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Study of gelatin-containing artificial skin V: fabrication of gelatin scaffolds using a salt-leaching method
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Biomaterials 26 (2005) 1961–1968 www.elsevier.com/locate/biomaterials
Study of gelatin-containing artificial skin V: fabrication of gelatin scaffolds using a salt-leaching method Sang Bong Leea, Yong Han Kima, Moo Sang Chonga, Seung Hwa Hongb, Young Moo Leea, a
School of Chemical Engineering, College of Engineering, Hanyang University, Haengdang-dong, Seungdong-ku, Seoul 133–791, Republic of Korea b Korea FDA Biologics Evaluation Department, Blood Products Division, Nokburn-Dong 5 Eunpyung-Ku, Seoul 122–070, South Korea Received 16 December 2003; accepted 14 June 2004 Available online 29 July 2004
Abstract Porous gelatin scaffolds were prepared using a salt-leaching method and these were compared to scaffolds fabricated using a freeze-drying method. The salt-leached gelatin scaffolds were easily formed into desired shapes with a uniformly distributed and interconnected pore structure with an average pore size of around 350 mm. The mechanical strength and the biodegradation rate of the scaffolds increased with the porosity, and were easily modulated by the addition of salt. After 1 week of in vitro culturing, the fibroblasts in salt-leached scaffolds were mainly attached on the surface of the pores in the scaffold, whereas cells seeded on freezedried scaffolds were widely distributed and aggregated on the top and the bottom of the scaffold. After 14 d of culturing, the fibroblasts showed a good affinity to, and proliferation on, the gelatin scaffolds without showing any signs of biodegradation. An in vivo study of cultured artificial dermal substitutes showed that an artificial dermis containing the fibroblasts enhanced the reepithelialization of a full-thickness skin defect when compared to an acellular scaffold after 1 week. r 2004 Elsevier Ltd. All rights reserved. Keywords: Artificial skin; Gelatin; Salt leaching; Scaffold
1. Introduction Gelatin, a natural polymer extracted from collagen, can be used as a scaffold for tissue engineering [1–3]. We have previously shown the efficacy of cross-linked gelatin-based sponges composed of gelatin and polysaccharides for wound-dressing materials [1–4]. Crosslinked gelatin sponges have been also investigated for their potential application as a component of artificial skin or tissue transplants to promote epithelialization and granulation tissue formation in wounds. However, gelatin sponges prepared by the conventional freezedrying method were not ideal for a cultured artificial dermal substitute in terms of their mechanical strength Corresponding author. Tel.: +82-222-91-9683; fax: +82-2-22915982. E-mail address: [email protected] (Y.M. Lee).
0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.06.032
and morphological microstructure. Therefore, a new method needs to be developed to prepare scaffolds without changing the desired gelatin construction material. Many methods to prepare porous three-dimensional biodegradable scaffolds have been developed in tissue engineering, including gas forming [5,6], three-dimensional printing [7], phase separation [8], emulsion freezedrying [9], and porogen-leaching [10]. Note that polysaccharide scaffolds are usually prepared by freeze-drying because the freeze-drying technique is beneficial for polysaccharides in aqueous media, whereas synthetic polymer scaffolds are prepared using organic solvents. To improve the properties of gelatin sponges, the salt-leaching method has been used to prepare porous gelatin scaffolds employing excess NaCl crystals as a porogen, although NaCl particles can be dissolved in water. For example, Ma et al. [11] prepared
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a chitosan scaffold using a porogen-leaching method in aqueous solution. The purpose of this study was to prepare gelatin scaffolds using the salt-leaching technique and to compare these with scaffolds fabricated using the conventional freeze-drying method in terms of their morphological, mechanical, and biodegradable properties. Furthermore, a cultured artificial dermal substitute was evaluated in terms of its in vitro cell culture and in vivo wound healing ability.
solutions were poured into a polystyrene Petri dish, frozen at 76 1C, and then lyophilized. The dried scaffolds were cross-linked by immersion into 20 ml of an acetone:water mixture (9:1 by volume) containing 30% EDC by gelatin weight, and slowly shaken at room temperature for 1 d. The cross-linked scaffolds were then rinsed with deionized water five times to remove any residual chemicals. The rinsed scaffolds were then frozen at 76 1C, lyophilized, and sterilized with ethylene oxide gas for cell culturing. 2.3. Preparation of salt-leached gelatin scaffolds
2. Materials and methods 2.1. Materials The gelatin used was derived from bovine skin, and was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and Type IV collagenase with a 388 units/mg digestion activity were also purchased from Sigma Chemical Co. The human fibroblast cells used were aseptically isolated from a foreskin donated by the Urology Department of Hanyang University Hospital in Seoul, Korea. The dermal fibroblast culture medium used was composed of Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco BRL, Rockville, MD, USA) and 10% fetal bovine serum (FBS, Gibco BRL). Dulbecco’s phosphate buffered saline (PBS), trypsin–EDTA (0.05% trypsin 5.3 mM EDTA 4Na), and the antibiotic agent used, penicillin–streptomycin (100 U/ml) were purchased from Gibco BRL. The trypan blue (0.4% 100 ml), T-8154, and sodium bicarbonate used were purchased from Sigma Chemical Co. The water used was doubly deionized using the Milli-Q System (Waters, Millipore, USA). All the other chemicals used were of reagent grade, and were used without any further purification.
Gelatin (2.5 g) was dissolved in doubly deionized water (7.5 g) at 50 1C at a concentration of 25 wt%. Crystals of sodium chloride (average particle size= 300–500 mm from sieving) were added at various weight ratios (100–175 g) into the gelatin solution, as listed in Table 1, and the components were homogeneously mixed. The solution containing the NaCl crystals was poured into a Teflon mold (thickness=2 mm), uniformly packed, and then dried in a vacuum oven at 50 1C for 24 h to remove the water. The ensuing preparative stages, such as cross-linking, lyophilizing, and sterilizing, were performed using the same protocols as discussed in Section 2.2 on the freezedrying method. 2.4. Morphologies of the scaffolds The morphology of the scaffolds was investigated using scanning electron microscopy (SEM, JEOL6400F, Kyoto, Japan). The scaffold microstructures were evaluated from geometrical measurements on the scanning electron micrographs. The morphological features, such as the porosity and average pore size of the scaffolds, were investigated using an image analyzer (Bum-Mi Universe Co. Ltd., Seoul, Korea).
2.2. Preparation of freeze-dried gelatin scaffolds
2.5. Mechanical properties and enzymatic biodegradation
Gelatin was dissolved in various concentrations in doubly deionized water at 50 1C, as listed in Table 1. The
A universal testing machine (UTM, INSTRON No. 4465, NY, USA) was used to determine the
Table 1 Composition, porosity, and pore size of gelatin scaffolds prepared by freeze-drying and salt-leaching methods Freeze-drying method
Salt-leaching method
Sample code
Concentration (wt%)
Porosity (%)a
Sample code
NaCl/gelatinb (wt/wt)
Porosity (%)a
Pore size (mm)c
Gf-0.5 Gf-1.0 Gf-1.5 Gf-2.0
0.5 1.0 1.5 2.0
90.0970.9 74.0771.5 66.8271.0 62.6971.3
Gs-40 Gs-50 Gs-60 Gs-70
40 50 60 70
72.9672.3 76.6672.9 83.3171.2 88.3272.6
344.26734.12 364.57722.90 340.59739.68 354.68742.13
a
Porosity was obtained from area analysis between the pore zone and matrix zone by the image analyzer program. Concentration of gelatin solutions: 25 (wt/wt) %. c Average pore size was calculated by measuring the size of 30 pores using the image analyzer tools. b
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mechanical strength of the scaffolds (dimensions=5.0 1.5 0.2 cm3) at a constant tensile deformation rate of 1 mm/min in the dry state at 25 1C. To assess biodegradation, gelatin scaffolds (dimensions=1.5 1.5 0.2 cm3) were placed in a pH=7.4 PBS solution with 60 mg/ml collagenase, and shaken in a water bath maintained at 37 1C. After the determined time, the scaffolds were removed from the collagenase solution, and washed three times with deionized water. The remaining weight of the scaffold was measured after lyophilizing the scaffold. The remaining weight (%) of the gelatin scaffold was calculated using the following equation:
USA) was used to sew the Tegaderm. The dressed wound was bandaged with an adhesive plaster (FIX ROLLs, Young Chemical Co. Ltd., Busan, Korea). The mice were sacrificed after postoperative periods of 1 (n ¼ 3) and 2 (n ¼ 3) weeks, and the wound tissues were embedded in paraffin for staining with Masson’s trichrome.
remaining weight ð%Þ ¼ W t =W 0 100;
Fig. 1 shows the preparative process used for the gelatin scaffolds employing the salt-leaching technique. In aqueous media, salts such as NaCl exist as solid particles in the mixing solution when above the saturation concentration (about NaCl=2.7 g/25 wt% gelatin solution=10 g). Therefore, some NaCl was dissolved in the mixing solution and was recrystallized as solid particles in the matrix during solvent evaporation. The excess and recrystallized NaCl particles were washed out either during or after the cross-linking process of the matrix. During this procedure, macropores were formed in the spaces previously occupied by the NaCl particles, which did not react with any of the functional groups or other chemicals. Ma et al. [11] used a similar technique to prepare chitosan scaffolds dissolved in an aqueous solution. The salt or porogens added to the solution were NaCl, glucose, and sucrose. However, the mass of added porogen was so low that all the salt and porogens could be dissolved in the solution below the saturation concentration. The pores formed were from the recrystallized salts or porogens after the solvent was evaporated. Note that the scaffold morphologies of our method depended on both the excess and the recrystallized NaCl particles.
where W 0 is the initial weight of scaffold and W t is the weight of the scaffold after degradation with collagenase. 2.6. In vitro cell culture Human fibroblasts were subcultured in DMEM supplemented with 10% (v/v) FBS. The cells were harvested using 0.05% trypsin–EDTA after washing with PBS. After the cultured fibroblasts were suspended in a DMEM medium containing 10% FBS, the cell concentration was adjusted to 2.5 106 cells/cm2 and spread on each scaffold (dimensions=1.5 1.5 0.2 cm3). Fresh medium was added to each culture dish, and the scaffold containing the cells was then incubated at 37 1C in a 5% CO2 atmosphere incubator. Shrinkage values were measured using the following equation: shrinkage value ð%Þ ¼ ½At =Ai 100; where At and Ai are the surface area of scaffold at the shrinkaged and initial states, respectively. The fibroblasts cultured in the scaffold for 1 and 2 weeks were fixed using a 10% formaldehyde solution in PBS at room temperature for 2 h. The samples were embedded in paraffin for staining with hematoxylin–eosin (H&E) and Masson’s trichrome, and then photographed. 2.7. Animal tests with an artificial dermis Under intraperitoneal anesthesia with Nembutal, a full-thickness wound, 1.5 cm in diameter, was experimentally prepared on the back of athymic mice (BALB/ c Slc-nu, 4 weeks old, Japan SLC Inc., Hamamatsu, Japan). After disinfection of the wound with povidoneiodide, a cellular artificial dermis and an acellular scaffold of sample Gs-50 were grafted onto an excised wound (n ¼ 6). A transparent Tegaderm film (3M Health Care, St. Paul, MN) was used to cover the artificial dermis to prevent the grafted cellular artificial dermis from drying out, and a 6–0 nylon suture (Ethicon Inc., a Johnson and Johnson Company, Somerville, NJ,
3. Results 3.1. Preparation of salt-leached scaffolds
3.2. Morphology of the scaffolds Fig. 2 shows a cross-sectional structure of a gelatin scaffold prepared using the freeze-drying method with various concentrations. Gelatin scaffolds appeared in the interconnected network pore configuration, characterized by its membrane-like structure. The structure of the vertical channels was uniformly distributed, and formed an interconnected pore structure. Exceptionally, the cross-sectional morphology of Sample Gf-0.5 formed a web-like structure, due to the low gelatin concentration in the initial solution. Fig. 3 shows a cross-sectional structure of a scaffold prepared using the salt-leaching method with different mixing concentrations of NaCl. The scaffolds exhibited interconnected pore structures, but did not form a membrane-like structure between pores. Sample Gs-4,
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structures were shown in Samples Gs-50, Gs-60, and Gs-70, whereas Sample Gs-40 had a relatively dense surface (data not shown). Table 1 shows that the porosity of the scaffolds increased with increasing NaCl concentration in the saltleached scaffolds and with increasing gelatin concentration in the feed solution of freeze-dried scaffolds. The pore size of salt-leached scaffolds maintained the original NaCl particle size (300–500 mm by sieving), and the pores were evenly distributed, regardless of the concentration of salt added to the gelatin solution. 3.3. Mechanical strength and in vitro biodegradation Figs. 4(a) and (b) show the strain–stress curves of the freeze-dried and salt-leached scaffolds, respectively, used to evaluate the mechanical strength of the scaffolds prepared using the two methods. The initial modulus of the scaffolds increased with decreasing NaCl content in the salt-leached scaffold. The mechanical strength of Sample Gs-40 sharply improved due to the formation of a dense layer on the surface of the scaffold, as shown in Fig. 3(a). In the freeze-dried scaffolds, the mechanical strength increased with increasing gelatin content of the solution. Fig. 5 shows the in vitro biodegradation of the gelatin scaffolds, which was determined by measuring the decrease in weight of the biopolymer caused by hydrolytic or enzymatic degradation. Sample Gf-0.5 was completely degraded within 12 h and we could not measure its weight loss any further. The biodegradation rate of Samples Gf-1.0 and Gf-1.5 rapidly increased up to 24 h and then leveled off. Sample Gf-2.0 degraded slowly in comparison with the other freeze-dried scaffolds. In the enzymatic degradation studies, the degradation rates of the scaffolds prepared using the salt-leaching method were slower than those of the scaffolds prepared using the freeze-drying method. The percent weight remaining for Samples Gs-40 and Gs-70 declined to 63% and 40%, respectively, at 72 h. 3.4. In vitro cell culture
Fig. 1. Preparative process of gelatin scaffolds using salt-leaching technique: (a) 25 wt% gelatin solution (10 g), (b) adding NaCl particles (100–175 g) into the solution, (c–d) sunk NaCl particles above saturated concentration (about NaCl=2.7 g/25 wt% gelatin solution=10 g) and drying it at 50 1C vacuum oven, and (e) leaching the salts with water after cross-linking the gelatin with EDC.
containing a relatively low NaCl content, appeared to show a bilayer morphology composed of dense and porous parts. Results for the surface morphologies of the salt-leached scaffolds reveal that the porous
Fig. 6 shows the morphologies of the cell-cultured Gs50 scaffolds after 1 and 2 weeks. The fibroblasts after 1 week of culturing showed a good affinity to the scaffold, and were mainly distributed on the surface of the macropores formed in the Gs-50 scaffold. The fibroblasts attached to the surface of the pores were well proliferated on the entire areas of scaffold after 2 weeks of culturing (see Fig. 6(b)). 3.5. Histological evaluation Fig. 7 shows the histological results from the acellular and cellular Gs-50 scaffolds applied to the dorsal
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Fig. 2. Cross-sectional SEM images of freeze-dried gelatin scaffold: (a) Gf-0.5, (b) Gf-1.0, (c) Gf-1.5, and (d) Gf-2.0.
Fig. 3. Cross-sectional SEM images of salt-leached gelatin scaffold: (a) Gs-40, (b) Gs-50, (c) Gs-60, and (d) Gs-70.
skin wound of athymic mice after dressing for 1 and 2 weeks. The skin defects for the cellular Gs-50 samples containing fibroblasts showed only a central defect after 1 week, whereas the wound dressed by a cellular Gs-50 sample had after 2 weeks almost re-epithelialized and completely regenerated, as shown in Figs. 7(a) and
(b). On the other hand, in the case of the acellular Gs-50 sample, the surface of the wound was covered with a thick zone of acute inflammatory exudates with an underlying exuberant granulation tissue that had not yet epithelialized after 2 weeks (see Figs. 7(c) and (d)).
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Weight remaining (%)
Stress (kPa)
100
Gs-40
8
Gs-50
6
4
Gs-60
2
Gf-2.0 Gf-1.5 Gf-1.0 Gf-0.5
80 60 40 20
Gs-70
0 0.00
0 0.01
0.02
0.03
0.04
0.05
0.06
0.07
8
0
0.08
10
20
(a)
Strain (mm/mm)
(a)
30
40
50
60
Gs-40 Gs-50 Gs-60 Gs-70
Stress (kPa)
4
Gf-0.5
2
0 0.00
Weight remaining (%)
Gf-1.5 Gf-1.0
80
100
Gf-2.0
6
70
Time (hours)
80 60 40 20 0
0.01
(b)
0.02
0.03
0.04
0.05
0.06
0.07
Strain (mm/mm)
0 (b)
10
20
30
40
50
60
70
80
Time (hours)
Fig. 4. Mechanical strength of gelatin scaffolds (5.0 1.5 0.2 cm3) at a constant tensile deformation rate of 1 mm/min in the dry state at 25 1C.
Fig. 5. Weight remaining of gelatin scaffolds (1.5 1.5 0.2 cm3) by enzymatic biodegradation (pH 7.4, PBS with 60 mm/ml collagenase): (a) freeze-drying and (b) salt-leaching methods.
4. Discussion
gelatin scaffolds with the aim of benefiting cell culturing. The salt-leaching method has not been used in watersoluble polymer systems, because porogens such as NaCl and KCl are easily dissolved in water. However, the method is usually used for polymers such as PLGA dissolved in organic solvents, which do not dissolve inorganic salts. The porosity and the particle size of the scaffolds were regulated by the number and size of the NaCl crystals. Compared with the freezedried scaffolds, the salt-leached scaffolds had larger interconnected pores and did not form a membranelike structure. However, insufficient NaCl crystals existing in the solid form caused a bilayer-scaffold formation, because the NaCl crystals sank to the bottom of the mold. The bilayer structure also affected the mechanical strength. Fig. 4(b) shows that the initial modulus of Sample Gs-40 increased steeply due to the bilayer structure. However, compared with the freezedried scaffolds, the mechanical properties of the saltleached scaffolds did not improve with excess NaCl crystals in the solution. Note that the NaCl content
The freeze-drying method is usually used to prepare gelatin scaffolds, because gelatin is a water-soluble natural polysaccharide. However, gelatin-based sponges formed using the freeze-drying method have a dense surface that does not allow cells to penetrate into the inner areas when the cell suspension is seeded on the scaffold [1–3]. This dense gelatin layer may contribute to the uniaxial orientation of the pore channels in the upper part of the scaffold, because the upper part of the sponge is directly exposed to the vacuum, and has a different structure to the lower region in contact with the mold surface. A dense surface on the wounddressing material is suitable for protecting the scaffold from evaporating exudates from the wound, and from the invasion of bacteria into the wound. However, a film-like surface has a detrimental effect on cell migration in seeding cells on the surface of a scaffold. Thus, we felt it was necessary to introduce another technique, such as the salt-leaching method, to prepare
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Fig. 7. Masson’s trichrome staining of the wounds implanting cellular gelatin with human fibroblasts scaffolds on the back of athymic mice for 1 week (a) and 2 weeks (b); acellular gelatin scaffolds implanted for (a) 1 week and (b) 2 weeks (original magnification, 40).
Fig. 6. Histological evaluation of human fibroblasts-cultured gelatin scaffold in vitro (2.5 106 cells/cm2) of Gs-50 on (a) 1 week and (b) 2 weeks (Masson’s trichrome staining, original magnification, 40).
added to the polymer solution was 40–70-fold that of gelatin. To maintain the scaffold construction after seeding the cells, the scaffolds have to be mechanically strong, because the dermis has to provide physical strength and flexibility to the skin as well as the scaffold to support extensive vasculatures, the lymphatic system, nerve bundles, and other structures in the skin. However, the mechanical strength of the Gf scaffolds was not sufficient in that the attached cells shrank the scaffold. Morphologically, the 0.8, 0.9, and 1.0 wt% Gf scaffolds maintained their original size after seeding cell, whereas low concentration gelatin scaffolds (0.5, 0.6, and 0.7 wt%) were contracted by seeded fibroblasts (shrinkage values=40, 40, and 63%, respectively). Furthermore, the high concentration of gelatin required to avoid the shrinkage of the Gf scaffolds inhibited cell migration after cell seeding. On the other hand, the Gs scaffolds had satisfactory mechanical properties after seeding with the same number of cells, because the high
initial concentration of gelatin (25 wt%) formed a smaller number of thicker walls between the pores of the scaffolds, whereas the Gf scaffolds had a relatively large number of walls, which were thinner than those of the Gs scaffolds. A highly porous scaffold structure assists both cell penetration and polymer degradation. The rate of degradation is affected by the morphology of the scaffold, and a large surface area speeds up the diffusion of water molecules into the bulk of the polymers when they are placed in an aqueous environment (e.g., in vivo) [12–18]. Thus, the degradation behavior was investigated as a function of the degree of porosity. As shown in Fig. 5, the rate of weight loss decreased with increasing porosity of the gelatin scaffold. The higher degradation in scaffolds Gf-0.5 and Gs-70 was probably due to the high porosity of the matrix and subsequent increase in the accessibility of active sites to the enzyme, whereas the relatively slow degradation rate of scaffolds Gf-2.0 and Gs-40 was probably due to the dense surface and the closed pores that retarded the migration of the enzyme. Note that the remaining weight (%) in the Gs scaffolds did not rapidly decrease and remained relatively constant compared to that of the Gf scaffold. The in vivo tests showed that the artificial dermis, rather than the acellular sponge, improved the reepithelialization on the full-thickness skin defect. This was caused by the effect of the fibroblasts on the artificial dermis. Several studies have indicated that the presence of fibroblasts in a dermal equivalent
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stimulates epidermal differentiation [19–21]. It is assumed that the presence of fibroblasts in a dermal equivalent accelerates the healing process by reducing the time needed for the fibroblasts to invade the wound tissue, and by the early synthesis of new skin tissue, because the fibroblasts on the artificial dermis can release biologically active substances, such as cytokines [19,21]. These cytokines are stimulated by the increased proteolytic activity leading to the fragmentation of more proteins and the activation of larger numbers of latent growth factors, resulting in a higher chemotactic activity of the wound tissue [19].
5. Conclusions A new gelatin scaffold was designed and prepared using the salt-leaching method. The scaffolds prepared had interconnected pore structures, but did not form a membrane-like structure between the pores. The mechanical strength and the rate of biodegradation were easily modulated by the addition of salt, which affected the porosity of the scaffold. The fibroblasts after 1 week of in vitro culturing also showed a good affinity to the scaffold, and were mainly distributed on the surface of the macro-pores formed in the salt-leached scaffold. The fibroblasts attached on the surface of pores were proliferated over the entire area of the scaffold after 2 weeks. In vivo, the artificial dermis rather than the acellular sponge improved the re-epithelialization on a full-thickness skin defect.
Acknowledgements Financial support from the Korea Food and Drug Administration (KFDA) under the program year 2003 is greatly appreciated. SBL and YHK appreciate the fellowship from BK21 program. References [1] Choi YS, Hong SR, Lee YM, Song KW, Park MH, Nam YS. Study on gelatin-containing artificial skin: I. Preparation and characteristics of navel gelatin-alginate sponge. Biomaterials 1999;20:409–17. [2] Choi YS, Hong SR, Lee YM, Song KW, Park MH, Nam YS. Study on gelatin-containing artificial skin: II. Preparation and characterization of cross-linked gelatin-hyaluronate sponge. J Biomed Mater Res 1999;48(5):631–9. [3] Choi YS, Lee SB, Hong SR, Lee YM, Song KW, Park MH, Nam YS. Studies on gelatin-based sponges. Part III: a comparative study of cross-linked gelatin/alginate, gelatin/hyaluronate and chitosan/hyaluronate sponges and their application as a wound dressing in full-thickness skin defect of rat. J Mater Sci Mater M 2001;12:67–73.
[4] Hong SR, Lee SJ, Shim JW, Choi YS, Lee YM, Song KW, Park MH, Nam YS, Lee SI. Study on gelatin-containing artificial skin IV: a comparative study on the effect of antibiotic and EGF on cell proliferation during epidermal healing. Biomaterials 2001;22:2777–83. [5] Mooney DJ, Baldwin DF, Suh NP, Vacanti JP, Langer R. Novel approach to fabricate porous sponges of poly(D,L-lactic-coglycolic acid) without the use of organic solvents. Biomaterials 1996;17:1417–22. [6] Murphy WL, Kohn DH, Mooney DJ. Growth of continuous bonelike mineral within porous poly(lactic-co-glycolide) scaffolds in vitro. J Biomed Mater Res 2000;50(1):50–8. [7] Sherwood JK, Riley SL, Palazzolo R, Brown SC, Monkhouse DC, Coates M, Griffith LG, Landeen LK, Ratcliffe A. A threedimensional osteochondral composite scaffold for articular cartilage repair. Biomaterials 2002;23:4739–51. [8] Hua FJ, Park TG, Lee DS. A facile preparation of highly interconnected macroporous poly(D,L-lactic acid-co-glycolic acid) (PLGA) scaffolds by liquid–liquid phase separation of a PLGA–dioxane–water ternary system. Polymer 2003;44:1911–20. [9] Whang K, Thomas CH, Healy KE. A novel method to fabricate bioabsorable scaffolds. Polymer 1995;36:837–42. [10] Hou Q, Grijpma DW, Feijen J. Porous polymeric structures for tissue engineering prepared by a coagulation, compression moulding and salt leaching technique. Biomaterials 2003;24: 1937–47. [11] Ma J, Wang H, He B, Chen J. A preliminary in vitro study on the fabrication and tissue engineering applications of a novel chitosan bilayer material as a scaffold of human neofetal dermal fibroblasts. Biomaterials 2001;22:331–6. [12] Chen G, Ushida T, Tateishi T. A biodegradable hybrid sponge nested with collagen microsponges. J Biomed Mater Res 2000; 51:273–9. [13] Freed LE, Grand DA, Lingbin Z, Emmanual J, Marquis JC, Langer R. Joint resurfacing using allograft chondrocytes and synthetic biodegradable polymer scaffolds. J Biomed Mater Res 1994;28:891–9. [14] Freed LE, Marquis JC, Nohria A, Emmanual J, Mikos AC, Langer R. Neocartilage formation in vitro and in vivo using cells cultured on synthetic biodegradable polymers. J Biomed Mater Res 1993;27:11–23. [15] Oberpenning F, Meng J, Yoo JJ, Atala A. De novo reconstitution of a functional mammalian urinary bladder by tissue engineering. Nat Biotechnol 1999;17:149–55. [16] Zacchi V, Soranzo C, Cortivo R, Radice M, Brun P, Abatangelo G. In vitro engineering of human skin-like tissue. J Biomed Mater Res 1998;40:187–94. [17] Ishaug SL, Crane GM, Miller MJ, Yasko AW, Yaszemski MJ, Cusick RA, Lee H, Sano K, Pollok JM, Utsunomiya H, Ma PX, Langer R, Vacanti JP. The effect of donor and recipient age on engraftment of tissue-engineered liver. J Pediatr Surg 1997; 32:357–60. [18] Dunn MG, Liesch JB, Tiku ML, Zawadsky JP. Development of fibroblast-seeded ligament analogs for ACL reconstruction. J Biomed Mater Res 1995;29:1363–71. [19] Quirk RA, Chen WC, Davies MC, Tendler SJB, Shakesheff KM. Poly(L-lysine)-GRGDS as a biomimetic surface modifier for poly(lactic acid). Biomaterials 2001;22:865–72. [20] Lee DY, Ahn HT, Cho KH. A new skin equivalent model: dermal substrate that combines de-epidermized dermis with fibroblast-populated collagen matrix. J Dermatol Sci 2000;23: 132–7. [21] Bernard C, Corinne L, Louis D. Influence of human dermal fibroblasts on epidermalization. J Invest Dermatol 1989;92: 122–5.
Biomaterials 26 (2005) 1961–1968 www.elsevier.com/locate/biomaterials
Study of gelatin-containing artificial skin V: fabrication of gelatin scaffolds using a salt-leaching method Sang Bong Leea, Yong Han Kima, Moo Sang Chonga, Seung Hwa Hongb, Young Moo Leea, a
School of Chemical Engineering, College of Engineering, Hanyang University, Haengdang-dong, Seungdong-ku, Seoul 133–791, Republic of Korea b Korea FDA Biologics Evaluation Department, Blood Products Division, Nokburn-Dong 5 Eunpyung-Ku, Seoul 122–070, South Korea Received 16 December 2003; accepted 14 June 2004 Available online 29 July 2004
Abstract Porous gelatin scaffolds were prepared using a salt-leaching method and these were compared to scaffolds fabricated using a freeze-drying method. The salt-leached gelatin scaffolds were easily formed into desired shapes with a uniformly distributed and interconnected pore structure with an average pore size of around 350 mm. The mechanical strength and the biodegradation rate of the scaffolds increased with the porosity, and were easily modulated by the addition of salt. After 1 week of in vitro culturing, the fibroblasts in salt-leached scaffolds were mainly attached on the surface of the pores in the scaffold, whereas cells seeded on freezedried scaffolds were widely distributed and aggregated on the top and the bottom of the scaffold. After 14 d of culturing, the fibroblasts showed a good affinity to, and proliferation on, the gelatin scaffolds without showing any signs of biodegradation. An in vivo study of cultured artificial dermal substitutes showed that an artificial dermis containing the fibroblasts enhanced the reepithelialization of a full-thickness skin defect when compared to an acellular scaffold after 1 week. r 2004 Elsevier Ltd. All rights reserved. Keywords: Artificial skin; Gelatin; Salt leaching; Scaffold
1. Introduction Gelatin, a natural polymer extracted from collagen, can be used as a scaffold for tissue engineering [1–3]. We have previously shown the efficacy of cross-linked gelatin-based sponges composed of gelatin and polysaccharides for wound-dressing materials [1–4]. Crosslinked gelatin sponges have been also investigated for their potential application as a component of artificial skin or tissue transplants to promote epithelialization and granulation tissue formation in wounds. However, gelatin sponges prepared by the conventional freezedrying method were not ideal for a cultured artificial dermal substitute in terms of their mechanical strength Corresponding author. Tel.: +82-222-91-9683; fax: +82-2-22915982. E-mail address: [email protected] (Y.M. Lee).
0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.06.032
and morphological microstructure. Therefore, a new method needs to be developed to prepare scaffolds without changing the desired gelatin construction material. Many methods to prepare porous three-dimensional biodegradable scaffolds have been developed in tissue engineering, including gas forming [5,6], three-dimensional printing [7], phase separation [8], emulsion freezedrying [9], and porogen-leaching [10]. Note that polysaccharide scaffolds are usually prepared by freeze-drying because the freeze-drying technique is beneficial for polysaccharides in aqueous media, whereas synthetic polymer scaffolds are prepared using organic solvents. To improve the properties of gelatin sponges, the salt-leaching method has been used to prepare porous gelatin scaffolds employing excess NaCl crystals as a porogen, although NaCl particles can be dissolved in water. For example, Ma et al. [11] prepared
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a chitosan scaffold using a porogen-leaching method in aqueous solution. The purpose of this study was to prepare gelatin scaffolds using the salt-leaching technique and to compare these with scaffolds fabricated using the conventional freeze-drying method in terms of their morphological, mechanical, and biodegradable properties. Furthermore, a cultured artificial dermal substitute was evaluated in terms of its in vitro cell culture and in vivo wound healing ability.
solutions were poured into a polystyrene Petri dish, frozen at 76 1C, and then lyophilized. The dried scaffolds were cross-linked by immersion into 20 ml of an acetone:water mixture (9:1 by volume) containing 30% EDC by gelatin weight, and slowly shaken at room temperature for 1 d. The cross-linked scaffolds were then rinsed with deionized water five times to remove any residual chemicals. The rinsed scaffolds were then frozen at 76 1C, lyophilized, and sterilized with ethylene oxide gas for cell culturing. 2.3. Preparation of salt-leached gelatin scaffolds
2. Materials and methods 2.1. Materials The gelatin used was derived from bovine skin, and was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and Type IV collagenase with a 388 units/mg digestion activity were also purchased from Sigma Chemical Co. The human fibroblast cells used were aseptically isolated from a foreskin donated by the Urology Department of Hanyang University Hospital in Seoul, Korea. The dermal fibroblast culture medium used was composed of Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco BRL, Rockville, MD, USA) and 10% fetal bovine serum (FBS, Gibco BRL). Dulbecco’s phosphate buffered saline (PBS), trypsin–EDTA (0.05% trypsin 5.3 mM EDTA 4Na), and the antibiotic agent used, penicillin–streptomycin (100 U/ml) were purchased from Gibco BRL. The trypan blue (0.4% 100 ml), T-8154, and sodium bicarbonate used were purchased from Sigma Chemical Co. The water used was doubly deionized using the Milli-Q System (Waters, Millipore, USA). All the other chemicals used were of reagent grade, and were used without any further purification.
Gelatin (2.5 g) was dissolved in doubly deionized water (7.5 g) at 50 1C at a concentration of 25 wt%. Crystals of sodium chloride (average particle size= 300–500 mm from sieving) were added at various weight ratios (100–175 g) into the gelatin solution, as listed in Table 1, and the components were homogeneously mixed. The solution containing the NaCl crystals was poured into a Teflon mold (thickness=2 mm), uniformly packed, and then dried in a vacuum oven at 50 1C for 24 h to remove the water. The ensuing preparative stages, such as cross-linking, lyophilizing, and sterilizing, were performed using the same protocols as discussed in Section 2.2 on the freezedrying method. 2.4. Morphologies of the scaffolds The morphology of the scaffolds was investigated using scanning electron microscopy (SEM, JEOL6400F, Kyoto, Japan). The scaffold microstructures were evaluated from geometrical measurements on the scanning electron micrographs. The morphological features, such as the porosity and average pore size of the scaffolds, were investigated using an image analyzer (Bum-Mi Universe Co. Ltd., Seoul, Korea).
2.2. Preparation of freeze-dried gelatin scaffolds
2.5. Mechanical properties and enzymatic biodegradation
Gelatin was dissolved in various concentrations in doubly deionized water at 50 1C, as listed in Table 1. The
A universal testing machine (UTM, INSTRON No. 4465, NY, USA) was used to determine the
Table 1 Composition, porosity, and pore size of gelatin scaffolds prepared by freeze-drying and salt-leaching methods Freeze-drying method
Salt-leaching method
Sample code
Concentration (wt%)
Porosity (%)a
Sample code
NaCl/gelatinb (wt/wt)
Porosity (%)a
Pore size (mm)c
Gf-0.5 Gf-1.0 Gf-1.5 Gf-2.0
0.5 1.0 1.5 2.0
90.0970.9 74.0771.5 66.8271.0 62.6971.3
Gs-40 Gs-50 Gs-60 Gs-70
40 50 60 70
72.9672.3 76.6672.9 83.3171.2 88.3272.6
344.26734.12 364.57722.90 340.59739.68 354.68742.13
a
Porosity was obtained from area analysis between the pore zone and matrix zone by the image analyzer program. Concentration of gelatin solutions: 25 (wt/wt) %. c Average pore size was calculated by measuring the size of 30 pores using the image analyzer tools. b
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mechanical strength of the scaffolds (dimensions=5.0 1.5 0.2 cm3) at a constant tensile deformation rate of 1 mm/min in the dry state at 25 1C. To assess biodegradation, gelatin scaffolds (dimensions=1.5 1.5 0.2 cm3) were placed in a pH=7.4 PBS solution with 60 mg/ml collagenase, and shaken in a water bath maintained at 37 1C. After the determined time, the scaffolds were removed from the collagenase solution, and washed three times with deionized water. The remaining weight of the scaffold was measured after lyophilizing the scaffold. The remaining weight (%) of the gelatin scaffold was calculated using the following equation:
USA) was used to sew the Tegaderm. The dressed wound was bandaged with an adhesive plaster (FIX ROLLs, Young Chemical Co. Ltd., Busan, Korea). The mice were sacrificed after postoperative periods of 1 (n ¼ 3) and 2 (n ¼ 3) weeks, and the wound tissues were embedded in paraffin for staining with Masson’s trichrome.
remaining weight ð%Þ ¼ W t =W 0 100;
Fig. 1 shows the preparative process used for the gelatin scaffolds employing the salt-leaching technique. In aqueous media, salts such as NaCl exist as solid particles in the mixing solution when above the saturation concentration (about NaCl=2.7 g/25 wt% gelatin solution=10 g). Therefore, some NaCl was dissolved in the mixing solution and was recrystallized as solid particles in the matrix during solvent evaporation. The excess and recrystallized NaCl particles were washed out either during or after the cross-linking process of the matrix. During this procedure, macropores were formed in the spaces previously occupied by the NaCl particles, which did not react with any of the functional groups or other chemicals. Ma et al. [11] used a similar technique to prepare chitosan scaffolds dissolved in an aqueous solution. The salt or porogens added to the solution were NaCl, glucose, and sucrose. However, the mass of added porogen was so low that all the salt and porogens could be dissolved in the solution below the saturation concentration. The pores formed were from the recrystallized salts or porogens after the solvent was evaporated. Note that the scaffold morphologies of our method depended on both the excess and the recrystallized NaCl particles.
where W 0 is the initial weight of scaffold and W t is the weight of the scaffold after degradation with collagenase. 2.6. In vitro cell culture Human fibroblasts were subcultured in DMEM supplemented with 10% (v/v) FBS. The cells were harvested using 0.05% trypsin–EDTA after washing with PBS. After the cultured fibroblasts were suspended in a DMEM medium containing 10% FBS, the cell concentration was adjusted to 2.5 106 cells/cm2 and spread on each scaffold (dimensions=1.5 1.5 0.2 cm3). Fresh medium was added to each culture dish, and the scaffold containing the cells was then incubated at 37 1C in a 5% CO2 atmosphere incubator. Shrinkage values were measured using the following equation: shrinkage value ð%Þ ¼ ½At =Ai 100; where At and Ai are the surface area of scaffold at the shrinkaged and initial states, respectively. The fibroblasts cultured in the scaffold for 1 and 2 weeks were fixed using a 10% formaldehyde solution in PBS at room temperature for 2 h. The samples were embedded in paraffin for staining with hematoxylin–eosin (H&E) and Masson’s trichrome, and then photographed. 2.7. Animal tests with an artificial dermis Under intraperitoneal anesthesia with Nembutal, a full-thickness wound, 1.5 cm in diameter, was experimentally prepared on the back of athymic mice (BALB/ c Slc-nu, 4 weeks old, Japan SLC Inc., Hamamatsu, Japan). After disinfection of the wound with povidoneiodide, a cellular artificial dermis and an acellular scaffold of sample Gs-50 were grafted onto an excised wound (n ¼ 6). A transparent Tegaderm film (3M Health Care, St. Paul, MN) was used to cover the artificial dermis to prevent the grafted cellular artificial dermis from drying out, and a 6–0 nylon suture (Ethicon Inc., a Johnson and Johnson Company, Somerville, NJ,
3. Results 3.1. Preparation of salt-leached scaffolds
3.2. Morphology of the scaffolds Fig. 2 shows a cross-sectional structure of a gelatin scaffold prepared using the freeze-drying method with various concentrations. Gelatin scaffolds appeared in the interconnected network pore configuration, characterized by its membrane-like structure. The structure of the vertical channels was uniformly distributed, and formed an interconnected pore structure. Exceptionally, the cross-sectional morphology of Sample Gf-0.5 formed a web-like structure, due to the low gelatin concentration in the initial solution. Fig. 3 shows a cross-sectional structure of a scaffold prepared using the salt-leaching method with different mixing concentrations of NaCl. The scaffolds exhibited interconnected pore structures, but did not form a membrane-like structure between pores. Sample Gs-4,
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structures were shown in Samples Gs-50, Gs-60, and Gs-70, whereas Sample Gs-40 had a relatively dense surface (data not shown). Table 1 shows that the porosity of the scaffolds increased with increasing NaCl concentration in the saltleached scaffolds and with increasing gelatin concentration in the feed solution of freeze-dried scaffolds. The pore size of salt-leached scaffolds maintained the original NaCl particle size (300–500 mm by sieving), and the pores were evenly distributed, regardless of the concentration of salt added to the gelatin solution. 3.3. Mechanical strength and in vitro biodegradation Figs. 4(a) and (b) show the strain–stress curves of the freeze-dried and salt-leached scaffolds, respectively, used to evaluate the mechanical strength of the scaffolds prepared using the two methods. The initial modulus of the scaffolds increased with decreasing NaCl content in the salt-leached scaffold. The mechanical strength of Sample Gs-40 sharply improved due to the formation of a dense layer on the surface of the scaffold, as shown in Fig. 3(a). In the freeze-dried scaffolds, the mechanical strength increased with increasing gelatin content of the solution. Fig. 5 shows the in vitro biodegradation of the gelatin scaffolds, which was determined by measuring the decrease in weight of the biopolymer caused by hydrolytic or enzymatic degradation. Sample Gf-0.5 was completely degraded within 12 h and we could not measure its weight loss any further. The biodegradation rate of Samples Gf-1.0 and Gf-1.5 rapidly increased up to 24 h and then leveled off. Sample Gf-2.0 degraded slowly in comparison with the other freeze-dried scaffolds. In the enzymatic degradation studies, the degradation rates of the scaffolds prepared using the salt-leaching method were slower than those of the scaffolds prepared using the freeze-drying method. The percent weight remaining for Samples Gs-40 and Gs-70 declined to 63% and 40%, respectively, at 72 h. 3.4. In vitro cell culture
Fig. 1. Preparative process of gelatin scaffolds using salt-leaching technique: (a) 25 wt% gelatin solution (10 g), (b) adding NaCl particles (100–175 g) into the solution, (c–d) sunk NaCl particles above saturated concentration (about NaCl=2.7 g/25 wt% gelatin solution=10 g) and drying it at 50 1C vacuum oven, and (e) leaching the salts with water after cross-linking the gelatin with EDC.
containing a relatively low NaCl content, appeared to show a bilayer morphology composed of dense and porous parts. Results for the surface morphologies of the salt-leached scaffolds reveal that the porous
Fig. 6 shows the morphologies of the cell-cultured Gs50 scaffolds after 1 and 2 weeks. The fibroblasts after 1 week of culturing showed a good affinity to the scaffold, and were mainly distributed on the surface of the macropores formed in the Gs-50 scaffold. The fibroblasts attached to the surface of the pores were well proliferated on the entire areas of scaffold after 2 weeks of culturing (see Fig. 6(b)). 3.5. Histological evaluation Fig. 7 shows the histological results from the acellular and cellular Gs-50 scaffolds applied to the dorsal
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Fig. 2. Cross-sectional SEM images of freeze-dried gelatin scaffold: (a) Gf-0.5, (b) Gf-1.0, (c) Gf-1.5, and (d) Gf-2.0.
Fig. 3. Cross-sectional SEM images of salt-leached gelatin scaffold: (a) Gs-40, (b) Gs-50, (c) Gs-60, and (d) Gs-70.
skin wound of athymic mice after dressing for 1 and 2 weeks. The skin defects for the cellular Gs-50 samples containing fibroblasts showed only a central defect after 1 week, whereas the wound dressed by a cellular Gs-50 sample had after 2 weeks almost re-epithelialized and completely regenerated, as shown in Figs. 7(a) and
(b). On the other hand, in the case of the acellular Gs-50 sample, the surface of the wound was covered with a thick zone of acute inflammatory exudates with an underlying exuberant granulation tissue that had not yet epithelialized after 2 weeks (see Figs. 7(c) and (d)).
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Weight remaining (%)
Stress (kPa)
100
Gs-40
8
Gs-50
6
4
Gs-60
2
Gf-2.0 Gf-1.5 Gf-1.0 Gf-0.5
80 60 40 20
Gs-70
0 0.00
0 0.01
0.02
0.03
0.04
0.05
0.06
0.07
8
0
0.08
10
20
(a)
Strain (mm/mm)
(a)
30
40
50
60
Gs-40 Gs-50 Gs-60 Gs-70
Stress (kPa)
4
Gf-0.5
2
0 0.00
Weight remaining (%)
Gf-1.5 Gf-1.0
80
100
Gf-2.0
6
70
Time (hours)
80 60 40 20 0
0.01
(b)
0.02
0.03
0.04
0.05
0.06
0.07
Strain (mm/mm)
0 (b)
10
20
30
40
50
60
70
80
Time (hours)
Fig. 4. Mechanical strength of gelatin scaffolds (5.0 1.5 0.2 cm3) at a constant tensile deformation rate of 1 mm/min in the dry state at 25 1C.
Fig. 5. Weight remaining of gelatin scaffolds (1.5 1.5 0.2 cm3) by enzymatic biodegradation (pH 7.4, PBS with 60 mm/ml collagenase): (a) freeze-drying and (b) salt-leaching methods.
4. Discussion
gelatin scaffolds with the aim of benefiting cell culturing. The salt-leaching method has not been used in watersoluble polymer systems, because porogens such as NaCl and KCl are easily dissolved in water. However, the method is usually used for polymers such as PLGA dissolved in organic solvents, which do not dissolve inorganic salts. The porosity and the particle size of the scaffolds were regulated by the number and size of the NaCl crystals. Compared with the freezedried scaffolds, the salt-leached scaffolds had larger interconnected pores and did not form a membranelike structure. However, insufficient NaCl crystals existing in the solid form caused a bilayer-scaffold formation, because the NaCl crystals sank to the bottom of the mold. The bilayer structure also affected the mechanical strength. Fig. 4(b) shows that the initial modulus of Sample Gs-40 increased steeply due to the bilayer structure. However, compared with the freezedried scaffolds, the mechanical properties of the saltleached scaffolds did not improve with excess NaCl crystals in the solution. Note that the NaCl content
The freeze-drying method is usually used to prepare gelatin scaffolds, because gelatin is a water-soluble natural polysaccharide. However, gelatin-based sponges formed using the freeze-drying method have a dense surface that does not allow cells to penetrate into the inner areas when the cell suspension is seeded on the scaffold [1–3]. This dense gelatin layer may contribute to the uniaxial orientation of the pore channels in the upper part of the scaffold, because the upper part of the sponge is directly exposed to the vacuum, and has a different structure to the lower region in contact with the mold surface. A dense surface on the wounddressing material is suitable for protecting the scaffold from evaporating exudates from the wound, and from the invasion of bacteria into the wound. However, a film-like surface has a detrimental effect on cell migration in seeding cells on the surface of a scaffold. Thus, we felt it was necessary to introduce another technique, such as the salt-leaching method, to prepare
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Fig. 7. Masson’s trichrome staining of the wounds implanting cellular gelatin with human fibroblasts scaffolds on the back of athymic mice for 1 week (a) and 2 weeks (b); acellular gelatin scaffolds implanted for (a) 1 week and (b) 2 weeks (original magnification, 40).
Fig. 6. Histological evaluation of human fibroblasts-cultured gelatin scaffold in vitro (2.5 106 cells/cm2) of Gs-50 on (a) 1 week and (b) 2 weeks (Masson’s trichrome staining, original magnification, 40).
added to the polymer solution was 40–70-fold that of gelatin. To maintain the scaffold construction after seeding the cells, the scaffolds have to be mechanically strong, because the dermis has to provide physical strength and flexibility to the skin as well as the scaffold to support extensive vasculatures, the lymphatic system, nerve bundles, and other structures in the skin. However, the mechanical strength of the Gf scaffolds was not sufficient in that the attached cells shrank the scaffold. Morphologically, the 0.8, 0.9, and 1.0 wt% Gf scaffolds maintained their original size after seeding cell, whereas low concentration gelatin scaffolds (0.5, 0.6, and 0.7 wt%) were contracted by seeded fibroblasts (shrinkage values=40, 40, and 63%, respectively). Furthermore, the high concentration of gelatin required to avoid the shrinkage of the Gf scaffolds inhibited cell migration after cell seeding. On the other hand, the Gs scaffolds had satisfactory mechanical properties after seeding with the same number of cells, because the high
initial concentration of gelatin (25 wt%) formed a smaller number of thicker walls between the pores of the scaffolds, whereas the Gf scaffolds had a relatively large number of walls, which were thinner than those of the Gs scaffolds. A highly porous scaffold structure assists both cell penetration and polymer degradation. The rate of degradation is affected by the morphology of the scaffold, and a large surface area speeds up the diffusion of water molecules into the bulk of the polymers when they are placed in an aqueous environment (e.g., in vivo) [12–18]. Thus, the degradation behavior was investigated as a function of the degree of porosity. As shown in Fig. 5, the rate of weight loss decreased with increasing porosity of the gelatin scaffold. The higher degradation in scaffolds Gf-0.5 and Gs-70 was probably due to the high porosity of the matrix and subsequent increase in the accessibility of active sites to the enzyme, whereas the relatively slow degradation rate of scaffolds Gf-2.0 and Gs-40 was probably due to the dense surface and the closed pores that retarded the migration of the enzyme. Note that the remaining weight (%) in the Gs scaffolds did not rapidly decrease and remained relatively constant compared to that of the Gf scaffold. The in vivo tests showed that the artificial dermis, rather than the acellular sponge, improved the reepithelialization on the full-thickness skin defect. This was caused by the effect of the fibroblasts on the artificial dermis. Several studies have indicated that the presence of fibroblasts in a dermal equivalent
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stimulates epidermal differentiation [19–21]. It is assumed that the presence of fibroblasts in a dermal equivalent accelerates the healing process by reducing the time needed for the fibroblasts to invade the wound tissue, and by the early synthesis of new skin tissue, because the fibroblasts on the artificial dermis can release biologically active substances, such as cytokines [19,21]. These cytokines are stimulated by the increased proteolytic activity leading to the fragmentation of more proteins and the activation of larger numbers of latent growth factors, resulting in a higher chemotactic activity of the wound tissue [19].
5. Conclusions A new gelatin scaffold was designed and prepared using the salt-leaching method. The scaffolds prepared had interconnected pore structures, but did not form a membrane-like structure between the pores. The mechanical strength and the rate of biodegradation were easily modulated by the addition of salt, which affected the porosity of the scaffold. The fibroblasts after 1 week of in vitro culturing also showed a good affinity to the scaffold, and were mainly distributed on the surface of the macro-pores formed in the salt-leached scaffold. The fibroblasts attached on the surface of pores were proliferated over the entire area of the scaffold after 2 weeks. In vivo, the artificial dermis rather than the acellular sponge improved the re-epithelialization on a full-thickness skin defect.
Acknowledgements Financial support from the Korea Food and Drug Administration (KFDA) under the program year 2003 is greatly appreciated. SBL and YHK appreciate the fellowship from BK21 program. References [1] Choi YS, Hong SR, Lee YM, Song KW, Park MH, Nam YS. Study on gelatin-containing artificial skin: I. Preparation and characteristics of navel gelatin-alginate sponge. Biomaterials 1999;20:409–17. [2] Choi YS, Hong SR, Lee YM, Song KW, Park MH, Nam YS. Study on gelatin-containing artificial skin: II. Preparation and characterization of cross-linked gelatin-hyaluronate sponge. J Biomed Mater Res 1999;48(5):631–9. [3] Choi YS, Lee SB, Hong SR, Lee YM, Song KW, Park MH, Nam YS. Studies on gelatin-based sponges. Part III: a comparative study of cross-linked gelatin/alginate, gelatin/hyaluronate and chitosan/hyaluronate sponges and their application as a wound dressing in full-thickness skin defect of rat. J Mater Sci Mater M 2001;12:67–73.
[4] Hong SR, Lee SJ, Shim JW, Choi YS, Lee YM, Song KW, Park MH, Nam YS, Lee SI. Study on gelatin-containing artificial skin IV: a comparative study on the effect of antibiotic and EGF on cell proliferation during epidermal healing. Biomaterials 2001;22:2777–83. [5] Mooney DJ, Baldwin DF, Suh NP, Vacanti JP, Langer R. Novel approach to fabricate porous sponges of poly(D,L-lactic-coglycolic acid) without the use of organic solvents. Biomaterials 1996;17:1417–22. [6] Murphy WL, Kohn DH, Mooney DJ. Growth of continuous bonelike mineral within porous poly(lactic-co-glycolide) scaffolds in vitro. J Biomed Mater Res 2000;50(1):50–8. [7] Sherwood JK, Riley SL, Palazzolo R, Brown SC, Monkhouse DC, Coates M, Griffith LG, Landeen LK, Ratcliffe A. A threedimensional osteochondral composite scaffold for articular cartilage repair. Biomaterials 2002;23:4739–51. [8] Hua FJ, Park TG, Lee DS. A facile preparation of highly interconnected macroporous poly(D,L-lactic acid-co-glycolic acid) (PLGA) scaffolds by liquid–liquid phase separation of a PLGA–dioxane–water ternary system. Polymer 2003;44:1911–20. [9] Whang K, Thomas CH, Healy KE. A novel method to fabricate bioabsorable scaffolds. Polymer 1995;36:837–42. [10] Hou Q, Grijpma DW, Feijen J. Porous polymeric structures for tissue engineering prepared by a coagulation, compression moulding and salt leaching technique. Biomaterials 2003;24: 1937–47. [11] Ma J, Wang H, He B, Chen J. A preliminary in vitro study on the fabrication and tissue engineering applications of a novel chitosan bilayer material as a scaffold of human neofetal dermal fibroblasts. Biomaterials 2001;22:331–6. [12] Chen G, Ushida T, Tateishi T. A biodegradable hybrid sponge nested with collagen microsponges. J Biomed Mater Res 2000; 51:273–9. [13] Freed LE, Grand DA, Lingbin Z, Emmanual J, Marquis JC, Langer R. Joint resurfacing using allograft chondrocytes and synthetic biodegradable polymer scaffolds. J Biomed Mater Res 1994;28:891–9. [14] Freed LE, Marquis JC, Nohria A, Emmanual J, Mikos AC, Langer R. Neocartilage formation in vitro and in vivo using cells cultured on synthetic biodegradable polymers. J Biomed Mater Res 1993;27:11–23. [15] Oberpenning F, Meng J, Yoo JJ, Atala A. De novo reconstitution of a functional mammalian urinary bladder by tissue engineering. Nat Biotechnol 1999;17:149–55. [16] Zacchi V, Soranzo C, Cortivo R, Radice M, Brun P, Abatangelo G. In vitro engineering of human skin-like tissue. J Biomed Mater Res 1998;40:187–94. [17] Ishaug SL, Crane GM, Miller MJ, Yasko AW, Yaszemski MJ, Cusick RA, Lee H, Sano K, Pollok JM, Utsunomiya H, Ma PX, Langer R, Vacanti JP. The effect of donor and recipient age on engraftment of tissue-engineered liver. J Pediatr Surg 1997; 32:357–60. [18] Dunn MG, Liesch JB, Tiku ML, Zawadsky JP. Development of fibroblast-seeded ligament analogs for ACL reconstruction. J Biomed Mater Res 1995;29:1363–71. [19] Quirk RA, Chen WC, Davies MC, Tendler SJB, Shakesheff KM. Poly(L-lysine)-GRGDS as a biomimetic surface modifier for poly(lactic acid). Biomaterials 2001;22:865–72. [20] Lee DY, Ahn HT, Cho KH. A new skin equivalent model: dermal substrate that combines de-epidermized dermis with fibroblast-populated collagen matrix. J Dermatol Sci 2000;23: 132–7. [21] Bernard C, Corinne L, Louis D. Influence of human dermal fibroblasts on epidermalization. J Invest Dermatol 1989;92: 122–5.