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Bioengineered muscle constructs and tissue-based therapy for cardiac disease
Progress in Pediatric Cardiology 21 (2006) 167 – 171 www.elsevier.com/locate/ppedcard
Bioengineered muscle constructs and tissue-based therapy for cardiac disease Herman H. Vandenburgh * Department of Pathology, Brown Medical School/Miriam Hospital, RISE Research Bldg., 164 Summit Ave., Providence, R.I. 02906, USA Available online 8 February 2006
Abstract Bioengineering of contractile muscle tissues and organs for disease treatment is currently in the preclinical experimental stage of development. Cell biology over the last several decades has primarily focused on breaking down tissues and cells to smaller and smaller components to understand their functioning at the molecular level. We are just beginning to understand how to reassemble these components back into larger functional units. The merging of the fields of traditional engineering, biomaterials, cell biology, and computer science will lead to the ex vivo engineering tissues and organs for many cardiovascular applications in the future. For pediatric cardiology, bioengineered contractile tissues may serve as force generating cardiac patches, heart valve papillary muscle substitutes, or as living therapeutic protein delivery Fdevices_ when bioengineered from genetically engineered muscle cells. D 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Tissue engineering; Force generation; Gene therapy; Therapeutic protein delivery
1. Introduction
2. In vitro bioengineering
Bioengineering is a new discipline for the in vitro construction of implantable tissues such as pancreatic islets, liver, skin, cartilage, bone, muscle, and blood vessels [1,2]. The primary goal of bioengineering is to replace defective organs, but with current technology, bioengineering human skeletal and cardiac muscle for structural repair of the damaged heart is not possible. Organized contractile forces of sufficient magnitude required for supplementing the failing forces generated by diseased or aged muscle is not currently possible, and will require the development of new, more advanced bioengineering processes. In this short review, the progress that has been made to date in engineering of contractile muscle will be outlined and areas requiring further study indicated. A more complete review of skeletal muscle tissue engineering has been recently published [3]. The potential use of current technologies for non-force generating clinical applications of striated muscle such as therapeutic protein delivery will be summarized.
The isolation and growth of skeletal and cardiac muscle cells in monolayer tissue cultures is well established for rodent and human skeletal muscle cells [4] and for rodent neonatal [5] and adult [6] cardiomyocytes. Primary human cardiomyocytes are much more difficult to isolate and grow in vitro than skeletal muscle cells, making the current interest in embryonic and adult stem cell transformation into functioning cardiomyocytes [7,8] an attractive alternative cell source for future bioengineering applications. Striated muscle cells can be reorganized in tissue culture into in vivo like two-dimensional structures by several methods. Neonatal rodent cardiomyocytes grown on an elastic substratum and mechanically stretched can be aligned into thin sheets of organized rod-shaped contractile cells [9,10]. Adult rodent cardiomyocytes are also aligned parallel to each other using mechanical forces, but they are difficult to maintain in vitro in their rod-shaped differentiated contractile state for more than several days [11]. Electrical field forces can also be used to organize rodent cardiomyocytes [12]. Skeletal muscle cells grown in monolayers can also be aligned into parallel arrays of
* Tel.: +1 401 793 4273; fax: +1 401 793 4534. E-mail address: [email protected]. 1058-9813/$ - see front matter D 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ppedcard.2005.11.003
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contractile multi-nucleated muscle fibers (myofibers) by mechanical forces [13]. In all cases, many variables determine the response of the cells to these organizing mechanical forces which are similar to those which occur during embryonic development. The pattern of mechanical loading, percent stretch, rate of stretch and rest periods are all important factors, and each cell type responds differently to these parameters. Future bioengineering studies will be required to understand the mechanisms underlying the cellto-cell and cell-to-extracellular matrix interactions involved in tissue organization, and to utilize this knowledge in the bioengineering of functional organs [14]. Contractile muscle cells have also been bioengineered into three-dimensional cardiac [15 – 18] and skeletal muscle tissues [19 – 21] with some of the functional and structural properties associated with cardiac and skeletal muscle. A standard method used for bioengineering these tissues is to suspend several million cells in a small volume of ice cold extracellular matrix collagen solution and cast the cell matrix mixture into a mold of the desired shape (Fig. 1). When warmed to 37 -C, the collagen matrix gels and over several days undergo cell cytoskeletal-induced gel contracture. If Fattachment_ sites are provided in the mold, the contracted cell:gel mixture will remain under passive tension, and this tension will align the proliferating cells parallel to each other in the direction of the matrix tension vector (Fig. 1, lower left insert). As the differentiating cells interact with their neighboring cells and the matrix, they
form parallel arrays of postmitotic skeletal muscle fibers or cardiomyocytes linked by intercalated disks, allowing directed force generation when stimulated. Repetitive mechanical forces can be applied in vitro to these Fbioartificial muscle_ (BAM) tissues using various electromechanical stimulation hardware, and chronic repetitive loading improves their functionality [22,23]. But we still do not have the capability to generate adult striated muscle cells in vitro of the appropriate size and cell packing density to generate the directed forces necessary to perform functional work in vivo. The best active forces generated to date are only 2– 5% of those generated by in vivo adult muscle [18,20] (Vandenburgh et al, unpublished data). The limited force generating capacity of bioengineered muscle results partly from the current ability to provide nutrients to the multilayered in vitro tissues only through diffusion since they lack a vasculature. Because of striated muscles’ high metabolic needs when performing work, skeletal and cardiac muscles are very highly vascularized in vivo, with approximately one capillary associated with each cell. Manipulation of the tissue culture medium, growth factor supplements, perfusion conditions, electrical stimulation, mechanical loading/conditioning, and co-culturing with blood vessel forming cells [24 – 26] or neurons [27] will eventually generate more functional muscle tissues for transplantation. Currently, these parameters have to be individually determined by trial and error. In the future, computer-controlled feed back systems will be developed to
Fig. 1. Bioengineering a bioartificial muscle (BAM). Passive internal forces generated by proliferating muscle cells cast in a collagen extracellular matrix gel causes the cell:gel mixture to contract away from the mold walls and remain attached to end support structures (stainless steel screening, lower right photo). The cell:gel mixture is under passive tension, as seen when one end of the construct is detached from its end support (photo, lower left). The passive tension aligns the fusing muscle cells with the matrix tension vector, causing them to form aligned contractile muscle fibers which express sarcomeric tropomyosin (whole mount immunostain, middle left photo). In cross section (photo, upper left), a high packing density of muscle fibers (myosin immunostained) occurs if the BAM diameter is 200 Am or less.
H.H. Vandenburgh / Progress in Pediatric Cardiology 21 (2006) 167 – 171
optimize the bioengineering process, based on maximizing force generating capacity of the tissue. Enhancing tissues through genetic engineering may also help circumvent some of the current roadblocks to bioengineering tissues in vitro for their successful transplantation and functionality in vivo. For example, genetic engineering of BAMs to locally secrete growth factors such as insulin-like growth factor-1 significantly increases the number of BAM skeletal muscle fibers and their force generation ability (Shansky et al., submitted for publication; Lee et al., in preparation). In another example, genetic engineering of bioengineered muscle tissues to locally secrete the angiogenic protein vascular endothelial growth factor (VEGF) stimulates their rapid in vivo vascularization [28] and muscle cell survival [29]. These results indicate an important research area for future bioengineering with genetically modified cells.
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human skeletal muscle myoblasts injected into the damaged heart for functional repair [34 – 36]. Only a small percent of the injected cells survives and partially differentiates, while the large majority of cells (> 90%) undergoes rapid apoptosis and die. As with in vitro bioengineering, genetically enhanced muscle cells offer an interesting option to improve muscle cell transplantation protocols for this ‘‘in vivo bioengineering’’ approach. For example, skeletal muscle myoblasts genetically engineered to express the intercalated disc protein connexin43 form better intercellular gap junctions in vitro [37], and cells engineered to express either the angiogenic protein VEGF [38], or a fibrotic inhibitor protein IL-1 inhibitor [39] performed better when injected into an ischemic heart model. The later IL-1 inhibitor also improved muscle cell survival when implanted into the heart [40]. This combination of genetic engineering of cells for transplantation survival and functionality holds much promise for future in vivo bioengineering for clinical cardiovascular applications.
3. In vivo bioengineering We are currently able to isolate adult human skeletal muscle stem cells (satellite cells), expand them to billions of myogenic cells (myoblasts) in tissue culture, and reconstitute them in vitro into organized tissue containing partially differentiated contractile myofibers as outlined above. A similar readily available source of adult cardiac stem cell source is not available, although there is evidence of their in vivo existence [30,31]. Proliferating skeletal myoblasts have been shown to successfully generate functional contractile tissue (in vivo bioengineering) when injected into damaged heart tissue in animal studies [32,33], and there are a number of current clinical trials underway using adult
4. Bioengineering striated muscle for therapeutic protein delivery While the structural use of the bioengineered contractile tissue is a distant goal, several near term goals are more feasible. These include their use for therapeutic drug testing for muscle wasting diseases [19,41], high content drug screening in vitro [23,42], and as implantable protein delivery Fdevices_ following genetic engineering of the cells to secrete a therapeutic protein (Fig. 2). We have successfully engineered skeletal muscle BAMs to secrete systemically therapeutic levels of recombinant proteins such as
Fig. 2. Autologous bioengineered tissue delivery of a therapeutic protein.
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growth hormone (GH), vascular endothelial growth factor (VEGF), and the blood clotting Factor 9 (F9) for up to 6 months in mice. The use of genetically modified myoblasts to bioengineer implantable BAMs for the chronic delivery of therapeutic proteins would have numerous potential advantages over other forms of muscle cell and gene protein therapy. First, myoblast fusion efficiency into myofibers in vitro is several orders of magnitude greater than that which occurs in vivo from injected myoblasts. Thus, while in vivo fusion efficiencies in human myoblast cell therapy are on the order of 0.5% to 5% [43], in vitro fusion efficiencies of myoblasts are 60% to 80%. The majority of the myofiber nuclei in BAMs formed from these myoblasts therefore contain the foreign transgene, making a more reproducible, high-secreting delivery implant from non-dividing cells [44]. Second, myoblast viral transduction efficiencies are more targeted and generally greater in vitro (90 –100%) than in vivo [29]. Third, formation of BAMs in vitro allows the preimplantation monitoring of protein secretory levels, leading to predictive protein dosage delivery in vivo [28,44]. Fourth, implantation of non-migratory, non-proliferating postmitotic cells containing the foreign transgene reduces the possibility of proliferating cell transformation, migration, and tumor formation [45]. Finally, implanting a defined organ-like structure allows surgical removal of the implant, if necessary [44]. The recent instance of in vitro retroviral gene therapy-related leukemia in several SCID infants (which resulted from the specific nature of the transgene inserted and stem cell type utilized [46]), warrants a cautious approach for any new gene therapy protocols. Thus, potential retrievability of implanted genetically engineered cells is critically important for any new viral vector transgene insertional approach along with the potential future use of nonviral gene transfer protocols [47]. The feasibility of long term recombinant protein delivery from BAMs was initially established using murine myoblasts stably transduced with the GH transgene [44]. These cells were tissue engineered in vitro into BAMs containing organized postmitotic skeletal myofibers secreting high levels of GH. Subcutaneous (subQ) implantation of the BAMs under tension into syngeneic mice led to the rapid and stable appearance of physiological levels of GH in the serum for up to six months. The implanted BAMs retained their preimplantation structure, allowing surgical removal, and rapid disappearance of GH from the blood [44]. Maintenance of tension on the myofibers in the BAMs was critical for preventing myofiber atrophy and a > 70% reduction in recombinant protein output [44]. When implanted subQ under tension into hindlimb-unloaded mice, the atrophy of the host soleus and plantaris muscles was significantly attenuated compared to animals implanted with non-GH secreting BAMs [48]. The BAMs secreting GH were more effective in attenuating muscle atrophy than daily injections of GH. Similar studies have been performed with murine BAMs delivering VEGF to an ischemic limb [28], and autologous sheep BAMs delivering VEGF to the sheep
heart [49]. Bioengineered tissue delivery of therapeutic proteins is therefore conceptually proven, but scale-up to a clinically useful product in humans is still a major challenge. Recently, human BAMs (HBAMs) bioengineered with FDA approved materials were shown to deliver F9 systemically for greater than eighty days in a predictive manner in an immunodeficient mouse [50], a preclinical animal model accepted by the FDA for hemophilia gene therapy [51]. This predictable and reproducible delivery of rF9 from in vitro engineered and implanted HBAMs is in marked contrast to the poor predictability and tumorgenicity of implanted embryonic stem cells genetically engineered in vitro to express F9 [52]. Coupling the idea of genetically enhancing implanted tissue survival with systemic therapeutic protein delivery, we showed that localized VEGF from implanted HBAMs elevated several-fold the systemic long term delivery of F9 [29]. Major challenges in this application of engineered tissues include improved cell survival following transplantation and increased therapeutic protein secretion levels that would be useful in humans. Future technological developments in HBAMs as protein delivery devices will provide useful information for eventual bioengineering of skeletal muscle implants as structural contractile tissue substitutes. For pediatric cardiology, bioengineered contractile tissues may serve as force generating cardiac patches, heart valve papillary muscle substitutes, or as living therapeutic protein delivery Fdevices_ when bioengineered from genetically engineered muscle cells. We are just beginning to learn to use the tools of cell and molecular biology for bioengineering tissues which will eventually revolutionize medical treatment for many cardiovascular disorders.
Acknowledgements Support for this work was provided by a NIH Senior Fellowship (HL079767) and NASA Life Sciences Grant NCC2-1062. The author thanks the Miriam Hospital for sabbatical support during which this review was written.
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H.H. Vandenburgh / Progress in Pediatric Cardiology 21 (2006) 167 – 171 [7] Hassink RJ, Dowell JD, Brutel R, Doevendans PA, Field LJ. Stem cell therapy for ischemic heart disease. Trends Mol Med 2003;9: 436 – 41. [8] Hughes S. Cardiac stem cells. J Pathol 2002;197:468 – 78. [9] Vandenburgh HH, Solerssi R, Shansky J, Adams J, Henderson SA. Mechanical stimulation of organogenic cardiomyocyte growth in vitro. Am J Physiol (Cell Physiol) 1996;270:C1284 – 92. [10] Decker ML, Janes DM, Barclay MM, Harger L, Decker RS. Regulation of adult cardiocyte growth: effects of active and passive mechanical loading. Am J Physiol Heart Circ Physiol 1997;272:H2902 – 18. [11] Samuel J-L, Vandenburgh HH. Mechanically induced orientation of adult rat cardiac myocytes in vitro. In Vitro 1990;26:905 – 14. [12] Radisic M, Park H, Shing H, et al. Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds. Proc Natl Acad Sci U S A 2004;101:18129 – 34. [13] Vandenburgh HH. Dynamic mechanical orientation of skeletal myofibers in vitro. Dev Biol 1982;93:438 – 43. [14] Lutolf MP, Hubbell JA. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat Biotechnol 2005;23:47 – 55. [15] Papadaki M, Bursac N, Langer R, Merok J, Vunjak-Novakovic G, Freed LE. Tissue engineering of functional cardiac muscle: molecular, structural, and electrophysiological studies. Am J Physiol Heart Circ Physiol 2001;280:H168 – 78. [16] Bursac N, Papadaki M, Cohen RJ, et al. Cardiac muscle tissue engineering: toward an in vitro model for electrophysiological studies. Am J Physiol Heart Circ Physiol 1999;277:H433 – 44. [17] Zimmermann WH, Fink C, Kralisch D, Remmers U, Weil J, Eschenhagen T. Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes. Biotechnol Bioeng 2000;68:106 – 14. [18] Zimmermann WH, Schneiderbanger K, Schubert P, et al. Tissue engineering of a differentiated cardiac muscle construct. Circ Res 2002;90:223 – 30. [19] Vandenburgh HH, Swasdison S, Karlisch P. Computer aided mechanogenesis of skeletal muscle organs from single cells in vitro. FASEB J 1991;5:2860 – 7. [20] Dennis RG, Kosnik PE. Excitability and isometric contractile properties of mammalian skeletal muscle constructs engineered in vitro. In Vitro Cell Dev Biol Anim 2000;6(5):327 – 35. [21] Okano T, Matsuda T. Tissue engineered skeletal muscle: preparation of highly dense, highly oriented hybrid muscular tissues. Cell Transplant 1998;7(1):71 – 82. [22] Fink C, Ergu¨n S, Kralisch D, Remmers U, Weil J, Eschenhagen T. Chronic stretch of engineered heart tissue induces hypertrophy and functional improvement. FASEB J 2000;14:669 – 79. [23] Powell C, Smiley B, Mills J, Vandenburgh H. Mechanical stimulation improves tissue-engineered human skeletal muscle. Am J Physiol Cell Physiol 2002;283:C1557 – 65. [24] Okano T, Matsuda T. Muscular tissue engineering: capillary-incorporated hybrid muscular tissues in vivo tissue culture. Cell Transplant 1998;7(5):435 – 42. [25] Ennett AB, Mooney DJ. Tissue engineering strategies for in vivo neovascularisation. Expert Opin Biol Ther 2002;2:805 – 18. [26] Levenberg S, Rouwkema J, Macdonald M, et al. Engineering vascularized skeletal muscle tissue. Nat Biotechnol 2005 [advanced online publication]. [27] Bach AD, Beier JP, Stark GB. Expression of Trisk-a´51, agrin and nicotinic-acetycholine receptor e-subunit during muscle development in a novel three-dimensional muscle-neuronal co-culture system. Cell Tissue Res 2003;314:263 – 74. [28] Lu Y, Shansky J, Del Tatto M, Ferland P, Wang X, Vandenburgh H. Recombinant vascular endothelial growth factor secreted from tissueengineered bioartificial muscles promotes localized angiogenesis. Circulation 2001;104(5):594 – 9. [29] Thorrez L, Vandenburgh H, Collen D, Shansky J, VandenDriessche T, Chuah M. Enhanced factor IX delivery from bioengineered hybrid human skeletal muscle co-expressing VEGF. Am Soc Gene Therapy 8th Annual Mtg. (abstract).
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[30] Urbanek K, Quaini F, Tasca G, et al. Intense myocyte formation from cardiac stem cells in human cardiac hypertrophy. Proc Natl Acad Sci U S A 2003;100(18):10440 – 5. [31] Kajstura J, Leri A, Finato N, Di Loreto C, Beltrami CA, Anversa P. Myocyte proliferation in end-stage cardiac failure in humans. Proc Natl Acad Sci U S A 1998;95(15):8801 – 5. [32] Murry CE, Wiseman RW, Schwartz SM, Hauschka SD. Skeletal myoblast transplantation for repair of myocardial necrosis. J Clin Invest 1996;98:2512 – 23. [33] Taylor DA, Atkins BZ, Hungspreugs P, et al. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nat Med 1998;4:929 – 33. [34] Hagege AA, Carrion C, Menasche P, et al. Viability and differentiation of autologous skeletal myoblast grafts in ischaemic cardiomyopathy. Lancet 2003;361:491 – 2. [35] Menasche P. Skeletal muscle satellite cell transplantation. Cardiovasc Res 2003;58:351 – 7. [36] Menasche P. Skeletal myoblast for cell therapy. Coron Artery Dis 2005;16(2):105 – 10. [37] Suzuki K, Brand NJ, Allen S, et al. Overexpression of connexin 43 in skeletal myoblasts: relevance to cell transplantation to the heart. J Thorac Cardiovasc Surg 2001;122(4):759 – 66. [38] Suzuki K, Murtuza B, Smolenski RT, et al. Cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor-expressing skeletal myoblasts. Circulation 2001;104(12 Suppl 1):I207 – 12. [39] Murtuza B, Suzuki K, Bou-Gharios G, et al. Transplantation of skeletal myoblasts secreting an IL-1 inhibitor modulates adverse remodeling in infarcted murine myocardium. Proc Natl Acad Sci U S A 2004;101(12):4216 – 21. [40] Suzuki K, Murtuza B, Beauchamp JR, et al. Role of interleukin-1beta in acute inflammation and graft death after cell transplantation to the heart. Circulation 2004;110(11 Suppl 1):II219 – 24. [41] Vandenburgh H, Chromiak J, Shansky J, DelTatto M, LeMaire J. Space travel directly induces skeletal muscle atrophy. FASEB J 1999;13:1031 – 8. [42] Wakatsuki T, Fee JA, Elson EL. Phenotypic screening for pharmaceuticals using tissue constructs. Curr Pharm Biotech 2004;5:181 – 9. [43] Gussoni E, Blau HM, Kunkel LM. The fate of individual myoblasts after transplantation into muscles of DMD patients. Nat Med 1997;3:970 – 7. [44] Vandenburgh HH, Del Tatto M, Shansky J, et al. Tissue engineered skeletal muscle organoids for reversible gene therapy. Hum Gene Ther 1996;7:2195 – 200. [45] Vile RG, Russell SJ. Retroviruses as vectors. Br Med Bull 1995; 51:12 – 30. [46] Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. LMO2associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003;302(5644):415 – 9. [47] Lu QL, Bou-Gharios G, Partridge TA. Non-viral gene delivery in skeletal muscle: a protein factory. Gene Ther 2003;10:131 – 42. [48] Vandenburgh H, Del Tatto M, Shansky J, et al. Attenuation of skeletal muscle wasting with recombinant human growth hormone secreted from a tissue-engineered bioartificial muscle. Hum Gene Ther 1998; 9:2555 – 64. [49] Lu Y, Shansky J, Del Tatto M, et al. Therapeutic potential of implanted tissue-engineered bioartificial muscles delivering recombinant proteins in the sheep heart. Ann N Y Acad Sci 2001;961:78 – 82. [50] Vandenburgh H, Thorrez L, Collen D, Shansky J, VandenDriessche T, Chuah MKL. Long-term transgene expression in vivo following implantation of adult human skeletal muscle stem cells transduced with lentiviral vectors. TESI Annual Mtg, vol. 181. Abstract. [51] Vandenburgh HH. Factor VIIIdeltaB Transduced Autologous Muscle Analog. Pre-IND Information Package for CBER Meeting. [52] Fair JH, Cairns BA, LaPaglia MA, et al. Correction of factor IX deficiency in mice by embryonic stem cells differentiated in vitro. Proc Natl Acad Sci U S A 2005;102:2958 – 63.
Bioengineered muscle constructs and tissue-based therapy for cardiac disease Herman H. Vandenburgh * Department of Pathology, Brown Medical School/Miriam Hospital, RISE Research Bldg., 164 Summit Ave., Providence, R.I. 02906, USA Available online 8 February 2006
Abstract Bioengineering of contractile muscle tissues and organs for disease treatment is currently in the preclinical experimental stage of development. Cell biology over the last several decades has primarily focused on breaking down tissues and cells to smaller and smaller components to understand their functioning at the molecular level. We are just beginning to understand how to reassemble these components back into larger functional units. The merging of the fields of traditional engineering, biomaterials, cell biology, and computer science will lead to the ex vivo engineering tissues and organs for many cardiovascular applications in the future. For pediatric cardiology, bioengineered contractile tissues may serve as force generating cardiac patches, heart valve papillary muscle substitutes, or as living therapeutic protein delivery Fdevices_ when bioengineered from genetically engineered muscle cells. D 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Tissue engineering; Force generation; Gene therapy; Therapeutic protein delivery
1. Introduction
2. In vitro bioengineering
Bioengineering is a new discipline for the in vitro construction of implantable tissues such as pancreatic islets, liver, skin, cartilage, bone, muscle, and blood vessels [1,2]. The primary goal of bioengineering is to replace defective organs, but with current technology, bioengineering human skeletal and cardiac muscle for structural repair of the damaged heart is not possible. Organized contractile forces of sufficient magnitude required for supplementing the failing forces generated by diseased or aged muscle is not currently possible, and will require the development of new, more advanced bioengineering processes. In this short review, the progress that has been made to date in engineering of contractile muscle will be outlined and areas requiring further study indicated. A more complete review of skeletal muscle tissue engineering has been recently published [3]. The potential use of current technologies for non-force generating clinical applications of striated muscle such as therapeutic protein delivery will be summarized.
The isolation and growth of skeletal and cardiac muscle cells in monolayer tissue cultures is well established for rodent and human skeletal muscle cells [4] and for rodent neonatal [5] and adult [6] cardiomyocytes. Primary human cardiomyocytes are much more difficult to isolate and grow in vitro than skeletal muscle cells, making the current interest in embryonic and adult stem cell transformation into functioning cardiomyocytes [7,8] an attractive alternative cell source for future bioengineering applications. Striated muscle cells can be reorganized in tissue culture into in vivo like two-dimensional structures by several methods. Neonatal rodent cardiomyocytes grown on an elastic substratum and mechanically stretched can be aligned into thin sheets of organized rod-shaped contractile cells [9,10]. Adult rodent cardiomyocytes are also aligned parallel to each other using mechanical forces, but they are difficult to maintain in vitro in their rod-shaped differentiated contractile state for more than several days [11]. Electrical field forces can also be used to organize rodent cardiomyocytes [12]. Skeletal muscle cells grown in monolayers can also be aligned into parallel arrays of
* Tel.: +1 401 793 4273; fax: +1 401 793 4534. E-mail address: [email protected]. 1058-9813/$ - see front matter D 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ppedcard.2005.11.003
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H.H. Vandenburgh / Progress in Pediatric Cardiology 21 (2006) 167 – 171
contractile multi-nucleated muscle fibers (myofibers) by mechanical forces [13]. In all cases, many variables determine the response of the cells to these organizing mechanical forces which are similar to those which occur during embryonic development. The pattern of mechanical loading, percent stretch, rate of stretch and rest periods are all important factors, and each cell type responds differently to these parameters. Future bioengineering studies will be required to understand the mechanisms underlying the cellto-cell and cell-to-extracellular matrix interactions involved in tissue organization, and to utilize this knowledge in the bioengineering of functional organs [14]. Contractile muscle cells have also been bioengineered into three-dimensional cardiac [15 – 18] and skeletal muscle tissues [19 – 21] with some of the functional and structural properties associated with cardiac and skeletal muscle. A standard method used for bioengineering these tissues is to suspend several million cells in a small volume of ice cold extracellular matrix collagen solution and cast the cell matrix mixture into a mold of the desired shape (Fig. 1). When warmed to 37 -C, the collagen matrix gels and over several days undergo cell cytoskeletal-induced gel contracture. If Fattachment_ sites are provided in the mold, the contracted cell:gel mixture will remain under passive tension, and this tension will align the proliferating cells parallel to each other in the direction of the matrix tension vector (Fig. 1, lower left insert). As the differentiating cells interact with their neighboring cells and the matrix, they
form parallel arrays of postmitotic skeletal muscle fibers or cardiomyocytes linked by intercalated disks, allowing directed force generation when stimulated. Repetitive mechanical forces can be applied in vitro to these Fbioartificial muscle_ (BAM) tissues using various electromechanical stimulation hardware, and chronic repetitive loading improves their functionality [22,23]. But we still do not have the capability to generate adult striated muscle cells in vitro of the appropriate size and cell packing density to generate the directed forces necessary to perform functional work in vivo. The best active forces generated to date are only 2– 5% of those generated by in vivo adult muscle [18,20] (Vandenburgh et al, unpublished data). The limited force generating capacity of bioengineered muscle results partly from the current ability to provide nutrients to the multilayered in vitro tissues only through diffusion since they lack a vasculature. Because of striated muscles’ high metabolic needs when performing work, skeletal and cardiac muscles are very highly vascularized in vivo, with approximately one capillary associated with each cell. Manipulation of the tissue culture medium, growth factor supplements, perfusion conditions, electrical stimulation, mechanical loading/conditioning, and co-culturing with blood vessel forming cells [24 – 26] or neurons [27] will eventually generate more functional muscle tissues for transplantation. Currently, these parameters have to be individually determined by trial and error. In the future, computer-controlled feed back systems will be developed to
Fig. 1. Bioengineering a bioartificial muscle (BAM). Passive internal forces generated by proliferating muscle cells cast in a collagen extracellular matrix gel causes the cell:gel mixture to contract away from the mold walls and remain attached to end support structures (stainless steel screening, lower right photo). The cell:gel mixture is under passive tension, as seen when one end of the construct is detached from its end support (photo, lower left). The passive tension aligns the fusing muscle cells with the matrix tension vector, causing them to form aligned contractile muscle fibers which express sarcomeric tropomyosin (whole mount immunostain, middle left photo). In cross section (photo, upper left), a high packing density of muscle fibers (myosin immunostained) occurs if the BAM diameter is 200 Am or less.
H.H. Vandenburgh / Progress in Pediatric Cardiology 21 (2006) 167 – 171
optimize the bioengineering process, based on maximizing force generating capacity of the tissue. Enhancing tissues through genetic engineering may also help circumvent some of the current roadblocks to bioengineering tissues in vitro for their successful transplantation and functionality in vivo. For example, genetic engineering of BAMs to locally secrete growth factors such as insulin-like growth factor-1 significantly increases the number of BAM skeletal muscle fibers and their force generation ability (Shansky et al., submitted for publication; Lee et al., in preparation). In another example, genetic engineering of bioengineered muscle tissues to locally secrete the angiogenic protein vascular endothelial growth factor (VEGF) stimulates their rapid in vivo vascularization [28] and muscle cell survival [29]. These results indicate an important research area for future bioengineering with genetically modified cells.
169
human skeletal muscle myoblasts injected into the damaged heart for functional repair [34 – 36]. Only a small percent of the injected cells survives and partially differentiates, while the large majority of cells (> 90%) undergoes rapid apoptosis and die. As with in vitro bioengineering, genetically enhanced muscle cells offer an interesting option to improve muscle cell transplantation protocols for this ‘‘in vivo bioengineering’’ approach. For example, skeletal muscle myoblasts genetically engineered to express the intercalated disc protein connexin43 form better intercellular gap junctions in vitro [37], and cells engineered to express either the angiogenic protein VEGF [38], or a fibrotic inhibitor protein IL-1 inhibitor [39] performed better when injected into an ischemic heart model. The later IL-1 inhibitor also improved muscle cell survival when implanted into the heart [40]. This combination of genetic engineering of cells for transplantation survival and functionality holds much promise for future in vivo bioengineering for clinical cardiovascular applications.
3. In vivo bioengineering We are currently able to isolate adult human skeletal muscle stem cells (satellite cells), expand them to billions of myogenic cells (myoblasts) in tissue culture, and reconstitute them in vitro into organized tissue containing partially differentiated contractile myofibers as outlined above. A similar readily available source of adult cardiac stem cell source is not available, although there is evidence of their in vivo existence [30,31]. Proliferating skeletal myoblasts have been shown to successfully generate functional contractile tissue (in vivo bioengineering) when injected into damaged heart tissue in animal studies [32,33], and there are a number of current clinical trials underway using adult
4. Bioengineering striated muscle for therapeutic protein delivery While the structural use of the bioengineered contractile tissue is a distant goal, several near term goals are more feasible. These include their use for therapeutic drug testing for muscle wasting diseases [19,41], high content drug screening in vitro [23,42], and as implantable protein delivery Fdevices_ following genetic engineering of the cells to secrete a therapeutic protein (Fig. 2). We have successfully engineered skeletal muscle BAMs to secrete systemically therapeutic levels of recombinant proteins such as
Fig. 2. Autologous bioengineered tissue delivery of a therapeutic protein.
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growth hormone (GH), vascular endothelial growth factor (VEGF), and the blood clotting Factor 9 (F9) for up to 6 months in mice. The use of genetically modified myoblasts to bioengineer implantable BAMs for the chronic delivery of therapeutic proteins would have numerous potential advantages over other forms of muscle cell and gene protein therapy. First, myoblast fusion efficiency into myofibers in vitro is several orders of magnitude greater than that which occurs in vivo from injected myoblasts. Thus, while in vivo fusion efficiencies in human myoblast cell therapy are on the order of 0.5% to 5% [43], in vitro fusion efficiencies of myoblasts are 60% to 80%. The majority of the myofiber nuclei in BAMs formed from these myoblasts therefore contain the foreign transgene, making a more reproducible, high-secreting delivery implant from non-dividing cells [44]. Second, myoblast viral transduction efficiencies are more targeted and generally greater in vitro (90 –100%) than in vivo [29]. Third, formation of BAMs in vitro allows the preimplantation monitoring of protein secretory levels, leading to predictive protein dosage delivery in vivo [28,44]. Fourth, implantation of non-migratory, non-proliferating postmitotic cells containing the foreign transgene reduces the possibility of proliferating cell transformation, migration, and tumor formation [45]. Finally, implanting a defined organ-like structure allows surgical removal of the implant, if necessary [44]. The recent instance of in vitro retroviral gene therapy-related leukemia in several SCID infants (which resulted from the specific nature of the transgene inserted and stem cell type utilized [46]), warrants a cautious approach for any new gene therapy protocols. Thus, potential retrievability of implanted genetically engineered cells is critically important for any new viral vector transgene insertional approach along with the potential future use of nonviral gene transfer protocols [47]. The feasibility of long term recombinant protein delivery from BAMs was initially established using murine myoblasts stably transduced with the GH transgene [44]. These cells were tissue engineered in vitro into BAMs containing organized postmitotic skeletal myofibers secreting high levels of GH. Subcutaneous (subQ) implantation of the BAMs under tension into syngeneic mice led to the rapid and stable appearance of physiological levels of GH in the serum for up to six months. The implanted BAMs retained their preimplantation structure, allowing surgical removal, and rapid disappearance of GH from the blood [44]. Maintenance of tension on the myofibers in the BAMs was critical for preventing myofiber atrophy and a > 70% reduction in recombinant protein output [44]. When implanted subQ under tension into hindlimb-unloaded mice, the atrophy of the host soleus and plantaris muscles was significantly attenuated compared to animals implanted with non-GH secreting BAMs [48]. The BAMs secreting GH were more effective in attenuating muscle atrophy than daily injections of GH. Similar studies have been performed with murine BAMs delivering VEGF to an ischemic limb [28], and autologous sheep BAMs delivering VEGF to the sheep
heart [49]. Bioengineered tissue delivery of therapeutic proteins is therefore conceptually proven, but scale-up to a clinically useful product in humans is still a major challenge. Recently, human BAMs (HBAMs) bioengineered with FDA approved materials were shown to deliver F9 systemically for greater than eighty days in a predictive manner in an immunodeficient mouse [50], a preclinical animal model accepted by the FDA for hemophilia gene therapy [51]. This predictable and reproducible delivery of rF9 from in vitro engineered and implanted HBAMs is in marked contrast to the poor predictability and tumorgenicity of implanted embryonic stem cells genetically engineered in vitro to express F9 [52]. Coupling the idea of genetically enhancing implanted tissue survival with systemic therapeutic protein delivery, we showed that localized VEGF from implanted HBAMs elevated several-fold the systemic long term delivery of F9 [29]. Major challenges in this application of engineered tissues include improved cell survival following transplantation and increased therapeutic protein secretion levels that would be useful in humans. Future technological developments in HBAMs as protein delivery devices will provide useful information for eventual bioengineering of skeletal muscle implants as structural contractile tissue substitutes. For pediatric cardiology, bioengineered contractile tissues may serve as force generating cardiac patches, heart valve papillary muscle substitutes, or as living therapeutic protein delivery Fdevices_ when bioengineered from genetically engineered muscle cells. We are just beginning to learn to use the tools of cell and molecular biology for bioengineering tissues which will eventually revolutionize medical treatment for many cardiovascular disorders.
Acknowledgements Support for this work was provided by a NIH Senior Fellowship (HL079767) and NASA Life Sciences Grant NCC2-1062. The author thanks the Miriam Hospital for sabbatical support during which this review was written.
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