Biomedical subjects

L E Freed

Publications and source records attributed to L E Freed.

18 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

Joint resurfacing using allograft chondrocytes and synthetic biodegradable polymer scaffolds.

Cartilage implants which could potentially be used to resurface damaged joints were created using rabbit articular chondrocytes and synthetic, biodegradable polymer scaffolds. Cells were serially passaged and then cultured in vitro on fibrous polyglycolic acid (PGA) scaffolds. Cell-PGA constructs were implanted in vivo as allografts to repair 3-mm diameter, full thickness defects in the knee joints of adult rabbits, and cartilage repair was assessed histologically over 6 months. In vitro, chondrocytes proliferated on PGA and regenerated cartilaginous matrix. Collagen and glycosaminoglycan (GAG) represented 20 to 8% of the implant dry weight (dw), respectively, at the time of in vivo implantation; the remainder was PGA and unspecified components. Implants based on passaged chondrocytes had 1.7-times as much GAG and 2.6-times as much collagen as those based on primary chondrocytes. In vivo, cartilaginous repair tissue was observed after implantation of PGA both with and without cultured chondrocytes. Six month repair was qualitatively better for cell-PGA allografts than for PGA alone, with respect to: 1) surface smoothness, 2) columnar alignment of chondrocytes, 3) spatially uniform GAG distribution, 4) reconstitution of the subchondral plate, and 5) bonding of the repair tissue to the underlying bone. These pilot studies demonstrate that it is feasible to use cell-polymer allografts for joint resurfacing in vivo.

Animals↗

PubMed1994

Wetting of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) foams for tissue culture.

Biodegradable foams of hydrophobic polymers can be efficiently wet by two-step immersion in ethanol and water, which overcomes the hindered entry of water into air-filled pores. Ethanol readily enters into the porous polymer, after which it is diluted and replaced by water. This method was evaluated for porous disks of poly(L-lactic acid) (PLLA) and poly(DL-lactic-co-glycolic acid) (PLGA) foams of copolymer ratios 85:15 and 50:50. For PLLA disks of 0.88 porosity and 1730 microns thickness, prewetting with ethanol for 1 h increased the percentage of void volume filled with water after 48 h from 23 to 79%. The same enhanced entry of water was also observed for prewet PLGA 85:15 disks of 0.86 porosity and 1300 microns thickness, which exhibited an increase from 59 to 97% void volume occupied by water. Furthermore, the water entry even after 1 h was very close to its plateau value for all prewet polymers tested. In recent studies, this method has been useful in uniformly seeding three-dimensional biodegradable polymer substrates for cell and tissue culture.

Culture Techniques↗

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

Neocartilage formation in vitro and in vivo using cells cultured on synthetic biodegradable polymers.

Cartilaginous implants for potential use in reconstructive or orthopedic surgery were created using chondrocytes grown on synthetic, biodegradable polymer scaffolds. Chondrocytes isolated from bovine or human articular or costal cartilage were cultured on fibrous polyglycolic acid (PGA) and porous poly(L)lactic acid (PLLA) and used in parallel in vitro and in vivo studies. Samples were taken at timed intervals for assessment of cell number and cartilage matrix (sulfated glycosaminoglycan [S-GAG], collagen). The chondrocytes secreted cartilage matrix to fill the void spaces in the polymer scaffolds that were simultaneously biodegrading. In vitro, chondrocytes grown on PGA for 6 weeks reached a cell density of 5.2 x 10(7) cells/g, which was 8.3-fold higher than at day 1, and equalled the cellularity of normal bovine articular cartilage. In vitro, the cell growth rate was approximately twice as high on PGA as it was on PLLA; cells grown on PGA produced S-GAG at a high steady rate, while cells grown on PLLA produced only minimal amounts of S-GAG. These differences could be attributed to polymer geometry and biodegradation rate. In vivo, chondrocytes grown on both PGA and PLLA for 1-6 months maintained the three-dimensional (3-D) shapes of the original polymer scaffolds, appeared glistening white macroscopically, contained S-GAG and type II collagen, and closely resembled cartilage histologically. These studies demonstrate the feasibility of culturing isolated chondrocytes on biodegradable polymer scaffolds to regenerate 3-D neocartilage.

Animals↗

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↗

PubMed1993

Bioreactor based on suspended particles of immobilized enzyme.

A bioreactor for blood detoxification was developed in which oscillation-induced secondary flows suspend particles of immobilized enzyme in a reactor operating at clinically useful flowrates. Torsional oscillation of the reactor about its axis created a pair of counterrotating toroidal vortices which were readily observed in flow-visualization studies. Oscillation frequencies were selected to provide spatially uniform particle dispersion, as assessed visually. As a model system, blood deheparinization by reactors containing heparinase immobilized to agarose particles was investigated. Identical deheparinization profiles were observed in the continuous-flow reactor and in independent batch studies, done in well mixed test tubes of blood, demonstrating that the oscillating reactor design minimizes external mass transfer limitations. Identical heparin neutralization profiles and rates were also observed in the first and the second of consecutive heparin neutralization studies (0-2 h and 2-4 h, respectively) demonstrating an effective half-life of the immobilized enzyme in the oscillating reactor of at least 4 h. No significant decrease in red or white blood cell count, platelet count, or hematocrit, and clinically acceptable levels of plasma hemoglobin and activated complement were observed with 2 h (20 passes) of in vitro recirculation of human blood through the reactor. High, stable efficacy, operational stability, and excellent biocompatibility are attributed to secondary flow induced liquid-particle mixing within the oscillating reactor.

Biocompatible Materials↗

PubMed1993

Kinetics of immobilized heparinase in human blood.

Immobilized enzyme reactors can form the basis of useful blood detoxification systems. One such reactor was developed for heparin neutralization by immobilized heparinase. In this article, reactor kinetics were studied under clinically relevant conditions. Heparin neutralization was assessed in vitro in whole human blood using (a) a well-mixed batch reactor, and (b) an oscillating, continuous-flow reactor. The kinetics of heparin neutralization in human blood were first order over the entire range of heparin and enzyme concentrations and particle fractions tested. The kinetic rate was not sensitive to physiological variations in the concentration of antithrombin, a heparin binding protein in blood. Enzyme activity did not decrease significantly over the 2 hour test period. Kinetic control of the system with minimal intraparticle diffusional limitations was suggested by the Thiele moduli (0.11-0.67) and effectiveness factors (0.98 +/- 0.01). The ratio kcat/Km obtained in batch studies was 0.0028 +/- 0.0008 cm3/microgram-min. A continuous-flow oscillating reactor within a closed recirculation loop performed as a single well mixed batch reactor; there was a short mixing time of recirculating blood when compared to reaction time. A model based on this mixing pattern and the kinetics obtained in independent batch studies accurately predicted heparin neutralization profiles observed in the continuous-flow system.

Antithrombins↗

PubMed1988

Lipogenesis from ketone bodies in perfused livers from streptozocin-induced diabetic rats.

Production of ketone bodies and their contribution to lipogenesis were measured in isolated livers from normal and streptozocin-induced diabetic (STZ-D) rats perfused with tracer amounts of 3H2O and (R)-3-hydroxy[3-14C]butyrate. Diabetes decreased by 80-95% the total rates of fatty acid and 3-beta-hydroxysterol synthesis in perfused livers and livers of live rats. The activity of cytosolic acetoacetyl-CoA synthetase was slightly (17%) decreased in livers from STZ-D rats. The incorporation of ketone bodies into fatty acids and sterols was markedly inhibited in perfused livers from STZ-D rats despite the stimulation of ketogenesis by diabetes and the presence of oleate. Treatment of the rats with insulin before liver perfusion led to a normalization of the rates of ketogenesis and fatty acid synthesis. The rates of sterol synthesis were only partially normalized by insulin treatment. We conclude that in STZ-D, ketosis does not stimulate hepatic lipogenesis via cytosolic activation of acetoacetate.

3-Hydroxybutyric Acid↗

PubMed1982

Metabolism of plasma mevalonate in rats and humans.

A circadian rhythm in plasma mevalonate was identified in human subjects. This variation, over a 5-fold range, is paralleled by a rhythm in urinary excretion. No such diurnal change in plasma mevalonate was observed in schedule-fed, light-cycled rats, despite the presence of a pronounced rhythm in liver HMG-Coa reductase and sterol synthesis. A linear correlation was found between liver HMG-CoA reductase activity and the rate of hepatic sterol synthesis. Sterol synthesis accounted for 59% of the HMG-CoA reductase activity. A 4-fold increase in plasma mevalonate following bilateral nephrectomy did not feed back on liver HMG-CoA reductase. Turnover rates for circulating R- and S-mevalonate were determined by the kinetics of tritiated tracers. S-Mevalonate exhibited first-order kinetics with a T 1/2 of 19 to 23 min, while R-mevalonate kinetics could be resolved into two phases with half-lives of 9 and 42 min. The renal uptake of circulating mevalonate was measured by the initial rate of increase in plasma mevalonate immediately following bilateral nephrectomy; this was confirmed by determination of the renal arterio-venous difference. This value ranges between 500 and 600 pmol/min for a 250-g rat.

Adult↗

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↗

PubMedDate not supplied

A novel bioreactor based on suspended particles of agarose-immobilized species.

Bioreactors often contain porous particles of agarose because these provide an enormous surface area (50 m2/cc gel) onto which enzymes or antibodies can be immobilized. Although many investigators claim that contact with agarose induces significant blood damage, we find that the biocompatibility of immobilized agarose is significantly improved when a novel system is used to fluidize the particles within the bioreactor vessel. We have built a prototype device that is oscillated vigorously about the axis of fluid flow. This action produces secondary flow patterns within the vessel that suspend the particles. In our model system, the bioreactor contains agarose immobilized heparinase. The system is biocompatible for 2 hours in vitro (in human blood at 37 degrees C) as follows: 1) hematocrit, white cell, and platelet counts do not change, 2) levels of plasma hemoglobin increase to 15-34 mg/dl, and 3) levels of complement component C3a increase to 0.64-1.4 micrograms/cc. We hope these studies lead to the development of a heparin removal system that can improve the safety of a variety of extracorporeal procedures. In addition, the techniques and approach used are sufficiently general to permit their extension to any immobilized species bioreactor for blood detoxification.

Biocompatible Materials↗