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Biological characterization of EDC-crosslinked collagen–hyaluronic acid matrix in dermal tissue restoration
Biomaterials 24 (2003) 1631–1641
Biological characterization of EDC-crosslinked collagen–hyaluronic acid matrix in dermal tissue restoration Si-Nae Parka, Hye Jung Leeb, Kwang Hoon Leeb, Hwal Suha,c,* a
Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-ku, Seoul 120-752, South Korea b Department of Dermatology, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-ku, Seoul 120-752, South Korea c Brain Korea 21 Project Team for Medical Science, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-ku, Seoul 120-752, South Korea Received 23 April 2002; accepted 28 October 2002
Abstract Porous collagen matrices crosslinked with various amounts of hyaluronic acid (HA) by 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) were developed as scaffolds for dermal tissue regeneration. The effect of HA on cells in accordance with HA concentrations in the collagenous matrices was investigated using cultures of fetal human dermal fibroblasts, and the effect of EDC– crosslinked collagen–HA matrix on wound size reduction was also evaluated in vivo. scanning electron microscopic views of the matrices demonstrated that all of the collagen–HA matrices had interconnected pores with mean diameters of 150–250 mm. An HA matrix retention test showed that the concentration of HA decreased slowly after an initial rapid decrease over 24 h. Fetal human dermal fibroblasts adhered well to all of the collagen-based matrices as compared with the Porous polyurethane matrix used as a control. An 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide based proliferation test and the hematoxylin and eosin staining of a 2 week cultured matrix showed that the proliferation of fibroblasts was enhanced on a 9.6% HA contained collagen matrix. No significant difference was in terms of fibroblast migration into the various types of scaffolds as HA content was increased. In vivo testing showed that dermis treated with collagen or collagen–HA matrix was thicker than the control, and epithelial regeneration was accelerated, and collagen synthesis increased. However, no significant effect of HA on wound size reduction was found. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Collagen; Hyaluronic acid; EDC crosslinking; Fetal dermal fibroblast; Wound healing
1. Introduction The role of collagen at each phase of wound healing is well understood and appreciated. The major applications of collagen-based dressings introduced to date include, collagen films, collagen gels and collagen sponges [1–4]. Hyaluronic acid (HA), an important component of extracellular matrix, has been used as, a viscoelastic biomaterial for medical purposes, in cosmetics because of its high water retention capacity, and in drug delivery systems because of its biodegradability.
*Corresponding author. Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-ku, Seoul 120-752, South Korea. Tel.: +82-2-361-5406; fax: +82-2-363-9923. E-mail address: [email protected] (H. Suh).
HA exists in high concentrations during fetal skin development, is involved in cell migration and differentiation, and is the first macromolecule to appear in the extracellular matrix during wound healing [5,6]. It has been reported that the addition of HA promotes cell division in human fibroblasts by their passage though cell cycles when incorporated within a collagen matrix [7]. In contrast, it has also been reported that exogenous HA significantly inhibits fetal fibroblast proliferation. Moreover, HA was found to induce, inhibit or not to affect fibroblasts migration, depending on the tissue origin of fibroblasts, while fibroblast proliferation was never affected [8]. In these reports, the effects of soluble HA in the medium on fibroblasts were discussed, whereas little information was available on HA concentrations in the collagenous matrix. In a recent study, we fabricated porous collagenous matrix containing HA using a freeze-drying technique,
0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 2 ) 0 0 5 5 0 - 1
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and crosslinked the system with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) to achieve matrix mechanical stability [9]. Membranes produced from this material had good resistance to enzymatic degradation and acceptable toxicologically. The aim of this study was to determine the optimal concentration of HA in EDC-crosslinked porous collagen matrices used as a scaffold for dermal tissue restoration. Biological characteristics of the HA crosslinked in collagen matrices in terms of, the enhancement of attachment, proliferation and migration of fibroblasts in vitro, and upon wound size reduction after placement on full-thickness dermal defects in an animal model were also observed.
2. Materials and methods 2.1. Fabrication and crosslinking of porous collagen– hyaluronic acid matrix The porous collagen–HA matrix was fabricated as previously reported [9] with some modification. Briefly, HA (sodium salt, MW=120,000–150,000, Hanwha group R&E Center, Daejeon, Korea) as an aqueous solution was added to a 1% type I atelocollagen (RBC I, Regenmed, Seoul, Korea) dispersion, to produce 0%, 5%, 10%, and 20% (w/w) HA/collagen mixtures. The mixtures were homogenized at 8000 rpm for 3 min at 41C using a homogenizer (T25 basic, IKA Works, Selangor, Malaysia) and the pH was adjusted to 7.4 by dropping 1 n NaOH. The resulting slurry was poured into a polystyrene Petri dish, frozen at 701C and lyophilized at 501C for 24 h. The dried porous collagen–HA matrices were immersed in a 50 mm EDC (Sigma Chemical Co., St. Louis, MO, USA) solution (H2Oethanol=595) for 24 h to orient the crosslinking. The matrices produced were thoroughly washed in distilled water using a sonicator, and then relyophilized at 501C. Porous polyurethane (PU) matrix, which has been widely used as a wound dressing or skin substitute, was used as the control and prepared by a solventcasting particulate-leaching technique [10]. PU (Tecoflexs, Thermedics Inc., Woburn, MA, USA) was dissolved in chloroform (Sigma, St. Louis, MO, USA) to a final concentration of 12% (w/w). Sieved NaCl particles (150–250 mm), were added in a 12% PU solution and the manually mixed dispersion was cast in a glass Petri dish. The solvent was allowed to evaporate from the covered Petri dish over 48 h in a vacuum oven, and the resulting PU/salt composite membranes were immersed in distilled water on a shaker at room temperature for 48 h to leach out remaining salt.
2.2. Morphological characterization of crosslinked collagen matrices containing various amounts of HA To determine the amount of HA remaining in a collagen–HA matrix after the crosslinking process, the glucosamine contents of the HA molecules in the matrices were analyzed using the p-dimethylaminobenzaldehyde reaction [11]. The concentration of glucosamine was assayed spectrophotometrically (UV-1601S, Pharmacia Biotech, Uppsala, Sweden) at 527 nm against a reagent blank. The porosities of matrices containing various contents of HA after crosslinking were characterized under the scanning electron microscope (SEM) (S-800, HITACHI, Tokyo, Japan) using an image analyzing system (Escan 4000, Bum-Mi Universe Co., Ltd., Ansan, Korea). The ( membranes were coated with an ultrathin layer (300 A) of gold/Pt in an ion sputter (E1010, HITACHI, Tokyo, Japan). The image analysis program was used to determine the average diameter of pores and at least 40 pores were assessed. 2.3. HA retention in crosslinked collagen–HA matrices and water uptake ability To predict the elution rate of HA from the collagen– HA matrices under culture conditions, membranes containing various amounts of HA were prepared as disks (9 mm in diameter and 2 mm in thickness). The matrices were then immersed in 5 ml of Dulbecco’s modified Eagle’s medium (DMEM, JBI Inc, Daejeon, Korea) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/amphotericin-B, and incubated at 371C in an atmosphere of 5% CO2 and 95% air. Samples were withdrawn, freeze-dried and weighed after 3, 7, 14, 21 and 28 days of incubation. The glucosamine contents of the retained HA molecules in the matrices were analyzed by using the p-dimethylaminobenzaldehyde reaction, as described above. For water uptake ability testing, the EDC-crosslinked matrices or the PU matrix were separately immersed in distilled water at room temperature for 10 min. After being removed from the water, they were hung over a table for 1 min until no water dripped from them and then weighed. The water uptake of the matrices was calculated by the following equation: Water uptake ð%Þ ¼ ½ðWs Wd Þ=Ws 100; where Wd is the weight of the dry matrix and Ws is the weight of the swollen matrix (Table 1). 2.4. Cell culture Fetal human dermal fibroblasts were obtained from the Department of Plastic Surgery at the Yonsei Medical Center, having obtained permission from the Medical
S.-N. Park et al. / Biomaterials 24 (2003) 1631–1641 Table 1 The change of HA content in collagen–HA matrices during the crosslinking process and the water uptake of various types of matrices Samples
HA 0% matrix HA 3.2% matrix HA 5.4% matrix HA 9.6% matrix PU matrix
HA content in the matrices (wt%) Before crosslinking (%)
After crosslinking
0 5 10 20
0% 3.2%70.18 5.4%70.28 9.6%70.24
Water uptake
98.4370.072 98.3370.060 98.3270.068 98.2670.149 51.9771.57
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A porous collagen matrix was seeded with a high density of dermal fibroblasts (1 106/membrane) and cultured for 2 weeks. This cell populated matrix (CPM) was then overlaid with a cell-free porous collagen–HA or a cell-free porous PU matrix. A PTFE-disk (weight=0.95 g) was placed onto the loaded matrix to prevent it from floating in the culture medium. After 24 h of additional culture, the CPM and the PTFE disk were removed, and cells that had migrated into the overlaid collagen–HA or PU matrices were quantified using the MTT test. 2.7. In vivo animal testing
Material Control and Management Committee of Yonsei University; the donor’s age was 19 weeks. Outgrown cells were taken from primary culture, following established methods. Cells were cultured in 175 cm2 tissue culture flasks (NUNC, Roskilde, Denmark) using growth medium composed of DMEM (JBI Inc, Daejeoun, Korea), 1% penicillin/streptomycin/ amphotericin-B, and 10% FBS. Cultures were incubated in a humidified incubator in an atmosphere of 5% CO2 and 95% air. Confluent monolayers were propagated by trypsinization (0.25% trypsin, 0.02% EDTA solution) and replating at 12 dilution. For the experiments, fibroblasts were used in the fifth–seventh passages. 2.5. Cell attachment and proliferation testing Cell suspension containing 1 105 cells were placed onto the PU matrix and each crosslinked collagen matrix (as matrices of 10 mm in diameter, 1 mm in thickness). Culture medium (1 ml) was added 1 h after seeding, and cells were allowed to attach for 4 h. For proliferation testing, 5 104 cells were seeded onto each of the matrices and cultures were harvested after 1, 3, 7 or 14 days, the cell attached matrices were gently washed three times with phosphate buffered saline solution, and the attached or proliferated cells were then quantified by the 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay [12,13]. Fifty microliters of MTT solution (5 mg/ml in 0.9% NaCl, filter-sterilized) and 200 ml of medium were added to each culture well. After incubation for 4 h, the MTT reaction medium was removed, and 300 ml of dimethylsulfoxide and 25 ml of Sorensesn’s glycine buffer (0.1 m glycine, 0.1 m NaCl adjusted to pH 10.5 with 1 m NaCl) was added. Optical densities were determined by ELISA reader (Spectra Max 340, Molecular Device Inc., CA, USA) at a wavelength of 570 nm. 2.6. Cell migration test A three-dimensional migration system was employed to investigate the ability of fibroblasts to migrate.
Two types of matrix specimens, based on an EDCcrosslinked collagen matrix and an EDC-crosslinked collagen–HA matrix containing 9.6% of HA were investigated for the treatment of full thickness skin defects in a guinea pig model. The animal experiment procedure was managed in accordance with the Guidelines and Regulations for Use and Care of Animals in Yonsei University. Guinea pigs, weighing 200–400 g, were anesthetized with a ketamine (Yuhan Co. Soul, Korea) by intraperitoneal injection, and the dorsal hair were shaved. After disinfecting the skin with Betadines (Hyundai Pharm., Seoul, Korea), two full thickness round shaped skin defects (2 cm in diameter) were prepared by excising the dorsal skin of each guinea pig. The wound was covered with each type of sterilized matrix or uncovered as a control. Commercial polyurethane matrix (Allevyns, Smith & Nephew Healthcare Ltd., Hull, UK) was used to protect the specimens. 3, 7, 14 or 21 days after implantation, the guinea pigs were sacrificed, and wound sizes were measured. The percentage wound size reduction was calculated according to the following formula Cn ¼ ½ðS0 Sn ÞS0 = 100; where Cn is the percentage of wound size reduction on 3, 7, 14 or 21 days after implantation, S0 the initial wound size, Sn the wound size on 3, 7, 14 or 21 days after implantation. For histological examination, tissue specimens from animal wounds were obtained by excising the wound including the surrounding rim of normal skin and underlying fascia, and muscle. The tissue specimens were then fixed in 10% neutral-buffered formalin solution, embedded in paraffin wax, sectioned at 3 mm, and stained with hematoxylin–eosin (H–E) in the conventional manner. Inflammation, neovascularization, and fibroblast proliferation in the lesion were evaluated under the microscope using a grading system as delineated in Table 2. Measurements were carried out at a magnification of 400 .
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2.8. Statistical analysis Significance levels were determined by ANOVA or the Student’s t-test. Multiple comparisons were carried out using the Tukey method. All statistical calculations were performed on the SAS system for Windows (version 8.00, SAS Institute Inc., Cary, NC, USA). p-values of o0.05 were considered significant.
3. Results and discussion 3.1. Morphological characterization of crosslinked collagen matrices containing various amounts of HA The porous collagen matrices with different HA contents were fabricated by freeze-drying and crosslinked using EDC. The loss of HA, a soluble polysaccharide, could be expected during crosslinking and washing processes. For this reason, the final HA content in the matrices was analyzed by determining the percentage of glucosamine present in the EDC-crosslinked membrane. The result revealed that HA content of the crosslinked matrices, in which the initial HA content was 5%, 10% or 20% (w/w) had decreased to 3.2%, 5.4% or 9.6% (w/w), respectively (Table 1). HA concentrations exceeding ca. 15% made handling of the dried uncrosslinked matrices difficult in a humidified atmosphere. Therefore, crosslinked collagen matrices with 0–9.6% HA content were selected for the experiments described in the present study. SEM observation demonstrated that the cross-section and surfaces of the membranes containing various amounts of HA had interconnected pores (Fig. 1 (A–D and F–I). Generally, the inner three-dimensional structure of such structures, including pore size and porosity has been shown to modulate cell behavior [14]. Allemann et al. [15] reported that the addition of HA influenced the density and the pore size of collagen–HA matrices. The result may have been because the concentration of the HA solution used (1% w/v) was higher than that of collagen solution (0.5% w/v) which would have caused the increment of the density of the mixed solution containing HA. To minimize pore size changes, we used a 1% (w/v) collagen dispersion and a
1% (w/v) HA solution for fabricating membranes. Image analysis revealed that all the fabricated membranes had nearly the same distribution of pore sizes (Fig. 2). Although the pore structure of membranes became more irregular and fibrous as the HA level was increased, the HA contained in the membrane did not significantly affect the pore size. Fig. 1 (E and J) shows the surface and cross-sectional morphology of a porous PU membrane matrix manufactured using 150–250 mm sodium salt as a porogen. Porous PU membrane was used as a control material for cell attachment, proliferation and migration testing in the present study. 3.2. Water uptake, HA retention and weight changes of collagen–HA membranes during culture Ikada et al. [16] reported that the crosslinking through the esterification of its carboxylic groups like this yields a product with the low water content such as 60– 90 wt%. In their experiment, the water content of HA film crosslinked with 50 mm EDC in 75% ethanol was about 95%. This might explain that our result where HA contained in the matrices did not affect the water uptake significantly (Table 1). In the HA retention test (Fig. 3), the HA content of the matrices decreased only slowly after the initial rapid release. After 4 weeks of incubation, the HA content of the matrices that had contained 3.2%, 5.4% and 9.6% w/w HA decreased to 0.8%, 1.0% and 1.5% w/w in the dried samples, respectively. HA elution might be accelerated initially by the rapid hydrolysis of ester bond between HA molecules and by the high water uptake into the porous collagen–HA matrices (Table 1). As described in our previous paper [9], EDC mediates acid anhydride formation between two carboxyl groups of HA and the resultant acid anhydride may readily react with a hydroxyl group of HA to yield an ester bond, which functions as a crosslink of polysaccharide molecule and can be easily attacked by water molecules. Fig. 4 shows the degradation profile of collagen-based membranes containing various ratios of HA under in vitro culture conditions. The weight of the collagen–HA matrices containing 0%, 3.2%, 5.4% or 9.6% (w/w) of
Table 2 Indices of inflammation, new vascularization and fibroblast proliferation in histological examination Inflammation
New vascularization
Fibroblast proliferation
Classification
The number of infiltrated leukocytes
+ ++ +++ ++++
0–10 11–100 101–500 >501
The number of lumen-included RBC or lumen lined with endothelial cells 0–10 11–20 21–30 >31
The number of proliferated fibroblasts 0–100 101–500 501–1000 >1001
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Fig. 1. (A–E) Scanning electron micrographs of the surface of EDC-crosslinked collagen matrices with various contents of HA (A) collagen (HA 0%) matrix; (B) HA 3.2% (w/w) matrix; (C) HA 5.4% (w/w); and (D) HA 9.6% matrix, and the surface of the PU matrix (E). (F–J) Scanning electron micrographs of the cross-sections of EDC-crosslinked collagen matrices with various contents of HA(F) collagen (HA 0%) matrix; (G) HA 3.2% (w/w) matrix; (H) HA 5.4% (w/w); and (I) HA 9.6%, and the cross-section of the PU matrix (J).
HA decreased to 93%, 92.5%, 85% or 81% (w/w) of the original weights after a 3-week period. The masses of the 0% and 3.2% HA matrices decreased to about 94% of the original mass over the first 3 days, and then plateaued. Matrices containing 5.4% or 9.6% (w/w) HA degraded to 86–80% of the original weights gradually over a 2-week culture period. These results indicated that collagen in the matrix was more resistant to the hydrolytic degradation compared with HA and that the weight changes of collagen–HA matrices primarily reflect the solubilization of free HA [17]. Although the fabricated matrices had been washed by sonication, it was very hard to eliminate perfectly the free or weakly bound molecules from the matrices because the washing for longer time could cause the disruption of the matrices. The sonication in fresh distilled water for 30 s five times could remove the residual EDC from the matrices and no signs of cytotoxic effect of residual EDC was observed as described in our previous paper [9]. However, it might be expected that the unbound or weakly crosslinked collagen and HA molecules could be remained in the matrices. 3.3. Attachment and proliferation of fetal human dermal fibroblasts on the various types of matrix Fetal fibroblasts attached better to all the collagenbased matrices than to the PU matrix (po0:05) (Fig. 5). However, no significant effect of HA content in the
Fig. 2. The effect of HA contents on pore size of the collagen–HA scaffold. &: pore size at the surface of matrices; ’: pore size of matrix cross-section. At least 40 pores were assessed, values were statistically analyzed and expressed as means7standard deviation.
collagen matrices on cell attachment was found. SEM observations revealed that the fibroblasts attached evenly either on the collagen or collagen–HA matrices (Fig. 6A and B). The majority of the cells in the PU matrix were aggregated and localized in the pores (Fig. 6C). This may have been because the water uptake capacity of PU matrix was less than that of collagen– HA matrices, as a PU surface consists of both hydrophilic and hydrophobic phases. This property might limit cell adsorption, and non-adsorbed fibroblasts might then adhere to each other.
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Fig. 3. Remaining weight (wt%) of HA in EDC-crosslinked collagen– HA matrices versus culture time. Three matrices of each type were examined on each occasion.
Fig. 4. Degradation rate of EDC-crosslinked collagen–HA matrices under culture conditions. There are significant differences between 5.4% or 9.6% HA contained matrices and 0% HA contained matrix in the remaining weights on 1, 7, 14 and 21 days after incubation. (npo0.05) ’: collagen matrix (HA 0%); E: HA 3.2% (w/w) matrix; m: HA 5.4% (w/w) matrix; and K: HA 9.6% (w/w) matrix.
It has been repeatedly reported that HA might modulate cell proliferation [18,19]. Greco et al. reported that the addition of HA promotes cell division in human fibroblasts by accelerating their passage though the cell cycle, when it is incorporated within a collagen matrix [7]. However, others have reported that exogenous HA at 1–100 mg/ml inhibits the proliferation of rabbit dermal fibroblasts [20]. Additional HA of high molecular weights or high concentrations, inhibited the proliferation of rabbit synovial cells in culture [21], and HA also inhibited the multiplication of embryonic chick skin fibroblasts in different environments [22]. The MTT test as used in this study is a nonradioactive cell proliferation assay that identifies living
Fig. 5. Cell attachment assessed by MTT testing performed 4 h after seeding. The data reported for the control (PU), collagen (HA 0%), HA 3.2%, HA 5.4% and HA 9.6% matrices are means7SD for n ¼ 5: Increased cell attachment on collagen or collagen–HA matrices was significantly different from the control (PU matrix) (po0.05).
cells, and is based on the cellular conversion of a tetrazolium salt into a formazan product, a chromaphore, which can be quantified by enzyme-linked immunosorbent assay (ELISA) reader [22]. This MTTbased proliferation test revealed that cell proliferation on the PU matrix, collagen and collagen–HA matrices was enhanced significantly compared with that on the control (tissue culture plate, TCP) and cells proliferated more rapidly on the 9.6% HA contained collagen–HA matrix than other matrices 2 weeks after seeding (po0.005) (Fig. 7). In particular, fibroblasts exhibited an outstanding proliferation rate in the 9.6% HA containing matrix, as they increased approximately fivefold during 2 weeks in culture. These 2-week cultured matrices showed a progressive penetration of predominantly elongated cells throughout the collagen network and an increased cell density in the collagen–HA matrix as compared with the collagen matrix (Fig. 8). The proliferation rates were not different significantly between the 0% and 5.6% HA containing matrices. The effect of HA on cell proliferation is usually discussed in terms of its mechanical effects on cells and the extracellular matrix. The physiological effect of HA may lie in its ability to act as an accessory receptor in cooperative ligand-binding pathways. HA may not only bind peptide including growth factors, which increase the effective concentration of these factors at the cell surface, but also attract integrins to the cell surfaces [23]. Cell number was maintained in culture on the tissue culture plate (TCP) 1 week after seeding probably because of the relatively limited space. The cell proliferation into a PU matrix has a similar growth pattern to that on TCP, but the relatively higher cell population in the PU matrix might be due to its
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Fig. 6. Scanning electron micrographs of cells attached on a collagen matrix (A); a collagen matrix containing 9.6% HA (B); and a PU matrix (C). (magnification 100).
3.4. Migration of dermal fibroblasts from cell populated collagen matrix to various other types of matrix
Fig. 7. Proliferation curves for fetal human dermal fibroblasts on various types of matrices. E: TCP (tissue culture plate); ’: PU matrix; m: collagen (HA 0%) matrix; &: HA 3.2% matrix; * : HA 5.4% matrix; and K: HA 9.6% matrix.
Fig. 8. Routine histological observations of cell proliferation in matrices. Proliferation of dermal fibroblasts in collagen matrices containing HA 0% (A) or HA 9.6% (B) after 2 weeks of culture. (Magnification 40).
three-dimensional structure, which may provide sufficient space for cell growth as compared to twodimensional culture on TCPs.
HA is known to stimulate the migration of a large variety of cell types. Stimulation of cell migration by HA has been explained to occur by different mechanisms. HA was shown to specifically bind to cell surface receptors, and this inhibition of cellular HA receptor function was demonstrated to decrease cell migration and tumor growth [24]. The increase or decrease of HA during specific phases in embryogenesis, under certain pathological conditions, and the discovery that the great majority of cells possess HA receptors, is also consistent with a role for HA in cell migration and function [25– 28]. HA accumulates in the early phases of inflammation and wound repair, where it might provide a matrix favoring cell adhesion and migration [29]. We used a three-dimensional migration system to investigate the early migration of dermal fibroblasts from wounded dermal tissues to the regenerative matrices. The basis of this system was a cell populated collagen matrix (CPM), which had been cultivated for 2 weeks in a static state and was used as a dermis-like tissue. The effect of HA on the migration of dermal fibroblasts from the CPM is shown in Fig. 9. Our results revealed that the migrations of cells to the collagen matrix and the collagen–HA matrices were enhanced compared with the PU matrix, and that collagen–HA matrices containing 5.4% or 9.6% HA increased the number of migrated cells significantly (p ¼ 0:00421 and 0.00034, respectively). 3.5. Effect of crosslinked collagen–HA matrices on wound size reduction in vivo The fetal wound-healing matrix is exceptionally rich in HA. Fetal wounds heal without scarring or contraction. Shepard et al. reported that adult dermal
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Fig. 9. Migration of fetal human dermal fibroblasts from cell populated matrices (CPMs) to various other types of matrices. PU: cell migration to a PU matrix; 0% HA: cell migration to a collagen (HA 0%) matrix; 3.2% HA: cell migration to an HA 3.2% matrix5.4% HA: cell migration to an HA 5.4% HA matrix; and 9.6% HA: cell migration to a 9.6% HA matrix. Cell migration to the 5.4% HA matrix and the 9.6% HA matrix was higher than that to a PU matrix (n ¼ 7; npo0.05).
fibroblasts cultivated in the presence of 5 mg/ml of exogenous HA changed in morphology, and that the wound closed more quickly than control cultures [23]. In our in vitro experiments, the 9.6% HA contained matrix showed the better effects on proliferation and migration compared with other matrices. Therefore, in vivo study, the effects of collagen matrix, 9.6% HA contained collagen–HA matrix were compared with a control group. Fig. 10 shows changes in the mean wound area in groups undergoing full-thickness skin grafting at various times after treatment with collagen or collagen–HA matrices. In this entire group, the wound size reduction increased gradually to reach about 95% 2 weeks after the creation of the skin defects. On postoperative days 3 and 7, wound contraction in the collagen–HA matrix treated group increased to 53.2% and 73.6% of the original area, respectively. On the other hand, wound contraction increased to 53.2% and 62.8% in the collagen matrix treated group and to 42.4% and 60.3% in control groups, respectively. However, no significance of HA upon wound size reduction was found in this in vivo study. Possible reasons proposed for these results by authors were attributed to this animal model using guinea pigs and the matrix stability. The rate of wound healing was so fast that the wound size was reduced to above >50% in 3 days and the wounds were healed completely within 2 weeks. Therefore, it was rather difficult to find the significant difference in the wound size reduction during healing process in this study. It was also likely that the degradation of the collagen–HA matrix could be
Fig. 10. Wound contraction of a full thickness dermal defect in guinea pig. No significant effect of treatment on wound contraction was found (means7standard error of the mean). E: no treatment (control); ’: EDC-crosslinked collagen matrix; m: EDC-crosslinked collagen–HA matrix.
accelerated in vivo and that result in the higher initial release of HA from the crosslinked matrix compared with in vitro degradation. The histological appearance at low-power magnification of the wound tissue in the three groups is shown in Figs. 11–14. With a collagen matrix or a collagen–HA matrix, inflammatory response tended to be pronounced on postoperative day 3 (Fig. 11), and then gradually subsided. On day 7, as shown in Fig. 12, capillaries appeared and new epithelialization was initiated from the edge of the wound. After 14 days, a proliferation of fibroblasts and thickened collagen fibers were seen, blood vessels were decreased and the regeneration of epithelium was observed (Fig. 13). Dermis treated with regenerative matrices was thicker than the control; epithelial regeneration was accelerated and collagen synthesis increased compared with the control (Table 3). Histological analysis revealed that 21 days after wounding, epithelialization was complete in all wounds (Fig. 14). Wounds treated with collagen–HA matrix showed a similar histology to that of the collagen matrix treated group.
4. Conclusion Porous collagen–HA matrix containing interconnected pores with mean diameters of 150–250 mm was fabricated by freeze-drying and EDC crosslinking. In culture conditions, the HA concentration in the matrices decreased slowly after an initial rapid decrease. Fibroblasts attached well to all the collagen-based matrices
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Fig. 11. Photomicrographs of biopsy specimens from an untreated wound (A); from a wound treated with a crosslinked collagen matrix (B); a collagen–HA matrix (C) on postoperative day 3 (magnification 40).
Fig. 12. Photomicrographs of biopsy specimens from an untreated wound (A); from a wound treated with a crosslinked collagen matrix (B); or treated with a crosslinked a collagen–HA matrix (C) on postoperative day 7 (magnification 40).
Fig. 13. Photomicrographs of biopsy specimens from an untreated wound (A); from a wound treated with a crosslinked collagen matrix (B); or treated with a crosslinked a collagen–HA matrix (C) on postoperative day 14 (magnification 40).
Fig. 14. Photomicrographs of biopsy specimens from an untreated wound (A); from a wound treated with a crosslinked collagen matrix (B); or treated with a crosslinked a collagen–HA matrix (C) on postoperative day 21 (magnification 40).
compared with the PU matrix and the proliferation of fibroblasts was enhanced by the HA 9.6% matrix as compared with the PU matrix or TCP, 14 days after cell
seeding. Cell migration from CPM to collagen matrix or collagen–HA matrices was enhanced compared with that from CPM to the PU matrix. Collagen–HA
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Table 3 Histologic examination of tissue 3, 7 and 14 days after implantation Days
Control
Collagen matrix
Collagen–HA matrix
Inflammation
3 7 14 21
+++ + + +
+++ + + +
+++ + + +
New vascularization
3 7 14 21
++ +++ + +
+++ ++++ ++ +
+++ ++++ + +
Fibroblasts proliferation
3 7 14 21
++ +++ +++ +++
+++ ++++ ++++ +++
+++ +++ ++++ +++
matrices containing 5.4% or 9.6% HA increased migrating cell numbers significantly. In animal testing, however, no significant effect of HA upon wound size reduction was found.
[10]
[11]
Acknowledgements This work was supported by a grant from the Ministry of Information and Communication (Project no. 01-PJ11-PG9-01NT00-0045), Korea.
[12]
[13]
References [1] Balasubamani M, Kumar TR, Babu M. Skin substitutes: a review. Burns 2001;27:534–44. [2] Maeda M, Kadota K, Kajihara M, Sano A, Fugioka K. Sustained release of human growth hormone (hGH) from collagen film and evaluation of effect on wound healing in db/db mice. J Control Release 2001;77:261–72. [3] Mian M, Aloisi R, Benetti D, Rosini S, Fantozzi R. Potential role of heterologous collagen in promoting cutaneous wound repair in rats. Int J Tissue React 1992;14 suppl:43–52. [4] Grinnell F, Lamke CR. Reorganization of hydrated collagen lattices by human skin fibroblasts. J Cell Sci 1984;66:51–63. [5] Ruiz-Cardona L, Sanzgiri YD, Benedetti LM, Stella VJ, Topp EM. Application of benzyl hyaluronate membranes as potential wound dressings: evaluation of water vapor and gas permeabilities. Biomaterials 1997;17:1639–43. [6] Alexander SA, Donoff RB. The glycosaminoglycans of open wounds. J Surg Res 1980;29:422–9. [7] Greco RM, Iocono JA, Ehrlich HP. Hyaluronic acid stimulates human fibroblast proliferation within a collagen matrix. J Cell Physiol 1998;177:465–73. [8] Andreutti D, Geinoz A, Gabbiani G. Effect of hyaluronic acid on migration, proliferation and alpha smooth muscle actin expression by cultured rat and human fibroblasts. J Submicrosc CytoPathol 1999;31:173–7. [9] Park SN, Park JC, Kin HO, Song MJ, Suh H. Characterization of porous collagen/hyaluronic acid scaffold modified by 1-ethyl-3-(3-
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dimethylaminopropyl)carbodiimide cross-linking. Biomaterials 2002;23:1205–12. Suh H, Hwang YS, Song MJ, Lee WS, Han CD, Park JC. Behavior of osteoblast-like cells (MC 3T3-E1) on collagen grafted porous poly l-lactic aid (PLLA) membranes with various pore sizes. Biomater Res 2000;4:37–44. Swann DA, Balazs EA. Determination of the hexosamine content of macromolecules with manual and automated techniques using the p-dimethylaminobenzadehyde reaction. Biochim Biophys Acta 1966;130:112–29. Sung HW, Liang IL, Chen CN, Huang RN, Liang HF. Stability of a biological tissue fixed with a naturally occurring crosslinking agent (genipin). J Biomed Mater Res 2001;55:538–46. Chevally B, Abdul-Malak N, Herbage D. Mouse fibroblasts in long-term culture within collagen three-dimensional scaffolds: influence of crosslinking with diphenylphosphorylazide on matrix reorganization, growth, and biosynthetic and proteolytic activities. J Biomed Mater Res 2000;49:448–59. Schoof H, Apel J, Heshel I, Rau G. Control of pore structure and size in freeze-dried collagen sponges. J Biomed Mater Res 2001; 58:352–7. Allemann F, Mizuno S, Eid K, Yates KE, Zaleske D, Glowacki J. Effects of hyaluronan on engineered articular cartilage extracelluar matrix gene expression in 3-dimensional collagenscaffolds. J Biomed Mater Res 2001;55:13–9. Tomihata K, Ikada Y. Crosslinking of hyaluronic acid with water-soluble carbodiimide. J Biomed Mater Res 1997;37:243–51. Liu LS, Thompson AY, Heidaran MA, Poser JW, Spiro RC. An osteoconductive collagen/hyaluronate matrix for bone regeneration. Biomaterials 1999;20:1097–108. Yoneda M, Yanagata M, Suzuki S, Kimata K. Hyaluronic acid modulates proliferation of mouse dermal fibroblasts in culture. J Cell Sci 1988;90:265–73. Goldberg RL, Toole BP. Hyaluronate inhibition of cell proliferation. Arthritis Rheum 1987;30:769–78. Mast BA, Diegelmann RF, Krummel TM, Cohen IK. Hyaluronic acid modulates proliferation, collagen and protein synthesis of cultured fetal fibroblasts. Matrix 193;13:441–6. Bodo M, Pezzetti F, Baroi T, Carinci F, Arena N, Nicoletti I, Becchetti E. Hyaluronic acid modulates growth, morphology and cytoskeleton in embryonic chick skin fibroblasts. Int J Dev Biol 1993;37:349–52. Petermann AT, Hidromura K, Vlonski N, Pippin F, Monski M, Pippin J, Monkawa T, Durvasula R, Couser WG, Shankland SJ.
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Mechanical stress reduces podocyte proliferation in vitro. Kidney Int 2002;61:41–50. Shepard S, Becker H, Hartmann JX. Using hyaluronic acid to create a fetal-like environment in vitro. Ann Plast Surg 1996; 36:65–9. Nephis V, Hayen W. Are hyaluronan receptors involved in three-dimensional cell migration? Histol Histopathol 2000;15: 629–36. Laurent TC, Fraser JR. Hyaluronan. FASEB 1992;6:2397–404. Rockey DC, Chung JJ, McKee CM, Nobel PW. Stimulation of inducible nitric oxide synthase in rat liver by hyaluronan fragments. Hepatology 1998;27:86092.
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[27] Savanti RC, Wang C, Yang B, Zhang S, Kinsella MG, Wight TN, Stern R, Nanve DN, Turley EA. Migration of bovine aortic smooth muscle cells after wounding injury. The role of hyaluronan and RHAMM. J Clin Invest 1995;95:1158–68. [28] Trochon V, Mabilat C, Bertrand P, Legrand Y, Smadja-Joffe F, Soria C, Delpeck B, Lu H. Evidence of involvement of CD44 in endothelial cell proliferation, migration and angiogenesis in vitro. Int J Cancer 1996;66:664–8. [29] Croce MA, Dyne K, Boraldi F, Quaglino Jr., D, Cetta G, Tiozzo R, Ronchetti IP. Hyarluronan affects protein and collagen synthesis by in vitro human skin fibroblasts. Tissue Cell 2001;33:326–31.
Biological characterization of EDC-crosslinked collagen–hyaluronic acid matrix in dermal tissue restoration Si-Nae Parka, Hye Jung Leeb, Kwang Hoon Leeb, Hwal Suha,c,* a
Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-ku, Seoul 120-752, South Korea b Department of Dermatology, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-ku, Seoul 120-752, South Korea c Brain Korea 21 Project Team for Medical Science, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-ku, Seoul 120-752, South Korea Received 23 April 2002; accepted 28 October 2002
Abstract Porous collagen matrices crosslinked with various amounts of hyaluronic acid (HA) by 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) were developed as scaffolds for dermal tissue regeneration. The effect of HA on cells in accordance with HA concentrations in the collagenous matrices was investigated using cultures of fetal human dermal fibroblasts, and the effect of EDC– crosslinked collagen–HA matrix on wound size reduction was also evaluated in vivo. scanning electron microscopic views of the matrices demonstrated that all of the collagen–HA matrices had interconnected pores with mean diameters of 150–250 mm. An HA matrix retention test showed that the concentration of HA decreased slowly after an initial rapid decrease over 24 h. Fetal human dermal fibroblasts adhered well to all of the collagen-based matrices as compared with the Porous polyurethane matrix used as a control. An 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide based proliferation test and the hematoxylin and eosin staining of a 2 week cultured matrix showed that the proliferation of fibroblasts was enhanced on a 9.6% HA contained collagen matrix. No significant difference was in terms of fibroblast migration into the various types of scaffolds as HA content was increased. In vivo testing showed that dermis treated with collagen or collagen–HA matrix was thicker than the control, and epithelial regeneration was accelerated, and collagen synthesis increased. However, no significant effect of HA on wound size reduction was found. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Collagen; Hyaluronic acid; EDC crosslinking; Fetal dermal fibroblast; Wound healing
1. Introduction The role of collagen at each phase of wound healing is well understood and appreciated. The major applications of collagen-based dressings introduced to date include, collagen films, collagen gels and collagen sponges [1–4]. Hyaluronic acid (HA), an important component of extracellular matrix, has been used as, a viscoelastic biomaterial for medical purposes, in cosmetics because of its high water retention capacity, and in drug delivery systems because of its biodegradability.
*Corresponding author. Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-ku, Seoul 120-752, South Korea. Tel.: +82-2-361-5406; fax: +82-2-363-9923. E-mail address: [email protected] (H. Suh).
HA exists in high concentrations during fetal skin development, is involved in cell migration and differentiation, and is the first macromolecule to appear in the extracellular matrix during wound healing [5,6]. It has been reported that the addition of HA promotes cell division in human fibroblasts by their passage though cell cycles when incorporated within a collagen matrix [7]. In contrast, it has also been reported that exogenous HA significantly inhibits fetal fibroblast proliferation. Moreover, HA was found to induce, inhibit or not to affect fibroblasts migration, depending on the tissue origin of fibroblasts, while fibroblast proliferation was never affected [8]. In these reports, the effects of soluble HA in the medium on fibroblasts were discussed, whereas little information was available on HA concentrations in the collagenous matrix. In a recent study, we fabricated porous collagenous matrix containing HA using a freeze-drying technique,
0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 2 ) 0 0 5 5 0 - 1
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and crosslinked the system with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) to achieve matrix mechanical stability [9]. Membranes produced from this material had good resistance to enzymatic degradation and acceptable toxicologically. The aim of this study was to determine the optimal concentration of HA in EDC-crosslinked porous collagen matrices used as a scaffold for dermal tissue restoration. Biological characteristics of the HA crosslinked in collagen matrices in terms of, the enhancement of attachment, proliferation and migration of fibroblasts in vitro, and upon wound size reduction after placement on full-thickness dermal defects in an animal model were also observed.
2. Materials and methods 2.1. Fabrication and crosslinking of porous collagen– hyaluronic acid matrix The porous collagen–HA matrix was fabricated as previously reported [9] with some modification. Briefly, HA (sodium salt, MW=120,000–150,000, Hanwha group R&E Center, Daejeon, Korea) as an aqueous solution was added to a 1% type I atelocollagen (RBC I, Regenmed, Seoul, Korea) dispersion, to produce 0%, 5%, 10%, and 20% (w/w) HA/collagen mixtures. The mixtures were homogenized at 8000 rpm for 3 min at 41C using a homogenizer (T25 basic, IKA Works, Selangor, Malaysia) and the pH was adjusted to 7.4 by dropping 1 n NaOH. The resulting slurry was poured into a polystyrene Petri dish, frozen at 701C and lyophilized at 501C for 24 h. The dried porous collagen–HA matrices were immersed in a 50 mm EDC (Sigma Chemical Co., St. Louis, MO, USA) solution (H2Oethanol=595) for 24 h to orient the crosslinking. The matrices produced were thoroughly washed in distilled water using a sonicator, and then relyophilized at 501C. Porous polyurethane (PU) matrix, which has been widely used as a wound dressing or skin substitute, was used as the control and prepared by a solventcasting particulate-leaching technique [10]. PU (Tecoflexs, Thermedics Inc., Woburn, MA, USA) was dissolved in chloroform (Sigma, St. Louis, MO, USA) to a final concentration of 12% (w/w). Sieved NaCl particles (150–250 mm), were added in a 12% PU solution and the manually mixed dispersion was cast in a glass Petri dish. The solvent was allowed to evaporate from the covered Petri dish over 48 h in a vacuum oven, and the resulting PU/salt composite membranes were immersed in distilled water on a shaker at room temperature for 48 h to leach out remaining salt.
2.2. Morphological characterization of crosslinked collagen matrices containing various amounts of HA To determine the amount of HA remaining in a collagen–HA matrix after the crosslinking process, the glucosamine contents of the HA molecules in the matrices were analyzed using the p-dimethylaminobenzaldehyde reaction [11]. The concentration of glucosamine was assayed spectrophotometrically (UV-1601S, Pharmacia Biotech, Uppsala, Sweden) at 527 nm against a reagent blank. The porosities of matrices containing various contents of HA after crosslinking were characterized under the scanning electron microscope (SEM) (S-800, HITACHI, Tokyo, Japan) using an image analyzing system (Escan 4000, Bum-Mi Universe Co., Ltd., Ansan, Korea). The ( membranes were coated with an ultrathin layer (300 A) of gold/Pt in an ion sputter (E1010, HITACHI, Tokyo, Japan). The image analysis program was used to determine the average diameter of pores and at least 40 pores were assessed. 2.3. HA retention in crosslinked collagen–HA matrices and water uptake ability To predict the elution rate of HA from the collagen– HA matrices under culture conditions, membranes containing various amounts of HA were prepared as disks (9 mm in diameter and 2 mm in thickness). The matrices were then immersed in 5 ml of Dulbecco’s modified Eagle’s medium (DMEM, JBI Inc, Daejeon, Korea) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/amphotericin-B, and incubated at 371C in an atmosphere of 5% CO2 and 95% air. Samples were withdrawn, freeze-dried and weighed after 3, 7, 14, 21 and 28 days of incubation. The glucosamine contents of the retained HA molecules in the matrices were analyzed by using the p-dimethylaminobenzaldehyde reaction, as described above. For water uptake ability testing, the EDC-crosslinked matrices or the PU matrix were separately immersed in distilled water at room temperature for 10 min. After being removed from the water, they were hung over a table for 1 min until no water dripped from them and then weighed. The water uptake of the matrices was calculated by the following equation: Water uptake ð%Þ ¼ ½ðWs Wd Þ=Ws 100; where Wd is the weight of the dry matrix and Ws is the weight of the swollen matrix (Table 1). 2.4. Cell culture Fetal human dermal fibroblasts were obtained from the Department of Plastic Surgery at the Yonsei Medical Center, having obtained permission from the Medical
S.-N. Park et al. / Biomaterials 24 (2003) 1631–1641 Table 1 The change of HA content in collagen–HA matrices during the crosslinking process and the water uptake of various types of matrices Samples
HA 0% matrix HA 3.2% matrix HA 5.4% matrix HA 9.6% matrix PU matrix
HA content in the matrices (wt%) Before crosslinking (%)
After crosslinking
0 5 10 20
0% 3.2%70.18 5.4%70.28 9.6%70.24
Water uptake
98.4370.072 98.3370.060 98.3270.068 98.2670.149 51.9771.57
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A porous collagen matrix was seeded with a high density of dermal fibroblasts (1 106/membrane) and cultured for 2 weeks. This cell populated matrix (CPM) was then overlaid with a cell-free porous collagen–HA or a cell-free porous PU matrix. A PTFE-disk (weight=0.95 g) was placed onto the loaded matrix to prevent it from floating in the culture medium. After 24 h of additional culture, the CPM and the PTFE disk were removed, and cells that had migrated into the overlaid collagen–HA or PU matrices were quantified using the MTT test. 2.7. In vivo animal testing
Material Control and Management Committee of Yonsei University; the donor’s age was 19 weeks. Outgrown cells were taken from primary culture, following established methods. Cells were cultured in 175 cm2 tissue culture flasks (NUNC, Roskilde, Denmark) using growth medium composed of DMEM (JBI Inc, Daejeoun, Korea), 1% penicillin/streptomycin/ amphotericin-B, and 10% FBS. Cultures were incubated in a humidified incubator in an atmosphere of 5% CO2 and 95% air. Confluent monolayers were propagated by trypsinization (0.25% trypsin, 0.02% EDTA solution) and replating at 12 dilution. For the experiments, fibroblasts were used in the fifth–seventh passages. 2.5. Cell attachment and proliferation testing Cell suspension containing 1 105 cells were placed onto the PU matrix and each crosslinked collagen matrix (as matrices of 10 mm in diameter, 1 mm in thickness). Culture medium (1 ml) was added 1 h after seeding, and cells were allowed to attach for 4 h. For proliferation testing, 5 104 cells were seeded onto each of the matrices and cultures were harvested after 1, 3, 7 or 14 days, the cell attached matrices were gently washed three times with phosphate buffered saline solution, and the attached or proliferated cells were then quantified by the 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay [12,13]. Fifty microliters of MTT solution (5 mg/ml in 0.9% NaCl, filter-sterilized) and 200 ml of medium were added to each culture well. After incubation for 4 h, the MTT reaction medium was removed, and 300 ml of dimethylsulfoxide and 25 ml of Sorensesn’s glycine buffer (0.1 m glycine, 0.1 m NaCl adjusted to pH 10.5 with 1 m NaCl) was added. Optical densities were determined by ELISA reader (Spectra Max 340, Molecular Device Inc., CA, USA) at a wavelength of 570 nm. 2.6. Cell migration test A three-dimensional migration system was employed to investigate the ability of fibroblasts to migrate.
Two types of matrix specimens, based on an EDCcrosslinked collagen matrix and an EDC-crosslinked collagen–HA matrix containing 9.6% of HA were investigated for the treatment of full thickness skin defects in a guinea pig model. The animal experiment procedure was managed in accordance with the Guidelines and Regulations for Use and Care of Animals in Yonsei University. Guinea pigs, weighing 200–400 g, were anesthetized with a ketamine (Yuhan Co. Soul, Korea) by intraperitoneal injection, and the dorsal hair were shaved. After disinfecting the skin with Betadines (Hyundai Pharm., Seoul, Korea), two full thickness round shaped skin defects (2 cm in diameter) were prepared by excising the dorsal skin of each guinea pig. The wound was covered with each type of sterilized matrix or uncovered as a control. Commercial polyurethane matrix (Allevyns, Smith & Nephew Healthcare Ltd., Hull, UK) was used to protect the specimens. 3, 7, 14 or 21 days after implantation, the guinea pigs were sacrificed, and wound sizes were measured. The percentage wound size reduction was calculated according to the following formula Cn ¼ ½ðS0 Sn ÞS0 = 100; where Cn is the percentage of wound size reduction on 3, 7, 14 or 21 days after implantation, S0 the initial wound size, Sn the wound size on 3, 7, 14 or 21 days after implantation. For histological examination, tissue specimens from animal wounds were obtained by excising the wound including the surrounding rim of normal skin and underlying fascia, and muscle. The tissue specimens were then fixed in 10% neutral-buffered formalin solution, embedded in paraffin wax, sectioned at 3 mm, and stained with hematoxylin–eosin (H–E) in the conventional manner. Inflammation, neovascularization, and fibroblast proliferation in the lesion were evaluated under the microscope using a grading system as delineated in Table 2. Measurements were carried out at a magnification of 400 .
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2.8. Statistical analysis Significance levels were determined by ANOVA or the Student’s t-test. Multiple comparisons were carried out using the Tukey method. All statistical calculations were performed on the SAS system for Windows (version 8.00, SAS Institute Inc., Cary, NC, USA). p-values of o0.05 were considered significant.
3. Results and discussion 3.1. Morphological characterization of crosslinked collagen matrices containing various amounts of HA The porous collagen matrices with different HA contents were fabricated by freeze-drying and crosslinked using EDC. The loss of HA, a soluble polysaccharide, could be expected during crosslinking and washing processes. For this reason, the final HA content in the matrices was analyzed by determining the percentage of glucosamine present in the EDC-crosslinked membrane. The result revealed that HA content of the crosslinked matrices, in which the initial HA content was 5%, 10% or 20% (w/w) had decreased to 3.2%, 5.4% or 9.6% (w/w), respectively (Table 1). HA concentrations exceeding ca. 15% made handling of the dried uncrosslinked matrices difficult in a humidified atmosphere. Therefore, crosslinked collagen matrices with 0–9.6% HA content were selected for the experiments described in the present study. SEM observation demonstrated that the cross-section and surfaces of the membranes containing various amounts of HA had interconnected pores (Fig. 1 (A–D and F–I). Generally, the inner three-dimensional structure of such structures, including pore size and porosity has been shown to modulate cell behavior [14]. Allemann et al. [15] reported that the addition of HA influenced the density and the pore size of collagen–HA matrices. The result may have been because the concentration of the HA solution used (1% w/v) was higher than that of collagen solution (0.5% w/v) which would have caused the increment of the density of the mixed solution containing HA. To minimize pore size changes, we used a 1% (w/v) collagen dispersion and a
1% (w/v) HA solution for fabricating membranes. Image analysis revealed that all the fabricated membranes had nearly the same distribution of pore sizes (Fig. 2). Although the pore structure of membranes became more irregular and fibrous as the HA level was increased, the HA contained in the membrane did not significantly affect the pore size. Fig. 1 (E and J) shows the surface and cross-sectional morphology of a porous PU membrane matrix manufactured using 150–250 mm sodium salt as a porogen. Porous PU membrane was used as a control material for cell attachment, proliferation and migration testing in the present study. 3.2. Water uptake, HA retention and weight changes of collagen–HA membranes during culture Ikada et al. [16] reported that the crosslinking through the esterification of its carboxylic groups like this yields a product with the low water content such as 60– 90 wt%. In their experiment, the water content of HA film crosslinked with 50 mm EDC in 75% ethanol was about 95%. This might explain that our result where HA contained in the matrices did not affect the water uptake significantly (Table 1). In the HA retention test (Fig. 3), the HA content of the matrices decreased only slowly after the initial rapid release. After 4 weeks of incubation, the HA content of the matrices that had contained 3.2%, 5.4% and 9.6% w/w HA decreased to 0.8%, 1.0% and 1.5% w/w in the dried samples, respectively. HA elution might be accelerated initially by the rapid hydrolysis of ester bond between HA molecules and by the high water uptake into the porous collagen–HA matrices (Table 1). As described in our previous paper [9], EDC mediates acid anhydride formation between two carboxyl groups of HA and the resultant acid anhydride may readily react with a hydroxyl group of HA to yield an ester bond, which functions as a crosslink of polysaccharide molecule and can be easily attacked by water molecules. Fig. 4 shows the degradation profile of collagen-based membranes containing various ratios of HA under in vitro culture conditions. The weight of the collagen–HA matrices containing 0%, 3.2%, 5.4% or 9.6% (w/w) of
Table 2 Indices of inflammation, new vascularization and fibroblast proliferation in histological examination Inflammation
New vascularization
Fibroblast proliferation
Classification
The number of infiltrated leukocytes
+ ++ +++ ++++
0–10 11–100 101–500 >501
The number of lumen-included RBC or lumen lined with endothelial cells 0–10 11–20 21–30 >31
The number of proliferated fibroblasts 0–100 101–500 501–1000 >1001
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Fig. 1. (A–E) Scanning electron micrographs of the surface of EDC-crosslinked collagen matrices with various contents of HA (A) collagen (HA 0%) matrix; (B) HA 3.2% (w/w) matrix; (C) HA 5.4% (w/w); and (D) HA 9.6% matrix, and the surface of the PU matrix (E). (F–J) Scanning electron micrographs of the cross-sections of EDC-crosslinked collagen matrices with various contents of HA(F) collagen (HA 0%) matrix; (G) HA 3.2% (w/w) matrix; (H) HA 5.4% (w/w); and (I) HA 9.6%, and the cross-section of the PU matrix (J).
HA decreased to 93%, 92.5%, 85% or 81% (w/w) of the original weights after a 3-week period. The masses of the 0% and 3.2% HA matrices decreased to about 94% of the original mass over the first 3 days, and then plateaued. Matrices containing 5.4% or 9.6% (w/w) HA degraded to 86–80% of the original weights gradually over a 2-week culture period. These results indicated that collagen in the matrix was more resistant to the hydrolytic degradation compared with HA and that the weight changes of collagen–HA matrices primarily reflect the solubilization of free HA [17]. Although the fabricated matrices had been washed by sonication, it was very hard to eliminate perfectly the free or weakly bound molecules from the matrices because the washing for longer time could cause the disruption of the matrices. The sonication in fresh distilled water for 30 s five times could remove the residual EDC from the matrices and no signs of cytotoxic effect of residual EDC was observed as described in our previous paper [9]. However, it might be expected that the unbound or weakly crosslinked collagen and HA molecules could be remained in the matrices. 3.3. Attachment and proliferation of fetal human dermal fibroblasts on the various types of matrix Fetal fibroblasts attached better to all the collagenbased matrices than to the PU matrix (po0:05) (Fig. 5). However, no significant effect of HA content in the
Fig. 2. The effect of HA contents on pore size of the collagen–HA scaffold. &: pore size at the surface of matrices; ’: pore size of matrix cross-section. At least 40 pores were assessed, values were statistically analyzed and expressed as means7standard deviation.
collagen matrices on cell attachment was found. SEM observations revealed that the fibroblasts attached evenly either on the collagen or collagen–HA matrices (Fig. 6A and B). The majority of the cells in the PU matrix were aggregated and localized in the pores (Fig. 6C). This may have been because the water uptake capacity of PU matrix was less than that of collagen– HA matrices, as a PU surface consists of both hydrophilic and hydrophobic phases. This property might limit cell adsorption, and non-adsorbed fibroblasts might then adhere to each other.
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Fig. 3. Remaining weight (wt%) of HA in EDC-crosslinked collagen– HA matrices versus culture time. Three matrices of each type were examined on each occasion.
Fig. 4. Degradation rate of EDC-crosslinked collagen–HA matrices under culture conditions. There are significant differences between 5.4% or 9.6% HA contained matrices and 0% HA contained matrix in the remaining weights on 1, 7, 14 and 21 days after incubation. (npo0.05) ’: collagen matrix (HA 0%); E: HA 3.2% (w/w) matrix; m: HA 5.4% (w/w) matrix; and K: HA 9.6% (w/w) matrix.
It has been repeatedly reported that HA might modulate cell proliferation [18,19]. Greco et al. reported that the addition of HA promotes cell division in human fibroblasts by accelerating their passage though the cell cycle, when it is incorporated within a collagen matrix [7]. However, others have reported that exogenous HA at 1–100 mg/ml inhibits the proliferation of rabbit dermal fibroblasts [20]. Additional HA of high molecular weights or high concentrations, inhibited the proliferation of rabbit synovial cells in culture [21], and HA also inhibited the multiplication of embryonic chick skin fibroblasts in different environments [22]. The MTT test as used in this study is a nonradioactive cell proliferation assay that identifies living
Fig. 5. Cell attachment assessed by MTT testing performed 4 h after seeding. The data reported for the control (PU), collagen (HA 0%), HA 3.2%, HA 5.4% and HA 9.6% matrices are means7SD for n ¼ 5: Increased cell attachment on collagen or collagen–HA matrices was significantly different from the control (PU matrix) (po0.05).
cells, and is based on the cellular conversion of a tetrazolium salt into a formazan product, a chromaphore, which can be quantified by enzyme-linked immunosorbent assay (ELISA) reader [22]. This MTTbased proliferation test revealed that cell proliferation on the PU matrix, collagen and collagen–HA matrices was enhanced significantly compared with that on the control (tissue culture plate, TCP) and cells proliferated more rapidly on the 9.6% HA contained collagen–HA matrix than other matrices 2 weeks after seeding (po0.005) (Fig. 7). In particular, fibroblasts exhibited an outstanding proliferation rate in the 9.6% HA containing matrix, as they increased approximately fivefold during 2 weeks in culture. These 2-week cultured matrices showed a progressive penetration of predominantly elongated cells throughout the collagen network and an increased cell density in the collagen–HA matrix as compared with the collagen matrix (Fig. 8). The proliferation rates were not different significantly between the 0% and 5.6% HA containing matrices. The effect of HA on cell proliferation is usually discussed in terms of its mechanical effects on cells and the extracellular matrix. The physiological effect of HA may lie in its ability to act as an accessory receptor in cooperative ligand-binding pathways. HA may not only bind peptide including growth factors, which increase the effective concentration of these factors at the cell surface, but also attract integrins to the cell surfaces [23]. Cell number was maintained in culture on the tissue culture plate (TCP) 1 week after seeding probably because of the relatively limited space. The cell proliferation into a PU matrix has a similar growth pattern to that on TCP, but the relatively higher cell population in the PU matrix might be due to its
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Fig. 6. Scanning electron micrographs of cells attached on a collagen matrix (A); a collagen matrix containing 9.6% HA (B); and a PU matrix (C). (magnification 100).
3.4. Migration of dermal fibroblasts from cell populated collagen matrix to various other types of matrix
Fig. 7. Proliferation curves for fetal human dermal fibroblasts on various types of matrices. E: TCP (tissue culture plate); ’: PU matrix; m: collagen (HA 0%) matrix; &: HA 3.2% matrix; * : HA 5.4% matrix; and K: HA 9.6% matrix.
Fig. 8. Routine histological observations of cell proliferation in matrices. Proliferation of dermal fibroblasts in collagen matrices containing HA 0% (A) or HA 9.6% (B) after 2 weeks of culture. (Magnification 40).
three-dimensional structure, which may provide sufficient space for cell growth as compared to twodimensional culture on TCPs.
HA is known to stimulate the migration of a large variety of cell types. Stimulation of cell migration by HA has been explained to occur by different mechanisms. HA was shown to specifically bind to cell surface receptors, and this inhibition of cellular HA receptor function was demonstrated to decrease cell migration and tumor growth [24]. The increase or decrease of HA during specific phases in embryogenesis, under certain pathological conditions, and the discovery that the great majority of cells possess HA receptors, is also consistent with a role for HA in cell migration and function [25– 28]. HA accumulates in the early phases of inflammation and wound repair, where it might provide a matrix favoring cell adhesion and migration [29]. We used a three-dimensional migration system to investigate the early migration of dermal fibroblasts from wounded dermal tissues to the regenerative matrices. The basis of this system was a cell populated collagen matrix (CPM), which had been cultivated for 2 weeks in a static state and was used as a dermis-like tissue. The effect of HA on the migration of dermal fibroblasts from the CPM is shown in Fig. 9. Our results revealed that the migrations of cells to the collagen matrix and the collagen–HA matrices were enhanced compared with the PU matrix, and that collagen–HA matrices containing 5.4% or 9.6% HA increased the number of migrated cells significantly (p ¼ 0:00421 and 0.00034, respectively). 3.5. Effect of crosslinked collagen–HA matrices on wound size reduction in vivo The fetal wound-healing matrix is exceptionally rich in HA. Fetal wounds heal without scarring or contraction. Shepard et al. reported that adult dermal
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Fig. 9. Migration of fetal human dermal fibroblasts from cell populated matrices (CPMs) to various other types of matrices. PU: cell migration to a PU matrix; 0% HA: cell migration to a collagen (HA 0%) matrix; 3.2% HA: cell migration to an HA 3.2% matrix5.4% HA: cell migration to an HA 5.4% HA matrix; and 9.6% HA: cell migration to a 9.6% HA matrix. Cell migration to the 5.4% HA matrix and the 9.6% HA matrix was higher than that to a PU matrix (n ¼ 7; npo0.05).
fibroblasts cultivated in the presence of 5 mg/ml of exogenous HA changed in morphology, and that the wound closed more quickly than control cultures [23]. In our in vitro experiments, the 9.6% HA contained matrix showed the better effects on proliferation and migration compared with other matrices. Therefore, in vivo study, the effects of collagen matrix, 9.6% HA contained collagen–HA matrix were compared with a control group. Fig. 10 shows changes in the mean wound area in groups undergoing full-thickness skin grafting at various times after treatment with collagen or collagen–HA matrices. In this entire group, the wound size reduction increased gradually to reach about 95% 2 weeks after the creation of the skin defects. On postoperative days 3 and 7, wound contraction in the collagen–HA matrix treated group increased to 53.2% and 73.6% of the original area, respectively. On the other hand, wound contraction increased to 53.2% and 62.8% in the collagen matrix treated group and to 42.4% and 60.3% in control groups, respectively. However, no significance of HA upon wound size reduction was found in this in vivo study. Possible reasons proposed for these results by authors were attributed to this animal model using guinea pigs and the matrix stability. The rate of wound healing was so fast that the wound size was reduced to above >50% in 3 days and the wounds were healed completely within 2 weeks. Therefore, it was rather difficult to find the significant difference in the wound size reduction during healing process in this study. It was also likely that the degradation of the collagen–HA matrix could be
Fig. 10. Wound contraction of a full thickness dermal defect in guinea pig. No significant effect of treatment on wound contraction was found (means7standard error of the mean). E: no treatment (control); ’: EDC-crosslinked collagen matrix; m: EDC-crosslinked collagen–HA matrix.
accelerated in vivo and that result in the higher initial release of HA from the crosslinked matrix compared with in vitro degradation. The histological appearance at low-power magnification of the wound tissue in the three groups is shown in Figs. 11–14. With a collagen matrix or a collagen–HA matrix, inflammatory response tended to be pronounced on postoperative day 3 (Fig. 11), and then gradually subsided. On day 7, as shown in Fig. 12, capillaries appeared and new epithelialization was initiated from the edge of the wound. After 14 days, a proliferation of fibroblasts and thickened collagen fibers were seen, blood vessels were decreased and the regeneration of epithelium was observed (Fig. 13). Dermis treated with regenerative matrices was thicker than the control; epithelial regeneration was accelerated and collagen synthesis increased compared with the control (Table 3). Histological analysis revealed that 21 days after wounding, epithelialization was complete in all wounds (Fig. 14). Wounds treated with collagen–HA matrix showed a similar histology to that of the collagen matrix treated group.
4. Conclusion Porous collagen–HA matrix containing interconnected pores with mean diameters of 150–250 mm was fabricated by freeze-drying and EDC crosslinking. In culture conditions, the HA concentration in the matrices decreased slowly after an initial rapid decrease. Fibroblasts attached well to all the collagen-based matrices
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Fig. 11. Photomicrographs of biopsy specimens from an untreated wound (A); from a wound treated with a crosslinked collagen matrix (B); a collagen–HA matrix (C) on postoperative day 3 (magnification 40).
Fig. 12. Photomicrographs of biopsy specimens from an untreated wound (A); from a wound treated with a crosslinked collagen matrix (B); or treated with a crosslinked a collagen–HA matrix (C) on postoperative day 7 (magnification 40).
Fig. 13. Photomicrographs of biopsy specimens from an untreated wound (A); from a wound treated with a crosslinked collagen matrix (B); or treated with a crosslinked a collagen–HA matrix (C) on postoperative day 14 (magnification 40).
Fig. 14. Photomicrographs of biopsy specimens from an untreated wound (A); from a wound treated with a crosslinked collagen matrix (B); or treated with a crosslinked a collagen–HA matrix (C) on postoperative day 21 (magnification 40).
compared with the PU matrix and the proliferation of fibroblasts was enhanced by the HA 9.6% matrix as compared with the PU matrix or TCP, 14 days after cell
seeding. Cell migration from CPM to collagen matrix or collagen–HA matrices was enhanced compared with that from CPM to the PU matrix. Collagen–HA
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Table 3 Histologic examination of tissue 3, 7 and 14 days after implantation Days
Control
Collagen matrix
Collagen–HA matrix
Inflammation
3 7 14 21
+++ + + +
+++ + + +
+++ + + +
New vascularization
3 7 14 21
++ +++ + +
+++ ++++ ++ +
+++ ++++ + +
Fibroblasts proliferation
3 7 14 21
++ +++ +++ +++
+++ ++++ ++++ +++
+++ +++ ++++ +++
matrices containing 5.4% or 9.6% HA increased migrating cell numbers significantly. In animal testing, however, no significant effect of HA upon wound size reduction was found.
[10]
[11]
Acknowledgements This work was supported by a grant from the Ministry of Information and Communication (Project no. 01-PJ11-PG9-01NT00-0045), Korea.
[12]
[13]
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