American Journal of Pathology, Vol. 140, No. 6, June 1992 Coptright © American Association of Pathologists

Platelet-derived Growth Factor (BB Homodimer), Transforming Growth Factor-r1, and Basic Fibroblast Growth Factor in Dermal Wound Healing Neovessel and Matrix Formation and Cessation of Repair

Glenn F. Pierce,* John E. Tarpley,* Donna Yanagihara,* Thomas A. Mustoe,t Gary M. Fox, and Arlen Thomasoni1 From the Departments of Experimental Pathology;* Biology

and Biochemistr,j* and Molecular Biology,11 Amgen Inc., Thousand Oaks, California, and the Division of Plastic Surgery, Department of Surger, Northuestern University, Chicago, Illinois

Recombinant platelet-derived growth factor (BB homodimer, rPDGF-BB), transforming growthfactor 41 (rTGF-31), and basic fibroblast growth factor (rbFGF) can accelerate healing of soft tissues. However, little information is available characterizing the components of wound matrix induced by these growth factors and the molecular mechanisms underlying accelerated repair and wound maturation. In this study, the composition, quantity, and rate of extracellular matrix deposition within growth factor-treated lapine ear excisional wounds were analyzed at different stages of healing using specific histochemical and immunohistochemical stains, coupled with image analysis techniques. Single application of optimal concentrations of each growth factor accelerated normal healing by 30% (P < 0. 0003); rPDGF-BB markedly augmented early glycosaminoglycan (GAG) and fibronectin deposition, but induced significantly greater levels of collagen later in the repair process, compared with untreated wounds. rTGF-41 treatment led to rapidly enhanced collagen synthesis and maturation, without increased GAG deposition. In contrast, rbFGF treatment induced a predominantly angiogenic response in wounds, with a marked increase in endothelia and neovessels (P < 0.0001), and increased wound col-

lagenolytic activity (P < 0.03). rbFGF-treated wounds did not evolve into collagen-containing scars and continued to accumulate only provisional matrix well past wound closure. These results provide new evidence that growth factors influence wound repair via different mechanisms: 1) rPDGFBB accelerates deposition ofprovisional wound matrix; 2) rTGF-31 accelerates deposition and maturation of collagen; and 3) rbFGF induces a profound monocellular angiogenic response which may lead to a marked delay in wound maturation, and the possible loss of the normal signal(s) required to stop repair. These results suggest that specific growth factors may selectively regulate components of the repair response by differing mechanisms, offering the potentialfor targeted therapeutic intervention. (AmJ Pathol 1992, 140:1375-1388)

Recombinant DNA-derived polypeptide growth factors are promising therapeutics for healing of chronic dermal wounds in humans. Many of these growth factors are secreted from platelets, macrophages, endothelial cells, and fibroblasts, cells that are essential to the repair process.13 The cellular and molecular components of normal dermal repair are not well defined; however, healing is initiated during an acute inflammatory phase and deposition of provisional extracellular matrix, followed by collagen synthesis and remodeling phases. When fully healed, unknown signals trigger cessation of the repair response and the transition to a quiescent scar. Platelet-derived growth factor (PDGF) and transforming growth factor 431 (TGF-,B1) are potent vulnerary Accepted for publication January 6, 1992. Address reprint requests to Dr. Glenn F. Pierce, Amgen Inc., Amgen Center, Thousand Oaks, CA 91320.

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growth factors that stimulate soft tissue repair in animal models.i14 In fibroblast lines, PDGF selectively stimulates fibronectin, glycosaminoglycan (GAG), and hyaluronic acid synthesis;'1--17 TGF-,B1 triggers GAG, fibronectin, and procollagen type synthesis.17-22 Although rPDGF-BB and rTGF-,11 accelerate in vivo repair via markedly different mechanisms, both growth factors ultimately accelerate collagen synthesis, an essential requirement for normal healing.23'24 Early in repair, rPDGFBB augments the acute inflammatory response, specifically recruiting and activating wound macrophages, whereas rTGF-,1l appears to influence fibroblasts more directly to accelerate collagen synthesis.10 However, little is known concerning the time course and types of matrix deposited as growth factor-accelerated healing progresses toward a maturing scar, and the rates of healing induced in growth factor-treated wounds; these processes have been technically difficult to analyze and quantitate in previously described wound-healing models. A third growth factor, basic fibroblast growth factor (bFGF), is a recognized potent stimulator of endothelial cells and neovessel formation in in vitro and in vivo models of angiogenesis.1 In addition to stimulating endothelial cell proliferation, bFGF also induces the production of proteases, including collagenases in vitro.1'25'26 Since collagenases are required for neovessel penetration through the extracellular matrix,26 bFGF might be expected to alter the normal accumulation of collagen in treated wounds. bFGF has been reported to induce granulation tissue in several wound-repair models although detailed analyses of extracellular matrix deposited in these models have not been performed.' 112,27-29 Despite the previous identification of bFGF as a potent angiogenic agent in the rabbit cornea and chick chorioallantoic membrane assays,1 characterization of its possible role in neovessel formation within actual wounds has received little attention. Recently, Tsuboi and Rifkin demonstrated some increased capillaries within granulation tissue of open wounds treated with bFGF in diabetic

mice.29 Thus, different growth factors may selectively influence specific components of the repair sequence (e.g., acute inflammation, collagen synthesis, repair cessation). Specific growth factors may trigger faster deposition of granulation tissue, greater accumulation of granulation

tissue, and/or alter the composition of newly formed granulation tissue. In view of the potential therapeutic importance of rPDGF-BB, rTGF-13, and rbFGF, we developed techniques to qualitatively and quantitatively investigate the time-dependent sequence of tissue repair in an open excisional wound on the rabbit ear treated by these growth factors. This model was previously used to identify growth factors capable of augmenting granulation tis-

sue, and permitted an initial evaluation of matrix deposition within growth factor-treated granulating wounds.30'31 In this manuscript, we characterize differences observed in growth factor-accelerated matrix deposition and scar maturation.

Methods Growth Factors All growth factors were human, recombinant DNAderived, purified to homogeneity, and endotoxinfree. A 1 19 amino acid form of human PDGF-B was expressed in E. coli and converted in vitro to the active BB dimer. Purified E. co/i-derived rPDGF-BB was compared with Chinese hamster ovary (CHO) cell-derived rPDGF-BB and platelet-purified PDGF3 (and Thomason et al, manuscript in preparation) and found to be functionally equivalent. Half-maximal proliferation of normal rat kidney (NRK) fibroblasts was observed at approximately 0.5 ng/ ml. Homodimeric rTGF-,1l was produced in CHO cells and was tested for its ability to inhibit proliferation of mink lung epithelial cells. Half-maximal activity was observed at approximately 100 pg/ml. Recombinant serine-bFGF (r-ser-bFGF) had serine substitutions for cysteine residues at positions 70 and 88 of the natural bFGF.32 These substitutions do not affect the biological activity of bFGF, although in vitro experiments indicate that r-ser-bFGF is more stable than the natural molecule32 (and G. M. Fox, T. Arakawa, unpublished observations). r-ser-bFGF was assayed for its ability to stimulate DNA synthesis in NIH3T3 cells, and had half-maximal activity at 200 pg/ml. Growth factors were formulated in 0.01 mol/l ammonium acetate, 0.15 mol/l NaCI, 0.25% human serum albumin (pH 4.0) (rPDGF-BB), 0.02 mol/l sodium citrate, 0.1 mol/l NaCI (pH 5.0) (r-ser-bFGF), or phosphate-buffered saline (PBS) (pH 7.2) (rTGF-p1l).

Rabbit Ear Dermal Ulcer Model Full thickness excisional punch biopsies were done on the rabbit ear to the bare cartilage as previously described.30 Under sterile conditions, four wounds were made on each ear using a calibrated 6 mm trephine (Roboz Surgical Instrument Co., Washington, DC), leaving intact cartilage, with the perichondrium removed. Specific concentrations of growth factors were applied to each wound in approximately 5 [I aqueous buffer, and wounds were covered with an occlusive dressing (Tegaderm, 3M, Minneapolis, MN). Wounds were splinted by the cartilage, and hence, were noncontracting.3031 Growth factors were applied to wounds a single time. Approximately 50% of each growth factor remained in

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the wound bed after 1 hour; little remained after 1 day31 (and unpublished observations). Hence, initial biological activities observed up to 3 weeks postwounding are likely due to the endogenous local biological responses (e.g., growth factors) triggered by the growth factors within the first day of repair.

Morphometric Analyses of Wound Matrix Wounds were harvested on the specified days after surgery, bisected, fixed in OmniFix 11 (An-Con Genetics Inc., Melville, NY) for 2.5 hours, and processed by routine methods. Morphometric analyses were performed on bisected hematoxylin and eosin-stained sections. Deposition of new granulation tissue within the wound was quantified using a calibrated lens micrometer, as previously described.30 Briefly, the depth of new tissue at the leading edge of the bisected wound, and the influx of new tissue into the wound bed from the original wound margins were measured. Approximate volume of new granulation tissue was then calculated, and either expressed directly or as a percentage of the tissue deposited in control wounds. All morphometric analyses were performed on blinded samples. Wounds were considered fully healed when they were completely reepithelialized and a bed of new granulation tissue was deposited on the cartilage. In control wounds, this occurred an average of 14 days after wounding.

Lectin Histochemistry Neovascularization was evaluated using Masson's trichrome stain and Bandieria (Griffonia) simplicifolia lectin (BSL) staining using biotinylated BSL 1-isolectin B4 (Vector Laboratories, Burlingame, CA), streptavidin horseradish peroxidase (BRL, Gaithersburg, MD), and diaminobenzidine (DAB) as the chromogen. Incubation with 0.2 mol/l ot-D-galactose (Sigma, St. Louis, MO) before staining served as a specific negative control.33 Wound sections were evaluated for single endothelial cells and neovessel content independently by two individuals blinded to the treatments on a scale from 0 to 4. Vascularity of unwounded dermis was ranked 0, while a maximal response was considered a 4. Functionality of the vessels could not be assessed due to lysis of erythrocytes by the fixative.

GAG Histochemistry Total GAG content (hyaluronic acid and sulfated GAGs) was evaluated by alcian blue (Polysciences, Warrington,

PA) staining at pH 2.5. In previous experiments, staining at pH 4.0 or enzyme digestions was done to demonstrate that both hyaluronic acid and sulfated GAGs were identified when staining was performed at pH 2.5.31,34

Fibronectin Immunohistochemistry Fibronectin deposition into wounds was assessed on tissue sections using a biotinylated goat anti-rabbit fibronectin IgG (Calbiochem, La Jolla, CA) (gift of J. Vande Berg, UCSD), followed by streptavidin-peroxidase (BRL) and incubation with DAB, as described earlier for BSL staining.

Collagen Histochemistry Collagen bundles and fibrils were evaluated in 12 p.m thick sections using Sirius red F3BA (Roboz Surgical Instruments) staining with polarization optics.36 Large, wellorganized mature bundles stained orange-red to red, whereas thin, less-organized fibrils stained green to yellow.37 The color of birefringence was not correlated with collagen type. The influx of collagen bundles entering the wound from the wound margins was measured under polarized light. The ratio of collagen influx (Sirius Red) to total granulation tissue influx (as assessed on H&Estained serial sections) penetrating into the wound bed was calculated and is presented as the percentage of collagen matrix deposited in the wound. Collagenolysis of normal dermis bordering the wound was quantitated using Sirius red staining. The extent of loss of collagen from normal dermis at the wound borders was measured using a lens micrometer.

Image Analysis The Quantimet 520 image analyzer (Leica, Inc., Deerfield, IL) coupled to a Nikon Optiphot microscope was used to quantitate GAG and fibronectin deposition in wounds. For GAG quantitation, a 600 nm filter (Oriel Corp., Stratford, CT) with a 10 nm bandwidth was used to measure alcian blue deposition at the leading edge of the wound as a function of total wound area. A similar analysis was completed for fibronectin staining (DAB deposition) utilizing a 540 nm filter. Results are presented as the total area of positive staining in a bisection of the wound, as described.w

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(0.4-2.0 jig/cm2), consistent with, although tenfold more potent than a previous report in which it was administered to wounds in a collagen vehicle.30 r-ser-bFGF also appeared to induce a marked bimodal effect on granulation tissue formation (Figure 1). r-ser-bFGF triggered peak tissue deposition at 2 jig/ wound (8 jig/cm2), in marked contrast to its previously observed minimal influence on cell influx and matrix deposition at comparable doses when incorporated into a collagen vehicle.'

Statistical Analyses For each variable measured, 5-10 wounds treated by each growth factor were analyzed. On H&E-stained bisected wound sections, an unpaired Student's t-test was used to analyze absolute amounts of new tissue formation and the percent of new tissue relative to controls (one group analysis) (Statview 11, Abacus Concepts, Berkeley, CA). Regression analysis of healing as a function of time was performed; rates of healing and the 50% healing point were obtained from the slope and regression line, respectively. Nonparametric analysis was performed on noncontinuous data (BSL staining) using the MannWhitney U test. Differences in wound GAG and fibronectin content between groups were analyzed using oneway analysis of variance (ANOVA) and the unpaired t-test.

Kinetics of Granulation Tissue Formation Induced by Growth Factors Once optimal doses for aqueous formulations were established, wounds were treated with rPDGF-BB (5 jig), rTGF-,B1 (0.5 jLg), or r-ser-bFGF (2 jig), and harvested at days 3, 5, 7, 10, and 14 postsurgery to analyze rates of granulation tissue formation. After a lag phase of approximately 3 days, control wounds demonstrated a linear healing rate through day 14, when they were completely healed (fully closed) (Figure 2). Wounds were considered fully healed when they were completely reepithelialized and contained a bed of new granulation tissue overlying the cartilage. At this point, they contained approximately 15 mm3 of new tissue (Figure 2). The single application of rPDGF-BB or rTGF-p1 induced nearly identical accelerations of tissue deposition, diverging from controls between days 5 and 7, and resulting in a 4-day acceleration of healing by 10 days postwounding (30%). Minimal differences were detected in wounds at 3 days, indicating the growth factors are not detectably shortening the lag phase of repair in this model. r-ser-bFGF also accelerated repair by 4 days during the first 10 days (30%, Figure 2). In contrast to the self-

Results

Vulnerary Effects of rPDGF-BB, rTGF-11, and r-ser-bFGF Since previous studies were carried out using a collagen

vehicle,30 aqueous formulations of rPDGF-BB, rTGF-I1 and r-ser-bFGF first were evaluated in the rabbit ear ulcer model to determine optimal doses for maximal vulnerary potential (Figure 1). The volume of new granulation tissue deposited in wounds 7 days after surgery and the single application of different doses of growth factor in aqueous buffer is expressed as a percentage of new tissue found in untreated wounds (Figure 1). rPDGF-BB induced peak responses at 0.5-5.0 jg/wound (2-20 jg/cm2). rTGF-31 treatment appeared to have a bimodal effect on granulation tissue formation, peaking at 0.1-0.5 ,ug/wound, uJ

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Figure 1. New granulation tissue induced in rPDGF-BB, rTGF-1l, or r-ser-bFGF treated wounds after 7 davs. Growth factors in aqueous buffer were applied to wounds once at the time of surgery, and wounds were covered with an occlusive dressing. New matrix deposition in the wound was quantified histologically on bisected cross-sections. Each group contained at least ten wounds. A one group t-test was used to assess significance. For each treatment group, wound volumes were calculated, and are expressed as a percentage of untreated (control) wounds. Each doseresponse curve was repeated twice.

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limiting acceleration induced by rPDGF-BB and rTGF-B1, which ceased when the wounds were fully closed, granulation tissue deposition induced by r-ser-bFGF continued at a linear rate through day 14. By day 14, r-serbFGF-treated wounds had approximately 200% of the new tissue generated in rPDGF-BB, rTGF-,1l, and control wounds, resulting in a marked palpable mass on the ear. Although closed by day 10, r-ser-bFGF-treated wounds continued to accumulate matrix and remained nearly twice the volume of controls through at least 21 days postwounding. To more precisely define the rates of accelerated healing, regression analyses were performed on the growth factor-induced healing responses observed in Figure 2, revealing a calculated healing rate (in mm3 per day) for rPDGF-BB, rTGF-,1, and r-ser-bFGF of approximately 200% of control wounds between days 5 and 10 postwounding (Table 1). The day on which wounds achieved 50% healing was also accelerated by 2.5 days in growth factor-treated wounds (Table 1).

Glycosaminoglycan Deposition GAGs are a critical initial component of the provisional extracellular matrix deposited within wounds. In previous experiments, rPDGF-BB enhanced, whereas rTGF-p1 Table 1. Healing Rates of Growth Factor-treated Wounds* Control

Dose (,ug) Healing (mm3/day) % Control healing 50% Healing (days)t Acceleration to 50% healing (days)

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appeared to decrease absolute GAG levels within 10day open wounds.31 In the present study, using concentrations of growth factors found to be optimal for matrix deposition and wound closure (Figure 2), GAG content of growth factor-treated and untreated wounds after 7 or 21 days was quantitated by image analysis. GAGs consisted of both hyaluronic acid and proteoglycan sulfates, as assessed by differential pH staining and enzymatic digestion.31 At 7 days, when wounds are not yet fully reepithelialized, rTGF-pl-treated wounds contained markedly decreased amounts of both hyaluronic acid and proteoglycan sulfates, compared with rPDGF-BBtreated wounds (P < 0.005; Figure 3), which contained more than threefold more GAG than control wounds (P = 0.001, Figure 3). No increased GAGs were observed in rTGF-,1 -treated wounds at days 3 or 5 (data not shown), indicating that rTGF-p1 is not simply accelerating provisional matrix deposition. Similar to rPDGF-BB-treated wounds, r-ser-bFGF-treated wounds had a GAG content significantly higher than untreated wounds at day 7 (P = 0.024). Representative 7-day wounds showing differences in GAG deposition are shown in Figure 4A. At day 21, when wounds are closed and when maturing scar is present in controls, r-ser-bFGF-treated wounds contained almost threefold more GAG than controls (6.5 1.4 vs. 2.3 0.4 mm2, P = 0.006). In contrast, rPDGF-BB and rTGF-,1 -treated wounds had GAG ±

rPDGF-BB 5 2.35 194 6.79 2.5

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* Regression analysis was performed on the healing curves depicted in Figure 2, for the healing rate between days 5-10 postwounding. N = 10 wounds per group. t 50% healed wounds contained approximately 7.5 mm3 new tissue.

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Fibronectin Content of Wounds Since fibronectin also is a critical component of the provisional extracellular matrix of wounds,39'40 and is required for organized collagen deposition,41'42 growth factor-treated wounds were quantitatively analyzed for fibronectin deposition. Using fibronectin specific immunohistochemical and computerized image analyses, markedly increased fibronectin deposition was detected in all growth factor-treated wounds at day 7, compared with controls (Figures 4B, 5). At day 21, fibronectin continued to be deposited in r-ser-bFGF-treated wounds, and was almost threefold greater than levels in control wounds (6.2 1.2 vs. 2.3 0.5 mm2, P = 0.006). By day 21, rPDGF-BB- and rTGF-1l -treated wounds had fibronectin contents similar to controls (-2.0 mm2) (Figure 5). ±

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Deposition of Collagen in Growth Factor-treated Wounds Previous results indicated rTGF-f31 selectively increased collagen deposition in 10-day wounds.31 In the present studies, the degree of collagen influx into wounds induced throughout the entire repair process (through day 21) was quantitatively compared for rPDGF-BB-, rTGF,B-, r-ser-bFGF-treated and control wounds, using concentrations found to be optimal for matrix deposition and wound closure (Figure 6). rTGF-,1 induced a marked, early, and rapid increase in wound collagen (P = 0.001, Figure 6). The leading

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treated wounds after 7 days, and only in r-ser-bFGF-treated wounds after 21 days. At 21 days, GAGs were significantly decreased in rPDGF-BB- and TGF-1l-treated wounds, compared with controls, suggesting more rapid wound maturation in these wounds. Each group contained 5-10 wounds. Oneway analysis of variance and an unpaired t-test were used to assess significance (global

ANOVA P value = 0. 002 and 0.0001 at 7 and 21 days, respectively).

front of new matrix deposition in rTGF-pl treated 7-day wounds was comprised almost entirely of large, mature collagen bundles (Figure 4C). In contrast, rPDGF-BBtreated wounds had a significantly lower influx of new collagen than control wounds through day 14 postwounding (P < 0.02), indicating that the enhanced provisional matrix deposition (e.g., GAGs fibronectin) at the leading edge of new tissue were responsible for accelerated wound closure. However, by day 21, rPDGF-BBtreated wounds had a significantly greater deposition of new collagen within the scar compared with control wounds (P = 0.02). Surprisingly, despite continued deposition of matrix, r-ser-bFGF-treated wounds had a minimal accumulation of new collagen through day 21 postwounding, and had significantly less new collagen than controls at days 14 (P = 0.01) and 21 (P = 0.03). The absence of collagen in the r-ser-bFGFaugmented wound granulation tissue through day 21 (11 days after wound closure) prompted a search for potential in vivo collagenolytic activity in r-ser-bFGF-treated wounds. Morphometric analysis of Sirius red stained sections revealed significant erosion of mature collagen bundles in the normal unwounded dermis bordering day 7 r-ser-bFGF-treated wounds (Table 2, Figure 4C). Some erosion of unwounded dermal collagen occurs normally in the wound-healing process, as cells migrate through the dermis and into the wound bed; however, erosion of collagen was increased almost 50% in the dermis surrounding r-ser-bFGF-treated wounds. Increased collagen degradation was not observed in the dermis bordering rPDGF-BB or rTGF-pl -treated wounds, compared with control wounds (Figure 4C). New collagen fibrils normally appear at the leading edge of new matrix deposition within control wounds by day 7 (Figure 4C); these were essentially absent in r-ser-bFGF-treated wounds (Table 2), confirming the lack of new collagen bundle deposition within r-ser-bFGF-treated wounds observed in the time course experiments (Figure 6).

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Figure 4. Differences in extracellular matrix deposition in 7-day-old rPDGF-BB, rTGF-11, r-ser-bFGF treated, and (control) wounds. A: Alcian blue stain for GAGs. B: Fibronectin immunohistochemistry. C: Sirius red stain for collagen. Note theuntreated decreased GAGs in TGF-f1-treated wounds, and the increased collagen degradation of normal dermis bordering the wound in ser-b-FGF-treated (arrowhead). A, x 16; B, x 160; C, X80 original magnifications. Arrows, leading edges of new tissue deposition; C, cartilage; D, dermiswounds belou' cartilage; E, epidermis.

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Recombinant ser-bFGF-induced Neovessel Formation Routine histologic analysis suggested increased neovascularization in r-ser-bFGF-treated wounds (Figure 4A). The absence of significant collagen accumulation in r-ser-bFGF-treated wounds suggested a loss of normal time-dependent wound provisional matrix maturation into a collagenous scar. Therefore, a specific endothelial cell marker, Bandieria simplicifolia lectin, which binds endo10: 0

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thelial cell surface galactose residues, was used to characterize r-ser-bFGF-induced granulation tissue. Unexpectedly, BSL staining revealed a matrix containing almost entirely single endothelial cells and neovessels in r-ser-bFGF-treated wounds (Figure 7). In comparison, rPDGF-BB and rTGF-31 -treated wounds demonstrated supportive neovascularization, fewer single endothelial cells, and contained numerous non-BSL stained fibroblasts at 7 days. Morphometric analysis of BSL-stained wound sections revealed significant increases of single 0.0.006 006

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in fibronectin deposiFigure 5. tion in rPDGF-BB, r-ser-bFGF, and control wounds after 7 and 21 days. Total fibronectin content within wounds was quantitated using anti-fibronectin immunohistochemistry coupled with computerized image analysis Fibronectin was increased in all growth factor-treated wounds at 7 days, and only in r-ser-bFGF-treated wounds at 21 days. Each group contained 7-10 wounds. Oneway ANOVA and an unpaired t-test were used to assess significant differences between groups (global ANOVA P value = 0.029 and 0.007 at 7 and 21 days, respectively).

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DAYS POSTWOUNDING Figure 6. Collagen deposition in rPDGF-BB, rTGF- 131, and r-ser-bFGF treated wounds. The ratio of mature collagen bundle influx to overall extracellular matrix influx in healing wounds was assessedfrom 7 through 21 dayspostwounding using the Sirius red stain andpolanzation optics. An unpaired, two-tailed t-test was used to detect differences in collagen content between growth factor treated and control wounds for each day analyzed.

endothelial cells and neovessels in r-ser-bFGF-treated compared with both control (P = 0.0001) and rPDGFBB-treated wounds (P < 0.03; Figure 8) The endothelial cell activation and neovessel formation induced by r-serbFGF appeared reversible; by day 21 the wounds consisted of a nonmaturing provisional matrix (e.g., fibronectin, GAGs) containing primarily fibroblastic-like cells. BSL staining revealed neovessels and endothelia present in somewhat decreased proportions compared with earlier timepoints, indicating regression of neovessels. Significantly, collagen deposition in r-ser-bFGF-treated wounds remained absent at this time (Figure 6).

Discussion The mechanisms underlying the failure of chronic dermal wounds to heal are largely unknown. Although endogenous growth factor levels have not been measured in chronic wounds, and thus deficiencies have not been reported, pharmacologic doses of polypeptide growth factors trigger processes in vivo considered essential for normal tissue repair. Provisional matrix deposition, fol-

lowed by collagen deposition, and appropriate neovascularization of the developing scar are essential for normal repair, and agents that can safely and transiently accelerate these processes might have therapeutic value. Adequate in vivo wound-healing models that permit quantitation of a broad spectrum of repair processes (e.g., reepithelialization, angiogenesis, and matrix deposition) and detailed analysis of the normal repair sequence have been difficult to achieve experimentally. Table 2. Absence of New Collagen Fibrils and Erosion of Collagen from Normal Dermis Bordering r-ser-bFGFtreated Wounds*

Treatment

N

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Erosion of collagen (>m) 811 ± 96 553 t 75 0.03t

Wounds containing new collagen fibrils at leading edge 1 6 0.05t

P value < * Sirius red stained wound sections were analyzed 7 days after wounding and r-ser-bFGF treatment (2 Rg). The horizontal depth of collagen loss in unwounded dermis surrounding the wound was measured using a lens micrometer. Mean + SE presented. t Student's unpaired t-test.

t Chi-square analysis.

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Figure 7. Identification of endothelial cells and neovessels in r-serbFGF and rPDGF-BB-treated wounds after 7 davs. Wound sections u'ere stained with Bandieria simplicifolia lectin (brown chromagen) and counterstained with hematoxylin. A, r-ser-bFGF, 2 pLg per wound; B, rPDGF-BB, 5 Fg per wound; C, untreated wounds. Original magnification, X320.

The rabbit ear model permits a precise, reproducible, and more comprehensive analysis of each of these processes. In the present study, rPDGF-BB, rTGF-13l, and r-ser-bFGF each accelerated matrix deposition, and thus wound closure, via different mechanisms, predicted only in part from the analysis of their specific effects on cells in vitro. Detailed analyses of matrix deposition were performed only at the growth factor doses that induced maximal deposition of new tissue; conceivably other concentrations might induce different patterns of matrix deposition than observed in the present experiments.

rPDGF-BB accelerated wound closure via augmenting provisional matrix deposition at the leading edge of new granulation tissue, resulting in an absence of new collagen accumulation until later in the repair process (Figure 9). These observations are consistent with results obtained using cultured fibroblasts, in which PDGF has been shown to stimulate fibronectin, hyaluronic acid, and GAG synthesis.'117 Our previous results have shown that rPDGF-BB augments the acute inflammatory phase of repair, likely through stimulating a cascade of endogenous growth factor activities,810,13 consistent with its

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known potent in vitro effects on monocyte and fibroblast chemotaxis and activation.43 4 rPDGF-BB may stimulate increased collagenase production, as it does in vitro4546; however, no evidence for enhanced collagenolytic activity in rPDGF-BB-treated wounds was observed in the present experiments. Thus, rPDGF-BB appears to accel-

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wound fibroblast phenotype directly (Figure 9). Minimal GAG deposition was noted, although TGF-j1 has been shown to stimulate GAGs in vitro.18 In cultured fibroblasts, TGF-,1 also has been reported to trigger fibronectin and procollagen type synthesis, to decrease synthesis of metalloproteases and plasminogen activator, and to increase synthesis of metalloprotease inhibitor and plasminogen activator inhibitor, resulting in a net stabilization of matrix.19'22'47'48 Unlike TGF-l,1 PDGF-BB, and bFGF have not been shown to directly stimulate induction of procollagen type mRNA;21 however, PDGFBB also can induce metalloprotease inhibitor.46 A requirement for a fibronectin matrix has been established for extracellular collagen assembly, thus rTGF-,1l is directly influencing two processes critical for scar maturation.41'42 Recently, Quaglino and coworkers demonstrated induction of collagen and fibronectin mRNA, and repression of metalloproteinase mRNA in situ within rTGF-pl -treated pig wounds,49 further supporting a critical role for TGF-pl in stabilization and maturation of the scar. Conceivably, circumvention of the normal sequence of repair could lead to excessive scarring or inappropriate cellular activation.50 However, TGF-,1ltreated wounds had only transient acceleration of repair in this model. Recombinant ser-bFGF treatment resulted in a closed wound containing essentially no collagen (Figure 9). Collagen is essential for wound integrity and strength;5'10'23'24 its absence would suggest a weak immature scar at increased risk of dehiscence. Loss of collagen in the dermis surrounding the wound and absence of fibrils at the leading edge of the wound indicate that r-ser-bFGF may have induced collagenolysis, likely required for the marked neovessel response,25'26.51 but contributing, at least in part, to inhibition of net collagen deposition through day 21. Surprisingly, bFGF-mediated induction of endothelial cell proliferation and influx coupled with neovessel formation within an open wound bed has not been previously quantified, although bFGF is considered the prototypical angiogenic agent. In our previous studies, binding of rbFGF to its collagen vehicle may have prevented expression of its full biologic potential.30 In those studies, rbFGF demonstrated inconsistent angiogenesis only at high concentrations (>50 ,ug rbFGF per wound).30 In the present studies, using aqueous formulated r-ser-bFGF, the highly specific effect on endothelial cells confirms its role in angiogenesis in vivo and directly supports the observed direct induction of capillary sprouts in in vitro and in vivo models of angiogenesis. 1 25.26 Previously reported in vivo models of angiogenesis were semiquantitative at best.1 The present results suggest that the rabbit ear model will be a useful quantitative in vivo assay of putative angiogenic factors and their mechanisms of action.

Interestingly, TGF-pl, which has been proposed to both inhibit endothelial cell proliferation and to directly induce capillary-like structures in vitro,252 triggered only supportive angiogenesis similar to that induced by rPDGFBB. Thus, at the concentrations used, rTGF-,1 and rPDGF-BB also are potent angiogenic agents in this system, although they may function indirectly. However, in light of recent observations that PDGF-BB can stimulate microvascular endothelial cells directly,5'35 our experiments cannot rule out a direct influence on neovessel formation by rTGF-,B1 or rPDGF-BB. Unlike the self-limited acceleration induced by rPDGF-BB and rTGF-,1l, provisional matrix continued to be deposited in r-ser-bFGF-treated wounds long past wound closure, resulting in the development of macroscopic nodules resembling capillary hemangiomas by day 10. The signals that result in the cessation of repair in healed wounds are unknown. Treatment with supraphysiologic concentrations of r-ser-bFGF may interrupt or delay the potential negative feedback loop(s) that are initiated within healed scars to stop repair; rPDGF-BB and rTGF-pl do not seem to influence this normal homeostatic mechanism, which may involve apoptosis of fibroblasts and endothelial cells during the transition to a quiescent scar.55'56 Alternatively, matrix constituents of the maturing scar, such as collagen, may signal repair to stop; their absence in r-ser-bFGF-treated wounds may permit continued matrix deposition. Although pharmacologic doses of r-ser-bFGF used in the present study may influence or delay an apoptotic event, regulated levels of endogenous bFGF may be essential for normal tissue repair, as observed recently using specific antibodies to inhibit wound bFGF.57 Recently, TGF-p1 was shown to enhance apoptosis of cultured rabbit uterine epithelial cells, suggesting a role in reproductive regulation.58 Continued study of bFGF- and TGF-,1-induced repair may provide new insights on the transition of active wounds to quiescent scars.

Acknowledgments The authors thank Kelly Doria, Diane Duryea, Leslie Edwards, Stephanie McCarter, and Ed Shatzen for outstanding technical assistance; Joan Bennett for manuscript preparation; and Tsutomu Arakawa, Philip Hsieh, Sylvia Hu, and Keith Westcott for the highly purified recombinant proteins.

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Platelet-derived growth factor (BB homodimer), transforming growth factor-beta 1, and basic fibroblast growth factor in dermal wound healing. Neovessel and matrix formation and cessation of repair.

Recombinant platelet-derived growth factor (BB homodimer, rPDGF-BB), transforming growth factor beta 1 (rTGF-beta 1), and basic fibroblast growth fact...
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