session 5 - semantic scholar · the current state of the art. mass transport issues bioreactor...

Proceedings of the WTEC Workshop on Tissue Engineering Research in the United States

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SESSION 5

ENGINEERING DESIGN ASPECTS

David Mooney

INTRODUCTION

A number of engineering issues are clearly critical to the successful development of tissue engineeredproducts and a tissue engineering industry. These issues include elements of mass transport, biomechanics,biomaterials, and bioelectronics. The focus in this chapter is the mass transport and biomechanics designaspects, as the biomaterials and bioelectronics issues are covered in other chapters.

The specific mass transport aspects deemed critical for tissue engineering include:

1. Adaptation of existing bioreactor technology for large-scale cell expansion

2. Assuring sufficient oxygen and other nutrient availability to transplanted cells and those in bioreactors

3. Development of delivery vehicles for growth factors and other macromolecules to induce blood vesselformation

4. Identification of appropriate techniques for preserving both cells and engineered tissues.

Relevant biomechanics issues include:

1. Evaluating the critical mechanical properties of the tissues one wishes to replace

2. Determination of the minimum values of these properties required for an engineered tissue

3. Identifying the role of externally applied mechanical stimuli in the development and function ofengineered tissues.

The following sections contain a very brief overview of a few of these issues. The accompanying articles inthis section typically provide more extensive background to these issues, and a more in depth-discussion ofthe current state of the art.

MASS TRANSPORT ISSUES

Bioreactor technology

Bioreactors are utilized in tissue engineering both as a tool to generate cells for subsequent transplantation, togrow three-dimensional tissues prior to transplantation, and directly as organ support devices (see article byW. Miller). Many tissue engineering strategies rely on multiplying cells from a small biopsy or startingtissue source, and subsequently harvesting these cells for transplantation directly or on a polymeric scaffold.Traditional bioreactor technologies, which focused on growing single cells or small cell clusters, provide asuitable basis for this type of cell expansion work, which is done by a number of tissue engineering


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companies, including Advanced Tissue Sciences (La Jolla, CA), Organogenesis (Canton, MA), andReprogenesis (Cambridge, MA). In certain situations, however, simultaneous culture of multiple cell typesmay be required, and this requires more complex bioreactor design (Emerson et al. 1991). In addition, thecultivation of three-dimensional tissue constructs places great demands on the capability of the bioreactorsystem to provide sufficient nutrient transport, and is the basis for significant research (Obradovic et al.1999). The use of bioreactors as support devices for liver or kidney function provides another layer ofcomplexity, as transport between the cells in the device and fluids flowing through or in partial contact withthese cells must be optimized (McLaughlin et al. 1999; Nikolovski et al. 1999).

Oxygen transport

It is critical that transplanted cells or cells in bioreactors have sufficient nutrient and waste exchange withtheir surroundings in order to survive, function appropriately, and become integrated with host tissuefollowing implantation. Oxygen transport is typically considered the limiting factor for nutrient exchange(see article by C. Colton). Tissues in the body overcome issues of mass transport by containing closelyspaced capillaries that provide conduits for convective transport of nutrients and waste products to and fromthe tissues. It is similarly considered critical for any engineered tissue of significant size to becomevascularized. Oxygen transport is a critical feature of bioreactor design as well, and was briefly discussed inthe last section.

There are three approaches currently being investigated to promote vascularization of engineered tissues.First, scaffolds utilized for cell transplantation are designed to promote invasion of host fibrovascular tissueby the inclusion of large, interconnected pores (Mikos et al. 1993). However, fibrovascular ingrowth into thescaffolds occurs at a rate less than 1 mm/day, and typically takes one to two weeks to completely penetrateeven relatively thin (e.g., 3 mm thick) scaffolds. The second, more active approach to promotevascularization of engineered tissues is the delivery of angiogenic growth factors (e.g., VEGF, bFGF) to theimplant site. It has recently been demonstrated that these factors may be directly included within the tissueengineering scaffolds for a sustained delivery at the desired site (Sheridan et al. 2000). It may also bepossible to utilize local gene therapy to promote vascularization by release of plasmid DNA encoding thegrowth factors from the tissue engineering scaffold (Shea et al. 1999). A third approach to enhanceangiogenesis in engineered tissues is to co-transplant endothelial cells along with the primary cell type ofinterest. The endothelial cells seeded into a tissue engineering scaffold form capillaries that can merge withcapillaries growing into the scaffold from the host tissue (Nor et al. 1999).

Cryopreservation

Cells, macromolecular biologically active drugs, and three-dimensional tissues grown in bioreactors will alllikely be important tissue engineering products. In all three cases, it will be critical to develop technologiesfor the long-term, stable storage of these products following production and prior to clinical utilization.Storage typically involves reducing or removing water (e.g., lyophilization of protein solutions). Thecontrolled transport of water from the proteins, cells, and tissues is a complex mass transfer problem. Long-term storage of protein products is an important issue in the biotechnology and pharmaceutical industries,and has received extensive attention by these industries. Cryopreservation of cells and tissues, however, isstill an emerging field with many challenges (see article by M. Toner).

BIOMECHANICS ASPECTS

Many of the tissues for which one may desire to engineer a replacement tissue have a mechanical function(s)(e.g., blood vessels, bone, cartilage). However, at the current time the mechanical properties of many ofthese tissues have not been precisely defined. In addition, it is unclear which of the properties, and to whatmagnitude, are important to use as design parameters for the engineered replacement tissues (see articles byGuilak and Nerem for full discussion of this issue).

Externally applied mechanical signals are clearly regulators in the development and function of a variety oftissues. Increasing evidence from basic biology studies indicate cells mediate the response of mechanicalsignals. However, the increasing amount of basic information available from these studies is just now being


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utilized in the design of engineered tissues. The mechanical properties of many engineered tissues areinferior to the native tissue, and it has been widely hypothesized that appropriate mechanical stimulation ofengineered tissues may contribute to a more natural structure and mechanical properties. Recent studies withengineered cartilage (Carver and Heath 1999) and blood vessels (Niklason et al. 1999) support thishypothesis, as mechanically stronger tissues could be formed with appropriate mechanical input.

SUMMARY

A large number of engineering design aspects must be considered to engineer fully functional tissues. Therehas been considerable work recently in many of these areas, with promising results. However, significantwork remains in each of these areas. It may be particularly important in the future to consider how thesevariables may interact with each other to control the function of engineered tissues. For example, thebiomaterials and biomechanics design issues may need to be considered together in certain situations. It hasrecently been demonstrated that engineered smooth muscle tissues only respond to mechanical stimuli andform stronger tissues when adherent to specific types of adhesion molecules on the scaffolds (Kim et al.1999). In addition, the mass transfer issues may have significant impact on the mechanical properties ofengineered tissues, as recently described for cartilage grown in vitro (Vunjak-Novakovic et al. 1999).

REFERENCES

Carver, S.E. and C.A. Heath. 1999. Increasing extracellular matrix production in regenerating cartilage with intermittentphysiological pressure. Biotechnology & Bioengineering. 62(2):166-74.

Emerson, S.G., B.O. Palsson, and M.F. Clarke. 1991. The construction of high efficiency human bone marrow tissue exvivo. Journal of Cellular Biochemistry. 45(3):268-72.

Kim, B.S., J. Nikolovski, J. Bonadio, and D.J. Mooney. 1999. Cyclic mechanical strain regulates the development ofengineered smooth muscle tissue. Nature Biotechnology. 17(10):979-983.

McLaughlin, B.E, C.M. Tosone, L.M. Custer, and C. Mullon. 1999. Overview of extracorporeal liver support systemsand clinical results. Annals of the New York Academy of Sciences. 875:310-25.

Mikos, A.G., G. Sarakinos, M. Lyman, D.E. Ingber, J. Vacanti, and R. Langer. 1993. Prevascularization of porousbiodegradable polymers. Biotech. Bioeng. 42:716-723.

Niklason, L.E., J. Gao, W.M. Abbot, K.K. Hirschi, S. Houser, R. Marini, and R. Langer. 1999. Functional arteries grownin vitro. Science 284:489-493.

Nikolovski, J., E. Gulari, and H.D Humes. 1999. Design engineering of a bioartificial renal tubule cell therapy device.Cell Transplantation. 8(4):351-64.

Nor, J.E., J. Christensen, D.J. Mooney, and P.J. Polverini. 1999. VEGF enhances the survival of endothelial cells andsustains angiogenesis by inducing expression of Bcl-2. Am. J. Pathol. 154:375-384.

Obradovic, B., R.L. Carrier, G. Vunjak-Novakovic, and L.E. Freed. 1999. Gas exchange is essential for bioreactorcultivation of tissue engineered cartilage. Biotechnology & Bioengineering. 63(2):197-205.

Shea, L.D., E. Smiley, J. Bonadio, and D.J. Mooney. 1999. DNA delivery from polymer matrices for tissue engineering.Nature Biotechnology. 17(6):551-554.

Sheridan, M.H., L.D. Shea, M.C. Peters, and D.J. Mooney. 2000. Bioadsorbable polymer scaffolds for tissue engineeringcapable of sustained growth factor delivery. Journal of Controlled Release. 64(1-3):91-102.

Vunjak-Novakovic, G., I. Martin, B. Obradovic, S. Treppo, A.J. Grodzinsky, R. Langer, and L.E. Freed. 1999. Bioreactorcultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage. Journal ofOrthopaedic Research. 17(1):130-8.


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BIOREACTOR DESIGN CONSIDERATIONS FOR CELL THERAPIES AND

TISSUE ENGINEERING

William M. Miller

Bioreactors can be employed for many diverse applications at various scales of operation in cell therapiesand tissue engineering, including:

• Cell production and transduction on both (i) patient-specific clinical-scale (e.g., T-cells, hematopoieticstem and progenitor cells) and (ii) large-scale (e.g., embryonic stem cells, neural stem and progenitorcells, PC 12 cells, mature blood cells).

• Tissue production on both (i) patient-specific clinical-scale (e.g., cartilage, tendons, ligaments) and (ii)universal-donor clinical-scale (e.g., bioartificial skin).

• Functional extracorporeal or implanted bioartificial organs (e.g., liver, kidney, urologic tissues, pancreas,blood vessels).

• Model systems for toxicity and efficacy testing (e.g., cornea, liver).

Many different types of bioreactors, including homogeneous and heterogeneous systems, have beendeveloped for these and other applications. This analysis will focus on general bioreactor design issues,rather than evaluating the bioreactor status for each of the respective applications.

All bioreactor systems should be designed to meet a number of universal requirements:

• Control the physicochemical environment (e.g., pO2, pH, pCO2, shear rate). There is in vitro evidence tosuggest that gradients in pO2 and pH regulate the balance between cell proliferation and differentiation invivo. For example, pO2 has a marked effect on cytotrophoblast (Genbacev et al. 1997) andmegakaryocyte (Mostafa et al. 1998) differentiation. Similarly, pH modulates erythrocyte (McAdams etal. 1998) and granulocyte (Hevehan et al. 2000) differentiation. In addition to direct effects, pO2 and pHalso modulate cytokine production by accessory cells.

• Ensure aseptic feeding and sampling.

• Make effective use of expensive growth factors and medium components.

• Facilitate monitoring of cell and/or tissue “quality” (e.g., cell function, tissue structure).

• Ensure consistent cell and/or tissue quality in the face of donor cell variability. This is especiallyproblematic when cells used to seed a bioreactor are obtained from patient tissues.

• Materials must be compatible with the cells and processing steps. Materials approved for use in vivo orin blood processing equipment may not be suitable for ex vivo culture, especially for more primitive cellsand with serum-free media (LaIuppa et al. 1997; Koller et al. 1998).

• Maximize use of automated processing steps to increase reproducibility of bioreactor operation.

Homogeneous agitated reactor systems are well suited to addressing many of the design requirements notedabove, and there is evidence to suggest that stirred reactors can be used effectively to culture lymphoid cells,hematopoietic stem and progenitor cells, and embryonic and neural stem cells. Stirred reactors are ideallysuited for maintaining a uniform physicochemical environment, and for obtaining representative cellsamples. The latter property facilitates making changes to culture conditions or harvest time to account forvariability in the culture inocula from different donors. The design of large-scale systems for production ofmature blood cells for transfusions or for expansion of embryonic stem cells, as well as other cells that can be


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used for multiple recipients, can directly incorporate the many advances that have been made in large-scalecell culture for recombinant protein and vaccine production. These include automated pO2 and pH control,methods for effective oxygenation without exposing cells to damaging shear stress, and a variety ofautomated feeding protocols to maximize productivity. Also, due to their low surface-area-to-volume (S/V)ratio, material compatibility is less of an issue for large-scale systems. Patient-specific reactors are morechallenging because (i) components that come into contact with cells or medium should be disposable, (ii)the S/V ratio is much larger, (iii) automation costs are much higher on a per patient basis, and (iv) thebioreactor operators are likely to have other responsibilities, especially in a clinical setting. To date, clinicaltrials with hematopoietic cells have employed culture in flasks, bags, or stroma-associated heterogeneousperfusion cassettes in an automated culture system (Nielsen 1999). However, several companies aredeveloping agitated or other stroma-free clinical-scale patient-specific culture systems(www.t-therapeutics.com, www.nexellinc.com, www.wavebiotech.com, www.viacord.com/VPpress.htm).

Although homogeneous reactors facilitate cell sampling, it may not be possible to directly measure thedesired cell properties on a time-scale that is useful for process control or harvesting decisions. Examplesinclude evaluating the concentration of hematopoietic stem cells (defined by their ability to reconstitutehematopoiesis in vivo) or the ability of cultured neural stem cells to differentiate into various lineages inresponse to the appropriate stimuli. In some cases, the desired cell properties may correlate closely with cellsurface marker expression or metabolic activity. For example, hematopoietic stem cells are believed toexpress both CD34 and Thy-1 (Young et al. 1999). Also, hematopoietic progenitor cell content has beenshown to correlate with the lactate production rate and/or the oxygen uptake rate (Collins et al. 1997; 1998).

Agitated culture systems are also used to prepare tissue constructs such as bioartificial cartilage (Freed et al.,1997) and hepatocyte spheroids used to seed a bioartificial liver (Wu et al. 1996). These systems exhibitadded complexity because cells in the interior of the construct are exposed to pO2 and pH values lower thanthose at the construct surface. The potential effects of pO2 and pH on proliferation and maturation provideadditional degrees of freedom for tissue design. However, a number of complications must be addressed.First, the sampling system must be large enough to accommodate the tissue construct. Second, one mustoperate the system at a sufficiently high pO2 (and pH)—or else limit the construct size—to avoid forming anecrotic core. Finally, although samples may be readily removed from the reactor, it is typically necessary touse invasive assays to characterize the tissue structure and function.

Many tissue engineering applications require heterogeneous bioreactor systems with one or a few tissueconstructs in each reactor chamber or cartridge. As for the aggregates discussed above, there will begradients in pO2, pH, metabolites, and growth factors across the tissue constructs in these systems.Depending on the medium circulation pattern and flow rate, there may also be substantial gradients across thereactor, and hence across the exterior of the tissue construct. These external gradients can be minimized byoptimizing the flow distribution within the bioreactor chamber and by increasing the medium recirculationrate. The flow distribution can be predicted using a mathematical model (Horner et al. 1998; Ledezma et al.1999), and can be evaluated by measuring the residence time distribution or by visualizing the flow ofrheoscopic particles (Pasternak and Miller 1996). Small dead spots in the flow that could result in locallyadverse conditions may be detected by visualization with dye flow or rheoscopic particles. The mediumrecirculation rate should be adjusted such that the conditions (pH, metabolite and growth factorconcentrations, etc.) at the exit of the chamber provide a similar growth and differentiation environment asthose at the entrance of the chamber. However, the chamber design must be such that the requiredrecirculation rate does not result in an adverse shear environment. Oxygen is normally the limiting nutrientdue to its low solubility in culture medium. Thus, one way to decrease the required recirculation rate is toprovide for oxygen transport along the reactor chamber. Large-scale production of individually packagedtissue models, such as for bioartificial skin replacements (Naughton 1997), introduces the added challenge ofproviding equal medium flow to many parallel chambers.

Obtaining kinetic information on tissue structure and function during the course of a culture is a majorproblem for the heterogeneous bioreactor systems described above. Off-line analysis requires that a reactorchamber be harvested for each sample. Non-invasive, on-line sampling may allow for more detailed kineticanalysis. This would be especially advantageous for tissue constructs seeded with cells from patients orpatient-specific donors, because these constructs may exhibit substantial patient-to-patient variability.Analysis of the medium exiting a perfused culture chamber can provide valuable information regarding a


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tissue construct. Metabolic activity (e.g., O2 or glucose consumption) may be used to estimate the cellconcentration. However, metabolic patterns (e.g., the balance between oxidative and glycolytic metabolism)will vary with pO2 and pH across a tissue construct, and may also vary with cell differentiation. Changes inmetabolic patterns are often evidenced by changes in metabolic yield ratios such as lactate produced perglucose consumed, O2 consumed per glucose consumed, and ammonia produced per glutamine consumed.Therefore, much more information is obtained by measuring at least two metabolic parameters. Tissue-specific metabolic activity may also be evaluated using medium samples. For example, bioartificial liverconstructs can be evaluated in terms of albumin synthesis, ammonia clearance, urea synthesis, galactoseelimination, and cytochrome P450 system activity (e.g., in response to exogenous lidocaine).

Direct analysis of tissue structure and function may be problematic, but it provides valuable information. Forexample, on-line measurement of transepithelial electrical resistance in a culture chamber with defined apicaland basolateral regions (Pasternak and Miller 1996) can be used to evaluate epithelial barrier function.Impedance analysis (Fey-Lamprecht et al. 1998) provides additional information on the uniformity of anepithelial construct. Larger fluorescent tracer molecules can be used to follow the early development ofepithelial barrier function, as well as the permeability of leakier tissues, such as bioartificial vascular grafts.Optical clarity is a key requirement for a bioartificial cornea. It should be possible to assess tissue clarity in aspecially designed bioreactor chamber, perhaps using optical fibers for remote access.

Evaluation of cross-tissue pO2 and pH gradients, as well as the local tissue structure, are more difficultproblems, but have wide applicability. Jain and co-workers developed methods to obtain high-resolutionmeasurements of interstitial pH and pO2 profiles between adjacent blood vessels in tissue, using fluorescenceratio imaging and phosphorescence quenching microscopy (Helmlinger et al. 1997). Adaptation of theirsystem, perhaps in conjunction with confocal microscopy, for analysis of pH and pO2 gradients in perfusedtissue constructs would have tremendous utility. NMR can also be used to evaluate pH and pO2. Forexample, the 1H chemical shift of water is very sensitive to pH. Similarly, the 19F spin-lattice relaxationrates of fluorocarbon tracers are sensitive to pO2. NMR spectroscopy has been used to measure pH and pO2

in hollow fiber bioreactors, cells entrapped in alginate beads, perfused organs, and organs in vivo. NMRspectroscopy and microimaging have recently been used to follow changes in tissue volume, cellularity,bioenergetics, macromolecular content, and tissue heterogeneity during cartilage formation in a hollow fiberbioreactor (Petersen et al. 1997; Potter et al. 1998). Tissue constructs that incorporate multiple cell typespresent additional problems because the extent and nature of the interactions between the different cell typesare often crucial for effective tissue function. Development of a punch biopsy system for a tissue constructthat does not compromise bioreactor integrity would facilitate repeated sampling of the same construct forkinetic analysis, and may even have potential applicability for a quality control product release assay.

Extracorporeal bioartificial kidneys (BAKs) and livers (BALs) that are used as a bridge to organ transplantwill normally operate under conditions of kidney and liver failure, respectively. As a result, cells inextracorporeal BAKs and BALs are likely to be exposed to high levels of toxic metabolites that will result ina deterioration of bioartificial organ function over time. One approach to maintaining organ function is tocycle two identical bioartificial organs between on-line (exposed to patient fluids) and off-line (perfused withculture medium to restore cell function) configurations. Another option is to simultaneously expose cells toseparate flow circuits of patient fluids and a nutritive medium. This approach has been employed in a three-compartment BAL (Nyberg et al. 1993). A similar approach has been employed for an in vitro toxicityassay, in which the apical side of an epithelial construct is exposed to a test agent, while the basolateral sideis perfused with culture medium (Pasternak and Miller 1996).

REFERENCES

Collins, P.C., L.K. Nielsen, S.D. Patel, E.T. Papoutsakis, W.M. Miller, Biotechnol. Prog., 14:466 (1998).

Collins, P.C., L.K. Nielsen, C.-K. Wong, E.T. Papoutsakis, W.M. Miller, Biotechnol. Bioeng., 55:693 (1997).

Fey-Lamprecht, F., U. Gross, T.H. Groth, W. Albrecht, D. Paul, M. Fromm, A.H. Gitter, J. Mater. Sci. Mater. Med.,9:711 (1998).

Freed, L.E., R. Langer, I. Martin, N.R. Pellis, G. Vunjak-Novakovic, Proc. Nat. Acad. Sci. USA, 94:13885 (1997).


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Genbacev, O., Y. Zhou, J.W. Ludlow, S.J. Fisher, Science, 277:1669 (1997).

Helmlinger, G., F. Yuan, M. Dellian, R.K. Jain, Nature Med., 3:177 (1997).

Hevehan, D.L., E.T. Papoutsakis, W.M. Miller, Exp. Hematol., 28:267 (2000).

Horner, M., W.M. Miller, J.M. Ottino, E.T. Papoutsakis, Biotechnol. Prog., 14:689 (1998).

Koller, M.R., M.A. Palsson, I. Manchell, R.J. Maher, B.Ø. Palsson, Biomater., 19:1963 (1998).

LaIuppa, J.A., T.A. McAdams, E.T. Papoutsakis, W.M. Miller, J. Biomed. Mater. Res., 36:347 (1997).

Ledezma, G.A., A. Folch, S.N. Bhatia, U.J. Balis, M.L. Yarmush, M. Toner, J. Biomech. Eng., 121:58 (1999).

McAdams, T.A., W.M. Miller, E.T. Papoutsakis, Br. J. Haematol., 103: 317 (1998).

Mostafa, S.S., E.T. Papoutsakis, W.M. Miller, Blood, 92 (Supplement 1): 438a (1998).

Naughton, G.K., in Principles of Tissue Engineering, R. Lanza, R. Langer, W. Chick (eds.), R.G. Landes Co., p. 769(1997).

Nielsen, L.K., Annu. Rev. Biomed. Eng., 1:129 (1999).

Nyberg, S.L., R.A. Shatford, M.V. Peshwa, J.G. White, F.B. Cerra, W.-S. Hu, Biotechnol. Bioeng., 41:194 (1993).

Pasternak, A.S., W.M. Miller, Biotechnol. Bioeng., 50:568 (1996).

Petersen, E., K. Potter, J. Butler, K.W. Fishbain, W. Horton, R.G.S. Spencer, E.W. McFarland, Int. J. Imag. Sys.Technol., 8:285 (1997).

Potter, K., J.J. Butler, C. Adams, K.W. Fishbain, E.W. McFarland, W. Horton, R.G.S. Spencer, Matrix Biol., 17:513(1998).

Wu, F.J., J.R. Friend, C.C. Hsiao, M.J. Zilliox, W.-J. Ko, F.B. Cerra, W.-S. Hu, Biotechnol. Bioeng., 50:404 (1996).

Young, J.C., K. Lin, G. Hansteen, M. Travis, L.J. Murray, L. Jaing, R. Scollay, B.L. Hill, Exp. Hematol., 27:994 (1999).


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Bioreactor Design Considerationsfor Cell Therapies and Tissue Engineering

William M. Miller

Chemical Engineering Department

Northwestern University

OutlineApplications and Types of Bioreactors

pO2 and pH ConsiderationsCell and Tissue Characterization

Bioreactor Applicationsin Cell therapies and Tissue Engineering

• Cell production and transduction* patient-specific clinical-scale

T-cells hematopoietic stem and progenitor cells

* large-scale neural stem and progenitor cells cell lines (e.g., PC 12 cells) mature blood cells

• Tissue production* patient-specific clinical-scale

cartilage tendons and ligaments

* universal-donor clinical-scale bioartificial skin

• Functional extracorporeal or implanted bioartificialorgans* liver and kidney* urologic tissues and blood vessels* pancreas

• Model systems for toxicity and efficacy testing* cornea* liver


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Universal Bioreactor Requirements

• Control the physicochemical environment (e.g.,pO2, pH, pCO2, shear rate)

• Ensure aseptic feeding and sampling

• Make effective use of expensive growth factors andmedium components

• Facilitate monitoring of cell and/or tissue "quality"(e.g., cell function, tissue structure)

• Ensure consistent cell and/or tissue quality in theface of donor cell variability

• Maximize use of automated processing steps toincrease reproducibility of bioreactor operation

• Materials must be compatible with the cells andprocessing steps; materials approved for use in vivomay not be suitable for ex vivo culture, especiallyfor more primitive cells and with serum-free media(LaIuppa et al., 1997; Koller et al., 1998)

Homogeneous Agitated Reactors

• Ideally suited for maintaining a uniformphysicochemical environment

• Ideally suited for obtaining representative cell samples,which facilitates making changes to culture conditions orharvest time

• Large-scale systems for production of mature blood cellsor expansion of cells used for multiple recipients canbuild on cell culture advances for protein and vaccineproduction* automated pO2 and pH control* oxygenation without damaging shear stress* feeding protocols to maximize productivity

• Patient-specific reactors are more challenging because* components in contact with cells should be disposable* materials compatibility is more stringent (larger S/V)* automation costs are higher on a per patient basis* bioreactor operators are likely to have other duties

• Clinical trials with hematopoietic cells and T-cells haveemployed flasks, bags, or stroma-associated perfusioncultures

• Several companies are developing agitated or stroma-free clinical-scale patient-specific culture systems


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Agitated Systems for Tissue Constructs

• Examples include* bioartificial cartilage (Freed et al., 1997)* hepatocyte spheroids (Wu et al., 1996)

• Uniform physicochemical environment in the bulk liquid

• pO2 and pH gradients across the tissue construct

• Must operate at high pO2 (and pH) or limit construct sizeto avoid forming a necrotic core

• Sampling system must accommodate the tissue construct

Heterogeneous Bioreactor Systems

• Typically one or a few tissue constructs in eachreactor chamber or cartridge

• Gradients in pO2, pH, metabolites, and growthfactors across the tissue constructs

• There may also be substantial gradients across thereactor, and hence across the exterior of the tissueconstruct

• External gradients can be minimized by increasingthe medium recirculation rate and optimizing theflow distribution

• Required medium recirculation rate can often bedecreased by providing O2 transport along a reactorchamber

• Flow distribution can be predicted usingmathematical models and evaluated by measuringthe RTD or visualizing the flow of rheoscopicparticles or dye

• Large-scale production of individually-packagedtissue models (e.g., ATS bioartificial skinreplacements) adds the challenge of providing equalmedium flow to many parallel chambers


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Extracorporeal Bioartificial Organs

• Bioartificial kidneys (BAKs) and livers (BALs) thatare used as a bridge to organ transplant willnormally operate under conditions of kidney andliver failure, respectively

• Thus, cells in BAKs and BALs are likely to beexposed to high levels of toxic metabolites that willresult in a deterioration of organ function over time

• One approach to maintaining organ function is tocycle two identical bioartificial organs between on-line (exposed to patient fluids) and off-line(perfused with culture medium to restore cellfunction) configurations

• Another option is to simultaneously expose cells toseparate flow circuits of patient fluids and anutritive medium* three-compartment BAL (Nyberg et al., 1993)* in vitro toxicity assay with the apical side of an

epithelial construct exposed to a test agent, while thebasolateral side is perfused with culture medium(Pasternak and Miller, 1996)

pO2 and pH Gradients in Tissues

• Mean pO2 in bone marrow is ~50 mm Hg. (Ishikawa& Ito 1988)

• Rat mesentery tissue pO2 values decrease by ~16mm Hg thirty µm from a blood vessel, and continueto decrease at greater distances. (Yaegashi et al. 1996)

• The lowest pO2 and pH values in tumors areobserved farthest from blood vessels. (Helmlinger etal. 1997)

• pH values in normal subcutaneous tissue decreaseas the distance from a blood vessel (pH 7.4) isincreased from 10 µm (pH 7.25) to 30 µm (pH 7.1).(Martin & Jain 1994)


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pO2 and pH Considerations

• pO2 and pH gradients are physiologically relevant

• There is in vitro evidence to suggest that pO2 andpH affect cell growth and differentiation in vivo

• pO2 has a marked effect on cytotrophoblast(Genbacev et al., 1997) and megakaryocyte(Mostafa et al., 1998) differentiation, in a mannerconsistent with tissue physiology

• pH modulates erythrocyte (McAdams et al., 1998)and granulocyte (Hevehan et al., 2000)differentiation, in a manner consistent with tissuephysiology

• pO2 and pH may also be useful for manipulatingcell differentiation in engineered tissue constructs

Measurement & Control of pO2 & pH Gradientsin Tissue Constructs

• NMR can be used to evaluate pH and pO2

* 1H chemical shift of water is very sensitive to pH* 19F spin-lattice relaxation rate of fluorocarbon

tracers is sensitive to pO2

• NMR spectroscopy has been used to measure pHand pO2 in HFBRs, cells in alginate beads, perfusedorgans, organs in vivo

• Fluorescence ratio imaging and phosphorescencequenching microscopy has been used to obtainhigh-resolution pH and pO2 profiles between bloodvessels in vivo (Helmlinger et al., 1997)

• Adaptation of this system for analysis of pH andpO2 gradients in perfused tissue constructs wouldhave tremendous utility

• It should be possible to produce better defined pO2

profiles in tissue constructs by controlling pO2 atseveral locations


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Tissue Characterizationin Heterogeneous Bioreactors

• Kinetic information on tissue structure and functionis important, especially for constructs seeded withcells from patients or patient-specific donors

• Obtaining such kinetic information is a majorchallenge

• Off-line analysis requires that a reactor chamber beharvested for each sample

• Culture medium samples can provide usefulinformation

• Metabolic activity (e.g., qgluc) may be used toestimate the cell concentration, but metabolicpatterns will vary with pO2 and pH across a tissueconstruct, and may also vary with differentiation

• Changes in metabolic patterns are evidenced bychanges in metabolic yield ratios (e.g., qgluc / qO2),so it is important to measure at least two metabolicparameters

• Tissue-specific metabolic activity (e.g., albuminand urea synthesis, ammonia and galactoseclearance, cytochrome P450 activity) may also beevaluated using medium samples


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Direct Analysis of Tissue Function

• Direct analysis may be problematic, but providesvaluable information

• TER measurement in a perfusion chamber withdefined apical and basolateral regions can be usedto evaluate epithelial barrier function (Pasternak andMiller, 1996)

• Impedance analysis provides additional informationon the uniformity of an epithelial construct (Fey-Lamprecht et al., 1998)

• Large MW fluorescent tracers can be used to followearly development of epithelial barrier function, aswell as the permeability of leakier tissues, such asvascular grafts

• Optical clarity of a bioartificial cornea could beevaluated in a specially-designed bioreactorchamber

Characterization of Tissue Structure

• NMR spectroscopy and microimaging haverecently been used to follow changes in tissuevolume, cellularity, bioenergetics, macromolecularcontent, and tissue heterogeneity during cartilageformation in a HFBR (Petersen et al., 1997; Potter etal. 1998)

• Tissue constructs that incorporate multiple celltypes present additional problems because theextent and nature of the interactions between thedifferent cell types are often crucial for effectivetissue function

• A punch biopsy system that does not compromisebioreactor integrity would facilitate repeatedsampling of the same tissue construct for kineticanalysis, and has potential for a quality controlproduct harvest and release assay


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Use of Surrogate Markers

• It may not be possible to measure desired cellproperties on a time-scale useful for process controlor harvesting decisions* stem cells that reconstitute hematopoiesis in vivo* neural stem cells that differentiate into various

lineages

• Desired cell properties may correlate with cellsurface marker expression or metabolic activity

• Hematopoietic stem cells appear to express CD34and Thy-1 (Young et al., 1999)

• Hematopoietic progenitor cell content has beenshown to correlate with qlac and qO2 (Collins et al.,1997; 1998)


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MASS TRANSFER ISSUES IN TISSUE ENGINEERING

Clark K. Colton

Mass transfer plays a crucial role in the survival and function of tissues that are composed of cells havinghigh metabolic rates and that are normally well vascularized. The same applies to tissue-engineeredconstructs from these cells, which require adequate supply of nutrients and oxygen, removal of wastes andmetabolic intermediates, and release of secreted products. These mass transfer limitations are important indifferent contexts, including: (1) tissue-engineered constructs used ex vivo or implanted in vivo; (2) and thesame constructs, or the cells and tissues thereof, maintained or expanded in vitro. In addition, in allogeneicand xenogeneic implantations with immunobarrier materials, the transport of antigens and of components ofthe humoral immune response are also important. All of these issues are significant to different extents in avariety of applications, including pancreas, liver and muscular tissues (e.g., heart). What follows will beillustrated largely in the context of the bioartificial pancreas, but the concepts and principles apply to otherapplications as well.

IMPLANTED CONSTRUCTS

In the absence of immune rejection, cell viability and function is limited by the supply of nutrients andoxygen, of which the latter is thought to be critical. The problem is most severe in immunoisolation (1-4)because oxygen must diffuse from the nearest blood supply through adjacent tissue, immunobarrier materials,and the implanted tissue itself, where it is consumed. Oxygen gradients and the distance that oxygen candiffuse before decreasing to lethal levels depends, among other things, upon the oxygen consumption rate perunit volume of tissue and the packing density of tissue. Thus, the problem is most severe under theconditions of high packing density, which is desirable in order to minimize the size of the implant.

Theoretical analyses have been carried out of oxygen consumption and diffusion in rectilinear, cylindrical,and spherical geometries, together with illustrative sample calculations to determine how much tissue can besupported under defined conditions (5,6). Despite the importance of this issue, there is remarkably littlequantitative experimental data in the literature. Results from a variety of studies, some unpublished, indicatethat, without inducing neovascularization at the host-implant surface, the use of high density tissue inimplants leads rapidly to death of most of the tissue. The only way in which virtually all of the tissueimplanted has survived is through use of very low tissue densities, i.e. tissue volume fractions in the range of0.5 – 5% (3,4). In only one study (7,8), with a planar diffusion chamber using vascularizing membranes, hasthe volume of tissue contained after loading and after specified periods of implantation been carefullyquantitated. The results suggest substantial loss of viable islet mass, primarily during the first few days aftertransplantation as a result of the hypoxic environment around the immunobarrier device, which presents themost severe oxygen-supply limitations.

The problem has also been observed in naked transplantation (9) in a study in which syngeneic islets weretransplanted under the kidney capsule of normal and diabetic mice. In grafts harvested one and three daysafter transplantation, islets had undergone substantial damage, as evidenced by the presence of apoptoticnuclei, large fused masses with central necrosis, and reduced graft insulin content. Tissue remodeling wasevident after seven days, and good vascularization and oxygenation of the remaining islets were achieved atday fourteen. Thus, the hypoxic environment for several days following transplantation is a problemcommon to both naked and immunobarrier transplantation.

A breakthrough in allogeneic human islet transplantation for treating Type 1 diabetes was recently reported(NY Times 5/27/00) in which eight out of eight islet recipients became insulin independent. A key feature of


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these transplants was that the islet mass used for each patient was the total amount collected from two orthree cadavers. Once published, these important results will be a driver for further developments in severalways: (1) They will increase interest and drive further work in islet transplantation; (2) they heighten theneed for alternative tissue sources, including tissue-engineered constructs, because cadaver islet supplies areinsufficient for meeting the need by more than an order of magnitude; and (3) they further substantiate thecase for initially poor engraftment and loss of precious tissue, presumably because of the initial hypoxicenvironment at the transplantation site.

A variety of techniques have recently been brought to bear on this problem. Some investigators havedeveloped ways to study transport to implanted devices in situ using perfusion and microdialysis methods (10-15). Others have applied methods to measure oxygen partial pressure, in one case with a microelectrodeunder the kidney capsule in which islets were implanted and in another with 19F nuclear magnetic resonance(NMR) imaging of perfluorocarbon-loaded alginate capsules in various body spaces.

New approaches are needed to address the problems associated with oxygen supply limitations and earlysteps have been taken in this direction. The first was discovery of a class of microporous membranes thatinduce neovascularization at the material-host tissue interface by virtue of the membrane microarchitecture(18) by a process that takes 2 to 3 weeks (19) to bring blood vessels close to the implant, thereby improvingoxygen delivery. Other have used release or infusion of angiogenic proteins through nonvascularizing (20)or vascularizing (21) membranes in order to increase vascularity near the device interface. Another wayinvestigated to increase oxygen supply to the tissue is by local in situ oxygen generation by electrolyticdecomposition of water in an electrolyzer in the form of a thin, multiplayer sheet within which theelectrolysis reactions take place (22). An entirely different approach was to employ insulin-secreting tissuethat are resistant to hypoxia, either by virtue of their natural properties (23) or by genetically engineering thecells to overexpress anti-apoptotic genes (24). Lastly, a new method for vascularizing a tissue-engineeredconstruct made use of a machined branching vascular bed (25).

TISSUES IN VITRO

Tissues and tissue-engineered constructs in vitro provide useful vehicles for examining mass transfer-relatedissues. For example, both theoretical models and in vitro experiments have been employed to examine theeffect of diffusional limitations of the observed kinetics of glucose-stimulated insulin secretion inintravascular devices (26) and spheric microcapsules (27). In vitro experiments have also examined theeffects of gradients in oxygen partial pressure on insulin secretion rate by islets (28) and insulin-secreting βcell lines (29), as well as the effects of the presence of nutrients in the culture medium on oxygenconsumption rate (30). Experiments with β cell lines encapsulated in alginate beads using a variety oftechniques including NMR spectroscopy (31–34) have been useful in demonstrating changes in metabolismthat occur in the encapsulation environment.

Primary tissue such as islets can be damaged during the isolation process and during subsequent culture aswell. It is critical that practical and rapid techniques be developed to assess the quality of tissue preparationsprior to transplantations. For this purpose we suggest (1) measurement of oxygen consumption rate as anindicator of the volume of viable tissue in the preparation, and (2) examination of NMR spectra using ahighly sensitive technique such as magic angle spinning in order to assess the presence of markers of celldamage. For example, recent studies with 1H NMR spectroscopy (35) have developed a correlation betweenapoptotic cell death and changes in relative heights of certain peaks. Methods such as these may be criticalto determine the volume of viable tissue and/or presence of damaged tissue in each preparation prior totransplantation. Because dead tissue is likely to be highly immunogenic, its presence in significant amountmay rule out clinical use of a particular preparation.

IMMUNOISOLATION

The desire to prevent or minimize immune rejection through use of immunobarrier materials poses additionalmass transfer issues that need to be resolved. For example, what should be the barrier permeation properties?Presumably, large proteins of the humoral immune response (IgG and larger) should be retained, whereas


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proteins like albumin and transferrin should be able to reach the cells. If the rate of antigen passage fromimplanted to host tissue induces a florid local immune reaction around the implant, then the immunoisolatedcells could be destroyed by other mechanisms: (1) consumption of nutrients and oxygen by surroundingimmune cells, leading to starvation of immunoisolated cells, and (2) passage of cytokines across theimmunobarrier, followed by stimulation of pathways leading to cell death.

There is almost a complete lack of data in the literature to provide understanding of the relationship betweenbarrier permeation properties and immunoisolation effectiveness. Furthermore, there are varying degrees ofsuccess reported between laboratories, even with use of similar tissue preparations and methods. Fortunately,there are some successes from which useful conclusions can be drawn. For example, vascularizingmicroporous membranes in planar diffusion chambers (36) have prevented immune rejection by allogeneicand autoimmunity mechanisms in NOD mice exhibiting spontaneous diabetes. These results suggest thatunder these conditions it is necessary only to prevent cell-cell contact, at least in rodents. Preventingrejection of xenogeneic tissue is more difficult. However, success has been reported in at least one studywith porcine islets encapsulated in alginate-poly-L-lysine microcapsules and implanted in spontaneouslydiabetic cynomologus monkeys (37). More work is needed to rationally design immunobarrier materialswith appropriate permeation properties.

REFERENCES

1. Aebischer, P., and Lysaght, M.J. (1995). Immunoisolation and cellular xenotransplantation. Xenotransplantation 3,43-48.

2. Colton, C.K. and Avgoustiniatos, E.S. (1995). Bioengineering in development of the hybrid artificial pancreas. J.Biomech. Eng. 113, 152-170.

3. Colton, C.K. (1995). Implantable bioartificial organs. Cell Transplant. 4, 415-436.

4. Avgoustiniatos, E.S., W.H., and Colton, C.K. (2000). Engineering challenges in immunoisolation devicedevelopment. In “Principles of Tissue Engineering” 2nd ed. (R.P. Lanza, R. Langer, and J. Vacanti, eds.), pp. 321-350, Academic Press, New York.

5. Avgoustiniatos, E.S. and Colton, C.K. (1997). Design considerations in immunoisolation. In “Principles of TissueEngineering” (R.P. Lanza, R. Langer, and W.L. Chick, eds.), pp. 336-346, R.G. Landes, Austin, TX.

6. Avgoustiniatos, E.S. and Colton, C.K. (1997). Effect of external oxygen mass transfer resistances on viability ofimmunoisolated tissue. Ann. N.Y. Acad. Sci. 831, 145-167.

7. Suziki, K., Bonner-Weir, S., Hollister-Lock, J., Colton, C.K., and Weir, G.C. (1998). Number and volume of isletstransplanted in immunobarrier devices. Cell Transplant. 7, 47-52.

8. Suziki, K., Bonner-Weir, S., Trivedi, N., Yoon, K.-H. Hollister-Lock, J., Colton, C.K., and Weir, G.C. (1998).Function and survival of macroencapsulated syngeneic islets transplanted into streptozotocin-diabetic mice.Transplantation 66, 21-28.

9. Davalli, A., Scaglia, L., Zangen, D., Hollister, J., Bonner-Weir, S., and Weir, G.C. (1996). Vulnerability of islets inthe immediate posttransplantation period – Dynamic changes in structure and function. Diabetes 45, 1161-1167.

10. Rafael, E., Wernerson, A., Arner, P., Wu, G.S., and Tibell A. (1999). In vivo evaluation of glucose permeability ofan immunoisolation device intended for islet transplantation: a novel application of the microdialysis technique. CellTransplant. 8, 317-326.

11. Rafael, E., Wernerson, A., Arner, P., and Tibell A. (1999). In vivo evaluation of insulin permeability of animmunoisolation device intended for islet transplantation: using the microdialysis technique. Eur. Surg. Res. 31,249-258.

12. Rafael, E., Gazelius, B., Wu, G.S., and Tibell A. (2000). Longitudinal studies on the microcirculation around theTheraCyte immunoisolation device, using the laser Doppler technique. Cell Transplant. 9, 107-113.

13. Sarver, J.G., Fournier, R.L, Goldblatt, P.J., Phares, T.L., Mertz, S.E., Baker, A.R., Mellon R.J., Horner, J.M., andSelman, S.H. (1995). Tracer technique to measure in vivo chemical transport rates within an implantable celltransplantation device. Cell Transplant. 4, 201-217.

14. Whalen, D.W., Ding, Z., and Fournier, R.L. (1999). Method for measuring in vivo oxygen transport rates in abioartificial organ. Tissue Eng. 5, 81-89.


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15. Krohn, J.A., Baker, A.R., Long, J.L., Fournier, R.L., and Byers, J.P. (1999). In vivo measurement of solute transportrates in a bioartificial organ. Tissue Eng. 5, 197-206.

16. Carlsson, P.-O., Palm, F., Andersson, A., and Liss, P. (2000). Chronically decreased oxygen tension in rat pancreaticislets transplanted under the kidney capsule. Transplantation 69, 761-766.

17. Noth, U., Grohn, P., Jork, A., Zimmermann, U., Haase, A., and Lutz, J. (1999). 19F-MRI in vivo determination of thepartial oxygen pressure in perfluorocarbon-loaded alginate capsules implanted into the peritoneal cavity anddifferent tissues. Magn. Reson. Med. 42, 1039-1047.

18. Brauker, J.H., Carr-Brendel, V.E., Martinson, L.A., Crudele, J., Johnston, W.D., and Johnson, R.C. (1995).Neovascularization of synthetic membranes directed by membrane microarchitecture. J. Biomed. Mater. Res. 29,1517-1524.

19. Padera, R.F., and Colton, C.K. (1996). Time course of membrane microarchitecture-driven neovascularization.Biomaterials 17, 277-284.

20. Hunter, S.K., Kao, J.M., Wang, Y., Benda, J.A., and Rodgers, V.G.J. (1999). Promotion of neovascularizationaround hollow fiber bioartificial organs using biologically active substances. ASAIO J. 45, 37-40.

21. Trivedi, N., Steil, G.M., Colton, C.K., Bonner-Weir, S., and Weir, G.C. (2000). Improved vascularization of planarmembrane diffusion devices following continuous infusion of vascular endothelial growth factor. Cell Transplant. 9,115-124.

22. Wu., H., Avgoustiniatos, E.S., Swette, L., Bonner-Weir, S., Weir, G.C., and Colton, C.K. (1999). In situelectrochemical oxygen generation with an immunoisolation device. Ann. N.Y. Acad. Sci. 875, 105-125.

23. Wright Jr., J.R., Yang, H., and Dooley, K.C. (1998). Tilapia-A source of hypoxia-resistant islet cells forencapsulation. Cell Transplant. 3, 299-307.

24. Dupraz, P, Rinsch, C., Pralong, W., Rolland, E., Zufferey, R., Trono, D., and Thorens, B. (1999). Lentiviral deliveryof anti-apoptotic genes in β-cells improves their resistance to hypoxia and cytokines. Cell Transplant. 8, 175.

25. Kaihara, S., Borenstein, J., Kohn, R., Lalan, S., Ochoo, E.R., Ravens, M., Pien, H., Cunningham, B., and Vacanti,J.P. (2000). Silicon micromachining to tissue engineer branched vascular channels for liver fabrication. Tissue Eng.6, 105-118.

26. Colton, C.K., and Weinless, N.L. (1987). A theoretical model for insulin secretion rate in a hybrid artificialpancreas. In “Artificial Organs”. (J.D. Andrade et al., eds.), pp. 641-665, VCH Publishers, New York.

27. Tziampazis, E., and Sambanis, A. (1995). Tissue engineering of a bioartificial pancreas: Modeling the cellenvironment and device function. Biotechnol. Prog. 11, 115-126.

28. Dionne, K.E., Colton, C.K., and Yarmush, M.L. (1993). Effect of hypoxia on insulin secretion by isolated rat andcanine islets of Langerhans. Diabetes 42, 12-21.

29. Papas, K.K, Long Jr., R.C., Constantinidis, I., and Sambanis, A. (1996). Effects of oxygen on metabolic andsecretory activities of βTC3 cells. Biochim. Biophys. Acta. 1291, 163-166.

30. Papas, K.K., and Jarema, M.A.C. (1998). Glucose-stimulated insulin secretion is not obligatorily linked to anincrease in O

2 consumption in βHC9 cells. Am. J. Physiol. 275, E1100-1106.

31. Constantinidis, I., and Sambanis, A. (1995). Towards the development of artificial endocrine tissues: 31P NMRspectroscopic studies of immunoisolated, insulin-secreting AtT-20 cells. Biotechnol. Bioeng. 47, 431-443.

32. Constantinidis, I., Mukundan, N.E., Gamcsik, M.P., and Sambanis, A. (1997). Towards the development of abioartificial pancreas: A 13C NMR study on the effects of alginate/poly-L-lysine/alginate entrapment on glucosemetabolism by βTC3 mouse insulinoma cells. Cell. Mol. Biol. 43, 721-729.

33. Papas, K.K, Long Jr., R.C., Sambanis, A., and Constantinidis, I. (1999). Development of a bioartificial pancreas: I.Long-term propagation and basal and induced secretion from entrapped βTC3 cell cultures. Biotechnol. Bioeng. 66,219-230.

34. Papas, K.K, Long Jr., R.C., Sambanis, A., and Constantinidis, I. (1999). Development of a bioartificial pancreas: II.Effects of oxygen on long-term entrapped βTC3 cell cultures. Biotechnol. Bioeng. 66, 231-237.

35. Blankenberg, F.G., Katsikis, P.D., Storrs, R.W., Beaulieu, C., Spielman, D., Chen, J.Y., Naumovski, L., and Tait,J.F. (1997). Quantitative analysis of apoptotic cell death using proton nuclear magnetic resonance spectroscopy.Blood 89, 3778-3786.


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36. Loudovaris, T., Jacobs, S., Young, S., Maryanov, D., Brauker, J., and Johnson, R.C. (1999). Correction of diabeticnod mice with insulinomas implanted within Baxter immunoisolation devices. J. Mol. Med. 72, 219-222.

37. Sun, Y., Ma, X., Zhou, D., Vacek, I., and Sun, A.M. (1996) Normalization of diabetes in spontaneously diabeticcynomologus monkeys by xenografts of microencapsulated porcine islets without immunosuppression. J. Clin.Invest. 98, 1417-1422.


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THE ENGINEERING OF VASCULAR SUBSTITUTES AND THE ROLE OF

BIOMECHANICS

Robert M. Nerem

INTRODUCTION

Many of the tissues being addressed today in tissue engineering research laboratories are ones which functionnormally in a mechanical environment and which have a biomechanical function. These include cartilage,bone, blood vessels, and heart valves. For each of these there are important biomechanical considerations; infact, there is a major biomechanical function for each of these.

In Atlanta the Georgia Tech/Emory Center (GTEC) for the Engineering of Living Tissues was established bythe National Science Foundation in 1998 with a five-year Engineering Research Center Award. The goal ofGTEC is to develop the core, enabling technologies which can provide a foundation for the emerging tissueengineering industry. GTEC’s research program has been organized into three programmatic areas. Withineach of these there are three core technology thrusts. The three programmatic areas are: (1) cardiovascularsubstitutes, with the main focus being the development of a blood vessel substitute; (2) encapsulated celltechnologies, with the main application here being the bioartificial pancreas; and (3) orthopaedic tissueengineering, with applications being both bone constructs for the healing of bone defects and tissue-engineered cartilage.

Although in each of these programmatic areas there is a strong influence of mechanical or physical factors, itis the first of the three, i.e. cardiovascular substitutes, which is the focus of this discussion. Furthermore, inthe application of tissue engineering to the cardiovascular system, there obviously are a number of differentpossible targets. This includes myocardial patches, small diameter blood vessel substitutes, and heart valves.Although each of these is important in the context of patient need, and taken together represent the basicelements required in the tissue engineering of an entire heart, in this presentation it is the small diameterblood vessel substitute which will be used as the example.

VASCULAR BIOLOGIC RESPONSES

For native arteries, as well as for veins, there is a biomechanical environment imposed by the hemodynamicsof the cardiovascular system. This will be equally well true for a blood vessel substitute. For an artery, vein,or an arterial substitute, the vessel wall and the cells incorporated within it undergo a condition of cyclicstrain imposed by the pulsatile pressure. The endothelium, on the other hand, resides in a more complexbiomechanical environment, one in which an endothelial cell sees pressure directly, rides on a basementmembrane which is being cyclically stretched, and is also exposed to a time-varying viscous stress imposedby the pulsatile flow.

Over the several decades there has been extensive research into the influence of these biomechanical factorson vascular biology [1]. This has included the study of the different cell types as well as tissue responses.

CRITICAL ISSUES

For many in the cardiovascular research field, the successful development of a small diameter blood vesselsubstitute represents the holy grail. As such there is a long history of research and development aimed at this


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goal, and the application of tissue engineering represents a somewhat recent entry. This started with theinvestigation of the endothelial seeding of synthetic material vascular grafts [2], with the purpose ofproviding a non-thrombogenic lining, and more recently efforts have moved to even more biologicalapproaches. These latter attempts include those based on collagen-gel technology [3-4], those employingcell-seeded scaffold approaches [5-6], the cell self assembly technique of Auger and his co-workers [7-8],and various acellular concepts [9]. Although many of these concepts have shown considerable promise, thereare critical issues which still remain unresolved for all of them.

Some of the critical issues for tissue-engineered blood vessel substitutes are as follows.

• Immune acceptability, off-the-shelf availability

• Functional three-dimensional construct

• Short in vitro culture period

• Control of biological responses

• Validated test beds to predict clinical efficacy

In the above a critical issue is the engineering of a functional three-dimensional substitute or construct. To dothis requires providing the following characteristics.

• Sufficient mechanical strength

• Elastic mechanical properties

• Vasoactivity with contractile phenotype SMC

• Adherent, quiescent endothelium

All of these in some way represent a role for biomechanics in tissue engineering, and in GTEC’s researchprogram these are being addressed through the three core technologies as described next.

CORE TECHNOLOGIES

As noted earlier, each of the three programmatic areas of GTEC’s program, including that of cardiovascularsubstitutes, is structured to include three core technology thrusts. These are cell technology, constructtechnology, and the technologies for integration into living systems.

The goal of cell technology is to address the issue of cell sourcing and to develop the technologies necessaryfor the manipulation of cell function. The desirable function of cells in a tissue-engineered blood vesselsubstitute may involve the production of extracellular matrix, the secretion of proteins either constitutively orin response to physiological stimuli, or the expression of other specific differentiated cell functions. Asalready noted, a critical issue is that of cell sourcing. GTEC’s philosophy is that the broad-based availabilityof a tissue-engineered blood vessel substitute will only happen through the use of allogeneic cells. In this,genetic engineering can very much be an ally, being used to enhance certain functional cell characteristics.Long term, the future of tissue engineering may depend on the development of stem cell technology [10].Whatever the case, if non-autologous cells are employed, then the issue of immune acceptance must beaddressed.

The goal of construct technology is to develop the technology base required for the fabrication of a bloodvessel substitute with a three-dimensional architecture and functional characteristics which mimic that of anative artery. Blood vessels are complex, multicellular composites serving multifunctional needs through athree-dimensional matrix architecture, responsive to environmental cues both at the macroscopic andmicroscopic cellular levels, and with interfaces to their surroundings. In regard to the functionalcharacteristics described in the previous section, these are being addressed as part of construct technology.Clearly a blood vessel substitute must have sufficient strength, i.e. a burst pressure which is comparable tothat of the native vessel being replaced. The mechanical properties of a blood vessel substitute, however,must be more than simply ones exhibiting sufficient strength. Such a construct must possess elastic, evenvisco-elastic, mechanical properties which match those of a native vessel. This is because of the belief thatthere will be increased patency if the impedance of the implant matches that of the tissue being replaced.


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None of the current blood vessel substitute concepts exhibits this, with a major “flaw” apparently being thelack of elastin and thus the elasticity which goes with it. The ideal tissue-engineered blood vessel substitutealso must exhibit vasoactivity. This is another mechano-functional characteristic. To do this will requiresmooth muscle cells of a contractile phenotype.

Finally, as the interface between flowing blood and the underlying vessel wall, the endothelium is a criticalelement in the construct. It must not only serve to provide non-thrombogenicity, but also be the signalinginterface. To do this it obviously must be adherent and confluent; however, it also must exhibit the normalendothelial responsiveness to the mechanical environment. As such it must be quiescent, i.e. not activatedfrom a surface molecular expression point of view.

Construct technology extends to providing for the manufacturing of the product. A critical issue is the scaleup of the fabrication process, for it is one thing to make one-of-a-kind of a certain construct on the bench top,but far different to be able to manufacture 1000 a week with the reproducible quality which would berequired by FDA. Furthermore, once manufactured, how does one preserve a living cell product so as toprovide the off-the-shelf availability desired by clinicians? Also, in addition to the adaptation and remodelingwhich will take place when you implant a tissue-engineered blood vessel substitute, a construct can undergoremodeling in vitro when exposed to a prescribed biochemical/biomechanical environment. At Georgia Techthis has been investigated using a tubular, cell-seeded collagen construct. Using a human aortic smoothmuscle cell construct as an example, the application of a 10 percent strain, 1 Hz frequency strain to conditionthe construct for four days results in an remodeled morphology, altered collagen and elastin mRNAexpression, and an enhanced mechanical strength. Initial studies suggest that there is an important role formatrix metalloproteinases in the remodeling induced by mechanical conditioning. Although the results areobviously specific to the specific cell-seeded construct which we are using as a model system, we believethat there is wider applicability to these results. Furthermore, these studies suggest that in vitro remodeling ofa tissue construct can be induced, and that it will be important to determine the culture conditions that will beoptimal for achieving the desired construct characteristics.

Finally, the goal of integration into living systems is to develop the technologies required to evaluate andbetter integrate tissue-engineered blood vessel substitutes into the in vivo system. This thrust focuses on thedevelopment of quantitative assays to test tissue-engineered constructs for viability and function, theacquisition of performance data after biological incorporation, and the determination of the healing orimmunologic response of the living host to the construct. When a substitute is implanted into a living system,there can be a variety of biological responses as part of the adaptation and remodeling which takes place. Inthe case of a blood vessel substitute, and as with many other different tissues, the biomechanical environmentcan have important effects on these biological responses. This is particularly true of intimal thickening andplatelet deposition.

CONCLUDING DISCUSSION

Biomechanics has long been recognized to have a significant role in regulating vascular biology and thepathobiologic responses associated with the genesis and progression of disease. It equally has an importantrole in tissue engineering. This is particularly true for the engineering of a blood vessel substitute, oneinvolving living endothelial and smooth muscle cells. This is because many of the critical issues are ones inwhich there are strong biomechanical influences. This is particularly true when one attempts to address thefunctional characteristics of any blood vessel substitute. It thus is important that biomechanics becomeinvolved in tissue engineering and accept the challenges presented by this emerging area.

REFERENCES

1. Ku, D.N., and Nerem, R.M., “Hemodynamic Influences on Vascular Injury, Repair, and Remodeling,”Cardiovascular Toxicology, eds. S.P. Bishop and W.D. Kerns, Elsevier Science, New York, pp. 241-253, 1997.

2. Zilla, P. and Greisler, H.L. Tissue Engineering of Vascular Prosthetic Grafts, R.G. Landes Company, Austin, Texas,1999.


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3. Weinberg C.B., Bell E., A Blood Vessel Model Constructed from Collagen and Cultured Vascular Cells. Science.231:397-99, 1986.

4. Seliktar, D., Black, R.A., Vito, R.P., Nerem, R.M., “Dynamic Mechanical Conditioning of Collagen Gel BloodVessel Constructs Induces Remodeling in vitro,” Accepted for publication in Ann of Biomed Eng, 2000.

5. Niklason L.E., Gao J., Abbott W.M., Hirschi K.K., Houser S., Marini R., and Langer R. Functional Arteries Growninto Vitro. Science. 284:489-493, April 16, 1999.

6. Landeen, L.K., Graham, D.A., Alexader H.G., Garcia A., Fino M.R., Lee, A.A., and Ratcliffe A., Effects of ScaffoldDesign on Tissue Formation in Tissue-Engineered Vascular Grafts, Proceedings 3rd Annual Hilton Head Workshopon Tissue Engineering, Gene Delivery, and Regenerative Healing, Georgia Tech, Atlanta, GA, 1999.

7. L’Heureux N., Germain L., Labbe R., and Auger, F.A. In vitro Construction of a Human Blood Vessel fromCultured Vascular Cells: A morphological study. Journal of Vascular Surgery. 17(3):4099-509, 1993.

8. L’Heureux N., Paquet S., Labbe R., and Auger, F.A. A Completely Biological Tissue-engineered Human BloodVessel. FASEB. 12(1):47-56, January 1998.

9. Hyunh, T., Abraham G., Murray J., Brockbank K., Hagen P.O., and Sullivan S., Nature Biotechnology 17:1083-1086, 1999.

10. Solter, D., and Gearhart, J., Putting Stem Cells to Work. Science, 283:1468-1470, 1999.


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FUNCTIONAL TISSUE ENGINEERING OF ARTICULAR CARTILAGE : THE

ROLE OF BIOMECHANICS

Farshid Guilak

ABSTRACT

Tissue engineering approaches have used implanted cells, scaffolds, DNA, protein and/or protein fragmentsto replace or repair injured or diseased tissues and organs. Despite early successes, tissue engineers havefaced challenges in repairing or replacing tissues that serve a predominantly biomechanical function, such asarticular cartilage. An evolving discipline called “functional tissue engineering” (FTE) seeks to address thesechallenges. In this presentation, articular cartilage is used as a paradigm to present some of the generalprinciples of functional tissue engineering that may serve to improve the rational engineering of tissues forthe replacement or repair of load-bearing structures of the body.

INTRODUCTION

Damage or injury to the articular cartilage of the joints is the cause of significant pain and disability to thepopulation, yet few treatment options are available to promote cartilage repair. Tissue engineeringapproaches for cartilage repair have used implanted cells, scaffolds, and other methods in an attempt toreplace or repair injured or diseased tissue. Tissue engineering merges aspects of engineering and biology.Many rapid achievements in this field have arisen in part from significant advances in cell and molecularbiology (e.g., the isolation and manipulation of cells, genes, and growth factors), biomaterials (new andinnovative delivery vehicles), and the integration of biology and materials to deliver viable cells incompatible support structures.

Many of the tissues and organs to be replaced serve important biomechanical functions, and despite earlysuccesses, tissue engineers have faced challenges in repairing or replacing tissues that serve predominantlybiomechanical roles in the body, such as articular cartilage. In fact, the properties of these tissues are criticalto their proper function in vivo. An evolving discipline called “functional tissue engineering” (FTE) seeks toaddress these challenges. Here, the example of articular cartilage as a biomechanical structure in the body isused as a paradigm to present some of the general principles of functional tissue engineering.

In order for tissue engineers to effectively repair or replace these load-bearing structures, they must address anumber of significant questions on the interactions of engineered constructs with mechanical forces, both invivo and in vitro. To address these issues, the United States National Committee on Biomechanics (USNCB)formed a subcommittee in 1998 that adopted the concept of “Functional Tissue Engineering”, or FTE. TheUSNCB’s goals in advancing FTE were to: 1) increase awareness among tissue engineers about theimportance of restoring “function” when engineering tissue constructs; 2) identify the critical structural andmechanical requirements needed for each tissue engineered construct; and 3) encourage tissue engineers toincorporate these functional criteria in design and manufacturing processes to optimize the overall success ofengineered tissues.

What constitutes “success” will be expected to differ among tissues. For example, tissues or systems that aredesigned to prolong life may tolerate a lower margin for error than those that are designed to improve thequality of life. The difficulty in performing a procedure, and the duration of a specific treatment, may alsofactor into its perceived success. For example, therapies of replacement or regeneration of blood vessels or


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bone might be expected to last the lifetime of the individual, while replacement of cartilage may beconsidered successful if it delays total joint replacement for five to ten years.

FUNCTIONAL TISSUE ENGINEERING

Several principles of functional tissue engineering have recently been proposed in a general format that ismeant to be applicable to a variety of tissues [1]. These principles are not considered to be complete, but areproposed as an initial set of questions to be addressed and are expected to evolve along with the field oftissue engineering. It is hoped that the answers to these questions will significantly influence the quality ofthe implants that tissue engineers design and the repair outcomes after surgery.

1. What are the in vivo stress and/or strain histories in normal tissues for a variety of activities?

Knowing the mechanical “thresholds” that normal tissues encounter for different in vivo activities is criticalto effectively designing tissue repairs/replacements that can meet functional demands after surgery. Whilethese measurements can be difficult to make, they establish the patterns of activity and bounds of expectedusage. In many tissues (e.g., blood vessels, bone, tendon), there is considerable information on the in vivostresses and strains that are encountered under different conditions [1,2,3]. For other tissues such as cartilage,however, there is a lack of information on the normal in vivo mechanical environment. Peak stresses inarticular cartilage loading against an endoprosthesis have been shown to exceed 18 MPa [4], but stresses in anormal joint have been more difficult to measure. Experiments using pressure-sensitive films suggest thatnormal stresses may range from 5 to 10 MPa in vivo [5]. Little information has been reported on thedeformation behavior of cartilage in vivo. One of the few reports of this nature utilized sequential planarradiographs to show that cartilage deforms no more than 15-20% under physiologic conditions [6]. Becauseof the difficulties involved in measuring the in situ loads and deformations of cartilage, many investigatorshave used theoretical models of joint contact to predict these parameters [7]. This is an area that requiresfurther study and will likely benefit from both theoretical and experimental approaches.

2. What are the complete mechanical properties of the native tissues?

Due to their complex structure and composition, most biological tissues can be classified from a materialstandpoint as inhomogeneous, viscoelastic, nonlinear, and anisotropic materials. The fundamental basis forthese behaviors is not fully understood, and may differ among different tissues. Importantly, it remains to bedetermined which aspects of these mechanical properties are essential for the normal, healthy function ofdifferent tissues, as well as for successful tissue-engineered replacements. To effectively design tissue-engineered implants, it is important to understand the sub-failure and failure properties of the native tissue.Sub-failure properties can be measured within the bounds of expected loading conditions. However,knowledge of the failure properties of native tissues may prove crucial, especially if tissue engineeredimplants are to be designed with safety factors like the native tissue. Such failure testing provides bothstructural and material properties of the tissue to be replaced. “Structural” properties allow comparison oftissues or constructs to a baseline functional level, and incorporate the role of important morphologicalparameters, such as tissue geometry or joint congruence. “Material” properties are valuable in that they maybe determined in simplified loading configurations, in combination with physiologically relevant theoreticalmodels, but may be used to describe the mechanical response of a material to any loading history. Articularcartilage, for example, normally exhibits little or no wear with millions of cycles of loading that may reachten times body weight. Its unique mechanical and tribological properties, which are unparalleled in man-made bearings, have been attributed to the complex structure and composition of extracellular tissue matrix[8]. It is now well accepted that the primary mechanism of viscoelasticity in cartilage results from frictionalinteractions between the solid and fluid phases, although there is evidence that the solid matrix exhibitsintrinsic viscoelasticity. Cartilage also exhibits highly nonlinear mechanical properties such as strain-dependent moduli, strain-dependent hydraulic permeability, and a difference of nearly two orders ofmagnitude in tensile and compressive moduli. These properties are also anisotropic, particularly in tension,and vary significantly with distance from the tissue surface and with site on the joint. More complex butequally important mechanical behaviors include the presence of internal swelling pressures that give rise toinhomogeneous residual stresses within normal articular cartilage. Finally, cartilage possesses importantgeometric and material characteristics that endow it with unique frictional properties. This low coefficient of


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friction, coupled with fluid-dependent mechanisms of load support, allow for minimal tissue wear under arelatively harsh mechanical environment. A thorough understanding of these intrinsic properties may benecessary before a repair tissue can be designed in a rational manner.

3. Which subset of these mechanical properties should be prioritized as design parameters inengineered tissues?

The abundance of data now available in the literature from biomechanical testing does not, however, answerone important question facing the tissue engineer: Which, if any, of these parameters should be used whendesigning an engineered repair or replacement? Realistically, it will be difficult if not impossible to matchall of the material properties and structure of native tissues with tissue engineered constructs. At this point,the relative importance of these different characteristics in the design of an engineered repair tissue is notknown. Thus, it would be advantageous to prioritize the multitude of complex properties that are sought. Fewstudies have reported quantitative measures of the material properties of tissue-engineered cartilage. Of thefew that have, focus has been placed on the compressive moduli and hydraulic permeability of cartilage [9-12]. While these properties are likely the most logical starting points, the relative importance of recreatingthe tissue’s compressive properties in comparison to tensile properties, failure properties, or frictionalproperties, for example, are not yet known. On a structural basis, many questions remain regarding therelative importance of recreating the native tissue and joint architecture. For example, most attempts atarticular cartilage regeneration have sought complete integration between host and repair tissues [13,14].Complete graft integration has been used as a “gold standard” of cartilage repair, yet the long-termimplications of either complete or incomplete tissue integration are not fully understood. Additional factorssuch as the congruence of opposing cartilage surfaces in a joint may have important implications for thestress environment within the joint [15]. At this point, however, few tissue-engineering approaches are ableto precisely control the structure and geometry of newly formed cartilage. An important consideration insuch studies may also be the choice of an animal model and how representative the native tissue structure andproperties are compared to the human [16]. Since all of these complex issues are unlikely to be addressed atonce, it becomes important to prioritize their relative influence on the overall success of a given procedure.

4. What standards define the “success” of an engineered repair or replacement?

Assessment of the outcome of successful functional tissue engineering will require quantitative measures ofgraft properties, structure, and composition. Some aspects of the repair outcome may be inferior, but othermechanical factors of the repairs and replacements might be suitable. With an emphasis on the materialproperties and structure of tissue-engineered grafts, it will be necessary to quantify and report outcomemeasures directly related to the functional behavior of the tissues. Given the biomechanical nature of manytissue-engineered products, there have been surprisingly few reports of the material or structural properties ofengineered tissues. In articular cartilage, for example, several investigators have reported either mechanicalproperties of grafts prior to implantation [11] or at sacrifice [9,10,17]. An important direction for the fieldwill be the development of new methodologies that will allow assessment of the material or structuralproperties of engineered tissues in a non-invasive or minimally invasive manner. For example, the use ofbiological markers of tissue metabolism [18], in vivo (arthroscopic) biomechanical probes [19], magneticresonance imaging [20], and other techniques such as CT, ultrasound, or DEXA to assess tissue function mayprove to be critical in longitudinal studies of tissue engineered repair, particularly in the clinical setting.

5. What are the effects of mechanical factors in vivo on the physiology of engineered tissues?

Once implanted in the body, engineered constructs of cells and/or matrices will be subjected to a complexbiomechanical environment, potentially consisting of time-varying changes in stresses, strains, fluid pressure,fluid flow, and cellular deformation [21]. It is now well accepted that these various physical factors have thecapability to influence the biological activity of normal tissues, and therefore, may play an important role inthe eventual success or failure of engineered grafts. In this regard, it would be important to bettercharacterize the diverse array of physical signals that engineered cells may experience in vivo, as well as theirbiological response to such potential stimuli. This information may provide important insights into the long-term capabilities of engineered constructs to maintain the proper cellular phenotype. For example, thechemical, mechanical and architectural properties of the scaffold over time in vivo will affect thephysiological response of the cells. As a result, the mechanical influence on the cells will be related to the


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mechanical properties of the scaffold, the mechanical boundary conditions acting on the construct, and theinteractions between the cells and the scaffold. In addition, the shape and morphology of the cells will berelated to the cell/scaffold interactions. All of these factors may contribute to the cell’s ability to respond toboth mechanical and biologic signals, and subsequently to synthesize and express extracellular matrix.

6. What are the effects of physical factors on engineered cells or tissues in vitro?

Mechanical stress is an important modulator of cell physiology, and there is significant evidence thatphysical factors may be used to improve or accelerate tissue regeneration and repair in vitro. For example,early studies showed that cyclic mechanical stretch of skeletal myofibers increased the alignment ofmyotubes that assembled into “organoids” in culture [22]. In other studies, mechanical stretch has beenshown to increase cellular alignment, proliferation, and protein synthesis in many different cell types [23].As the cells used in the tissue-engineered construct must be organized to rapidly synthesize the desiredextracellular matrix, control of cellular alignment a priori may provide important advantages in controllingmatrix deposition, and presumably, a more rapid development of functional biomechanical properties. Morerecently, mechanical “bioreactors” have been used to increase matrix deposition in tissue-engineeredcartilage by exposing chondrocytes to fluid flow [24], simulated hypogravity [12], and cyclic compression[25,26]. Recent studies have shown improved success of tissue-engineered systems such as blood vessels bypreconditioning grafts with pulsatile fluid flow and pressure [27].

FUTURE DIRECTIONS

Clearly, the field of tissue engineering needs to establish functional criteria that will help those who seek todesign and manufacture these repairs and replacements. Scale-up, packaging, storage and handlingproperties are also critical. The implants must be capable of retaining their mechanical, structural, andbiological integrity during large-scale production, packaging, storage, and importantly, during surgicalimplantation. Understanding those conditions that preserve the character of the implants may be essential forthe success of tissue-engineered products. Other rapidly evolving new technologies may have a significantimpact on functional tissue engineering, and it is important to consider the principles of functional tissueengineering in light of the role of novel growth factors, new biomaterials, gene therapy, and other changingtechnologies.

ACKNOWLEDGMENTS

Supported by NIH grants AR43876 and AG15768. The author would like to thank Drs. David Butler, StevenGoldstein, and David Mooney for many significant contributions to the concepts outlined in this paper.

REFERENCES

[1] Butler, D., Goldstein, S.A., and Guilak, F., 2000. J Biomech Engng, Vol. 122, to appear.

[2] Komi, P. V., 1990. J Biomech, Vol. 23, pp. 23-34.

[3] Malaviya, P., Butler, D.L., Korvick, D.L. and Proch, F.S., 1998. J Biomech, Vol. 31, pp. 1043-9.

[4] Hodge, W.A., Carlson, K.L., Fijan, R.S., et al. J Bone Jt Surg, Vol. 71A, pp. 1378-1386.

[5] Ronsky, J.L., Herzog, W., Brown, T.D., et al., 1995. J Biomech, Vol. 28, pp. 977-983.

[6] Armstrong, C.G., Bahrani, A.S., and Gardner, D.L., 1979, J Bone Jt Surg, Vol. 61A, pp. 744-55.

[7] Donzelli, P.S., Spilker, R.L., Ateshian, G.A. et al., 1999. J Biomech, Vol. 32, pp. 1037-47.

[8] Mow, V.C., Ratcliffe, A., and Poole, A.R., 1992, Biomaterials, Vol. 13, pp. 67-97.

[9] Kwan, M.K., Coutts, R.D., Woo, S.L., and Field, F.P., 1989, J Biomech, Vol. 22, pp. 921-930.

[10] Mow, V.C., Ratcliffe, A., Rosenwasser, M.P., et al., 1991, J Biomech Eng, Vol. 113, pp. 198-207.

[11] Vunjak-Novakovic, G., Martin, I., Obradovic, B., et al, 1999, J Orthop Res, Vol. 17, pp. 130-138.


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[12] Freed, L.E., Langer, R., Martin, I., et al, 1997, Proc Nat Acad Sci, Vol. 94, pp. 13885-13890.

[13] Hunziker, E.B., and Rosenberg, L.C., 1996, J Bone Joint Surg Am, Vol. 78, pp, 721-733.

[14] Ahsan,T., and Sah, R.L., 1999, Osteoarthritis Cartilage, Vol. 7, pp. 29-40.

[15] Ateshian, G. A., Rosenwasser, M. P., and Mow, V. C., 1992, J. Biomech., Vol. 25, pp. 591-607.

[16] Hunziker, E. B., 1999, Clin Orthop, Vol. 367, pp. S135-146.

[17] Wakitani, S., Goto, T., Pineda, S.J., et al. J Bone Joint Surg, Vol. 76A, pp. 579-592.

[18] Lohmander, L.S. and Felson, D.T., 1997, J Rheumatol, Vol. 24, pp. 782-785.

[19] Lyyra, T., Jurvelin, J., Pitkanen, P., et al., 1995, Med Eng Phys, Vol. 17, pp. 395-399.

[20] Karvonen, R.L., Negendank, W.G., Fraser, S.M., et al., 1990, Ann Rheum Dis, Vol. 49, pp. 672-5.

[21] Guilak, F., Sah, R.L., and Setton, L.A., 1997, In: Basic Orthopaedic Biomechanics. Mow, VC and Hayes, WC.(Eds.) Philadelphia, Lippincott Raven, pp. 179-207.

[22] Vandenburgh, H.H., 1982, Dev Biol, Vol. 93, pp. 438-443.

[23] Buckley, M.J., Banes, A.J., Levin, L.G., et al., 1988, Bone Min, Vol. 4, pp. 225-236.

[24] Wu, F., Dunkelman, N., Peterson, A., et al., 1999, Ann New York Acad Sci, Vol. 875, pp. 405-11.

[25] Buschmann, M.D., Gluzband, Y., Grodzinsky, A.J., et al., 1995, J Cell Sci, Vol. 108, pp. 1497-08.

[26] Mauck, R.L., Soltz, M.A., Wang, C.B., et al. 2000, J Biomech Engng, Vol. 122, to appear.

[27] Niklason, L.E., Gao, J., Abbott, W.M., et al., 1999, Science, Vol. 284, pp. 489-493.


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CRYOPRESERVATION1

Jens O.M. Karlsson and Mehmet Toner

1 “Cryopreservation” by Jens O.M. Karlsson and Mehmet Toner from PRINCIPLES OF TISSUE ENGINEERING,Second Edition, edited by R.P. Lanza et al., copyright © by Academic Press, reprinted by permission of the publisher.All rights of reproduction in any form reserved. Submitted for this workshop proceedings by Mehmet Toner.


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