4.1 Genetically engineered elastin-like polypeptides for cartilage tissue regeneration

B2 otherwise be immunogenic. These issues have by no means been SFTPMWFE  BT JU JT EJG¾DVMU UP EFWJTF OPOIVNBO FYQFSJNFOUT UIBU provide clear guidance on this issue. However, for many reasons cartilage would be a good candidate for trial, as the chondrocytes even in an immature ECM, are in an environment devoid of blood vessels and away from the circulation that contains the cells necessary to initiate an immune response. So it might be expected to be a site of low immunogenic risk, requiring minimal immuno-suppression. It is therefore an intriguing challenge to establish if stem cells from unrelated individuals can be used for the repair of tissues like cartilage, as this would open the way to their use on a much broader scale because of the economies possible with a banked cell supply; but that is a hope for tomorrow and not a promise for today. References: 1. von der Mark K, Gauss V, von der Mark H, Muller P. Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture. Nature 1977;267(5611):531-2. 2. Bi W, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B. Sox9 is required for cartilage formation. Nat Genet 1999;22(1):85-9. 3. Li Y, Tew SR, Russell AM, Gonzalez K, Hardingham TE, Hawkins RE. Transduction of human articular chondrocytes with adenoviral, retroviral and lentiviral vectors and the effects of enhanced expression of SOX9. Tissue Eng 2004;10(3/4):575-84. 4. Tew SR, Li Y, Pothacharoen P, Tweats LM, Hawkins RE, Hardingham TE. Retroviral transduction with SOX9 enhances re-expression of the chondrocyte phenotype in passaged osteoarthritic human articular chondrocytes. Osteoarthritis Cartilage 2005;13(1):80-9. 5. Hardingham TE, Oldershaw RA, Tew SR. Cartilage, SOX9 and Notch signals in chondrogenesis. J Anat 2006;209(4):469-80. 6. Yoo JU, Barthel TS, Nishimura K, et al. The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Joint Surg Am 1998;80(12):1745-57. 7. Barry FP, Murphy JM, English K, Mahon BP. Immunogenicity of adult mesenchymal stem cells: lessons from the fetal allograft. Stem Cells Dev 2005;14(3):252-65. 8. Batten P, Sarathchandra P, Antoniw JW, et al. Human mesenchymal stem cells induce T cell anergy and downregulate T cell allo-responses via the TH2 pathway: relevance to tissue engineering human heart valves. Tissue Eng 2006;12(8):2263-73.

Abstracts of ICRS 2007, Warsaw, Poland

4.1 Genetically engineered elastin-like polypeptides for cartilage tissue regeneration L.A. Setton, D.L. Nettles, D.W. Lim, F. Guilak, A. Chilkoti, United States of America Injectable, in situ forming scaffolds have long been of interest for promoting regeneration of focal cartilage defects. With liquid-like properties that allow mixing with cells or other factors, these materials DBO CF JOKFDUFE UP FOBCMF DPNQMFUF ¾MMJOH PG JSSFHVMBSMZTIBQFE defects and the delivery of cell or bioactive drugs that could enhance integration of newly generated tissue with the existing native tissue. Additional advantages are the opportunity for these scaffolds to contribute to preservation of mechanical integrity and maintenance of cell phenotypes. Many hydrogels capable of in situ formation or crosslinking have been evaluated for potential to promote cartilage matrix regeneration, including alginate23, poly(ethylene oxide)8, poly(N-isopropylacrylamide)27  ¾CSJO26, hyaluronic acid21, collagen gels18, 33, chitosan7,19, poly(propylene fumarate)28, and poly(vinyl alcohol)24. These polymers share the characteristics that they form three-dimensional hydrogels in situ of high water content that support rounded cell morphologies and rapid diffusion of soluble nutrients. However, other properties of these hydrogels differ including biocompatibility, degradation, surface interaction, and cell recognition characteristics. In situ forming materials also have the potential to facilitate tissue regeneration in 2nd and 3rd generation procedures for autologous chondrocyte delivery, where various biological or polymer membranes or scaffolds have been used to preserve retention of the delivered cells6, 14. Our laboratory has developed a class of polypeptide gels for the purpose of promoting cartilage matrix regeneration, biocompatible cell delivery, and tissue integration. ELPs consist of oligomeric repeats of the pentapeptide sequence Val-Pro-Gly-Xaa-Gly, where Xaa is any amino acid except proline30. This is a naturally occurring sequence in the protein elastin contained in muscle, ligaments, cartilage and numerous other soft tissues. ELPs have been shown not to illicit an antibody response upon implantation in multiple animal and human applications31, and have been considered to be non-immunogenic. ELPs are environmentally responsive, as they exhibit a reversible inverse phase transition at a temperature (Tt) above which they undergo hydrophobic collapse and form intermolecular associations that result in an “aggregated” mass11. By designing the transition to occur at physiological temperatures, the environmental responsiveness of ELPs enables in situ formation and physical property changes that contribute to load-bearing and cell entrapment3-5. ELPs may be synthesized genetically, and so designed at the genetic level to control molecular weight, reactive crosslinking sites, and chemistry of the amino acid residues16,17. In this paper, we review our experience with uncrosslinked and crosslinkable ELPs for an ability to entrap chondrocytes and adult stem cells, contribute to mechanical load-bearing properties, and promote matrix integration in a goat model of osteochondral defect¾MMJOH5IFSFTVMUTJMMVTUSBUFUIFGBWPSBCMFDIBSBDUFSJTUJDTBOEGBDJMF synthesis of an in situ forming, biocompatible hydrogel that can assist cartilage matrix regeneration in multiple ways. Elastin-Like Polypeptide Gene Design and Synthesis. Genes have been designed to encode a wide range of ELP proteins with different guest residue sequences and combinations thereof17. For a given &-1 HFOFTFODPEJOHUIFUBSHFUFEQFOUBQFQUJEFTFRVFODFBSF¾STU constructed from synthetic oligonucleotides, and oligomerized by recursive directional ligation17. In our laboratory, peptides IBWF CFFO FYQSFTTFE JO & DPMJ &-1T NBZ CF FBTJMZ QVSJ¾FE CZ inverse transition cycling. This procedure consists of a series of cyclic temperature changes that drive the transitioning of the ELP sequence, but not other proteins resulting from the expression. After only two sequences of temperature shifts above and below the Tt (generally between 28-34°C) the purity of the resulting ELP product has been shown to be excellent, with endotoxin levels far below FDA allowable limits for medical devices15. Chondrocyte and Stem Cell Encapsulation for In Vitro Culture. ELPs were designed with Val, Gly and Ala in a 5:2:3 ratio, respectively, at the guest residue position of the pentapeptide (36 kDa MW). These ELPs give rise to a target Tt of 35°C at a solution concentration of 50 mg/ml and so enable their use for in situ forming gels at physiological temperatures5. Physical characteristics of the ELPs were obtained by evaluating the rheological properties above and below the Tt using viscometry experiments. Below Tt, the apparent viscosity |Ș*| and dynamic shear modulus |G*| were sensitive to ELP concentration and exhibited a 2-fold increase in magnitude with a 4-fold increase in concentration. However, by increasing Tt to 37°C,

Osteoarthritis and Cartilage Vol. 15, Supplement B

Extended Abstracts

the ELP experienced a 1000-fold increase in values for viscosity and moduli5. This dramatic increase in stiffness and viscosity for the uncrosslinked ELP gels alone may be functionally important for cell delivery to tissue defects, and fall within the range of values reported for other hydrogel materials.

for ELPs containing periodic glutamine and valine, or lysine and valine in the guest residue positions. When the two ELPs were mixed together, they were capable of crosslinking following addition of tTG. Primary porcine chondrocytes were added to a mixture of ELP solutions (1:1 ratio of the polymers) and placed in a 37 °C incubator to trigger the ELP phase transition and cell encapsulation process. The resulting pellet was re-solubilized at room temperature and NJYFE XJUI QVSJ¾FE U5( JO DBMDJVNGSFF CVGGFS DBMDJVN DBO USJHHFS the activation of tTG). Crosslinking was then initiated by the addition of 10 mM CaCl2 at 37°C followed by culture of the ELP gelcell constructs. Histological staining of the ELP gel-cell constructs showed accumulation of type II collagen-containing cartilaginous matrix in the vicinity of encapsulated cells, similar to that seen in the uncrosslinked ELPs. There was no evidence of staining for the anticollagen I antibody indicating that the accumulated matrix is clearly more representative of hyaline cartilage, as in articular cartilage, than ¾CSPDBSUJMBHF5IFSFXBTBMTPFWJEFODFPGBTUFBEZJODSFBTFJO4("( per construct dry weight that increased, but to a maximum of 1-2% only. In summary, we observed ELP gel formation and chondrocyte encapsulation in a biocompatible process, with formation of ELP-cell constructs that supported the chondrocyte phenotype and cartilage matrix synthesis in vitro, just as for the uncrosslinked form.

In one study5, primary chondrocytes from a porcine source were FOUSBQQFE JO BO &-1 TPMVUJPO BU WFSZ IJHI FG¾DJFODZ  PG UIF chondrocytes at 1 x 106 cells/ml ELP at ~50 mg/ml). The cells were mixed with ELP at room temperature, and allowed to incubate at 37°C for a one-hour period. When cultured in vitro for 2 weeks, ELP/chondrocyte constructs were found to promote the rounded chondrocyte morphology and to stain positively for toluidine blue BOE .BTTPOµT USJDISPNF  SF¿FDUJOH UIF BDDVNVMBUJPO PG OFXMZ synthesized sulfated glycosaminoglycans (S-GAG) and collagenous QSPUFJOT 5IJT XBT DPO¾SNFE CZ NFBTVSFT PG 4("( BOE DPMMBHFO content, corresponding to 10 µg/µg DNA (~ 1% dry wt.), and ~4.4 µg collagen /µgDNA, values lower than that reported for pre-formed scaffolds of poly(glycolic) acid25 or silk34. This work was focused on a short incubation time, however, and was important for demonstrating that ELPs support the viability of chondrocytes, and do not elicit DZUPUPYJDJUZ XIJDIJTDPOTJTUFOUXJUIUIFJSQSP¾MFBTCJPDPNQBUJCMF  natural materials. In additional work with the uncrosslinked ELPs, we turned our interests to an adult stem cell source derived from adipose tissue (hADAS) shown to contain multipotent progenitor cells that can differentiate into phenotypic chondrocytes9, 10, 35. The potential use of these cells for tissue engineering is attractive, since adipose tissue can be readily obtained, and autologous cells are immunocompatible. hADAS cells were obtained and expanded for 2-3 passages as described previously2, and encapsulated in ELP solutions as described above. An important objective of this work was to compare constructs cultured in “standard media” consisting of media, serum and ascorbate to promote chondrogenesis, versus media with the added “chondrogenic” supplements of TGF-ȕ1 and dexamethasone believed necessary to promote stem cell differentiation into chondrocytes. In measures of biochemical composition and histological appearance, constructs were not found to differ between constructs grown in chondrogenic or standard media after 2 weeks of culture. Values for biochemical composition also compared well with previous reports for hADAS cells cultured in alginate under chondrogenic conditions, XJUI ¾OEJOHT UIBU GFMM BSPVOE  •H 4("(•H %/"  BOE _ •H collagen/µg DNA. Immunohistochemical labeling of collagens type I and II showed that the majority of matrix formed by constructs consisted of type II collagen, with no detectable amount of type I DPMMBHFO 5IF ¾OEJOHT GSPN UIJT TUVEZ EFNPOTUSBUFE UIF BCJMJUZ of ELPs to induce and support the chondrocytic differentiation of human adipose tissue-derived adult stem cells in vitro in the absence of exogenous chondrogenic supplements, promoting interest in the use of ELPs as a carrier for stem cell delivery to cartilage defect sites to promote cell-mediated matrix regeneration. $IFNJDBM BOE &O[ZNBUJD $SPTTMJOLJOH PG &-1T 8IJMF UIFTF ¾STU studies showed that ELPs provide a physical environment conducive for cell viability, chondrocyte differentiation and cartilage matrix synthesis, a major limitation of the ELP gels was felt to be their inability to support high loads typical of cartilage weight-bearing in vivo. Thus, we explored several methods to crosslink ELPs in an attempt to increase mechanical integrity. An advantage of ELPs is an ability to incorporate moieties for crosslinking through genetic engineering, as well as sites for controlled degradation or ligand QSFTFOUBUJPO*OB¾STUTUVEZ HFOFTGPS&-1XFSFEFTJHOFEUPFODPEF GPS B QSFDJTFMZ TQFDJ¾FE OVNCFS PG BNJOF HSPVQT -ZT  UIBU XFSF periodically arranged along the primary amino acid sequence of the biopolymer29. ELPs were chemically crosslinked using a trifunctional crosslinker (tris-succinimidyl amino-triacetate, TSAT) to generate hydrogels of high stiffness, about two orders of magnitude greater than uncrosslinked ELPs. As organic solvents were necessary to create these chemically crosslinked gels, however, a biocompatible crosslinking process was sought that would enable cell encapsulation or in situ polymerization. In a later study, we chose enzymatic crosslinking to promote formation of ELP hydrogels. While many enzymes have been JEFOUJ¾FE BT DBUBMZTUT PG DSPTTMJOLJOH SFBDUJPOT  XF DIPTF IVNBO tissue transglutaminase (tTG), which catalyzes a calcium-dependent reaction that generates a Ȗ-glutamyl-İ-lysyl covalent bond between peptide-bound glutamine residues and primary amines13. tTG is expressed in a variety of cartilage tissues1 and many components PGDBSUJMBHFNBUSJY FH¾CSPOFDUJOBOEDPMMBHFOT** *** 7 BOE9* BSF crosslinking substrates for this enzyme. Two genes were synthesized

B3

Biocompatible Crosslinking of ELPs. While the intention of tTG initiated crosslinking was to increase the mechanical integrity of the ELP gels formed in situ, the dynamic shear stiffness of tTG crosslinked ELP gels was less than 5 kPa, and thus not considered suitable for mechanical load-bearing in a cartilage repair application. 5IJTSFTVMUFEGSPNQPPSBDUJWBUJPOPGUIFFO[ZNF MBDLPGTQFDJ¾DJUZ for the substrate, and relatively slow enzyme kinetics. In an effort to promote rapid and biocompatible crosslinking, Lys-containing ELPs were crosslinked with an amine-reactive trifunctional crosslinker, (ȕ-[Tris(hydroxymethyl) phosphino] propionic acid (betaine), THPP. The byproducts of this reaction are water and chemically stable aminomethyl-phosphines and so are biocompatible for crosslinking with cells 12. ELPs crosslinked with this system result in gels having mechanical stiffnesses 2-3 orders of magnitude greater than those reported for uncrosslinked ELP 12, with compressive stiffness values up to 50 kPa particularly for higher polypeptide concentration, lower periodicity of crosslinking sites (higher crosslinking density), and higher crosslinker-to-reactive amine ratio. These values are still one order-of-magnitude lower than corresponding values for articular cartilage of 500-1000 kPa, but are close to reported values for meniscus and intervertebral disc. Importantly, cells can be mixed with both polypeptide and crosslinker, and scaffolds will form in times on the order of seconds to minutes, depending on ELP sequence, concentration, MW, solution pH, ionic strength, and starting temperature. Our group has also led an effort to develop rapid and non-destructive in vitro “screens” of ELP hydrogels for their ability to synthesize cartilage matrix. In these tests, eleven different formulations of ELPs were used to construct chondrocyte-laden gels crosslinked with THPP for in vitro culture, followed by measures of construct production and consumption of metabolites (glucose and lactate), BOE%/"BOE4("(DPOUFOUTBUFBSMZUJNFQPJOUT ’EBZT 'SPN these data and numerical modeling analyses using neural networks, we can conclude that cells are more metabolically active in hydrogels at the lower ELP concentrations and accumulate higher amounts of 4("(BGUFSEBZTJODVMUVSF5IFTF¾OEJOHTJMMVTUSBUFBGVOEBNFOUBM challenge in working with hydrogels for cell-driven matrix repair, that ELP formulations optimal for mechanical load-bearing (higher concentrations or higher crosslink densities) are not simultaneously appropriate for supporting biosynthesis. The approaches developed in our group permit the use of both short term in vitro “screens” and a computational model to develop selection criteria that will simultaneously optimize mechanical and biological characteristics of crosslinked ELPs in longer-term culture settings, in order to move forward with a scaffold for cartilage regeneration. $SPTTMJOLFE &-1T &WBMVBUFE JO (PBU 0TUFPDIPOESBM %FGFDU ¾MMJOH Model. While both crosslinked and uncrosslinked ELP gels were found to be supportive of cartilage matrix synthesis for chondrocytes and stem cells in vitro, it was not known how these results would translate to the in vivo setting of a cartilage or osteochondral defect¾MMJOH  XIFSF NVMUJQMF DFMM QPQVMBUJPOT BOE WBTDVMBS GBDUPST DPVME contribute to long-term ELP performance. We undertook studies PG &-1 PTUFPDIPOESBM EFGFDU¾MMJOH JO B MBSHF EFGFDU NPEFM JO UIF goat 20, as developed by Jackson and co-workers (2001). Defects (6 x 4 mm) were generated on the medial femoral condyle of both knees in skeletally mature goats (n=7); defects in one joint were ¾MMFE XJUI &-1 NJYFE XJUI DSPTTMJOLFS XIJMF UIF DPOUSBMBUFSBM KPJOU was left empty as a control. We selected a high molecular weight ELP with a moderate density of crosslinking sites (Lys:Val=1:6, 200

B4 mg/ml) for delivery to the defect without any cell supplementation. Our goal was to achieve a highly elastic and stiff formulation that was still suitably viscous to inject into the defect site; since this was an acellular strategy, ELP parameters that contribute to cell viability and metabolism were not priorities. The ELP/crosslinker solution was easily delivered to osteochondral EFGFDUT BOE DSPTTMJOLFE JO MFTT UIBO ¾WF NJOVUFT 3FTVMUT XFSF evaluated via grading of gross appearance, histology and staining GPS UZQFT * BOE ** DPMMBHFO VTJOH B NPEJ¾FE *$34 TDIFNF "U  NPOUIT &-1¾MMFEEFGFDUTTIPXFETJHOJ¾DBOUMZCFUUFSJOUFHSBUJPOCZ evaluation of gross and histological appearance, as well as improved total and types I & II collagen scores compared to controls. At 6 NPOUIT IPXFWFS UIFIJTUPMPHJDBMBQQFBSBODFPG&-1¾MMFEEFGFDUT TDPSFE MPXFS GPS BSDIJUFDUVSF BOE QSPUFPHMZDBOT UIBO VO¾MMFE controls. ELP was observed in defects 7 days after surgery, but was largely absent at 3 months, suggesting that ELP-based hydrogels were degraded in vivo, consistent with our prior work showing that THPP-crosslinked ELPs are susceptible to proteolysis 22. Summary of Studies and Future Directions. Our experience with genetically engineered ELPs has illustrated some favorable features of in situ forming scaffolds for assisting cartilage regeneration. The liquid-like property of ELPs in aqueous solutions below the transition temperature makes mixing with cells and cell FODBQTVMBUJPO FG¾DJFOU BOE FYUSFNFMZ TJNQMF 0VS JO WJUSP TUVEJFT demonstrate that ELPs promote cell viability and a chondrocytic phenotype as demonstrated by an ability to synthesize cartilageTQFDJ¾D FYUSBDFMMVMBS NBUSJY 8IJMF TPNF WBMVFT GPS CJPDIFNJDBM composition synthesized in vitro compare well with values reported for chondrocytes in alginate gels, retention of newly synthesized proteoglycan components in the ELP gels can be improved. In our work, we have not made use of bioreactors or bioactive factors, both shown to be important for promoting more rapid cartilage matrix synthesis 32*OEFFE XFIBWFTQFDJ¾DBMMZDIPTFOUPTUVEZTUFNDFMM culture in ELP gels without bioactive factors (e.g., TGF-ȕ) in order to determine the effects of the polypeptide alone for stem cell differentiation. We believe that the strength and unique advantages of an in situ forming, or in situ crosslinking hydrogel like ELPs will lie in its ability to function as an injectable scaffold for cartilage tissue engineering through delivery of cells and/or bioactive factors. In comparison with synthetic polymers, ELP-derived scaffolds are particularly attractive from a macromolecular perspective, because their physico-chemical properties are genetically encodable. Thus, the transition temperature, the types and number of crosslinking sites, and the molecular weight of ELPs may be precisely controlled at the amino acid sequence level. This suggests that the important mechanical properties of viscosity, stiffness and dissipation for both uncrosslinked ELP gels and crosslinked constructs may be similarly controlled at the genetic level, permitting unique and precise UBSHFUJOH PG NFDIBOJDBM BOE CJPMPHJDBM EFTJHO TQFDJ¾DBUJPOT GPS B variety of cartilage tissues. Indeed, the animal model results have been useful for illustrating the favorable biocompatibility, integration and resorption characteristics of the crosslinked ELPs when used as in situ crosslinking injectable materials. Despite the high protein concentrations and crosslink densities used in the animal model, DFMMTXFSFGPVOEUPJO¾MUSBUFUIFIZESPHFMTBTFBSMZBTUISFFNPOUIT DPO¾SNJOH UIF PCTFSWBUJPO UIBU SFTPSQUJPO BTTJTUT TVQQPSU PG DFMM viability and new matrix synthesis. Future work will focus on the need to optimize both mechanical and biological design criteria for BQBSUJDVMBSEFGFDU¾MMJOHBQQMJDBUJPO BTXFMMBTFWBMVBUJPOTPGDFMM assisted cartilage regeneration through the delivery of cells, and incorporation of cell recognition sequences or bioactive drugs that can be directly designed into the ELP sequence. Acknowledgments. This work was supported by NIH EB002263, AR49294 and AR49790. The authors thank the many contributors who assisted with gene design, mechanical and cell culture tests, and animal surgeries.

Abstracts of ICRS 2007, Warsaw, Poland derived adult stem cells in elastin-like polypeptide, Biomaterials. 27 (2006) pp. 91-99. 5. H. Betre et al. Characterization of a genetically engineered elastinlike polypeptide for cartilaginous tissue repair, Biomacromolecules. 3 (2002) pp. 910-916. 6. M. Brittberg et al., Articular cartilage engineering with autologous chondrocyte transplantation. A review of recent developments, J Bone Joint Surg Am. 85-A Suppl 3 (2003) pp. 109-115. 7. A. Chenite et al., Novel injectable neutral solutions of chitosan form biodegradable gels in situ, Biomaterials. 21 (2000) pp. 21552161. 8. J. Elisseeff et al., Photoencapsulation of chondrocytes in poly(ethylene oxide)- based semi-interpenetrating networks, Journal of Biomedical Materials Research. 51 (2000) pp. 164-171. 9. G. R. Erickson et al., Chondrogenic potential of adipose tissuederived stromal cells in vitro and in vivo, Biochem Biophys Res Commun. 290 (2002) pp. 763-769. 10. F. Guilak et al., Clonal analysis of the differentiation potential of human adipose-derived adult stem cells, J Cell Physiol. 206 (2006) pp. 229-237. 11. B. Li et al., The molecular basis for the inverse temperature transition of elastin, J Mol Biol. 305 (2001) pp. 581-592. 12. D. W. Lim et al., Rapid Cross-Linking of Elastin-like Polypeptides with (Hydroxymethyl)phosphines in Aqueous Solution, Biomacromolecules (2007) pp. 13. L. Lorand and R. M. Graham, Transglutaminases: Crosslinking enzymes with pleiotropic functions, Nature Reviews Molecular Cell Biology 4(2003) pp. 140-156. 14. S. Marlovits et al., Cartilage repair: generations of autologous chondrocyte transplantation, Eur J Radiol. 57 (2006) pp. 24-31. 15. M. K. McHale et al., Synthesis and in vitro evaluation of enzymatically cross-linked elastin-like polypeptide gels for cartilaginous tissue repair., Tissue Engineering. 11 (2005) pp. 17681779.  % 5 .D1IFSTPO FU BM  1SPEVDUJPO BOE QVSJ¾DBUJPO PG B recombinant elastomeric polypeptide, G-(VPGVG)19-VPGV, from Escherichia coli, Biotechnol. Prog. 8 (1992) pp. 347-352. %&.FZFSBOE"$IJMLPUJ 1VSJ¾DBUJPOPGSFDPNCJOBOUQSPUFJOT by fusion with thermally- responsive polypeptides, Nat. Biotechnol. 17 (1999) pp. 1112-1115. 18. S. Nehrer et al., Canine chondrocytes seeded in type I and type II collagen implants investigated in vitro [published erratum appears in J Biomed Mater Res 1997 Winter;38(4):288], Journal of Biomedical Materials Research. 38 (1997) pp. 95-104. 19. D. L. Nettles et al., Potential use of chitosan as a cell scaffold material for cartilage tissue engineering, Tissue Engineering. 8 (2002) pp. 1009-1016. 20. D. L. Nettles et al., Chemically crosslinked elastin-like polypeptides as injectable hydrogels for articular cartilage repair in a goat model of an osteochondral defect., 6th Symposium of the International Cartilage Repair Society (2006) pp. 264. 21. D. L. Nettles et al., Photocrosslinkable hyaluronan as a scaffold for articular cartilage repair, Ann Biomed Eng. 32 (2004) pp. 391397.

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22. S. R. Ong et al., Epitope tagging for tracking elastin-like polypeptides, Biomaterials. 27 (2006) pp. 1930-1935.

1. D. Aeschlimann et al., Expression of Tissue Transglutaminase in Skeletal Tissues Correlates with Events of Terminal Differentiation of Chondrocytes, J. Cell Biol. 120 (1993) pp. 1461-1470.

23. K. T. Paige et al., Injectable cartilage, Plastic & Reconstructive Surgery. 96 (1995) pp. 1390-1398; discussion 1399-1400.

2. L. Aust et al., Yield of human adipose-derived adult stem cells from liposuction aspirates, Cytotherapy. 6 (2004) pp. 7-14.

24. R. H. Schmedlen et al., Photocrosslinkable polyvinyl alcohol IZESPHFMTUIBUDBOCFNPEJ¾FEXJUIDFMMBEIFTJPOQFQUJEFTGPSVTF in tissue engineering, Biomaterials. 23 (2002) pp. 4325-4332.

3. H. Betre et al., A thermally responsive biopolymer for intra-articular drug delivery, J Control Release. 115 (2006) pp. 175-182. 4. H. Betre et al., Chondrocytic differentiation of human adipose-

25. J. O. Seidel et al., Long-term culture of tissue engineered cartilage in a perfused chamber with mechanical stimulation, Biorheology. 41 (2004) pp. 445-458.

Osteoarthritis and Cartilage Vol. 15, Supplement B

Extended Abstracts

26. R. P. Silverman et al., Injectable tissue-engineered cartilage VTJOHB¾CSJOHMVFQPMZNFS 1MBTU3FDPOTUS4VSH  QQ 1818.

5.1

27. R. A. Stile et al., Synthesis and characterization of injectable poly(N-isopropylacrylamide)-based hydrogels that support tissue formation in vitro, Macromolecules. 32 (1999) pp. 7370-7379. 28. J. S. Temenoff and A. G. Mikos, Review: tissue engineering for regeneration of articular cartilage, Biomaterials. 21 (2000) pp. 431440. 29. K. Trabbic-Carlson, L. A. Setton, and A. Chilkoti, Swelling and mechanical behaviors of chemically cross-linked hydrogels of elastinlike polypeptides, Biomacromolecules. 4 (2003) pp. 572-580. 30. D. W. Urry, Free energy transduction in polypeptides and proteins based on inverse temperature transitions, Progress in Biophysics & Molecular Biology. 57 (1992) pp. 23-57. 31. D. W. Urry, T. M. Parker, M. C. Reid, and D. C. Gowda, Biocompatibility of the Bioelastic Materials, Poly(Gvgvp) and Its Gamma-Irradiation Cross-Linked Matrix - Summary of Generic Biological Test-Results, Journal of Bioactive and Compatible Polymer. 6 (1991) pp. 263-282. 32. G. Vunjak-Novakovic et al., Dynamic cell seeding of polymer scaffolds for cartilage tissue engineering, Biotechnol. Prog. 14 (1998) pp. 193-202. 33. S. Wakitani et al., Repair of large full-thickness articular cartilage defects with allograft articular chondrocytes embedded in a collagen gel, Tissue Engineering. 4 (1998) pp. 429-444. 34. Y. Wang et al., Cartilage tissue engineering with silk scaffolds and human articular chondrocytes, Biomaterials. 27 (2006) pp. 4434-4442. 35. P. A. Zuk et al., Multilineage cells from human adipose tissue: implications for cell-based therapies, Tissue Eng. 7 (2001) pp. 211228.

B5

What is the patient’s opinion of their knee? Experience from using the knee injury and osteroarthritis outcome score (KOOS) E. Roos, Sweden The Knee injury and Osteoarthritis Outcome Score (KOOS) is increasingly used for evaluation of the patient’s perspective of knee injury and knee osteoarthritis. The rational for and the development of the KOOS is outlined. Psychometric properties important for use in clinical trials are discussed and examples from use in cartilage repair, ACL reconstruction, and other interventions are given. Development of the KOOS The Knee injury and Osteoarthritis Outcome Score (KOOS) was developed as an extension of the WOMAC Osteoarthritis Index with the purpose of evaluating short-term and long-term symptoms and function in subjects with a variety of knee injuries possibly resulting JOPTUFPBSUISJUJT5IF,004IPMET¾WFTFQBSBUFMZTDPSFETVCTDBMFT Pain, other Symptoms, Function in daily living (ADL), Function in Sport and Recreation (Sport/Rec), and knee-related Quality of Life (QOL). The KOOS has been validated for several orthopaedic interventions such as anterior cruciate ligament reconstruction (1), meniscectomy (2), post-traumatic osteoarthritis (3) and total knee replacement (4). In addition the instrument has been used to evaluate a variety of other interventions including cartilage repair (5-9), tibial osteotomy (10, 11), physical therapy (12), nutritional supplementation (13) and glucosamine supplementation (14). The effect size is generally largest for the subscale QOL followed by the subscale Pain. The KOOS is a valid, reliable and responsive self-administered instrument that can be used for short-term and long-term follow-up of several types of knee injury including osteoarthritis. The main reason for developing a single instrument with the purpose of covering several types of knee injury and including osteoarthritis (OA), was that traumatic knee injuries often causes concomitant damage to multiple structures (ligaments, menisci, cartilage, etc.) and frequently lead to the later development of OA. To be able to follow patients after a trauma and to gain insight into the change of symptoms, function etc. over time, a questionnaire which covers both the short-term and long-term consequences is needed. Prior instruments such as the Lysholm knee scoring scale (15) have focused only on the short-term consequences and instruments such as the WOMAC Osteoarthritis Index (16) only on the long-term consequences. An instrument intended for follow-up of these patients needs to adequately monitor both the acute injury consequences in the physically active and younger patients, and the chronic outcome in the older. To ensure content validity for subjects with ACL injury, meniscus injury, and early OA, we reviewed the literature, consulted an expert panel, and conducted a pilot study. The literature indicated three principal areas of patient-relevant outcomes: symptoms, functional status, and satisfaction. An expert panel comprised of patients referred to physical therapy because of knee injuries, orthopaedic surgeons, and physical therapists from both Sweden and the United States, was asked to identify short- and long-term symptoms and functional disabilities resulting from a meniscus or ACL injury. Seven GBDUPST XFSF JEFOUJ¾FE CZ UIF QBOFM QBJO  FBSMZ EJTFBTFTQFDJ¾D TZNQUPNT MBUFEJTFBTFTQFDJ¾DTZNQUPNT FHTZNQUPNTPG0"

 function, quality of life, activity level, and satisfaction. A pilot study was then conducted to identify the subjectively most relevant factors among patients with post-traumatic osteoarthritis. 4FWFOUZ¾WF JOEJWJEVBMT XIP IBE IBE NFOJTDVT TVSHFSZ  ZFBST previously were asked to respond to two questionnaires, both selfadministered. The participants ranged in age from 35 to 76 (mean 

 BOE TIPXFE SBEJPMPHJDBM TJHOT PG LOFF 0"  EF¾OFE BT KPJOU space narrowing and osteophytes. One of the questionnaires, by Flandry et al. (17), was constructed to assess symptoms of anterior cruciate ligament (ACL) injury and the other, WOMAC Osteoarthritis Index (16), for assessing symptoms of knee OA Questions that most frequently received high responses, and were thus considered to SF¿FDU UIF NPTU QSFEPNJOBOU TZNQUPNT  JODMVEFE UIPTF SFMBUJOH to pain, swelling, stiffness, and the ability to run, jump, kneel, and squat. 5IF,004JTBDPNQSFIFOTJWFJOTUSVNFOUJODMVEJOH¾WFTVCTDBMFT assessing aspects of knee injury and knee OA considered important by patients. Most other instruments used for acute knee injury aggregate items measuring different aspects into one score. This QSPDFEVSF ¿BUUFOT UIF SFTVMUT BOE NBLFT JOUFSQSFUBUJPO NPSF EJG¾DVMUTJODFUIFJODMVEFEJUFNTEPOPUBMXBZTDPSSFMBUF5IF,004 JTTFMGBENJOJTUFSFEBOEUBLFTBQQSPYJNBUFMZNJOVUFTUP¾MMPVU