Combination Treatment of Artificial Dermis and Basic Fibroblast Growth Factor for Skin Defects: A Histopathological Examination

This study examined a combination of artificial dermis and basic fibroblast growth factor (bFGF) to treat skin defects in clinical cases, and it histopathologically examined the effects on the conditions of recipient beds. Materials and Methods. The subjects were 11 patients with skin defects from burn ulcers or traumatic ulcers. In each subject, debridement was performed and subsequently artificial dermis was applied to the defect. The bFGF was used on 1 side (combination therapy) of the artificial dermis and not used on the other side (artificial dermis monotherapy). A histopathological examination was performed on the granulation tissue collected from the recipient bed. The authors also measured skin hardness 6 months after the skin graft. Results. Histologically, the combination therapy site had more extensive capillary angiogenesis than the monotherapy site. The combination therapy site also had capillary walls consisting of thick, large endothelial cells; fibroblast proliferation and activation; and more severe infiltration of inflammatory cells. Skin hardness after the graft was also much softer in the combination therapy. Conclusion. The results suggest the usefulness of this combination therapy in the preparation of skin graft beds to improve skin hardness after skin grafts in clinical cases. 

Skin defects are generally treated using artificial dermis after the recipient bed is in satisfactory condition prior to skin grafting. Recent use of basic fibroblast growth factor (bFGF) formulations to treat skin ulcers not only enhances granulation, but also minimizes wound contracture and inhibits scar formation, allowing near-normal dermis to form.^1-5 Studies are currently underway, primarily in animals, in the hopes of proving a synergistic effect when artificial dermis is used together with bFGF preparations.^6-9 This study used a combination of artificial dermis and bFGF in clinical practice and histopathologically examined the effects on the conditions of recipient beds. The usefulness of bFGF was examined as a cell growth factor in the preparation of skin graft beds.

The subjects were 11 patients who had skin defects from burn injuries or traumatic wounds and who needed wound closure with a skin graft or flap. The age range was 18-74 years (mean: 40.5 years). Six patients had burn wounds and 5 had traumatic wounds (Table 1). In each patient, the necrotic tissue was debrided and subsequently artificial dermis (PELNAC, Smith & Nephew, Tokyo, Japan) was applied to the defect. The bFGF (Fiblast Spray, Kaken Pharmaceutical Co Ltd, Tokyo, Japan) was used on 1 side (combination therapy) of the artificial dermis and not used on the other side (monotherapy). In 1 case (case 11), an artificial dermis made of antecollagen (TERUDERMIS, Olympus Terumo Biomaterials, Tokyo, Japan) was used. When a patient had only 1wound, the authors divided the wound area into 2 parts: bFGF spray was used on 1 side (combination therapy) and artificial dermis alone was used on the other side (monotherapy). The patients in cases 1, 3, and 8 had 2 isolated wounds. The authors designated 1 wound to receive combination therapy and the other to receive monotherapy. 

For the combination therapy, bFGF was sprayed directly onto the artificial dermis at the time the dermis was applied to the defect. Subsequently, bFGF was sprayed through meshlike holes made in the silicon membrane of the artificial dermis once a day for at least 2 weeks (Figure 1). Five sprays of bFGF were used per application onto an area of 25 cm² (~1 µg per 1 cm²). The state of granulation was macroscopically evaluated 2 weeks after the artificial dermis was applied. Subsequently, skin grafting was performed in all of the cases except case 11, where the skin graft did not take because the patient failed to follow postoperative care instructions, such as rest. 

A few cubic millimeters of granulation tissue were collected from the recipient bed before skin grafting. Hematoxylin and eosin staining (H&E) and immunostaining (CD34 and vimentin) were performed for histopathological examination. For each tissue, the following features were evaluated: capillary angiogenesis, capillary endothelial activation, fibroblast proliferation, inflammatory cell infiltration, and collagen fiber proliferation. The number of capillaries per mm² stained with H&E and CD34 were counted and the number of fibroblasts per mm² stained with vimentin were counted. These measurements were compared between the monotherapy and combination treatments and statistically analyzed. 

The authors also measured skin hardness 6 months after the skin graft on cases 1-10 using an elasticity measurement device (durometer) (Cutometer dual MPA 580, CK Electronic, Cologne, Germany). The device is designed to measure elasticity of the upper skin layer using negative pressure, which mechanically deforms the skin. The maximum amplitude (R0) represents the passive behavior of the skin firmness. The authors measured 3 points on each of the monotherapy sites, the combination sites, and normal skin around the graft as a control. Measurements were performed 3 times each, and the average was calculated.

When a single wound was divided into a monotherapy site and a combination therapy site, the monotherapy site was covered while spraying bFGF so that the growth factor would not affect that side. Moreover, since there may have been a 2 mm to 3 mm overlap between the 2 sites, care was taken to avoid the borderline areas when skin biopsies were taken for histopathological exams or to measure skin hardness 6 months after the skin grafts. This study was carried out according to the guidelines of the official of the hospital ethics committee and patient consent was obtained.

The results were findings at approximately 2 weeks after the application of artificial dermis to the defect. Macroscopically, all patients had satisfactory granulation tissue formation in both the combination therapy and monotherapy sites. Histologically, the combination therapy site had more extensive capillary angiogenesis than the monotherapy site. The combination therapy site also had capillary walls consisting of thick, large endothelial cells; fibroblast proliferation, activation, and swelling; and more severe infiltration of the inflammatory cells. Overall, satisfactory recipient beds with high blood perfusion were obtained that were suitable for skin grafts (Tables 2, 3, and 4). 

The number of capillaries stained with H&E was counted per mm². Proliferation of capillaries was observed with combination therapy compared with monotherapy in all the cases except for 1 case (Figure 2A). There was a statistically significant difference in the average number of capillaries per mm² (P = 0.047). 

Values are expressed as mean ± SD with P < 0.05 vs control group. However, when analyzing the capillaries stained with CD34, proliferation of capillaries was observed in only 6 cases with combination therapy compared with monotherapy (Figure 2B). Values are expressed as mean ± SD, and there were no significant differences (P = 0.205). 

On the other hand, when the fibroblasts stained with vimentin were counted per mm², more proliferation of fibroblasts was observed with combination therapy compared to monotherapy in most of the cases (Figure 3). The average number of fibroblasts per mm² are expressed as mean ± SD (P = 0.031). Values are expressed as mean ± SD, with P < 0.05 vs control group. There was a statistically significant difference in the average number of fibroblasts per mm². 

The authors also measured skin hardness 6 months after the skin grafts in cases 1-10 using a durometer. The average maximum amplitude (R0) was higher in 6 of the combination therapy areas compared with 4 monotherapy areas (Table 5). 

Case study 1. The patient was an 18-year-old man who suffered an injury in a car crash. Traffic ulcers (9 cm x 35 cm) developed, reaching muscle and bone of the left dorsum of the hand and arm (Figure 4A). The artificial dermis was applied to the ulcers. Postoperatively, the arm was treated with monotherapy, and the dorsum of the hand was treated with combination therapy. Two weeks after the artificial dermis was applied, the hand treated with combination therapy had satisfactory granulation tissue formation with clearly red coloration compared with the arm treated with monotherapy (Figure 4B). Histopathologically, the hand (combination therapy) had more marked capillary and fibroblast proliferation compared with the arm (monotherapy). In addition, the combination therapy site had severe inflammation characterized by a mixture of lymphocytes, plasma cells, and polymorphonuclear leukocytes. The combination therapy site had swollen vascular endothelial cells and vascular walls that were thicker than those of the monotherapy site (Figures 4 C, D). When capillaries per mm² were counted, there was a higher number in the combination therapy site (266.7 capillaries/mm² with H&E stain, 235.5 capillaries/mm² with CD34 stain) compared with the monotherapy site (79.6 capillaries/mm² with H&E stain, 102.2 capillaries/mm² with CD34 stain) (Figures 4C-F). On the other hand, when fibroblasts per mm² were counted, there was a higher number in the combination therapy site (398.3 fibroblasts/mm²) compared with the monotherapy site (311.7 fibroblasts/mm²) (Figures 4 G, H). A split-thickness skin graft harvested from the lateral thigh was used. No obvious scar contracture was observed in the arm or hand after skin grafting, and the findings at follow-up were satisfactory (Figure 4I).

Case study 7. A 74-year-old man sustained deep dermal burns on both feet caused by hot water (Figure 5A). Three weeks after the injury, these lesions were surgically debrided. The wounds on the right and left feet were of similar depth, and artificial dermis was applied to both feet. Postoperatively, the bFGF was sprayed daily on the right foot. Two weeks after the artificial dermis was applied, the right foot, treated with combination therapy, showed signs of healthy granulation with clear red coloration compared to the left foot that had only been treated with artificial dermis monotherapy (Figure 5B). Histopathologically, the right foot had more marked capillary and fibroblast proliferation compared to the left foot. In addition, the right foot was severely inflamed, characterized by a mixture of lymphocytes, plasma cells, and polymorphonuclear leukocytes. The right foot had swollen vascular endothelial cells and vascular walls that were thicker than those of the left foot (Figures 5 C, D). When capillaries per mm² were counted, there were higher numbers on the combination therapy side at 69.9 capillaries/mm² with H&E stain, and 126.9 capillaries/mm² with CD34 stain, compared with the monotherapy side at 38.7 capillaries/mm² with H&E stain, and 64.5 capillaries/mm² with CD34 stain. On the other hand, when fibroblasts per mm² were counted, there were higher numbers in the combination therapy side at 701.3 fibroblasts/mm² compared with the monotherapy side at 545.5 fibroblasts/mm². A split-thickness skin graft harvested from the lateral thigh was used. No obvious scar contracture was observed in the left or right foot after skin grafting, and the findings seen at follow-up were satisfactory. 

The cell growth factor bFGF promotes the proliferation of cells of ectodermal and endodermal origin such as fibroblasts, vascular endothelial cells, epidermal cells, osteoblasts, and chondrocytes. The bFGF also promotes wound healing by promotion of angiogenesis, granulation formation, and epithelialization.¹ Artificial dermis is often clinically used in Japan to cover dermal and soft tissue defects. It consists of a collagen spongy form with a silicone membrane on the top. The collagen sponge acts as a scaffold for dermal tissue structure and induces vascular endothelial cells and fibroblasts, resulting in the formation of dermislike tissue.¹⁰

The authors’ study was conducted to examine whether combination therapy of bFGF and artificial dermis can result in the preparation of a satisfactory skin-graft recipient bed and in a shortened period of granulation tissue proliferation. The combination therapy was used in clinical cases, and histological and pathological examinations were performed to study the combined effects of bFGF (such as on the condition of skin graft bed, skin graft survival, and scarring after skin grafting) in the preparation of skin graft beds after artificial dermis was applied.

There have been previous reports on the combination therapy of bFGF and artificial dermis. In an animal study, Kawai and colleagues⁶ found combination therapy promoted wound healing. In Ono and coauthors’⁷ animal study, new tissue formed in the artificial dermis and featured collagen with a uniformly horizontal arrangement, resembling normal dermis. In addition, formation of thick epithelium was observed. There was also more marked inhibition of wound contraction in a group treated with the combination therapy than in the control group. Moreover, Hamuy and colleagues⁸ reported an investigation with nude rats where they used bFGF in combination with artificial dermis. By applying a split-thickness skin graft with an artificial dermis, they achieved improved postgraft elasticity, better fibroblast maturation, and stabilization of structures to enhance blood flow with less bacterial invasion.⁸ Another study⁹ showed controlled-released bFGF into the artificial dermis promoted fibroblast proliferation and angiogenesis, and intermittent or continuous administration of bFGF resulted in accelerated tissue formation.⁹ 

The combination therapy has been clinically applied to diabetic toe ulcers¹¹; intractable ulcers on the sacrum and wrist¹²; burn, traumatic, or ischemic ulcers on the fingertips¹³; and traumatic skin defects of the heel of the foot.¹⁴ Various reports have shown good results using combination therapy, the authors have reported the hopes of proving a synergistic effect as well.¹⁵ Case reports have accounted for the majority of reports on combination therapy in clinical cases. 

There have not been any reports on its effectiveness that include histological details. In all the cases, the collagen layer of the artificial dermis was replaced by dermislike tissue. In the present study, the combination therapy of bFGF and artificial dermis resulted in larger amounts of granulation tissue with clearly red coloration and in macroscopically superior skin-graft recipient bed compared with the artificial dermis monotherapy. These findings were due to angiogenesis and granulation tissue formation caused by bFGF.  As in previous case reports, this study showed that combination therapy enabled shorter preparation time for skin-graft recipient beds, improved safety of skin graft survival, and improved stability of the wound healing process. In a histopathological study of skin-graft beds, the combination therapy promoted capillary angiogenesis and inflammatory cell infiltration more than the monotherapy. The combination therapy also resulted in swelling of vascular endothelial cells and thickening of capillary walls. Swelling of vascular endothelial cells leads to their activation and proliferation, while thickening of capillary walls increases blood flow. These effects are believed to be due to bFGF. 

In addition, the combination therapy group showed marked fibroblast proliferation and activation. It seems these results are due to cell activation by bFGF. When the capillary count and fibroblast count per mm² were compared between combination therapy and monotherapy, the combination therapy resulted in significantly higher counts compared with the monotherapy in most of the patients.

In this study, the authors used H&E staining, CD34 staining, and vimentin staining to evaluate the tissue. An increase in capillary vessels was seen in all of the bFGF treatments by H&E staining, except for case 9. By CD34 staining, an increase in capillary vessels was not seen in the bFGF group in cases 3 and 4. There was no particular difference in cases 3, 4, and 9 in terms of the patients’ age, site and depth of wounds, or past history. As these are clinical trials, tissue sampling was limited. When formation of granulation tissue showed a difference, the worst one was selected to receive combination therapy due to ethical reasons. On the other hand, the number of fibroblasts was found to significantly increase upon combination therapy in 10 out of 11 patients. These results demonstrated combination therapy was useful for wound healing. The results also suggest combination therapy can shorten the time from application of artificial dermis until a satisfactory skin-graft recipient bed is obtained. 

It has been reported that when bFGF is administered on the surface of a skin ulcer or suture wound, wound contraction is minimized, scar formation is inhibited, and soft skin tissue resembling normal skin is formed.^2-5 Akita and coauthors^2,16 found that by applying bFGF before a skin graft for trauma-induced ulceration, not only does the postoperative skin graft feel more natural in terms of hardness, but pigmentation can even be minimized. It was speculated that bFGF administration caused cell proliferation and angiogenesis and that apoptosis was induced after convergence of a series of reactions.  Abe and colleagues¹⁷ reported that bFGF may inhibit lengthening of keloid-derived fibroblasts and induce apoptosis.

A recent study has shown that fibroblasts express alpha-smooth muscle actin (alpha-SMA) and become myofibroblasts, resulting in wound contracture.¹⁸ Akasaka and coauthors¹⁹ conducted animal studies and reported that bFGF inhibited alpha-SMA expression and wound contracture. Ishiguro et al²⁰ reported the combination therapy of artificial dermis and bFGF inhibited alpha-SMA expression. Akita et al² found that burn wounds treated with clinically approved bFGF might contribute to a better cutaneous wound quality using a durometer to measure scar hardness. The results of the current study, using artificial dermis and bFGF, also demonstrated that combination therapy improved the hardness of the skin after grafting. In future clinical experiments, the authors hope to examine factors related to wound contracture such as alpha-SMA after combination therapy and to show combination therapy enables the formation of soft skin that more closely resembles normal skin in the period of scar formation after healing.

This study histopathologically examined the effects of combination therapy of bFGF and artificial dermis on skin defects in 11 clinical cases. The combination therapy promoted angiogenesis and fibroblast proliferation in the artificial dermis, and it histologically demonstrated its usefulness in the preparation of skin-graft beds in clinical cases.

From the Department of Plastic, Reconstructive and Aesthetic Surgery, Chiba University, Chiba, Japan; Department of Pathology, St. Mary’s Hospital, Fukuoka, Japan; and Department of Plastic and Reconstructive Surgery, Showa University School of Medicine, Tokyo, Japan

Address correspondence to:
Nobuyuki Mitsukawa, MD
Department of Plastic, Reconstructive and Aesthetic Surgery
Chiba University,
Faculty of Medicine
1-8-1, Inohana, Chuo-ku, Chiba City, Chiba, 260-8670 Japan
nmitsu@air.linkclub.or.jp

Disclosure: The authors disclose no financial or other conflicts of interest.