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Improvement in wound healing by a novel synthetic collagen-gel dressing in genetically diabetic mice
Asian Journal of Oral and Maxillofacial Surgery 22 (2010) 61–67
Contents lists available at ScienceDirect
Asian Journal of Oral and Maxillofacial Surgery journal homepage: www.elsevier.com/locate/ajoms
Original Research
Improvement in wound healing by a novel synthetic collagen-gel dressing in genetically diabetic mice Daichi Chikazu a,∗ , Tetsushi Taguchi b , Hiroyuki Koyama c , Hisako Hikiji a , Hisako Fujihara a , Hideyuki Suenaga a , Hideto Saijo a , Yoshiyuki Mori a , Ichiro Seto a , Mitsuyoshi Iino a , Tsuyoshi Takato a a
Department of Oral and Maxillofacial Surgery, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan c Department of Vascular Regeneration, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan b
a r t i c l e
i n f o
Article history: Received 23 May 2009 Accepted 13 January 2010 Available online 24 July 2010 Keywords: Wound healing Collagen-gel dressing Diabetic mice
a b s t r a c t Our study aimed to develop an intelligent wound dressing containing elements that promote wound healing. Unlike conventional dressings, this is a gel-type dressing that contains basic factors required by cells in a complex of alkaline-treated collagen and atelocollagen. Full-thickness 8-mm skin defects were made with a DermaPunch® in the left and right sides of the back of 8-week-old genetically diabetic mice (db/db). Collagen gel was applied to the fascia on the right side, while the left (control) side was left untreated. Both wounds was coated and fixed with Tegaderm® . On days 3, 7 and 14, macroscopic appearance and percent decrease in wound area were assessed histopathologically. On the collagen-gel side, granulation tissue had formed, with wound contraction caused by migration of epidermal cells from the surrounding tissue. By day 14, wound area had decreased to 37% for the untreated side as compared to 22% on the collagen-gel side, indicating that collagen gel was associated with significantly greater wound contraction. Moreover, there were a lot of blood vessels on the collagen-gel side. Collagen-gel dressing is most effective for dry, open wounds, i.e., those with minimal effusion, and it may also be effective for depressed open wounds. Collagen-gel dressing is expected to be very useful in treating wounds such as skin defects and skin ulcers. © 2010 Asian Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
1. Introduction In the past, it was erroneously believed that wounds heal best when in a dry environment, but at present, it is accepted that optimal wound healing occurs with a proper level of moisture. Moist wound healing is a technique that maintains a moist environment, and materials that cover wounds to create an environment suitable for wound healing are called dressings. Currently, a burn blister is considered one of the ideal environments for wound healing because a moist environment is maintained at the wound surface, and as long as the blister does not break, the wound is sealed from the outside environment to block out bacteria and foreign materials. Moreover, the liquid inside the blister contains white blood cells, macrophages, and various growth factors. In 1943, Cope proposed a treatment that preserved the blister membrane as a biological wound covering [1]. In 1957, Gimbel and colleagues studied burn blisters and reported that epithelialization was about 40% faster when blisters were left intact when
∗ Corresponding author. Tel.: +81 3 3815 5411x33714; fax: +81 3 5800 8669. E-mail address: [email protected] (D. Chikazu).
compared to suctioning or removing the liquid [2]. Winter conducted animal studies to compare moist and dry environments in 1962 [3], and proved that when a film was placed over porcine skin defects to trap exudate, wounds healed twice as fast when compared to dry wound healing. In 1963, Hinman and colleagues examined human skin wounds and showed that wounds healed faster when a moist environment was maintained [4]. Many subsequent studies have shown that a moist environment facilitates wound healing. As compared with conventional dressings such as gauze, dressings used to promote moist wound healing are often referred to as modern dressings. Film dressing materials (Opsite® ; Smith & Nephew plc, London, UK) were marketed as modern dressings in 1971. A transparent polyurethane film is coated with adhesive on one side, allowing the passage of carbon dioxide and oxygen, but not water. A moist environment can therefore be maintained by wound closure. Hydrocolloid dressing (DuoDerm® ; ConvaTec, New York, NY, USA) was launched in 1983. This dressing adheres to the skin when it contacts the moist wound surface, covers the wound, and absorbs exudate. It turns into a gel and maintains a moist environment. Subsequently, many modern dressings have been developed, manufactured, and marketed.
0915-6992/$ – see front matter © 2010 Asian Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ajoms.2010.01.001
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In the field of oral surgery, extensive resection of tumors arising in the oral cavity, including the tongue, requires primary suture closure. Wound constriction and cicatricial contracture can severely restrict movement of the tongue, and if they occur at the mucoperiosteal defects after palatoplasty, these complications are an important cause of impaired future growth of the jaws [5]. To prevent wound constriction and cicatrical contracture, atelocollagen sponge (Terudermis® , Olympus Terumo Biomaterials Corp., Tokyo, Japan; Pelnac® , Gunze Medical Materials Center, Kyoto, Japan), is often used. When atelocollagen sponge is applied to the wound surface, fibroblasts and capillaries from the wound surface infiltrate and proliferate into the pores of the sponge [6,7]. As new collagen fibers are produced by proliferating fibroblasts, the collagen sponge is degraded and absorbed, turning into regenerated skin-like tissue. However, this formation of skin-like tissue requires 2–3 weeks. This prolonged treatment period is a disadvantage because almost defects usually become uneven and irregularly shaped. Hence, close contact between the collagen sponge and the wound surface is frequently difficult to achieve. The aim of the present study was to develop a viscous gel-type wound dressing that can maintain close contact with the surface of uneven, irregularly shaped wounds and serve as a framework for the infiltration and proliferation of fibroblasts and capillaries from the wound surface. The wound dressing should also be promptly absorbed and replaced by healthy tissue after cell infiltration. Another aim was to study the effectiveness of the wound dressing in vivo.
Fig. 1. The new collagen-gel dressing with collagen crosslinked with citric acid derivative (CAD).
of 5, has carboxyl groups generated by the hydrolysis of residual amide groups in its asparagine and glutamine components. Citric acid (CA), N-hydroxysuccinimide (HOSu), tetrahydrofuran (THF), 2,4,6-trinitrobenzenesulfonic acid (TNBS), disodium hydrogen phosphate, sodium hydrogen phosphate, toluidine blueO, dimethyl sulfoxide (DMSO), ethanol, tert-butyl alcohol, HCl, acetic acid, 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 10% formalin solution, and NaOH were purchased from Wako Pure Chemical Industrials Ltd. (Osaka, Japan). Dicyclohexylcarbodiimide (DCC) was purchased from Kokusan Chemical Co. Ltd. (Tokyo, Japan). Other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
2. Materials and methods
2.2. Preparation of CAD
2.1. Materials
Citric acid derivative (CAD) was prepared by methods previously reported [8–10]. Briefly, CA was first dissolved in THF, and then HOSu and DCC were added. The resulting mixture was stirred for 30 min and then concentrated with rotary evaporation under a
AlCol and AtCol derived from pig’s tissue were provided by Nitta Gelatin Inc. (Osaka, Japan). AlCol, which has an isoelectric point
Fig. 2. Macroscopic appearance of the wound healing of skin defects. The collagen-gel dressing was applied on the right side, and the untreated side was on the left. Tegaderm® was placed over both wounds. The wound area on the right side decreased with time.
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two glass plates for 24 h at 37 ◦ C. The AlCol-CAD was subsequently immersed in excess pure water for 48 h at 37 ◦ C to remove DMSO from the AlCol-CAD matrices. AtCol gel was prepared by mixing 0.3% (w/v) AtCol, 10 M phosphate-buffered saline (PBS) (pH 7.4), and NaOH-HEPES buffer solution. 2.4. Animals
Fig. 3. Percent decrease in the wound area due to the collagen-gel dressing. Taking the wound area on day 0 as 100%, and the decrease in the wound area in each group was calculated relative to this value. The wound area decreased significantly on the collagen-gel dressing side. Data are mean ± S.E.M. (n = 6). # Significant difference from day 0 control group, p < 0.01. * Significant effect of collagen-gel dressing, p < 0.01.
Genetically diabetic male mice (BKS.Cg− + Leprdb /+Leprdb /Jcl, db/db) were purchased from CLEA Japan Inc. (Tokyo, Japan) at the age of 8 weeks. The mice were housed at room temperature (24–25 ◦ C) and a relative humidity of 55%, with a circadian light rhythm of 12 h. Food consisted of commercial standard diet pellets (MF; Sankyo Corp, Tokyo, Japan; calcium content, 1.1%; phosphorus content, 0.83%). Water was available ad libitum. All animal experiments were performed according to the guidelines of the International Association for the Study of Pain [11]. In addition, the experimental protocol was reviewed and approved by the Animal Care Committee of the University of Tokyo. 2.5. Wounding
reduced pressure to remove the THF. The final mixture was recrystallized to yield pure CAD. 2.3. Preparation of AlCol-CAD and AtCol gel AlCol was first dissolved in DMSO, and then CAD solution was added to AlCol solution. This AlCol mixture was then stirred and put into a mold with a 1-mm thick silicone rubber spacer between
The mice were anesthetized with ether, and the back was shaved with an electric shaver on the day before wound surgery. On the day of wound surgery, a collagen-gel dressing was prepared. AlColCAD (1%, w/v) and AtCol (2%, w/v), which were preserved at 4 ◦ C, were dissolved in a water bath heated to 37 ◦ C. Combining these ingredients in a 1:1 (v/v) ratio results in the formation of a CAD crosslinked, paste-like, adhesive collagen-gel (Fig. 1). Next, the skin was sterilized with 70% ethanol. A full-thickness wound was made
Fig. 4. Histopathological examination on day 3 (hematoxylin and eosin staining). Inflammatory-cell infiltration was evident at the wound margins on the collagen-gel dressing side. (A) and (C) show the untreated side. (B) and (D) show the collagen-gel side. Magnifications of (A) and (B) are 20×. (C) and (D) are at greater magnification (100×). ES: excised site.
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Fig. 5. Histopathological examination on day 7 (hematoxylin and eosin staining). Extensive infiltration of fibroblasts, extracellular-matrix formation, and angiogenesis were evident. (A) and (C) show the untreated side. (B) and (D) show the collagen-gel side. Magnifications of (A) and (B) are 20×. (C) and (D) are at greater magnification (100×). ES: excised site.
by excising both the skin and panniculus carnosus, with the use of a 8-mm diameter template. The collagen-gel dressing was applied on the right wound surface, and a transparent occlusive dressing (Tegaderm® ; 3M Health Care, St. Paul, MN, USA) was placed over both wounds. Hence, the left wound surface without the collagengel dressing acted as the control side. Six mice in each group were killed under general anesthesia 0, 3, 7 and 14 days after wounding.
with T-PBS and PBS, the peroxidase-labeled streptoavidin regent was dropped on the sections and was incubated at room temperature for 5 min. A solution of DAB was applied after the third washing procedure with T-PBS and PBS, followed by washing with 50 mM Tris–HCl, pH 7.0. The sections were washed with water after the 3-min reaction.
2.6. Evaluation of wound healing
Means of groups were compared by analysis of variance (ANOVA). The statistical significance of differences was determined by post hoc testing with the Bonferroni method.
Decrease in wound area at the skin defects was assessed by taking images with a digital camera at the same magnification. The wound area was determined with the use of NIH Image 1.62 (National Institutes of Health, Bethesda, MD, USA). Taking the wound area on day 0 as 100%, decreases in wound area on the collagen-gel side (right) and on the untreated side (left) were calculated on days 3, 7, and 14. In addition, the skin including the wound site was excised for histological evaluation. The skin was fixed in 10% buffered formalin solution. The tissues were embedded in paraffin and sectioned in 4-m thick slices. The sections were stained with hematoxylin and eosin. On the other hand, the sections were washed with flowing water and equilibrated with 0.01 M PBS (pH 7.2) for immunostaining using a Histofine SABPO(R) kit. The sections ware blocked for 10 min by 1–2 drops of blocking reagent before a 1 h treatment with monoclonal antimouse ␣-smooth muscle actin (␣-SMA) antibodies at 37 ◦ C, and were then washed 3 times with 0.05% Tween 20/PBS (T-PBS) and PBS, respectively, and were reacted with the second antibody for 10 min at room temperature. After the same washing procedure
2.7. Statistical analysis
3. Results 3.1. Effect of the collagen-gel dressing on decrease in wound area Wound area on the untreated side did not decrease significantly during the first 7 days after surgery but had decreased to 37% by day 14. In contrast, wound area on the collagen-gel dressing side gradually decreased from 3 days after surgery and had decreased significantly to 62% by day 7 and to 22% by day 14. Decrease in wound area on day 14 was significantly greater on the collagen-gel dressing side than on the untreated side (Figs. 2 and 3). 3.2. Effect of the collagen-gel dressing on histologic findings By day 3, infiltration of neutrophils and other types of inflammatory cells were noted at the wound margins on the collagen-gel side. By day 7, there was marked evidence of extensive infiltration
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Fig. 6. Histopathological examination on day 14 (hematoxylin and eosin staining). The numbers of fibroblasts and capillaries and the amount of undulating collagen had increased. The structure was similar to that of normal skin. (A) and (C) show the untreated side. (B) and (D) show the collagen-gel side. Magnifications of (A) and (B) are 20×. (C) and (D) are at greater magnification (100×). ES: excised site.
of fibroblasts, extracellular-matrix formation, and angiogenesis. These findings were scant on the untreated side. By day 14, the numbers of fibroblasts and capillaries and the amount of undulating collagen had increased on the collagen-gel side. The structure was very similar to that of normal skin. Similar structures were seen on the untreated side; however, undulating collagen was limited. Immunostaining with anti-␣-SMA antibody on day 14 revealed that in the collagen-gel side, more new blood vessels were able to be seen compared with the untreated side (Figs. 4–7).
4. Discussion The effect on wound healing of a novel synthetic collagengel dressing was examined using the full-thickness wound model in genetically diabetic male mice. In genetically diabetic male mice, wound healing is clearly slower than in healthy mice; diabetes delays wound healing because of weak initial inflammatory reactions and the degeneration of collagen in connective tissue due to glycosylation [12]. In full-thickness wounds in genetically diabetic mice, the collagen-gel dressing significantly
Fig. 7. Immunohistochemical localization of ␣-SMA at the wound on day 14. The full-thickness wound sections were immunoreacted with anti-␣-SMA antibody as described in Section 2. In the collagen-gel side, more new blood vessels were able to be seen compared with the untreated side. (A) and (B) show the untreated side and the collagen-gel side, respectively. Magnifications of (A) and (B) are 100×.
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accelerated wound healing when compared to control conditions. Reconstruction of the dermis with artificial skin utilizing collagen has markedly improved since Yannas and Burke used collagen dressings for stage I wounds [13,14] and at present, Terudermis® and Pelnac® are used widely. When artificial skin is applied to a skin defect, fibroblasts and capillaries invade from the wound surface into the sponge gaps. As proliferating fibroblasts form new collagen fibers, the collagen sponge is degraded, absorbed, and replaced with regenerated dermis-like tissue. However, the currently available artificial skins consisting of collagen sponge are not necessarily perfect. The chemical cross-linking and long-term heatdehydrated cross-linking that are designed to maintain sponge structure and collagen stability in the tissue [15] hinder tissue affinity and enhance foreign body recognition [16], resulting in unnecessary inflammatory-cell infiltration and cicatricial contracture [17]. In order to overcome these obstacles, the collagen-gel dressing is made by physical crosslinking between aterocollagen and citricbearing alkali-treated collagen. We previously developed a novel tissue adhesive composed of CAD and collagen [8]. It was clarified that the resulting adhesive possessed both high bonding strength and biocompatibility. Moreover, physico-chemical properties of alkali-treated collagen crosslinked with CAD were evaluated by means of spectrometric method [18]. From these experiments, water-soluble citric-bearing alkali-treated collagen (anionic collagen) was generated when the excess amount of CAD was added to the solution of alkali-treated collagen. Using this citric acid-bearing alkali-treated collagen (anionic polymer), we developed a novel injectable collagnen gel dressing by the combination with aterocollagen (cationic collagen) via electrostatic interaction. As clarified from the present study, the developed collagen-gel dressing quickly induced favorable granulation, and fibroblasts and capillaries are attracted due to the dressing’s superior cell affinity. These excellent results were due to that collagen was crosslinked by just only electrostatic interaction (not covalent crosslinking). Various cytokines are involved in wound healing, and wound healing is accelerated when certain stimuli are applied. Basic fibroblast growth factor (bFGF) induces the growth of not only fibroblasts, but also various endodermal and ectodermal cells such as chondrocytes, osteoblasts, keratinocytes, and endovascular cells, and it potently induces neovascularization and accelerates wound healing via granulation or epithelialization [19–21]. bFGF preparations (e.g., Trafermin, FIBLAST® spray: Kaken Pharmaceutical Co. Ltd., Kyoto, Japan) have been used clinically to treat skin ulcers, and their efficacy has been confirmed. However, since bFGF is a protein, its biological half-life is short and it is quickly degraded in the body to lose its activity. Therefore, local intermittent administration [22] or sustained release [13,24] is required. Tabata et al. prepared artificial skin with sustained bFGF release by impregnating bFGF into gelatin microspheres [25]. With our collagen-gel dressing, the gel has a negative charge and can hold bFGF, which is a basic protein, and with the resulting sustained bFGF release, fibroblast proliferation and higher neovascularization should accelerate dermis-like tissue formation. However, further investigations are needed to confirm this. We also plan to investigate the involvement of cyclooxygenase (COX)-2 in the collagen-gel dressing’s facilitation of wound healing. When COX-2 inhibitor was administered to animals, or when tumors were grafted to COX-2 knockout mice (COX-2−/− ), intratumoral neovascularization was more haphazard when compared to the control. Prostaglandin (PG) E2 produced by COX-2 reacts with EP2 and EP3 receptors to induce the production of bFGF and vascular endothelial growth factor (VEGF) [26,27]. Moreover, PGE2 increases the migratory capacity of vascular endothelial cells [28], activates MAP, and accelerates vascular structure formation
[29]. Another study found that COX-2-selective non-steroidal antiinflammatory drugs (NSAIDs) delay wound healing [30], but moist wound healing was not assessed. Therefore, if COX-2 is shown to play a significant role in neovascularization in dermis-like tissue using the collagen-gel dressing, then this is new information that sheds new light on wound healing. In summary, the present study has shown that a novel synthetic Collagen-gel dressing is most effective for rather dry, open wounds, i.e., wound surfaces with minimal effusion, and may be also effective for depressed open wounds. Collagen-gel dressing is expected to be a very useful material for the treatment of wounds such as skin defects and skin ulcers. Acknowledgments We appreciate the excellent technical assistance of Satomi Ogura. Funding: This study was supported in part by the 2004 Industrial Technology Research Grant Program from the New Energy and Industrial Technology Development Organization (NEDO) of Japan. Conflict of interests: We have no conflict of interests to declare. Ethical approval: The experimental protocol was reviewed and approved by the Animal Care Committee of the University of Tokyo. References [1] Cope O. Treatment of surface burns. Ann Surg 1943;117:885–93. [2] Gimbel NS, Kapetansky DI, Weissman F, Pinkus HKB. A study of epithelization in blistered burns. AMA Arch Surg 1957;74:800–3. [3] Winter GD. Formation of scab and rate of epithelialization of superficial wounds in the skin of the young domestic pig. Nature 1962;193:293–4. [4] Hinman CD, Maibach H. Effect of air exposure and occlusion on experimental human skin wounds. Nature 1963;200:377–8. [5] Friede H, Enemark H, Semb G, Paulin G, Abyholm F, Bolund S, et al. Craniofacial and occlusal characteristic in unilateral cleft lip and palate patients from four Scandinavian centers. Scand J Plast Reconstr Hand Surg 1991;25:269–76. [6] Koide M, Osaki K, Konishi J, Oyamada K, Katakura T, Takahashi A. A new type of biomaterial for artificial skin; dehydrothermally cross-linked composites of fibrillar and denatured collagens. J Biomed Mater Res 1993;27:79–87. [7] Soejima K, Nozaki M, Sasaki K, Takeuchi M, Negishi N. Reconstruction of burn deformity using artificial dermis combined with thin split-skin grafting. Burns 1997;23:501–4. [8] Taguchi T, Saito H, Uchida Y, Sakane M, Kobayashi H, Kataoka K, et al. Bonding of soft tissues using a novel tissue adhesive consisting of a citric acid derivative and collagen. Mater Sci Eng C 2004;24:775–80. [9] Saito H, Taguchi T, Aoki H, Murabayashi S, Mitamura Y, Tanaka J, et al. pH-responsive swelling behavior of collagen gels prepared by novel crosslinkers based on naturally derived di- or tricarboxylic acids. Acta Biomater 2007;3:89–94. [10] Saito H, Taguchi T, Kobayashi H, Kataoka K, Tanaka J, Murabayashi S, et al. Physicochemical properties of gelatin gels prepared using citric acid derivative. Mater Sci Eng C 2004;24:781–5. [11] Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983;16:109–10. [12] Delbridge L, Ctertcteko GH, Fowler C, Reeve TS, Le Quesne LP. The aetiology of diabetic neuropathic ulceration of the foot. Br J Surg 1985;72:1–6. [13] Yannas IV, Burke JF. Design of an artificial skin. I. Basic design principles. J Biomed Mater Res 1980;14:65–81. [14] Burke JF, Yannas IV, Quinby Jr WC, Bondoc CC, Jung WK. Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury. Ann Surg 1981;194:413–28. [15] Stenzel KH, Miyata T, Rubin AL. Collagen as a biomaterial. Annu Rev Biophys Bioeng 1974;3:231–53. [16] McPherson JM, Ledger PW, Sawamura S, Conti A, Wade S, Reihanian H, et al. The preparation and physicochemical characterization of an injectable form of reconstituted, glutaraldehyde cross-linked, bovine corium collagen. J Biomed Mater Res 1986;20:79–92. [17] Yoshizato K, Taira T, Yamamoto N. Growth inhibition of human fibroblasts by reconstituted collagen fibrils. Biomed Res 1985;6:61–71. [18] Saito H, Murabayashi S, Mitamura Y, Taguchi T. Unusual cell adhesion and antithrombogenic behavior of citric acid-cross-linked collagen matrices. Biomacromolecules 2007;8:1992–8. [19] McGee GS, Davidson JM, Buckley A, Sommer A, Woodward SC, Aquino AM, et al. Recombinant basic fibroblast growth factor accelerates wound healing. J Surg Res 1988;45:145–53. [20] Rifkin DB, Moscatelli D. Recent developments in the cell biology of basic fibroblast growth factor. J Cell Biol 1989;109:1–6.
D. Chikazu et al. / Asian Journal of Oral and Maxillofacial Surgery 22 (2010) 61–67 [21] Klingbeil CK, Cesar LB, Fiddes JC. Basic fibroblast growth factor accelerates tissue repair in models of impaired wound healing. Prog Clin Biol Res 1991;365:443–58. [22] Okumura M, Okuda T, Nakamura T, Yajima M. Acceleration of wound healing in diabetic mice by basic fibroblast growth factor. Biol Pharm Bull 1996;19:530–5. [24] Yamada K, Tabata Y, Yamamoto K, Miyamoto S, Nagata I, Kikuchi H, et al. Potential efficacy of basic fibroblast growth factor incorporated in biodegradable hydrogels for skull bone regeneration. J Neurosurg 1997;86:871–5. [25] Kawai K, Suzuki S, Tabata Y, Ikada Y, Nishimura Y. Accelerated tissue regeneration through incorporation of basic fibroblast growth factor-impregnated gelatin microspheres into artificial dermis. Biomaterials 2000;21:489–99. [26] Sonoshita M, Takaku K, Sasaki N, Sugimoto Y, Ushikubi F, Narumiya S, et al. Acceleration of intestinal polyposis through prostaglandin receptor EP2 in ApcD716 knockout mice. Nat Med 2001;7:1048–51.
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[27] Leahy KM, Ornberg RL, Wang Y, Zweifel BS, Koki AT, Masferrer JL. Cyclooxygenase-2 inhibition by celecoxib reduces proliferation and induces apoptosis in angiogenic endothelial cells in vivo. Cancer Res 2002;62:625–31. [28] Jiang H, Weyrich AS, Zimmerman GA, Mclntyre TM. Endothelial cell confluence regulates cyclooxygenase-2 and prostaglandin E2 production that modulate motility. J Biol Chem 2004;279:55905–13. [29] Jones MK, Wang H, Peskar BM, Levin E, Itani RM, Sarfeh IJ, et al. Inhibition of angiogenesis by nonsteroidal anti-inflammatory drugs: insight into mechanisms and implications for cancer growth and ulcer healing. Nat Med 1999;5:1418–23. [30] Kamoshita E, Ikeda Y, Fujita M, Amano H, Oikawa A, Suzuki T, et al. Recruitment of a prostaglandin E receptor subtype, EP3 -expressing bone marrow cells is crucial in wound induced angiogenesis. Am J Pathol 2006;169: 1458–72.
Contents lists available at ScienceDirect
Asian Journal of Oral and Maxillofacial Surgery journal homepage: www.elsevier.com/locate/ajoms
Original Research
Improvement in wound healing by a novel synthetic collagen-gel dressing in genetically diabetic mice Daichi Chikazu a,∗ , Tetsushi Taguchi b , Hiroyuki Koyama c , Hisako Hikiji a , Hisako Fujihara a , Hideyuki Suenaga a , Hideto Saijo a , Yoshiyuki Mori a , Ichiro Seto a , Mitsuyoshi Iino a , Tsuyoshi Takato a a
Department of Oral and Maxillofacial Surgery, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan c Department of Vascular Regeneration, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan b
a r t i c l e
i n f o
Article history: Received 23 May 2009 Accepted 13 January 2010 Available online 24 July 2010 Keywords: Wound healing Collagen-gel dressing Diabetic mice
a b s t r a c t Our study aimed to develop an intelligent wound dressing containing elements that promote wound healing. Unlike conventional dressings, this is a gel-type dressing that contains basic factors required by cells in a complex of alkaline-treated collagen and atelocollagen. Full-thickness 8-mm skin defects were made with a DermaPunch® in the left and right sides of the back of 8-week-old genetically diabetic mice (db/db). Collagen gel was applied to the fascia on the right side, while the left (control) side was left untreated. Both wounds was coated and fixed with Tegaderm® . On days 3, 7 and 14, macroscopic appearance and percent decrease in wound area were assessed histopathologically. On the collagen-gel side, granulation tissue had formed, with wound contraction caused by migration of epidermal cells from the surrounding tissue. By day 14, wound area had decreased to 37% for the untreated side as compared to 22% on the collagen-gel side, indicating that collagen gel was associated with significantly greater wound contraction. Moreover, there were a lot of blood vessels on the collagen-gel side. Collagen-gel dressing is most effective for dry, open wounds, i.e., those with minimal effusion, and it may also be effective for depressed open wounds. Collagen-gel dressing is expected to be very useful in treating wounds such as skin defects and skin ulcers. © 2010 Asian Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
1. Introduction In the past, it was erroneously believed that wounds heal best when in a dry environment, but at present, it is accepted that optimal wound healing occurs with a proper level of moisture. Moist wound healing is a technique that maintains a moist environment, and materials that cover wounds to create an environment suitable for wound healing are called dressings. Currently, a burn blister is considered one of the ideal environments for wound healing because a moist environment is maintained at the wound surface, and as long as the blister does not break, the wound is sealed from the outside environment to block out bacteria and foreign materials. Moreover, the liquid inside the blister contains white blood cells, macrophages, and various growth factors. In 1943, Cope proposed a treatment that preserved the blister membrane as a biological wound covering [1]. In 1957, Gimbel and colleagues studied burn blisters and reported that epithelialization was about 40% faster when blisters were left intact when
∗ Corresponding author. Tel.: +81 3 3815 5411x33714; fax: +81 3 5800 8669. E-mail address: [email protected] (D. Chikazu).
compared to suctioning or removing the liquid [2]. Winter conducted animal studies to compare moist and dry environments in 1962 [3], and proved that when a film was placed over porcine skin defects to trap exudate, wounds healed twice as fast when compared to dry wound healing. In 1963, Hinman and colleagues examined human skin wounds and showed that wounds healed faster when a moist environment was maintained [4]. Many subsequent studies have shown that a moist environment facilitates wound healing. As compared with conventional dressings such as gauze, dressings used to promote moist wound healing are often referred to as modern dressings. Film dressing materials (Opsite® ; Smith & Nephew plc, London, UK) were marketed as modern dressings in 1971. A transparent polyurethane film is coated with adhesive on one side, allowing the passage of carbon dioxide and oxygen, but not water. A moist environment can therefore be maintained by wound closure. Hydrocolloid dressing (DuoDerm® ; ConvaTec, New York, NY, USA) was launched in 1983. This dressing adheres to the skin when it contacts the moist wound surface, covers the wound, and absorbs exudate. It turns into a gel and maintains a moist environment. Subsequently, many modern dressings have been developed, manufactured, and marketed.
0915-6992/$ – see front matter © 2010 Asian Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ajoms.2010.01.001
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In the field of oral surgery, extensive resection of tumors arising in the oral cavity, including the tongue, requires primary suture closure. Wound constriction and cicatricial contracture can severely restrict movement of the tongue, and if they occur at the mucoperiosteal defects after palatoplasty, these complications are an important cause of impaired future growth of the jaws [5]. To prevent wound constriction and cicatrical contracture, atelocollagen sponge (Terudermis® , Olympus Terumo Biomaterials Corp., Tokyo, Japan; Pelnac® , Gunze Medical Materials Center, Kyoto, Japan), is often used. When atelocollagen sponge is applied to the wound surface, fibroblasts and capillaries from the wound surface infiltrate and proliferate into the pores of the sponge [6,7]. As new collagen fibers are produced by proliferating fibroblasts, the collagen sponge is degraded and absorbed, turning into regenerated skin-like tissue. However, this formation of skin-like tissue requires 2–3 weeks. This prolonged treatment period is a disadvantage because almost defects usually become uneven and irregularly shaped. Hence, close contact between the collagen sponge and the wound surface is frequently difficult to achieve. The aim of the present study was to develop a viscous gel-type wound dressing that can maintain close contact with the surface of uneven, irregularly shaped wounds and serve as a framework for the infiltration and proliferation of fibroblasts and capillaries from the wound surface. The wound dressing should also be promptly absorbed and replaced by healthy tissue after cell infiltration. Another aim was to study the effectiveness of the wound dressing in vivo.
Fig. 1. The new collagen-gel dressing with collagen crosslinked with citric acid derivative (CAD).
of 5, has carboxyl groups generated by the hydrolysis of residual amide groups in its asparagine and glutamine components. Citric acid (CA), N-hydroxysuccinimide (HOSu), tetrahydrofuran (THF), 2,4,6-trinitrobenzenesulfonic acid (TNBS), disodium hydrogen phosphate, sodium hydrogen phosphate, toluidine blueO, dimethyl sulfoxide (DMSO), ethanol, tert-butyl alcohol, HCl, acetic acid, 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 10% formalin solution, and NaOH were purchased from Wako Pure Chemical Industrials Ltd. (Osaka, Japan). Dicyclohexylcarbodiimide (DCC) was purchased from Kokusan Chemical Co. Ltd. (Tokyo, Japan). Other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
2. Materials and methods
2.2. Preparation of CAD
2.1. Materials
Citric acid derivative (CAD) was prepared by methods previously reported [8–10]. Briefly, CA was first dissolved in THF, and then HOSu and DCC were added. The resulting mixture was stirred for 30 min and then concentrated with rotary evaporation under a
AlCol and AtCol derived from pig’s tissue were provided by Nitta Gelatin Inc. (Osaka, Japan). AlCol, which has an isoelectric point
Fig. 2. Macroscopic appearance of the wound healing of skin defects. The collagen-gel dressing was applied on the right side, and the untreated side was on the left. Tegaderm® was placed over both wounds. The wound area on the right side decreased with time.
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two glass plates for 24 h at 37 ◦ C. The AlCol-CAD was subsequently immersed in excess pure water for 48 h at 37 ◦ C to remove DMSO from the AlCol-CAD matrices. AtCol gel was prepared by mixing 0.3% (w/v) AtCol, 10 M phosphate-buffered saline (PBS) (pH 7.4), and NaOH-HEPES buffer solution. 2.4. Animals
Fig. 3. Percent decrease in the wound area due to the collagen-gel dressing. Taking the wound area on day 0 as 100%, and the decrease in the wound area in each group was calculated relative to this value. The wound area decreased significantly on the collagen-gel dressing side. Data are mean ± S.E.M. (n = 6). # Significant difference from day 0 control group, p < 0.01. * Significant effect of collagen-gel dressing, p < 0.01.
Genetically diabetic male mice (BKS.Cg− + Leprdb /+Leprdb /Jcl, db/db) were purchased from CLEA Japan Inc. (Tokyo, Japan) at the age of 8 weeks. The mice were housed at room temperature (24–25 ◦ C) and a relative humidity of 55%, with a circadian light rhythm of 12 h. Food consisted of commercial standard diet pellets (MF; Sankyo Corp, Tokyo, Japan; calcium content, 1.1%; phosphorus content, 0.83%). Water was available ad libitum. All animal experiments were performed according to the guidelines of the International Association for the Study of Pain [11]. In addition, the experimental protocol was reviewed and approved by the Animal Care Committee of the University of Tokyo. 2.5. Wounding
reduced pressure to remove the THF. The final mixture was recrystallized to yield pure CAD. 2.3. Preparation of AlCol-CAD and AtCol gel AlCol was first dissolved in DMSO, and then CAD solution was added to AlCol solution. This AlCol mixture was then stirred and put into a mold with a 1-mm thick silicone rubber spacer between
The mice were anesthetized with ether, and the back was shaved with an electric shaver on the day before wound surgery. On the day of wound surgery, a collagen-gel dressing was prepared. AlColCAD (1%, w/v) and AtCol (2%, w/v), which were preserved at 4 ◦ C, were dissolved in a water bath heated to 37 ◦ C. Combining these ingredients in a 1:1 (v/v) ratio results in the formation of a CAD crosslinked, paste-like, adhesive collagen-gel (Fig. 1). Next, the skin was sterilized with 70% ethanol. A full-thickness wound was made
Fig. 4. Histopathological examination on day 3 (hematoxylin and eosin staining). Inflammatory-cell infiltration was evident at the wound margins on the collagen-gel dressing side. (A) and (C) show the untreated side. (B) and (D) show the collagen-gel side. Magnifications of (A) and (B) are 20×. (C) and (D) are at greater magnification (100×). ES: excised site.
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Fig. 5. Histopathological examination on day 7 (hematoxylin and eosin staining). Extensive infiltration of fibroblasts, extracellular-matrix formation, and angiogenesis were evident. (A) and (C) show the untreated side. (B) and (D) show the collagen-gel side. Magnifications of (A) and (B) are 20×. (C) and (D) are at greater magnification (100×). ES: excised site.
by excising both the skin and panniculus carnosus, with the use of a 8-mm diameter template. The collagen-gel dressing was applied on the right wound surface, and a transparent occlusive dressing (Tegaderm® ; 3M Health Care, St. Paul, MN, USA) was placed over both wounds. Hence, the left wound surface without the collagengel dressing acted as the control side. Six mice in each group were killed under general anesthesia 0, 3, 7 and 14 days after wounding.
with T-PBS and PBS, the peroxidase-labeled streptoavidin regent was dropped on the sections and was incubated at room temperature for 5 min. A solution of DAB was applied after the third washing procedure with T-PBS and PBS, followed by washing with 50 mM Tris–HCl, pH 7.0. The sections were washed with water after the 3-min reaction.
2.6. Evaluation of wound healing
Means of groups were compared by analysis of variance (ANOVA). The statistical significance of differences was determined by post hoc testing with the Bonferroni method.
Decrease in wound area at the skin defects was assessed by taking images with a digital camera at the same magnification. The wound area was determined with the use of NIH Image 1.62 (National Institutes of Health, Bethesda, MD, USA). Taking the wound area on day 0 as 100%, decreases in wound area on the collagen-gel side (right) and on the untreated side (left) were calculated on days 3, 7, and 14. In addition, the skin including the wound site was excised for histological evaluation. The skin was fixed in 10% buffered formalin solution. The tissues were embedded in paraffin and sectioned in 4-m thick slices. The sections were stained with hematoxylin and eosin. On the other hand, the sections were washed with flowing water and equilibrated with 0.01 M PBS (pH 7.2) for immunostaining using a Histofine SABPO(R) kit. The sections ware blocked for 10 min by 1–2 drops of blocking reagent before a 1 h treatment with monoclonal antimouse ␣-smooth muscle actin (␣-SMA) antibodies at 37 ◦ C, and were then washed 3 times with 0.05% Tween 20/PBS (T-PBS) and PBS, respectively, and were reacted with the second antibody for 10 min at room temperature. After the same washing procedure
2.7. Statistical analysis
3. Results 3.1. Effect of the collagen-gel dressing on decrease in wound area Wound area on the untreated side did not decrease significantly during the first 7 days after surgery but had decreased to 37% by day 14. In contrast, wound area on the collagen-gel dressing side gradually decreased from 3 days after surgery and had decreased significantly to 62% by day 7 and to 22% by day 14. Decrease in wound area on day 14 was significantly greater on the collagen-gel dressing side than on the untreated side (Figs. 2 and 3). 3.2. Effect of the collagen-gel dressing on histologic findings By day 3, infiltration of neutrophils and other types of inflammatory cells were noted at the wound margins on the collagen-gel side. By day 7, there was marked evidence of extensive infiltration
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Fig. 6. Histopathological examination on day 14 (hematoxylin and eosin staining). The numbers of fibroblasts and capillaries and the amount of undulating collagen had increased. The structure was similar to that of normal skin. (A) and (C) show the untreated side. (B) and (D) show the collagen-gel side. Magnifications of (A) and (B) are 20×. (C) and (D) are at greater magnification (100×). ES: excised site.
of fibroblasts, extracellular-matrix formation, and angiogenesis. These findings were scant on the untreated side. By day 14, the numbers of fibroblasts and capillaries and the amount of undulating collagen had increased on the collagen-gel side. The structure was very similar to that of normal skin. Similar structures were seen on the untreated side; however, undulating collagen was limited. Immunostaining with anti-␣-SMA antibody on day 14 revealed that in the collagen-gel side, more new blood vessels were able to be seen compared with the untreated side (Figs. 4–7).
4. Discussion The effect on wound healing of a novel synthetic collagengel dressing was examined using the full-thickness wound model in genetically diabetic male mice. In genetically diabetic male mice, wound healing is clearly slower than in healthy mice; diabetes delays wound healing because of weak initial inflammatory reactions and the degeneration of collagen in connective tissue due to glycosylation [12]. In full-thickness wounds in genetically diabetic mice, the collagen-gel dressing significantly
Fig. 7. Immunohistochemical localization of ␣-SMA at the wound on day 14. The full-thickness wound sections were immunoreacted with anti-␣-SMA antibody as described in Section 2. In the collagen-gel side, more new blood vessels were able to be seen compared with the untreated side. (A) and (B) show the untreated side and the collagen-gel side, respectively. Magnifications of (A) and (B) are 100×.
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accelerated wound healing when compared to control conditions. Reconstruction of the dermis with artificial skin utilizing collagen has markedly improved since Yannas and Burke used collagen dressings for stage I wounds [13,14] and at present, Terudermis® and Pelnac® are used widely. When artificial skin is applied to a skin defect, fibroblasts and capillaries invade from the wound surface into the sponge gaps. As proliferating fibroblasts form new collagen fibers, the collagen sponge is degraded, absorbed, and replaced with regenerated dermis-like tissue. However, the currently available artificial skins consisting of collagen sponge are not necessarily perfect. The chemical cross-linking and long-term heatdehydrated cross-linking that are designed to maintain sponge structure and collagen stability in the tissue [15] hinder tissue affinity and enhance foreign body recognition [16], resulting in unnecessary inflammatory-cell infiltration and cicatricial contracture [17]. In order to overcome these obstacles, the collagen-gel dressing is made by physical crosslinking between aterocollagen and citricbearing alkali-treated collagen. We previously developed a novel tissue adhesive composed of CAD and collagen [8]. It was clarified that the resulting adhesive possessed both high bonding strength and biocompatibility. Moreover, physico-chemical properties of alkali-treated collagen crosslinked with CAD were evaluated by means of spectrometric method [18]. From these experiments, water-soluble citric-bearing alkali-treated collagen (anionic collagen) was generated when the excess amount of CAD was added to the solution of alkali-treated collagen. Using this citric acid-bearing alkali-treated collagen (anionic polymer), we developed a novel injectable collagnen gel dressing by the combination with aterocollagen (cationic collagen) via electrostatic interaction. As clarified from the present study, the developed collagen-gel dressing quickly induced favorable granulation, and fibroblasts and capillaries are attracted due to the dressing’s superior cell affinity. These excellent results were due to that collagen was crosslinked by just only electrostatic interaction (not covalent crosslinking). Various cytokines are involved in wound healing, and wound healing is accelerated when certain stimuli are applied. Basic fibroblast growth factor (bFGF) induces the growth of not only fibroblasts, but also various endodermal and ectodermal cells such as chondrocytes, osteoblasts, keratinocytes, and endovascular cells, and it potently induces neovascularization and accelerates wound healing via granulation or epithelialization [19–21]. bFGF preparations (e.g., Trafermin, FIBLAST® spray: Kaken Pharmaceutical Co. Ltd., Kyoto, Japan) have been used clinically to treat skin ulcers, and their efficacy has been confirmed. However, since bFGF is a protein, its biological half-life is short and it is quickly degraded in the body to lose its activity. Therefore, local intermittent administration [22] or sustained release [13,24] is required. Tabata et al. prepared artificial skin with sustained bFGF release by impregnating bFGF into gelatin microspheres [25]. With our collagen-gel dressing, the gel has a negative charge and can hold bFGF, which is a basic protein, and with the resulting sustained bFGF release, fibroblast proliferation and higher neovascularization should accelerate dermis-like tissue formation. However, further investigations are needed to confirm this. We also plan to investigate the involvement of cyclooxygenase (COX)-2 in the collagen-gel dressing’s facilitation of wound healing. When COX-2 inhibitor was administered to animals, or when tumors were grafted to COX-2 knockout mice (COX-2−/− ), intratumoral neovascularization was more haphazard when compared to the control. Prostaglandin (PG) E2 produced by COX-2 reacts with EP2 and EP3 receptors to induce the production of bFGF and vascular endothelial growth factor (VEGF) [26,27]. Moreover, PGE2 increases the migratory capacity of vascular endothelial cells [28], activates MAP, and accelerates vascular structure formation
[29]. Another study found that COX-2-selective non-steroidal antiinflammatory drugs (NSAIDs) delay wound healing [30], but moist wound healing was not assessed. Therefore, if COX-2 is shown to play a significant role in neovascularization in dermis-like tissue using the collagen-gel dressing, then this is new information that sheds new light on wound healing. In summary, the present study has shown that a novel synthetic Collagen-gel dressing is most effective for rather dry, open wounds, i.e., wound surfaces with minimal effusion, and may be also effective for depressed open wounds. Collagen-gel dressing is expected to be a very useful material for the treatment of wounds such as skin defects and skin ulcers. Acknowledgments We appreciate the excellent technical assistance of Satomi Ogura. Funding: This study was supported in part by the 2004 Industrial Technology Research Grant Program from the New Energy and Industrial Technology Development Organization (NEDO) of Japan. Conflict of interests: We have no conflict of interests to declare. Ethical approval: The experimental protocol was reviewed and approved by the Animal Care Committee of the University of Tokyo. References [1] Cope O. Treatment of surface burns. Ann Surg 1943;117:885–93. [2] Gimbel NS, Kapetansky DI, Weissman F, Pinkus HKB. A study of epithelization in blistered burns. AMA Arch Surg 1957;74:800–3. [3] Winter GD. Formation of scab and rate of epithelialization of superficial wounds in the skin of the young domestic pig. Nature 1962;193:293–4. [4] Hinman CD, Maibach H. Effect of air exposure and occlusion on experimental human skin wounds. Nature 1963;200:377–8. [5] Friede H, Enemark H, Semb G, Paulin G, Abyholm F, Bolund S, et al. Craniofacial and occlusal characteristic in unilateral cleft lip and palate patients from four Scandinavian centers. Scand J Plast Reconstr Hand Surg 1991;25:269–76. [6] Koide M, Osaki K, Konishi J, Oyamada K, Katakura T, Takahashi A. A new type of biomaterial for artificial skin; dehydrothermally cross-linked composites of fibrillar and denatured collagens. J Biomed Mater Res 1993;27:79–87. [7] Soejima K, Nozaki M, Sasaki K, Takeuchi M, Negishi N. Reconstruction of burn deformity using artificial dermis combined with thin split-skin grafting. Burns 1997;23:501–4. [8] Taguchi T, Saito H, Uchida Y, Sakane M, Kobayashi H, Kataoka K, et al. Bonding of soft tissues using a novel tissue adhesive consisting of a citric acid derivative and collagen. Mater Sci Eng C 2004;24:775–80. [9] Saito H, Taguchi T, Aoki H, Murabayashi S, Mitamura Y, Tanaka J, et al. pH-responsive swelling behavior of collagen gels prepared by novel crosslinkers based on naturally derived di- or tricarboxylic acids. Acta Biomater 2007;3:89–94. [10] Saito H, Taguchi T, Kobayashi H, Kataoka K, Tanaka J, Murabayashi S, et al. Physicochemical properties of gelatin gels prepared using citric acid derivative. Mater Sci Eng C 2004;24:781–5. [11] Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983;16:109–10. [12] Delbridge L, Ctertcteko GH, Fowler C, Reeve TS, Le Quesne LP. The aetiology of diabetic neuropathic ulceration of the foot. Br J Surg 1985;72:1–6. [13] Yannas IV, Burke JF. Design of an artificial skin. I. Basic design principles. J Biomed Mater Res 1980;14:65–81. [14] Burke JF, Yannas IV, Quinby Jr WC, Bondoc CC, Jung WK. Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury. Ann Surg 1981;194:413–28. [15] Stenzel KH, Miyata T, Rubin AL. Collagen as a biomaterial. Annu Rev Biophys Bioeng 1974;3:231–53. [16] McPherson JM, Ledger PW, Sawamura S, Conti A, Wade S, Reihanian H, et al. The preparation and physicochemical characterization of an injectable form of reconstituted, glutaraldehyde cross-linked, bovine corium collagen. J Biomed Mater Res 1986;20:79–92. [17] Yoshizato K, Taira T, Yamamoto N. Growth inhibition of human fibroblasts by reconstituted collagen fibrils. Biomed Res 1985;6:61–71. [18] Saito H, Murabayashi S, Mitamura Y, Taguchi T. Unusual cell adhesion and antithrombogenic behavior of citric acid-cross-linked collagen matrices. Biomacromolecules 2007;8:1992–8. [19] McGee GS, Davidson JM, Buckley A, Sommer A, Woodward SC, Aquino AM, et al. Recombinant basic fibroblast growth factor accelerates wound healing. J Surg Res 1988;45:145–53. [20] Rifkin DB, Moscatelli D. Recent developments in the cell biology of basic fibroblast growth factor. J Cell Biol 1989;109:1–6.
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