Tissue Engineering and the Challenges Within

Transcription

1 Cell Transplantation, Vol. 15, Supplement 1, pp. S11 S15, /06 $ Printed in the USA. All rights reserved. E-ISSN Copyright 2006 Cognizant Comm. Corp. Tissue Engineering and the Challenges Within Sara L. Wargo, Thangappan Ravi Kumar, and Alan J. Russell McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA Researchers face many challenges, both scientific and societal, in the field of tissue engineering. Herein we discuss the challenges in material design, selection of therapeutic cell source, the in vitro culturing of cells and materials, and finally the integration of the cultured construct into the body. We focus special attention on a new approach to the design of a biomaterial that would bridge synthetic and biologic materials seamlessly. The scaffolds we have developed serve as a transitional material between biotic and abiotic systems. Key words: Tissue engineering; Biomaterials; Cell source; Gradient scaffolds the first aspects taken into consideration is what type of material should be used. Many scientists work with biologically derived materials, some choose to work with synthetic polymers, and others combine polymers with biologically derived materials (8). There are definite advantages and disadvantages for using each of these material types. It is not possible to design a construct a priori that mimics exactly the remarkably delicate balance of structural and biological properties that natural matrices ex- hibit. Many tissues, when looked at under high magnification, appear to have a random order. However, it is clear in the way that nature functions that everything has order. It is for this reason that biologically derived materials have some substantial advantages over synthetic polymers. The inherent structure of biologically derived materials allows for effective cellular adherence and infiltration (8). These materials are also innately nontoxic. When using synthetic polymers these issues must be addressed. Much research has been performed with acellular tissue matrices, such as urinary bladder or small intestinal submucosa (8,11). These materials pre- serve the order designed by nature, something that scientists using synthetic polymers have yet to achieve. That said, to decellularize and sterilize the constructs conditions must be used that likely diminish what could be achieved with untreated extracellular matrices. Biologically derived matrices, however, lack the pro- cessability of synthetic polymers. Synthetic polymer constructs can be made into various shapes and sizes via numerous processing methods, including thermally induced phase separation (TIPS), injection molding, and electrospinning. The processing method chosen will de- Tissue engineering is a rapidly evolving, multidisciplinary field that faces many challenges, both societal and academic. It has been defined specifically as a field working towards the development of artificial organs and more broadly such that the definition includes the development of tissues and biomaterials ( mecanada.ca/gcglossaire/glossaire/index.asp, and www. hc-sc.gc.ca/english/organandtissue/glossary/). One of the most inclusive definitions of the field describes tissue engineering as the use of cells, engineering materials, and suitable biochemical factors to improve or replace biological functions in an effort to effect the advancement of medicine ( engineering). Scientists and clinicians have united in an unprecedented manner to pursue tissue engineering. It is a field in which the multidisciplinary efforts of biologists and chemists, clinicians and engineers, as well as scientists from numerous other fields are required for its advancement. Many challenges face these scientists and their diverse backgrounds, and expertise is what has allowed this field to progress to where we are today. Scientists who are developing technologies that will improve or replace biological functions face many academic challenges. For example, they must decide upon the best material for building the tissue engineered construct. They must also decide what cells to use, how to culture those cells in vitro in the presence of the matrix, and how to integrate the now living construct in vivo (2 4,9,15). Interestingly, as we solve the scientific challenges we also find ourselves confronted by new societal challenges. When designing a tissue engineered construct, one of Address correspondence to Alan J. Russell, McGowan Institute for Regenerative Medicine, 100 Technology Drive, Suite 200, University of Pittsburgh, Pittsburgh, PA 15219, USA. Tel: (412) ; Fax: (412) ; russellaj@upmc.edu S11

2 S12 WARGO, KUMAR, AND RUSSELL fine the morphological structure of the construct. For decade about both embryonic and adult stem cells has example, TIPS (5,10,19) and electrospinning (14,17,18) been dramatic, it is still outweighed by what we do not will produce a porous construct whereas injection mold- know. Claims that we know which cell type will be best ing (1,13,16) can, but this technique can also be used to suited to a given disease are usually driven by an advo- make a solid construct. cacy position rather than science. The reality is that cellbased The biodegradability of synthetic constructs can also therapies are challenging no matter the source of be tailored by processing or chemistry. Biologically de- the cells. For example, the use of embryonic stem cells rived materials are also biodegradable in their natural in immune-incompetent mice can produce teratomas (7). state and they have the advantage that their degradation Indeed, many theologians have suggested embryonic products are well tolerated in vivo. stem cells are dangerous to use for this reason. Unfortu- An implanted tissue engineered construct must be nately, there is also a growing recognition that even able to maintain mechanically its structure while cells adult stem cells bear some risk of generating tumors divide and remodel the matrix. Synthetic polymers can (12). Only when we are able to overcome the disparate be made equally as strong and sometimes stronger than challenges of each cell source will we be able to make natural tissues. valuable decisions on which cell source to use for a Finally, cells must be able to readily adhere to the given therapeutic target. construct. Scaffolds made from biologically derived ma- Table 1 summarizes the pros and cons of adult and terials do not have problems with cellular adherence (8). embryonic stem cells as a source for tissue engineering However, cells are not always attracted to synthetic constructs. Indicated in the table are areas where the polymer constructs, which can be hydrophobic in nature. challenges have been addressed, difficult and perhaps In these cases, the surface can be modified to incorpo- intractable problems, and intermediate issues. Embryonic rate cell binding sequences of fibronectin, such as the stem cells have unlimited potential. These cells well-known RGD peptide (arginine-glycine-aspartic acid). have the ability to differentiate into any cell type found Despite all of the available materials for tissue engineering, in the body, regardless of what lineage the cell was deered. the perfect biomaterial has yet to be discov- rived from. However, scientists do not fully understand Researchers have already begun to incorporate how to manipulate these cells and force differentiation synthetic polymers with biologically derived materials effectively (5). Further, these cells will continue to selfrenew in an attempt to combine the strengths of these materials. until they have differentiated. Prior to therapeutic Finding a suitable material is just one of the chal- use, therefore, embryonic stem cells must be differentiated; lenges in tissue engineering. Next, researchers must decide otherwise they have been known to cause teratolenges upon what kind of cells to use. There are many mas. Adult stem cells have been found in nearly every available sources for cells, both human and mammalian organ in the body. These cells are rare, but have the (7). Tissue engineered constructs could be preseeded potential to differentiate into lineage-specific cell lines. with cells from the recipient. This is the ideal scenario as Adult stem cells are much less controversial than embry- the chance for an immunogenic response is eliminated. onic stem cells but they are challenging to identify and However, this would result in a time delay to allow the even more challenging to culture and expand. cells the opportunity to adhere to the surface and infil- Finally, researchers are faced with the challenge of trate into the construct. For this reason, it may not always incorporating the tissue engineered construct into bio- be feasible for the recipient to also be the donor. logical systems. It is essential that these foreign materi- In allogeneic cell-based therapies the construct can als must be able to transition seamlessly into the funcbe seeded well in advance, thereby decreasing the time tioning biological system, fusing the abiotic with biotic. to implantation. In some instances, the recipient would In our laboratory, we are designing a tissue engineering then have the risk of suffering an immunogenic response, construct that can function as a transitioning scaffold. causing his or her own body to reject the implanted construct. We are achieving this goal by making scaffolds comnosuppressant This may also require the recipient to take immu- posed of a gradient, illustrated in Figure 1. The scaffolds drugs, increasing the vulnerability to infection. are initially composed of a purely nondegradable mate- That said, tissue engineered allogeneic skin rial and gradually transition to a completely degradable equivalents have been shown to be very effective and material. The degradable region of this scaffold has a the rejection of the allogeneic cells does not appear to vast network of interconnecting pores that transition to diminish the effectiveness of the therapy. a sparsely porous network in the nondegradable region. Tissue engineers have a choice of many different cell The degradable region is replaced by tissue, which continues types. The ongoing heated debate surrounding the use to infiltrate into the nondegradable region via the of embryonic stem cells serves to remind us that not all pores. This creates an interlocking network between the the challenges in the field are scientific (7,15). Although tissue and the scaffold. the knowledge that researchers have learned in the last We have performed preliminary studies using poly

3 TISSUE ENGINEERING AND THE CHALLENGES WITHIN S13 Table 1. Comparison Between Adult and Embryonic Stem Cells The green regions represent areas where challenges have been addressed, such as the isolation of embryonic stem cells. Difficult and perhaps intractable problems, such as addressing the immunogenicity of embryonic stem cells or proliferating adult stem cells without differentitation, are red. Intermediate issues are yellow. An example of this would be our basic understanding of embryonic stem cells due to decades of laboratory research on mice. (lactic) acid (PLLA) and poly(lactic-co-glycolic) acid (PLGA). These materials are commonly used biomaterials and FDA approved for biomedical devices. We synthesized rectangular-shaped scaffolds and created a porous network through TIPS. Degradation studies as well as gel permeation chromatography were performed to ensure that we had synthesized scaffolds with a gradient along the length scale. We then studied these materials in vitro. These studies revealed the following. As expected all regions supported cellular growth. The region composed purely of PLGA showed the greatest amount of cellular infiltration, followed by the gradient region and then that composed purely of PLLA. Histochemical analysis of the samples showed that all regions promoted extracellular matrix production. We screened the samples for type I and type IV collagen as well as fibronectin. Again, we saw that the cells in PLGA region produced the greatest amount of ECM proteins, followed by the gradient re- gions, and the smallest amount was observed in the PLLA region. We also tested these materials in an animal model. Figure 1. The figure represents a schematic of our gradient scaffolds. The outer regions of the scaffold are highly porous and rapidly degradable. As the scaffold transitions to the interior regions the degradability decreases the porosity becomes more sparse.

4 S14 WARGO, KUMAR, AND RUSSELL All of the results that we observed in the in vitro study large tissue segments and perhaps even whole complex were confirmed in vivo. Further, we stained samples organs. Regenerative medicine has been surrounded by with a cell surface marker to look for an inflammatory extraordinary hype for many years and scientific discovery response. We found that all regions stained negatively is the road we must take to convert that promise into for inflammation. a reality. If we could predict how long the path will be Whereas many positive results came out of these experiments, and which turn to take when, there would be no chalrigid we also learned that these materials were too lenges other than time. In the real world, however, com- and brittle to be used as a seamless interface-generating plex tissue engineering will revolve around our under- material. Currently we are working on synthesiz- standing of which materials and cells to use and how to ing gradient scaffolds from degradable and nondegradable culture them ex vivo prior to integration in vivo. polyurethanes in which we can marry the elastic properties needed with gradients in biodegradability. REFERENCES We synthesized biologically compatible polyurethanes 1. Abu Bakar, M. S.; Cheng, M. H. W.; Tang, S. M.; Yu, with a hard segment of hexane diisocyanate and butane S. C.; Liao, K.; Tan, C. T.; Khor, K. A.; Cheang, P. Tendiamine. The degradable polyurethane had a soft segsponse sile properties, tension-tension fatigue and biological rement of polyetheretherketone-hydroxyapatite compos- of poly(caprolactone) diol and the soft segment of ites for load-bearing orthopedic implants. Biomaterials 24: the nondegradable polyurethane was composed on poly ; (tetramethylene ether glycol) (6). 2. Bonassar, L. J.; Vacanti, C. A. Tissue engineering: The We processed these polymers into microfibers via first decade and beyond. J. Cell. Biochem. Suppl. 30/31: electrospinning. Circular scaffolds will be created with ; the PTMEG-based polyurethane as the core transitioning 3. Chapekar, M. S. Tissue engineering: Challenges and op- portunities. J. Biomed. Mater. Res. 53: ; radially into the PCL-based polyurethane. These scaf- 4. Griffith, L. G.; Naughton, G. Tissue engineering current folds will address some of the challenges researchers challenges and expanding opportunities. Science 295: currently face in choosing a suitable biomaterial. They ; allow for the transition between materials that are com- 5. Guan, J.; Fujimoto, K. L.; Sacks, M. S.; Wagner, W. R. pletely natural to those that are synthetic. Gradient scafgradable polyurethane scaffolds for soft tissue applica- Preparation and characterization of highly porous, biodefolds can incorporate natural biomaterials, such as colla- tions. Biomaterials 26: ; gen or growth factors, to enhance tissue ingrowth and 6. Guan, J.; Sacks, M. S.; Beckman, E. J.; Wagner, W. R. degradation rates, while maintaining mechanical strength. Synthesis, characterization, and cytocompatibility of elas- Further, because the scaffolds are made from synthetic tomeric, biodegradable poly(ester-urethane)ureas based on materials, the processing methods are boundless. poly(caprolactone) and putrescine. J. Biomed. Mater. Res. 61: ; We believe that these novel scaffolds will be an im- 7. Health, C. A. Cells for tissue engineering. Trends Biotechportant addition to the already existing biomaterials in nol. 18:17 19; the quest for attaining perfection. The close interactions 8. Kim, B. S.; Baez, C. E.; Atala, A. Biomaterial for tissue between the synthetic materials and natural materials engineering. World J. Urol. 18:2 9; will merge their unique advantages into one tissue engilenges. Pharm. Res. 14: ; Langer, R. Tissue engineering: A new field and its chal- neered construct. As we continue to learn, the field of 10. Nam, Y. S.; Park, T. G. Porous biodegradable polymeric tissue engineering continues to evolve and gradient scaf- scaffolds prepared by thermally induced phase separation. folds might become the next generation of biomaterials. J. Biomed. Mater. Res. 47:8 17; Within the last decade regenerative medicine has 11. Rosso, F.; Marino, G.; Giordano, A.; Barbarisi, M.; Paremerged from the laboratory into the clinic and is now meggiani, D.; Barbarisi, A. Smart materials as scaffolds for tissue engineering. J. Cell. Physiol. 203: ; ready for application to traumatic injuries, such as those caused by war. Devices, cells, and degradable materials 12. Rubio, D.; Garcia-Castro, J.; Martín, M. C.; de la Fuente, can be combined to reverse the functional losses that R.; Cigudosa, J. C.; Lloyd, A. C.; Bernad, A. Spontaneous are treated today through the rehabilitative process. The human adult stem cell transformation. Cancer Res. 65: innate capacity of the body to heal itself can now be ; Sousa, R. A.; Kalay, G.; Reis, R. L.; Cunha, A. M.; Bevis, accelerated and the age of regenerative rehabilitation is M. J. Injection molding of a starch/evoh blend aimed as upon us. The tools through which we can deliver regen- an alternative biomaterial for temporary applications. J. erative medicine will continue to develop over time. To- Appl. Polym. Sci. 77: ; day we can envisage regenerating skin, cartilage, and 14. Stankus, J. J.; Guan, J.; Wagner, W. R. Fabrication of biobone. Hollow internal organs can be tissue engineered degradable elastomeric scaffolds with sub-micron mor- phologies. J. Biomed. Mater. Res. 70: ; clinically. A wide variety of organ insufficiencies can 15. Stock, U. A.; Vacanti, J. P. Tissue engineering: Current be addressed through cell-based therapies. In the future state and prospects. Annu. Rev. Med. 52: ; we will be in a position to regenerate in vitro and in vivo 16. Sundback, C.; Hadlock, T.; Cheney, M.; Vacanti, J. Man-

5 TISSUE ENGINEERING AND THE CHALLENGES WITHIN S15 ufacture of porous polymer nerve conduits by a novel low- 18. Zeng, J.; Xu, X.; Chen, X.; Liang, Q.; Bian, X.; Yang, L.; pressure injection molding process. Biomaterials 24:819 Jing, X. Biodegradable electrospun fibers for drug deliv- 830; ery. J. Controlled Release 92: ; Yoshimoto, H.; Shin, Y. M.; Terai, H.; Vacanti, J. P. A 19. Zhang, R.; Ma, P. X. Poly(α-hydroxyl acids)/hydroxyapabiodegradable nanofiber scaffold by electrospinning and tite porous composites for bone-tissue engineering. I. its potential for bone tissue engineering. Biomaterials 24: ; Preparation and morphology. J. Biomed. Mater. Res. 44: ; 1999.