- Email: [email protected]
A novel approach for the cryodesiccated preservation of tissue-engineered skin substitutes with trehalose
Materials Science and Engineering C 60 (2016) 60–66
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
A novel approach for the cryodesiccated preservation of tissue-engineered skin substitutes with trehalose Mei Sun 1, Man Jiang 1, Jihong Cui, Wei Liu, Lu Yin, Chunli Xu, Qi Wei, Xingrong Yan, Fulin Chen ⁎ Lab of Tissue Engineering, the College of Life Sciences, Northwest University, Taibai North Rd 229, Xi'an 710069, PR China
a r t i c l e
i n f o
Article history: Received 30 June 2015 Received in revised form 23 September 2015 Accepted 20 October 2015 Available online 21 October 2015 Keywords: Tissue-engineered skin Freeze-drying Trehalose Wound healing
a b s t r a c t Tissue-engineered skin (TES) holds great promise for wound healing in the clinic. However, optimized preservation methods remain an obstacle for its wide application. In this experimental work, we developed a novel approach to preserve TES in the desiccated state with trehalose. The uptake of trehalose by fibroblasts under various conditions, including the trehalose concentration, incubation temperature and time, was studied. The cell viability was investigated by the MTT assay and CFSE/PI staining after cryodesiccation and rehydration. TES was then prepared and incubated with trehalose, and the wound healing effect was investigated after desiccated preservation. The results showed that the optimized conditions for trehalose uptake by fibroblasts were incubation in 200 mM trehalose at 37 °C for 8 h. Cryodesiccated cells and TES maintained 37.55% and 28.31% viabilities of controls, respectively. Furthermore, cryodesiccated TES exhibited a similar wound healing effect to normal TES. This novel approach enabled the preservation and transportation of TES at ambient temperature with a prolonged shelf time, which provides great advantages for the application of TES. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Tissue-engineered skin (TES) is an optimized alternative for the treatment of skin defects resulting from trauma, inflammation and tumor excision [1]. The strategy involves the combination of keratinocytes or fibroblasts with certain scaffolds [2]. Cells seeded into the scaffold secrete various growth factors and extracellular matrix proteins, which could stimulate the proliferation and differentiation of adjacent epithelial tissue and accelerate skin defect repair [3]. Several TES products, including Apligraf ® and Dermagraf ®, have been authorized by the FDA [4–6]. To facilitate the application of TES in emergent situations, a relatively large storage of TES is needed, because the preparation of TES is time consuming. However, some TES products, such as Apligraf ®, should be preserved at 4 °C and the shelf time is very short, usually ranging from 7 to 10 days because of the difficulty in keeping cell viability [7,8]. Furthermore, conditioned transportation is also necessary to deliver these living TES products [9]. Cryopreservation of cells in suspension has been demonstrated to be effective under the protection of DMSO [10,11]. However, the technique is not applicable for the cryopreservation of tissues or organs with relatively large volume due to their low thawing rate [12–15]. Therefore, it is necessary to develop novel approaches to preserve and deliver TES.
⁎ Corresponding author. E-mail address: chenfl@nwu.edu.cn (F. Chen). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.msec.2015.10.057 0928-4931/© 2015 Elsevier B.V. All rights reserved.
Some organisms have developed specialized adaptations to protect their cellular components from the damage caused by desiccation and rehydration. For example, the larvae of the sleeping chironomid can survive complete dehydration in a “cryptobiosis” or “anhydrobiosis” state [16]. Water bear, a tardigrade, can be revived after extreme desiccation for decades [17]. One mechanism, common to all such organisms, is the accumulation of disaccharides, especially trehalose, within cells and tissues at the onset of dehydration. Trehalose is a non-reducing disaccharide that is primarily used for drying or freeze-drying of proteins and other biological compounds [18]. Trehalose could form hydrogen-bonds to polar groups on proteins and lipids and prevent the occurrence of fusion or denaturation [19–21]. Chen et al. [22] demonstrated that trehalose could significantly improve human keratinocyte viability in suspension and tissue-engineered cell sheets during cryopreservation. When transplanted into nude mice, trehalose-cryopreserved keratinocyte sheets repaired skin defects as efficiently as that of non-cryopreserved controls, indicating that trehalose could maintain the function of cryopreserved keratinocyte sheets. Trehalose could also replace water molecules, thereby maintaining the structure of plasma membrane and preventing protein denaturation and aggregation during the process of desiccation [23]. Wolkers et al. [24] reported that trehalose could be rapidly taken up by human platelets, and 85% of freeze-dried platelets could be recovered after rehydration. Importantly, the function of the recovered platelets was almost identical to that of fresh platelets. McGinnis et al. [25] treated mouse sperm with trehalose and injected the desiccated sperms (stored at 4 °C for 1 month) into oocytes and found that the percentage of blastocyst formation was up to 63%. Gordon et al. [26] incubated
M. Sun et al. / Materials Science and Engineering C 60 (2016) 60–66
human mesenchymal stem cells with trehalose, and then air dried the cells. Further tests showed that the cells maintained high viability and proliferation capacity, expressed mRNA for characteristic stromal factor, and responded to reagents known to induce differentiation. To provide the best clinical outcome, TES must be processed and stored in a manner that maintains the viability and structural integrity until needed for transplantation. In the current study, we hypothesized that TES could be cryodessicated and functionally preserved in the dried state with trehalose. The ability to preserve and transport TES products in the dried state at ambient temperature provides a significant advantage for their application.
61
−50 °C with a LGJ-25 vacuum Freeze Drier (Xiangyi, China) and were stored in vacuum condition at room temperature for 1 week before use. 2.3.2. Rehydration Samples were rehydrated 1 week after cryodesiccation. For the trehalose + DMSO group and the trehalose group, cells were rehydrated by adding 200 μL cell culture medium containing 200 mM trehalose and were incubated at 37 °C and 5% CO2 for 45 min. While for the DMSO group and control group, cells were rehydrated by 200 μL normal culture medium. 2.4. Cell viability assay
2. Materials and methods 2.1. Mouse fibroblast cell culture Mouse fibroblast cells (L929 cell line) were purchased from the Shanghai Institute for Biological Sciences, Chinese Academy of Sciences Institute of Cell Resource Center. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 1% non-essential amino acids (Hyclone, USA), and 100 U/100 μg penicillin/streptomycin (Hyclone, USA) at 37 °C with 5% CO2. Cells were subcultured at a 1:4 split ratio by 0.25% trypsin when reached approximately 70–80% confluent for further experiments. 2.2. Trehalose loading and quantization Approximately 4 × 104 fibroblasts in 200 μL cell culture medium were plated into each well of 96-well plates and cultured overnight for adhesion. To measure the trehalose uptake by fibroblasts, the medium was removed, and the cells were incubated in fresh medium with different conditions, including trehalose concentration, temperature and incubation time. For the concentration series, cells were cultured in medium containing 50, 100, 200, 300 or 400 mM of trehalose at 37 °C for 8 h (n = 5). For the temperature series, cells were incubated at 4 °C, 20 °C, 37 °C and 4–37 °C (4 °C for 10 min and then transferred to 37 °C) in medium with 200 mM trehalose for 8 h (n = 5). For the time course series, fibroblasts were incubated in medium containing 200 mM trehalose at 37 °C for 0.5, 1, 2, 4, 6 and 8 h (n = 5). Cell morphology was observed by phase contrast microscopy during the experiment. Cells were collected by centrifugation at 1000 rpm for 10 min after digestion with trypsin. Cells were then mixed with 4 mL of 80% methanol and incubated at 85 °C for 1 h to extract the intracellular trehalose. After collecting the supernatant by centrifugation and evaporating in a vacuum drier, the dry residue was dissolved in 1 mL distilled water. Trehalose quantification was performed by the anthrone reaction [27]. Briefly, the samples were mixed with 3 mL of 2% anthrone reagent (Sigma-Aldrich, USA) and heated at 100 °C for 10 min. The absorbance at 630 nm was measured on a spectrophotometer (Thermo, Multiskan FC, USA) and compared with a standard curve (n = 5 for each group). 2.3. Cryodesiccation and rehydration 2.3.1. Cryodesiccation Approximately 4 × 104 fibroblasts in 200 μL of medium were plated into each well of 96-well plates and cultured overnight for adhesion. The cells were then incubated in fresh medium containing the following: (1) 200 mM trehalose +10% DMSO (Sigma-Aldrich, USA), (2) 200 mM trehalose, (3) 10% DMSO. Fresh cell culture medium acted as control (n = 5 for each group). After incubation at 37 °C for 8 h, the medium was removed, and the cells were transferred immediately into a programmed freezing container (Ruobilin, RBL-PA, China) and frozen at cooling speed of 1 °C/min to −50 °C. Cells were freeze-dried for 7 h at
2.4.1. Fluorescent labeling The viability of the rehydrated cells after cryodesiccation was determined by 5- or 6-(N-succinimidyloxycarbonyl)-3′,6′-O,O′diacetylfluorescein (CFSE, Sigma-Aldrich, USA)/fluorescent propidium iodide (PI, Sigma-Aldrich, USA) labeling, according to the manufacturer's protocol. Briefly, the cells were incubated in the medium with 20 μL PI solution at 37 °C. After 15 min, the cells were washed twice with PBS and were added by 2.5 μM CFSE prepared in PBS for 15 min incubation. The staining was stopped by replacing the medium with fresh DMEM and incubating for another 30 min. Cell fluorescence was then imaged by a fluorescent microscope (Olympus, FV1000, Japan). 2.4.2. MTT assay The viability of the rehydrated cells was further determined by the MTT assay following the manufacturer's instruction. A 10 μL volume of MTT solution was added into each well and incubated at 37 °C for 4 h. Then, the medium was gently removed, and 200 μL of DMSO was added. All the wells were measured immediately by a microplate reader (Versa Max, USA) at 490 nm. Normal fibroblasts acted as control (n = 5 for each group). 2.5. Cryodesiccation of TES and wound healing effect 2.5.1. Mouse fibroblast isolation and culture Mouse fibroblasts were isolated from newborn male BALB/c mice (2–3 days after birth). The sterilized skin samples were digested with 0.25% trypsin (Hyclone, USA) at 37 °C for 2 h and washed with PBS. Primary fibroblasts were suspended in DMEM supplemented with 10% FBS, 1% non-essential amino acids and 100 U/100 μg penicillin/ streptomycin at 37 °C with 5% CO2. Cells were subcultured at a 1:4 split ratio by 0.25% trypsin when reached approximately 70–80% confluent for further experiments. Cells of third passage were used for TES preparation. 2.5.2. Preparation of TES TES samples were prepared as described previously [28]. A 150 μL volume of 10 × DMEM was added into 1 mL of an ice cold collagen solution (4 mg/mL type I collagen from rat tail dissolved in 0.1% acetic acid). After neutralization with 500 μL 0.1% NaOH solution, 100 μL of cell suspension (5 × 106 cells) was then added and mixed immediately. The mixture was transferred into 48-well plates and incubated at 37 °C for gelling. 2.5.3. Cryodesiccation, rehydration and MTT assay The prepared TES were incubated in medium containing 200 mM trehalose + 10% DMSO, 200 mM trehalose and 10% DMSO for 8 h respectively. The TES specimens were processed for cryodesiccation and rehydration as described for mouse fibroblasts above. Rehydrated samples were first examined by gross inspection and handling test. The MTT assay was further applied to test the viability of the specimens 4 weeks after storage in vacuum condition at room temperature. Normal TES without treatment acted as the control (n = 5 for each group).
62
M. Sun et al. / Materials Science and Engineering C 60 (2016) 60–66
2.5.4. Wound healing effect Male BALB/c mice (4 weeks old) were purchased from the Experimental Animal Center of The Fourth Military Medical University and were maintained in accordance with the guidelines provided by the Institutional Ethics Committee of Northwest University. Circular skin defects of 1 cm in diameter were created on the back after anesthetization by intraperitoneal injection of pentobarbital sodium (20 mg/kg body weight). TES samples were grafted onto the defects (n = 5 for each group). Grafts were covered with vaseline gauze and adhesive bandages for 3 days. Wound healing was monitored at day 0, 3, 6, 9, 12, and 15 post-operation with a digital camera. The wound area was measured by tracing the wound margin and was calculated using Image-Pro Plus Software (Media Cybernetics LP, Silver Spring, MD, USA). The wound closure percentage was calculated as follows: (original defect area minus actual defect area)/original defect area × 100%. Freshly prepared normal TES and collagen gel (prepared one day before implantation) acted as control. Animals were sacrificed at day 15 and skin samples were embedded in paraffin for the following experiments. Sections of skin grafts were stained with a mouse panCytokeratin antibody (1:200; SANTA, USA), and a secondary AlexaFluor 594-labeled goat anti-mouse IgG (1:500; Invitrogen, USA) was used. Further detection was carried out by hematoxylin & eosin (H&E) staining.
2.6. Statistical analysis The data are expressed as the mean ± SD of at least three independent samples. Statistical comparisons between groups were performed with one-way ANOVA analysis, and p b 0.05 was considered significant.
3. Results 3.1. Concentration, temperature and time course influence fibroblast uptake of trehalose The uptake of trehalose by fibroblasts incubated under different sugar concentrations in medium at 37 °C for 8 h is presented in Fig. 1A. The results revealed that the absorption of trehalose was 18.47 ± 1.17 (50 mM group), 29.47 ± 1.86 (100 mM group), 52.67 ± 2.56 (200 mM group), 37.33 ± 1.83 (300 mM group), and 10.70 ± 1.15 μg/106 cells (400 mM group), respectively. The absorption between any one of the groups was significantly different (p b 0.01), indicating that trehalose loading depended on the sugar concentration in the medium. The highest intracellular trehalose concentration was obtained with 200 mM trehalose in medium. The cells maintained normal morphology when cultured in medium containing lower concentration of trehalose. When the trehalose concentration reached 300 mM and above, significant cell shrinkage could be observed (Fig. 1D). As shown in Fig. 1B, the uptake of trehalose was 17.33 ± 1.72, 19.21 ± 1.28, 47.67 ± 3.22 and 39.54 ± 2.64 μg/106 cells, when incubated at 4 °C, 20 °C, 37 °C and 4–37 °C. The result of the 37 °C group was significantly higher than any other groups (p b 0.01). Cell shrinkage was noticed when cultured at 4 °C and 20 °C (Fig. 1E). The time course was also an important factor that affected the uptake of trehalose. Fig. 1C showed the trehalose uptake by fibroblasts during incubation with 200 mM trehalose at 37 °C for different time courses. It could be observed that the uptake increased with the extension of the incubation time. The absorptive amounts of 0.5, 1, 2, 4, 6 and 8 h groups were 5.90 ± 1.45, 11.67 ± 2.25, 20.93 ± 1.68, 28.83 ± 2.50, 40.53 ± 3.02 and 43.57 ± 3.68 μg/106 cells, respectively. There was no significant difference between the 6 h group and the 8 h group, but
Fig. 1. Trehalose uptake by fibroblasts and cell morphology observation. (A–C) Incubation concentration, time and temperature dependence of trehalose uptake. (D) Cell morphology alteration under gradient trehalose concentration (37 °C for 8 h). a) 50 mM, b) 100 mM, c) 200 mM, d) 300 mM, e) 400 mM, f) control. (E) Cell morphology alteration at different incubation temperature (200 mM trehalose for 8 h). a) 4 °C, b) 20 °C, c) 37 °C, d) 4–37 °C. (F) Cell morphology alteration for different incubation time (200 mM trehalose at 37 °C). a) 0.5 h, b) 1 h, c) 2 h, d) 4 h, e) 6 h, f) 8 h. Error bars represent SDs, n = 5 for each group, **p b 0.01. Scale bar = 100 μm.
M. Sun et al. / Materials Science and Engineering C 60 (2016) 60–66
63
the results of these two groups were significantly higher than the other groups. The cells maintained normal morphology during 8 h incubation (Fig. 1F). Collectively, the optimum condition for trehalose uptake by fibroblasts was 200 mM at 37 °C for 8 h.
was 28.31% of normal TES in the 200 mM trehalose + 10% DMSO group, which was significantly higher than the 200 mM trehalose group and 10% DMSO group (5.28% and 19.27%, respectively. p b 0.01).
3.2. Viability of cells and TES after cryodesiccation under the protection of trehalose
3.3. Wound healing effect of cryodesiccated TES
We used CFSE and PI staining to observe the viable cells and necrotic cells. Live cells were stained green while dead cells were stained red. As shown in Fig. 2A, more cells stained green could be observed in the 200 mM trehalose + 10% DMSO group than in 200 mM trehalose group and 10% DMSO group. According to the MTT assay, the cell viability maintained 37.55% of the normal fibroblasts viability in 200 mM trehalose + 10% DMSO group, whereas the viabilities were 10.70% and 27.87% in 200 mM trehalose group and 10% DMSO group after cryodesiccation and rehydration. The cell viability of 200 mM trehalose + 10% DMSO group was significantly higher than 200 mM trehalose group and 10% DMSO group (p b 0.01) (Fig. 2B). Fig. 2C (b, c) showed gross appearance of the cryodesiccated TES. After rehydration, the specimen recovered its gross appearance of gel and could be handled conveniently with forceps, similar to normal TES. The MTT assay results (Fig. 2D) indicated that TES viability
All of the animals survived, and no inflammation occurred throughout the experiment. Gross inspections showed a reduction in the area of the defects in all groups after TES grafting (Fig. 3A). As shown in Fig. 3B, the wound repair rate after different TES grafting was quantitatively measured. The collagen gel without cells showed the lowest capacity for the repair of skin defects, and by day 15, the overall wound repair rate was 77.65 ± 4.81%, which was significantly lower than the other groups at this time point (p b 0.01). The normal TES group exhibited the highest wound closure rate, and the skin defects were almost completely repaired by day 15 (97.44 ± 0.82%). Similar to the normal TES group, the wound closure rate was 95.84 ± 1.23% in the 200 mM trehalose +10% DMSO group, and there was no significant difference compared with the normal TES group at this time point (p N 0.05). While the rate was 84.53 ± 3.04% in the 200 mM trehalose group and 89.61 ± 3.18% in the 10% DMSO group, significantly lower than that of 200 mM trehalose + 10% DMSO group (p b 0.01 and p b 0.05, respectively).
Fig. 2. Viability assay of rehydrated cells and TES after cryodesiccation. (A) Images of CFSE/PI labeling. (a) 200 mM trehalose incubated cells after cryodesiccation and rehydration (T). (b) 10% DMSO incubated cells after cryodesiccation and rehydration (D). (c) 200 mM trehalose + 10% DMSO incubated cells after cryodesiccation and rehydration (T + D). (d) Normal cells without treatment (Control). (B) Viability analysis of rehydrated cells after cryodesiccation. (C) Gross inspection of TES before and after rehydration. a) Normal TES. b, c) TES after cryodesiccation. d) Rehydrate TES. (D) Viability assay of cryodesiccated TES by MTT. Error bars represent SDs, n = 5 for each group, **p b 0.01. Scale bar = 100 μm.
64
M. Sun et al. / Materials Science and Engineering C 60 (2016) 60–66
Fig. 3. Wound healing effect investigation. (A) Gross inspection after TES grafting for each group and time point. (B) Wound healing effect of cryodesiccated TES for each group over the time course. Error bars represent SDs, n = 5 for each group, *p b 0.05 and **p b 0.01.
3.4. Histological and immunohistochemical evaluation of the epidermis After 15 days, excised grafts were sectioned and stained with pan-Cytokeratin reagent and H&E to observe tissue structure. PanCytokeratin immunostaining showed that cytokeratins existed in all groups, indicating the covering of epithelial tissues (Fig. 4). And the results of H&E staining revealed that, in the cell-free, 200 mM trehalose and 10% DMSO only group, the dermis was thin in the central area of the defect. While the defect of 200 mM trehalose + 10% DMSO grafting group was repaired by skin with normal epidermis and dermis layers, which was very similar to the result of normal TES grafting group.
4. Discussion Proper preservation and transportation of tissue engineered products with high viability are essential for their successful application [29]. In the current experimentation, we reported for the first time the
preservation of three dimensional tissue engineered skin grafts with trehalose and DMSO and their effect on the repair of mouse skin defects. DMSO prevented ice crystal formation and protected fibroblasts during freezing, whereas trehalose acted to inhibit protein denaturation and to maintain the plasma membrane structure during desiccation, thus keeping the viability and function of the engineered skin substitutes. The mechanism of trehalose uptake is fluid phase endocytosis, and the process is dependent on the incubation time, temperature and extracellular trehalose concentration. Gyana et al. [30] reported that for red blood cells, uptake was very low when the trehalose concentration was below 600 mM, whereas the uptake increased significantly when the concentration reached 800 mM. In platelets, the highest uptake was obtained with 52 mM extracellular trehalose, and at concentrations greater than 52 mM, the trehalose uptake decreased [24]. In our experiment, we found that the optimized trehalose concentration for fibroblasts was 200 mM (Fig. 1A). When the trehalose concentration was above 300 mM, fibroblasts exhibited morphological changes (Fig. 1D). Therefore, the concentration for trehalose uptake is cell type
M. Sun et al. / Materials Science and Engineering C 60 (2016) 60–66
65
Fig. 4. Histological and immunohistochemical evaluation of the excised grafts after 15 day. Pan-Cytokeratin staining indicates mouse keratinocytes within skin. Scale bar = 100 μm.
dependent and could be different from cell to cell. In the current experiments, the highest intracellular trehalose content reached 52.67 ± 2.56 μg/106 cells when the cells were incubated at 200 mM concentration at 37 °C for 8 h, and the optimized incubation time and temperature agreed with previous studies [24,26,30]. After incubation with 200 mM trehalose and 10% DMSO, fibroblasts were processed for cryodessicated preservation and rehydration. CFSE and PI staining showed that many cells were viable after rehydration (Fig. 2A). The MTT assay further indicated that cell viability maintained 37.55% of normal fibroblasts after treatment with trehalose and DMSO, which was significantly higher than that of the trehalose or DMSO group (Fig. 2B). The MTT assay is a colorimetric assay that depends on reducing the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide to its insoluble formazan form by cellular oxidoreductase enzymes. The result of trehalose group indicated that activity of cellular enzymes could be partially preserved by trehalose. In vivo experimentation demonstrated that cryodesiccated TES had an optimized effect on stimulating skin defect healing after 4-week preservation. Fifteen days after the grafting of a cryodesiccated graft, the healing rate reached 95.84%, and there was no significant difference compared with the normal TES grafting group (Fig. 3A). One may question why cryodesiccated TES had an optimized skin defect healing effect when the viability was only 28.31% compared to normal TES. The possible explanations include the following: 1) Collagen gel has certain effect on wound healing. As shown in Fig. 3A, the skin defect healing rate reached 77.65% 15 d after collagen gel grafting. 2) Paracrine growth factors have a direct effect on the proliferation and differentiation of adjacent epithelial tissues. Fibroblasts could express various growth factors, including FGF, PDGF, IGF, VEGF, TGF and EGF, in collagen gel. The activity of these growth factors was preserved by trehalose, which enabled the cryodesiccated TES to be used efficiently for skin defect repair. 3) Trehalose could improve the fast recovery of cellular EGF and TGF-β secretion after cryopreservation. According to Chen's experiment [22], three days after thawing, the EGF and TGF-β concentrations of trehalose + DMSO cryopreserved cells were 110% and 50% higher than that of DMSO treated cells. The gross inspection and histological evaluation indicated that the 200 mM trehalose + 10% DMSO group presented rapid and excellent repair effect, which may guarantee timely and effective skin regeneration. In the current study, we developed a novel approach for the cryodesiccated preservation of TES with trehalose. The approach enabled the preservation and transportation of TES at ambient temperature with
a prolonged shelf life and provides tremendous practical and economic advantages. However, the uptake of trehalose by fibroblasts was relatively low in the current experiment, and the approach was more suitable for TES, act as a temporary wound covering. In brief, for the cryodesiccated preservation of other types of tissue engineered products, in which cells should survive in and integrate with the host tissue, more efficient approaches should be developed to improve trehalose uptake and cell viability. Acknowledgments This work was supported by the National Natural Science Foundation of P. R. China (30970745 and 31300797). References [1] S. Bottcher-Haberzeth, T. Biedermann, E. Reichmann, Burns 36 (2010) 450–460. [2] T. Biedermann, S. Boettcher-Haberzeth, E. Reichmann, Eur. J. Pediatr. Surg. 23 (2013) 375–382. [3] J.G. Rheinwald, H. Green, Nature 265 (1977) 421–424. [4] J.N. Kearney, Burns 27 (2001) 545–551. [5] Y.M. Bello, A.F. Falabella, J. Wound Care 11 (2002) 182–183. [6] W.A. Marston, J. Hanft, P. Norwood, R. Pollak, G. Dermagraft Diabetic Foot Ulcer Study, Diabetes Care 26 (2003) 1701–1705. [7] J.F. Trent, R.S. Kirsner, Int. J. Clin. Pract. 52 (1998) 408–413. [8] H. Bannasch, M. Fohn, T. Unterberg, F. Knam, B. Weyand, G.B. Stark, Chirurg 74 (2003) 802–807. [9] G.K. Naughton, Ann. N. Y. Acad. Sci. 961 (2002) 372–385. [10] S. Renzi, T. Lombardo, S. Dotti, S.S. Dessi, P. De Blasio, M. Ferrari, Biopreserv. Biobanking 10 (2012) 276–281. [11] D.E. Pegg, Methods Mol. Biol. 1257 (2015) 3–19. [12] A. Ruffo, P. Rossotto, G. Motta, Minerva Med. 56 (1965) 3153–3157. [13] H.A. Hackensellner, P. Krietsch, G. Matthes, Acta Med. Pol. 19 (1978) 189–196. [14] P. Groscurth, M. Erni, M. Balzer, H.J. Peter, G. Haselbacher, Anat. Embryol. (Berl) 174 (1986) 105–113. [15] J. Saragusty, Methods Mol. Biol. 1257 (2015) 381–397. [16] T. Kikawada, N. Minakawa, M. Watanabe, T. Okuda, Integr. Comp. Biol. 45 (2005) 710–714. [17] J.H. Crowe, L.M. Crowe, Nat. Biotechnol. 18 (2000) 145–146. [18] J.H. Crowe, L.M. Crowe, D. Chapman, Science 223 (1984) 701–703. [19] L.M. Crowe, J.H. Crowe, A. Rudolph, C. Womersley, L. Appel, Arch. Biochem. Biophys. 242 (1985) 240–247. [20] T.D. Madden, M.B. Bally, M.J. Hope, P.R. Cullis, H.P. Schieren, A.S. Janoff, Biochim. Biophys. Acta 817 (1985) 67–74. [21] N. Tsvetkova, B. Tenchov, L. Tsonev, T. Tsvetkov, Cryobiology 25 (1988) 256–263. [22] F. Chen, W. Zhang, W. Wu, Y. Jin, L. Cen, J.D. Kretlow, W. Gao, Z. Dai, J. Wang, G. Zhou, W. Liu, L. Cui, Y. Cao, Biomaterials 32 (2011) 8426–8435. [23] R. Cornette, T. Kikawada, IUBMB Life 63 (2011) 419–429. [24] W.F. Wolkers, N.J. Walker, F. Tablin, J.H. Crowe, Cryobiology 42 (2001) 79–87.
66
M. Sun et al. / Materials Science and Engineering C 60 (2016) 60–66
[25] L.K. McGinnis, L. Zhu, J.A. Lawitts, S. Bhowmick, M. Toner, J.D. Biggers, Biol. Reprod. 73 (2005) 627–633. [26] S.L. Gordon, S.R. Oppenheimer, A.M. Mackay, J. Brunnabend, I. Puhlev, F. Levine, Cryobiology 43 (2001) 182–187. [27] K. Motegi, K. Shoji, Rinsho Byori (Suppl. 15) (1968) 71–77.
[28] S.G. Priya, H. Jungvid, A. Kumar, Tissue Eng. Part B Rev. 14 (2008) 105–118. [29] T.M. Fraser, Aerosp. Med. 39 (1968) 146–152. [30] G.R. Satpathy, Z. Torok, R. Bali, D.M. Dwyre, E. Little, N.J. Walker, F. Tablin, J.H. Crowe, N.M. Tsvetkova, Cryobiology 49 (2004) 123–136.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
A novel approach for the cryodesiccated preservation of tissue-engineered skin substitutes with trehalose Mei Sun 1, Man Jiang 1, Jihong Cui, Wei Liu, Lu Yin, Chunli Xu, Qi Wei, Xingrong Yan, Fulin Chen ⁎ Lab of Tissue Engineering, the College of Life Sciences, Northwest University, Taibai North Rd 229, Xi'an 710069, PR China
a r t i c l e
i n f o
Article history: Received 30 June 2015 Received in revised form 23 September 2015 Accepted 20 October 2015 Available online 21 October 2015 Keywords: Tissue-engineered skin Freeze-drying Trehalose Wound healing
a b s t r a c t Tissue-engineered skin (TES) holds great promise for wound healing in the clinic. However, optimized preservation methods remain an obstacle for its wide application. In this experimental work, we developed a novel approach to preserve TES in the desiccated state with trehalose. The uptake of trehalose by fibroblasts under various conditions, including the trehalose concentration, incubation temperature and time, was studied. The cell viability was investigated by the MTT assay and CFSE/PI staining after cryodesiccation and rehydration. TES was then prepared and incubated with trehalose, and the wound healing effect was investigated after desiccated preservation. The results showed that the optimized conditions for trehalose uptake by fibroblasts were incubation in 200 mM trehalose at 37 °C for 8 h. Cryodesiccated cells and TES maintained 37.55% and 28.31% viabilities of controls, respectively. Furthermore, cryodesiccated TES exhibited a similar wound healing effect to normal TES. This novel approach enabled the preservation and transportation of TES at ambient temperature with a prolonged shelf time, which provides great advantages for the application of TES. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Tissue-engineered skin (TES) is an optimized alternative for the treatment of skin defects resulting from trauma, inflammation and tumor excision [1]. The strategy involves the combination of keratinocytes or fibroblasts with certain scaffolds [2]. Cells seeded into the scaffold secrete various growth factors and extracellular matrix proteins, which could stimulate the proliferation and differentiation of adjacent epithelial tissue and accelerate skin defect repair [3]. Several TES products, including Apligraf ® and Dermagraf ®, have been authorized by the FDA [4–6]. To facilitate the application of TES in emergent situations, a relatively large storage of TES is needed, because the preparation of TES is time consuming. However, some TES products, such as Apligraf ®, should be preserved at 4 °C and the shelf time is very short, usually ranging from 7 to 10 days because of the difficulty in keeping cell viability [7,8]. Furthermore, conditioned transportation is also necessary to deliver these living TES products [9]. Cryopreservation of cells in suspension has been demonstrated to be effective under the protection of DMSO [10,11]. However, the technique is not applicable for the cryopreservation of tissues or organs with relatively large volume due to their low thawing rate [12–15]. Therefore, it is necessary to develop novel approaches to preserve and deliver TES.
⁎ Corresponding author. E-mail address: chenfl@nwu.edu.cn (F. Chen). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.msec.2015.10.057 0928-4931/© 2015 Elsevier B.V. All rights reserved.
Some organisms have developed specialized adaptations to protect their cellular components from the damage caused by desiccation and rehydration. For example, the larvae of the sleeping chironomid can survive complete dehydration in a “cryptobiosis” or “anhydrobiosis” state [16]. Water bear, a tardigrade, can be revived after extreme desiccation for decades [17]. One mechanism, common to all such organisms, is the accumulation of disaccharides, especially trehalose, within cells and tissues at the onset of dehydration. Trehalose is a non-reducing disaccharide that is primarily used for drying or freeze-drying of proteins and other biological compounds [18]. Trehalose could form hydrogen-bonds to polar groups on proteins and lipids and prevent the occurrence of fusion or denaturation [19–21]. Chen et al. [22] demonstrated that trehalose could significantly improve human keratinocyte viability in suspension and tissue-engineered cell sheets during cryopreservation. When transplanted into nude mice, trehalose-cryopreserved keratinocyte sheets repaired skin defects as efficiently as that of non-cryopreserved controls, indicating that trehalose could maintain the function of cryopreserved keratinocyte sheets. Trehalose could also replace water molecules, thereby maintaining the structure of plasma membrane and preventing protein denaturation and aggregation during the process of desiccation [23]. Wolkers et al. [24] reported that trehalose could be rapidly taken up by human platelets, and 85% of freeze-dried platelets could be recovered after rehydration. Importantly, the function of the recovered platelets was almost identical to that of fresh platelets. McGinnis et al. [25] treated mouse sperm with trehalose and injected the desiccated sperms (stored at 4 °C for 1 month) into oocytes and found that the percentage of blastocyst formation was up to 63%. Gordon et al. [26] incubated
M. Sun et al. / Materials Science and Engineering C 60 (2016) 60–66
human mesenchymal stem cells with trehalose, and then air dried the cells. Further tests showed that the cells maintained high viability and proliferation capacity, expressed mRNA for characteristic stromal factor, and responded to reagents known to induce differentiation. To provide the best clinical outcome, TES must be processed and stored in a manner that maintains the viability and structural integrity until needed for transplantation. In the current study, we hypothesized that TES could be cryodessicated and functionally preserved in the dried state with trehalose. The ability to preserve and transport TES products in the dried state at ambient temperature provides a significant advantage for their application.
61
−50 °C with a LGJ-25 vacuum Freeze Drier (Xiangyi, China) and were stored in vacuum condition at room temperature for 1 week before use. 2.3.2. Rehydration Samples were rehydrated 1 week after cryodesiccation. For the trehalose + DMSO group and the trehalose group, cells were rehydrated by adding 200 μL cell culture medium containing 200 mM trehalose and were incubated at 37 °C and 5% CO2 for 45 min. While for the DMSO group and control group, cells were rehydrated by 200 μL normal culture medium. 2.4. Cell viability assay
2. Materials and methods 2.1. Mouse fibroblast cell culture Mouse fibroblast cells (L929 cell line) were purchased from the Shanghai Institute for Biological Sciences, Chinese Academy of Sciences Institute of Cell Resource Center. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 1% non-essential amino acids (Hyclone, USA), and 100 U/100 μg penicillin/streptomycin (Hyclone, USA) at 37 °C with 5% CO2. Cells were subcultured at a 1:4 split ratio by 0.25% trypsin when reached approximately 70–80% confluent for further experiments. 2.2. Trehalose loading and quantization Approximately 4 × 104 fibroblasts in 200 μL cell culture medium were plated into each well of 96-well plates and cultured overnight for adhesion. To measure the trehalose uptake by fibroblasts, the medium was removed, and the cells were incubated in fresh medium with different conditions, including trehalose concentration, temperature and incubation time. For the concentration series, cells were cultured in medium containing 50, 100, 200, 300 or 400 mM of trehalose at 37 °C for 8 h (n = 5). For the temperature series, cells were incubated at 4 °C, 20 °C, 37 °C and 4–37 °C (4 °C for 10 min and then transferred to 37 °C) in medium with 200 mM trehalose for 8 h (n = 5). For the time course series, fibroblasts were incubated in medium containing 200 mM trehalose at 37 °C for 0.5, 1, 2, 4, 6 and 8 h (n = 5). Cell morphology was observed by phase contrast microscopy during the experiment. Cells were collected by centrifugation at 1000 rpm for 10 min after digestion with trypsin. Cells were then mixed with 4 mL of 80% methanol and incubated at 85 °C for 1 h to extract the intracellular trehalose. After collecting the supernatant by centrifugation and evaporating in a vacuum drier, the dry residue was dissolved in 1 mL distilled water. Trehalose quantification was performed by the anthrone reaction [27]. Briefly, the samples were mixed with 3 mL of 2% anthrone reagent (Sigma-Aldrich, USA) and heated at 100 °C for 10 min. The absorbance at 630 nm was measured on a spectrophotometer (Thermo, Multiskan FC, USA) and compared with a standard curve (n = 5 for each group). 2.3. Cryodesiccation and rehydration 2.3.1. Cryodesiccation Approximately 4 × 104 fibroblasts in 200 μL of medium were plated into each well of 96-well plates and cultured overnight for adhesion. The cells were then incubated in fresh medium containing the following: (1) 200 mM trehalose +10% DMSO (Sigma-Aldrich, USA), (2) 200 mM trehalose, (3) 10% DMSO. Fresh cell culture medium acted as control (n = 5 for each group). After incubation at 37 °C for 8 h, the medium was removed, and the cells were transferred immediately into a programmed freezing container (Ruobilin, RBL-PA, China) and frozen at cooling speed of 1 °C/min to −50 °C. Cells were freeze-dried for 7 h at
2.4.1. Fluorescent labeling The viability of the rehydrated cells after cryodesiccation was determined by 5- or 6-(N-succinimidyloxycarbonyl)-3′,6′-O,O′diacetylfluorescein (CFSE, Sigma-Aldrich, USA)/fluorescent propidium iodide (PI, Sigma-Aldrich, USA) labeling, according to the manufacturer's protocol. Briefly, the cells were incubated in the medium with 20 μL PI solution at 37 °C. After 15 min, the cells were washed twice with PBS and were added by 2.5 μM CFSE prepared in PBS for 15 min incubation. The staining was stopped by replacing the medium with fresh DMEM and incubating for another 30 min. Cell fluorescence was then imaged by a fluorescent microscope (Olympus, FV1000, Japan). 2.4.2. MTT assay The viability of the rehydrated cells was further determined by the MTT assay following the manufacturer's instruction. A 10 μL volume of MTT solution was added into each well and incubated at 37 °C for 4 h. Then, the medium was gently removed, and 200 μL of DMSO was added. All the wells were measured immediately by a microplate reader (Versa Max, USA) at 490 nm. Normal fibroblasts acted as control (n = 5 for each group). 2.5. Cryodesiccation of TES and wound healing effect 2.5.1. Mouse fibroblast isolation and culture Mouse fibroblasts were isolated from newborn male BALB/c mice (2–3 days after birth). The sterilized skin samples were digested with 0.25% trypsin (Hyclone, USA) at 37 °C for 2 h and washed with PBS. Primary fibroblasts were suspended in DMEM supplemented with 10% FBS, 1% non-essential amino acids and 100 U/100 μg penicillin/ streptomycin at 37 °C with 5% CO2. Cells were subcultured at a 1:4 split ratio by 0.25% trypsin when reached approximately 70–80% confluent for further experiments. Cells of third passage were used for TES preparation. 2.5.2. Preparation of TES TES samples were prepared as described previously [28]. A 150 μL volume of 10 × DMEM was added into 1 mL of an ice cold collagen solution (4 mg/mL type I collagen from rat tail dissolved in 0.1% acetic acid). After neutralization with 500 μL 0.1% NaOH solution, 100 μL of cell suspension (5 × 106 cells) was then added and mixed immediately. The mixture was transferred into 48-well plates and incubated at 37 °C for gelling. 2.5.3. Cryodesiccation, rehydration and MTT assay The prepared TES were incubated in medium containing 200 mM trehalose + 10% DMSO, 200 mM trehalose and 10% DMSO for 8 h respectively. The TES specimens were processed for cryodesiccation and rehydration as described for mouse fibroblasts above. Rehydrated samples were first examined by gross inspection and handling test. The MTT assay was further applied to test the viability of the specimens 4 weeks after storage in vacuum condition at room temperature. Normal TES without treatment acted as the control (n = 5 for each group).
62
M. Sun et al. / Materials Science and Engineering C 60 (2016) 60–66
2.5.4. Wound healing effect Male BALB/c mice (4 weeks old) were purchased from the Experimental Animal Center of The Fourth Military Medical University and were maintained in accordance with the guidelines provided by the Institutional Ethics Committee of Northwest University. Circular skin defects of 1 cm in diameter were created on the back after anesthetization by intraperitoneal injection of pentobarbital sodium (20 mg/kg body weight). TES samples were grafted onto the defects (n = 5 for each group). Grafts were covered with vaseline gauze and adhesive bandages for 3 days. Wound healing was monitored at day 0, 3, 6, 9, 12, and 15 post-operation with a digital camera. The wound area was measured by tracing the wound margin and was calculated using Image-Pro Plus Software (Media Cybernetics LP, Silver Spring, MD, USA). The wound closure percentage was calculated as follows: (original defect area minus actual defect area)/original defect area × 100%. Freshly prepared normal TES and collagen gel (prepared one day before implantation) acted as control. Animals were sacrificed at day 15 and skin samples were embedded in paraffin for the following experiments. Sections of skin grafts were stained with a mouse panCytokeratin antibody (1:200; SANTA, USA), and a secondary AlexaFluor 594-labeled goat anti-mouse IgG (1:500; Invitrogen, USA) was used. Further detection was carried out by hematoxylin & eosin (H&E) staining.
2.6. Statistical analysis The data are expressed as the mean ± SD of at least three independent samples. Statistical comparisons between groups were performed with one-way ANOVA analysis, and p b 0.05 was considered significant.
3. Results 3.1. Concentration, temperature and time course influence fibroblast uptake of trehalose The uptake of trehalose by fibroblasts incubated under different sugar concentrations in medium at 37 °C for 8 h is presented in Fig. 1A. The results revealed that the absorption of trehalose was 18.47 ± 1.17 (50 mM group), 29.47 ± 1.86 (100 mM group), 52.67 ± 2.56 (200 mM group), 37.33 ± 1.83 (300 mM group), and 10.70 ± 1.15 μg/106 cells (400 mM group), respectively. The absorption between any one of the groups was significantly different (p b 0.01), indicating that trehalose loading depended on the sugar concentration in the medium. The highest intracellular trehalose concentration was obtained with 200 mM trehalose in medium. The cells maintained normal morphology when cultured in medium containing lower concentration of trehalose. When the trehalose concentration reached 300 mM and above, significant cell shrinkage could be observed (Fig. 1D). As shown in Fig. 1B, the uptake of trehalose was 17.33 ± 1.72, 19.21 ± 1.28, 47.67 ± 3.22 and 39.54 ± 2.64 μg/106 cells, when incubated at 4 °C, 20 °C, 37 °C and 4–37 °C. The result of the 37 °C group was significantly higher than any other groups (p b 0.01). Cell shrinkage was noticed when cultured at 4 °C and 20 °C (Fig. 1E). The time course was also an important factor that affected the uptake of trehalose. Fig. 1C showed the trehalose uptake by fibroblasts during incubation with 200 mM trehalose at 37 °C for different time courses. It could be observed that the uptake increased with the extension of the incubation time. The absorptive amounts of 0.5, 1, 2, 4, 6 and 8 h groups were 5.90 ± 1.45, 11.67 ± 2.25, 20.93 ± 1.68, 28.83 ± 2.50, 40.53 ± 3.02 and 43.57 ± 3.68 μg/106 cells, respectively. There was no significant difference between the 6 h group and the 8 h group, but
Fig. 1. Trehalose uptake by fibroblasts and cell morphology observation. (A–C) Incubation concentration, time and temperature dependence of trehalose uptake. (D) Cell morphology alteration under gradient trehalose concentration (37 °C for 8 h). a) 50 mM, b) 100 mM, c) 200 mM, d) 300 mM, e) 400 mM, f) control. (E) Cell morphology alteration at different incubation temperature (200 mM trehalose for 8 h). a) 4 °C, b) 20 °C, c) 37 °C, d) 4–37 °C. (F) Cell morphology alteration for different incubation time (200 mM trehalose at 37 °C). a) 0.5 h, b) 1 h, c) 2 h, d) 4 h, e) 6 h, f) 8 h. Error bars represent SDs, n = 5 for each group, **p b 0.01. Scale bar = 100 μm.
M. Sun et al. / Materials Science and Engineering C 60 (2016) 60–66
63
the results of these two groups were significantly higher than the other groups. The cells maintained normal morphology during 8 h incubation (Fig. 1F). Collectively, the optimum condition for trehalose uptake by fibroblasts was 200 mM at 37 °C for 8 h.
was 28.31% of normal TES in the 200 mM trehalose + 10% DMSO group, which was significantly higher than the 200 mM trehalose group and 10% DMSO group (5.28% and 19.27%, respectively. p b 0.01).
3.2. Viability of cells and TES after cryodesiccation under the protection of trehalose
3.3. Wound healing effect of cryodesiccated TES
We used CFSE and PI staining to observe the viable cells and necrotic cells. Live cells were stained green while dead cells were stained red. As shown in Fig. 2A, more cells stained green could be observed in the 200 mM trehalose + 10% DMSO group than in 200 mM trehalose group and 10% DMSO group. According to the MTT assay, the cell viability maintained 37.55% of the normal fibroblasts viability in 200 mM trehalose + 10% DMSO group, whereas the viabilities were 10.70% and 27.87% in 200 mM trehalose group and 10% DMSO group after cryodesiccation and rehydration. The cell viability of 200 mM trehalose + 10% DMSO group was significantly higher than 200 mM trehalose group and 10% DMSO group (p b 0.01) (Fig. 2B). Fig. 2C (b, c) showed gross appearance of the cryodesiccated TES. After rehydration, the specimen recovered its gross appearance of gel and could be handled conveniently with forceps, similar to normal TES. The MTT assay results (Fig. 2D) indicated that TES viability
All of the animals survived, and no inflammation occurred throughout the experiment. Gross inspections showed a reduction in the area of the defects in all groups after TES grafting (Fig. 3A). As shown in Fig. 3B, the wound repair rate after different TES grafting was quantitatively measured. The collagen gel without cells showed the lowest capacity for the repair of skin defects, and by day 15, the overall wound repair rate was 77.65 ± 4.81%, which was significantly lower than the other groups at this time point (p b 0.01). The normal TES group exhibited the highest wound closure rate, and the skin defects were almost completely repaired by day 15 (97.44 ± 0.82%). Similar to the normal TES group, the wound closure rate was 95.84 ± 1.23% in the 200 mM trehalose +10% DMSO group, and there was no significant difference compared with the normal TES group at this time point (p N 0.05). While the rate was 84.53 ± 3.04% in the 200 mM trehalose group and 89.61 ± 3.18% in the 10% DMSO group, significantly lower than that of 200 mM trehalose + 10% DMSO group (p b 0.01 and p b 0.05, respectively).
Fig. 2. Viability assay of rehydrated cells and TES after cryodesiccation. (A) Images of CFSE/PI labeling. (a) 200 mM trehalose incubated cells after cryodesiccation and rehydration (T). (b) 10% DMSO incubated cells after cryodesiccation and rehydration (D). (c) 200 mM trehalose + 10% DMSO incubated cells after cryodesiccation and rehydration (T + D). (d) Normal cells without treatment (Control). (B) Viability analysis of rehydrated cells after cryodesiccation. (C) Gross inspection of TES before and after rehydration. a) Normal TES. b, c) TES after cryodesiccation. d) Rehydrate TES. (D) Viability assay of cryodesiccated TES by MTT. Error bars represent SDs, n = 5 for each group, **p b 0.01. Scale bar = 100 μm.
64
M. Sun et al. / Materials Science and Engineering C 60 (2016) 60–66
Fig. 3. Wound healing effect investigation. (A) Gross inspection after TES grafting for each group and time point. (B) Wound healing effect of cryodesiccated TES for each group over the time course. Error bars represent SDs, n = 5 for each group, *p b 0.05 and **p b 0.01.
3.4. Histological and immunohistochemical evaluation of the epidermis After 15 days, excised grafts were sectioned and stained with pan-Cytokeratin reagent and H&E to observe tissue structure. PanCytokeratin immunostaining showed that cytokeratins existed in all groups, indicating the covering of epithelial tissues (Fig. 4). And the results of H&E staining revealed that, in the cell-free, 200 mM trehalose and 10% DMSO only group, the dermis was thin in the central area of the defect. While the defect of 200 mM trehalose + 10% DMSO grafting group was repaired by skin with normal epidermis and dermis layers, which was very similar to the result of normal TES grafting group.
4. Discussion Proper preservation and transportation of tissue engineered products with high viability are essential for their successful application [29]. In the current experimentation, we reported for the first time the
preservation of three dimensional tissue engineered skin grafts with trehalose and DMSO and their effect on the repair of mouse skin defects. DMSO prevented ice crystal formation and protected fibroblasts during freezing, whereas trehalose acted to inhibit protein denaturation and to maintain the plasma membrane structure during desiccation, thus keeping the viability and function of the engineered skin substitutes. The mechanism of trehalose uptake is fluid phase endocytosis, and the process is dependent on the incubation time, temperature and extracellular trehalose concentration. Gyana et al. [30] reported that for red blood cells, uptake was very low when the trehalose concentration was below 600 mM, whereas the uptake increased significantly when the concentration reached 800 mM. In platelets, the highest uptake was obtained with 52 mM extracellular trehalose, and at concentrations greater than 52 mM, the trehalose uptake decreased [24]. In our experiment, we found that the optimized trehalose concentration for fibroblasts was 200 mM (Fig. 1A). When the trehalose concentration was above 300 mM, fibroblasts exhibited morphological changes (Fig. 1D). Therefore, the concentration for trehalose uptake is cell type
M. Sun et al. / Materials Science and Engineering C 60 (2016) 60–66
65
Fig. 4. Histological and immunohistochemical evaluation of the excised grafts after 15 day. Pan-Cytokeratin staining indicates mouse keratinocytes within skin. Scale bar = 100 μm.
dependent and could be different from cell to cell. In the current experiments, the highest intracellular trehalose content reached 52.67 ± 2.56 μg/106 cells when the cells were incubated at 200 mM concentration at 37 °C for 8 h, and the optimized incubation time and temperature agreed with previous studies [24,26,30]. After incubation with 200 mM trehalose and 10% DMSO, fibroblasts were processed for cryodessicated preservation and rehydration. CFSE and PI staining showed that many cells were viable after rehydration (Fig. 2A). The MTT assay further indicated that cell viability maintained 37.55% of normal fibroblasts after treatment with trehalose and DMSO, which was significantly higher than that of the trehalose or DMSO group (Fig. 2B). The MTT assay is a colorimetric assay that depends on reducing the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide to its insoluble formazan form by cellular oxidoreductase enzymes. The result of trehalose group indicated that activity of cellular enzymes could be partially preserved by trehalose. In vivo experimentation demonstrated that cryodesiccated TES had an optimized effect on stimulating skin defect healing after 4-week preservation. Fifteen days after the grafting of a cryodesiccated graft, the healing rate reached 95.84%, and there was no significant difference compared with the normal TES grafting group (Fig. 3A). One may question why cryodesiccated TES had an optimized skin defect healing effect when the viability was only 28.31% compared to normal TES. The possible explanations include the following: 1) Collagen gel has certain effect on wound healing. As shown in Fig. 3A, the skin defect healing rate reached 77.65% 15 d after collagen gel grafting. 2) Paracrine growth factors have a direct effect on the proliferation and differentiation of adjacent epithelial tissues. Fibroblasts could express various growth factors, including FGF, PDGF, IGF, VEGF, TGF and EGF, in collagen gel. The activity of these growth factors was preserved by trehalose, which enabled the cryodesiccated TES to be used efficiently for skin defect repair. 3) Trehalose could improve the fast recovery of cellular EGF and TGF-β secretion after cryopreservation. According to Chen's experiment [22], three days after thawing, the EGF and TGF-β concentrations of trehalose + DMSO cryopreserved cells were 110% and 50% higher than that of DMSO treated cells. The gross inspection and histological evaluation indicated that the 200 mM trehalose + 10% DMSO group presented rapid and excellent repair effect, which may guarantee timely and effective skin regeneration. In the current study, we developed a novel approach for the cryodesiccated preservation of TES with trehalose. The approach enabled the preservation and transportation of TES at ambient temperature with
a prolonged shelf life and provides tremendous practical and economic advantages. However, the uptake of trehalose by fibroblasts was relatively low in the current experiment, and the approach was more suitable for TES, act as a temporary wound covering. In brief, for the cryodesiccated preservation of other types of tissue engineered products, in which cells should survive in and integrate with the host tissue, more efficient approaches should be developed to improve trehalose uptake and cell viability. Acknowledgments This work was supported by the National Natural Science Foundation of P. R. China (30970745 and 31300797). References [1] S. Bottcher-Haberzeth, T. Biedermann, E. Reichmann, Burns 36 (2010) 450–460. [2] T. Biedermann, S. Boettcher-Haberzeth, E. Reichmann, Eur. J. Pediatr. Surg. 23 (2013) 375–382. [3] J.G. Rheinwald, H. Green, Nature 265 (1977) 421–424. [4] J.N. Kearney, Burns 27 (2001) 545–551. [5] Y.M. Bello, A.F. Falabella, J. Wound Care 11 (2002) 182–183. [6] W.A. Marston, J. Hanft, P. Norwood, R. Pollak, G. Dermagraft Diabetic Foot Ulcer Study, Diabetes Care 26 (2003) 1701–1705. [7] J.F. Trent, R.S. Kirsner, Int. J. Clin. Pract. 52 (1998) 408–413. [8] H. Bannasch, M. Fohn, T. Unterberg, F. Knam, B. Weyand, G.B. Stark, Chirurg 74 (2003) 802–807. [9] G.K. Naughton, Ann. N. Y. Acad. Sci. 961 (2002) 372–385. [10] S. Renzi, T. Lombardo, S. Dotti, S.S. Dessi, P. De Blasio, M. Ferrari, Biopreserv. Biobanking 10 (2012) 276–281. [11] D.E. Pegg, Methods Mol. Biol. 1257 (2015) 3–19. [12] A. Ruffo, P. Rossotto, G. Motta, Minerva Med. 56 (1965) 3153–3157. [13] H.A. Hackensellner, P. Krietsch, G. Matthes, Acta Med. Pol. 19 (1978) 189–196. [14] P. Groscurth, M. Erni, M. Balzer, H.J. Peter, G. Haselbacher, Anat. Embryol. (Berl) 174 (1986) 105–113. [15] J. Saragusty, Methods Mol. Biol. 1257 (2015) 381–397. [16] T. Kikawada, N. Minakawa, M. Watanabe, T. Okuda, Integr. Comp. Biol. 45 (2005) 710–714. [17] J.H. Crowe, L.M. Crowe, Nat. Biotechnol. 18 (2000) 145–146. [18] J.H. Crowe, L.M. Crowe, D. Chapman, Science 223 (1984) 701–703. [19] L.M. Crowe, J.H. Crowe, A. Rudolph, C. Womersley, L. Appel, Arch. Biochem. Biophys. 242 (1985) 240–247. [20] T.D. Madden, M.B. Bally, M.J. Hope, P.R. Cullis, H.P. Schieren, A.S. Janoff, Biochim. Biophys. Acta 817 (1985) 67–74. [21] N. Tsvetkova, B. Tenchov, L. Tsonev, T. Tsvetkov, Cryobiology 25 (1988) 256–263. [22] F. Chen, W. Zhang, W. Wu, Y. Jin, L. Cen, J.D. Kretlow, W. Gao, Z. Dai, J. Wang, G. Zhou, W. Liu, L. Cui, Y. Cao, Biomaterials 32 (2011) 8426–8435. [23] R. Cornette, T. Kikawada, IUBMB Life 63 (2011) 419–429. [24] W.F. Wolkers, N.J. Walker, F. Tablin, J.H. Crowe, Cryobiology 42 (2001) 79–87.
66
M. Sun et al. / Materials Science and Engineering C 60 (2016) 60–66
[25] L.K. McGinnis, L. Zhu, J.A. Lawitts, S. Bhowmick, M. Toner, J.D. Biggers, Biol. Reprod. 73 (2005) 627–633. [26] S.L. Gordon, S.R. Oppenheimer, A.M. Mackay, J. Brunnabend, I. Puhlev, F. Levine, Cryobiology 43 (2001) 182–187. [27] K. Motegi, K. Shoji, Rinsho Byori (Suppl. 15) (1968) 71–77.
[28] S.G. Priya, H. Jungvid, A. Kumar, Tissue Eng. Part B Rev. 14 (2008) 105–118. [29] T.M. Fraser, Aerosp. Med. 39 (1968) 146–152. [30] G.R. Satpathy, Z. Torok, R. Bali, D.M. Dwyre, E. Little, N.J. Walker, F. Tablin, J.H. Crowe, N.M. Tsvetkova, Cryobiology 49 (2004) 123–136.