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TGF-ß3 Decreases Type I Collagen and Scarring after Labioplasty

¹ Graduate School of Dental Science,
² Pediatric Dentistry, Division of Oral Health, Growth & Development, and
³ Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan; and
⁴ Cartilage Biology and Orthopaedics Branch, National Institutes of Health, Bethesda, MD, USA;

*corresponding author, m-ohishi{at}dent.kyushu-u.ac.jp

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Cleft lip is a common congenital malformation, and labioplasty performed on infants to repair such defects often results in severe scar formation. Since TGF-ß3 has been implicated in wound healing, we therefore hypothesized that TGF-ß3 functions to reduce scarring after cleft lip repair. In this investigation, we demonstrated that exogenous TGF-ß3 reduced scar formation in an incised and sutured mouse lip in vivo. During labioplasty, endogenous TGF-ß3 expression was also elevated. In vitro experiments showed that exogenous TGF-ß3 reduced type I collagen accumulation. Furthermore, TGF-ß3 inhibited alpha-smooth-muscle actin expression, a marker for myofibroblasts. In tandem, TGF-ß3 induced the expression and activity of MMP-9. Analysis of our data suggests that TGF-ß3 is normally secreted following labioplastic wound healing. An elevated level of TGF-ß3 reduces type I collagen deposition by restricting myofibroblast differentiation and thereby collagen synthesis, and by promoting collagen degradation by MMP-9. In combination, these events lead to TGF-ß3-mediated reduced scar formation.

KEY WORDS: cleft lip • CL/Fraser mouse • mesenchymal cells • myofibroblast • matrix metalloproteinase-9

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Cleft lip is one of the most common congenital malformations in humans. Cleft lip can arise at several stages of development due to the complexity in morphogenetic movements that form the mid- and lower face, and perturbations that alter mesenchymal cell migration, proliferation, differentiation, and matrix interactions (Young et al., 2000). Clinical management of cleft lip deformity offers a unique ongoing challenge in facial plastic surgery. Surgical repair of the cleft lip in neonates often results in severe residual scar formation due to deficiency in available skin tissue and wound contracture (Chen and Yeow, 1999). These scars are composed of heavily deposited type I collagen fibers derived primarily from myofibroblasts (Nedelec et al., 2000).

Mesenchymal cell migration and tissue remodeling during wound healing require the controlled degradation of the extracellular matrix. These processes are partly regulated by extracellular proteases, particularly those belonging to the serine protease and matrix metalloproteinase (MMP) families (Soo et al., 2000). MMPs are secreted as inactive zymogens and can degrade various components of scar tissue extracellular matrix, including type I collagen (Mauch et al., 1994). Therefore, the accumulation and organization of matrix components, and their modeling by MMPs, are instrumental for wound healing and associated scar formation.

Transforming growth factor-beta (TGF-ß) is a superfamily of multifunctional peptide growth factors (Roberts and Sporn, 1992). In particular, TGF-ßs participate in wound healing and tissue repair, and regulate the rate and extent of these processes (Branton and Kopp, 1999; Shukla et al., 1999). Although the three mammalian TGF-ß isoforms appear to behave similarly in most assays, they are differentially regulated, and their expressions in tissues during embryogenesis, fibrosis, and wound healing are different (Shah et al., 1995; Frank et al., 1996). TGF-ß3 has distinct roles in mediating fetal wound healing (Hsu et al., 2001).

The purpose of the present study is to elucidate the biological role of TGF-ß3 in the activities of mesenchymal cells and the formation of the extracellular matrix during cleft lip repair. We hypothesize that TGF-ß3 contributes to reduced scar formation after labioplasty of cleft lip by modulating matrix composition. Using both in vivo and in vitro approaches, we observed that TGF-ß3 is normally secreted during recovery after labioplasty of the cleft lip. TGF-ß3 functions to reduce scar formation by decreasing type I collagen formation, as well as enhancing type I collagen degradation. These mechanisms may mimic conditions for scarless healing of fetal wounds. Application of exogenous TGF-ß3 to decrease type I collagen accumulation and consequential scar formation provide an opportunity for the clinical augmentation of scar reduction after cleft lip repair.

	MATERIALS & METHODS

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Neonatal Mouse Lip Surgery
Animal studies conformed to guidelines from the Council on Animal Care at Kyushu University. One full-thickness incision of 5 mm in length, 2 mm in width, and at 5 mm lateral from the midline was made on the upper lip of an anesthetized CL/Fraser neonatal mouse. Animals were divided into four groups: (1) unoperated control, (2) sutured only, (3) sutured with phosphate-buffered saline (PBS) injection, and (4) sutured with TGF-ß3 injection. A 100-µL quantity of human recombinant TGF-ß3 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) at 0.1 ng/µL was injected along the suture site into group 4, while an equal volume of PBS was used in group 3. Injections were repeated every 12 hrs for 3 days. Animals were allowed to recover until post-natal day 24.

Organ Culture of Maxillary and Nasal Processes
The maxillary and nasal processes isolated by microdissection were cultured in a serum-free organ culture system as described (Slavkin et al., 2000). Exogenous TGF-ß3 was added 4 hrs after initial explantation at 10 or 100 ng/mL to experimental cultures.

Immunohistochemistry and Quantitation
Tissues were harvested and processed, and paraffin sections at 4-µm thickness were collected. Antibodies against TGF-ß3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), type I collagen (Cosmo Bio Co., Tokyo, Japan), MMP-9 (Santa Cruz Biotechnology), and alpha-smooth-muscle actin (Progen, Heidelberg, Germany) were used at 0.5, 0.2, 0.2, and 0.5 µg/mL, respectively. Immunohistochemistry and cell counting were performed as described (Kohama et al., 2002). The intensity of the immunoreactions for type I collagen was measured as described (Lehr et al., 1997). Five serial sections were obtained from the upper lip region from each animal, and 5 animals were used in each group.

Semi-quantitative Reverse-transcription/Polymerase Chain-reaction (RT-PCR)
Total RNA extraction, RT-PCR, and measurements were performed as described (Nonaka et al., 1999). Primer sequences, PCR annealing temperatures, and limited PCR cycle numbers were as follows: for TGF-ß3, 5'-GTCTTCCAGATACTTAGAC-3' and 5'-AGCAGTTCTCCTCCAGGTTG-3', at 58°C for 30 cycles; for MMP-9, 5'-GGGCAACTCGGCAGGAGAGC-3' and 5'-CCAGGTGACGGGCTGCTTGT-3', at 56°C for 35 cycles; for MMP-1, 5'-GATGATGATGACCTGTCTGAGGAAG-3' and 5'-TGTAGCCTTTGGAACTGCTTGTC-3', at 56°C for 30 cycles; for alpha-smooth-muscle actin, 5'-CTGGAGAAGAGCTACGAACTGC-3' and 5'-CTGATCCACATCTGCTGGAAGG-3', at 62°C for 30 cycles; and for type I collagen, 5'-CCCAGAGTGGAACAGCGATTAC-3' and 5'-TGTCTTGCCCCATTCATTTGTC-3', at 56°C for 30 cycles. Expression of these genes of interest was relative to ß-actin expression (primers from Genesetoligos, Kyoto, Japan). The unpaired two-tailed Student’s t test was performed, and statistical difference was taken at the 95% confidence level (p < 0.05).

Primary Cell Culture and Gelatinolytic Zymogram
CL/Fr neonatal mouse lips were dissociated in 0.25% trypsin EDTA (Gibco BRL, Gaithersburg, MD, USA) and 0.25 mg/mL collagenase (Wako, Osaka, Japan) in 0.1 M PBS for 10 min at 37°C. Ten microliters of the cell suspension at 2 x 10⁷ cells/mL were cultured in serum-free medium as described (Southerland and Lucas, 1995). Exogenous TGF-ß3 was added to the culture medium at 100 ng/mL. The conditioned media were sampled for gelatinolytic zymography as described (Nakada et al., 1999).

Bead Implantation and in situ Zymography
Affi-Gel blue agarose beads (BioRad Labs., Hercules, CA, USA) at 100 mesh (50 µm in diameter) were soaked overnight in 100 ng/µL TGF-ß3, or in PBS as control, and implanted into the upper lip explants as described (Nonaka et al., 1999). At the time of harvest, tissues were embedded without fixation, frozen sectioned, and processed for in situ zymography as described (Nakada et al., 1999).

Western Blot Analysis
Organ culture tissues were homogenized in lysis buffer and processed for Western blot analysis as described (Seki et al., 2002). Equal amounts of total proteins were loaded in each lane. Blots were incubated with polyclonal primary antibodies directed against TGF-ß3, MMP-9, or type I collagen (Santa Cruz Biotechnology) at dilutions of 1:200, 1:100, and 1:200, respectively. Immunodetection of the antigen was performed with use of the ECL^TM Western blotting detection kit (Pharmacia Biotech-Amersham, Buckinghamshire, UK). The intensity of the specific bands was quantitated by laser densitometry.

	RESULTS

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TGF-ß3 Reduced Scar Formation in vivo
We first documented the function of TGF-ß3 during neonatal wound healing in vivo. Animals with spontaneous cleft lip did not survive well, despite labioplasty, and were not suitable for extended in vivo studies. Therefore, we used a surgical cleft model in which the upper lips of normal neonatal animals (Fig. 1A) were incised, sutured, and allowed to recover for 24 days. Lips that were operated upon but not additionally treated with TGF-ß3 developed deep, sunken, and contractural scarring (Fig. 1B; 100% scarred, 10/10). The scar was composed of excessive extracellular matrix materials, with a loss of dermal elements such as salivary and sebaceous glands, and vibrissae follicles. The epidermis was deeply creviced (compare Fig. 1E with Fig. 1F). However, the wound that was treated with TGF-ß3 improved noticeably (Fig. 1C, 43% scarred, 6/14). The recovered area was covered with fur and whiskers (Fig. 1C), similar to unoperated controls (Fig. 1A). Sections showed abundant mesenchymal fibroblasts, capillaries, and less-fibrous connective tissues (Fig. 1G). The epidermal surface was smooth, albeit thicker than that of controls. Sutured wound that was injected with PBS was comparable with that of untreated wound (Figs. 1D, 1H; 80% scarred, 8/10).

View larger version (94K): [in this window] [in a new window]   Figure 1. TGF-ß3 reduced scar formation in vivo and was normally expressed in sutured cleft lip. The upper lip of the neonatal mouse was incised, sutured, and allowed to recover until post-natal day 24. Control animals that had not been operated upon showed smooth epidermis and dermal tissues (A,E). Animals that had been operated upon showed conspicuous macroscopic scar formation (B; between white arrowheads). Histological analysis revealed deeply creviced scarring, with abundant fibrous connective tissues in the dermis and loss of salivary and vibrissae follicles (F). Animals that had been operated upon and treated with exogenous TGF-ß3 showed a significant reduction in scar formation. The site of the surgery was identified by the presence of the suture thread (C; between white arrowheads). Histological evaluation showed smooth epidermis, and dermis with abundant mesenchymal cells and capillaries (G). Animals treated with PBS instead of TGF-ß3 (D,H) were similar to untreated controls (B,F). Dotted lines delineate site of surgery. Cleft lip of the CL/Fr neonatal mouse (I) was repaired by labioplasty (L). Black-and-white arrowheads indicate non-repaired and repaired cleft lips, respectively. Immunohistochemistry for TGF-ß3, 30 min after surgery, showed positive reaction (brown deposits) at the site of the surgery (M). Higher magnification of the boxed area in (M) represented in (N) identified TGF-ß3 expression in mesenchymal cells (white arrowheads). TGF-ß3 immunoreactivity was not detectable in unoperated controls (J, boxed area represented in K). Scale bars in A-D are 3 mm, in E-H are 340 µm, in I and L are 2 mm, in J and M are 350 µm, and in K and N are 140 µm. The number of mesenchymal cells positive for TGF-ß3 was significantly higher in the sutured group (7.2 ± 0.5, N = 5) compared with the non-sutured group (0.2 ± 0.2, N = 5) (O). Ten min following labioplasty, there was a significant increase in TGF-ß3 expression in the sutured cleft lip (10.8 ± 0.3, N = 5) as compared with the non-sutured cleft lip (8.4 ± 0.3, N = 5) (P). Numerical data are expressed as mean ± standard error of the mean. p < 0.05.

TGF-ß3 Reduced Type I Collagen Accumulation in Repaired Cleft Lip
We next studied the mechanism by which TGF-ß3 functions in reducing scar formation. Cleft lip of the CL/Fr mouse was sutured and explanted into ex vivo serum-free culture in the presence or absence of exogenous TGF-ß3. Sutured cleft lip accumulated excessive type I collagen at the site of surgery after 3 days in culture (Fig. 2B) when compared with control (Fig. 2A). However, in the presence of exogenous TGF-ß3, we observed an apparent decrease in type I collagen immunostaining (Figs. 2C, 2D). Using morphometric analyses, we detected a 2.5-fold increase (p < 0.05) in type I collagen immunoreactivity in unsupplemented cultures and a dose-dependent decrease in the presence of TGF-ß3 (Fig. 2E). In support of these protein data, RT-PCR showed that TGF-ß3 significantly attenuated the elevated expression of type I collagen after cleft lip repair (Fig. 2F).

View larger version (130K): [in this window] [in a new window]   Figure 2. TGF-ß3 reduced type I collagen accumulation in repaired cleft lip. CL/Fr mouse cleft lip was sutured, placed in serum-free organ culture, and assayed for type I collagen expression. Suturing of cleft lip (B) resulted in a significant increase in the accumulation of type I collagen (brown deposit indicates immunoreactivity) 3 days after explant culture, as compared with non-sutured cleft lip (A,E). Dotted lines and asterisks delineate site of surgery. Scale bars in A-D are 140 µm. In the presence of exogenous TGF-ß3, type I collagen immunoreactivity was reduced, and this decrease was dose-dependent on the concentration of TGF-ß3 (38.2 ± 0.5 for control, 95.2 ± 0.7 for 0 ng/mL, 66.1 ± 1.4 for 10 ng/mL, and 51.2 ± 1.4 for 100 ng/mL TGF-ß3, N = 5 for all groups) (C,D,E). Ten hrs after explant culture, a dose-dependent decrease in type I collagen message level was detected by RT-PCR (5.4 ± 0.6 for control, 8.6 ± 0.5 for 0 ng/mL, 5.2 ± 0.2 for 10 ng/mL, and 3.1 ± 0.4 for 100 ng/mL TGF-ß3, N = 5 for all groups) (F). Numerical data are expressed as mean ± standard error of the mean. p < 0.05 compared with non-sutured group; p < 0.05 compared with group with no TGF-ß3; p < 0.05 compared with group with 10 ng/mL TGF-ß3.

View larger version (112K): [in this window] [in a new window]   Figure 3. TGF-ß3 inhibited alpha-smooth-muscle actin expression. CL/Fr mouse cleft lip was sutured, and placed in serum-free organ culture, and assayed for alpha-smooth-muscle actin expression. Suturing of cleft lip (B) resulted in a significant increase in alpha-smooth-muscle actin (brown deposit indicates immunoreactivity) 3 days after explant culture, as compared with non-sutured cleft lip (A,E). Dotted lines and asterisks delineate site of surgery. Scale bars in A-D are 140 µm. In the presence of exogenous TGF-ß3, the number of mesenchymal cells immunopositive for alpha-smooth-muscle actin was reduced, and this decrease was dose-dependent on the concentration of TGF-ß3 (2.4 ± 0.2 for control, 8.8 ± 0.4 for 0 ng/mL, 7.4 ± 0.2 for 10 ng/mL, and 5.8 ± 0.4 for 100 ng/mL TGF-ß3, N = 5 for all groups) (C,D,E). Eight hrs after explant culture, a dose-dependent decrease in alpha-smooth-muscle actin message level was detected by RT-PCR (2.9 ± 0.3 for control, 11.9 ± 1.3 for 0 ng/mL, 7.1 ± 0.4 for 10 ng/mL, and 3.3 ± 0.5 for 100 ng/mL TGF-ß3, N = 5 for all groups) (F). Numerical data are expressed as mean ± standard error of the mean. p < 0.05 compared with non-sutured group; p < 0.05 compared with group with no TGF-ß3; p < 0.05 compared with group with 10 ng/mL TGF-ß3.

View larger version (62K): [in this window] [in a new window]   Figure 4. TGF-ß3 promoted MMP-9 expression and activity. CL/Fr mouse cleft lip was placed in serum-free organ culture and assayed for MMP-9. Suturing of cleft lip (B) resulted in a significant increase in MMP-9 (brown deposit indicates immunoreactivity) 10 hrs after explant culture, as compared with non-sutured cleft lip (A,E). Dotted lines and asterisks delineate site of surgery. Scale bars in A-D are 140 µm. In the presence of exogenous TGF-ß3, the number of mesenchymal cells immunopositive for MMP-9 was elevated, and this increase was dose-dependent on the concentration of TGF-ß3 (0.2 ± 0.2 for control, 7.4 ± 0.5 for 0 ng/mL, 14.6 ± 0.7 for 10 ng/mL, and 27 ± 0.8 for 100 ng/mL TGF-ß3, N = 5 for all groups) (C,D,E). Eight hrs after explant culture, a dose-dependent increase in MMP-9 message level was detected by RT-PCR (7.3 ± 0.1 for control, 8.4 ± 0.5 for 0 ng/mL, 11.1 ± 0.4 for 10 ng/mL, and 13.1 ± 0.9 for 100 ng/mL TGF-ß3, N = 5 for all groups) (F). In contrast, MMP-1 message level remained unchanged in the presence of TGF-ß3 (G). Numerical data are expressed as mean ± standard error of the mean. p < 0.05 compared with non-sutured group; p < 0.05 compared with group with no TGF-ß3; p < 0.05 compared with group with 10 ng/mL TGF-ß3. In situ zymography showed that TGF-ß3-soaked beads implanted into the upper lip resulted in a cleared gelatinolytic zone surrounding (asterisks) the bead in neonatal (H) and fetal (I) tissues. Control PBS beads had no effect (J). Scale bars in H-J are 140 µm. The upper lip was dissociated into primary cell culture, and the conditioned media were assayed for MMP expression and activity by gelatinolytic zymography (K). In the absence of TGF-ß3, latent pro-MMP2 (72 kDa) and active MMP-2 (62 kDa) were detected. No MMP-9 was present. In the presence of 100 ng/mL TGF-ß3, MMP-2 remained unchanged. However, the latent pro-MMP-9 (92 kDa) as well as the active MMP-9 (82 kDa) were detected. We performed Western analysis to quantitate the levels of protein expression for TGF-ß3, MMP-9, and type I collagen (L). Sutured cleft lip had a 3.3-fold increase in TGF-ß3 protein level (p < 0.05). Similarly, MMP-9 levels also increased by 3.5-fold and 5.8-fold at 10 and 100 ng/mL exogenous TGF-ß3, respectively (p < 0.05). Type I collagen levels decreased by 0.8-fold and 0.1-fold at 10 and 100 ng/mL exogenous TGF-ß3, respectively (p < 0.05).

To support our immunolocalization results and to quantitate changes in protein levels, we performed Western blot analysis for TGF-ß3, MMP-9, and type I collagen (Fig. 4L). We detected a 3.3-fold increase in band intensity for TGF-ß3 in the sutured cleft lip as compared with the non-sutured lip. MMP-9 protein levels exhibited dose-dependent increases in the sutured group—3.5-fold and 5.8-fold at 10 and 100 ng/mL TGF-ß3, respectively. In contrast, type I collagen levels decreased by 0.8-fold and 0.1-fold at 10 and 100 ng/mL TGF-ß3, respectively. These results were consistent with our immunolocalization and RT-PCR data.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
TGF-ß3 has been implicated in cutaneous wound healing and augments scarless recovery. In particular, TGF-ß3 is associated with scarless wound healing in the fetus (Hsu et al., 2001), and several studies have shown that cleft lip repair in the fetus heals without scar formation (Oberg et al., 1998; Stelnicki et al., 1999). However, the mechanisms of such developmental events are largely unresolved. Our previous studies show that microsurgical repair of cleft lip in the fetus that produced scarless fusion is mediated by TGF-ß3 regulation of mesenchymal cell proliferation and migration at the site of repair (Kohama et al., 2002). In this study, we demonstrate that TGF-ß3 is also instrumental for scarless repair of cleft lip in neonatal animals and functions by modulating matrix composition.

Analysis of our data suggests that TGF-ß3 is normally activated and expressed in mesenchymal cells following labioplasty, since the TGF-ß3 level is normally low in unoperated lip mesenchyme. This is a new and distinct role of TGF-ß3 in its regulation of the lip. In normal lip fusion—that is, during embryonic development and not associated with wound healing—TGF-ß3 is transiently expressed in the lip epithelium (Kohama et al., 2002). Epithelial mesenchymal transdifferentiation allows for the fusion of the upper lip halves (Sun et al., 2000). In our study, we observed an increase in the thickness of the lip epithelium following TGF-ß3 treatment. This increase may be due to increased cell proliferation (Fan et al., 1999).

Using the CL/Fr mouse model, and coupled with an organ culture system, we characterized the molecular events elicited by TGF-ß3 that eventually lead to reduce scar formation. Our findings suggest that TGF-ß3 reduces scar formation by changing the dynamic balance of type I collagen accumulation and degradation. These changes are consistent with several wounding models, in which an abundance of collagen fibers is associated with scar formation, and the presence of elastin reduced wound contracture (Berthod et al., 2001). Inhibition of type I collagen accumulation is mediated by TGF-ß3 inhibition of myofibroblast differentiation and consequential type I collagen production by these cells. This is particularly interesting, since TGF-ß1 and -ß2 have opposite effects on scar formation. Neutralization of TGF-ß1 or TGF-ß2 in cutaneous wounds of adult rat reduces scarring (Shah et al., 1995). TGF-ß1 induces fibroblasts to differentiate into myofibroblasts (Yokozeki et al., 1997), whereas neutralizing anti-TGF-ß1 antibody inhibits transdifferentiation of fibroblasts to myofibroblasts (Fan et al., 1999). Therefore, although we have not directly compared the effects of TGF-ß3 with those of TGF-ß1 and -ß2, our observations suggest that the differences among TGF-ß isoforms could be partly due to differences in their regulation of myofibroblast differentiation and the modulation of matrix materials.

We also observed that TGF-ß3 promotes MMP-9 expression and activity, which implicates the promotion of type I collagen degradation at the site of the surgery. MMPs are synthesized as latent molecules that can function only when cleaved into active forms. Our observations are consistent with previous data showing that TGF-ß regulates the activity of MMP-9 (Richiert and Ireland, 1999; Han et al., 2001). Moreover, MMP-9 activity is low or undetectable in hypertrophic scars where collagen is excessive (Neely et al., 1999).

In summary, the most significant findings support an instructive role of TG-ß3 in promoting scarless repair of cleft lip following labioplasty, and its mechanisms of action. The mechanisms of TGF-ß3 function are to modulate matrix composition, including reducing type I collagen accumulation. Since TGF-ß3 is normally secreted following labioplastic wound healing, we conclude that an elevated level of TGF-ß3 reduces type I collagen deposition by promoting degradation by MMP-9, and inhibiting synthesis by restricting myofibroblast differentiation. This duality leads to TGF-ß3-mediated reduced scar formation.

	ACKNOWLEDGMENTS

 
We thank Dr. Taisei Nomura, Osaka University, for his generous gift of CL/Fraser mouse breeding pairs. Thanks are also due to Dr. Kou Matsuo and Dr. Ieyoshi Kobayashi, Kyushu University, for technical support. The present study was supported by grants-in-aid 11470439, 14370677, 13672103, and 12557177 to M. Ohishi and 07557135 and 15390638 to K. Nonaka from the Ministry of Education, Culture, Sports, Science and Technology, Japan. This paper is based on a thesis submitted to the Graduate School of Dental Science, Kyushu University, in partial fulfillment of the requirements for the PhD degree for R. Hosokawa.

	FOOTNOTES

 
^* The first two authors contributed equally to this project and are considered co-first authors;

Received September 17, 2002; Last revision March 27, 2003; Accepted April 24, 2003

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