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For centuries scientists and technologists have sought artificial leg replacements that fully capture the versatility of their intact biological counterparts. However, biological gait requires coordinated volitional and reflexive motor control by complex afferent and efferent neural interplay, making its neuroprosthetic emulation challenging after limb amputation. Here we hypothesize that continuous neural control of a bionic limb can restore biomimetic gait after below-knee amputation when residual muscle afferents are augmented. To test this hypothesis, we present a neuroprosthetic interface consisting of surgically connected, agonist–antagonist muscles including muscle-sensing electrodes. In a cohort of seven leg amputees, the interface is shown to augment residual muscle afferents by 18% of biologically intact values. Compared with a matched amputee cohort without the afferent augmentation, the maximum neuroprosthetic walking speed is increased by 41%, enabling equivalent peak speeds to persons without leg amputation. Further, this level of afferent augmentation enables biomimetic adaptation to various walking speeds and real-world environments, including slopes, stairs and obstructed pathways. Our results suggest that even a small augmentation of residual muscle afferents restores biomimetic gait under continuous neuromodulation in individuals with leg amputation.

Tissue-engineered constructs, at the interface of material science, biology, engineering, and medicine, have the capacity to improve outcomes for cardiac patients by providing living cells and degradable biomaterials that can regenerate the native myocardium. With an ultimate goal of both delivering cells and providing mechanical support to the healing heart, we designed three-dimensional (3D) elastomeric scaffolds with (1) stiffnesses and anisotropy mimicking explanted myocardial specimens as predicted by finite-element (FE) modeling, (2) systematically varied combinations of rectangular pore pattern, pore aspect ratio, and strut width, and (3) structural features approaching tissue scale. Based on predicted mechanical properties, three scaffold designs were selected from eight candidates for fabrication from poly(glycerol sebacate) by micromolding from silicon wafers. Large 20×20 mm scaffolds with high aspect ratio features (5:1 strut height:strut width) were reproducibly cast, cured, and demolded at a relatively high throughput. Empirically measured mechanical properties demonstrated that scaffolds were cardiac mimetic and validated FE model predictions. Two-layered scaffolds providing fully interconnected pore networks were fabricated by layer-by-layer assembly. C2C12 myoblasts cultured on one-layered scaffolds exhibited specific patterns of cell elongation and interconnectivity that appeared to be guided by the scaffold pore pattern. Neonatal rat heart cells cultured on two-layered scaffolds for 1 week were contractile, both spontaneously and in response to electrical stimulation, and expressed sarcomeric α-actinin, a cardiac biomarker. This work not only demonstrated several scaffold designs that promoted functional assembly of rat heart cells, but also provided the foundation for further computational and empirical investigations of 3D elastomeric scaffolds for cardiac tissue engineering.

Human mesenchymal stem cells (hMSCs) and three-dimensional (3D) woven poly(ɛ-caprolactone) (PCL) scaffolds are promising tools for skeletal tissue engineering. We hypothesized that in vitroculture duration and medium additives can individually and interactively influence the structure, composition, mechanical, and molecular properties of engineered tissues based on hMSCs and 3D poly(ɛ-caprolactone). Bone marrow hMSCs were suspended in collagen gel, seeded on scaffolds, and cultured for 1, 21, or 45 days under chondrogenic and/or osteogenic conditions. Structure, composition, biomechanics, and gene expression were analyzed. In chondrogenic medium, cartilaginous tissue formed by day 21, and hypertrophic mineralization was observed in the newly formed extracellular matrix at the interface with underlying scaffold by day 45. Glycosaminoglycan, hydroxyproline, and calcium contents, and alkaline phosphatase activity depended on culture duration and medium additives, with significant interactive effects (all p< 0.0001). The 45-day constructs exhibited mechanical properties on the order of magnitude of native articular cartilage (aggregate, Young's, and shear moduli of 0.15, 0.12, and 0.033 MPa, respectively). Gene expression was characteristic of chondrogenesis and endochondral bone formation, with sequential regulation of Sox-9, collagen type II, aggrecan, core binding factor alpha 1 (Cbfα1)/Runx2, bone sialoprotein, bone morphogenetic protein-2, and osteocalcin. In contrast, osteogenic medium produced limited osteogenesis. Long-term culture of hMSC on 3D scaffolds resulted in chondrogenesis and regional mineralization at the interface between soft, newly formed engineered cartilage, and stiffer underlying scaffold. These findings merit consideration when developing grafts for osteochondral defect repair.

Pheochromocytomas are widely believed to induce cardiomyopathy via hypersecretion of catecholamines, including norepinephrine (NE). NE can have direct cardiomyocyte toxicity and/or can stimulate myocardial remodeling secondary to the induction of hypertension. Yet, the development of cardiomyopathy is not entirely related to catecholamine dose or the extent of hypertension. To explore these effects, we engineered a polymeric encapsulation system to control PC12 cell kinetics and NE release in vitroand in vivo. Primary neonatal rat cardiomyocytes incubated with pheochromocytoma-conditioned media exhibited greater cytoskeletal changes than myocytes cultured with identical doses of NE alone, including more profound dose-dependent decreases in desmin, β-tubulin, and vinculin and upregulation of dystrophin. Cardiomyocyte contractility was 29 ± 6% greater at given levels of NE release. Agarose-encapsulated PC12 cells retain cell viability and structural integrity in vivo. These implants induce a 30% greater degree of cardiac enlargement as compared to pumps releasing equivalent doses of NE. Protein level alterations observed in vitrowere mirrored in vivoafter implantation of encapsulated cells or NE pumps for 28 days. Together, these data suggest that pheochromocytoma-induced cardiomyopathy is not solely a catecholamine-mediated event; rather, the pathogenesis of this dilated cardiomyopathy appears to be dependent upon secondary factors unexamined to date.

Biochemical and mechanical signals enabling cardiac regeneration can be elucidated using in vitrotissue-engineering models. We hypothesized that insulin-like growth factor-I (IGF) and slow, bi-directional perfusion could act independently and interactively to enhance the survival, differentiation, and contractile performance of tissue-engineered cardiac grafts. Heart cells were cultured on three-dimensional porous scaffolds in medium with or without supplemental IGF and in the presence or absence of slow, bi-directional perfusion that enhanced transport and provided shear stress. Structural, molecular, and electrophysiologic properties of the resulting grafts were quantified on culture day 8. IGF had independent, beneficial effects on apoptosis (p< 0.01), cellular viability (p< 0.01), contractile amplitude (p< 0.01), and excitation threshold (p< 0.01). Perfusion independently affected the four aforementioned parameters and also increased amounts of cardiac troponin-I (p< 0.01), connexin-43 (p< 0.05), and total protein (p< 0.01) in the grafts. Interactive effects of IGF and perfusion on apoptosis were also present (p< 0.01). Myofibrillogenesis and spontaneous contractility were present only in grafts cultured with perfusion, although contractility was inducible by electrical field stimulation of grafts from all groups. Our findings demonstrate that multi-factorial stimulation of tissue-engineered cardiac grafts using IGF and perfusion resulted in independent and interactive effects on heart cell survival, differentiation, and contractility.

Human mesenchymal stem cells (hMSCs) isolated from bone marrow aspirates were cultured on silk scaffolds in rotating bioreactors for three weeks with either chondrogenic or osteogenic medium supplements to engineer cartilage- or bone-like tissue constructs. Osteochondral composites formed from these cartilage and bone constructs were cultured for an additional three weeks in culture medium that was supplemented with chondrogenic factors, supplemented with osteogenic factors or unsupplemented. Progression of cartilage and bone formation and the integration between the two regions were assessed by medical imaging (magnetic resonance imaging and micro-computerized tomography imaging), and by biochemical, histological and mechanical assays. During composite culture (three to six weeks), bone-like tissue formation progressed in all three media to a markedly larger extent than cartilage-like tissue formation. The integration of the constructs was most enhanced in composites cultured in chondrogenic medium. The results suggest that tissue composites with well-mineralized regions and substantially less developed cartilage regions can be generated in vitroby culturing hMSCs on silk scaffolds in bioreactors, that hMSCs have markedly higher capacity for producing engineered bone than engineered cartilage, and that chondrogenic factors play major roles at early stages of bone formation by hMSCs and in the integration of the two tissue constructs into a tissue composite.

This article contains the collective views expressed at the second session of the workshop "Tissue Engineering—The Next Generation,'' which was devoted to the tools of tissue engineering: scaffolds, bioreactors, and molecular and physical signaling. Lisa E. Freed and Farshid Guilak discussed the integrated use of scaffolds and bioreactors as tools to accelerate and control tissue regeneration, in the context of engineering mechanically functional cartilage and cardiac muscle. Edward Guo focused on the opportunities that tissue engineering generates for studies of mechanobiology and on the need for tissue engineers to learn about mechanical forces during tissue and organ genesis. Martha L. Gray focused on the potential of biomedical imaging for noninvasive monitoring of engineered tissues and on the opportunities biomedical imaging can generate for the development of new markers. Robert Tranquillo reviewed the approach to tissue engineering of a spectrum of avascular habitually loaded tissues— blood vessels, heart valves, ligaments, tendons, cartilage, and skin. Jeffrey W. Holmes offered the perspective of a "reverse paradigm''—the use of tissue constructs in quantitative studies of cell-matrix interactions, cell mechanics, matrix mechanics, and mechanobiology. Milica Radisic discussed biomimetic design of tissue-engineering systems, on the example of synchronously contractile cardiac muscle. Michael V. Sefton proposed a new, simple approach to the vascularization of engineered tissues. This session stressed the need for advanced scaffolds, bioreactors, and imaging technologies and offered many enlightening examples on how these advanced tools can be utilized for functional tissue engineering and basic research in medicine and biology.

We report that the functional assembly of engineered cardiac muscle can be enhanced by oxygen supply provided by mechanisms resembling those in normal vascularized tissues. To mimic the capillary network, cardiomyocytes and fibroblasts isolated from the neonatal rat hearts were cultured on a highly porous elastomer with a parallel array of channels that were perfused with culture medium. To mimic oxygen supply by hemoglobin, culture medium was supplemented with a perfluorocarbon (PFC) emulsion; constructs perfused with unsupplemented culture medium served as controls. In PFC-supplemented medium, the decrease in the partial pressure of oxygen in the aqueous phase was only 50% of that in control medium (28mmHg vs. 45mmHg between the construct inlet and outlet at a flow rate of 0.1mL/ min). Consistently, constructs cultivated in the presence of PFC contained higher amounts of DNA and cardiac markers (troponin I, connexin-43) and had significantly better contractile properties as compared to control constructs. In both groups, electron microscopy revealed open channels and the presence of cells at the channel surfaces as well as within constructs. Improved properties of cardiac constructs could be correlated with the enhanced supply of oxygen to the cells, by a combined use of channeled scaffolds and PFC.

Hybrid cardiac constructs with mechanical properties suitable for in vitro loading studies and in vivo implantation were constructed from neonatal rat heart cells, fibrin (Fn), and biodegradable knitted fabric (Knit). Initial (2-h) constructs were compared with native heart tissue, studied in vitro with respect to mechanical function (stiffness, ultimate tensile strength [UTS], failure strain εf, strain energy density E) and compositional remodeling (collagen, DNA), and implanted in vivo. For 2-h constructs, stiffness was determined mainly by the Fn and was half as high as that of native heart, whereas UTS, εf, and E were determined by the Knit and were, respectively, 8-, 7-, and 30-fold higher than native heart. Over 1 week of static in vitro culture, cell-mediated, serum-dependent remodeling was demonstrated by a 5-fold increase in construct collagen content and maintenance of stiffness not observed in cell-free constructs. Cyclic stretch further increased construct collagen content in a manner dependent on loading regimen. The presence of cardiac cells in cultured constructs was demonstrated by immunohistochemistry (troponin I) and Western blot (connexin 43). However, in vitro culture reduced Knit mechanical properties, decreasing UTS, εf, and E of both constructs and cell-free constructs and motivating in vivo study of the 2-h constructs. Constructs implanted subcutaneously in nude rats for 3 weeks exhibited the continued presence of cardiomyocytes and blood vessel ingrowth by immunostaining for troponin I, connexin 43, and CD-31. Together, the data showed that hybrid cardiac constructs initially exhibited supraphysiologic UTS, εf, and E, and remodeled in response to serum and stretch in vitro and in an ectopic in vivo model.

We tested the hypothesis that cardiomyocytes maintained their phenotype better if cultured as three-dimensional tissue constructs than if cultured as confluent monolayers. Neonatal rat cardiomyocytes were cultured on biomaterial scaffolds in rotating bioreactors for 1 week, and resulting tissue constructs were compared with confluent monolayers and slices of native ventricular tissue with respect to proteins involved in cell metabolism (creatine kinase isoform MM), contractile function (sarcomeric myosin heavy chain), and intercellular communication (connexin 43), as well as action potential characteristics (e.g., membrane resting potential, maximum depolarization slope, and action potential duration), and macroscopic electrophysiological properties (maximum capture rate). The molecular and electrophysiological properties of cardiomyocytes cultured in tissue constructs, although inferior to those of native neonatal ventricles, were superior to those of the same cells cultured as monolayers. Construct levels of creatine kinase, myosin heavy chain, and connexin 43 were 40-60% as high as ventricle levels, whereas monolayer levels of the same proteins were only 11-20% as high. Construct action potential durations were 1.8-fold higher than those in ventricles, whereas monolayer action potential durations were 2.4-fold higher. Pharmacological studies using 4-aminopyridine showed that prolonged action potential duration and reduced maximum capture rate in tissue constructs as compared with native ventricles could be explained by decreased transient outward potassium current.

Controlled in vitrostudies of cells and tissues under the conditions of microgravity (simulated on Earth, or actual in space) can improve our understanding of gravity sensing, transduction, and responses in living cells and tissues. This paper discusses the scientific results and practical implications of three NASA-related biotechnology projects: ground and space studies of microgravity tissue engineering (JSC-Houston), and the development of the cell culture unit for use aboard the International Space Station (ARC-Ames).

We hypothesized that the mechanically active environment present in rotating bioreactors mediates the effectiveness of three‐dimensional (3D) scaffolds for cartilage tissue engineering. Cartilaginous constructs were engineered by using bovine calf chondrocytes in conjunction with two scaffold materials (SM) (benzylated hyaluronan and polyglycolic acid); three scaffold structures (SS) (sponge, non‐woven mesh, and composite woven/non‐woven mesh); and two culture systems (CS) (a bioreactor system and petri dishes). Construct size, composition [cells, glycosaminoglycans (GAG), total collagen, and type‐specific collagen mRNA expression and protein levels], and mechanical function (compressive modulus) were assessed, and individual and interactive effects of model system parameters (SM, SS, CS, SM∗CS and SS∗CS) were demonstrated. The CS affected cell seeding (higher yields of more spatially uniform cells were obtained in bioreactor‐grown than dish‐grown 3‐day constructs) and subsequently affected chondrogenesis (higher cell numbers, wet weights, wet weight GAG fractions, and collagen type II levels were obtained in bioreactor‐grown than dish‐grown 1‐month constructs). In bioreactors, mesh‐based scaffolds yielded 1‐month constructs with lower type I collagen levels and four‐fold higher compressive moduli than corresponding sponge‐based scaffolds. The data imply that interactions between bioreactors and 3D tissue engineering scaffolds can be utilized to improve the structure, function, and molecular properties of in vitro‐generated cartilage.

To test the hypothesis that engineered cartilage can provide a mechanically functional template capable of undergoing orderly remodeling during the repair of large osteochondral defects in adult rabbits, as assessed by quantitative structural and functional methods. Engineered cartilage generated in vitro from chondrocytes cultured on a biodegradable scaffold was sutured to a subchondral support and the resulting composite press-fitted into a 7-mm long, 5-mm wide, 5-mm deep osteochondral defect in a rabbit knee joint. Defects left empty (group 1) or treated with cell-free composites (group 2) served as controls for defects treated with composites of engineered cartilage and the support, without or with adsorbed bone marrow (groups 3 and 4, respectively). Engineered cartilage withstood physiologic loading and remodeled over 6 months into osteochondral tissue with characteristic architectural features and physiologic Young's moduli. Composites integrated well with host bone in 90% of cases but did not integrate well with host cartilage. Structurally, 6-month repairs in groups 3 and 4 were superior to those in group 2 with respect to histologic score, cartilage thickness, and thickness uniformity, but were inferior to those in unoperated control tissue. At 6 months, Young's moduli in groups 2, 3, and 4 (0.68, 0.80, and 0.79 MPa, respectively) approached that in unoperated control tissue (0.84 MPa), whereas the corresponding modulus in group 1 (0.37 MPa) was significantly lower. Composites of tissue-engineered cartilage and a subchondral support promote the orderly remodeling of large osteochondral defects in adult rabbits.

The repair of articular cartilage lesions remains a clinical problem. Two novel approaches to cartilage formation, gene transfer and tissue engineering, have been limited by short-term transgene expression in transplanted chondrocytes and inability to deliver regulatory signals to engineered tissues according to specific temporal and spatial patterns. We tested the hypothesis that the transfer of a cDNA encoding the human insulin-like growth factor I (IGF-I) can provide sustained gene expression in cell-polymer constructs in vitro and in vivo and enhance the structural and functional properties of tissue-engineered cartilage. Bovine articular chondrocytes genetically modified to overexpress human IGF-I were seeded into polymer scaffolds, cultured in bioreactors in serum-free medium, and implanted subcutaneously in nude mice; constructs based on nontransfected or lacZ-transfected chondrocytes served as controls. Transgene expression was maintained throughout the duration of the study, more than 4 weeks in vitro followed by an additional 10 days either in vitro or in vivo. Chondrogenesis progressed toward the formation of cartilaginous tissue that was characterized by the presence of glycosaminoglycans, aggrecan, and type II collagen, and the absence of type I collagen. IGF-I constructs contained increased amounts of glycosaminoglycans and collagen and confined-compression equilibrium moduli as compared with controls; all groups had subnormal cellularity. The amounts of glycosaminoglycans and collagen per unit DNA in IGF-I constructs were markedly higher than in constructs cultured in serum-supplemented medium or native cartilage. This enhancement of chondrogenesis by spatially defined overexpression of human IGF-I suggests that cartilage tissue engineering based on genetically modified chondrocytes may be advantageous as compared with either gene transfer or tissue engineering alone.

Concentration gradients associated with the in vitro cultivation of engineered tissues that are vascularized in vivo result in the formation of only a thin peripheral tissue-like region (e.g., ~100 μm for engineered cardiac muscle) around a relatively cell-free interior. We previously demonstrated that diffusional gradients within engineered cardiac constructs can be minimized by direct perfusion of culture medium through the construct. In the present study, we measured the effects of medium perfusion rate and local oxygen concentration (pO2) on the in vitro reconstruction of engineered cardiac muscle. Neonatal rat cardiomyocytes were seeded onto biodegradable polymer scaffolds (fibrous discs, 1.1 cm diameter × 2 mm thick, made of polyglycolic acid, 24 × 10⁶ cells per scaffold). The resulting cell-polymer constructs were cultured for a total of 12 days in serially connected cartridges (n = 1–8), each containing one construct directly perfused with culture medium at a flow rate of 0.2–3.0 mL/min. In all groups, oxygen concentration decreased due to cell respiration, and depended on construct position in the series and medium flow rate. Higher perfusion rates and higher pO2 correlated with more aerobic cell metabolism, and higher DNA and protein contents. Constructs cultured at pO2 of 160 mm Hg had 50% higher DNA and protein contents, markedly higher expression of sarcomeric α-actin, better organized sarcomeres and cell junctions, and 4.5-fold higher rate of cell respiration as compared to constructs cultured at pO2 of 60 mm Hg. Contraction rates of the corresponding cardiac cell monolayers were 40% higher at pO2 of 160 than 60 mm Hg. The control of oxygen concentration in cell microenvironment can thus improve the structure and function of engineered cardiac muscle. Experiments of this kind can form a basis for controlled studies of the effects of oxygen on the in vitro development of engineered tissues. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 617–625, 2002.

Cardiac muscle with a certain threshold thickness, uniformity of tissue architecture, and functionality would expand the therapeutic options currently available to patients with congenital or acquired cardiac defects. Cardiac constructs cultured in well-mixed medium had an approximately 100-μm-thick peripheral tissue-like region around a relatively cell-free interior, a structure consistent with the presence of concentration gradients within the tissue. We hypothesized that direct perfusion of cultured constructs can reduce diffusional distances for mass transport, improve control of oxygen, pH, nutrients and metabolites in the cell microenvironment, and thereby increase the thickness and spatial uniformity of engineered cardiac muscle. To test this hypothesis, constructs (9.5-mm-diameter, 2-mm-thick discs) based on neonatal rat cardiac myocytes and fibrous polyglycolic acid scaffolds were cultured either directly perfused with medium or in control spinner flasks. Perfusion improved the spatial uniformity of cell distribution and enhanced the expression of cardiac-specific markers, presumably due to the improved control of local microenvironmental conditions within the forming tissue. Medium perfusion could thus be utilized to better mimic the transport conditions within native cardiac muscle and enable in vitro engineering of cardiac constructs with clinically useful thicknesses.

The effects of four regulatory factors on tissue-engineered cartilage were examined with specific focus on the ability to increase construct growth rate and concentrations of glycosaminoglycans (GAG) and collagen, the major extracellular matrix (ECM) components. Bovine calf articular chondrocytes were seeded onto biodegradable polyglycolic acid (PGA) scaffolds and cultured in medium with or without supplemental insulin-like growth factor (IGF-I), interleukin-4 (IL-4), transforming growth factor-β1 (TGF-β1) or platelet-derived growth factor (PDGF). IGF-I, IL-4, and TGF-β1 increased construct wet weights by 1.5-2.9-fold over 4 weeks of culture and increased amounts of cartilaginous ECM components. IGF-I (10-300 ng/mL) maintained wet weight fractions of GAG in constructs seeded at high cell density and increased by up to fivefold GAG fractions in constructs seeded at lower cell density. TGF-β1 (30 ng/mL) increased wet weight fractions of total collagen by up to 1.4-fold while maintaining a high fraction of type II collagen (79 ± 11% of the total collagen). IL-4 (1-100 ng/mL) minimized the thickness of the GAG-depleted region at the construct surfaces. PDGF (1-100 ng/mL) decreased construct growth rate and ECM fractions. Different regulatory factors thus elicit significantly different chondrogenic responses and can be used to selectively control the growth rate and improve the composition of engineered cartilage.

Bone marrow stromal cells BMSC are pluripotent progenitor cells that can regenerate different skeletal tissues in response to environmental signals. In this study, we used highly porous, structurally stable threedimensional polymer foams in conjunction with specific regulatory molecules to selectively differentiate mammalian BMSC into either cartilaginous or bonelike tissues. Bovine BMSC were expanded in monolayers and cultured on 5mmdiameter, 2mmthick foams made of polylacticcoglycolic acid and polyethylene glycol. Constructs maintained their original size and shape for up to 4 weeks of culture and supported BMSC growth and production of extracellular matrix ECM. By proper use of chondrogenic dexamethasone, insulin, transforming growth factorβ1 or osteogenic dexamethasone, βglycerophosphate medium supplements, we could control whether the generated ECM was cartilaginous containing collagen type II and sulfated glycosaminoglycans or bonelike containing osteocalcin, osteonectin, and mineralized foci. After 4 weeks of cultivation, cartilaginous and bonelike ECM were uniformly distributed throughout the construct volume and respectively represented 34.2 ± 9.3 and 12.6 ± 3.2 of the total available area. BMSC culture on polylacticcoglycolic acidpolyethylene glycol foams provides a threedimensional model system to study the development of mesenchymal tissues in vitroand has potential applications in engineering autologous grafts for skeletal tissue repair. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 55: 229–235, 2001

Functional cartilaginous constructs for scientific research and eventual tissue repair were cultivated in bioreactors starting from chondrocytes immobilized on polymeric scaffolds. The scaffolds gradually degraded as the cells regenerated tissue matrix consisting of glycosaminoglycan (GAG) and type II collagen. To facilitate data interpretation and optimize cultivation conditions, a mathematical model was developed which yields the concentrations of oxygen and GAG as functions of time and position in growing tissue. Calculated GAG concentrations were qualitatively and quantitatively consistent with profiles measured via high‐resolution image processing of tissue samples cultured at two different oxygen tensions for various periods of time.

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 &lt; 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 &lt; 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.

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 × 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 (~40 mmHg) and low pH (~6.7) were associated with anaerobic cell metabolism (yield of lactate on glucose, YL/G, of 2.2 mol/mol) while higher oxygen tension (~80 mmHg) and higher pH (~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. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 197–205, 1999.

This paper discusses a novel approach to tissue engineering involving seeding mammalian cells onto biodegradable polymer scaffolds, growing the cells on these scaffoldsin vitro, and then implanting the cell-polymer constructs in vivo. Approaches for in vitrocultivation of cells are first discussed. The types of polymer scaffolds that are utilized and the kinds of bioreactor conditions for growing cells are then delineated. The application of this approach in vitro and in vivofor producing cartilage, tendon, liver, urothelial tissue, intestine, and bone are then examined.

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‐μm 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.

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 μm 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°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 × 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.

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 vitrodifferentiation 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 vitrosystem studied, the same population could be selectively induced to regenerate either cartilaginous or bonelike tissue.

A heat-stable microparticle platform improves oral micronutrient delivery.