Tissue ENGINEERING BIoREACTORS Lisa E, Freed and Gordana Vunjak-Novakovie INTRODUCTION ne approach to tissue engineering isto create an in vitro environment that embodies the bio- chemical and mechanical signals that regulate tissue development and maintenance in viv. Figure 13.1 shows our in vir tissue engincering system, which has three major components (Freed and Vanjak-Novakovic, 1998): (1) metabolically ative cells able to express their differentiated phenotype, (2) polymeric scaffolds that provide a three-dimensional (3D) structure for cell at- tachment and tissue growth, and (3) bioreactor culture vessels that provide an in vitro envicon- ‘ment in which cell-polymer constructs can develop into functional rssues. These constructs can potentially be used én ito, for controlled studies of tissue growth and function, of in vive, for tis- sue repair. As compared to in vivo implantation of dissociated cells and/or biodegradable mateti- als alone, the implantation of a Functional engineered tissue can improve the localization of cell delivery and promote graf fixation and survival. In addition, in vitro bioreactor studies can be de- signed to distinguish the eects of specific biochemical and physical signals from the complex in vo milieu (eg. host endocrinologic and immunologic responses) thus providing useful, com- plementary information regarding the process of the formation of 3D tissues starting from isolat- edcalls In this chapter, we focus on tssue engineering bioreactors, which will be defined asin vito caleure systems designed to perform a last one ofthe following four functions: (1) establish spa- tially uniform cell disteibutions on 3D scaffolds, (2) maintain desired concentrations of gases and ‘usrients inthe culture medium, (3) provide efficient mass transfer to the growing tissue, and (4) expose developing tissues to physical stimuli. We furcher focus this chapter on engineered tissues based on biodegradable cell culture templates, although bioreactors are also being utilized in con- junction with nondegradabl cll culture substrates (e-., microcariers, hollow fibers, membranes) ‘0 study the assembly of cells into 3D structures (eg, Jessup e¢ al, 1993; Unsworth and Lelkes, 1998; Qiu eal, 1999) and to develop hybrid bioartificial organs (., Jauregui etal, 1997; Lan- waand Chick, 1997; Bader etal, 1995). ‘The dimensional and functional requirements of the tssue to be engineered determine the specific design requirements for the cell-polymer-bioreactor system. In this chapter, we will use ‘wo examples of engineered tissues, cartilage and cardiac muscle, to illustrate general and tssue- specific principles of bioreactor design and operation. CELL-POLYMER CONSTRUCTS Call sypes studied using our model system (Fig. 13.1) were obtained from cartilage, bone mar- ‘ow, or cardiac muscle of developmentally immature animals. In particular, we have studied artic- tar chondrocytes from 2- to 4-week-old bovine calves (Freed etal, 1994b) and 2- to 8-month- old rabbits (Freed et al, 1994), bone marrow stromal cells from 16-day-old embryonic chicks (Martin er ad, 1998) and 2- to 4-week-old bovine calves (Martin etal, 19992), and cardiac cells from 14-day-old embryonic chicks and 1- to 2-day-old neonatal rats (Carrier eral, 1999). Calls Bice cnet “020 Awa Po Aight eee ym CHAPTER 13 143146 Fig. 13.1, Tissue engineering system. Cells. isolated from Cartilage or cardiac tissue) are seeded onto three-dimensional polymer scaffolds (e.g, fibrous meshes) using bioreactors (eg, ‘spinner flasks, rotating vessels, Or perfused cartridges). The re- sulting. engineered tissue con- structs can be used either for in vitro research or in vivo implan- tation (Freed and Vunjak-Nova- kkovic, 1998). Freed and Vunjak-Novakovic Cells In Vitro Studies 0 — | > Caltvaton Polymer Scaffold 7 Implantation _were isolated by enzymatic digestion, in some cases serially expanded in monolayers, and inocu- lated into bioreactors containing 3D polymer scaffolds. “The polymer scaffold we have best characterized and most used ro date is made of polyely- colicacid (PGA), processed into 297% porous. nonwoven mesh of fibers 13 um in diameter (Freed tal, 19940). The average distance berween the fibers was calculated to be 62, wm (Vunjak-No- vakovic etal, 1998). This scaffold decreases its mass by 50 of 70% after 4 or 8 weeks of culiva- tion, respectively (Freed etal, 1994c). In this chapter, we consider our “scaffold” ro bea fibrous PGA disk with a diameter of 5 mm and a thickness of 2mm. Seructural assessments of engincered tissues include (1) presence and spatial arrangement of cellsand extracellular mattix (ECM), determined histologically, immunohistochemically, and by tleetron microscopy (e.g. Riesle er al, 1998; Cartier etal, 1999), (2) quantitative distributions ‘of specific components, determined by computer-based image analysis of histological sections (Martin et a, 1999b; Vunjak-Novakovic er al, 1998), (3) cell numbes, determined from the amount of DNA (Kim eal, 1988), (4) sulfated glycosaminoglycan (GAG) content, determined by dimethylmethylene blue dye binding (Farndale etal, 1986), (5) total protein content, deter mined by a Biorad protein assay kit (Career eal, 1999), (6 sta collagen content, determined from hydroxyproline (Woessner, 1961), (7) typeI collagen content, determined by inhibition en- zymelinked immunosorbent assay (ELISA) (Freed ct al, 1998), and (8) amounts of type IX col- lagen, creatine kinase, and sarcomeric myosin, determined by densitomenic analysis of Western blots (Rice eal, 1998; Papadaki er a, 1999). Functional assessments of engincered tissues include (1) cell metabolism, assesed from ghi- cose consumption and lactate production (Obradovic etal, 1999) and fiom enzymatic conver- Sion of 3-(45-dimethylthiazol-2-y))-2,5-diphenyl tetrazolium bromide (MTT) (Caster et al, 1999), (2) call damage, assessed from medium concentrations of lactate dehydrogenase (LDH) (Carrier et al, 1999), (3) ECM synthesis rates, determined by macromolecular incorporation of radiolabeled tracers (Freed etal, 1998), (4) cartilage mechanical properties (equilibrium modu- tus, dynamic stiffness, hydraulic permeability, streaming potential), determined in static and dy- ‘namie radially confined compression (Vanjak-Novakovic eal, 1999a), and (5) cardiac tissue elec- ‘tophysiological properties (conduction velocity signal amplitude, excitation threshold, maximum ‘apture rat), assessed from macroscopic impulse propagation studies using an array ofexracellu- lar electrodes (Bursac etal, 1999). BIOREACTOR TECHNOLOGIES (Cou SEEDING all seeding of 3D scaffolds i the first step of bioreactor cultivation of engineered tissues Seeding requirement include (1) high yield, to maximize cell ueiliztion, (2) high kinetic rate, 0 minimize the time in suspension for anchorage-dependent and shear-senstive cells, and (3) high and spatially uniform distribucion of attached cells for rapid and uniform tissue growth, Th the ease of engineered cartilage, these requirements are best met using mixed flasks (Fig-145 Fig. 13.2. Dynamic cell seeding. Schematic: mixed flask system used to sced cells ‘onto 3D polymer scaffolds. Graph: normalized concentration of cells remaining in the ‘culture medium as a function of time following flask inoculation (Reprinted with ‘permission from Vunjak.Novakovic et al, 1998. Copyright 1998 American Chemical > os ‘Tine Society. 13.2) containing scaffolds threaded onto needles embedded inthe flask stopper (up to 12 scaffolds ‘on four needles per flask). Isolated cells and culture medium are added (120 ml medium, 5 X 108 calls per scaffold) and the fas is magnetically sired at 50~80 spn. Under these conditions, es- sentially all cls atach throughout the scaffold volume in less than 24 br (Fig. 13.2) and main- tain their spherical shape (Vanjak-Novakovic eral, 1998). In chis system, magnetic string main- ‘tains a uniform cell suspension and provides relative velocity between the cells and the scaffolds at an average intensity of turbulence below that causing cell damage or detachment (Cherry and Pa- poutsakis, 1988). The kinetics and possible mechanisms of cell seeding were related to the forma- tion of cell aggregates using a simple mathematical model, which can be sed to optimize sceding conditions for specifi scaffold sizes and cell seeding densities (VunjakeNovakovic etal, 1998). ‘The above cel seeding requirements can also be met using rotating vessels. These bioreactors ae filled with culture medium, to which prewetted scaffolds and cells are added, and the vessel is rotated a a solid body in a horizontal plane at 1215 rpm (Freed and Vanjak-Novakovic, 1995, 19972). Cardiac constructs seeded in rotating vesels had higher metabolic acivty indexes than those seeded in spinner asks, implying that optimal hydrodynamic conditions fr cell ceding de- pend on call rype (Carrier eral, 1999). Tissue Courivation Approximately 1~3 days after seeding, clpolymer constructs are transferred into a variety of cultivation vessels, including static and mixed flasks, and rotating and perfused vessels, as de- picted in Fig. 13.3. The flask system contains 120 ml of culture medium and up o 12 tissue tis- sue constructs that are fixed in place (Fig. 13.3) Flasks cither are operate statically or are mixed at 50-80 rpm using a 4-cm-long magnetic sti bar. Medium is exchanged batchwrse ata rate of 5096 crety 2-3 days or 3 ml per construct per day), whereas gas exchange is provided by surface acration of culture medium va loosened sidearm caps. Rotating bioreactors with two different geometries, che slow-turning lateral vessel (STLV) (Big. 13.36) (Schwarz et al, 1992) and the high-aspect-atio vessel (HARV) (Fig. 13.3c) (Prewert tal, 1993), have been used to engineer cartilage and cardiac tissues in ground-based studics (cg, Freed and Vanjak-Novakovic, 1995, 19972). The STLY is configured as the annular space between, to concentric eyinders, the inner of which isa silicone gas exchange membrane, whereas the HARV isa cylindrical vessel with a gas exchange membrane at its base. These vessels contain 100— 110 ml ofculeure medium and up to 12 tissue constructs, and are operated by solid-body rotation in horizontal plane at rates of 15~40 rpm. Viscous coupling induces a rotational flow field that can suspend tissue constructs without external fisation (Freed and Vunjak-Novakovic, 1995). Ves- sel rotational speed is adjusted such that constructs remain suspended close toa stationary point within the vese, relative to an observer on the ground, due to a dynamic equilibrium beeween the ‘acting gravitational, centrifugal, and drag forces. Medium is exchanged batchwise (ata rate of 50% ‘every 2-3 days of 3 ml per construct per day), and is equilibrated with gas continuously. A rovating-vall perfused vessel (RWPV) (Fig. 13.3d) developed at the National Aeronautics ‘and Space Administration (NASA) (Jessup et a, 1996) was used to engineer cartilage in the mi- ‘togravity environment of space and in a control study on Earth (Freed etal, 1997). The RWPV is configured as an annular space berween two concentric eyinders, with the medium inlet at onesuojypuos Buperedo jo Areununs y soquieyo pasnyad () pue “wuinjo> pos jt 40 2135) YS (2) 5]2559A UMM 4OPDERHOG sad fssan ype 40 UoM “rasna) 5591 pasnyad em-Burpes (p) (ASH) [862A onerredseBiy (2) (AUS) worries 098) tase asa coe) sopsiosaapaum 26 ma mu erase ee 2 | MYL TG HRA 08 0) nei ng) “sons foals13 Bioreactors ‘end and the medium outlet via a filter on the central cylinder. A flat disk atone end of the central cojlinder serves sa viscous pump. The RWPV contains 125 ml of culture medium and holds up 10 10 tissue constructs. In microgravity the inner and outer eylinders were differentially rotated at rates of 10 and I rpm, respectively; on Earth, the vessel was rotated as 2 solid body at 28 rpm. ‘Medlium was periodically recirculated between the culture vessel and an external membrane gas ‘exchanger at arate of 4 ml/min for 20 min four times per day, and was exchanged at arate of 5 10 ml per construct per day. Perfused columns were designed to allow nondestructive assessment of tissue development cover the course of cultivation using magnetic resonance imaging (Fig. 13.36) (Obradovic etal, 19975 Williams etal, 1998) The columns have an inle a the base, an outlet atthe top, and con- tain 15 ml of culrure medium and up to four constructs that are fixed to a centrally positioned needle, Medium is continuously recirculated berween the column and aa external membrane gas ‘exchanger at a rate of 2~3 ml/h, and is exchanged at rates of 2=15 ml per construct per day. Perfused chambers (Fig, 13.34) were designed to allow tissue culture in the microgravity en- vironment of space, and for contol studies under conditions of une gravity on Earth or atificial savy in space (Vunjak Novakovic etal, 1999), The chamber is configured as a central com- pparument bounded by a porous membrane and an annular space. The medium inlet sin the cen tral tissue culture space and the medium outlet is in the peripheral cell-free space. Chambers con- tain 3, 10, or 30 ml of medium and up to five tissue constructs. Medium is continuously secizculated between the chamber and an external membrane gas exchanger at rates of 1-30 ml ‘min, and is exchanged at rates of 1~30 ml per construct per day. Chambers ate mounted on a cir- cular tray that allows automated sampling and on-line microscopy. Hypropynamic Conprrions 1 BiorEAcToRs In static flasks, mass transfer in che bulk medium occurs by molecular diffusion, and there is no fluid flow at the surfces of the tissue constructs. In mixed flasks, mass transfer is by convec- tion, and fluid low atthe surfaces ofthe tissue constructs i turbulent, such cha che smallest tur- bbulent eddies have sizes of250 jum and velocities of 0.4 em/see (Vanjak-Novakovic eal, 1996). {In rotating vessels, laminar flow at the construct surfaces was demonstcated by flow visual- ination conducted using tracer particles illuminated by a sheet of laser light (Neitzl etal, 1998) (Fig. 13a). Particle image velocimetry (PIV) studies, done by cithercros-cotration of particle images in window of the flow field or by individual particle tracking, allowed quantitation of the velocity vector at every location in the plane of the ligheshect (Fig. 13.4b) (Neitz etal, 1998). ‘These data were used to calculate maximal shea stresses at the construct surface, which were om the order of .8 dyn/cm? fora 120-ml rotating vessel containing one model construct and oper- ated such that the inner and outer cylinders were rotated at 13 and 37 rpm, respectively. The ob- served velocity fields were predicted numerically (Fig. 13-4) and were used to calculate the rates ‘of mass transport of chemical species through the medium to the construct. Residence time dis- tribution studies demonstrated efficient convective mixing due to gravitational construct setding in rotating vessels (Freed and Vunjak-Novakovic, 19976). In particular, mixing efficiencies were ‘comparable for an STLY containing 12 model constructs and rotated at 15~28 rpm and a spin- ner flask containing 12 eonstrues and sired at 50~80 rpm. In perfused columns and chambers, fluid flow i laminar and mass transfer is by convection due o recirculation. BIOREACTOR MODULATION OF TISSUE FORMATION In this section, we describe several studies that demonstrate how bioreactors can modulate 3D ‘issue formation in vitro, General design principles for tissue engineering bioreactors include main- taining desired levels of chemical species in the bulk medium, providing ficient mass transfer fiom the medium tothe tissue surfaces, and exposing developing tissues to physical stimuli. These bioreactor functions can result in increased size and improved structure and function of enginecred tissues, as follows, The effects of biochemical, hydrodynamic, and mechanical factors were stud- for cngincered cartilage cultured in rotating bioreactors (Fig. 13.5). In one case, medium oxy- {gen tension, 70, and pH were varied using STLYs operated under the same hydrodynamic com ditions (Fig. 13:52); in another case, hydrodynamic and mechanical factors were varied using RWPVs operated with comparable medium compositions (Fig. 13.5b). The specific effects of hy- 7(0661 “ye ¥ [2ZION) voreymuss feayewnu Aq -ayBtysa5e| pur s2j2qued 1038 BuIsn pjay Moy pInyy 24230 uojep pay A20j0n (2) dnowD0}aa a8eUu aed Aq Aequauedke pouluusdap prey £420) nezensy (2) sopseeyong Sunejo ® U! YannsuaD papuadsns e punole suonpUOD MOLL "FEL “By (wo)13 Bioreactors z 3 ; - a _ 10 : a 6 BS We = os ae a6 i 06 i i 4 Eu 2 . 4 a 2 oa 1 5 © © '0 250. 500 750 1000 1250 1500 ‘Mir ‘Depth from surface [um] Fig. 13.5. (a) Effects of biochemical factors on engineered cartilage. Comparison of constructs cul- tivated in medium with relatively high pO, and pH, or with lower pO, and pH (due to higher oF lower rates of bioreactor gas exchange). Histology: safranin-O staining for glycosaminoglycan. Graph: glycosaminoglycan fraction as a function of depth from the consiruct surface From Obradovic et al,, 1999, Oxygen is essential for bioreactor cultivation of tissue engineered cart- lage. Biotechnol. Bioeng. 63, 197-205. Reprinted by permission of Wiley-Liss, Inc., a subsidiary cof John Wiley & Sons, In). (b) Effects of hydrodynamic and mechanical factors on engineered cartilage. Comparison of constructs cultivated in the presence of higher levels of hydrodynamic ‘and mechanical stimuli (due to gravitational settling on Earth) or relatively quiescenty (aboard the ‘Mir Space Station). Histology: safranin-O staining for glycosaminaglycan. Graphs: glycosamino- _slycan fraction and equilibrium modulus (Freed et al, 1997). Data are the average *SD of three {four independent measurements. ddrodynamic factors and the accompanying changes in mass transfer were further studied by sys- tematically.comparing engineered cartilage and cardiac muscle culeured in different bioreactors ig. 13.6. Biochemical factors were vatied by establishing two groups: (I) pO, = 87 mm Hg, pH = 7.0 and (2) 20, = 43 mm Hg, pH = 6,7 in STLVs operated with or without an internal gas ex- change membrane (Obradovic eral, 1999). Cartilaginous constructs were compared with respect to several paraineters, including yield of lactate to glucose (¥;,,), construc thickness, and the amounts and distributions of glycosaminoglycan. In group 2, low pO, and pH were associated. with anaerobic cell metabolism (Y,j< of 2.2 mol/mol) as compared to group 1, in which higher values of 0, and pH were asociated with more aerobie cell metabolism (Y,,¢, of 171.8 mol! ‘mol). Constructs grown aerobically for 5 weeks were approximately 2.5 mm thick and had a high fraction [~6% of wet weight (ww)] of uniformly distributed GAG, except for a 460-im-thick. cuter capsule (Fig. 13.5a), In contrast, construets grown anaerobically were only about 1.6 mm thick and liad markedly lower GAG fractions (1.5~3.5% ww) chat decreased with increasing depth. One key bioreactor function is thus to provide efficient gas exchange for control of medi- tum 0, and pH. Hydrodynamic conditions and mechanical factors were varied by utilizing rotating bioreac- tor vesels on Earth and in the microgravity environment of space, aboard the Mir Space Staion, (Freed of al, 1997). In particular, cartilaginous constructs were first culeured in STLVs for 3 Mir 149150 Lactate/glucose (moVmol) Lactate/glucose (mol/mol) Freed and Vunjak-Novakovic Giycosaminoglycan (sewet weight) 28 ‘Equilibrium modus (KP) 3 so | ; ° ‘Static Mixed Rotating 0 Static Mixed Rotating oe Mg Me cae ee Se z i 30. E250 i z= 5” ® 150. j 2 0 3 100 ¢ 2 5 é 2, a Io otating ‘Stale Mixed Rotating vessel flask flask vessel Fig. 13.6 fects of hydrodynamic factors (a) Effects on engineered cartilage. Molar ratio off {ate production and glucose utilization (actate/glucose), ghycosaminoglycan fraction, and eat, ee oereesdlus in constructs cultured in static or mixed flasks, or in rotating vessels (STLVs) fObudovie et al, 1997; Vunjak-Novakovic etal, 1999). (b) fects on engineered cardiac tissue ese Msctate ratio, DNA content, and metabolic activity index (MIT/DNA) of consructs cul err ic or mixed flask, or in rotating vessels (HARVs) (From Carrier et al,, 1999) Cardiac thee engineering: Cell seeding, cultivation parameters and tissue construct characterization ies fe Boog 64, 500-509. Reprinted by permission of Wiley-Liss, Inc. a subsidiary of ohn {atlas & Sons Inc). (ab) Horizontal ines represent lactateglucose ratios of 2 and I, which cor wend to theoretical values fr anaerobic and aerobic metabolism, respectively. Data are the av- ‘rage #:5D of three to four independent measurements. ‘months on Earth and then in RWPVs for an additional 4 months either on Earth or aboard Mir. ‘During culation on Earth, construets were exposed to rclatively higher levels of hydrodynamic hen lue to gravitational seing) and mechanical stimuli (ue to construct collisions withthe toeating ved walls) as compared toon Mir, where constructs floated freely inthe culture medi- ae Madium values of pH and pO, were comparable in the ewo groups, Cartilaginows constructs ‘wore compared with respect to eeveral parameters, including dimensions, wet weight factions of GAG, and equilibrium moduli in radially confined compression. Constructs grown for7 months co Buith resid thei inital discoid shape, were large (429 mg ww), and had high GAG frac- ‘dons (8.89% vow) and equilibrium moduli (0.93 MPa) (Fig. 13.56). In contrast, constructs grown, for monehy on Earth and then 4 months on Mir tended to become more spherical, were small- G30 mg won, and had lower GAG fractions (3.6% wr) and equilibrium modul (0.31 MPs} Other important functions of ssi engineering bioteactor are chs to provide appropriats levels cof hydrodynamic and mechanical stimuli \Canilaginous constructs seeded in mixed flasks (Fig. 13.2) and then cultured for 6 weeks in seatc of mised flasks (Fig. 13.3a) or in rotating vessels (STLV3) (Fig, 13.36) were compared with Tapectto several parameters including ¥ gy wet weight factions GAG, and equilibrium mod-13. Bioreactors ulus in radially confined compression (Obradovic etal, 1997; Vanjak-Novakovic al, 19992), ‘Values of Fc for constructs cultured statically, in mixed flasks, and in rotating vessels were, re- spectively 1.7, 1.3,and 1.3 mol/mol, implying that cell metabolism was more aerobic under mixed, than static culture conditions (Fig. 13.6a) (Obradovic etal, 1997). GAG fractions and equilib ‘um moduli were significantly higher for constructs grown in rotating vessels as compared to either static or mixed flasks (Fig. 13.6a) (Vunjak-Novakovic et al, 1999). In static flasks, diffusional constraints of mass transport resulted in chin, fragile constructs in which GAG accumulated main- ly ina I-mm-thick outer region. In mixed flasks, urbulent conditions apparently induced the for- mation of capsules at the construct surfaces that were up t9 400 jim thick and consisted of mul- tiple lyers of elongated cells and litle GAG. In rotating vessels, dynamic laminar flow patterns ‘permitted the growth of constructs with high, spatially uniform GAG fractions. These studies demonstrate that hytrodynamic factors, sch as mixing pattern and flow regime, play a key role in determining the structure and function of engineered cartilage. ‘Cardiaclike constructs seeded in mixed flasks (Fig. 13.2) and then cultured for 2 weeks in static or mixed flasks (Fig. 13.3a) or in rotating vessels (ARVs) (Fig. 13.30) were compared with respect to several parameters, including J; cellularity (DNA per construct), and metabolic ac- tivity index (MTT units/mg DNA) (Caster et al, 1999). Values of ¥ for constructs cultured statically, in mixed flasks, and in rotating vessels were, respectively, 2.0, 1.6, nd 1.0 mol/mol, im- plying anaerobic, partally aerobic, and aerobic cell metabolism (Fig. 13.66). Cellulatties and metabolic activity indexes of constructs cultured statically were markedly lower than those of con- seruets cultured in either mixed flasks or rotating vesls (Fig. 13.66) (Caries eral, 1999). These studies demonstrate that hydrodynamic factors affect the structure and function of engineered car- diac tissue in a manner consistent with that observed for engineered cartilage (Fig, 13.62). BIOREACTOR CULTIVATION OF FUNCTIONAL TISSUES Carmace Cantlaginous constructs cultured for 6 weeks in rotating bioreactors (STLVs) were continu- ously carlaginous over their entire cross-sectional areas (6.7 mm in diameter X Smm thick) (reed et al, 1998) and contained an interconnected network of collagen fibers that resembled that of articular collagen with respect to overall organization, fiber diameter (Fig, 13.7a) (Riese e+ aL, 1998), and molecular structure (type II represented more than 90% of the tora collagen) (Freed etal, 1998). As compared to native bovine caf articular cartilage, G-weck constructs had comparable cellularcies, 68% as much GAG, and 33% as much collagen type Ik per unit wet ‘weight (Freed er al, 1998). Extracellular matrix synthesis rates decreased to approximately 60% ofnital over 6 weeks, and the fraction of newly synthesized macromolecules released into the cul- ‘ure medium decreased from about 25% of total at 4 days to less than 4% at 6 weeks. Over 7 ‘months of invite cultivation, the GAG fraction and equilibrium modulus reached or exceeded values measured for native cartilage (Fg. 13.72). However, other properties of 7-month constructs remained subnormal (¢g., collagen fraction, the concentration of pyridinium cross-links, and dy- namic stiffness wer, respectively, 34, 30, and 46% as high asin native cartilage) Freed etal, 19975, Riedle er al, 1998). Construct mechanical properties (ie. equilibrium modulus, hydraulic per- meability, dynamic stifness, and streaming potential) correlated with the wet weight fractions of GAG, collagen, and water (Vunjak-Novakovie etal, 1999). Canprac Tissue, Cardiac like tissues cultivated for 1 weelsin mixed flasks were 5 mm in diameter and 1.3 mm thick and ad a50- to 70-qum-thick tssuelike region a the constrict peeiphery in which cells ex- pressed the muscle-specific protein sacomeric tropomyosin and exhibited cardiac-specifi ulra- structural features, including intercalated disks and sarcomeres (Bursac etal, 1999) (Fig. 13.7b. ‘As compared to native neonatal rat ventricles, I-week constructs had 1696 as much DNA pet unit ‘wet weight (Fg. 13.7b), and similar indexes of cll size (protein/DNA) and metabolic activity (MIT/DNA). The petiphera region of constructs was electrically excitable and could be captured over a wide range of pacing frequencies (80-270 beats/min) (Fig. 13.74) (Bussac etal, 1999). Tmpulses propagated at conduction veloccies that were haf of those in native neonatal rat venti- des Bursac eral, 1999) (Fig. 13.76). Contractile cardia-lke tissues were also grown in rotting 151152 Freed and Vunjak-Novakovic 6 wk construct 5 Fo 2 z 2 & 3 ge i Bs 4 Ho f . i g 2 i Bo ° Biay Gk 3m0 7 m0 “aay Gwk 3m07m0 Native cartiags Tm, Cultivation ine Cultivation ie Cetularty (mg DNA/g wet weight) ‘Construct. Neonatal ventricle coll ‘Construct Neonatal ventricle ‘Conduction velocity (ms) a8 Fig. 13.7. Bioreactor cultivation of functional tissues. (a) Cartilage. Micrographs show the ultra- siructural appearance of collagen networks ina 6-week construct and in native calf articular car- tilage(Riesle eta, 1998, Collagen in tissue engineered cartilage: Types, structure and cross-links. J. Cell. Biochem. 71, 313-327. Reprinted by permission of Wiley-Liss In., a subsidiary of John Wiley & Sons, In). Graphs: giycasaminoglycan fraction and equilibrium modulus of constructs cultivated in rotating vessels (STLVs) for 3 days, 6 weeks, and 3 or 7 months (Feed et al, 1997; ‘Vunjak-Novakovic etal, 1999a). Dotted lines represent ranges of parameters measured for native cartilage. (b) Cardiac muscle. Recording: electrophysiological response of construct to electrical Stimulation at a rate of 80 bpm. Micrographs: ultrastructural demonstration of sarcomeres (outer photo) and an intercalated disk (inset). Graphs: DNA content and conduction velocity of -week ‘constructs and native neonatal rat ventricles (Bursac et al,, 1999). Data are the average *SD of three to six independent measurements vessels (HARVs), and were shown to contain the gap-junctional protein connexin-43, creatine ki- ‘nase isoform MM, sarcomeric myosin heavy-chain protein (Papadaki eral, 1999), muscle desmin, ‘cardiac troponin T, and sarcomeric tropomyosin (Carter eral, 1999). ‘TISSUE ENGINEERING BIOREACTORS: STATE OF THE ART Tn contrast to a large numberof tissue engineering studies focusing on scaffold design and in tiv issue repair using cells and/or biomaterial, there has been relatively lice work inthe area of bioreactors. As descibed in this chapter, specific bioreactor desig features can be exploited to in- prove the structure and function of engineered tissues, including in particular (1) flow and mix- ing patterns that can resultin eficient and spatially uniform seeding of polymer scaffolds, (2) en-13 Bioreactors hhanced mass transport of chemical species through the medium and to the tissue surfaces, and (3) physical simulation ofthe tissue during its development. ‘Ouray studies demonstrated increased thickness and improved structure of engineered car- tilage when scaffolds were seeded in orbitally mixed petri dishes as compared to static dishes (Freed tal, 19948). The concept of maintaining a uniform suspension of isolated cells and providing relative velocity between cells and the scaffold during seeding was improved by using mixed flasks and rotating vessels Freed and Vanjak-Novakovic, 1995, 1997a; Vunjak-Novakovic e al, 1996, 1999). The kinetics and mechanisms of seeding in mixed flasks, which are at ths time the pre- {erred system for call ceding of 3D scaffolds, were characterized in detail (Vunjak-Novakovic al, 1998). Engineered cartilage cultured in mixed flasks was structurally superior to that growa in of bitally mixed dishes, which was in turn superior to that grown statically (Vanjak-Novakovic eal, 1996). Efficient convective mixing demonstrated for mixed flasks and rotating vessels (rd and ‘Vunjak-Novakovic, 19976) implies that both bioreactors can maintain constant concentrations of chemical species in the bulk medium. The hydrodynamic environment present in rotating ves (Fig. 13.4) stimulated the development of engineered cartilage that was metabolically and me- chanicaly functional (Fig. 13.5). Engineered tissues (cartilage, cardiac) grown in rotating vesscls ‘were structurally and functionally superior to construct grown in either static or mixed flasks (Fig. 13.6). Although the mechanisms underlying these effects are yet to be determined, others have hy- pothesized thar hydrodynamic frees affect cultured cells via pressure fluctuations, stretching the cell membrane, and/or shear stress (Berthiaume and Frangos, 1993; Smith et a, 1995). ‘The bioreactor systems we have used to date (Fig, 13.3) promote mass transfer to the con- struct surfice, but do not enhance the relatively slow diffusion of chemical species to the construct interior. This has not been an overwhelming problem in the engineering of cartilage, an avascular tissue with low cellularity (only about 4~5% of the wet weight), which can be cultivated in biore- actors ta thickness exceeding that of native articular cartilage (Le, 5 mm) (Fred eal, 1998). However, diffusional limitations of mass transport have severely curtailed efforts to engineer tis- sues chat normally have high vascularity and/or cellularity. In particular, only very low thickness- cs were reported to date for engineered bone (250-500 jum) (Ishaug etal, 1997; Martin etal, 1998) and cardiac tissue (50-180 jum) (Eschenhagen etal, 1997; Bursac et aly 1999 Carrer et al, 1999). ‘Other groups have demonstrated advantages of using culture systems that provide continu ‘ous perfusion and mechanical stimulation during cultivation. In one system, growing constructs were directly perfused with culeure medium within 1.2 ml cartridges (Dunkcelman etal, 1995). Engineered cartilage cultivated in these bioreactors for 2~4 weeks repaired osteochondral defects and integrated with underlying bone in a 2-year rabbic study (Schreiber eal, 1999). In other sys- tems, cartilage (Grande er al, 1997) and skeletal muscle (Chromiak et al, 1998) constructs were surrounded by culture medium chat was continuously recirculated through a closed loop. In all the above cases, perfusion of medium reportedly improved tissue growth and metabolism by en hrancing mass transfer and reducing the variations in medium concentrations of gases, nutrients, metabolites, and regulatory factors that occur in periodically refed cultures. In addition, closed- loop perfused bioreactors reduce the risk of contamination during long-term cell cultivation. “The fac that physical stimuli modulaté tissue development has motivated the design of sev- «ral bioreactor systems in which growing tissues are exposed to mechanical fores. A system that exposed cartilaginous constructs to dynamic compression at physiological frequencies enhanced ECM synthesis rates as compared to statically loaded controls (Buschmann et al, 1995), Likewise, system that exposed cartilaginous constructs to physiological levels of intermittent hydrostatic pressure enhanced GAG deposition as compared to nonpressurzed controls (Carver and Heath, 1999). A system that exposed skeletal muscle constructs to static and dynamic tension was associ- ated with increased size and improved function (Vandenburgh et al, 1991). The concept often sie loading was extended by exploiting the native contractile properties of collagenous cell culure substrates. Examples include engineered tissue containing genetically modified cells that secreted human groweh hormone for up to 3 months in mice (Vandenbugh etal, 1996), engineered car- diac muscle that contracted and in some cases secreted recombinant B-galactosidase for up to 10 days in vitro (Eschenhagen etal, 1997), and engineered tendon that repaired gap defects over 3 ‘months in rabbits (Young eral, 1998). Inall ofthe above studies, the presence of mechanical forces 153154 Freed and Vunjak-Novakovie (excernally applied or internally generated) during cultivation stimulated the development of en- sgincered tissues, presumably by providing physical stimuli normally present in vive. ‘Anovel bioreactor sytem for vascular tissue cngincering elegantly demonstrate the combined benefits of continuous perfusion and mechanical stimulation (Niklason etal, 1999). Tubular poly- mer scaffolds seeded with smooth muscle cells were placed ina sired vesel and cultivated in the presence of pulsatile radial stress, applied via highly dsrensble silicone tubing in the construct lu sen. Under these conditions, small-cliber (<6 mm in diameter) arcries with high rupture strengths and high suture retention strengths could be cultivated over 2 months in vrs these re ‘mained patent for up to 24 days following in viv implantation in swine, SUMMARY AND RESEARCH NEEDS In this chapter, we have discussed the design and operating principles of tissue engineering bioreactors developed forthe cultivation of engineered tissues starting from isolated cells and 3D biodegradable scaffolds. Bioreactor technologies can be utilized to grow functional tissues for in vie implantation, and for controlled in vit seudies of how biochemical and physical factors reg ‘ulate tissue development. In general, the functions ofthe bioreactor are to seed cells onto 3D scaf- folds and to provide appropriate stimuli during in wieo tissue development. Specific design re- quirements depend on the dimensions, complexity, and application ofthe tissue tobe engineered. Further advances in bioreactor technologies that ae likely havea major impact on the field of tis- sue engineering include (1) improved control over culture medium composition, (2) new meth- ods to provide physiological levels of mechanical stimuli to engineered tissues in vitro, (3) new techniques to induce and/or mimic vascularization of engineered tissues in vitro, (4) determina- tion of appropriate conditions and duration of in stv culture prior to implantation, and (5) ex- tension from animal model systems to human cel sources and scales. ACKNOWLEDGMENTS ‘This work was supported by the National Aeronautics and Space Administration (Grants ‘NAG9-836 amd NCC8-174). The authors thank G. P. Neivl, J. B. Brown, and M. Smith for the bioreactor fluid dynamics data shown in Fig. 13.4, and R. Langer for helpful advice. REFERENCES der, A, Kaop , Frucha,N, Crome, 0. Bock, K, Chisians, U, Oldhafr, K, Rings By Pichlmaye, Rand Sewing, KF (1995). 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