Supramolecular design of self-assembling SPECIAL FEATURE for cartilage regeneration

Ramille N. Shaha,b, Nirav A. Shahc, Marc M. Del Rosario Limd, Caleb Hsieha,b, Gordon Nubere, and Samuel I. Stuppa,b,f,g,1

aInstitute for BioNanotechnology in Medicine, Northwestern University, 303 E. Superior Street 11th floor, Chicago, IL 60611; bDepartment of Materials Science and Engineering, Northwestern University, 2220 Campus Drive, Evanston, IL 60208; cDepartment of Orthopaedic Surgery, Northwestern University, 676 N. Saint Clair, Suite 1350, Chicago, IL 60611; dDepartment of Biomedical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208; eNorthwestern Orthopaedic Institute, 680 N. Lakeshore Dr., Suite 1028, Chicago, IL 60611; fDepartment of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208; and gDepartment of Medicine, Northwestern University, 251 East Huron Street, Suite 3-150, Chicago, IL 60611

Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved December 23, 2009 (received for review June 12, 2009)

Molecular and supramolecular design of bioactive biomaterials planted through minimally invasive means that localizes and main- could have a significant impact on regenerative medicine. Ideal tains cells and growth factors within the defect site, promotes stem regenerative therapies should be minimally invasive, and thus chondrogenic differentiation, and stimulates biosynthesis. the notion of self-assembling biomaterials programmed to trans- In prior work, we developed self-assembling biomaterials form from injectable liquids to solid bioactive structures in tissue based on a broad class of molecules known as amphi- is highly attractive for clinical translation. We report here on a coas- philes (PAs) that self-assemble from aqueous media into supra- sembly system of peptide (PA) molecules designed to molecular nanofibers of high aspect ratio. These molecules, form nanofibers for cartilage regeneration by displaying a high targeted to serve as the components of artificial extracellular ma- density of binding epitopes to transforming growth factor β-1 trices, consists of a peptide segment covalently bonded to a more (TGFβ-1). Growth factor release studies showed that passive hydrophobic segment such as an alkyl tail (9–12). PAs are nor- release of TGFβ-1 was slower from PA containing the growth mally charged molecules so that screening ions in the biological factor binding sites. In vitro experiments indicate these materials environment can trigger self-assembly into cylindrical nanofibers, support the survival and promote the chondrogenic differentiation which form by hydrogen bonding among peptide segments into SCIENCES of human mesenchymal stem cells. We also show that these β-sheets and the hydrophobic collapse of their alkyl segments

materials can promote regeneration of articular cartilage in a full (13). Furthermore, PAs can have a terminal biosignaling peptide APPLIED BIOLOGICAL thickness chondral defect treated with microfracture in a rabbit domain, which upon self-assembly becomes exposed in very high model with or even without the addition of exogenous growth densities on the surfaces of the nanofibers. At specific pH values factor. These results demonstrate the potential of a completely and PA concentrations, these amphiphilic molecules can assem- synthetic bioactive biomaterial as a therapy to promote cartilage ble into self-supporting gels made up of an interconnected net- regeneration. work of nanofibers (12). Over time, PA gels should biodegrade into amino acids and lipids that can be safely cleared by the body. self-assembling biomaterials ∣ chondral defects ∣ microfracture ∣ We had previously discovered by phage display a peptide se- peptide ∣ transforming growth factor quence (HSNGLPL) with a binding affinity to transforming growth factor β1 (TGF-β1), which is known to be important in amaged articular cartilage in our joints is an important the formation of connective tissues and for other biological Dregenerative medicine target because adults lack the ability functions (14). Prior work has demonstrated that TGF-β1 plays to effectively form cartilage with the architecture and morphol- a significant role in the regulatory network of growth factors that ogy of the native tissue. This can eventually lead to joint pain with maintains articular cartilage in the differentiated phenotype (15) loss of physical function (1–3), a serious health care issue in an and is a critical factor for inducing chondrogenesis in marrow- aging and physically active global population. Full thickness focal derived MSCs (16). Furthermore, in articular cartilage tissue chondral lesions may progress to osteoarthritis that has an engineering TGF-β1 has been shown to increase collagen and estimated economic impact approaching $65 billion in the US proteoglycan production and inhibit matrix breakdown (17). alone when considering healthcare costs, loss of wages, and so- The effectiveness of TGF-β1, however, has also been demon- cietal impact costs (4). With the limited natural healing capability strated to be significantly dependent on the delivery kinetics of articular cartilage, clinical intervention is necessary to prevent and/or simultaneous delivery with other proteins (18, 19). further articular cartilage degradation and early progression of In this study, we designed and evaluated in vivo a unique self- degenerative osteoarthritis. assembling PA molecule, which includes a TGF-binding domain Microfracture is a common clinical procedure used for the re- (Fig. 1A) for specific use in articular cartilage regeneration. This pair of cartilage defects (5). The benefits of microfracture include particular element in artificial matrix design, that is, the binding the fact that it is a single-stage procedure, is relatively simple from of growth factors to components of the extracellular space such as a technical point of view, is cost-effective with low patient morbid- collagens and heparin, have been shown to occur naturally in ity, and involves the patients’ own mesenchymal stem cells (MSCs) biological systems (20, 21). The matrix studied here also con- as a cell source to stimulate cartilage repair. The reparative pro- tained a nonbioactive PA of smaller molecular dimensions cess in microfracture involves a clot of pluripotent MSCs that (Fig. 1B) that coassembles with the TGF-binding molecules. adhere to the subchondral bone. Histological assessment of microfracture in animal (6, 7) and clinical testing (5) have shown Author contributions: R.N.S., N.A.S., and G.N. designed research; R.N.S., N.A.S., M.M.D.R.L., that most lesions form fibrous cartilage with predominant type I and C.H. performed research; R.N.S., N.A.S., M.M.D.R.L., and C.H. analyzed data; and collagen and a limited amount of type II collagen present. Addi- R.N.S., N.A.S., and S.I.S. wrote the paper. tionally, there is a significant drop in clinical outcome scores after The authors declare no conflict of interest. 18 months as well as in patients older than 40 years (8). This sug- This article is a PNAS Direct Submission. gests that there is deficient bioactivity, quantity, quality, and reten- 1To whom correspondence should be addressed at: Northwestern University, Cook Hall, tion of chondrocyte phenotype within the repair tissue. An ideal Room 1127, 2220 Campus Drive, Evanston, IL 60208. E-mail: [email protected]. regenerative medicine solution to augment this current clinical This article contains supporting information online at www.pnas.org/cgi/content/full/ procedure would be a bioactive scaffold capable of being im- 0906501107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0906501107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 25, 2021 microscopy (SEM) revealed cells extended their processes to interact with the surrounding PA matrix (Fig. 2B). When grown in chondrogenic media, most MSCs obtained a more rounded phenotype as expected, indicating that the PA gels supported and did not inhibit MSC differentiation. Gene expression analysis for cartilage markers (aggrecan and type II collagen) at 3 and 4 wks showed upregulation of these genes in both filler and TGFBPA that incorporated 100 ng∕mL rhTGF-β1 within the gels (Fig. 2C and Fig. S1). At 3 wks, glyco- saminoglycan (GAG) content in the scaffolds was also assessed and showed significantly higher (p < 0.05) GAG production in the PA gels that contained supplemented growth factor (Fig. 2D), but there was no apparent difference between the filler and TGFBPA at this time point. It is expected that the presence of supplemented TGFβ-1 in both the filler and TGFBPA would result in chondrogenic differentiation of MSCs as long as the therapeutic amount of growth factor is present. At 4 wks, how- Fig. 1. Design of PAs for articular cartilage regeneration. Chemical structure ever, there was a significantly higher aggrecan expression level of (A) TGF-binding PA and (B) nonbioactive filler PA. (C) Illustration of co- for the TGFBPA (10 mol%) compared to the filler PA assembly of the TGF-binding PA and the filler PA showing binding epitopes (p < 0.03). This most likely is the result of prolonged and more exposed on the surface of the nanofiber. (D) ELISA results showing TGFβ-1 localized delivery of the growth factor from the TGFBPA com- release up to 72 hours from filler PA and 10 mol% TGF-binding PA (TGFBPA). 100 ng∕mL of TGFβ-1 were loaded in all gels. Error bars equal standard deviation, n ¼ 4.

The supramolecular concept in designing bioactivity in these materials was to coassemble both molecules so that the binding epitope could adequately capture and display the growth factor for signaling (Fig. 1C). In vitro studies were performed to estab- lish the ability of these PA systems to support mesenchymal stem cell viability and chondrogenic differentiation. Furthermore, we utilized an in vivo chondral defect microfracture model in rabbits to test the ability of these systems to promote hyaline cartilage regeneration. To our knowledge, this is the first study to investi- gate the potential use of designed (growth factor binding) self- assembling PA systems for the treatment of articular cartilage defects in an in vivo model. Results and Discussion Growth Factor Release Kinetics. Growth factor release studies were performed to determine if the presence of binding epitopes to TGFβ-1 on the PA nanofibers are able to slow the release of the growth factor from the . Gels (n ¼ 4) containing 10 mol % TGFBPA (mixed with the filler PA without the binding epi- tope) were compared to gels made up of 100% filler PA. PA gels were loaded with 1 μg∕mL of TGFβ-1 and incubated in a buffer solution. The buffer was collected and replaced at 6, 24, 48, and 72 h and the quantity of TGFβ-1 in the collected aliquots was assayed by ELISA. Results revealed release of the growth factor from both the filler and TGFBPA gels, with a slower release of growth factor from gels containing the TGF-binding epitopes (Fig. 1D). After 72 h, the cumulative percentage of TGFβ-1 re- leased from the filler PA gel was 3-fold greater (approximately 60%) compared to the TGFBPA gel (approximately 20%). The slower release of TGFβ-1 from TGFBPA gels suggests successful binding is occurring between the growth factor and epi- topes, which may help localize the growth factor and prolong its release at defect sites for enhancing tissue regeneration.

In Vitro Viability and Differentiation of Human MSCs Cultured Within Peptide Amphiphile Gels. In vitro studies were necessary to ensure that PA gels did not cause cell death or inhibit MSC differentia- Fig. 2. In vitro viability and differentiation of hMSCs cultured in PA scaf- tion. MSCs were cultured in gels of nonbioactive filler PA or gels folds. (A) Live/dead images of cells cultured in PA gels (green ¼ live; ¼ containing 5 or 10 mol% TGF-binding PA (TGFBPA) mixed with red dead). (B) SEM of hMSC on nanofiber gel surface. (C) Aggrecan gene expression from hMSCs cultures in PA gels at 2, 3, and 4 wks. filler PA. Gels alone or gels mixed with 100 ng∕mL of recombi- ¼ 100 ∕ ¼ 100 ¼ β Filler PA filler; ng mL of TGF TGF; 5 mol% TGFBPA nant human (rh)TGF- 1 were also compared. A Live/Dead stain 5%TGFBPA; 10 mol% TGFBPA ¼ 10%TGFBPA. (D) GAG per DNA quantifica- (Invitrogen) showed that MSCs remained viable within PA gels tion in digested PA gels at 3 wks for filler, filler þ 100TGF, and 10%TGFBPA þ throughout the culture period (Fig. 2A). Scanning electron 100TGF groups. Error bars equal standard deviation, n ¼ 3.

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.0906501107 Shah et al. Downloaded by guest on September 25, 2021 pared to the filler PA (as supported by the growth factor release study, none of the rabbits in any of the groups developed grossly experiments), and it is logical that the biological effect of this apparent degeneration or synovial hypertrophy of the joint at SPECIAL FEATURE difference is observed at later time points when therapeutic doses 12 wks. Macroscopic observation of defects after 12 wks revealed of growth factor are still maintained within the TGFBPA. no obvious differences between defects treated with the growth Collagen type II expression results revealed a significant effect factor alone and ones treated with the nonbioactive filler PA with of time (p < 0.0001) and rhTGF-β1 supplementation (p < regard to tissue fill or appearance of repair tissue (Fig. 4A and B). 0.0001), but there were no significant differences in type II In these groups, defects in the trochlea were still very obvious, collagen expression between the PA gels with and without the with defined defect boundaries and noticeable color and texture TGF-binding epitope at 4 wks (Fig. S1). It is speculated, however, differences compared to the surrounding cartilage tissue. In great that longer term in vitro culture may lead to differential type II contrast, defects treated with the TGF-binding PA with and with- collagen expression between the PA groups. Based on the gene out growth factor (TGFBPA/TGF and TGFBPA) showed nearly expression data, the filler PA and the 10 mol% TGF-binding complete tissue fill in most defects, and the formed tissue was PA were chosen for in vivo evaluation. similar in color and texture to the surrounding cartilage (Fig. 4C and D). In some of these defects, the defect boundaries could In Vivo Evaluation of PAs in a Full Thickness Articular Cartilage Defect barely be distinguished, indicating excellent integration of the Rabbit Model Treated with Microfracture. Full thickness chondral regenerated tissue to the surrounding cartilage. defects treated with microfracture in the trochlea of adult rabbits Qualitative assessment of histological sections of the growth were used to evaluate the potential of PAs to promote cartilage factor alone treated and filler PA/TGF groups revealed incom- regeneration in the presence of bone marrow-derived stem cells plete fill of the defects and repair tissue that was not integrated (Fig. 3). 10% pyrene-labeled PA was used in preliminary studies with the surrounding cartilage and/or the subchondral bone to assess the retention and adherence of the PA within the defects (Fig. 5A and B). Tissue in defects treated with growth factor alone after application. Fluorescence was detected under a UV lamp resembled more fibrocartilage with cells having a fibroblast-like and revealed that the PA is successfully contained within the morphology, abnormal cell density and organization, and little to defect following application (Fig. 3D). A preliminary 4-week no staining for glycosaminoglans (GAGs, Safranin-O stain) study showed no obvious chronic inflammatory response in (Fig. 5A) and type II collagen (Fig. 5E). Defects treated with PA-treated defects—no apparent swelling, redness, or synovial the filler PA with growth factor also showed little staining for

hypertrophy of the joint, as well as negative staining of the tissue GAGs (Fig. 5B), but did show some positive staining for type SCIENCES sections for CD4, an inflammatory cell marker specifically II collagen (Fig. 5F). In comparison to the TGF group, some for T helper cells (Fig. S2). cells in the repair tissue of the filler PA/TGF group had a more APPLIED BIOLOGICAL A 12-week study was performed comparing the following rounded chondrocyte-like morphology, cells located in lacunae, i μ β groups: ( ) control group treated with 10 L of rhTGF- 1 and some cell clustering. Cell density and organization within this 100 ∕ ii ( ng mL) per defect (TGF); ( ) defects treated with the group, however, was still abnormal compared to the surrounding þ 100 ∕ β iii nonbioactive filler PA ng mL rhTGF- 1 (filler/TGF); ( ) cartilage (Fig. 5B and F). These results indicate that the presence defects treated with 10 mol% TGFBPA mixed with the filler of a nanofiber scaffold without any bioactive epitope can still þ 100 ∕ β iv PA ng mL rhTGF- 1 (TGFBPA/TGF); and ( ) defects have an enhanced effect on cell differentiation and synthesis treated with 10 mol% TGFBPA mixed with the filler PA without of articular cartilage specific matrix molecules (in this case type growth factor (TGFBPA). At the end of the study, all rabbits in II collagen). This may be due to increased localization of the each group appeared to have full range of motion of their knees. growth factor within the defect (mechanically trapped within One rabbit knee was noted to have a dislocation of the patella the PA gel) compared to treatment with growth factor solu- that was not recognized until the day of sacrifice, and therefore tion alone. this sample was not included in the analysis. As in the 4-week

Fig. 3. Full thickness articular cartilage defect microfracture rabbit model. Surgical procedure creating (A) full thickness articular cartilage defects in rabbit trochlea using a microcurette and (B) microfracture holes through the subchondral bone using a microawl to induce bleeding into the defect. Fig. 4. Observation of articular cartilage defects 12 wks after treatment. (C) PA gel in defect after injection (Arrow). (D) Pyrene-labeled PA gel illus- Macroscopic views of articular cartilage defects after 12 wks postop treated trating containment of the gel within articular cartilage defects after with (A) 100 ng∕mL TGF-β1 (100TGF), (B) filler PA þ 100TGF, (C) 10%TGFBPAþ injection. 100TGF, and (D) 10%TGFBPA alone.

Shah et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 25, 2021 Fig. 5. Histological evaluation of sample sections 12 wks after treatment. Safranin-O staining for glycosaminoglycans (A–D) and type II collagen staining (E–H) in articular cartilage defects treated with (A, E) 100 ng∕mL TGF-β1 (100TGF), (B, F) filler PA þ 100TGF, (C, G) 10%TGFBPA þ 100TGF, and (D, H) 10%TGFBPA alone 12 wks postop.

As in the macroscopic observations, there was a significant en- with (21.9 1.2) and without (20.8 2.1) growth factor, how- hanced effect on tissue regeneration when using the PA system ever, had higher scores in each O’Driscoll category and about containing the TGF-binding epitope (with and without TGFβ- 1.5-fold higher total histological scores compared to the other 1), resulting in more hyaline-like tissue formation (Fig. 5C and two groups (p < 0.0001). Interestingly, there was no significant D). In these groups, tissue formed within the defect space showed difference between the groups treated with the TGF-binding nearly complete fill to the level of the undamaged surrounding PA with or without growth factor. The significant enhancement cartilage tissue. Furthermore, voids between the regenerated in hyaline cartilage formation in defects without the addition tissue and surrounding cartilage or subchondral bone were not of exogenous TGF-β1 may indicate that binding events of endo- apparent, indicating excellent integration. GAG (Fig. 5C and genous TGF-β1 (i.e., from the bleeding marrow through the D) and collagen type II (Fig. 5G and H) staining in these samples microfracture holes or from the surrounding synovial fluid) to nearly matched the intensity and quantity of the surrounding the epitopes displayed by the supramolecular PA nanofibers undamaged cartilage. Additionally, cell morphology resembled are occurring in vivo, and at a level that increases the local a chondrocyte morphology (rounded and in lacunae), cells were concentration of the protein within the defect space to see a re- organized in a columnar architecture, and cell density and clus- generative response. Based on our in vitro growth factor release tering were similar to the undamaged native cartilage. In some studies, there is greater retention (slower release) of TGF-β1 samples, the boundaries of the defects could not even be identi- within PA gels containing TGF-β1 growth factor binding sites fied due to the close similarity in architecture and morphology to compared to the filler PA alone. This may be the reason for the surrounding cartilage. the significant enhanced regeneration in the TGFBPA treated Quantitative scoring of histological sections (stained with defects compared to the ones treated with the filler PA. It is plau- Safranin-O) was performed using a modified O’Driscoll (22) sible that as long as the TGFBPA is present within the defects, 24-point scoring system (see Table S1) that is commonly used there is continuous localization of TGF-β1 that can encourage for evaluating the quality of new tissue formation in articular car- chondrogenic differentiation of mesenchymal stem cells from tilage defects in vivo. The major categories scored in this system the bone marrow and subsequent production of cartilage matrix include assessment of cellular morphology, Safranin-O staining, molecules. Future studies are warranted to investigate if binding surface regularity, structural integrity, thickness, bonding to of TGF-β1 to the PAs would be affected in the presence of other adjacent cartilage, hypocellularity, chondrocyte clustering, and growth factors or matrix molecules in vivo. freedom from degenerative changes in adjacent cartilage. The Not only is it possible for the TGF-binding epitope to prolong average scores in each category and total scores are presented the localization of growth factor within the defect, but it may also in Table 1 and the spread of the scores in each group is also pre- help preserve the integrity and activity of the growth factor by sented in Fig. 6 (n ¼ 8–10 defects). There was no statistically sig- protecting it from proteolytic degradation through the molecu- nificant difference in histological scoring between groups treated larly specific binding events. For example, it is known that growth with growth factor alone (15.5 4.7) or with filler PA and growth factors that bind to heparin in the extracellular space (e.g., angio- factor (15.1 3.7). Both groups treated with the TGF-binding PA genic growth factors) through highly specific heparin binding

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.0906501107 Shah et al. Downloaded by guest on September 25, 2021 Table 1. Histological scores for TGF, filler PA þ TGF, 10% TGFBPA þ TGF, and 10% TGFBPA groups at 12 wks SPECIAL FEATURE TGF Filler PA þ TGF 10% TGFBPA þ TGF 10% TGFBPA Category Mean SD Mean SD Mean SD Mean SD Cellular morphology 2.56 1.43 2.67 1.38 3.93 0.22 3.80 0.45 Safranin-O staining of the matrix 1.48 0.60 1.21 0.67 2.44 0.24 2.30 0.58 Surface regularity 2.00 0.80 1.67 0.69 2.41 0.49 2.37 0.51 Structural integrity 1.07 0.62 0.88 0.40 1.70 0.31 1.57 0.32 Thickness 1.15 0.60 1.29 0.55 1.85 0.18 1.77 0.32 Bonding to the adjacent cartilage 1.33 0.53 1.13 0.62 1.81 0.18 1.80 0.23 Hypocellularity 1.96 0.72 2.08 0.53 2.89 0.17 2.63 0.40 Chondrocyte clustering 1.26 0.40 1.46 0.25 1.93 0.15 1.73 0.31 Freedom From Degenerative Changes in Adjacent Cartilage 2.70 0.35 2.75 0.24 2.89 0.33 2.83 0.24 Total 15.52 4.74 15.13 3.66 21.85 1.19 20.80 2.06

domains are protected from enzymatic degradation (23). One gene expression of cartilage specific markers over prolonged additional observation in our study is the narrow spread in his- times. We demonstrated in vivo that PAs synthesized with a pep- tological scores for specimens treated with the TGF-binding tide binding sequence to TGF-β1 significantly enhanced the re- PA compared to the broader spread in scores for the growth fac- generative potential of microfracture-treated chondral defects. tor alone or filler plus growth factor groups (Fig. 6). This suggests Most importantly, we were able to induce a significant regenera- more consistent tissue formation within defects treated with tive response in vivo in the presence of marrow-derived mesen- the TGF-binding PA, potentially demonstrating the dependable chymal cells, without the addition of exogenous growth factor. bioactivity of this PA system. Our findings demonstrate the potential of molecularly designed These results show that the healing of chondral defects treated supramolecular biomaterials in promoting a specific biological with microfracture can be accelerated and enhanced with de- response without the need for exogenous growth factors or trans- signed PA systems. Results from the TGFβ1 growth factor alone planted cells. The chemical versatility of these peptide based self- þ assembling systems and the ability to potentially apply these

group (without any gel) and the filler PA growth factor treated SCIENCES groups were similar, and demonstrated that the PA gels do not therapies through minimally invasive injection into the joint space

impede healing. The addition of the TGFβ1 binding epitopes makes them promising therapeutic candidates to improve current APPLIED BIOLOGICAL on the PA molecules, however, significantly promoted the forma- clinical cartilage repair strategies. tion of cartilage matrix rich in GAG with similar histological ap- pearance to the native cartilage tissue. These promising results Materials and Methods warrant future in vivo studies in larger defects as well as in larger Peptide Synthesis and Purification. The synthesis of linear peptide sequences was performed using standard solid phase methods in an Applied animal models to further assess the regenerative potential of this Biosystems 433A automated peptide synthesizer. Filler PA with sequence technology. H3CðCH2Þ14CO-VVVAAAEEE, was grown on a preloaded glutamic acid Wang resin, using 3.8 M equivalents of HBTU, 6.0 equivalents of diisopropylethyla- Conclusions mine (DIEA) and 4 equivalents of each amino acid. A palmitoyl alkyl tail was We have observed extensive cartilage regeneration in the pre- added to the N-terminus of the peptide manually, by adding 4 M equivalents sence of supramolecular nanofibers designed to bind the growth of palmitic acid and the same molar equivalents of HBTU and DIEA as above. factor TGFβ-1 relative to a nonbioactive system. Our in vitro stu- The PA was cleaved from the resin and amino acid side groups were dies showed that self-assembling PA scaffolds can support hMSC deprotected in 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane, viability and chondrogenic differentiation and that the presence 2.5% deionized water. TFA was removed in a rotary evaporator and of the binding epitope at a specific concentration (10 mol%) can were collected by precipitation in cold diethyl ether. β TGF-binding sequences were previously designed in the Stupp laboratory better retain TGF -1 within gels, which can lead to upregulated using phage display methods (24, 25). For the TGFBPA with sequence HSNGLPLGGGSEEEAAAVVVðKÞ-COðCH2Þ10CH3, a lysine with a dodecylamine side chain was reacted with a Rink amide resin. The remainder of the peptide was then synthesized as described above, but yielding a PA with reverse po- larity (9). The resulting products were purified using standard preparative HPLC methods.

Growth Factor Release Studies. One weight percent solutions of PA were used for growth factor release studies by dissolving PA in aqueous solutions containing 1 μg∕mL of TGFβ-1. Filler PA gels were compared with gels con- taining 10 mol% TGFBPA. PA gels were made by injecting 100 μLofthePA solution in 200 μL PBS containing 25 mM calcium chloride and 50 μg∕mL of BSA and incubating at 37 °C for 1 h. After 1 h, 200 μL of buffer (PBS contain- ing 50 μg∕mL BSA) was added to each gel. The buffer was collected and replaced at 6, 24, 48, and 72 h. TGFβ-1 released in the buffer solutions was quantified by an Enzyme-Linked Immunosorbent Assay (eBioscience).

In Vitro Cell Viability and Differentiation Studies. Human mesenchymal stem cells (hMSCs) from one donor (Lonza) were used at passage 5 for culture with- in PA scaffolds at 40,000 cells per 20 μL gel (n ¼ 3). For cell encapsulation, cell Fig. 6. Histological scores of 12 wk in vivo samples showing significantly suspensions were mixed with aqueous PA solutions (pH 7) and 20 μL aliquots higher scores for the groups treated with the 10% TGF-binding PA with of the PA/cell suspension were injected into 200 μL of MSC growth media or without growth factor. Circles represent scores for individual specimens (Lonza) supplemented with 25 mM calcium chloride to induce gelation (in in each group (n ¼ 8–10). Of note is the narrow distribution of scores for a 96-well U-bottom plate). For differentiation studies, media was changed the defect groups treated with the 10% TGFBPA compared to the wider to a serum-free chondrogenic media containing high glucose DMEM (high spread in scores for those treated with 100 ng∕mL rhTGF-β1 (100TGF) alone glucose 4.5% without L-glutamine), 0.1 mM nonessential amino acids, or filler PA þ 100TGF. 10 mM Hepes buffer, 100 U∕mL penicillin, 100 μg∕mL streptomycin gluta-

Shah et al. PNAS Early Edition ∣ 5of6 Downloaded by guest on September 25, 2021 mate, ITSþ1 (100x, by Sigma Chemical), 0.1 mM ascorbic acid 2-phosphate, dilute calcium chloride solution. Calcium ions are used to screen the negative 1.25 mg∕mL BSA, 10 ng∕mL of TGF-β1, and 100 nM dexamethasone. Media charges on the PA molecules to induce self-assembly. Before the addition of was completely changed every 2–3 d (200 μL of media per change) and gels calcium chloride, gelation of the PA solution was already triggered by elec- were cultured up to 4 wks. trolytes naturally present in marrow blood from the microfracture holes Viability of cells was assessed by fluorescence microscopy using a Live/ (Fig. 3C). Postoperatively each rabbit was given IM antibiotics (Baytril 72 h Dead stain (Invitrogen). At the end of the culture period, total RNA was ex- duration) and IM pain medicine (Buprenex 24 h duration). Signs of infection tracted from each sample with TRIzol and reverse-transcribed into cDNA and the ability of the rabbits to bear weight and move within their cages using the SuperScript® III First-Strand Synthesis kit (Invitrogen). Real-time were evaluated. A 12-week study was performed comparing the four condi- ’ PCR was performed with the BioRad iQ5 Real-Time PCR system and Promega s tions described previously using 10 rabbits. For each trochlea, 2 defects were Plexor® qPCR kit. Expression of Aggrecan (AGC1) and Collagen II Alpha I created and treated the same, and in each rabbit two different conditions (COL2A1) was used to assess chondrogenic differentiation, and Glyceralde- were tested (i.e., one treatment in one knee and a different treatment in hyde 3-phosphate dehydrogenase was as the housekeeping gene. the other knee). Overall, there were 10 defects per experimental condition. For GAG quantification after 3 wks in culture, the PA gels containing MSCs At each end point, rabbits were euthanized by injection of pentobarbital were first digested in a papain solution (100 μL) consisting of 0.01 M intravenously and secondary measures of bilateral thoracotomy. The distal L-cysteine and 0.5% papain (25 mg∕mL) dissolved in phosphate buffered femur was harvested and processed for histological analysis. EDTA (0.04 M Na2HPO4, 0.06 NaH2PO4·H2O, 0.01 M Na2EDTA · 2H2O) ad- justed to a pH of 6.5. Samples were allowed to digest for 24 h in a 60 °C water bath. Following digestion, 20 μL from each sample was added to a 96 well Histological Analysis. Extracted specimens were fixed in 10% neutral buffered plate. 200 μL of a dimethylmethylene blue solution was added and mixed formalin, decalcified for 24 h, and tissue processed for paraffin embedding. per well, and absorbance was read at 535 nm. GAG quantities were obtained Four-micrometer-thick sections were obtained from the center cross sections μ using a chondroitin sulfate standard curve and normalized to DNA content of the defects (1,000 m from the defects interface) and histochemically obtained from a Picogreen assay (Invitrogen). stained for hemotoxylin and eosin and Safranin-O/Fast Green, and immuno- histochemically stained for type II collagen. Immunohistochemical staining In Vivo Full Thickness Articular Cartilage Rabbit Model Treated with Microfrac- for CD4þ cells was also used in a preliminary 4-week study to assess immune ture. Ten adult male New Zealand White Rabbits (3–3.5 kg, approximately response to the PA gels (5 rabbits were used in this experiment). For the 6 months old) were anesthetized by intramuscular injection of ketamine 12-week study, scoring of histological sections was performed by 3 indepen- at 30–40 mg∕kg and xylazine 5–7 mg∕kg. Isoflourane (1–3%) and oxygen dent, blinded observers using a 24-point scale (Table S1). were supplied by face mask for sedation and general anesthesia during the entire procedure. Under sterile aseptic technique, a midline 2 cm incision was Sample Size Determination and Statistical Analysis. Sample size was based on made with the knee flexed at about 20 ° and subsequently a medial para- an a priori power analysis using alpha <0.05 (CI ≥ 95%) and 1 − β ≥ 0.8.A patellar capsulotomy was performed and the patella was translated laterally minimal sample size of five (defects) was required per group. Significant to expose the articular surface of the trochlea. Two 2 mm diameter full differences were evaluated with ANOVA (F < 0.05) testing and a least thickness chondral defects followed by microfracture were created in the significant difference post hoc test ( p < 0.05). Each defect was considered rabbit trochlea (proximal–medial and distal–lateral). The articular cartilage, an independent sample. Data are expressed as mean SD. including the calcified cartilage layer, was removed with a micro-curette (Fig. 3A). Care was taken not to disrupt the underlying subchondral plate, ACKNOWLEDGMENTS. We thank A. Cheetham for PA synthesis, A. Mata for and sharp (perpendicular) defect edges were created with the aid of a dermal SEM, M. Seniw for assistance with illustrations, the Center for Comparative punch. To allow bone marrow MSCs into the defect space, microfracture was Medicine (Northwestern University) for assistance with animal surgeries, performed within the defects by creating 3 holes spaced equally apart and Pathology Core (Robert H. Lurie Comprehensie Cancer Center, Northwestern 2 mm deep into subchondral bone with a micro-awl (Fig. 3B). Marrow blood University) for histological processing, and the Institute for BioNanotechnol- was observed emerging out of each microfracture hole. After application of ogy in Medicine at Northwestern University for facilities support. This work the PA solution (1 wt%) within the defect, self-assembly of the supramo- was supported by the National Institutes of Health Grant 5-R01-EB003806 lecular nanofibers into a gel network is ensured by adding an aliquot of and Nanotope, Inc.

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