INTRODUCTION

Bone defects caused by periodontal diseases and traumatic injuries compromised patients’ quality of life. Tissue engineering has been emerging as an alternative way to cure bone diseases and defects1). Good designs of biomaterial scaffolds for mechanical functions and mass transport of nutrients have been proven important for tissue regeneration2,3). Porous scaffolds had several superior properties, especially for the exchange of nutrients and metabolites in and out of scaffolds and for promoting cell growth and differentiation on the scaffolds4-7). Various methods have been developed to fabricate porous scaffolds, such as solvent casting/particulate leaching8), high-pressure processing9), gas foaming/particulate leaching10), thermally induced phase separation11), etc.

Lyophilization (also called freeze-drying) has been proven as an effective method to fabricate porous scaffolds4,12-14). Porous collagen scaffolds were successfully fabricated using solvents such as ethanol and acetic acid, and glutaraldehyde for chemical crosslink reactions15). Gelatin scaffolds with controlled pore structures were also fabricated using lyophilization14). Glutaraldehyde treatment is by far the most used crosslinking treatment of collagen16,17). However, the major disadvantages of this method were potential toxic residuals remained in the scaffolds18-20), which compromised cell growth and differentiation. Removal of toxic residuals from the scaffolds by washing was time-consuming21) and might affect the 3-dimensional structures of porous scaffolds.

Fluvastatin, one kind of statin drugs, was reported

to promote bone regeneration via up-regulating expression of the bone morphogenetic protein-2 (BMP-2) gene22,23). In our previous studies, fluvastatin containing gelatin hydrogel promoted bone regeneration in rat calvarial defects24) and improved femur bone healing in low-turnover osteoporosis model mice25). Fluvastatin was released from the scaffold as the gelatin was degraded. However, the degradation of gelatin hydrogel was fast and uncontrollable in vivo, making it difficult to maintain its 3-dimensional structures and achieve controlled release of fluvastatin. Interestingly, collagen/gelatin sponges were reported to act as carriers for sustained releases of the basic fibroblast growth factor (bFGF)26-29). Atelocollagen and gelatin both showed good biocompatibility and low antigenicity30). Moreover, the degradation of atelocollagen was much slower than that of gelatin, which made it possible to maintain its 3-dimensional structures for a longer time. As atelocollagen is water-insoluble, whereas gelatin is water-soluble, how to fabricate atelocollagen/gelatin (ACG) sponge without toxic residuals and changes in pH value remains a challenge.

In this study, we developed an easier way to fabricate ACG sponge by using lyophilization. Lyophilization works by freezing the materials and then reducing the surrounding pressure to allow the frozen water (or ice crystals) in the materials to sublimate directly from the solid phase to the gas phase31). Because the ice crystals that form during the freezing process determine the pore shape and size in the scaffold12), any factor that influences ice crystals formation affects the pore structures. This study aimed to investigate the

Influence of lyophilization factors and gelatin concentration on pore structures of atelocollagen/gelatin sponge biomaterialLongqiang YANG1,2, Koji TANABE2,3, Tadashi MIURA2, Masao YOSHINARI2, Shinji TAKEMOTO2,4, Seikou SHINTANI1 and Masataka KASAHARA3

1 Department of Pediatric Dentistry, Tokyo Dental College, 2-9-18 Misaki-cho, Chiyoda-ku, Tokyo 101-0061, Japan2 Oral Health Science Center, Tokyo Dental College, 2-9-18 Misaki-cho, Chiyoda-ku, Tokyo 101-0061, Japan3 Department of Pharmacology, Tokyo Dental College, 2-1-14 Misaki-cho, Chiyoda-ku, Tokyo 101-0061, Japan4 Department of Dental Materials Science, Tokyo Dental College, 2-9-18 Misaki-cho, Chiyoda-ku, Tokyo 101-0061, JapanCorresponding authors, Koji TANABE; E-mail: kotanabe@tdc.ac.jp, Masataka KASAHARA; E-mail: mkasahar@tdc.ac.jp

This study aimed to investigate influences of lyophilization factors and gelatin concentration on pore structures of ACG sponge. ACG sponges of different freezing temperatures (−30, −80 and −196oC), freezing times (1, 2 and 24 h), gelatin concentrations (0.6%AC+0.15%G, 0.6%AC+0.6%G and 0.6%AC+2.4%G), and with 500 μM fluvastatin were fabricated. Pore structures including porosity and pore size were analyzed by scanning electron microscopy and ImageJ. The cytotoxic effects of ACG sponges were evaluated in vitro. Freezing temperature did not affect porosity while high freezing temperature (−30oC) increased pore size. The high gelatin concentration group (0.6%AC+2.4%G) had decreased porosity and pore size. Freezing time and 500 μM fluvastatin did not affect pore structures. The cytotoxicity and cell proliferation assays revealed that ACG sponges had no cytotoxic effects on human mesenchymal stromal cell growth and proliferation. These results indicate that ACG sponge may be a good biomaterial scaffold for bone regeneration.

Keywords: Atelocollagen, Gelatin, Pore size, Porosity, Lyophilization

Received Jun 29, 2016: Accepted Dec 5, 2016doi:10.4012/dmj.2016-242 JOI JST.JSTAGE/dmj/2016-242

Dental Materials Journal 2017; 36(4): 429–437

Fig. 1 Fabrication scheme of atelocollagen/gelatin (ACG) sponge with lyophilization (or freeze-drying). The water-soluble gelatin solution facilitated the dissolution of water-insoluble atelocollagen

powder without additives such as 1 mM phosphate acid. As for lyophilization, ice crystals (or particulates) determined the pore shape and size formed in the sponge. The heat crosslink reaction was employed instead of the chemical crosslink reaction to avoid the presence of potential toxic residuals as well as to sterilize the sponge. The final ACG sponge had no toxic residuals and changes in pH value. The pores were hexagonal in shape. SEM: scanning electron microscopy

influences of various lyophilization factors and gelatin concentration on the pore structures of ACG sponges. Pore structures including porosity and pore size were analyzed. The cytotoxic effects of ACG sponges were also evaluated in vitro.

MATERIALS AND METHODS

Generally, ACG sponge was fabricated using lyophilization as shown in Fig. 1. Various factors that might influence pore structures were identified, including freezing temperature, freezing time and gelatin concentration. As our previous studies found that the fluvastatin containing gelatin-hydrogels promoted bone regeneration24,25), the influences of fluvastatin on the pore structures of ACG sponge were also examined. The cytotoxic effects of ACG sponges were evaluated by observing the growth of human mesenchymal stromal cells (hMSCs) on ACG sponges, and measuring cell proliferation in ACG extracts in vitro. The ACG sponge that contained 0.6% (wt/v) atelocollagen/0.6% (wt/v) gelatin and was frozen at −80oC for 2 h was used as the control group for the entire study. The experimental groups of fabricated ACG sponges were listed in Table 1.

Preparation of ACG sponge samples at different freezing temperaturesBriefly, the 0.6% gelatin solution was obtained by

dissolving 120 mg of gelatin powder (Nitta Gelatin, Osaka, Japan) into 20 mL of Milli-Q water. Then 60 mg of atelocollagen powder (KOKEN, Tokyo, Japan) was slowly dissolved into 10 mL of the 0.6% gelatin solution under continuous mixing at 4oC overnight. The resulting ACG composite solution contained 0.6% atelocollagen and 0.6% gelatin. The ACG composite solution was poured into 96-well plates at 200 μL/well and frozen at −30, −80 and −196oC (liquid nitrogen) respectively for 2 h. The frozen ACG composite solution samples were freeze-dried at −50oC in the vacuum for 24 h. Finally, the freeze-dried ACG sponges were heat cross-linked at 125oC in the vacuum for 12 h.

Preparation of ACG sponge samples for different freezing timesThe same method was employed as described above. However, the freezing times were preset for 1, 2, and 24 h respectively at −80oC.

Preparation of ACG sponge samples of different gelatin concentrationsThe same method was used as described above. The gelatin concentration was adjusted to 0.15, 0.6 and 2.4% respectively, while the atelocollagen concentration remained 0.6%. All the samples were frozen at −80oC for 2 h.

430 Dent Mater J 2017; 36(4): 429–437

Table 1 Experimental groups of fabricated ACG sponges

Experimental Group

Variable Factors

FreezingTemperature (°C)

FreezingTime (h)

ACGConcentration* (wt/v)

Fluvastatin

Control group −80 2 0.6%:0.6% no

Freezing temperature−30−196

22

0.6%:0.6%0.6%:0.6%

nono

Freezing time−80−80

124

0.6%:0.6%0.6%:0.6%

nono

ACG concentration−80−80

2 2

0.6%:0.15%0.6%:2.4%

nono

Fluvastatin −80 2 0.6%:0.6% 500 μM

*ACG concentration: concentrations of atelocollagen and gelatin

Preparation of ACG sponge samples containing 500 μM fluvastatinFluvastatin sodium (MW=433.45, Toronto Research Chemical, Toronto, Canada) was dissolved into Milli-Q water to obtain 1 mM fluvastatin stock solution. The 500 μM fluvastatin/0.6% gelatin solution was obtained by dissolving 120 mg of gelatin powder into 10 mL of 1 mM fluvastatin stock solution and 10 mL of Milli-Q water. Then 60 mg of atelocollagen powder was slowly dissolved into 10 mL of the 500 μM fluvastatin/0.6% gelatin solution under continuous mixing at 4oC overnight. The resulting ACG composite solution contained 500 μM fluvastatin, 0.6% atelocollagen and 0.6% gelatin. The ACG composite solution was poured into 96-well plates at 200 μL/well and frozen at −80oC for 2 h. Then, the same parameters of freeze-drying and heat crosslink were used as described above.

Scanning electron microscopy (SEM) and image analysisThe pore structures of the ACG sponge samples in the different groups were examined using SEM. The ACG sponge samples were cut vertically to the long axis to obtain the horizontal cross-sectional surfaces. Sputter coating with gold/palladium (Au/Pd) alloy was performed in a sputter coater (SC 500A, Elminet, Tokyo, Japan). The cross-sectional surfaces were examined with scanning electron microscopy (HITACHI SU6600, Hitachi, Tokyo, Japan) at an accelerating voltage of 15 kV. The porosity and pore size (represented by pore area and Feret diameter) were analyzed with ImageJ 1.50g software (NIH, Bethesda, MD, USA). Three independent samples were selected for each ACG sponge experimental group and five fields (1.5 Kv×150 in size) were measured for each sample.

Cytotoxicity assay of ACG spongeHuman mesenchymal stromal cells (hMSCs) (Cell Applications, San Diego, CA, USA) at passage 7 were used for the cytotoxicity assay of ACG sponge in vitro.

The MEM Alpha (Gibco, Grand Island, NY, USA) containing 10% Fetal Bovine Serum (FBS, Biowest, Nuaillé, France) and 1% streptomycin/penicillin (Gibco) was used as the normal culture medium for the entire study. ACG sponge discs (diameter 6.0 mm, height 3.0 mm) were placed in each well of the 96-well plates. Cells subcultured at 37oC in a humidified atmosphere with 5% CO2 were suspended in MEM Alpha at a concentration of 1.25×105 cells/mL. For the experimental group, 20 μL of suspended cells (equaled 2,500 cells) was seeded onto the ACG sponge in each well. That equaled 2,500 cells/well. For the control group, 20 μL of culture medium was seeded onto the ACG sponge. The 96-well plates were incubated in a humidified atmosphere of 5% CO2

at 37oC for 1.5 h. Then, 130 μL of culture medium was added to each well to make the final total volume 150 μL. The culture medium was refreshed every two days. ACG sponge samples with or without hMSCs were harvested on day 3 and day 7. The samples were fixed with 10% neutral buffered formalin for 30 min at room temperature, dehydrated in each concentration of ethanol series (50, 70, 90 and 99.5%) for 15 min and frozen in 200 μL/well of 2-methyl-2-propanol (Wako Pure Chemical, Osaka, Japan) at −80oC for 1 h. Then frozen samples were freeze-dried at −10oC for 24 h in the vacuum. The freeze-dried samples were coated with Au/Pd alloy. SEM was performed to examine the changes in pore structure of ACG sponges and hMSCs’ growth on the ACG sponges.

Cell proliferation assayAs previously mentioned, the volume of each fabricated ACG sponge was 200 μL. The ACG extracts were obtained by immersing ACG sponge in 5 mL of culture medium at 4oC for 3 and 7 days, respectively. The hMSCs at passage 7 were seeded to the 96-well plates at 100 μL per well. The cell number per well was 2000. The 96-well plates were pre-incubated at 37oC and in a humidified atmosphere of 5% CO2 for 24 h. Then, the

431Dent Mater J 2017; 36(4): 429–437

Fig. 2 Influence of freezing temperature on pore structures of atelocollagen/gelatin (ACG) sponge. Scanning electron micrographs of ACG sponges frozen at −30oC (A, B), −80oC (C, D) and −196oC (E, F).

Quantitative analysis of porosity (G) and pore size including pore area (H) and Feret diameter (I) shown as mean±SD (n=3). Scale bars represent 1.00 mm for A, C, and E and 100 μm for B, D, and F. **p<0.001

culture medium was replaced with ACG extracts of day 3 and day 7. The culture medium was refreshed every two days with ACG extracts. The hMSCs cultured in normal culture medium were used as the control group. The cell proliferation of hMSCs was tested with Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacture’s protocol. Five parallel replicates were prepared. After incubation at 37oC for 1.5 h, the absorbance at 450 nm was determined using a Micro-plate Reader (SpectraMax M5e, Molecular Devices, CA, USA). The test was repeated for three times.

Statistical analysisAll the data were expressed as mean±standard deviation (SD). Statistical significance was determined using SPSS v20.0 software (SPSS, Chicago, IL, USA). Analysis of variance by Student’s t-test was used to determine significant differences between control and experimental groups. The p-value less than 0.05 were considered statistically significant.

RESULTS

Influence of freezing temperatures on pore structures of ACG spongeACG sponges were made for three different freezing temperature groups (−30, −80, and −196oC) (Figs. 2A, C, and E). Typical porous structures were observed on

the cross-sectional surfaces of both the −30 and −80oC groups. The pores were hexagonal in shape (Figs. 2B and D). ACG sponge frozen at −196oC had no regular pore formations (Fig. 2F). Because the pores in the −196oC group were extremely small, measurements of porosity and pore size were not performed. The −30 and −80oC groups had the same porosity, about 76.5% (Fig. 2G). However, the pore area and diameter were relatively larger in the −30oC group (p<0.001). The mean pore area was 6,334±94 μm2 and the diameter was 114±1 μm in the −30oC group, whereas the mean pore area was 4,286±63 μm2 and the diameter was 93±1 μm in the −80oC group (control group) (Figs. 2H and I).

Influence of freezing times on pore structures of ACG spongeACG sponges were made for different freezing time groups (1, 2 and 24 h) (Figs. 3A, C and E). Similar porous structures were observed in all three groups, and pores were hexagon in shape (Figs. 3B, D and F). As for porosity, no statistical significances were observed among the three groups. The mean porosity of the three groups was about 76.5% (Fig. 3G). In addition, the pore area and diameters among the three groups were not statistically different. The mean pore area was about 4,300 μm2 and the diameter was about 93 μm (Figs. 3H and I).

432 Dent Mater J 2017; 36(4): 429–437

Fig. 3 Influence of freezing time on pore structures of atelocollagen/gelatin (ACG) sponge. Scanning electron micrographs of ACG sponges frozen for 1 h (A, B), 2 h (C, D) and 24 h (E, F).

Quantitative analysis of porosity (G) and pore size including pore area (H) and Feret diameter (I) shown as mean±SD (n=3). Scale bars represent 1.00 mm for A, C, and E and 100 μm for B, D, and F.

Fig. 4 Influence of gelatin concentrations on pore structures of atelocollagen/gelatin (ACG) sponge. Scanning electron micrographs of ACG sponges in different gelatin concentrations: 0.6%AC+0.15%G

(A, B), 0.6%AC+0.6%G (C, D) and 0.6%AC+2.4%G (E, F). Quantitative analysis of porosity (G) and pore size including pore area (H) and Feret diameter (I) shown as mean±SD (n=3). Scale bars represent 1.00 mm for A, C and E, and 100 μm for B, D and F. AC: atelocollagen, G: gelatin. **p<0.001

433Dent Mater J 2017; 36(4): 429–437

Fig. 5 Influence of fluvastatin on pore structures of atelocollagen/gelatin (ACG) sponge. Scanning electron micrographs of ACG sponges without fluvastatin (A, B) and with 500 μM fluvastatin (C,

D). Quantitative analysis of porosity (E) and pore size including pore area (F) and Feret diameter (G) shown as mean±SD (n=3). Scale bars represent 1.00 mm for A and C and 100 μm for B and D. Flu: fluvastatin

Influence of gelatin concentrations on pore structures of ACG spongeThe 0.6%AC+0.15%G group showed no typical pore formations (Figs. 4A and B), whereas the 0.6%AC+0.6%G group (control group) (Figs. 4C and D) and 0.6%AC+2.4%G group (Figs. 4E and F) had regular hexagonal pores. Increased pore wall thickness was observed in the 0.6%AC+2.4%G group (Fig. 4F). The 0.6%AC+0.15%G group had the highest porosity (86.23±0.16)%, while the 0.6%AC+2.4%G group had the lowest porosity (66.40±0.22)%. The control group (0.6%AC+0.6%G) had a porosity of (76.38±0.34)%. All three groups were statistically different (p<0.001) (Fig. 4G). Furthermore, the 0.6%AC+0.6%G group had a larger pore area and diameter than those of the 0.6%AC+2.4%G group (pore area 3,261±48 μm2 and diameter 79±1 μm) (Figs. 4H and I). The pore area and diameter of the 0.6%AC+0.15%G group were not measured as no regular pores were formed (Fig. 4B).

Influence of fluvastatin on pore structures of ACG spongeACG sponges were obtained in both ACG groups without fluvastatin and with 500 μM fluvastatin (Figs. 5A and C). Similar porous structures were observed in both groups (Figs. 5B and D). The porosity was (76.38±0.34)% in the ACG without fluvastatin, and (75.78±0.11)% in the ACG with 500 μM fluvastatin. No statistical significance was observed between the two groups (Fig. 5E). The pore area and diameter showed no significant statistical differences between the two groups (Figs. 5F and G).

Cytotoxicity and cell proliferation assaysAlthough the pore structures were well preserved in the culture medium, pore shrinkage occurred and degradation of ACG sponge was observed by day 3 (Fig. 6A). More significant degradation of ACG sponge occurred by day 7 compared with that by day 3 (Fig. 6B). Seeded hMSCs were evenly distributed and well attached to the surface of ACG sponges by day 3 (Fig. 6C). After 7-d culture, hMSCs were still viable and evenly covered the surface of the ACG sponges. The numbers

434 Dent Mater J 2017; 36(4): 429–437

Fig. 6 Cytotoxicity assay of atelocollagen/gelatin (ACG) sponge. ACG sponge in culture medium for 3 days (A) and 7 days (B). Co-culture of ACG sponge and

hMSCs in culture medium for 3 days (C) and 7 days (D). Scale bars represent 100 μm.

Fig. 7 Proliferation of hMSCs cultured in ACG extracts of day 3 and day 7 analyzed by CCK-8 assay (n=5).

ACG extracts of day 3 and day 7 posed no cytotoxic effect on hMSCs’ proliferation. On day 5 and day 6, the ACG extract of day 3 group showed a bit higher proliferation rate compared with the control group.

*p<0.05

of hMSCs increased (Fig. 6D). The cell proliferation analyzed with CCK-8 assay revealed that the hMSCs cultured in the ACG extracts of day 3 and day 7 showed almost the same proliferation rate as the control group did. On day 5 and day 6, the ACG extract of day 3 group showed a bit higher proliferation rate compared with the control group (Fig. 7).

DISCUSSION

Lyophilization has been used to fabricate porous scaffolds for tissue engineering32). Organic solvents such as acetic acid and cytotoxic chemicals such as glutaraldehyde20) for chemical crosslink reaction were mainly used. The main problems encountered in lyophilization were the presence of potential toxic residuals that compromised cell growth and proliferation on the scaffolds18). In our case, an easier way was developed to fabricate ACG sponge using lyophilization. Milli-Q water was used instead of organic solvents. The gelatin was found to facilitate the dissolution of atelocollagen powder into Milli-Q water. Moreover, the chemical crosslink reaction was replaced with the heat crosslink reaction (at 125oC in the vacuum). Thus, no toxic residuals remained in the ACG sponge and the pH values were unchanged. The high temperature used for the heat crosslink reaction also served to sterilize the ACG sponge.

Several studies have reported that pore sizes were important factors that influenced cell adhesion and differentiation on the porous scaffolds4,15,33,34). In our study, the ACG sponge fabricated with lyophilization had a mean pore size of about 90–115 μm. At a higher freezing temperature such as −30oC, the pore size increased to 115 μm. This was almost the same size given in the report using collagen-glycosaminoglycan (GAG) scaffold4). These results suggested that higher freezing temperatures increased pore size, compared with pores fabricated at a low temperature of −80oC and an extremely low temperature of −196oC. This finding

435Dent Mater J 2017; 36(4): 429–437

was consistent with the previous report that a low temperature resulted in the rapid formation of small, dense ice crystals while a high temperature resulted in the slow formation of sparse and larger ice crystals35). In addition, the introduction of an annealing step during freeze-drying was found to result in a significant increase (40%) in pore size36). If combined with the gas foam technique10), achievement of a much larger pore size would be possible.

As reported by Zhang et al.37), high concentration reduced pore size and led to scaffolds with thick walls of recombinant human collagen-peptide-chitosan (RHCC) scaffolds, which was consistent with our study results. Although high gelatin concentrations led to higher mechanical strength, high gelatin concentrations compromised the porosity and pore formations, and increased the thickness of pore walls. In contrast, the porosity increased at low gelatin concentrations, but no regular pores were formed in the ACG sponge. The freezing time did not affect the porosity and pore size of ACG scaffolds, which might be attributed to the small size of the samples used in this study. For large-size samples, freezing time should be a factor requiring serious consideration.

The collagen/gelatin sponge was used as a drug delivery system of bFGF for the treatment of scarred vocal fold, and had no cytotoxic effects on vocal fold fibroblasts38,39). Fluvastatin released from poly(ethylene glycol)-based hydrogels triggered the osteogenic differentiation and modulated the function of hMSCs40). In our previous study, fluvastatin released from gelatin hydrogel promoted bone regeneration in the calvarial bone defects24). For that reason, the influence of fluvastatin on pore structures of ACG sponge was examined. Our results showed that 500 μM fluvastatin did not affect the porosity and pore size of ACG sponges. Together with our previous results24), these results indicated that ACG sponge might also act as a potential drug delivery system of fluvastatin while the pore structures remained unchanged.

Porous structures of tissue engineering scaffolds were essential for cell migration and diffusion of oxygen and nutrients41). Chondrocytes were reported to prefer scaffolds with a pore size of between 250 and 500 μm for better proliferation and ECM production42). In this part, we found that ACG sponges retained their pore structures to a reasonable extent in the culture medium for 7 days. Gradual degradation of the ACG sponges was observed in the pore walls. In addition, the hMSCs evenly distributed and attached well to the ACG sponges with an average pore size 93 μm for 7-d culture. ACG extracts of day 3 and day 7 were also shown to have almost the same proliferation rate of hMSCs as that of control group in vitro. These results suggested that ACG sponges had no cytotoxic effects and no toxic residuals remained in the sponges. O’Brien et al. 4) also reported that MC3T3-E1 cells attached most to the surface of collagen-GAG scaffold with a mean pore size of 96 μm. However, the optimal pore size for bone regeneration is still controversial43-46). The in vivo

biocompatibility and osteogenic effects of ACG sponges need further investigation.

CONCLUSION

Lyophilization is an easy and effective method of fabricating porous ACG sponges for bone regeneration. Freezing temperature and ACG concentrations influenced the porosity and pore size of ACG sponges. Freezing time and presence of fluvastatin did not change pore structures. The ACG sponges showed no cytotoxicity in vitro. Furthermore, ACG sponge may serve as a potential drug delivery system for fluvastatin, while remaining its pore structure unchanged. ACG sponge is a promising porous biomaterial scaffold for use in bone tissue engineering.

ACKNOWLEDGMENT

This work was supported by a Grant-in-Aid for Scientific Research (Young Scientists (B) 15K20488) from the Japan Society for the Promotion of Science.

REFERENCES

1) Langer R, Vacanti JP. Tissue engineering. Science 1993; 260: 920-926.

2) Peter SJ, Miller MJ, Yasko AW, Yaszemski MJ, Mikos AG. Polymer concepts in tissue engineering. J Biomed Mater Res 1998; 43: 422-427.

3) Hollister SJ. Porous scaffold design for tissue engineering. Nat Mater 2005; 4: 518-524.

4) O’Brien FJ, Harley BA, Yannas IV, Gibson LJ. The effect of pore size on cell adhesion in collagen-GAG scaffolds. Biomaterials 2005; 26: 433-441.

5) Griffon DJ, Sedighi MR, Schaeffer DV, Eurell JA, Johnson AL. Chitosan scaffolds: interconnective pore size and cartilage engineering. Acta Biomater 2006; 2: 313-320.

6) Yamane S, Iwasaki N, Kasahara Y, Harada K, Majima T, Monde K, Nishimura S, Minami A. Effect of pore size on in vitro cartilage formation using chitosan-based hyaluronic acid hybrid polymer fibers. J Biomed Mater Res A 2007; 81: 586-593.

7) Ko YG, Grice S, Kawazoe N, Tateishi T, Chen G. Preparation of collagen-glycosaminoglycan sponges with open surface porous structures using ice particulate template method. Macromol Biosci 2010; 10: 860-871.

8) Mikos AG, Thorsen AJ, Czerwonka LA, Bao Y, Langer R, Winslow DN, Vacanti JP. Preparation and characterization of poly(l-lactic acid) foams. Polymer 1994; 35: 1068-1077.

9) Mooney DJ, Baldwin DF, Suh NP, Vacanti JP, Langer R. Novel approach to fabricate porous sponges of poly(D,L-lactic-co-glycolic acid) without the use of organic solvents. Biomaterials 1996; 17: 1417-1422.

10) Nam YS, Yoon JJ, Park TG. A novel fabrication method of macroporous biodegradable polymer scaffolds using gas foaming salt as a porogen additive. J Biomed Mater Res 2000; 53: 1-7.

11) Nam YS, Park TG. Porous biodegradable polymeric scaffolds prepared by thermally induced phase separation. J Biomed Mater Res 1999; 47: 8-17.

12) Kang HW, Tabata Y, Ikada Y. Fabrication of porous gelatin scaffolds for tissue engineering. Biomaterials 1999; 20: 1339-1344.

13) Nanda HS, Chen S, Zhang Q, Kawazoe N, Chen G. Collagen scaffolds with controlled insulin release and controlled pore

436 Dent Mater J 2017; 36(4): 429–437

structure for cartilage tissue engineering. Biomed Res Int 2014; 2014: 623805.

14) Chen S, Zhang Q, Nakamoto T, Kawazoe N, Chen G. Gelatin scaffolds with controlled pore structure and mechanical property for cartilage tissue engineering. Tissue Eng Part C Methods 2016; 22: 189-198.

15) Zhang Q, Lu H, Kawazoe N, Chen G. Pore size effect of collagen scaffolds on cartilage regeneration. Acta Biomater 2014; 10: 2005-2013.

16) Khor E. Methods for the treatment of collagenous tissues for bioprostheses. Biomaterials 1997; 18: 95-105.

17) Imani R, Rafienia M, Emami SH. Synthesis and characterization of glutaraldehyde-based crosslinked gelatin as a local hemostat sponge in surgery: an in vitro study. Biomed Mater Eng 2013; 23: 211-224.

18) Huang-Lee LL, Cheung DT, Nimni ME. Biochemical changes and cytotoxicity associated with the degradation of polymeric glutaraldehyde derived crosslinks. J Biomed Mater Res 1990; 24: 1185-1201.

19) van Luyn MJ, van Wachem PB, Olde Damink LH, Dijkstra PJ, Feijen J, Nieuwenhuis P. Secondary cytotoxicity of cross-linked dermal sheep collagens during repeated exposure to human fibroblasts. Biomaterials 1992; 13: 1017-1024.

20) Jayakrishnan A, Jameela SR. Glutaraldehyde as a fixative in bioprostheses and drug delivery matrices. Biomaterials 1996; 17: 471-484.

21) Sun H, Zhu F, Hu Q, Krebsbach PH. Controlling stem cell-mediated bone regeneration through tailored mechanical properties of collagen scaffolds. Biomaterials 2014; 35: 1176-1184.

22) Mundy G, Garrett R, Harris S, Chan J, Chen D, Rossini G, Boyce B, Zhao M, Gutierrez G. Stimulation of bone formation in vitro and in rodents by statins. Science 1999; 286: 1946-1949.

23) Horiuchi N, Maeda T. Statins and bone metabolism. Oral Dis 2006; 12: 85-101.

24) Tanabe K, Nomoto H, Okumori N, Miura T, Yoshinari M. Osteogenic effect of fluvastatin combined with biodegradable gelatin-hydrogel. Dent Mater J 2012; 31: 489-493.

25) Ohira T, Tanabe K, Sasaki H, Yoshinari M, Yajima Y. Effect of locally applied fluvastatin in low-turnover osteoporosis model mouse with femur bone defect. J Hard Tissue Biol 2015; 24: 147-154.

26) Takemoto S, Morimoto N, Kimura Y, Taira T, Kitagawa T, Tomihata K, Tabata Y, Suzuki S. Preparation of collagen/gelatin sponge scaffold for sustained release of bFGF. Tissue Eng Part A 2008; 14: 1629-1638.

27) Kanda N, Morimoto N, Takemoto S, Ayvazyan AA, Kawai K, Sakamoto Y, Taira T, Suzuki S. Efficacy of novel collagen/gelatin scaffold with sustained release of basic fibroblast growth factor for dermis-like tissue regeneration. Ann Plast Surg 2012; 69: 569-574.

28) Morimoto N, Yoshimura K, Niimi M, Ito T, Aya R, Fujitaka J, Tada H, Teramukai S, Murayama T, Toyooka C, Miura K, Takemoto S, Kanda N, Kawai K, Yokode M, Shimizu A, Suzuki S. Novel collagen/gelatin scaffold with sustained release of basic fibroblast growth factor: clinical trial for chronic skin ulcers. Tissue Eng Part A 2013; 19: 1931-1940.

29) Ito R, Morimoto N, Liem PH, Nakamura Y, Kawai K, Taira T, Tsuji W, Toi M, Suzuki S. Adipogenesis using human adipose tissue-derived stromal cells combined with a collagen/gelatin sponge sustaining release of basic fibroblast growth factor. J Tissue Eng Regen Med 2014; 8: 1000-1008.

30) Lynn AK, Yannas IV, Bonfield W. Antigenicity and

immunogenicity of collagen. J Biomed Mater Res B Appl Biomater 2004; 71: 343-354.

31) Morais ARdV, Alencar ÉdN, Xavier Júnior FH, Oliveira CMd, Marcelino HR, Barratt G, Fessi H, do Egito ES, Elaissari A. Freeze-drying of emulsified systems: A review. Int J Pharm 2016; 503: 102-114.

32) Mandal BB, Kundu SC. Cell proliferation and migration in silk fibroin 3D scaffolds. Biomaterials 2009; 30: 2956-2965.

33) Nehrer S, Breinan HA, Ramappa A, Young G, Shortkroff S, Louie LK, Sledge CB, Yannas IV, Spector M. Matrix collagen type and pore size influence behaviour of seeded canine chondrocytes. Biomaterials 1997; 18: 769-776.

34) Oh SH, Park IK, Kim JM, Lee JH. In vitro and in vivo characteristics of PCL scaffolds with pore size gradient fabricated by a centrifugation method. Biomaterials 2007; 28: 1664-1671.

35) O’Brien FJ, Harley BA, Yannas IV, Gibson L. Influence of freezing rate on pore structure in freeze-dried collagen-GAG scaffolds. Biomaterials 2004; 25: 1077-1086.

36) Haugh MG, Murphy CM, O’Brien FJ. Novel freeze-drying methods to produce a range of collagen-glycosaminoglycan scaffolds with tailored mean pore sizes. Tissue Eng Part C Methods 2010; 16: 887-894.

37) Zhang J, Zhou A, Deng A, Yang Y, Gao L, Zhong Z, Yang S. Pore architecture and cell viability on freeze dried 3D recombinant human collagen-peptide (RHC)-chitosan scaffolds. Mater Sci Eng C Mater Biol Appl 2015; 49: 174-182.

38) Hiwatashi N, Hirano S, Mizuta M, Tateya I, Kanemaru S, Nakamura T, Ito J, Kawai K, Suzuki S. Biocompatibility and efficacy of collagen/gelatin sponge scaffold with sustained release of basic fibroblast growth factor on vocal fold fibroblasts in 3-dimensional culture. Ann Otol Rhinol Laryngol 2015; 124: 116-125.

39) Hiwatashi N, Hirano S, Mizuta M, Kobayashi T, Kawai Y, Kanemaru SI, Nakamura T, Ito J, Kawai K, Suzuki S. The efficacy of a novel collagen-gelatin scaffold with basic fibroblast growth factor for the treatment of vocal fold scar. J Tissue Eng Regen Med 2015.

40) Benoit DS, Nuttelman CR, Collins SD, Anseth KS. Synthesis and characterization of a fluvastatin-releasing hydrogel delivery system to modulate hMSC differentiation and function for bone regeneration. Biomaterials 2006; 27: 6102-6110.

41) Kock L, van Donkelaar CC, Ito K. Tissue engineering of functional articular cartilage: the current status. Cell Tissue Res 2012; 347: 613-627.

42) Lien SM, Ko LY, Huang TJ. Effect of pore size on ECM secretion and cell growth in gelatin scaffold for articular cartilage tissue engineering. Acta Biomater 2009; 5: 670-679.

43) Klawitter JJ, Bagwell JG, Weinstein AM, Sauer BW. An evaluation of bone growth into porous high density polyethylene. J Biomed Mater Res 1976; 10: 311-323.

44) Tsuruga E, Takita H, Itoh H, Wakisaka Y, Kuboki Y. Pore size of porous hydroxyapatite as the cell-substratum controls BMP-induced osteogenesis. J Biochem 1997; 121: 317-324.

45) Karageorgiou V, Kaplan D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 2005; 26: 5474-5491.

46) Murphy CM, Haugh MG, O’Brien FJ. The effect of mean pore size on cell attachment, proliferation and migration in collagen-glycosaminoglycan scaffolds for bone tissue engineering. Biomaterials 2010; 31: 461-466.

437Dent Mater J 2017; 36(4): 429–437