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. Author manuscript; available in PMC: 2012 Aug 7.
Published in final edited form as: Int J Mol Med. 2011 Mar 3;27(5):647–653. doi: 10.3892/ijmm.2011.641

Priming with a combination of proangiogenic growth factors improves wound healing in normoglycemic mice

Maximilian Ackermann 1, Tanja Wolloscheck 1, Axel Wellmann 2, Vincent W Li 3,4, William W Li 4, Moritz A Konerding 1,*
PMCID: PMC3413261  NIHMSID: NIHMS392402  PMID: 21373751

Abstract

Growth factors and/or angiogenic factors are supposed to improve wound healing. The aim of our study was to evaluate the effects of subcutaneous pretreatment with combinatory proangiogenic factors on wound closure, mechanical properties, vessel density, and morphology. Twenty-eight Balb/c mice were divided equally into two groups. A mixture of VEGF (35.0 μg), bFGF (2.5 μg), and PDGF (3.5 μg) was administered 3, 5, and 7 days subcutaneously to 14 mice before full thickness skin punch biopsy wounding, whereas 14 control animals received three times 0.2 ml saline solution. Wound sizes were assessed daily and the repaired tissues were harvested 7 days after complete wound closure. Complete closure (≥95% healing of initial wound area) was reached in all proangiogenic pretreated animals on day 10, whereas controls needed 13 days for complete closure. Tensile strengths were nearly twofold higher than in the controls (p≤0.01). The punch biopsy material revealed 4.2 fold higher vessel densities in the proangiogenic pretreated group. On day 17, the vessel densities in the proangiogenic pretreated wounds were also 3.2 fold higher than in the untreated controls. No significant differences were seen in the collagen ratio. Pretreatment with proangiogenic factors revealed several significant effects on wound healing: faster time to closure, a higher vessel density, better functional outcome. These results suggest a beneficial effect of pretreatment with combinatory growth factors in mouse skin wounds without impaired wound healing. This might be exploited in further investigations in diabetic healing as a therapeutic approach for elective surgery.

Introduction

Angiogenesis represents a fundamental control point in many pathological and physiological processes (1). The identification of numerous signal transduction pathways as well as the availability of different purified growth factors has enabled therapeutic approaches in various disease areas (2). Similarities in the morphology between wounds and tumors as ‘wounds that do not heal’ (3) helped to catalyze interest in manipulating wound healing by application of growth factors (4). These different processes of tumor vasculature have to be targeted for gaining unbiased results (5).

Impaired microcirculation, consequently also impaired wound healing, represents a central pathological comorbidity (6) of many diseases such as diabetes (7) or peripheral arterial disease (PAD) that tends to result in scarring, delay and comprosing mechanical skin healing (8). Wound healing is strongly dependant on the formation of granulation tissue, which in turn is intimately correlated with the induction of new vessel formation (9). Angiogenesis represents a complex process that relies on extracellular matrix in the wound bed as well as migration and mitogenic stimulation of endothelial cells (10). Therefore several investigations examined as to whether angiogenesis stimulation could promote wound healing.

Many proangiogenic growth factors rose interest in wound healing (11): Besides VEGF that facilitates tissue repair by proliferating pre-existing endothelial cells and increasing vascular permeability (2), FGF and PDGF regulate predominantly the synthesis and deposition of ECM components (12), but they also intensify wound angiogenesis. PDGF stimulates macrophages amd secrete other growth factors of importance in the healing process and several matrix molecules, like fibronectin, collagen, proteoglycans and other (13).

The therapeutic experiences with these proangiogenic growth factors have been ambivalent: mode and time of application (14) are thought to have great influence on boosting of wound healing. In addition, the application of one individual factor (15) may not do justice to the complex mechanisms involved in tissue repair (16,17). Not only the way of application, but also the time may reflect an important influence on the process of growth stimulation(18). Cross-talk and a complex redundancy of growth factors appear to be inherent in the tissue repair system (19).

Clinical studies have shown that growth factor stimulation does facilitate healing in diabetic patients (20). Other studies of other growth factors have been inconclusive, i.e. recent observations in mouse wound healing models suggest that topical application of single growth factors does not result in faster wound healing(21).

The combination of different key players acting on vessel formation, stabilization and the interaction with the extracellular matrix and the surrounding tissues seems to be a much more powerful approach.

Methods

Animals

Twenty-eight female Balb/c mice, 25–33g, were used in all experiments. They were obtained from the Central Animal Facilities of University of Mainz and housed in an approved animal care facility with 12-hour light cycles. Food and water were provided ad libitum. The care of the animals was consistent with the legal guidelines and all experiments were conducted prior approval by the local animal welfare authorities (Licence n°:1.5 177-07/041-2). Animals were kept in individual cages during the experiment in order to avoid biting and interference with the wounds. While the wounds were still open and wet, only soft tissue paper was used instead of conventional bedding in order to avoid wound irritation or contamination

Growth factor priming

The dorsal skin of the mice was shaved and depilated. 3, 5, and 7 days prior to surgery, a combination of 35.0 μg VEGF (R&D Systems, Minneapolis, MN), 2.5μg bFGF (R&D Systems, Minneapolis, MN) and 3.5μg PDGF (R&D Systems, Minneapolis, MN) dissolved in 0.2 ml saline solution was injected subcutaneuously 1.0 cm paramedian in normoglycemic mice. The control group animals received 0.2 ml saline solution (B. Braun, Melsungen, Germany). This combination of different growth factors and the respective dosage based on own preliminerary tests and on a review from existing literature (16,17,20).

Surgery

Mice were anesthetized with an intraperitoneal injection of avertin (1.5 ml/100g bodyweight, 2,2,2-tribromoethanol; Sigma-Aldrich, Munich, Germany). Immediately before biopsy an area of 15 × 15 mm was shaved again using disposable shavers followed by skin disinfections with 70% ethanol. Under sterile conditions 8-mm circular full thickness punch biopsy wounds were made using disposable punches (Stiefel, Offenbach, Germany) in the midline of the lumbar dorsal skin of the mice.

Wound size analysis

Digital photographs (JVC KY-F75U, JVC, Yokohama, Japan) were taken at time of surgery and thereafter daily in a distance of 18–20 cm. A ruler (length: 10 mm) was placed on the back of the animal for a reference. The wound areas were measured by tracing wound margins with a fine resolution computer stylus and were analyzed by means of a morphometry software (Diskus 4.80, Hilgers, Königswinter; Germany). Percentual wound areas were calculated as actual residuals in reference to the wound areas of day 1 after surgery. Wound closure was defined as 95% wound closure according to the FDA-guidance for industry ‘Chronic Ulcers and Burn Wounds – Developing Products for treatment’(22).

Infrared-thermography

To assess possible effects of angiogenic factors on local perfusion, an infrared thermographic system (Varioscan ST-3021, Jenoptik AG, Jena, Germany) was used. Thermograms were taken from non-anesthetized mice every three days post surgery at a distance of 10–12 cm from the camera. The computerized evaluation was carried out using Irbis® control software (version 2.2, InfraTec GmbH, Dresden, Germany). Temperatures were measured in the wound bed23, the wound margin and the adjacent unwounded tissue with line- and area scans. The wound itself was easily identified because of the relative local hyperthermia and was confirmed by comparing the IRT-image with the macroscopic appearances. A two mm wide area encircling the wound bed was defined as wound margin. Unwounded skin at least 4 mm distant from the wound margin was used for reference measurements.

Tissue sampling and histology

The animals were sacrificed on day 17 after surgery. The central parts of the wound area were used for maximum tensile strength measurements, and the adjacent parts for histology and immunochemistry. 5 μm sections of paraffin-embedded specimens were stained with hematoxylin-eosin and picrosirius red according to standard protocols. H&E-stained sections were used to assess the scar dimensions at the upper, the middle and the lower dermis, the scar thickness, the quality of granulation tissue and the progress of remodeling. Thicknesses of the individual skin layers were measured with image analyzing software Diskus 4.80 (Hilgers, Königswinter, Germany). All sections underwent blind quantifaction by an independent observer.

Picrosirius red staining was used to quantify the collagen content24, which was expressed as percentage of red-stained pixels using the image analyzing software KS300 (Kontron AG, Eching, Germany). Areas of green (type III collagen-like) and red (type I collagen-like) were calculated for each field captured. The same settings were used for all analyses.

Immunohistochemical staining of endothelial cells was performed using a monoclonal antibody against CD31 (BD Biosciences Pharmingen, Heidelberg, Germany). Antibody binding was visualized via a three-step staining procedure using a biotinylated polyclonal anti rat Ig-G secondary antibody (DakoCytomation GmbH, Hamburg, Germany) and the streptavidin horse-radish peroxidase reaction together with the DAB detection system. Vessel densities were assessed using a Weibel grid (25)and expressed as percentual vessel surface area.

Tensile strength measurements

Test strips were punched out from the harvested wound vertically to the craniocaudal axis. The test strips had a defined hour glass form with 3 mm width at the narrowest part constituting a predetermined breaking point. The design of the hour glass form was based on material testing standards. Test strips without the hour glass form would inevitably tear at the wedge grips, where the tissue is already bruised. The breaking strength test device consisted of two opposing gripping jaws which fixed the tissue strip. The electric motor driven gripping jaws were moved apart with a constant strain rate of 0.5 mm/s under displacement control. Time, force, and displacement were recorded for stretching up until failure. A position encoder (WA300) was used to register the stretching distance; a force transducer (S2, maximum value 150 N) was used to quantify the power applied to the tissue strip. The endpoint was the breaking strength in Newton [kg·m/s2]. The resulting values were recorded by a multiple channel PC measuring device (Spider 8, HBM Hottinger Baldwin Messtechnik, Darmstadt) and plotted as force-deflection curve (software: Catman 4.5, all HBM Hottinger Baldwin Messtechnik, Darmstadt, Germany). The maximum breaking strength was determined from the stress–strain curve.

Statistical analysis

Statistical analysis was based on measurements in at least 27 different mice. The unpaired Student’s t-test for samples of unequal variances was used to calculate statistical significance. The data was expressed as mean ± one standard deviation. The significance level for the sample distribution was defined as P<0.05.

Results

Gross appearance and wound closure

Despite identical lesion sizes, the proangiogenic pretreated animals seemed to react with a more pronounced wound contraction (Fig. 1). The priming of wounds with proangiogenic factors showed a temporally advantage in wound healing process: 50% wound closure was reached on day 3.9 in the tests versus day 5.2 in the controls (Fig. 2a). At day 8 we achieved a remaining wound area of 26.1 % in control group vs. 9.5% in proangiogenic pretreated group. All proangiogenic pretreated wounds were completely closed on day 10, whereas on the same day only 22% of the control group wounds were completely closed (Fig. 2b). All control wounds were completely closed on day 13. Fur growth and hypertrichosis were also accelerated and more pronounced in the proangiogenic pretreated animals. Already macroscopically more pronounced vessel density on the site of proangiogenic pretreated group is obvious (Fig. 1).

Figure 1.

Figure 1

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Macroscopic assessment of wound closure as a function of time in representative mice receiving either control treatment (20μl isotonic saline solution; upper panel) or proangiogenic treatment (a combination of 35.0 μg VEGF, 2.5μg bFGF and 3.5μg PDGF; lower panel) on days 3, 5 and 7 prior to surgery. Note the higher degree of wound contraction in proangiogenic primed group. Day 17 shows the backside of specimen biopsy. The black marker has a length of 5 mm.

Figure 2.

Figure 2

Figure 2

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Assessment of wound healing as a function of time after wound placement in mice receiving either control treatment (20μl isotonic saline solution; grean line) or proangiogenic treatment (a combination of 35.0 μg VEGF, 2.5μg bFGF and 3.5μg PDGF; red line) on days 3, 5 and 7 prior to surgery; a: remaining relative wound surface, b: percentage of animals with complete wound closure (≥95%).

Infrared thermography

Fig. 3 shows that the mean temperatures were significantly higher in proangiogenic pretreated group than in controls. The wound margins of the proangiogenic primed animals revealed the highest temperature levels. Here, the mean temperature was 0.59°C higher in than in the control group, whereas the proangiogenic pretreated wounds’ ground was 0.47°C warmer than the one of the controls. In the adjacent vicinity of the wound margin the temperatures of the tests were even 0.68°C higher.

Figure 3.

Figure 3

Figure 3

Figure 3

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High resolution infrared thermography of full thickness biopsy wounds. a shows a typical example for a line-scan (grey line) thermogram of wound area (dotted circle), wound margin (dashed circle) and unwounded tissue (dashed dotted circle). b illustrates the characteristic temperature profile with two prominent humps at wound margin and decreased temperatures in the wound bed. c shows that the temperature levels both in the wound bed, wound margin and adjacent areas are significantly higher in the pretreated tests. Box-whisker plot showing median, 5th, 10th, 25th, 75th, 90th, and 95th percentile.

Vessel density

The vessel densities were determined both in the wounds after sacrificing the animals as well as in the punched skin of the pretreated wound bed on the day of the surgery. Fig. 4a shows that pretreatment resulted in significantly higher percentual vessel densities at time of surgery: priming yields more than four fold higher vessel surface areas in pretreated group vs. controls (mean: 10.3% vs. 2.4% vascular surface area).

Figure 4.

Figure 4

Figure 4

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Microvessel density of harvested wound tissue in mice receiving either control treatment (20μl isotonic saline solution) or proangiogenic pretreatment (a combination of 35.0 μg VEGF, 2.5μg bFGF and 3.5μg PDGF) on days 3, 5 and 7 prior to surgery. a: day 0 (punched tissue), b: day 17 after wound.

After complete wound closure and harvesting vessel densities were assessed separately in the wound ground, the former wound margin and in the adjacent unwounded tissue. Figure 4b illustrates that the values were significantly higher in all measured areas in the proangiogenic pretreated group. Fig. 5 gives some typical examples.

Figure 5.

Figure 5

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Vascularization of excised skin punch biopsies in control- and pretreated animals on the day of surgery (0 d) and of the wound tissue one week after complete closure (17 d). Note the higher vessel density (red arrowheads highlights vessels by way of example) in the pretreated group on the day of surgery and on day 17 as well as the hypertrichosis in the newly formed, healed tissue. ↑ points exemplary to hair follicles. Anti-CD31 staining, bars = 500 μm.

Maximum tensile strength

Functional tensile strength testing revealed a significant advantage of the proangiogenic factor pretreatment in comparison to sham treated control group: we attained nearly twofold higher breaking strength results in the proangiogenic pretreated group (p<0.01, Fig. 6).

Figure 6.

Figure 6

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Comparison of tensile strength values obtained on day 17 after surgery. Mice received either control treatment (20μl isotonic saline solution) or proangiogenic treatment (a combination of 35.0 μg VEGF, 2.5μg bFGF and 3.5μg PDGF) on days 3, 5 and 7 prior to surgery. Box whisker plot showing median, 10th, 25th, 75th, 90th percentile.

Collagen contents

Figure 7 shows significant increases of the percentage of juvenile type III collagen-like fibers after surgery. The proangiogenic factor treated test group showed postoperatively significantly higher values than the controls (p<0.001). The levels of type I collagen-like fibers did not change to the same degree (data not shown).

Figure 7.

Figure 7

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Assessment of juvenile type III collagen-like fibers at day 17 after wounding using picrosirius red staining. Mice received either control treatment (20μl isotonic saline solution) or proangiogenic treatment (a combination of 35.0 μg VEGF, 2.5μg bFGF and 3.5μg PDGF) on days 3, 5 and 7 prior to surgery. Contents of juvenile type III collagen-like fibers in the resected tissue (day 0) and in the harvested wound (day 17) showing significantly higher values in the pretreated test group. Box-whisker plot showing median, 5th, 10th, 25th, 75th, 90th, and 95th percentile.

Discussion

The present study, to our knowledge, is the first to investigate effects of subcutaneous priming with proangiogenic factors on wound healing. It shows that priming with a combination of proangiogenic factors (VEGF, bFGF, and PDGF) can significantly accelerate wound healing. In particular, assessment of time-to-closure and functional outcome revealed a significant advantage for the proangiogenic pretreated group in comparison to control animals. In addition, we observed a higher degree of wound contraction immediately after wounding in the proangiogenic factors pretreated group. Healing by contraction is more predominant than epithelialization in loose skin animals and depends on vascularization, which is elevated in proangiogenic primed group and which correlates with myofibroblast activity (26).

This study was designed as pilot study focusing primarily on the functional outcome and the time to closure as primary endpoint. Histological assessments were carried out only on day 0 and seven days after complete closure. Infra-red thermography was used instead for monitoring the healing process non-invasively.

Factors contributing to the quality of wound healing include not only the time required for the wound to close, but also the functional properties of the new skin, an aspect which is receiving increased attention at present. Additionally, from the findings presented here, it would appear that angiogenesis seems to be strongly related to the tensile strength of the newly formed epidermis and dermis, as revealed by significantly higher breaking strength values.

Collagen is the major component of extracellular matrix and provides tensile strength to skin. Gabbiani et al. (27) found that granulation tissue in rat wounds contained proportionally more type III collagen. In our study, collagen deposition changes towards a higher level of juvenile type III collagen-like fibers following application of proangiogenic growth factors, suggesting intensified tissue remodeling by vascularized tissue like in granulation tissue. A stringent link between collagen content and functional outcome, in particular breaking strength, should, however, not be presumed. Our recent observations on abdominal wall nevertheless confirm the theory that cross-linking and interfacing of collagen bundles can be seen as a pivotal criterion influencing breaking strength (Konerding et al., submitted). Many studies show different results of PDGF-application on tensile strength: Ashraf et al. (28) reveal that a sustained release of PDGF leads to a paradoxical decrease of wound breaking strength in an impaired wound healing model, whereas Eming et al.(29) showed a significant increase of tensile strength by PDGF-gene transfer. Our priming experiments support the thesis of benefical effects of PDGF on breaking strength. In further experiments we will focus on priming on single proangiogenic growth factors as PDGF and VEGF and their influence on tensile strength in normal and impaired diabetic wound healing.

The application of VEGF has been seen to increase vascular permeability, with a maximum effect being seen 2–3 days after wound placement, primarily in postcapillary venules and small veins. Brown and colleagues (30) described leaky vessels which were initially seen at the wound margin, and later on became most prominent in the subepidermal granulation tissue. At that time microvessel density reaches its maximum increase during wound healing process (31). An increased permeability of microvessels is also an important facet necessary for interaction with the surrounding extracellular matrix and tissue remodeling (32). If new blood vessels and supporting connective tissue are to develop in wound tissue, e.g., as a response to the increased metabolic needs in the wound healing process, native tissue stroma must be replaced with a new matrix structure that supports migration of endothelial cells and fibroblasts (30). In this context, priming with angiogenic factors could allow a preconditioning of the wound bed: For example, application of VEGF leads on the one hand to a hyperpermeability of microvessels which facilitates the early steps of wound healing (extravasation, infiltration, granulation tissue) and on the other to the induction of angiogenesis prior to surgery so that when a wound is inflicted, a newly formed vessel network is already ‘waiting’ to cope with the inflicted insult to the tissue.

Pretreatment with proangiogenic factors in the present study led to higher tissue temperatures – measured using infrared thermography - and can be assumed to indicate a higher level of vascularization and local tissue perfusion. In addition, infrared thermography appears to be a reliable surrogate parameter for the assessment of local tissue perfusion in wounds (33). A correlation between levels of vessel densities and higher temperature warrants further discussion, especially since a high vessel density alone does not necessarily result in an increase in local tissue perfusion. In particular, the influence of vasoregulation on blood flow in remodeled tissue appears to be a crucial element in the control of perfusion (34,35). Normal blood vessels respond to specific physiological signals from the tissue they are supporting and from the perivascular microenvironment (36). Nevertheless, the stimulation of vascularization through application of angiogenic factors is thought to be capable of increasing significantly local tissue perfusion(36).

Wound healing is a well-regulated continuum of vessel induction and regression (31,37). In healing wounds, rising levels of proangiogenic growths factors induce a period of robust angiogenesis. After that, a period of vessel regression is seen during which capillarity density returns to a level comparable to that found in normal skin (18). Immature blood vessels have been reported to regress as a consequence of withdrawing vascular endothelial growth factor. Exogenous proangiogenic factors applied by priming in addition to endogenous growth factors released in wound healing process may improve stabilization of vessel networks by VEGF as ‘vessel survival factor’(30) and may effect a certain latent period of vessel regression.

Many studies (37,38) reviewing the therapeutic influence of proangiogenic growth factors on the success of wound healing have found a wide spectrum of results. In general, dosage and mode of application seems to represent the variables with the highest impact as far as boosting angiogenesis in wound healing is concerned. Where topically applied growth factors have been seen to be ineffective(20) this lack of effect may be due to a proteloytic degradation of factors at the wound site.

To conclude, priming could represent - in addition to gene therapeutic(37) concepts - a potential therapeutic approach for improving the vascularization of skin prior to elective surgery, especially in patients where a compromised wound healing might be expected. Following the therapeutic success of priming with proangiogenic factors in normoglycemic mice in the present pilot study, further research will focus on the effects of priming in a diabetic mice model, in which an assessment of morphological and functional outcome in diabetes-related impaired healing will be possible. Furthermore, an optimization of the growth factor combination and the ideal time points is needed.

Acknowledgments

This work was supported by grants of the German Army to MK and AW (M-SAB 1-5-A009) and The Angiogenesis Foundation. The authors acknowledge skillful technical assistance of Ms Kerstin Bahr and thank Dr. Bickes-Kelleher and Dr. George Broughton for critical review of the manuscript.

Footnotes

The authors state no conflict of interest.

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