Emergent study of a newly developedbilayer artificialskin Shako So, YoshitoIkada*
KazuyaMa~u~, ~ob~o
Iss~,
Yap
Tamda* and
Department of Plastic Surgery, Faculty of Medicine, and *Research Center for Medical fWymers and Biomaterials, Uyoto University, Shogoin, Sakyo-ku, Kyoto. 606 Japan (Received 75 May 1989; accepted 26 June 1989)
A bilayer artificial skin composed of an outer layer of silicone polymer and an inner sponge layer of collagen containing chondroitin 6-sulphate was developed by modifying the technique proposed by Yannas et al. The artificial skin was placed on to skin defects on the backs of rats. Histological observation indicated that fibroblasts and capillaries infiltrated into the pores and filled in lattice spaces, resulting in synthesis of the connective tissue matrix and absorption of the original network of collagen and chondroitin 6-sulphate. Epidermal cells migrated from the edge of the wound between the two layers. Post-operative cont~ctu~ in the wound with the artificial skin was significantly less than in the control. Keywords: Collagen, wound dressings, chondroitin 6-suiphate
Though a number of skin equivalents’-5 have been developed for coverage of a skin defect, resulting for instance from burn or trauma, most of them served only as a temporary skin substitute. A new biiayer artificial skin (stage 1 membrane), developed by Yannas et al.6-‘2, which is composed of an inner sponge layer of collagen and chondroitin 6-sulphate (a glycosaminoglycan, GAG), and an outer layer of silicone polymer, seems better than other skin equivalents. According to their reports, the stage 1 membrane, when placed on the wound, was infiltrated into the pores of the inner layer by the cellular tufts of fibroblasts and capillaries. These cells, which infiltrated the membrane pores, gradually produced synthesized connective tissue matrix similar to the true dermis, as the original network of collagen and GAG became biodegraded. Most clinical cases, they report, required thin split-thickness skin graft secondarily but the post-operative contracture was minimal because dermis-like tissues were reconstructed underneath. Permeability of water vapour through the outer layer of silicone polymer was fairly comparable to normal skin. This investigation set out to improve the artificial skin by modifying their technique.
MATERlAlS Preparation
AND
METHODS
of an artificial
skin
Soluble atelocollagen was used as the main material, to decrease antigenicity. Hydrochloric acid solution of pH 3.0 Correspondence to Dr S. Suzuki.
containing 3% atelocollagen (Nitta Gelatine Co.) was stirred with a refrigerated homogenizer at 1800-2000 rev/min for 60 min. An aqueous chondroitin 6-sulphate solution was added dropwise to the stirred collagen solution, until the weight ratio of ~hondro~tin 6-sulphate to collagen reached 8%. Following further stirring of the mixed solution for 5 min, the foaming solution was poured into a mould and quickly frozen. After that, the frozen content was directly freeze-dried in the mould for 48 h, to yield a highly porous sponge sheet. The thickness of the sheet was adjusted to 2 mm. The sponge sheet was then exposed to a vacuum oven at 105°C for 24 h so as to be cross-linked and sterilized. Silicone (Medical Adhesive Silicone Type A) was coated over thesheettothicknessof 25pm.Afterthesiliconewasdried, the bilayer sheet was further cross-linked by immersing in 0.05 M acetic acid solution containing 0.2 wt% of glutaraldehyde at 4°C for 24 h. The bilayer sheet was rinsed in phosphate buffer solution to remove any glutaraldehyde. The final concentration of glutaraldehyde in the sheet was only 0.576 p.p,m. The prepared bilayer artificial skin was stored in 70 ~01% ethanol solution at 4°C until use. When used, it was rinsed and washed in saline solution. Our modifications in preparing the artificial skin are summarized in Table 1.
Structure
Biomaterials
1990, Vol 11 July
skin
Observation of the inner sponge layer with a scanning electron microscope revealed that the pores had a size of 50100pm diameter and a pore volume fraction of 94-98% (Figure 7). These values were scarcely different from those 1990
356
of the artificial
Butterworth-Heinemann
Ltd. 0142-961
Z/90/050356-05
New btlaver artificial skm: S. Suzuki et al
Table 1 Difference in preparation methods for our artificial skin and the stage 1 membrane of Yannas et al. Artificial skin
Stage 1 membrane
Collagen materials
Soluble prg skin collagen insoluble bovine hide collagen
Production of sponge structure
Mixed solution of collagen and glycosaminoglycan is directly frozen and freeze-dried
of stage 1 membrane. silicone layer was 2.3 that of normal skin, Lamke et&l3 (Table
Table 2 Difference in structures membrane of Yannas et al.
Collagen and glycosamin~lycan are coprecipitated and the precipitate is freeze-dried
The moisture permeability of the outer mg cm-* hh’, which approximates to 1.2-2.0 mg cm-’ h-’ as reported by 2).
Inner layer Pore srze rn diameter Pore volume fraction Ourei layer Thickness Moisture permeability
of our artificial skin and the stage 1
Artifioal skrn
Stage 1 membrane
50-IOOflm
50 t 20pm
94-98%
96%
25qI
50-100pm
2.3 mg cm-’
h
’
l-lOmgcm~~Zh~’
The paired wounds were both unbandaged (in Bollman cages for 2 wk and in the ordinary cage for 8 wk). Ten weeks post-operatively, the re-epithelization area was measured for both wounds.
Experiment 1 Thirty Wistar rats each weighing 350-400g were used after shaving the hairs of their backs with a depilatory cream. Full-thickness skin defects, measuring 15 X 15 mm, were made on their backs, preserving the panniculus carnosus. The artificial skin was placed on to the skin defects and the wounds were unbandaged. Thereafter, the animals were fixed in the Bollman cage for 2 wk and then housed singly. Under general anaesthesia, the covered wounds were observed every 3-7 d for IO wk and the tissues that were not infected macroscopically were biopsied for histological examination.
Expe~ment 2 Twelve rats were used to make paired skin defects, measuring IO X 20 mm, on their backs (figure 2a). The artificial skin was placed on to one of the skin defects (figure 26) and sterile Vaseline was coated on to the other one. The artificial skin was placed on to both the cranial and caudal defects in equal numberto standardize the conditions.
Figure 1 Scanning electron microscopic view of the inner sponge fayet frnag~j~~at~on X 1.20].
Figure 2 (a) Paired skin defects (IO x 20 mm) with the panniculus camosus preserved wera made on the back of a rat. {b) The artificialskin was placed on to one of the skin defects, while sterile Vaseline was coated on to the other as a control. (cj 10 wk post-operative appearance. The re-epiihelized area where the artificial skin was placed is wider than that of the control.
Biomaterials
1990, Vol 1 I July
357
New bilayer artificial skin: S. Suzuki et al.
RESULTS Experiment 1 3 d post-operatively, the inner layer of the artificial skin did not completely adhere to the wound bed. Histological
findings indicated that mononuclear cells infiltrated into the lowest portion of the inner sponge layer (Figure 3a); 6 d post-operatively, cellular tufts consisting of mononuclear cells,fibroblasts and capillaries migrated intothe pores in the lower portion (Figure 3b); 9 d post-operatively, these cellular tufts further expanded and filled the lattice space in the
Figure 3 Histological appearances of the artificial skin grafted on to the skin defects on the backs of rats. (Haematoxylin and eosin staining.) (a) 3 d postoperative view. Mononuclear cells infiltrate the lower portion of the inner sponge layer (original magnification X 50). (b) 6 d post-operative view. Cellular tufts consisting of mononuclear cells, fibroblasts and capillaries migrate into the pores in the lower portion (original magnification X 50). (c) 9 dpost-operative view. Cellular tufts further expend and fill the lattice space in the middle-to-lower portion (original magnification X 50). (d) 12 d post-operative view. Cellular tufts expand into the middle-to-upper portion, and epidermal cells migrate from the edge of the wound towards the centre (original magnification X 25). (e) 17 dpostoperative view. Epithelization is almost completed and the inner layer isconverted into a tissue heavily populated with vessels, fibroblasts and newly synthesized connective tissue matrix, with the original network partially remaining (original magnification X 25). (f) 10 wk post-operative view. The inner layer is converted into a tissue similar to the true dermis (original magnification X 25).
358
Biomaterials
1990, Vol 11 July
New bilaver arttficial skin: S. Suzuki et al.
middle to lower portion (Qure 3~); 12 d post-operatively, migration of epidermal cells from the edge of the wound toward the centre was observed (Figure 3d); and 17 d postoperatively, epitheiization had been almost completed and the inner layer was converted into a tissue heavily populated with vessels, fibroblasts and newly synthesized connective tissue matrix, with the original network of collagen and GAG partially remaining (Figure 3e). In the later stages, the inner layer was converted into a tissue that resembled the true dermis under the maturation of the newly synthesized connective tissue (Figure 3f). Throughout the course, no notable foreign body reaction was recognized.
Experiment 2 10 wk post-operatively, the re-epithelized area was 125.8 f 10.3 mm2 (mean ?I SEM) in the wound where the artificial skin had been placed versus 74.2 rt 7.2 mm’ in the control, which indicated a significant difference (P < 0.01, Student’s t test) (Figures 2c and 4).
DISCUSSION Skin substitutes may be classified into two groups: those developed for covering a split-thickness skin defect or dermal burn, and those for a full-thickness skin defect or deep burn. The skin substitutes that belong to the former group, such as lyophilized porcine skin2s3 and polyurethane sheet4, are used to accelerate epithelization, prevent water loss and relieve pain. Most of them accord with these purposes to a certain extent. Those that belong to the latter group include PVF sponge’, Biobranee, and bilayer artificial skin like ours. The PVF sponge is useful as a temporary skin substitute but infection occasionally occurs during pro-
hm2)
100
longed usage. Although Biobrane, which has an affinity to the host tissue, seems to be a more advanced material than the PVF sponge, both are temporary. Consequently, pain and bleeding are inevitable when peeling. The present investigation indicates that the artificial skin we have made is outstanding in the following: The inner sponge layer of collagen and GAG has an affinity to the tissue and little antigenicity. In particular, the atelocollagen used in our artificial skin may have less antigenicity than the insoluble collagen used in the stage 1 membrane. The inner sponge layer does not need to be peeled off because the original network of collagen and GAG is biodegraded, with infiltration of cellular tufts of fibroblasts and capillaries and synthesis of connective tissue matrix. Epidermal cells migrate at a speed of approximately 0.5 mm per day from the edge of the wound into the plane between the newly synthesized connective tissue and the silicone layer. (A split-thickness skin graft is required secondarily in most clinical uses because the skin defects are too wide for epidermal cells to reach the centre in a few weeks. However, the secondary skin graft to be used clinically can be fairly thin so that killing of donors may be kept to a minimum and skin taken repeatedly at intervals from the same site.) 4. Since the inner layer is spontaneously converted into a new connective tissue like dermis, post-operative contracture is not significant. 5. Water vapour permeability of the outer silicone layer is comparable to that of the normal skin. A three-dimensional structure, including pore diameter and pore volume fraction, seemed to be more critical for infiltration and proliferation of fibroblasts and capillaries than the type of collagen used and the process of preparing the sponge layer. When the artificial skin was placed on to the defect with pressure, cell infiltration and proliferation were inhibited’ 4 (Figure 5). In our preliminan/ study, another artificial skin was prepared using gelatin instead of atelocollagen but cell infiltration and proliferation were poor (Figure 6); it remains unclear whether the failure was due to its three-dimensional structure or its nature. Yannas et a/.*.’ attempted to develop a stage 2 membrane by seeding the stage 1 membrane with epidermal
50
Artificial skin Figure 4 Mean re-epithelized area in the artificial skin and control groups. n = 12; P < 0.01, Student’s t test.
Figore 5 7 d post-operative histological appearance of artdicial skin grafted on to the skin defect under pressure (haematoxylin and eosin staining; original magnification X 25). Sponge structure is crushed and cell infiltration and proliferation are poor compared wth those shown in Figures 3b and c.
Biomaterials
1990, Vol I I Julv
359
New bilayer artificial skin: S. Suzuki et al.
reports of Yannas et al. that glycosaminoglycan incorporated in the collagen sponge contributed to the function of the artificial skin7. The role of the added GAG (chondroitin 6sulphate and others) has been examined to verify their effects and the results will be reported in a later paper17.
REFERENCES 1
2 Figure 6 7 d post-operative histological appearance of artificial skin composed of gelatin sponge (haematoxylin and eosin staining; original magnification X 25). The original network. which seems thicker than that of collagen, almost remains with few cells infiltrating.
3 4
5
cells, which dispenses with a secondary skin graft. However, the advantage of a stage 2 membrane may be questionable at present in terms of cost-performance and function. The material has to be mass-produced at a low cost to be applicable to many patients. Combined use of our artificial skin and cultured epidermis15 appears to be more practical in treating extensive burns. The artificial skin is placed on to the burned wound and simultaneously a piece of epidermis is taken for culture proliferation. While the epidermal cells are being proliferated to make an appropriate sheet of epidermis, a dermis-like tissue would have been prepared as a recipient bed for grafting the cultured epidermis sheet. The modifications we made in producing the artificial skin originally developed by Yannas et al. are as follows. The use of atelocollagen, instead of insoluble collagen, allowed us to minimize antigenicity. Instead of coprecipitation of collagen and GAG, the mixed solution was rapidly frozen and directly freeze-dried. This procedure permits the use of various kinds of GAGS instead of chondroitin 6-sulphate and regulation of the ratio of GAG to collagen. The thickness of the outer silicone layer was adjusted to 25 ,um, thinner than that of the stage 1 membrane but the moisture permeability was approximately equal to that of normal skin. Our artificial skin has already been used clinically. Though a thin split-thickness skin graft is required, postoperative appearance is satisfactory in almost all cases16. Though our artificial skin has several advantages, some problems remain to be solved. Chondroitin 6-sulphate was added to the collagen sponge in this study following the
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Chardack. W.M.. Brueske, D.A., Santomauro, A.P. and Fazekas, G., Experimental studies on synthetic substitutes for skin and their use in the treatment of burns, Ann. Surg., 1962, 155, 127-l 39 Chang, W.H.J.. Gometz, N.H. and Edelstein, L.M., Use of lyophilized pigskinfordonorsitecover,& J. Plast. Surg. 1973.26.147-149 Elliot, HA. Jr. and Hoehm, J.G., Use of commercial porcrne skin for wound dressings, Plast. Reconstr. Surg. 1973, 52, 40 l-405 James, J.H. and Watson, A.C.H., The use of opsite, a vapor permeable dressing on skin graft donor sues, Br. J. Plast. Surg. 1975, 28, 107-l 10 Tab% M.J., Thornton, J.W., Bartlett, R.H. et al., A new composite skin prosthesis, Burns 1980. 7, 123 Yannas. I.V. and Burke, J.F., Design of an arttficral skin. I. Basic design principles, J. Biomed. Mater. Res. 1980, 14, 65-8 1 Yannas. I.V.. Burke. J.F.. Gordon, P.L. et al., Destgn of an artificial skin. II. Control of chemical composition,J. Biomed. Mater. Res. 1980,14, 107-131 Yannas. I.V.. Burke, J.F., Warpehoski, M. et al., Prompt, long-term functional replacement of skin, Trans. Am. Sot. Artif Intern. Organs 1981, 27, 19-23 Yannas. I.V., Burke, J.F.. Orgill, D.P. and Skrabut, E.M., Wound tissue can utrlize a polymeric template to synthesize a functional extension of skin, Science 1982, 215. 174-l 76 Burke. J.F.. Yannas, I.V., Quinby, W.C. et al., Successful use of physiologically acceptable artrficral skin in the treatment of extensive burn injury, Ann. Surg., 1981, 194, 413-428 Burke, J.F.. Observations on the development of an artificial skin: presidential address, 1982 American Burn Association Meeting, J. Trauma 1983,23, 543-551 Dagalakis. N., Flink, J., Stasikelis, P. et al., Desrgn of an artificial skin. III. Control of pore structure, J. Biomed. Mater. Res. 1980, 14, 51 l-528 Lamke, L.O. and Ltljedahl, SO., Evaporatrve water loss from burns, grafts and donor sites,Scand. J. Plast. Reconstr. Surg. 1971,5,17-22 Suzuki. S.. Matsuda, K., Isshiki, N., Tamada, Y. and Ikada, Y., An artificial skin composed of an outer layer of silicone and an inner layer of collagen and GAG, Jap. J. Plast. Reconstr. Surg. 1988, 31, 298-305 O’Connor, N.E.. Mulliken. J.B., Banks-Schlegel, S. et al., Grafting of burns wrth cultured epitheltum prepared from autologous eprdermal cells, Lancet 1981, i, 75-78 Suzuki, S., Matsuda, K., Isshiki, N. et al., Clinical evaluation of a new bilayer ‘artificral skin’composed of collagen sponge and sillcone layer, Br. J. Plast. Surg. 1990. 43, 47-54 Matsuda, K., Suzukr, S., Isshikr, N., et al., Influence of glycosamrnoglycans on the collagen sponge component of a brlayer artificial skin, Biomaterials 1990, 11, 35 l-355
KazuyaMa~u~, ~ob~o
Iss~,
Yap
Tamda* and
Department of Plastic Surgery, Faculty of Medicine, and *Research Center for Medical fWymers and Biomaterials, Uyoto University, Shogoin, Sakyo-ku, Kyoto. 606 Japan (Received 75 May 1989; accepted 26 June 1989)
A bilayer artificial skin composed of an outer layer of silicone polymer and an inner sponge layer of collagen containing chondroitin 6-sulphate was developed by modifying the technique proposed by Yannas et al. The artificial skin was placed on to skin defects on the backs of rats. Histological observation indicated that fibroblasts and capillaries infiltrated into the pores and filled in lattice spaces, resulting in synthesis of the connective tissue matrix and absorption of the original network of collagen and chondroitin 6-sulphate. Epidermal cells migrated from the edge of the wound between the two layers. Post-operative cont~ctu~ in the wound with the artificial skin was significantly less than in the control. Keywords: Collagen, wound dressings, chondroitin 6-suiphate
Though a number of skin equivalents’-5 have been developed for coverage of a skin defect, resulting for instance from burn or trauma, most of them served only as a temporary skin substitute. A new biiayer artificial skin (stage 1 membrane), developed by Yannas et al.6-‘2, which is composed of an inner sponge layer of collagen and chondroitin 6-sulphate (a glycosaminoglycan, GAG), and an outer layer of silicone polymer, seems better than other skin equivalents. According to their reports, the stage 1 membrane, when placed on the wound, was infiltrated into the pores of the inner layer by the cellular tufts of fibroblasts and capillaries. These cells, which infiltrated the membrane pores, gradually produced synthesized connective tissue matrix similar to the true dermis, as the original network of collagen and GAG became biodegraded. Most clinical cases, they report, required thin split-thickness skin graft secondarily but the post-operative contracture was minimal because dermis-like tissues were reconstructed underneath. Permeability of water vapour through the outer layer of silicone polymer was fairly comparable to normal skin. This investigation set out to improve the artificial skin by modifying their technique.
MATERlAlS Preparation
AND
METHODS
of an artificial
skin
Soluble atelocollagen was used as the main material, to decrease antigenicity. Hydrochloric acid solution of pH 3.0 Correspondence to Dr S. Suzuki.
containing 3% atelocollagen (Nitta Gelatine Co.) was stirred with a refrigerated homogenizer at 1800-2000 rev/min for 60 min. An aqueous chondroitin 6-sulphate solution was added dropwise to the stirred collagen solution, until the weight ratio of ~hondro~tin 6-sulphate to collagen reached 8%. Following further stirring of the mixed solution for 5 min, the foaming solution was poured into a mould and quickly frozen. After that, the frozen content was directly freeze-dried in the mould for 48 h, to yield a highly porous sponge sheet. The thickness of the sheet was adjusted to 2 mm. The sponge sheet was then exposed to a vacuum oven at 105°C for 24 h so as to be cross-linked and sterilized. Silicone (Medical Adhesive Silicone Type A) was coated over thesheettothicknessof 25pm.Afterthesiliconewasdried, the bilayer sheet was further cross-linked by immersing in 0.05 M acetic acid solution containing 0.2 wt% of glutaraldehyde at 4°C for 24 h. The bilayer sheet was rinsed in phosphate buffer solution to remove any glutaraldehyde. The final concentration of glutaraldehyde in the sheet was only 0.576 p.p,m. The prepared bilayer artificial skin was stored in 70 ~01% ethanol solution at 4°C until use. When used, it was rinsed and washed in saline solution. Our modifications in preparing the artificial skin are summarized in Table 1.
Structure
Biomaterials
1990, Vol 11 July
skin
Observation of the inner sponge layer with a scanning electron microscope revealed that the pores had a size of 50100pm diameter and a pore volume fraction of 94-98% (Figure 7). These values were scarcely different from those 1990
356
of the artificial
Butterworth-Heinemann
Ltd. 0142-961
Z/90/050356-05
New btlaver artificial skm: S. Suzuki et al
Table 1 Difference in preparation methods for our artificial skin and the stage 1 membrane of Yannas et al. Artificial skin
Stage 1 membrane
Collagen materials
Soluble prg skin collagen insoluble bovine hide collagen
Production of sponge structure
Mixed solution of collagen and glycosaminoglycan is directly frozen and freeze-dried
of stage 1 membrane. silicone layer was 2.3 that of normal skin, Lamke et&l3 (Table
Table 2 Difference in structures membrane of Yannas et al.
Collagen and glycosamin~lycan are coprecipitated and the precipitate is freeze-dried
The moisture permeability of the outer mg cm-* hh’, which approximates to 1.2-2.0 mg cm-’ h-’ as reported by 2).
Inner layer Pore srze rn diameter Pore volume fraction Ourei layer Thickness Moisture permeability
of our artificial skin and the stage 1
Artifioal skrn
Stage 1 membrane
50-IOOflm
50 t 20pm
94-98%
96%
25qI
50-100pm
2.3 mg cm-’
h
’
l-lOmgcm~~Zh~’
The paired wounds were both unbandaged (in Bollman cages for 2 wk and in the ordinary cage for 8 wk). Ten weeks post-operatively, the re-epithelization area was measured for both wounds.
Experiment 1 Thirty Wistar rats each weighing 350-400g were used after shaving the hairs of their backs with a depilatory cream. Full-thickness skin defects, measuring 15 X 15 mm, were made on their backs, preserving the panniculus carnosus. The artificial skin was placed on to the skin defects and the wounds were unbandaged. Thereafter, the animals were fixed in the Bollman cage for 2 wk and then housed singly. Under general anaesthesia, the covered wounds were observed every 3-7 d for IO wk and the tissues that were not infected macroscopically were biopsied for histological examination.
Expe~ment 2 Twelve rats were used to make paired skin defects, measuring IO X 20 mm, on their backs (figure 2a). The artificial skin was placed on to one of the skin defects (figure 26) and sterile Vaseline was coated on to the other one. The artificial skin was placed on to both the cranial and caudal defects in equal numberto standardize the conditions.
Figure 1 Scanning electron microscopic view of the inner sponge fayet frnag~j~~at~on X 1.20].
Figure 2 (a) Paired skin defects (IO x 20 mm) with the panniculus camosus preserved wera made on the back of a rat. {b) The artificialskin was placed on to one of the skin defects, while sterile Vaseline was coated on to the other as a control. (cj 10 wk post-operative appearance. The re-epiihelized area where the artificial skin was placed is wider than that of the control.
Biomaterials
1990, Vol 1 I July
357
New bilayer artificial skin: S. Suzuki et al.
RESULTS Experiment 1 3 d post-operatively, the inner layer of the artificial skin did not completely adhere to the wound bed. Histological
findings indicated that mononuclear cells infiltrated into the lowest portion of the inner sponge layer (Figure 3a); 6 d post-operatively, cellular tufts consisting of mononuclear cells,fibroblasts and capillaries migrated intothe pores in the lower portion (Figure 3b); 9 d post-operatively, these cellular tufts further expanded and filled the lattice space in the
Figure 3 Histological appearances of the artificial skin grafted on to the skin defects on the backs of rats. (Haematoxylin and eosin staining.) (a) 3 d postoperative view. Mononuclear cells infiltrate the lower portion of the inner sponge layer (original magnification X 50). (b) 6 d post-operative view. Cellular tufts consisting of mononuclear cells, fibroblasts and capillaries migrate into the pores in the lower portion (original magnification X 50). (c) 9 dpost-operative view. Cellular tufts further expend and fill the lattice space in the middle-to-lower portion (original magnification X 50). (d) 12 d post-operative view. Cellular tufts expand into the middle-to-upper portion, and epidermal cells migrate from the edge of the wound towards the centre (original magnification X 25). (e) 17 dpostoperative view. Epithelization is almost completed and the inner layer isconverted into a tissue heavily populated with vessels, fibroblasts and newly synthesized connective tissue matrix, with the original network partially remaining (original magnification X 25). (f) 10 wk post-operative view. The inner layer is converted into a tissue similar to the true dermis (original magnification X 25).
358
Biomaterials
1990, Vol 11 July
New bilaver arttficial skin: S. Suzuki et al.
middle to lower portion (Qure 3~); 12 d post-operatively, migration of epidermal cells from the edge of the wound toward the centre was observed (Figure 3d); and 17 d postoperatively, epitheiization had been almost completed and the inner layer was converted into a tissue heavily populated with vessels, fibroblasts and newly synthesized connective tissue matrix, with the original network of collagen and GAG partially remaining (Figure 3e). In the later stages, the inner layer was converted into a tissue that resembled the true dermis under the maturation of the newly synthesized connective tissue (Figure 3f). Throughout the course, no notable foreign body reaction was recognized.
Experiment 2 10 wk post-operatively, the re-epithelized area was 125.8 f 10.3 mm2 (mean ?I SEM) in the wound where the artificial skin had been placed versus 74.2 rt 7.2 mm’ in the control, which indicated a significant difference (P < 0.01, Student’s t test) (Figures 2c and 4).
DISCUSSION Skin substitutes may be classified into two groups: those developed for covering a split-thickness skin defect or dermal burn, and those for a full-thickness skin defect or deep burn. The skin substitutes that belong to the former group, such as lyophilized porcine skin2s3 and polyurethane sheet4, are used to accelerate epithelization, prevent water loss and relieve pain. Most of them accord with these purposes to a certain extent. Those that belong to the latter group include PVF sponge’, Biobranee, and bilayer artificial skin like ours. The PVF sponge is useful as a temporary skin substitute but infection occasionally occurs during pro-
hm2)
100
longed usage. Although Biobrane, which has an affinity to the host tissue, seems to be a more advanced material than the PVF sponge, both are temporary. Consequently, pain and bleeding are inevitable when peeling. The present investigation indicates that the artificial skin we have made is outstanding in the following: The inner sponge layer of collagen and GAG has an affinity to the tissue and little antigenicity. In particular, the atelocollagen used in our artificial skin may have less antigenicity than the insoluble collagen used in the stage 1 membrane. The inner sponge layer does not need to be peeled off because the original network of collagen and GAG is biodegraded, with infiltration of cellular tufts of fibroblasts and capillaries and synthesis of connective tissue matrix. Epidermal cells migrate at a speed of approximately 0.5 mm per day from the edge of the wound into the plane between the newly synthesized connective tissue and the silicone layer. (A split-thickness skin graft is required secondarily in most clinical uses because the skin defects are too wide for epidermal cells to reach the centre in a few weeks. However, the secondary skin graft to be used clinically can be fairly thin so that killing of donors may be kept to a minimum and skin taken repeatedly at intervals from the same site.) 4. Since the inner layer is spontaneously converted into a new connective tissue like dermis, post-operative contracture is not significant. 5. Water vapour permeability of the outer silicone layer is comparable to that of the normal skin. A three-dimensional structure, including pore diameter and pore volume fraction, seemed to be more critical for infiltration and proliferation of fibroblasts and capillaries than the type of collagen used and the process of preparing the sponge layer. When the artificial skin was placed on to the defect with pressure, cell infiltration and proliferation were inhibited’ 4 (Figure 5). In our preliminan/ study, another artificial skin was prepared using gelatin instead of atelocollagen but cell infiltration and proliferation were poor (Figure 6); it remains unclear whether the failure was due to its three-dimensional structure or its nature. Yannas et a/.*.’ attempted to develop a stage 2 membrane by seeding the stage 1 membrane with epidermal
50
Artificial skin Figure 4 Mean re-epithelized area in the artificial skin and control groups. n = 12; P < 0.01, Student’s t test.
Figore 5 7 d post-operative histological appearance of artdicial skin grafted on to the skin defect under pressure (haematoxylin and eosin staining; original magnification X 25). Sponge structure is crushed and cell infiltration and proliferation are poor compared wth those shown in Figures 3b and c.
Biomaterials
1990, Vol I I Julv
359
New bilayer artificial skin: S. Suzuki et al.
reports of Yannas et al. that glycosaminoglycan incorporated in the collagen sponge contributed to the function of the artificial skin7. The role of the added GAG (chondroitin 6sulphate and others) has been examined to verify their effects and the results will be reported in a later paper17.
REFERENCES 1
2 Figure 6 7 d post-operative histological appearance of artificial skin composed of gelatin sponge (haematoxylin and eosin staining; original magnification X 25). The original network. which seems thicker than that of collagen, almost remains with few cells infiltrating.
3 4
5
cells, which dispenses with a secondary skin graft. However, the advantage of a stage 2 membrane may be questionable at present in terms of cost-performance and function. The material has to be mass-produced at a low cost to be applicable to many patients. Combined use of our artificial skin and cultured epidermis15 appears to be more practical in treating extensive burns. The artificial skin is placed on to the burned wound and simultaneously a piece of epidermis is taken for culture proliferation. While the epidermal cells are being proliferated to make an appropriate sheet of epidermis, a dermis-like tissue would have been prepared as a recipient bed for grafting the cultured epidermis sheet. The modifications we made in producing the artificial skin originally developed by Yannas et al. are as follows. The use of atelocollagen, instead of insoluble collagen, allowed us to minimize antigenicity. Instead of coprecipitation of collagen and GAG, the mixed solution was rapidly frozen and directly freeze-dried. This procedure permits the use of various kinds of GAGS instead of chondroitin 6-sulphate and regulation of the ratio of GAG to collagen. The thickness of the outer silicone layer was adjusted to 25 ,um, thinner than that of the stage 1 membrane but the moisture permeability was approximately equal to that of normal skin. Our artificial skin has already been used clinically. Though a thin split-thickness skin graft is required, postoperative appearance is satisfactory in almost all cases16. Though our artificial skin has several advantages, some problems remain to be solved. Chondroitin 6-sulphate was added to the collagen sponge in this study following the
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