Targeted delivery of progenitor cells for cartilage repair

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Journal of Ort hopaedic Research

Journal of Orthopaedic Research 22 (2004) 735-741

www.elsevier.com/locate/ort hres

Targeted delivery of progenitor cells for cartilage repair James E. Dennis

Nir Cohen

b,

Victor M. Goldberg ', Arnold I. Caplan ''

Abstract

An approach for promoting the adherence of chondrogenic progenitor cells to specific matrix molecules has been tested in a cartilage defect model. Culture-expanded pre-chondrocytesfluorescently labeled with a vital dye were coated by a two-step method wherein lipidated protein G was first allowed to intercalate into cell membranes, and a second incubation in a solution of antibodies to cartilage matrix antigens allowed the binding of the antibodies to the protein G, on the external surface of the cell. The coating technique (termed "cell painting") does not effect cell viability or inhibit growth and chondrogenic potential. Painted cells were then added to rabbit cartilage explants that had a partial-thickness defect, washed, and prepared for histological examination and for confocal microscopy. The histological observations and the confocal observations and fluorescent intensity quantification consistently demonstrated that progenitor cells painted with multiple antibodies were capable of preferential binding to the exposed cartilage matrix within the defect. These results demonstrate that painting cell membranes with antibodies to matrix molecules is an effective method for promoting the adherence of stem or progenitor cells to a cartilage injury site. 0 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. Kqw.ort/.s: Cartilage; Progenitor cells; Targeting; Repair

Introduction Advances in stem cell biology and tissue engineering are being applied to the repair of complex tissues with autologous progenitor cells. The precise delivery of these reparative cells to appropriate repair sites is an essential component of any progenitor cell-based tissue engineering strategy. One approach to engineering repair tissue is to combine stem or progenitor cells with natural or synthetic scaffolds that promote integration and differentiation in vivo. Another method is to introduce progenitor cells systemically or locally to affect repair by the progenitor cells alone. The efficiency of local delivery or adherence of stem cells, such as satellite cells of muscle [ 11, can be very low, compromising the efficacy of some cell-based therapies. A cell coating method has been tested for its ability to promote the attachment of cartilage repair cells to articular cartilage defects as a

*Corresponding author. Tel.: +I-216-368-3567; fax: +1-216-3684077. E-muii address: [email protected] (J.E. Dennis).

first step in the development of a scaffold-free cell delivery strategy. Articular cartilage is aneural, avascular, and has a limited capability for self-repair [ 121. Cartilage tissue was chosen as a test tissue for cell targeting based on this limited ability to self-repair, because of its well-characterized and abundant matrix molecules as potential targets, and because of an easily accessible supply of chondrogenic progenitor cells. Interestingly, cartilage is replete with cells that are capable of mitotic expansion and differentiation into chondrocytes, yet cartilage has minimal ability to regenerate, especially in adults [13]. It is postulated that chondrocytes in situ are either constrained from expansion and differentiation by the cartilage matrix [2] or that there is a scarcity of molecules stimulatory to chondrogenesis [7]. The paucity of functional progenitor cells is exacerbated by the avascularity of cartilage, which deprives it of vascular-derived sources of adult stem cells such as mesenchymal stem cells [3]. The experiments herein show that culture-expanded pre-chondrocytes can be directed to adhere to cartilage repair sites by pre-coating cells with antibodies to tissuespecific matrix molecules.

0736-0266/$ - see front matter 0 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved doi: 10.1016/j.orthres.2003.12.002

J . E. Denni,$~t (11. / Jriurnul of' Ortllopaetiic Resfarch 22 (2004) 735-741

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Protein G

Protein G Coating

Antibody Coating

mycin sulfate: 100 pglml; GibcolBRL). The cells were counted with a hemocytometer and plated in 100 mm petri-dishes at 3 . 4 10' ~ cells per cm2. The first medium change was done 48-72 h after plating, after which the medium was changed twice a week. One day prior to targeting experiments, rabbit chondrocytes were incubated in 10 pM Vybrant(tm) (Molecular Probes, Eugene, OR) in Hank's balanced salt solution for 15 min at 37 "C in 5% carbon dioxidel95'%i air and washed once with Hank's balanced salt solution and fresh D M E M containing 10% fetal bovine serum. Staining of the cells was verified by fluorescent microscopy after harvesting the cells and before the PPG-coating procedure. Cell cociting

Cell Targeting

Fig. I . Diagrammatic representation of the two-step coating method for targeting cells with antibodies. The matrix depicted here represents cartilage, which contains collagen I 1 and aggregate as the predominant matrix molecules to be targeted.

The methodology for coating cells with antibodies to cartilage extracellular matrix molecules is based on the capability of lipidated (palmitated) protein A to intercalate into cell membranes [S] and on the ability to then attach the Fc region of many antibodies to intercalated protein A [4]; the term "cell painting" was first introduced by Chen et al. [4]. In this study, the cell painting methodology was modified for the use of palmited protein G in place of protein A so that an additional range of antibody subtypes from different species could be used in the second painting step. Palmitated protein G was mixed with cells and intercalated into cell membranes (Fig. 1). In the second coating step, antibodies that bind to protein G were added to the PPG-coated cells and were bound to the PPG with the antigeiibinding domain extending outward from the cell surface. The goal was to coat cells with antibodies specific to repair sites of interest in order to direct progenitor cells or stem cells to specific repair sites.

Methods

Recombinant protein G (Sigma, St. Louis, MO) was derivatized with N-hydroxysuccinimide ester of palmitic acid (Sigma, St. Louis, MO), as previously described [8] for the palmitation of protein A. The lipid-derivatized protein G was purified as described previously [6] on a 10 ml Sephadex G-25 (Pharmacia, Piscataway, NJ) column equilibrated with PBS containing 0. I'%I deoxycholate (DOC) pH-7.4. The protein concentration was adjusted to 750 pglml by O D absorbance (UV-160 spectrophotometer, Shimadzu) at 280 nm according to standard curves, 20 pm filter sterilized, and stored at 4 "C until use. In vitro expanded chondrocytes were incubated in 0.2% trypsin, 0.25 mM EDTA for 5 min at 37 "C, collected, washed three times in serum free D M E M , and re-suspended at a density of 3 4 x IO'lml in DMEM. Varying concentrations of palmitated protein G (PPG) or non-derivatized protein G (as a negative control) were added to the cell suspension, and the mixture was incubated at 37 "C for 2 h with constant gentle mixing. The samples were washed in 2 ml of Hank's balanced salt solution (HBSS) three times, centrifuging at 400g for 5 min between each wash. The PPG-coated cells were then incubated in targeting antibody or antibodies at 100 pg/ml per antibody in PBS for 1 h at 4 "C. The targeting antibodies were: 2B6, an antibody to chondroitin-4-sulfate [5], 5D4, which recogniLes keratan sulfate [lo] and 11-116B3, an antibody to collagen type I1 [9]. To assess the incorporation of PPG onto cell surfaces, cells incubated in different concentrations of PPG in PBS plus O.I'%l DOC o r cells incubated in buffer alone for 2 h were washed twice in the buffer ~ cells) of and then incubated at 4 "C for I h with 100 pI (per 1 . 0 10' 100 pg/ml of FITC-human IgG (Sigma) diluted in PBS plus 0.I'%) DOC. Cells were washed three times in the buffer and analyzed at the (Coulter XL-MCL) Flow Cytometry Core Facility at Case Western Reserve University and by a fluorescent microscopy (Olympus BH-2RFCA). The toxicity of rising concentrations of PPG coating was assessed using propidium iodine uptake as quantified by FACS scan. Cell viability is based o n exclusion of propidium iodine DNA-binding fluorescent dye in intact cell. Cells that had been incubated in PPG and cells incubated in PBS plus O.I'%l DOC only were suspended in 500 pl of HBSS. and 5 p1 of a 50 pg/ml solution of propidium iodine (in PBS, pH 7.4) was added within 3 rnin of flow cytometry analysis. Cells that exclude the fluorescent propidium iodine were scored as viable; 20,000 cells were analyzed for each sample, and the experiment was run twice. Forward and side scatter parameters were set to window particles the size of cells. Aliquots of each sample and uncoated control cells were ~ cells per 10 cm plate ( 1 . 7 lo4 ~ cells per cm'). culplated at 1 . 0 loh tured in D M E M containing lo'%, FBS. One week later, the cells were harvested by trypsin digestion and counted in a hemacytometer.

Articultir. clionrirocyte culture O.~trocliondrulcsplunts

New Zealand rabbits were euthanized by phenobarbital overdose injection (2600 mg/kg; Fatal-Plus', Vortex Pharmaceuticals, Dearborn, MI). The distal femoral condyles were removed, and the articular cartilage layer was dissected off the condyle using a scalpel and placed into neutral buffered saline. The cartilage slices were minced into 1-2 mm pieces and incubated in Dulbecco's modified Eagle's medium (DMEM) containing 1% collagenase, 0.05% trypsin, and O.l'%, chondroitinase overnight at 37 "C with constant gentle mixing. The mixture was filtered through a 70 pm nitex filter, centrifuged at 400g for five minutes, and the supernatant discarded and replaced with fresh DMEM supplemented with 10% selected lots [ I ] of fetal calf serum (Gibco BRL, Gaithersburg, MD) and antibiotic-antimycotic solution (penicillin G sodium: 100 U/ml, amphotericin B: 0.5 pdrnl, strepto-

Osteochondral explants were harvested from 1-year-old male New Zealand white rabbits euthanized by intravenous phenobarbital overdose injection. The distal femoral condyles were sterilely harvested, and a 4.25 mm diameter trephine was used to manually drill approximately 3 4 mm into the articular surface at 4 different sites, 2 each on the lateral and medial condyles. After drilling into the articular surface, a defect was created by sliding a 1 rnm diameter ring curette along the cartilage surface with sufficient force to cut a U-shaped trough that extended down to the level of the tidemark or partially into the calcified cartilage region. The trough was approximately l mm in diameter and extended across the entire surface of the cylinder. After formation of the defect, the cylinder was snapped off at its bony base

J. E. Diwii.? et ul. I Joiirnul of Orthopaetlic Rrs~circli22 (2004) 735- 741

by applying lateral force with a 4 mm diameter disposable Uni-punch (Premier Biomedical Products, King-of-Prussia, PA). Explants were placed in a 96-well plate with the cartilage side facing up, and the Vybrant(tm)'*-stained cells coated with the different antibodies (as described in cell coritiiig) were added to the top of each well ~ cells were added per explant in a 100 pl above the explants: 1 . 5 lo6 volume. The explants were incubated for 45 min at 37 "C in 5% carbon dioxide/95'%1air and were then inverted. placed in an open-ended conical tube, placed into wells filled with fresh DMEM. and incubated overnight; the conical insert held the explant above the bottom of the well and allowed unattached cells to fall to the bottom of the well. The explants were then fixed in 10% neutral buffered formalin. decalcified, embedded, and analyzed with a fluorescent microscopy. Whole mount osteochondral explants where analyzed by inverted Ziess fluorescent microscopy, and image analysis was performed with Metamorph software.

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The collected optical slice images containing intensity values were then processed by Z-stacking using Zeiss LSM software to obtain measurable fluorescence intensity values throughout the depth of the defect and along the cartilage surface. The collected stacked images were then quantified with the use of Metamorph software. In Metamorph. an area of interest was drawn around regions of undamaged articular surface and around the defect. The intensity of fluorescence per unit area was determined for each specimen for the undamaged "surface" and "defect" areas. The data were from two separate experiments with duplicate samples with n = 4. The "surface" and "defect" areas were individually tested for significant differences by one-way ANOVA. and Dunnett t-tests were used for post-hoc multiple comparisons.

Results and discussion l~i~~ii~mol~i.~/oclIr,iii,sf~~~

Type I1 collagen immunohistochemistry staining was carried out as previously described [I I]. Sections were rehydrated in PBS for 5 min and digested with bovine testis hyaluronidase 8000 UIml (Sigma H3506) for 60 min. A second digestion was performed using pronase 1 inglml (Sigma P-5147) after which nonspecific adhesion sites were blocked using 3'%, BSA in PBS (BSA/PBS). Next, the sections were incubated in mouse anti-collagen type I 1 IgG (11-1 16B3) diluted in 3% BSAIPBS I :200 for 60 min. The slides were washed with 3'%1BSAlPBS and coated with a second layer of horseradish peroxidase-conjugate goat-anti-mouse IgG. Slides were washed in PBS and contrasted in a solution of Vector VIP Substrate (Vector labs; Burlingame, CA) according to the manufacturers instructions, washed, and counterstained with Fast Green. The slides were observed on an Olympus BH2 fluorescent microscope. A no first antibody control was included in each experiment. Aggregate cultures [ 141 were used to assess chondrogenic potential of antibody-coated cells. Cells were incubated with a range of concentrations of PPG (0-60 pglml) and a second coating with human FITC IgG antibody. Cells were placed in 0.5 ml of defined medium (Dulbecco's modified Eagle medium base supplemented with 6.25 pg/ ml insulin. 6.26 pglml transferring, 6.25 pglml selenious acid, 5.35 pg/ ml linoleic acid. 1.25 pglml bovine serum albumin (BSA), 1 mM ~ cells per 15 pyruvate, and 37.5 ng/ml ascorbate-2-phospate) at 2 . 0 10' ml polypropylene conical tube and centrifuged at 5009 for five minutes. The pellets were incubated at 37 "C in 5% carbon dioxide/95'%i air for one to three weeks with medium changes every other day. Within the first 24 h, the cells formed a free-floating pellet. At three weeks, the pellets were harvested fixed in 10% neutral buffered formalin for standard histology. Scunniiig clectron niicroscopj

Scanning electron microscopy (SEM) specimens were fixed in 1% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 for 4 h and stored in buffer overnight. The samples were then post-fixed in 1% osmium tetroxide for 1 h, washed, dehydrated in ethanol. and critically point dried with liquid carbon dioxide in a Balzers CPD-020 critical point dryer. The samples were sputter coated with approximately 200 pm of palladium-gold and observed in a JSM 840A SEM operated at 20 kV. Confocul tiiicroscopy

Cell-targeted explants were washed in HBSS, placed in 20% polyvinyl alcohol containing, lo'%, glycerol, containing 1.0 mglml of p-phenylenediamine, inverted. and observed with a Zeiss Axiomat microscope equipped with an argon/krypton laser. All of the samples were examined, and those with the greatest fluorescent signal were used to set the maximum value for collecting signal intensity data. Once set, all of the samples from a single experiment (I7 = 2 for each antibody or control condition) were observed during the same session with identical data collection parameters. T o sample fluorescence intensity, each sample was scanned from the articular surface to the bottom of the defect, with an interval of 30 pm between scans.

Initial experiments were conducted to determine coating parameters that maximized PPG integration into or onto cell surfaces without causing cell death. Rabbit pre-chondrocytes were incubated in a range of PPG concentrations, buffer only, or with non-palmitated protein G. Flow cytometric analysis showed a linear increase of mean fluorescence intensity in samples incubated in 10-60 pg/ml of PPG (Fig. 2). The PPG coating was tested up to a concentration nearly double that tested for palmitated protein A [4], but the intensity curve had not reached a plateau, indicating that the coating had not yet reached saturation. Flow cytometric analysis of propidium iodine uptake (Fig. 3A) showed that cells coated with concentrations of up to 6O-pglml PPG retained greater than 95% viability (propidium iodine exclusion); the percentage of cells that excluded dye in PPG-coated samples was indistinguishable from that of uncoated cells. Quantification of cell growth one week after coating showed no significant differences between PPG-coated and uncoated cells (Fig. 3A). To test the chondrogenic potential of PPG-coated cells, coated and uncoated prechondrocytes were incubated in a defined chondrogenic cell aggregate culture system [I41 and examined at 1, 2, and 3 weeks culture. Histological examination of toluidine blue-stained 5-pm sections of three week old aggregates showed rounded cells surrounded by abundant metachromatic stained matrix in both uncoated (Fig. 3B) and PPG-coated samples (Fig. 3C); the metachormatic staining indicates a matrix rich in cationic matrix, such as the sulfated glycosaminoglycans found in cartilage. Serial sections incubated with antibody to collagen type I1 showed a highly positive signal within the cell matrix in uncoated samples (Fig. 3D) and in samples incubated in 60 pg/ml PPG (Fig. 3E); Fig. 3F shows an uncoated sample with no first antibody. These results demonstrated that the PPG coating up to 60 pg/ ml concentration did not adversely effect cell viability, growth, or chondrogenic potential. The effect of antibody-coating (painting) on the adherence of pre-chondrocytes to cartilage matrix was tested on cylindrical rabbit articular cartilage explants

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lri 22 (2004) 735-741 J. E. Denr1i.c et LII. I Jorrrntil of Orth~ i p ~ i t ~ iRrreurcl~

Fig. 2. Effect of PPG concentration on intercalation of PPG into cell membranes. The fluorescence intensity for each coilcentration of PPG tested is plotted against the median fluorescence intensity (11 = 2). The insert above the graph shows a representative fluorescent micrograph of a single cell at each test PPG concentration.

Fig. 3. Effects of PPG coating on cell growth, viability, and chondrogeiiic potential; 11 = 2 for both the growth and viability experiments. Expansion and viability of pre-chondrogenic cells after coating with PPG is shown in (A). Histologic sections of 3-week-old aggregate cultures stained with toluidine blue showed metachromatic areas typical of cartilage matrix iii both uncoated samples (B) and in prechondrocytes coated with 60 pg/ml PPG (C). Cultured samples immunostained with antibody to type 11 collagenase shown in uiicoated pre-chondrocyte samples (D) and in PPG-coated pre-chondrocytes (E): a no first antibody negative control is shown in (F).

(4.25 mm diameter and 3 4 mm deep) obtained from adult rabbit femoral condyles, which had a 1 mm diameter full-thickness defect carved into the articular surface. A scanning electron micrograph (Fig. 4A) shows a representative defect viewed looking downward onto the articular surface, with the defect running north to south. Pre-chondrocytes incubated in a vital fluorescent dye were coated with the highest tested concentration of PPG (60 pg/ml), washed and incubated with different antibodies to cartilage matrix molecules. These antibody-coated (and control) pre-chondrocytes were then incubated with rabbit cartilage explants and examined for fluorescent signal. Histological sections of Vybrant(tm)-labeled cells incubated with cartilage explants were obtained (Fig. 4B-E). Greater numbers of fluorescent-positive cells were observed in explants incubated with pre-chondrocytes incubated with PPG at a concentration of 60 pg/ml plus antibodies to collagen 11 (Fig. 4C), chondroitin-4-sulfate (Fig. 4D), and a combination of anti-collagen I1 and chondroitin-2-sulftte antibodies (Fig. 4E) compared to PPG only control (Fig. 4B). Explants incubated with cells coated with multiple antibodies had the greatest amount of fluorescence compared to all other treatments. To observe the entire explant surface and to quantify the fluorescent signal, whole-mounts of explants that had been targeted with different combinations of antibody or controls were observed by confocal microscopy. Each image obtained was a collection of 10-16 optical slice images collapsed together with Z-stack software. Fig. 5 shows representative low magnification images for cells coated with antibodies to collagen 11, chondroitin4-sulfate, keratan sulfate, PPG only, and multi-antibodies (from the same experiment). Visual evaluation of fluorescent micrographs showed a pattern of increased fluorescence intensity in single- to double- to triplecoated cells, as was observed in the sectioned material. Cells coated with PPG only (Fig. 5B) had a greater affinity for cartilage defects than Vybrant(tm) only cells, while cells coated with single antibodies to either type I1 collagen (Fig. 5C), chondroitin-4-sulfate (Fig. 5D) and keratan sulfate (Fig. 5E) often showed greater fluorescent signal. Cells coated with two antibodies, such as anti-type I1 collagen and anti-chondroitin-4-sulfate (Fig. 5F), nearly always showed much greater intensity of fluorescence than PPG only or single antibody cell coating samples. The combination of 5D4 and type 11 collagen antibodies showed fluorescence similar to single antibody treatments (Fig. 5G), while triple coating with antibodies to type 11 collagen, chondroitin-4-sulfate, and keratan sulfate (Fig. 5H) showed a high fluorescence signal. To quantify the cell targeting results, the summed intensities of the Z-stacked optical slices were measured and expressed as fluorescence intensity per unit area of the defect and undamaged surface (Fig. 6). The quan-

Fig. 4. Targeting of pre-chondrocytes to cartilage explants. A scanned electron micrograph ( A ) shows the undamaged cartilage surface (asterisk), and the edges of the defect are indicated by arrowheads. Vybrant(tm)-stained pre-chondrogenic cells were incubated with rabbit cartilage explants, washed, fixed, and observed by fluorescence microscopy. Photomicrographs show examples of pre-chondrocytes coated with either PPG only (B). PPG plus anti-type 11 collagen antibody (C), PPG plus anti-chondroitin-4-sulfate antibody (D), or both antibodies (E). The arrow in (B) points to the tidemark, which delineates calcified and non-calcified cartilage. Arrows in (C-E) point to brightly fluorescent cells attached to a defect surface.

tified fluorescence results demonstrated that the doublecoating combination of chondroitin sulfate (2B6) and type I1 collagen antibody and triple-coating with all three antibodies showed a significant increase in fluorescence intensity in the cartilage defect region (p < 0.05) compared to Vybrant(tm) controls, but a significant difference was not detected for the undamaged articular surface region. In addition, coating with the single antibodies to keratan sulfate (5D4) and type I1 collagen (TIIC) showed a trend toward significance in the defect regions compared to Vybrant(tm) surface intensity at p < 0.10. In all cases, the defect zone contained higher fluorescence signal than did the surface zone. The diminished fluorescence intensity on the surface is partially accounted for by the geometry of the defect, for which no adjustment was made for defect curvature. As a result, the defect area measurements are underestimated. When adjusting the values for a semicircle, the change in area measured would only account for, at most, a 60'% increase in intensity; area measurements are 1/2 )(. (diameter) (length) for a semi-circle compared to (width) (length) for the cell surface. In most cases, the measured defect intensity was more than twice the intensity of that of the surface intensity. No mathematical correction of curvature was made because all of the measurements of the condyle surface were affected by uncontrollable curvature effects inherent in the sample surface, variations in the formation of the defect, and the inability to assure perfect alignment of the defect parallel to the confocal objective. The results of the double antibody-coating showed one unexpected result where double staining with type I1 antibody and keratan sulfate antibody was insignificantly different from either antibody alone. Similarly, the triple-coating results showed lower, but not significantly different, intensity than double-coating with anti-type I1 and anti-chondroitin-4-sulfate antibody. It appears that the combination of anti-type I1 antibody and anti-keratan sulfate antibody tends to cancel out the cell adherence properties. At this point, the mechanism of this inhibition can only be a matter of speculation. These studies demonstrate the feasibility of using a two-step painting technique to coat cells with antibodies that promote the binding of coated cells to cartilage extracellular matrix. The coating technique has no adverse effects on cell viability, growth, or potential to differentiate into chondrocytes. Coating of cells with a single antibody showed only a minimal increase in cell attachment to cartilage matrix, but the use of double and triple antibody-coating significantly increased the number of cells bound to the cartilage defect region compared to uncoated control cells. The lack of a significant increase in binding of double- and triple-coated cells to the undamaged articular surface may simply be a result of fewer target molecules being exposed on normal cartilage surface compared to damaged surfaces. It

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Fig. 5. Confocal imaging of targeted cartilage explants. All targcted cells were incubated in Vybrdnt(tm) to make them fluorescent. Confocal images of explants targeted with control cells with no cell coating are shown in ( A ) and cells coated with PPG only are shown in (B). Explants targeted with cells coated with single antibodies are shown in (C) type 11 collagen. (D) cliondroitin-4-sulfate and (E) keratan sulfate. Cells coated with two antibodies are shown in (F) anti-type I1 collagen and anti-cIiondroitiii-4-sulf~1teand (G) anti-type 11 collagen and anti-keratan sulfate, and a triple Vybrant(tm) only; PPG = PPG only: coated sample is shown i n ( H ) anti-type II collagen, choiidroitin-4-sulfte. and keratan sulfate. Vy : CTlI = anti-collagen type 11: C-4-S = anti-chondroitin-4-s~iIfate;KS = anti-kcratan sulfate.

M. Pendergast for confocal microscopy assistance. Supported by grants from NIH.

References

Cell Coating Fig. 6. Quantification of confocal microscopic fluorescent signal in rabbit cartilage explants. The relative intensity was determined as a function of the area of the defect (black bars) and the cartilage surface (gray bars): 4 individual sample results are shown for each treatment. The A indicates a trend in significance at p < 0.10 and the * indicates a significant difference from controls at p < 0.05 (ANOVA and Dunnett t-test post-hoc).

is proposed that this two-step coating method can be used as a paradigm for the delivery of any progenitor cell to a specific tissue site where appropriate progenitor cells and target molecules have been identified.

Acknowledgements We thank Drs. R.M. Nerem, R. Langer, and A. Mikos for critical comments on the manuscript, A. Awadallah for all of the histology preparation, M. Sramkoski for his assistance with flow cytometry, and

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