Biomedical subjects

G Vunjak-Novakovic

Publications and source records attributed to G Vunjak-Novakovic.

10 recordsLinked to original sources

PubMed1999

Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage.

Tissue engineered cartilage can be grown in vitro if the necessary physical and biochemical factors are present in the tissue culture environment. Cell metabolism and tissue composition were studied for engineered cartilage cultured for 5 weeks using bovine articular chondrocytes, polymer scaffolds (5 mm diameter x 2 mm thick fibrous discs), and rotating bioreactors. Medium pH and concentrations of oxygen, carbon dioxide, glucose, lactate, ammonia, and glycosoaminoglycan (GAG) were varied by altering the exchange rates of gas and medium in the bioreactors. Cell-polymer constructs were assessed with respect to histomorphology, biochemical composition and metabolic activity. Low oxygen tension ( approximately 40 mmHg) and low pH ( approximately 6.7) were associated with anaerobic cell metabolism (yield of lactate on glucose, YL/G, of 2.2 mol/mol) while higher oxygen tension ( approximately 80 mmHg) and higher pH ( approximately 7.0) were associated with more aerobic cell metabolism (YL/G of 1.65-1.79 mol/mol). Under conditions of infrequent medium replacement (50% once per week), cells utilized more economical pathways such that glucose consumption and lactate production both decreased, cell metabolism remained relatively aerobic (YL/G of 1.67 mol/mol) and the resulting constructs were cartilaginous. More aerobic conditions generally resulted in larger constructs containing higher amounts of cartilaginous tissue components, while anaerobic conditions suppressed chondrogenesis in 3D tissue constructs.

Aerobiosis↗

PubMed1999

Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage.

Cartilaginous constructs have been grown in vitro with use of isolated cells, biodegradable polymer scaffolds, and bioreactors. In the present work, the relationships between the composition and mechanical properties of engineered cartilage constructs were studied by culturing bovine calf articular chondrocytes on fibrous polyglycolic acid scaffolds (5 mm in diameter, 2-mm thick, and 97% porous) in three different environments: static flasks, mixed flasks, and rotating vessels. After 6 weeks of cultivation, the composition, morphology, and mechanical function of the constructs in radially confined static and dynamic compression all depended on the conditions of in vitro cultivation. Static culture yielded small and fragile constructs, while turbulent flow in mixed flasks yielded constructs with fibrous outer capsules; both environments resulted in constructs with poor mechanical properties. The constructs that were cultured freely suspended in a dynamic laminar flow field in rotating vessels were the largest, contained continuous cartilage-like extracellular matrices with the highest fractions of glycosaminoglycan and collagen, and had the best mechanical properties. The equilibrium modulus, hydraulic permeability, dynamic stiffness, and streaming potential correlated with the wet-weight fractions of glycosaminoglycan, collagen, and water. These findings suggest that the hydrodynamic conditions in tissue-culture bioreactors can modulate the composition, morphology, mechanical properties, and electromechanical function of engineered cartilage.

Animals↗

PubMed1998

Collagen in tissue-engineered cartilage: types, structure, and crosslinks.

The function of articular cartilage as a weight-bearing tissue depends on the specific arrangement of collagen types II and IX into a three-dimensional organized collagen network that can balance the swelling pressure of the proteoglycan/water gel. To determine whether cartilage engineered in vitro contains a functional collagen network, chondrocyte-polymer constructs were cultured for up to 6 weeks and analyzed with respect to the composition and ultrastructure of collagen by using biochemical and immunochemical methods and scanning electron microscopy. Total collagen content and the concentration of pyridinium crosslinks were significantly (57% and 70%, respectively) lower in tissue-engineered cartilage that in bovine calf articular cartilage. However, the fractions of collagen types II, IX, and X and the collagen network organization, density, and fibril diameter in engineered cartilage were not significantly different from those in natural articular cartilage. The implications of these findings for the field of tissue engineering are that differentiated chondrocytes are capable of forming a complex structure of collagen matrix in vitro, producing a tissue similar to natural articular cartilage on an ultrastructural scale.

Amino Acids↗

PubMed1998

Chondrogenesis in a cell-polymer-bioreactor system.

Chondrogenesis was studied under controlled in vitro conditions using a cell-polymer-bioreactor system. Bovine calf articular chondrocytes were seeded onto biodegradable polymer scaffolds and cultured in rotating bioreactor vessels. Concomitant increases in the amounts of glycosaminoglycan (GAG) and type II collagen resulted in cell-polymer constructs with continuous cartilaginous matrix over their entire cross sections (6.7 mm diameter x 5 mm thick) after 40 days of cultivation. As compared to natural calf cartilage, constructs had comparable cellularities, 68% as much GAG and 33% as much type II collagen per gram wet weight. The progression of chondrogenesis in chondrocyte-polymer constructs was similar to that suggested previously for precursor cells in vitro and developing limbs in vivo. In particular, the polymer scaffold provided a three-dimensional structure that could be seeded with chondrocytes at high cell densities in order to establish cell-to-cell contacts and initiate cartilage tissue development, whereas the bioreactor vessel provided a permissive microenvironment for chondrogenesis. This work demonstrates the promise of using tissue engineered constructs for in vitro studies of cell interactions and differentiation.

Animals↗

PubMed1998

In vitro differentiation of chick embryo bone marrow stromal cells into cartilaginous and bone-like tissues.

Bone marrow stromal cells, progenitor cells involved in repair of bone and cartilage, can potentially provide a source for autologous skeletal tissue engineering. We investigated which factors were required to induce in vitro differentiation of avian bone marrow stromal cells into three-dimensional cartilaginous and bone-like tissues. Bone marrow stromal cells from embryonic chicks were expanded in monolayers, seeded onto biodegradable polyglycolic acid scaffolds, and cultured for 4 weeks in orbitally mixed Petri dishes. Cell-polymer constructs developed an organized extracellular matrix containing glycosaminoglycans and collagen, whereas control bone marrow stromal cell pellet cultures were smaller and consisted predominantly of fibrous tissue. Bone marrow stromal cells expanded with fibroblast growth factor-2 and seeded onto polymer scaffolds formed highly homogeneous three-dimensional tissues that contained cartilage-specific molecular markers and had biochemical compositions comparable with avian epiphyseal cartilage. When cell-polymer constructs were cultured in the presence of beta-glycerophosphate and dexamethasone, the extracellular matrix mineralized and bone-specific proteins were expressed. Our work shows that cell expansion in the presence of fibroblast growth factor-2 and cultivation on a three-dimensional polymer scaffold allows differentiation of chick bone marrow stromal cells into three-dimensional cartilaginous tissues. In the in vitro system studied, the same population could be selectively induced to regenerate either cartilaginous or bone-like tissue.

Animals↗

PubMed1997

Tissue engineering of cartilage in space.

Tissue engineering of cartilage, i.e., the in vitro cultivation of cartilage cells on synthetic polymer scaffolds, was studied on the Mir Space Station and on Earth. Specifically, three-dimensional cell-polymer constructs consisting of bovine articular chondrocytes and polyglycolic acid scaffolds were grown in rotating bioreactors, first for 3 months on Earth and then for an additional 4 months on either Mir (10(-4)-10(-6) g) or Earth (1 g). This mission provided a unique opportunity to study the feasibility of long-term cell culture flight experiments and to assess the effects of spaceflight on the growth and function of a model musculoskeletal tissue. Both environments yielded cartilaginous constructs, each weighing between 0.3 and 0.4 g and consisting of viable, differentiated cells that synthesized proteoglycan and type II collagen. Compared with the Earth group, Mir-grown constructs were more spherical, smaller, and mechanically inferior. The same bioreactor system can be used for a variety of controlled microgravity studies of cartilage and other tissues. These results may have implications for human spaceflight, e.g., a Mars mission, and clinical medicine, e.g., improved understanding of the effects of pseudo-weightlessness in prolonged immobilization, hydrotherapy, and intrauterine development.

Animals↗

PubMed1997

Microgravity tissue engineering.

Tissue engineering studies were done using isolated cells, three-dimensional polymer scaffolds, and rotating bioreactors operated under conditions of simulated microgravity. In particular, vessel rotation speed was adjusted such that 10 mm diameter x 2 mm thick cell-polymer constructs were cultivated in a state of continuous free-fall. Feasibility was demonstrated for two different cell types: cartilage and heart. Conditions of simulated microgravity promoted the formation of cartilaginous constructs consisting of round cells, collagen and glycosaminoglycan (GAG), and cardiac tissue constructs consisting of elongated cells that contracted spontaneously and synchronously. Potential advantages of using a simulated microgravity environment for tissue engineering were demonstrated by comparing the compositions of cartilaginous constructs grown under four different in vitro culture conditions: simulated microgravity in rotating bioreactors, solid body rotation in rotating bioreactors, turbulent mixing in spinner flasks, and orbital mixing in petri dishes. Constructs grown in simulated microgravity contained the highest fractions of total regenerated tissue (as a percent of construct dry weight) and of GAG, the component required for cartilage to withstand compressive force.

Animals↗

PubMed1994

Biodegradable polymer scaffolds for tissue engineering.

Synthetic polymer scaffolds designed for cell transplantation were reproducibly made on a large scale and studied with respect to biocompatibility, structure and biodegradation rate. Polyglycolic acid (PGA) was extruded and oriented to form 13 microns diameter fibers with desired tenacity. Textile processing techniques were used to produce fibrous scaffolds with a porosity of 97% and sufficient structural integrity to maintain their dimensions when seeded with isolated cartilage cells (chondrocytes) and cultured in vitro at 37 degrees C for 8 weeks. Cartilaginous tissue consisting of glycosaminoglycan and collagen was regenerated in the shape of the original PGA scaffold. The resulting cell-polymer constructs were the largest grown in vitro to date (1 cm diameter x 0.35 cm thick). Construct mass was accurately predicted by accounting for accumulation of tissue components and scaffold degradation. The scaffold induced chondrocyte differentiation with respect to morphology and phenotype and represents a model cell culture substrate that may be useful for a variety of tissue engineering applications.

Biocompatible Materials↗

PubMed1993

Cultivation of cell-polymer cartilage implants in bioreactors.

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.

Biotechnology↗

PubMedDate not supplied

Dynamic cell seeding of polymer scaffolds for cartilage tissue engineering.

Cell seeding of three-dimensional polymer scaffolds is the first step of the cultivation of engineered tissues in bioreactors. Seeding requirements of large scaffolds to make implants for potential clinical use include: (a) high yield, to maximize the utilization of donor cells, (b) high kinetic rate, to minimize the time in suspension for anchorage-dependent and shear-sensitive cells, and (c) high and spatially uniform distribution of attached cells, for rapid and uniform tissue regeneration. Highly porous, fibrous polyglycolic acid scaffolds, 5-10 mm in diameter and 2-5 mm thick, were seeded with bovine articular chondrocytes in well-mixed spinner flasks. Essentially, all cells attached throughout the scaffold volume within 1 day. Mixing promoted the formation of 20-32-micron diameter cell aggregates that enhanced the kinetics of cell attachment without compromising the uniformity of cell distribution. The kinetics and possible mechanisms of cell seeding were related to the formation of cell aggregates by a simple mathematical model that can be used to optimize seeding conditions for cartilage tissue engineering.

Animals↗