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Research of PDGF-BB Gel on the Wound Healing of Diabetic Rats and Its Pharmacodynamics
Journal of Surgical Research 145, 41– 48 (2008) doi:10.1016/j.jss.2007.02.044
Research of PDGF-BB Gel on the Wound Healing of Diabetic Rats and Its Pharmacodynamics Haihong Li, M.D.,* Xiaobing Fu, M.D.,†,1 Lei Zhang, M.D.,* Qingjun Huang, M.D.,* Zhigu Wu, M.D.,† and Tongzhu Sun, M.D.† *Mental Health Center, ShanTou University Medical College, ShanTou, People’s Republic of China; †Wound Healing and Cell Biology Laboratory, Burns Institute, The First Affiliated Hospital, General Hospital of PLA, Trauma Center of Postgraduate Medical College, Beijing, People’s Republic of China Submitted for publication October 5, 2006
excisional wound of diabetic rats. The effects of topically applied PDGF-BB were dose-dependent. Conclusions. PDGF-BB is an important future clinical tool, particularly for stimulating soft tissue repair in patients with an impaired capacity for wound healing. © 2008 Elsevier Inc. All rights reserved. Key Words: platelet-derived growth factor; wound healing; pharmacodynamics; diabetes; rat.
Background. One of the leading causes of impaired wound healing is diabetes mellitus. In diabetic patients, a minor skin wound often leads to serious complications. Many experiments had demonstrated that the expression of platelet-derived growth factor (PDGF) and its receptor was decreased in wounds of healing-impaired diabetic mice, indicating that a certain expression level of PDGF is essential for normal repair. Materials and methods. The diabetic rats was induced by a single i.p. injection of streptozotocin and a 1.8 cm diameter full-thickness wound was made on each side of the rat mid-back. Then the rats were randomly divided into five groups, with eight animals in each group as follows: blank control, vehicle control, 3.5 g PDGF-BB/cm2 treatment group, 7 g PDGF-BB/cm 2 treatment group and 14 g PDGF-BB/cm 2 treatment group for either 7 or 14 consecutive days after wounding. Re-epithelialization area was measured by computerized planimetry, percentage wound closure and percentage wound contraction was calculated, granulation tissue and collagen formation was assessed by Masson trichrome, cell proliferation (proliferating cell nuclear antigen staining) and angiogenesis (Factor VIII related antigen staining) was assessed by immunohistological methods. Results. PDGF-BB treatment improved healing quality, enhanced angiogenesis, cell proliferation and epithelialization, and formed thicker and more highly organized collagen fiber deposition in full-thickness
INTRODUCTION
One of the leading causes of impaired wound healing is diabetes mellitus. Diabetic wound healing is a complex and lengthy process and the cost of it in the United States has been estimated at $1 billion per year, so the treatment of diabetic wounds is a formidable clinical challenge [1, 2]. In diabetic patients, a minor skin wound often leads to chronic, nonhealing ulcers and ultimately result in infection, gangrene, even amputation [3, 4]. Multiple factors contribute to the impairment of wound healing in the wound environment of diabetic animals and patients. One of the important reasons is that diabetic animals and patients do not produce enough growth factors or the destruction of growth factors is increased [5, 6]. Platelet-derived growth factor (PDGF) exerts its functions by binding to transmembrane tyrosine kinase receptors [7]. A series of studies suggested an important role of PDGF in the repair process [8 –10]. Upon injury, PDGF is released from degranulated platelets and presented in wound fluid, particularly early after injury. Interestingly, expression of PDGF and their receptors was reduced in wounds of healingimpaired diabetic animals, indicating that a certain expression level of PDGF and its receptors is essential for normal repair [11, 12].
1 To whom correspondence and reprint requests should be addressed at Wound Healing and Cell Biology Laboratory, Burns Institute, The First Affiliated Hospital (304th Hospital), General Hospital of PLA, Trauma Center of Postgraduate Medical College, 51 Fu Cheng Road, Beijing 100037, P.R. China. E-mail: fuxb@cgw. net.cn; [email protected].
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0022-4804/08 $34.00 © 2008 Elsevier Inc. All rights reserved.
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To investigate the effects of topical application of PDGF-BB and its pharmacodynamics on the wound healing in diabetic rats, we made full-thickness wounds on mid-back of diabetic rats, then we measured wound area and re-epithelialization area by computerized planimetry, calculated the percentage wound closure and wound contraction, and assessed granulation tissue, collagen formation (Masson trichrome), cell proliferation (proliferating cell nuclear antigen [PCNA] staining), and angiogenesis (Factor VIII related antigen staining) by immunohistological methods. MATERIALS AND METHODS Preparation of Diabetic Rats All procedures on rats were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Before and during experiments, all rats were housed in individual cages in a central animal care facility, maintained on a 12 h light-dark cycle, and given free access to standard
FIG. 2. Schematic representation of wound healing on day N after wounding.
rodent chow and water. Forty male Wistar rats, obtained from the Academy of Military Medical Sciences (Beijing, China), aged 2 mo with an initial body mass of approximately 180 g were used for the study. Diabetes was induced by a single i.p. injection of sterile 0.5% streptozotocin (STZ) (Sigma, ST. Louis, MO) in sodium citrate buffer (0.1 mol/L, pH 4.5) at a dose of 60 mg/kg body weight immediately before use. The diabetic state was confirmed 72 h after STZ injections by blood glucose levels exceeding 15 mmol/L. One week after the initial administration of STZ, studies were carried out as described below.
Wounding and Treatment The rats were anesthetized by ethylether inhalation. After clipping the hair on the back, the skin was sterilized with 75% alcohol. A sterile template of 1.8 cm in diameter was placed on each side of the mid-back and a full-thickness wound to deep fascia corresponding to the template was made by excising the skin (Fig. 1A). Then the rats were randomly divided into five groups: blank control, vehicle control, 3.5 g PDGF-BB/cm 2 treatment group, 7 g PDGF-BB/cm 2 treatment group, and 14 g PDGF-BB/cm 2 treatment group. Different concentration of PDGF-BB were mixed in a vehicle of sterilely filtered 5% polyethylene glycol and stored at 4°C until the day of use. In PDGF-BB-treatment groups and vehicle control group, 0.1 mL of the PDGF-BB plus vehicle or vehicle only was applied to each wound once daily for either 7 or 14 consecutive days starting at wounding by injecting it to the wound and allowing it to spread over the wound bed, while in blank control group, neither PDGF-BB nor vehicle was applied to the wound. Then the wounds were covered with one layer of petrolatum gauze and five layers of sterilized dressing, and stabilized with 3M adhesive tape.
Serial Analysis of Wound Healing
Blood Glucose In all groups, blood was obtained from tail vein and glucose content was analyzed using a blood glucose test meter (GlucoDr, Seoul, Korea) at the day of wounding and at day 3, 7, 10, and 14 after wounding.
Body Weight FIG. 1. Administration of PDGF-BB topically accelerated wound healing in diabetic rats. Full-thickness excisional wounds about 1.8 cm in diameter were created on the back of male diabetic rats, age 2 mo (A). Different concentration of PDGF-BB, or vehicle only, or neither of them, was administered topically once daily for either 7 or 14 consecutive days starting at wounding by injecting it to the wounds and allowing it to spread over the wound beds. The wounds were completely closed in 14 g PDGF-BB/cm 2 treatment group (F), while in blank control group (B), vehicle control group (C), 3.5 g PDGF-BB/cm 2 treatment group (D), and 7 g PDGF-BB/cm 2 treatment group (E), there were still some open wounds of varied extent. (Color version of figure is available online.)
Body weight was weighed using electronic balance (accuracy to g) on the day of wounding, as well as day 3, 7, 10, and 14 after wounding.
Wound Area and Re-epithelialization Area The edge of migration epithelium was easily discernible from the moist granulation tissue and the presence of the epithelial border was the edge of the healing wound. The edge of the wound was traced onto a glass microscope slide and the wound area was determined by planimetry using ImageMeasure (Phoenix Biotechnology, Seattle, WA). The trace taken immediately after wounding was used as the
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original area (day 0 area). Re-epithelialization area of the wound on day N was the area between the wound edge and the newly formed epithelial edge on day N (Fig. 2). Wounds were considered closed if moist granulation tissue was no longer apparent, and the wounds appeared covered with new epithelium.
number of new capillary sprouts was counted under 400⫻ visual field and at least three visual fields of granulation tissues or wounds were counted per slide.
Percentage Wound Closure and Percentage Wound Contraction
Data were analyzed using standard statistical software (SPSS 10.0; SPSS Inc., Chicago, IL). The values were expressed as mean ⫾ standard deviation. The statistical significance of values among groups was evaluated by one-way analysis of variance, followed by least significant difference t test. The difference was considered significant when the P-value was 0.05 or less.
Wounds were closed by both contraction and forming newly formed epithelium to cover the wounds. Wound closure was expressed as percentage closure of the original wound and was calculated by the following formula: % wound closure on day N ⫽ [(area on day 0-open area on day N)/area on day 0] ⫻ 100. % wound contraction on day N ⫽ % wound closure on day N ⫺ [(Re-epithelialization area on Day N/Area on Day 0) ⫻ 100].
Histological Analysis After the final wound tracing on either day 7 or day 14, the rats were given a lethal ethylether inhalation, and the entire wound, including a 5 mm margin of unwounded skin, was excised down to the fascia. The wound was divided in half through the least healed portion. One-half of the wound was stored in the liquid nitrogen for future molecular studies, the other half was fixed in 10% formalin for at least 24 h, followed by processing for conventional paraffin embedding and sectioned 5-m-thick on a Microme (Leica, Wetzlar, Germany). For morphological observations, sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome, and for immunohistochemical analysis, sections were processed using the two-step immunostaining kit (Zymed, San Francisco, CA).
H & E Staining Tissue sections were dewaxed in xylene, rehydrated through graded alcohols to phosphate-buffered saline, stained in H&E, and mounted in resin.
Statistical Analysis
RESULTS Preparation of Diabetic Rats and Wounding
Rats receiving STZ resulted in significant elevation in blood glucose level after 1 wk (data not shown) that was sustained throughout the duration of the study. On average, rats had a mean body weight of 181.0 g ⫾ 7.6 g) at the beginning of the study. The rats tolerated the wounding procedure without problems and they had no obvious changes in food consumption or weight loss after wounding. Effects of PDGF-BB on the Blood Glucose and Body Weight
Blood glucose was elevated at varied levels 3 d after wounding and reached its peak 7 d after wounding in all of the five groups, but there was no significant difference between groups at different time points (Fig. 3A). Body weight of the rats between groups and within-group had no significant differences at varied time points after wounding, but it indeed increased at different levels (Fig. 3B).
Masson’s Trichrome Sections were dewaxed and rehydrated conventionally, placed in Weigert’s hematoxylin stain for 1 h, rinsed under lukewarm water for 5 min, immersed in Masson solution for 15 min, and rinsed in deionized water before placing in phosphomolybdic acid for 10 min. Subsequently, sections were immersed in 2% aniline blue for 15 min, rinsed in 1% acetic acid, 95% ethanol, and absolute ethanol in turn, immersed in xylene for 10 min, and mounted with resin. Collagen fibers were stained blue, cytoplasm and erythrocyte were stained red, and nuclei were stained bluish brown.
Immunohistochemical Staining Sections were dewaxed and rehydrated, then were incubated in 3% hydrogen peroxide to block endogenous peroxidase. Sections for Factor VIII related antigen detection were incubated with 0.4% pepsin at 37°C for 30 min, while sections for PCNA detection were microwaved for 15 min at 95°C in citrate buffer. Subsequently, sections were incubated in rabbit anti-Factor VIII related antigen (NeoMarkers, Fremont, CA) and mouse anti-PCNA (NeoMarkers) monoclonal antibodies at a 1:100 dilution respectively, followed by incubation in horseradish peroxidase conjugated goat anti-rabbit IgG (Vector, Burlingame, CA) or goat anti-mouse IgG (Vector). Sections were finally developed in 3,3=-diaminobenzidine, counterstained with hematoxylin and mounted in resin. PCNA expressed in nuclei and Factor VIII related antigen in cytoplasm. The number of brown labeled nuclei was counted to evaluate the PCNA labeling index, namely, the percentage of immunoreactive nuclei in 100 cells and at least 2000 cells in the wounds were counted per slide. The
Effects of PDGF-BB on Percentage Wound Contraction, Re-epithelialization Area and Percentage Wound Closure in Diabetic Rats
An approximation of the extent of contraction was achieved by comparing the area bordered by the wound edge at 7, 10, and 14 d to the area of wound at day 0 respectively. The skin rapidly contracted to cover the defect and only a small region healed by development of granulation tissue and re-epithelialization in the diabetic rats in all of the groups. However, wound contraction contributed less to the healing of diabetic rats in 7 g PDGF-BB/cm 2 (86.3% ⫾ 7.0%) and 14 g PDGF-BB/cm 2 (86.2% ⫾ 3.4%) treatment groups than rats in vehicle control group (91.6% ⫾ 1.9%) or blank control group (91.2% ⫾ 1.2%) on day 14 after wounding (Fig. 4A). Roughly 91% of the wound closure in control groups was attributable to wound contraction, while only about 86% of the wound closure in 7 g PDGFBB/cm 2 and 14 g PDGF-BB/cm 2 treatment groups was due to contraction (Fig. 4A). There was still no obvious epithelium tissue growth 3 d after wounding. Thereafter, newly formed epithelium was seen to migrate from the edge of the wound.
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FIG. 3. Changes in blood glucose and body weight in diabetic rats in each group at varied time points. Blood glucose was elevated at varied levels 3 d after wounding and reached its peak 7 d after wounding in all of the five groups, but there was no significant difference between groups at different time points (A). The body weight of the rats between groups and within-group had no significant difference at varied time points after wounding, but it indeed increased at different extent (B). *P ⬍ 0.05 compared with blank control; ŒP ⬍ 0.05 compared with vehicle control.
The newly formed epithelium area was significantly improved in the three PDGF-BB-treated groups versus blank control (0.51 ⫾ 0.29 cm 2) on day 7, and 14 g PDGF-BB/cm 2 treated group versus either vehicle control or blank control on day 7 and day 14, as well as 7 g PDGF-BB/cm 2-treated group (0.46 ⫾ 0.19 cm 2) versus either vehicle control (0.33 ⫾ 0.06 cm 2) or blank control (0.26 ⫾ 0.07 cm 2) on day 14 (Fig. 4B). Treatment of wounds with PDGF-BB also enhanced the percentage wound closure when compared with vehicle treatment group and blank control group on day 7 after wounding. By day 14, all of the wounds had approached complete closure, so no difference was noted in the percentage wound closure, but the healing
quality of the diabetic rats in both 7 g PDGF-BB/cm 2 and 14 g PDGF-BB/cm 2 treatment groups was significantly improved compared with rats in vehicle control group and blank control group on that day. The effects of topically applied PDGF-BB were dose-dependent. No significant improvement in percentage wound closure and re-epithelialization area was observed in diabetic rats treated with PDGF-BB 3.5 g/cm 2 per day compared with rats receiving vehicle treatment or blank treatment at day 14 after wounding. In contrast, rats receiving PDGF-BB, either 7 g/cm 2 or 14 g/cm 2 per day, displayed significantly improved wound healing compared with rats receiving vehicle treatment or blank treatment on days 7 and 14 (Fig. 4C).
FIG. 4. Percentage wound contraction, re-epithelization area, and percentage wound closure in diabetic rats in each group at varied time points. Wound edges were traced onto glass microscope slides and image analysis performed to determine percentage wound contraction (%), re-epithelization area (cm 2), and percentage wound closure (%). Wound contraction contributed less to the healing of full-thickness skin excisions in 7 g PDGF/cm 2 (86.3% ⫾ 7.0%) or 14 g PDGF/cm2 (86.2% ⫾ 3.4%) treatment groups than those in vehicle control group (91.6% ⫾ 1.9%) or blank control group (91.2% ⫾ 1.2%) on day 14 after wounding (A). The newly formed epithelium area was significantly improved in both the three PDGF-treated groups versus blank control (0.51 ⫾ 0.29 cm 2) on day 7, and 14 g PDGF/cm 2 treated group versus either vehicle control or blank control on days 7 and 14, as well as 7 g PDGF/cm 2 treated group (0.46 ⫾ 0.19 cm 2) versus vehicle control (0.33 ⫾ 0.06 cm 2) or blank control (0.26 ⫾ 0.07 cm 2) on day 14 (B). Rats treated with PDGF-BB displayed significantly enhanced percentage wound closure compared with rats treated with vehicle or nothing on day 7 after wounding. By 14 days all of the wounds had approached complete closure, so no difference was noted in the percentage wound closure (C). *P ⬍ 0.05 compared with blank control; ŒP ⬍ 0.05 compared with vehicle control.
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FIG. 5. Expression of Factor VIII related antigen in wound tissue on both day 7 and day 14 after wounding in each group. Immunostaining of Factor VIII related antigen, a marker of vascular endothelial cell, in blank control group (A, F), vehicle control group (B, G), 3.5 g PDGF-BB/cm 2 treatment group (C, H), 7 g PDGF-BB/cm 2 treatment group (D, I) and 14 g PDGF-BB/cm 2 treatment group (E, F) on day 7 (A–E) and day 14 (F–J). The number of new capillary sprouts was counted under 400⫻ visual field and at least three visual fields of granulation tissue or wounds were counted per slide. Statistic analysis showed that there was a significant increase in new capillary sprouts in both the three PDGF-BB treatment groups compared with the two control groups on day 7, and 7 g PDGF-BB/cm 2 (47.1 ⫾ 21.3) or 14 g PDGF-BB/cm 2 (53.6 ⫾ 39.8) treatment groups compared with vehicle control group (29.5 ⫾ 19.7) or blank control group (28.3 ⫾ 12.9) on day 14 (H). (Original magnification: 200⫻). *P ⬍ 0.05 compared with blank control; ŒP ⬍ 0.05 compared with vehicle control. (Color version of figure is available online.)
Effects of PDGF-BB on Gross Appearance and Histological Evaluation in Diabetic Rats
There were no apparent granulation tissue formation and epithelium growth for the first 3 d, so the full-thickness wounds created on the back of diabetic rats did not show any gross evidence of healing. However, the wounds indeed became smaller by wound contraction during that time. On the third day after wounding, the wounds were fresh with hemorrhage in the three PDGF-BB treatment groups, whereas they were pale in the two control groups. The wounds filled with granulation tissue and the majority of the surface of the granulation tissue was 3 to 5 mm above the surface of the wounds, and there was newly formed epithelial growth at the edge of the wounds, but the wound contraction was also apparent on day 7 after wounding. Thereafter, surface of the granulation tissue reached the level of the wounds gradually. By day 14, all of the wounds had approached complete closure (Fig. 1B, C, D, E, F). Immunohistochemical analysis expression of Factor VIII related antigen, a marker of vascular endothelial cell, in the wounds showed that there was a statistically significant increase in the number of new capillary sprouts both in the three PDGF-BB treatment groups compared with the two control groups on day 7, and in 7 g PDGF-BB/cm 2 (47.1 ⫾ 21.3) or 14 g PDGF-BB/cm 2 (53.6 ⫾ 39.8) treatment groups compared with vehicle control group (29.5 ⫾ 19.7) or blank control group (28.3 ⫾ 12.9) on day 14 (Fig. 5). PCNAlabeling index, a marker of proliferation, was higher both in 7 g PDGF-BB/cm 2 (24.5% ⫾ 6.7%) or 14 g PDGF-BB/cm 2 (26.7% ⫾ 8.5%) treatment groups versus
the respective control on day 7, and in 7 g PDGF-BB/ cm 2 (27.6% ⫾ 9.3%) treatment groups versus vehicle control group (22.9% ⫾ 5.4%), as well as 14 g PDGFBB/cm 2 (32.5% ⫾ 4.2%) treatment groups versus the blank control group (23.7% ⫾ 4.8%) or vehicle control group (22.9% ⫾ 5.4%) on day 14 (Fig. 6). Masson trichrome staining and histological evaluation showed a close correlation between the gross and microscopic appearances of wounds. Masson trichrome staining showed that wounds developed vascular granulation tissue tended to be populated with inflammatory cells and fibroblasts, and partially formed newly formed epithelium at the edge of the wounds in all of the five groups at day 7 after wounding. At day 14, there was thicker and more highly organized collagen fiber deposition, populated with less inflammatory cells and more fibroblasts, in wounds treated with PDGF-BB, especially in 14 g PDGF-BB/cm 2 per day treatment group. However, the granulation tissue was thin, dominated by inflammatory cells, and collagen fiber alignment was disorganized in wounds of both blank control group and vehicle-treated group (Fig. 7). DISCUSSION
One of the leading causes of impaired wound healing is diabetes mellitus. In diabetic patients, a minor skin wound often leads to chronic, nonhealing ulcers and ultimately results in infection, gangrene, even amputation [3, 4]. Many experiments have demonstrated that the processes of wound repair were controlled by a wide variety of different growth factors and cytokines, such as PDGF, fibroblast growth factor, epidermal growth factor, insulin-like growth factor, and so on
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FIG. 6. Expression of PCNA in wound tissue on both day 7 and day 14 after wounding in each group. Immunostaining of Factor VIII related antigen in blank control group (A, F), vehicle control group (B, G), 3.5 g PDGF-BB/cm 2 treatment group (C, H), 7 g PDGF-BB/cm 2 treatment group (D, I), and 14 g PDGF-BB/cm 2 treatment group (E, F) on day 7 (A–E) and day 14 (F–J). Statistic analysis showed that PCNA-labeling index was higher in 7 g PDGF-BB/cm 2 (24.5% ⫾ 6.7%) or 14 g PDGF-BB/cm 2 (26.7% ⫾ 8.5%) treatment groups versus the two control on day 7, 7 g PDGF-BB/cm 2 (27.6% ⫾ 9.3%) treatment groups versus vehicle control group (22.9% ⫾ 5.4%) on day 14, and 14 g PDGF-BB/cm 2 (32.5% ⫾ 4.2%) treatment groups versus blank control group (23.7% ⫾ 4.8%) or vehicle control group (22.9% ⫾ 5.4%) on day 14. Original magnification: 200⫻. *P ⬍ 0.05 compared with blank control; ŒP ⬍ 0.05 compared with vehicle control. (Color version of figure is available online.)
[13–17]. Their expression dynamics showed characteristic temporal and spatial regulation, and changes in the expression pattern of growth factors were associated with impaired wound healing. Most importantly, alteration in the level of one factor was likely to affect the production of other growth factors and cytokines. Many experiments have demonstrated that both decreased growth factors production and increased destruction of growth factors contributed to the pathogenesis of impaired healing in diabetes mellitus [5, 6].
FIG. 7. Masson trichrome staining of the wounds on both day 7 and day 14 after wounding in each group. Masson trichrome stained tissues from blank control group (A, F), vehicle control group (B, G), 3.5 g PDGF-BB/cm 2 treatment group (C, H), 7 g PDGF-BB/cm 2 treatment group (D, I), and 14 g PDGF-BB/cm 2 treatment group (E, F) on day 7 (A–E) and day 14 (F–J). The staining results showed that wounds developed vascular granulation tissue, tended to be populated with inflammatory cells and fibroblasts, and partially formed newly formed epithelium at the edge of the wounds in all of the five groups on day 7 after wounding, while on day 14, there were thicker and more highly organized collagen fibers deposition in wounds treated with PDGF-BB, especially in 14 g PDGF-BB/cm 2 treatment group. Original magnification: 200⫻. (Color version of figure is available online.)
Among these growth factors, PDGF had been implicated as important regulatory factor in the wound healing disorders [3, 18]. PDGF is synthesized by many different cell types, and its expression is broad. Its synthesis is in response to external stimuli, such as injuries, or stimulation by other cytokines and growth factors. Upon injury, PDGF is released in large amounts from degranulating platelets, and it is present in wound fluid, particularly early after injury. PDGF comprises a family of homoor heterodimeric growth factor, including PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD [19]. It exerts its functions by binding to transmembrane tyrosine kinase receptors. PDGF induces tyrosine phosphorylation of PDGF receptor and numerous other intracellular proteins. Subsequently, PDGF receptor mediates fibroblast chemotaxis, proliferation, and induction of both the extracellular matrix and metalloproteinases, which are required for wound remodeling [7]. Interestingly, expression of PDGF and its receptor was decreased in wounds of healing-impaired diabetic mice, indicating that a certain expression level of PDGF is essential for normal repair [5, 6]. To investigate the pharmacodynamics effect of PDGF-BB gel on the wounding of diabetes mellitus (DM), we used accepted DM model rats injected with STZ. STZ is a widely used chemical inducer for insulindependent DM [20]. STZ had been shown to produce free radicals in the body that specifically cut DNA chains in the pancreatic  cells, resulting in disorder of the function of pancreatic  cell and, at a later phase, destruction of the  cells by necrosis [21]. Rats, after being induced with STZ, had higher blood glucose level compared with rats before being induced. Blood glucose and body weight of the diabetic rats
LI ET AL.: RESEARCH OF PDGF-BB GEL ON WOUND HEALING
among the blank control group, vehicle control group, 3.5 g PDGF-BB/cm 2 treatment group, 7 g PDGF-BB/ cm 2 treatment group, and 14 g PDGF-BB/cm 2 treatment group had no significant differences at varied time points after wounding. After wounding, the skin rapidly contracted to cover the defect and only a small region healed by development of granulation tissue and re-epithelialization in the diabetic rats in all of the five groups. Nevertheless, wound contraction contributed less to the healing of diabetic rats in both 7 g PDGF-BB/cm 2 and 14 g PDGF-BB/cm 2 treatment groups than animals in vehicle control group or blank control group on day 14 after wounding. The newly formed epithelium area was significantly improved in both the three PDGF-BBtreated groups versus blank control on day 7, and 14 g PDGF-BB/cm 2 treated group versus either vehicle control or blank control on days 7 and 14, as well as 7 g PDGF-BB/cm 2 treated group versus either vehicle control or blank control on day 14. Meanwhile, treatment of wounds with PDGF-BB also enhanced percentage wound closure at day 7 after wounding. The expression of Factor VIII related antigen, a marker of vascular endothelial cell, and PCNA, a marker of proliferation, in the wounds showed that there was a increase in the number of new capillary sprouts and PCNA-labeling index in the three PDGF-BB treatment groups at varied extent, especially in 14 g PDGF-BB/cm 2 groups, compared with that in blank control group or vehicle control group on day 7 and day 14 after wounding. At day 14, Masson trichrome staining showed there were thicker and more highly organized collagen fiber deposition, populated with less inflammatory cells and more fibroblasts, in wounds treated with PDGF-BB, especially in 14 g PDGF-BB/cm 2 treatment group. However, the granulation tissue was thin, dominated by inflammatory cells, and collagen fiber alignment was disorganized in wounds of both control group and vehicletreated group. The effects of topically applied PDGF-BB were dosedependent. Rats receiving PDGF-BB, either 7 g/cm 2 or 14 g/cm 2 per day, especially 14 g/cm 2 per day, had much higher percentage wound closure, re-epithelialization area, new capillary sprouts, and PCNAlabeling index compared with rats receiving PDGF-BB, 3.5 g/cm 2 per day. In summary, our current study using PDGF-BB improved healing quality, enhanced angiogenesis, cell proliferation and epithelialization, and formed thicker and more highly organized collagen fiber deposition in full-thickness excisional wounds of diabetic rats. The effects of topically applied PDGF-BB were dosedependent. PDGF-BB is an important future clinical tool, particularly for stimulating soft tissue repair in patients with an impaired capacity for wound healing.
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ACKNOWLEDGMENTS
This work was supported in part by the National Basic Science and Development Program (973 Program and 2005CB522603) and by the National Natural Science Foundation of China (30500194, 30672176). The authors declare that they have no competing financial interests.
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in corneal fibroblasts during corneal wound healing. Invest Ophthalmol Vis Sci 2006;47:591. 17. Haber M, Cao Z, Panjwani N, et al. Effects of growth factors (EGF, PDGF-BB and TGF- 1) on cultured equine epithelial cells and keratocytes: Implications for wound healing. Vet Ophthalmol 2003;6:211. 18. Keswani SG, Katz AB, Lim FY, et al. Adenoviral mediated gene transfer of PDGF-B enhances wound healing in type I and type II diabetic wounds. Wound Repair Regen 2004;12:497.
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Werner S, Grose R. Regulation of wound healing by growth factors and cytokines. Physiol Rev 2003;83:835. 20. Junod A, Lambert AE, Stauffacher W, et al. Diabetogenic action of streptozotocin: Relationship of dose to metabolic response. J Clin Invest 1969;48:2129. 21. Yamamoto H, Uchigata Y, Okamoto H. Streptozotocin and alloxan induce DNA strand breaks and poly (ADPribose) synthetase in pancreatic islets. Nature 1981;294: 284.
Research of PDGF-BB Gel on the Wound Healing of Diabetic Rats and Its Pharmacodynamics Haihong Li, M.D.,* Xiaobing Fu, M.D.,†,1 Lei Zhang, M.D.,* Qingjun Huang, M.D.,* Zhigu Wu, M.D.,† and Tongzhu Sun, M.D.† *Mental Health Center, ShanTou University Medical College, ShanTou, People’s Republic of China; †Wound Healing and Cell Biology Laboratory, Burns Institute, The First Affiliated Hospital, General Hospital of PLA, Trauma Center of Postgraduate Medical College, Beijing, People’s Republic of China Submitted for publication October 5, 2006
excisional wound of diabetic rats. The effects of topically applied PDGF-BB were dose-dependent. Conclusions. PDGF-BB is an important future clinical tool, particularly for stimulating soft tissue repair in patients with an impaired capacity for wound healing. © 2008 Elsevier Inc. All rights reserved. Key Words: platelet-derived growth factor; wound healing; pharmacodynamics; diabetes; rat.
Background. One of the leading causes of impaired wound healing is diabetes mellitus. In diabetic patients, a minor skin wound often leads to serious complications. Many experiments had demonstrated that the expression of platelet-derived growth factor (PDGF) and its receptor was decreased in wounds of healing-impaired diabetic mice, indicating that a certain expression level of PDGF is essential for normal repair. Materials and methods. The diabetic rats was induced by a single i.p. injection of streptozotocin and a 1.8 cm diameter full-thickness wound was made on each side of the rat mid-back. Then the rats were randomly divided into five groups, with eight animals in each group as follows: blank control, vehicle control, 3.5 g PDGF-BB/cm2 treatment group, 7 g PDGF-BB/cm 2 treatment group and 14 g PDGF-BB/cm 2 treatment group for either 7 or 14 consecutive days after wounding. Re-epithelialization area was measured by computerized planimetry, percentage wound closure and percentage wound contraction was calculated, granulation tissue and collagen formation was assessed by Masson trichrome, cell proliferation (proliferating cell nuclear antigen staining) and angiogenesis (Factor VIII related antigen staining) was assessed by immunohistological methods. Results. PDGF-BB treatment improved healing quality, enhanced angiogenesis, cell proliferation and epithelialization, and formed thicker and more highly organized collagen fiber deposition in full-thickness
INTRODUCTION
One of the leading causes of impaired wound healing is diabetes mellitus. Diabetic wound healing is a complex and lengthy process and the cost of it in the United States has been estimated at $1 billion per year, so the treatment of diabetic wounds is a formidable clinical challenge [1, 2]. In diabetic patients, a minor skin wound often leads to chronic, nonhealing ulcers and ultimately result in infection, gangrene, even amputation [3, 4]. Multiple factors contribute to the impairment of wound healing in the wound environment of diabetic animals and patients. One of the important reasons is that diabetic animals and patients do not produce enough growth factors or the destruction of growth factors is increased [5, 6]. Platelet-derived growth factor (PDGF) exerts its functions by binding to transmembrane tyrosine kinase receptors [7]. A series of studies suggested an important role of PDGF in the repair process [8 –10]. Upon injury, PDGF is released from degranulated platelets and presented in wound fluid, particularly early after injury. Interestingly, expression of PDGF and their receptors was reduced in wounds of healingimpaired diabetic animals, indicating that a certain expression level of PDGF and its receptors is essential for normal repair [11, 12].
1 To whom correspondence and reprint requests should be addressed at Wound Healing and Cell Biology Laboratory, Burns Institute, The First Affiliated Hospital (304th Hospital), General Hospital of PLA, Trauma Center of Postgraduate Medical College, 51 Fu Cheng Road, Beijing 100037, P.R. China. E-mail: fuxb@cgw. net.cn; [email protected].
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To investigate the effects of topical application of PDGF-BB and its pharmacodynamics on the wound healing in diabetic rats, we made full-thickness wounds on mid-back of diabetic rats, then we measured wound area and re-epithelialization area by computerized planimetry, calculated the percentage wound closure and wound contraction, and assessed granulation tissue, collagen formation (Masson trichrome), cell proliferation (proliferating cell nuclear antigen [PCNA] staining), and angiogenesis (Factor VIII related antigen staining) by immunohistological methods. MATERIALS AND METHODS Preparation of Diabetic Rats All procedures on rats were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Before and during experiments, all rats were housed in individual cages in a central animal care facility, maintained on a 12 h light-dark cycle, and given free access to standard
FIG. 2. Schematic representation of wound healing on day N after wounding.
rodent chow and water. Forty male Wistar rats, obtained from the Academy of Military Medical Sciences (Beijing, China), aged 2 mo with an initial body mass of approximately 180 g were used for the study. Diabetes was induced by a single i.p. injection of sterile 0.5% streptozotocin (STZ) (Sigma, ST. Louis, MO) in sodium citrate buffer (0.1 mol/L, pH 4.5) at a dose of 60 mg/kg body weight immediately before use. The diabetic state was confirmed 72 h after STZ injections by blood glucose levels exceeding 15 mmol/L. One week after the initial administration of STZ, studies were carried out as described below.
Wounding and Treatment The rats were anesthetized by ethylether inhalation. After clipping the hair on the back, the skin was sterilized with 75% alcohol. A sterile template of 1.8 cm in diameter was placed on each side of the mid-back and a full-thickness wound to deep fascia corresponding to the template was made by excising the skin (Fig. 1A). Then the rats were randomly divided into five groups: blank control, vehicle control, 3.5 g PDGF-BB/cm 2 treatment group, 7 g PDGF-BB/cm 2 treatment group, and 14 g PDGF-BB/cm 2 treatment group. Different concentration of PDGF-BB were mixed in a vehicle of sterilely filtered 5% polyethylene glycol and stored at 4°C until the day of use. In PDGF-BB-treatment groups and vehicle control group, 0.1 mL of the PDGF-BB plus vehicle or vehicle only was applied to each wound once daily for either 7 or 14 consecutive days starting at wounding by injecting it to the wound and allowing it to spread over the wound bed, while in blank control group, neither PDGF-BB nor vehicle was applied to the wound. Then the wounds were covered with one layer of petrolatum gauze and five layers of sterilized dressing, and stabilized with 3M adhesive tape.
Serial Analysis of Wound Healing
Blood Glucose In all groups, blood was obtained from tail vein and glucose content was analyzed using a blood glucose test meter (GlucoDr, Seoul, Korea) at the day of wounding and at day 3, 7, 10, and 14 after wounding.
Body Weight FIG. 1. Administration of PDGF-BB topically accelerated wound healing in diabetic rats. Full-thickness excisional wounds about 1.8 cm in diameter were created on the back of male diabetic rats, age 2 mo (A). Different concentration of PDGF-BB, or vehicle only, or neither of them, was administered topically once daily for either 7 or 14 consecutive days starting at wounding by injecting it to the wounds and allowing it to spread over the wound beds. The wounds were completely closed in 14 g PDGF-BB/cm 2 treatment group (F), while in blank control group (B), vehicle control group (C), 3.5 g PDGF-BB/cm 2 treatment group (D), and 7 g PDGF-BB/cm 2 treatment group (E), there were still some open wounds of varied extent. (Color version of figure is available online.)
Body weight was weighed using electronic balance (accuracy to g) on the day of wounding, as well as day 3, 7, 10, and 14 after wounding.
Wound Area and Re-epithelialization Area The edge of migration epithelium was easily discernible from the moist granulation tissue and the presence of the epithelial border was the edge of the healing wound. The edge of the wound was traced onto a glass microscope slide and the wound area was determined by planimetry using ImageMeasure (Phoenix Biotechnology, Seattle, WA). The trace taken immediately after wounding was used as the
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original area (day 0 area). Re-epithelialization area of the wound on day N was the area between the wound edge and the newly formed epithelial edge on day N (Fig. 2). Wounds were considered closed if moist granulation tissue was no longer apparent, and the wounds appeared covered with new epithelium.
number of new capillary sprouts was counted under 400⫻ visual field and at least three visual fields of granulation tissues or wounds were counted per slide.
Percentage Wound Closure and Percentage Wound Contraction
Data were analyzed using standard statistical software (SPSS 10.0; SPSS Inc., Chicago, IL). The values were expressed as mean ⫾ standard deviation. The statistical significance of values among groups was evaluated by one-way analysis of variance, followed by least significant difference t test. The difference was considered significant when the P-value was 0.05 or less.
Wounds were closed by both contraction and forming newly formed epithelium to cover the wounds. Wound closure was expressed as percentage closure of the original wound and was calculated by the following formula: % wound closure on day N ⫽ [(area on day 0-open area on day N)/area on day 0] ⫻ 100. % wound contraction on day N ⫽ % wound closure on day N ⫺ [(Re-epithelialization area on Day N/Area on Day 0) ⫻ 100].
Histological Analysis After the final wound tracing on either day 7 or day 14, the rats were given a lethal ethylether inhalation, and the entire wound, including a 5 mm margin of unwounded skin, was excised down to the fascia. The wound was divided in half through the least healed portion. One-half of the wound was stored in the liquid nitrogen for future molecular studies, the other half was fixed in 10% formalin for at least 24 h, followed by processing for conventional paraffin embedding and sectioned 5-m-thick on a Microme (Leica, Wetzlar, Germany). For morphological observations, sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome, and for immunohistochemical analysis, sections were processed using the two-step immunostaining kit (Zymed, San Francisco, CA).
H & E Staining Tissue sections were dewaxed in xylene, rehydrated through graded alcohols to phosphate-buffered saline, stained in H&E, and mounted in resin.
Statistical Analysis
RESULTS Preparation of Diabetic Rats and Wounding
Rats receiving STZ resulted in significant elevation in blood glucose level after 1 wk (data not shown) that was sustained throughout the duration of the study. On average, rats had a mean body weight of 181.0 g ⫾ 7.6 g) at the beginning of the study. The rats tolerated the wounding procedure without problems and they had no obvious changes in food consumption or weight loss after wounding. Effects of PDGF-BB on the Blood Glucose and Body Weight
Blood glucose was elevated at varied levels 3 d after wounding and reached its peak 7 d after wounding in all of the five groups, but there was no significant difference between groups at different time points (Fig. 3A). Body weight of the rats between groups and within-group had no significant differences at varied time points after wounding, but it indeed increased at different levels (Fig. 3B).
Masson’s Trichrome Sections were dewaxed and rehydrated conventionally, placed in Weigert’s hematoxylin stain for 1 h, rinsed under lukewarm water for 5 min, immersed in Masson solution for 15 min, and rinsed in deionized water before placing in phosphomolybdic acid for 10 min. Subsequently, sections were immersed in 2% aniline blue for 15 min, rinsed in 1% acetic acid, 95% ethanol, and absolute ethanol in turn, immersed in xylene for 10 min, and mounted with resin. Collagen fibers were stained blue, cytoplasm and erythrocyte were stained red, and nuclei were stained bluish brown.
Immunohistochemical Staining Sections were dewaxed and rehydrated, then were incubated in 3% hydrogen peroxide to block endogenous peroxidase. Sections for Factor VIII related antigen detection were incubated with 0.4% pepsin at 37°C for 30 min, while sections for PCNA detection were microwaved for 15 min at 95°C in citrate buffer. Subsequently, sections were incubated in rabbit anti-Factor VIII related antigen (NeoMarkers, Fremont, CA) and mouse anti-PCNA (NeoMarkers) monoclonal antibodies at a 1:100 dilution respectively, followed by incubation in horseradish peroxidase conjugated goat anti-rabbit IgG (Vector, Burlingame, CA) or goat anti-mouse IgG (Vector). Sections were finally developed in 3,3=-diaminobenzidine, counterstained with hematoxylin and mounted in resin. PCNA expressed in nuclei and Factor VIII related antigen in cytoplasm. The number of brown labeled nuclei was counted to evaluate the PCNA labeling index, namely, the percentage of immunoreactive nuclei in 100 cells and at least 2000 cells in the wounds were counted per slide. The
Effects of PDGF-BB on Percentage Wound Contraction, Re-epithelialization Area and Percentage Wound Closure in Diabetic Rats
An approximation of the extent of contraction was achieved by comparing the area bordered by the wound edge at 7, 10, and 14 d to the area of wound at day 0 respectively. The skin rapidly contracted to cover the defect and only a small region healed by development of granulation tissue and re-epithelialization in the diabetic rats in all of the groups. However, wound contraction contributed less to the healing of diabetic rats in 7 g PDGF-BB/cm 2 (86.3% ⫾ 7.0%) and 14 g PDGF-BB/cm 2 (86.2% ⫾ 3.4%) treatment groups than rats in vehicle control group (91.6% ⫾ 1.9%) or blank control group (91.2% ⫾ 1.2%) on day 14 after wounding (Fig. 4A). Roughly 91% of the wound closure in control groups was attributable to wound contraction, while only about 86% of the wound closure in 7 g PDGFBB/cm 2 and 14 g PDGF-BB/cm 2 treatment groups was due to contraction (Fig. 4A). There was still no obvious epithelium tissue growth 3 d after wounding. Thereafter, newly formed epithelium was seen to migrate from the edge of the wound.
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FIG. 3. Changes in blood glucose and body weight in diabetic rats in each group at varied time points. Blood glucose was elevated at varied levels 3 d after wounding and reached its peak 7 d after wounding in all of the five groups, but there was no significant difference between groups at different time points (A). The body weight of the rats between groups and within-group had no significant difference at varied time points after wounding, but it indeed increased at different extent (B). *P ⬍ 0.05 compared with blank control; ŒP ⬍ 0.05 compared with vehicle control.
The newly formed epithelium area was significantly improved in the three PDGF-BB-treated groups versus blank control (0.51 ⫾ 0.29 cm 2) on day 7, and 14 g PDGF-BB/cm 2 treated group versus either vehicle control or blank control on day 7 and day 14, as well as 7 g PDGF-BB/cm 2-treated group (0.46 ⫾ 0.19 cm 2) versus either vehicle control (0.33 ⫾ 0.06 cm 2) or blank control (0.26 ⫾ 0.07 cm 2) on day 14 (Fig. 4B). Treatment of wounds with PDGF-BB also enhanced the percentage wound closure when compared with vehicle treatment group and blank control group on day 7 after wounding. By day 14, all of the wounds had approached complete closure, so no difference was noted in the percentage wound closure, but the healing
quality of the diabetic rats in both 7 g PDGF-BB/cm 2 and 14 g PDGF-BB/cm 2 treatment groups was significantly improved compared with rats in vehicle control group and blank control group on that day. The effects of topically applied PDGF-BB were dose-dependent. No significant improvement in percentage wound closure and re-epithelialization area was observed in diabetic rats treated with PDGF-BB 3.5 g/cm 2 per day compared with rats receiving vehicle treatment or blank treatment at day 14 after wounding. In contrast, rats receiving PDGF-BB, either 7 g/cm 2 or 14 g/cm 2 per day, displayed significantly improved wound healing compared with rats receiving vehicle treatment or blank treatment on days 7 and 14 (Fig. 4C).
FIG. 4. Percentage wound contraction, re-epithelization area, and percentage wound closure in diabetic rats in each group at varied time points. Wound edges were traced onto glass microscope slides and image analysis performed to determine percentage wound contraction (%), re-epithelization area (cm 2), and percentage wound closure (%). Wound contraction contributed less to the healing of full-thickness skin excisions in 7 g PDGF/cm 2 (86.3% ⫾ 7.0%) or 14 g PDGF/cm2 (86.2% ⫾ 3.4%) treatment groups than those in vehicle control group (91.6% ⫾ 1.9%) or blank control group (91.2% ⫾ 1.2%) on day 14 after wounding (A). The newly formed epithelium area was significantly improved in both the three PDGF-treated groups versus blank control (0.51 ⫾ 0.29 cm 2) on day 7, and 14 g PDGF/cm 2 treated group versus either vehicle control or blank control on days 7 and 14, as well as 7 g PDGF/cm 2 treated group (0.46 ⫾ 0.19 cm 2) versus vehicle control (0.33 ⫾ 0.06 cm 2) or blank control (0.26 ⫾ 0.07 cm 2) on day 14 (B). Rats treated with PDGF-BB displayed significantly enhanced percentage wound closure compared with rats treated with vehicle or nothing on day 7 after wounding. By 14 days all of the wounds had approached complete closure, so no difference was noted in the percentage wound closure (C). *P ⬍ 0.05 compared with blank control; ŒP ⬍ 0.05 compared with vehicle control.
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FIG. 5. Expression of Factor VIII related antigen in wound tissue on both day 7 and day 14 after wounding in each group. Immunostaining of Factor VIII related antigen, a marker of vascular endothelial cell, in blank control group (A, F), vehicle control group (B, G), 3.5 g PDGF-BB/cm 2 treatment group (C, H), 7 g PDGF-BB/cm 2 treatment group (D, I) and 14 g PDGF-BB/cm 2 treatment group (E, F) on day 7 (A–E) and day 14 (F–J). The number of new capillary sprouts was counted under 400⫻ visual field and at least three visual fields of granulation tissue or wounds were counted per slide. Statistic analysis showed that there was a significant increase in new capillary sprouts in both the three PDGF-BB treatment groups compared with the two control groups on day 7, and 7 g PDGF-BB/cm 2 (47.1 ⫾ 21.3) or 14 g PDGF-BB/cm 2 (53.6 ⫾ 39.8) treatment groups compared with vehicle control group (29.5 ⫾ 19.7) or blank control group (28.3 ⫾ 12.9) on day 14 (H). (Original magnification: 200⫻). *P ⬍ 0.05 compared with blank control; ŒP ⬍ 0.05 compared with vehicle control. (Color version of figure is available online.)
Effects of PDGF-BB on Gross Appearance and Histological Evaluation in Diabetic Rats
There were no apparent granulation tissue formation and epithelium growth for the first 3 d, so the full-thickness wounds created on the back of diabetic rats did not show any gross evidence of healing. However, the wounds indeed became smaller by wound contraction during that time. On the third day after wounding, the wounds were fresh with hemorrhage in the three PDGF-BB treatment groups, whereas they were pale in the two control groups. The wounds filled with granulation tissue and the majority of the surface of the granulation tissue was 3 to 5 mm above the surface of the wounds, and there was newly formed epithelial growth at the edge of the wounds, but the wound contraction was also apparent on day 7 after wounding. Thereafter, surface of the granulation tissue reached the level of the wounds gradually. By day 14, all of the wounds had approached complete closure (Fig. 1B, C, D, E, F). Immunohistochemical analysis expression of Factor VIII related antigen, a marker of vascular endothelial cell, in the wounds showed that there was a statistically significant increase in the number of new capillary sprouts both in the three PDGF-BB treatment groups compared with the two control groups on day 7, and in 7 g PDGF-BB/cm 2 (47.1 ⫾ 21.3) or 14 g PDGF-BB/cm 2 (53.6 ⫾ 39.8) treatment groups compared with vehicle control group (29.5 ⫾ 19.7) or blank control group (28.3 ⫾ 12.9) on day 14 (Fig. 5). PCNAlabeling index, a marker of proliferation, was higher both in 7 g PDGF-BB/cm 2 (24.5% ⫾ 6.7%) or 14 g PDGF-BB/cm 2 (26.7% ⫾ 8.5%) treatment groups versus
the respective control on day 7, and in 7 g PDGF-BB/ cm 2 (27.6% ⫾ 9.3%) treatment groups versus vehicle control group (22.9% ⫾ 5.4%), as well as 14 g PDGFBB/cm 2 (32.5% ⫾ 4.2%) treatment groups versus the blank control group (23.7% ⫾ 4.8%) or vehicle control group (22.9% ⫾ 5.4%) on day 14 (Fig. 6). Masson trichrome staining and histological evaluation showed a close correlation between the gross and microscopic appearances of wounds. Masson trichrome staining showed that wounds developed vascular granulation tissue tended to be populated with inflammatory cells and fibroblasts, and partially formed newly formed epithelium at the edge of the wounds in all of the five groups at day 7 after wounding. At day 14, there was thicker and more highly organized collagen fiber deposition, populated with less inflammatory cells and more fibroblasts, in wounds treated with PDGF-BB, especially in 14 g PDGF-BB/cm 2 per day treatment group. However, the granulation tissue was thin, dominated by inflammatory cells, and collagen fiber alignment was disorganized in wounds of both blank control group and vehicle-treated group (Fig. 7). DISCUSSION
One of the leading causes of impaired wound healing is diabetes mellitus. In diabetic patients, a minor skin wound often leads to chronic, nonhealing ulcers and ultimately results in infection, gangrene, even amputation [3, 4]. Many experiments have demonstrated that the processes of wound repair were controlled by a wide variety of different growth factors and cytokines, such as PDGF, fibroblast growth factor, epidermal growth factor, insulin-like growth factor, and so on
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FIG. 6. Expression of PCNA in wound tissue on both day 7 and day 14 after wounding in each group. Immunostaining of Factor VIII related antigen in blank control group (A, F), vehicle control group (B, G), 3.5 g PDGF-BB/cm 2 treatment group (C, H), 7 g PDGF-BB/cm 2 treatment group (D, I), and 14 g PDGF-BB/cm 2 treatment group (E, F) on day 7 (A–E) and day 14 (F–J). Statistic analysis showed that PCNA-labeling index was higher in 7 g PDGF-BB/cm 2 (24.5% ⫾ 6.7%) or 14 g PDGF-BB/cm 2 (26.7% ⫾ 8.5%) treatment groups versus the two control on day 7, 7 g PDGF-BB/cm 2 (27.6% ⫾ 9.3%) treatment groups versus vehicle control group (22.9% ⫾ 5.4%) on day 14, and 14 g PDGF-BB/cm 2 (32.5% ⫾ 4.2%) treatment groups versus blank control group (23.7% ⫾ 4.8%) or vehicle control group (22.9% ⫾ 5.4%) on day 14. Original magnification: 200⫻. *P ⬍ 0.05 compared with blank control; ŒP ⬍ 0.05 compared with vehicle control. (Color version of figure is available online.)
[13–17]. Their expression dynamics showed characteristic temporal and spatial regulation, and changes in the expression pattern of growth factors were associated with impaired wound healing. Most importantly, alteration in the level of one factor was likely to affect the production of other growth factors and cytokines. Many experiments have demonstrated that both decreased growth factors production and increased destruction of growth factors contributed to the pathogenesis of impaired healing in diabetes mellitus [5, 6].
FIG. 7. Masson trichrome staining of the wounds on both day 7 and day 14 after wounding in each group. Masson trichrome stained tissues from blank control group (A, F), vehicle control group (B, G), 3.5 g PDGF-BB/cm 2 treatment group (C, H), 7 g PDGF-BB/cm 2 treatment group (D, I), and 14 g PDGF-BB/cm 2 treatment group (E, F) on day 7 (A–E) and day 14 (F–J). The staining results showed that wounds developed vascular granulation tissue, tended to be populated with inflammatory cells and fibroblasts, and partially formed newly formed epithelium at the edge of the wounds in all of the five groups on day 7 after wounding, while on day 14, there were thicker and more highly organized collagen fibers deposition in wounds treated with PDGF-BB, especially in 14 g PDGF-BB/cm 2 treatment group. Original magnification: 200⫻. (Color version of figure is available online.)
Among these growth factors, PDGF had been implicated as important regulatory factor in the wound healing disorders [3, 18]. PDGF is synthesized by many different cell types, and its expression is broad. Its synthesis is in response to external stimuli, such as injuries, or stimulation by other cytokines and growth factors. Upon injury, PDGF is released in large amounts from degranulating platelets, and it is present in wound fluid, particularly early after injury. PDGF comprises a family of homoor heterodimeric growth factor, including PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD [19]. It exerts its functions by binding to transmembrane tyrosine kinase receptors. PDGF induces tyrosine phosphorylation of PDGF receptor and numerous other intracellular proteins. Subsequently, PDGF receptor mediates fibroblast chemotaxis, proliferation, and induction of both the extracellular matrix and metalloproteinases, which are required for wound remodeling [7]. Interestingly, expression of PDGF and its receptor was decreased in wounds of healing-impaired diabetic mice, indicating that a certain expression level of PDGF is essential for normal repair [5, 6]. To investigate the pharmacodynamics effect of PDGF-BB gel on the wounding of diabetes mellitus (DM), we used accepted DM model rats injected with STZ. STZ is a widely used chemical inducer for insulindependent DM [20]. STZ had been shown to produce free radicals in the body that specifically cut DNA chains in the pancreatic  cells, resulting in disorder of the function of pancreatic  cell and, at a later phase, destruction of the  cells by necrosis [21]. Rats, after being induced with STZ, had higher blood glucose level compared with rats before being induced. Blood glucose and body weight of the diabetic rats
LI ET AL.: RESEARCH OF PDGF-BB GEL ON WOUND HEALING
among the blank control group, vehicle control group, 3.5 g PDGF-BB/cm 2 treatment group, 7 g PDGF-BB/ cm 2 treatment group, and 14 g PDGF-BB/cm 2 treatment group had no significant differences at varied time points after wounding. After wounding, the skin rapidly contracted to cover the defect and only a small region healed by development of granulation tissue and re-epithelialization in the diabetic rats in all of the five groups. Nevertheless, wound contraction contributed less to the healing of diabetic rats in both 7 g PDGF-BB/cm 2 and 14 g PDGF-BB/cm 2 treatment groups than animals in vehicle control group or blank control group on day 14 after wounding. The newly formed epithelium area was significantly improved in both the three PDGF-BBtreated groups versus blank control on day 7, and 14 g PDGF-BB/cm 2 treated group versus either vehicle control or blank control on days 7 and 14, as well as 7 g PDGF-BB/cm 2 treated group versus either vehicle control or blank control on day 14. Meanwhile, treatment of wounds with PDGF-BB also enhanced percentage wound closure at day 7 after wounding. The expression of Factor VIII related antigen, a marker of vascular endothelial cell, and PCNA, a marker of proliferation, in the wounds showed that there was a increase in the number of new capillary sprouts and PCNA-labeling index in the three PDGF-BB treatment groups at varied extent, especially in 14 g PDGF-BB/cm 2 groups, compared with that in blank control group or vehicle control group on day 7 and day 14 after wounding. At day 14, Masson trichrome staining showed there were thicker and more highly organized collagen fiber deposition, populated with less inflammatory cells and more fibroblasts, in wounds treated with PDGF-BB, especially in 14 g PDGF-BB/cm 2 treatment group. However, the granulation tissue was thin, dominated by inflammatory cells, and collagen fiber alignment was disorganized in wounds of both control group and vehicletreated group. The effects of topically applied PDGF-BB were dosedependent. Rats receiving PDGF-BB, either 7 g/cm 2 or 14 g/cm 2 per day, especially 14 g/cm 2 per day, had much higher percentage wound closure, re-epithelialization area, new capillary sprouts, and PCNAlabeling index compared with rats receiving PDGF-BB, 3.5 g/cm 2 per day. In summary, our current study using PDGF-BB improved healing quality, enhanced angiogenesis, cell proliferation and epithelialization, and formed thicker and more highly organized collagen fiber deposition in full-thickness excisional wounds of diabetic rats. The effects of topically applied PDGF-BB were dosedependent. PDGF-BB is an important future clinical tool, particularly for stimulating soft tissue repair in patients with an impaired capacity for wound healing.
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ACKNOWLEDGMENTS
This work was supported in part by the National Basic Science and Development Program (973 Program and 2005CB522603) and by the National Natural Science Foundation of China (30500194, 30672176). The authors declare that they have no competing financial interests.
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