The major challenge of tissue engineering is directing the cells to establish the physiological structure and function of the tissue being replaced across different hierarchical scales. To engineer myocardium, biophysical regulation of the cells needs to recapitulate multiple signals present in the native heart. We hypothesized that excitation-contraction coupling, critical for the development and function of a normal heart, determines the development and function of engineered myocardium. To induce synchronous contractions of cultured cardiac constructs, we applied electrical signals designed to mimic those in the native heart. Over only 8 days in vitro, electrical field stimulation induced cell alignment and coupling, increased the amplitude of synchronous construct contractions by a factor of 7, and resulted in a remarkable level of ultrastructural organization. Development of conductive and contractile properties of cardiac constructs was concurrent, with strong dependence on the initiation and duration of electrical stimulation.contraction ͉ excitation ͉ tissue engineering ͉ ultrastructure ͉ heart

Tissue engineered grafts may be useful in myocardial repair, however previous scaffolds have been structurally incompatible with recapitulating cardiac anisotropy. Utilizing microfabrication techniques, a novel accordion-like honeycomb microstructure was rendered in poly(glycerol sebacate) to yield porous, elastomeric 3-D scaffolds with controllable stiffness and anisotropy. Accordion-like honeycomb scaffolds with cultured neonatal rat heart cells demonstrated utility via: (1) closely matched mechanical properties compared to native adult rat right ventricular myocardium, with stiffnesses controlled by polymer curing time; (2) heart cell contractility inducible by electric field stimulation with directionally-dependent electrical excitation thresholds (p<0.05); and (3) greater heart cell alignment (p<0.0001) than isotropic control scaffolds. Prototype bilaminar scaffolds with 3-D interconnected pore networks yielded electrically excitable grafts with multi-layered neonatal rat heart cells. Accordion-like honeycombs can thus overcome principal structural-mechanical limitations of previous scaffolds, promoting the formation of grafts with aligned heart cells and mechanical properties more closely resembling native myocardium.

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.

Tissue engineering seeks to repair or regenerate tissues through combinations of implanted cells, biomaterial scaffolds and biologically active molecules. The rapid restoration of tissue biomechanical function remains an important challenge, emphasizing the need to replicate structural and mechanical properties using novel scaffold designs. Here we present a microscale 3D weaving technique to generate anisotropic 3D woven structures as the basis for novel composite scaffolds that are consolidated with a chondrocyte-hydrogel mixture into cartilage tissue constructs. Composite scaffolds show mechanical properties of the same order of magnitude as values for native articular cartilage, as measured by compressive, tensile and shear testing. Moreover, our findings showed that porous composite scaffolds could be engineered with initial properties that reproduce the anisotropy, viscoelasticity and tension-compression nonlinearity of native articular cartilage. Such scaffolds uniquely combine the potential for load-bearing immediately after implantation in vivo with biological support for cell-based tissue regeneration without requiring cultivation in vitro.

Summary: Cartilaginous constructs have been grown in v i f m 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 glycosarninoglycan 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.Articular cartilage derives its form and mechanical function from its matrix. which consists of tissue fluid and a framework of structural macromolecules (collagens, proteoglycans, and noncollagenous proteins and glycoproteins). During the development, maintenance, and remodeling of cartilage, chondrocytes synthesize appropriate types and amounts of macromolecules and assemble thcm into a highly organized matrix (4). Adult articular cartilage has a limited capacity to repair damage resulting from injury or disease, and there have been many different approaches to restore tissue composition, structure, and function. including the development of engineered cartilage for potential implantation (5.27). Cartilaginous constructs have been grown in vitro with use of isolated chondrocytes. biodegradable polymer scaffolds, and bioreactors and implanted in vivo to form subcutaneous cartilage or to promote joint repair (16). Fibrous polyglycolic acid scaffolds permitted chondrocytes to Received April 3,1998; accepted September 30. lYY8. Address correspondence and reprint requests to G. VunjakNovakovic at Massachusetts Institute of Technology E25-342. 45 Carleton Street, Carnbridgc, MA 02139, U.S.A. E-mail: gordana@ rnit.edu maintain their differentiated phenotype and provided a three-dimensional framework for tissue regeneration (14), and bioreactors provided control over the conditions of cell seeding and tissuc cultivation and affectcd construct structures and compositio...

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.

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.

Cardiac tissue engineering has been motivated by the need to create functional tissue equivalents for scientific studies and cardiac tissue repair. We previously demonstrated that contractile cardiac cell–polymer constructs can be cultivated using isolated cells, 3‐dimensional scaffolds, and bioreactors. In the present work, we examined the effects of (1) cell source (neonatal rat or embryonic chick), (2) initial cell seeding density, (3) cell seeding vessel, and (4) tissue culture vessel on the structure and composition of engineered cardiac muscle. Constructs seeded under well‐mixed conditions with rat heart cells at a high initial density ((6–8) × 106 cells/polymer scaffold) maintained structural integrity and contained macroscopic contractile areas (approximately 20 mm2). Seeding in rotating vessels (laminar flow) rather than mixed flasks (turbulent flow) resulted in 23% higher seeding efficiency and 20% less cell damage as assessed by medium lactate dehydrogenase levels (p < 0.05). Advantages of culturing constructs under mixed rather than static conditions included the maintenance of metabolic parameters in physiological ranges, 2–4 times higher construct cellularity (p ≤ 0.0001), more aerobic cell metabolism, and a more physiological, elongated cell shape. Cultivations in rotating bioreactors, in which flow patterns are laminar and dynamic, yielded constructs with a more active, aerobic metabolism as compared to constructs cultured in mixed or static flasks. After 1–2 weeks of cultivation, tissue constructs expressed cardiac specific proteins and ultrastructural features and had approximately 2–6 times lower cellularity (p < 0.05) but similar metabolic activity per unit cell when compared to native cardiac tissue. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 580–589, 1999.