasts attached to the scaffolds, and spread along the long axis of the collagen fibers. ACL fibroblasts adhered better than other fibroblast types; however, the ACL fibroblasts proliferated at the slowest rate. Patellar tendon fibroblasts proliferated at the most rapid rate. All four of the fibroblast types secreted protein and collagen within the collagen scaffolds. Preliminary in vivo studies suggest that fibroblasts seeded onto collagen scaffolds can remain viable following reimplantation into the donor rabbit. Ongoing studies will elucidate the role of autogenous seeded fibroblasts in neoligament formation/remodeling. These ‘ligament analogs’ are potentially useful for clinical ACL reconstruction: fibroblasts would be obtained from biopsy, cultured, seeded onto a collagen scaffold, and implanted as an ACL substitute into the same patient.