Clinical evaluation of allogeneic cultured dermal substitutes for intractable skin ulcers after tumor resection

ARTICLE

Auteur(s) : Yoichi MOROI¹, Shohei FUJITA¹, Shuji FUKAGAWA¹, Toshihiko MASHINO¹, Takako GOTO¹, Teiichi MASUDA¹, Kazunori URABE¹, Kentaro KUBO², Hiromichi MATSUI², Shizuko KAGAWA², Yoshimitsu KUROYANAGI², Masutaka FURUE¹

¹ Department of Dermatology, Graduate School of Medical Science, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan
² R&D Center for Artificial Skin, School of Allied Health Sciences, Kitasato University, Kitasato 1-15-1, Sagamihara, Kanagawa 228-8555, Japan

Article accepted on 15/3/2004

Avariety of skin substitutes have been developed and some of them are commercially available in the United States. A representative engineered product is autologous cultured epidermal substitute (CES), which is composed of stratified keratinocytes (Epicel^®; Genzyme Tissue Repair, Cambridge, MA, USA) [1-4]. Two types of allogeneic cultured dermal substitute (CDS) (Dermagraft^® and Trans Cyte^®; Advanced Tissue Sciences, La Jolla, CA, USA) are composed of fibroblasts on a scaffold [5-10]. Another skin substitute is allogeneic cultured skin substitute (CSS) (Apligraf^®; Organogenesis, Canton, MA, USA), which is composed of keratinocytes and fibroblasts on a scaffold [11-13]. Allogeneic CDS and CSS require an appropriate matrix, i.e., a scaffold for fibroblasts and/or keratinocytes. Recently, however, the commercialization of these allogeneic products was discontinued. There seem to be some problems in the design of these products.

Kuroyanagi and colleagues [16-19] developed an allogeneic CDS composed of a spongy collagen containing fibroblasts. The efficacy of this allogeneic CDS on wound healing has been assessed in animal studies and in preliminary clinical trials [20, 21]. Based on this concept, a second version of the CDS was developed through cultivation of fibroblasts on a two-layered spongy matrix of hyaluronic acid (HA) and atelo-collagen (Col). The HA molecule plays a critical role in several functions such as migration and proliferation of various types of cells by promoting adhesion and disadhesion on the tissue substrate [23-26]. The collagen molecules also play a pivotal role in the wound healing process. Collagen and collagen-derived peptides act as chemoattractants for fibroblasts in vitro and may have a similar activity in vivo [27]. 
A multi-center clinical study on the use of allogeneic CDS was performed in 30 hospitals across Japan as the Regenerating Medical Millennium Project of the Ministry of Health, Labor and Welfare of Japan. The Department of Dermatology of Kyushu University Hospital is a member of this project. This clinical study was designed to evaluate the usefulness and efficacy of allogeneic CDS in the treatment of full-thickness skin defects after the resection of skin tumors.

Materials and methods

Preparation of spongy matrix composed of hyaluronic acid (HA) and atelo-collagen (Col)

Spongy matrix was prepared by the method described previously [20, 21]. An aqueous solution of HA with cross-linking agent was poured into a polystyrene dish (11 cm × 10 cm), in which a sheet of hydrated, cellulose, non-woven fabric had been attached at the bottom. The HA solution in the dish was frozen and then lyophilized to obtain the HA sponge. After the sponge was thoroughly rinsed with distilled water to remove free cross-linking agent, the hydrated HA sponge was frozen and lyophilized to obtain the purified HA sponge. The purified HA sponge was punched mechanically to produce many holes. The Col solution was poured into a polystyrene dish (11 cm × 10 cm). The HA sponge with many holes was carefully immersed into the dish containing Col solution, keeping the sheet of non-woven fabric on the HA sponge facing upward. The hydrated HA sponge was frozen and lyophilized to obtain a two-layered sponge of HA and Col. Both surfaces of the two-layered sponge were irradiated with an ultraviolet lamp to induce intermolecular cross-linking of Col molecules.

Establishment of cell banking

Cell banking was established by the procedure described previously [21, 22]. A small piece of skin was donated from a 3-month-old patient during the surgical excision of excrescence. The patient was free from infectious viruses such as HBV, HCV, HIV, and HTLV, and also negative on the treponema pallidum hemagglutination test (TPHA), in compliance with the ethical guidelines of St. Marianna Medical College (Kawasaki, Kanangawa, Japan). Fibroblasts were isolated by enzymatic treatment. The successive cultivation of fibroblasts was initiated in culture medium to establish cell banking. The cells were tested for viruses including HBV, HCV, HIV, HTLV, and Parvovirus, and were found to be negative.

Preparation of cultured dermal substitute (CDS)

The allogeneic CDS was prepared by the method described previously [21, 22]. Prior to seeding fibroblasts on the two-layered sponge of HA and Col, 50 ml of culture medium was poured into a polystyrene dish (11 cm × 10 cm), and the two-layered sponge (10.5 cm × 9.5 cm) was immersed in the culture medium to hydrate and neutralize the acidic two-layered sponge. Excess culture medium was carefully removed from each dish by suction. The fibroblasts that had been obtained in successive cultivations from the cryopreserved cells were seeded on the two-layered sponge. Five ml of the cellular suspension was added dropwise on the collagen surface of the two-layered sponge. The number of fibroblasts on the two-layered sponge was adjusted to 1.0 × 10⁵ cells/cm². This sponge was placed in an incubator overnight, and then 50 ml of culture medium was added, followed by culturing for 1 week. The fibroblasts used in each CDS that was produced, were previously tested for mycoplasma and confirmed to be negative. The culture medium used in each CDS that was produced, was previously tested for bacteria and confirmed to be negative.

Cryopreserving and thawing procedures

Cryopreservation of CDS was performed according to the method described previously [21, 22]. The CDS was frozen in DMEM supplemented with 10% DMSO and 20% FBS from 4 °C to – 60 °C at a rate of – 1 °C/min, and then cryopreserved in a freezer at – 15 °C. The cryopreserved CDS was placed in a foam polystyrene box containing dry ice, shipped to hospitals, and then preserved at – 85 °C. Prior to clinical application, the polystyrene dish containing CDS was placed in a foam polystyrene box at room temperature for 30 min and then floated in a water bath at 37 °C; then, the CDS was rinsed with lactated Ringer’s solution to remove DMSO and FBS.

Clinical study

A clinical study on the use of allogeneic CDS was conducted in accordance with the study protocol and the ethical guidelines of Kitasato University Hospital and Kyushu University Hospital. Twelve patients aged 27-87 years (mean age, 57.5 years) with skin defects after the resection of skin tumors were included in this study. Informed consent for CDS treatment was obtained from all patients. Table I shows the background of the 12 patients. Application of allogeneic CDS was performed for the following conditions, (1) postoperative ulcer (n = 9), (2) covering on a mesh graft (n = 2), (3) other (n = 1; ulcer on orbital born that developed after total resection of orbital tissue and split-thickness skin graft). The wound surface was rinsed with saline solution. The allogeneic CDS that had been rinsed with lactated Ringer’s solution after thawing, was placed cell-seeded side down on the wound surface, and conventional ointment-gauze dressing was used to protect the CDS. A new CDS was applied every 3 to 5 days. Clinical evaluation was performed according to the study protocol. Epithelization and granulation tissue formation were graded according to the following scale: excellent (+ + +), good (+ +), fair (+), poor (–). The size of the ulcers was presented as long diameter x short diameter (cm). In all cases with postoperative ulcer (type 1), skin graft was performed on the resultant healthy granulation tissue.

Results

The clinical results of application of allogeneic CDS in patients with skin ulcers are shown in Table II. In 11 of the 12 cases, healthy granulation tissue developed rapidly. Full-thickness skin defects, especially those in which tendon and/or bone is exposed after surgical resection of a malignant tumor, are not suitable for immediate free skin graft. In cases of delayed surgery, it takes a long time for granulation tissue to form before secondary skin grafting can be performed. The application of CDS, however, resulted in rapid granulation tissue formation acceptable for skin graft in 10 cases (Cases 1, 4, 5, 7-12). The mesh skin graft is very useful for unhealthy and/or non-flat graft beds. Although a highly expanded mesh graft shows good adaptation, completion of epithelization takes a long time. In Cases 2 and 6, rapid epithelization was observed after CDS was applied on the mesh skin graft that had been placed over a large postoperative ulcer. Case 3 was an unusual case in which the patient had undergone total orbital tissue resection followed by split-thickness skin graft for a malignant melanoma on her conjunctiva. A dry ulcer with exposed bone was found on the orbital cavity and was subjected to CDS treatment. Although this dry ulcer was small, its size did not change even after applying CDS ten times. Treatment with CDS was discontinued. This case suggests that for a bone-exposing ulcer surrounding a thin skin graft CDS application may not induce the production of a sufficient number of endogenous fibroblasts to form granulation tissues.

Application of CDS was found to be useful in the majority of cases. Allergic reactions to the CDS treatments such as erythema, itchiness around the ulcer or anaphylactic shock, were not observed in any patient. The healing process in two representative cases who underwent allogeneic CDS is described.
Case 1 was a 68-year-old man with eccrine poroma on the dorsum of his foot (Fig. 1a). Preoperative biopsy suggested malignant transformation, and surgical resection of the tumor left a full- thickness skin defect of 9.3 cm × 7.8 cm in size, with exposed tendons. Allogeneic CDS was applied to the skin defect. Granulation tissue formed rapidly (Fig. 1b-1e). Skin grafting could be performed as soon as 10 days after tumor resection, and graft adaptation was excellent (Fig. 1f). Imunohistochemical analysis of the entire excised tumor led us to make the diagnosis of benign eccrine poroma..
Case 7 was a 50-year-old woman with a moderately differentiated squamous cell carcinoma on the dorsum of her foot that had arisen on a burn scar (Fig. 2a). Surgical resection of the tumor left a full-thickness skin defect of 14.0 cm × 8.0 cm in size, with exposed tendons (Fig. 2b). CDS was then applied to the skin defect (Fig. 2c). Granulation tissue rapidly developed (Fig. 2d-2g). Seventeen days after beginning CDS application, a skin graft was successfully performed and graft adaptation was complete (Fig. 2f). Observation for 1.5 years revealed no local recurrence of malignant tumor and no contracture of the foot joint after skin the graft.

Discussion

Autologous CES which is composed of stratified keratinocytes, is able to permanently take on the patient’s own skin defect and to form the epidermis on the resulting neodermis. On the other hand, allogeneic CDS which is composed of fibroblasts and a material as a scaffold, fails to permanently take on a patient’s skin defect. However, the cells in the CDS are able to produce a variety of biologically active substances including cell growth factor and extracellular matrix, which are necessary for wound healing. The efficacy of allogeneic CDS depends on the function of the cells and the function of the material that is used as a scaffold. Therefore, it is very important to use materials that have the ability to promote wound healing themselves. The two-layered spongy matrix of HA and Col was found to have higher potency for promoting wound healing, compared with a collagen spongy matrix, in a preliminary animal study [20]. When the CDS is applied on the wound surface in clinical use, the spongy structure degrades within about 1 week [20]. Both HA and Col molecules seem to be involved in the process of wound healing. Furthermore, fibroblasts also play multiple roles in the complex process of wound healing. They release a number of biologically active substances including growth factors and angiogenic factors. Neovascularization is essential for wound healing, in particular, for healing of chronic and poor-healing deeper wounds. VEGF plays a critical role in this process [28-30]. CDS releases a substantial amount of VEGF over a cultivation period of 1 week [21, 31]. Incubation with CDS enhanced proliferation of vascular endothelial cells in a dose-dependent fashion, and the addition of anti-human VEGF antibody reduced their proliferative activity [31]. Another important function of fibroblasts is production of extracellular matrix including collagen and fibronectin. Fibronectin serves several critical functions in effective wound healing. In practice, immunohistochemical staining for fibronectin in excised CDS showed substantial deposition of fibronectin on the spongy structure of CDS [21].
To date, CDS has been used in 12 patients at our hospital. Although CDS application was discontinued in one patient, 11 patients had an excellent response to CDS application. Our clinical results suggest that CDS is extremely useful for the treatment of deep wounds such as full-thickness skin defects. Upon application of CDS, all 9 cases with surgical skin defects due to resection of a malignant tumor showed rapid granulation tissue formation acceptable for secondary skin grafting. Granulation tissue formation upon treatment with CDS appeared to occur faster than that upon conventional ointment therapies. In the two cases in which CDS was applied to cover a mesh graft, quick epithelial formation was observed, and CDS seems to contribute to epithelial formation.
There were no adverse effects and no allergic reaction to CDS treatment was noticed in any patient. After application of a CDS, allogeneic fibroblasts in the CDS are gradually rejected depending on the wound condition. However, repeated application of CDS might lead to an allo-specific immune response and the materials that remain in the culture medium of CDS also could induce immune reactions. No patient complained of itchiness, pain nor any other sensation. In addition, we had paid special attention to signs of infection of the wound during the clinical course, because CDS can serve as an infectious basement. Although sufficient debridement should be performed before starting treatment with CDS, some cases showed bacterial colonization on their ulcers before and during the CDS treatment. We continue to use the CDS and no patient has had to discontinue this treatment due to bacterial infection. The resistance of CDS against bacterial infection remains to be elucidated.
The topical basic fibroblast growth factor (b-FGF) product has been used for a variety of skin ulcers in Japan, and its effectiveness was confirmed [32]. However, this product cannot be used for skin defects after tumor resection, because b-FGF could serve as a growth factor for the malignant tumor. However, upon application of CDS on postoperative ulcers after resection of malignant tumor, none of our patients developed local recurrence of tumor during an observation period ranging 1 to 3 years.
This study showed that CDS is useful for the treatment of intractable skin ulcers, and CDS could be a safe and powerful tool for the treatment of postoperative skin ulcers. n

References

1. Rheinwald JG, Green H. Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma. Cell 1975; 6: 317-30.

2. Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975; 6: 331-43.

3. Green H, Kehinde O, Thomas J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci USA 1979; 76: 5665-8.

4. Gallico G, O’Connor NE, Compton CC et al. Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med 1984; 311: 448-51.

5. Cooper ML, Hansbrough JF, Spielvogel RL et al. In vivo optimization of a living dermal substitute employing cultured human fibroblasts on a biodegradable polyglycolic acid or polyglactin mesh. Biomaterials 1991; 12: 243-8.

6. Hansbrough JF, Christine D, Hansbrough WB. Clinical trials of a living dermal tissue replacement placed beneath meshed, split-thickness skin grafts on excised burn wounds. J Burn Care Rehabil 1992; 13: 519-29.

7. Hansbrough JF, Cooper ML, Cohen R et al. Evaluation of a biodegradable matrix containing cultured human fibroblasts as a dermal replacement beneath meshed skin grafts on athymic mice. Surgery 1992; 111: 438-46.

8. Gentzkow GD, Iwasaki SD, Hershon KS et al. Use of dermagraft, a cultured human dermis, to treat diabetic foot ulcers. Diabetes Care 1996; 19: 350-4.

9. Hansbrough JF, Mozingo DW, Kealey GP et al. Clinical trials of a biosynthetic temporary skin replacement, Dermagraft-Transitional Covering, compared with cryopreserved human cadaver skin for temporary coverage of excised burn wounds. J Burn Care Rehabil 1997; 18: 43-51.

10. Hansbrough JF, Morgan J, Greenleaf G. Development of a temporary living skin replacement composed of human neonatal fibroblasts cultured in Biobrane, a synthetic dressing material. Surgery 1994; 115: 633-44.

11. Bell E, Ivarsson B, Merrill C. Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc Natl Acad Sci USA 1979; 211: 1274-8.

12. Bell E, Ehrlich HP, Buttle DJ, Nakatsuji T. Living tissue formed in vitro and accepted as skin-equivalent tissue of full thickness. Science 1981; 211: 1052-4.

13. Bell E, Ehrlich HP, Sher S, Merrill C et al. Development and use of a living skin equivalent. Plast Reconstr Surg 1981; 67: 386-92.

14. Bell E, Sher S, Hull B et al. The reconstitution of living skin. J Invest Dermatol 1983; 81 (Suppl.): S2-10.

15. Naughton G. The Advanced Tissue Sciences story. Sci Am 1999; 280: 84-5.

16. Yamada N, Shioya N, Kuroyanagi Y. Evaluation of an allogeneic cultured dermal substitute composed of fibroblasts within a spongy collagen matrix as a wound dressing. Scand J Plast Reconstr Surg 1995; 29: 211-9.

17. Tanaka M, Nakakita N, Kuroyanagi Y. Allogeneic cultured dermal substitute composed of spongy collagen containing fibroblasts: evaluation in animal test. J Biomater Sci Polymer Edn 1999; 10: 433-53.

18. Yamada N, Uchinuma N, Kuroyanagi Y. Clinical evaluation of an allogeneic cultured dermal substitute composed of fibroblasts within a spongy collagen matrix. Scand J Plast Reconstr Surg 1999; 33: 147-54.

19. Kuroyanagi Y, Yamada N, Yamashita R, Uchinuma E. Tissue-engineered product: allogeneic cultured dermal substitute composed of spongy collagen with fibroblasts. Artificial Organs 2001; 25: 180-6.

20. Kubo K, Kuroyanagi Y. Development of cultured dermal substitute composed of spongy matrix of hyaluronic acid and atelo-collagen combined with fibroblasts: fundamental evaluation. J Biomater Sci Polym Ed 2003; 14: 625-41.

21. Kubo K and Kuroyanagi Y. Characterization of cultured dermal substitute composed of spongy matrix of hyaluronic acid and collagen combined with fibroblasts. J Artif Organs 2003; 6: 138-44.

22. Kuroyana gi Y, Kubo K, Matsui H et al. Establishment of banking system for allogeneic cultured dermal substitute. Artif Organs 2004; 28: 13-21.

23. Culp LA, Murray BA, Rollins BJ. Fibronectin and proteoglycans as determinants of cell-substratum adhesion. J Supramol Struct 1979; 11: 401-27.

24. Lark MW, Laterra J, Culp LA. Close and focal contact adhesions of fibroblasts to a fibronectin-containing matrix. Fed Proc 1985; 44: 394-403.

25. Rollins BJ, Culp LA. Glycosaminoglycans in the substrate adhesion sites of normal and virus-transformed murine cells. Biochemistry 1979; 18: 141-8.

26. Benedetti L, Cortivo R, Berti T et al. Biocompatibility and biodegradation of different hyaluronan derivatives (Hyaff) implanted in rats. Biomater 1993; 14: 1154-60.

27. Postlethwaite AE, Seyer JM, Kang AH. Chemotactic attraction of human fibroblasts to type I, II, and III collagens and collagen-derived peptides. Proc Natl Acad Sci USA 1978; 75: 871-5.

28. Nissen NN, Polverini PJ, Koch AE et al. Vascular endothelial growth factor mediates angiogenic activity during the proliferative phase of wound healing. Am J Pathol 1998; 152: 1445-52.

29. Ferrara N, Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 1989; 161: 851-8.

30. Kumar R, Yoneda J, Fidler IJ. Regulation of distinct steps of angiogenesis by different angiogenic molecules. Int J Oncol 1998; 12: 749-57.

31. Kubo K, Kuroyanagi Y. Effects of vascular endothelial growth factor released from cultured dermal substitute on proliferation of vascular endothelial cells in vitro. J Artif Organs 2003; 6: 267-72.

32. Aragane Y, Okamoto T, Yajima A et al. Hydroxyurea-induced foot ulcer successfully treated with a topical basic fibroblast growth factor product. Br J Dermatol 2003; 148: 599-600.