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Characteristics of PLGA–gelatin complex as potential artificial nerve scaffold
Colloids and Surfaces B: Biointerfaces 57 (2007) 198–203
Characteristics of PLGA–gelatin complex as potential artificial nerve scaffold Xiao-kun Li a,d , Shao-xi Cai b , Bin Liu b,c , Zhi-ling Xu b,e , Xiao-zhen Dai b , Kai-wang Ma b,∗ , Shao-qiang Li d , Li Yang b,e , K.L. Paul Sung e,f , Xiao-bing Fu g a
Engineering Research Center of Bioreactor and Pharmaceutical Development, Ministry of Education, Jilin Agricultural University, Changchun, 130118, PR China b College of Bioengineering, Chongqing University, Chongqing, 400044, PR China c College of Life sciences, Southwest University, Chongqing, 400716, PR China d School of Pharmacy, Wenzhou Medical College, Wenzhou, 325027, PR China e China-USA International Collaborative Lab, Chongqing University and UCSD, College of Bioengineering, Chongqing University, Chongqing, 400044, PR China f Departments of Orthopaedics and Bioengineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0412, USA g Wound Healing and Cell Biology Laboratory Institute of Basic Medical Science, PLA General Hospital, Postgraduate Medical College, Beijing, 100853, PR China Received 21 December 2006; received in revised form 31 January 2007; accepted 2 February 2007 Available online 13 February 2007
Abstract The segmentation lesion of peripheral nerve will seriously impair the motion and sensation of the patients, and the satisfactory recovery of segmented peripheral nerve by autograft or allograft is still a great challenge posing to the neurosurgery. Apart from autograft for nerve repair, different allograft has been studying. In this study, a scaffold fabricated with polylactic acid-co-glycolic acid (PLGA) copolymer and gelatin was evaluated to be a potential artificial nerve scaffold in vitro. The effect of different mass ratio between PLGA and gelatin upon the characteristics of PLGA–gelatin scaffolds such as microstructure, mechanical property, degradation behavior in PBS, cell adhesion property were investigated. The results showed the homogeneity and mechanical property of the scaffolds became poor with the increase of gelatin, and the rate of max water-uptake and the mass loss of scaffolds increases with the increase of gelatin, and the cells could adhere to the scaffolds. Those indicated the scaffolds fabricated by the PLGA–gelatin complex had excellent biocompatibility, suitable mechanical property and sustained-release characteristics, which would meet the requirements for artificial nerve scaffold. © 2007 Elsevier B.V. All rights reserved. Keywords: Nerve scaffold; Nerve repair; PLGA; Gelatin
1. Introduction The human being peripheral nerve possesses a strong regeneration potentiality. The disrupted peripheral nerve could successfully regenerate [1] if a proper environment and route were provided. At present, to recover the disrupted nerve is clinically achieved with autograft [2], but there still exist certain limits and disadvantages: the donator source limitation for
∗
Corresponding author at: College of Bioengineering, Chongqing University, Chongqing, 400044, PR China. Tel.: +86 23 65102507; fax: +86 23 65112097. E-mail address: [email protected] (K.-w. Ma). 0927-7765/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2007.02.010
autograft [3] and the accompanying risks in harvesting autograft. To avoid those disadvantages, artificial nerve scaffold has been developed and shown to regenerate the disrupted nerve successfully [1,4]. Poly (lactic acid and glycolic acid) copolymer (PLGA), due to its excellent biocompatibility, biodegradable and suitable mechanical properties, has been extensively used in tissue engineering [5–8]. Gelatin, a kind of excellent natural biomaterial, has also been used in nerve repair [9,10] for it was considered to promote cell proliferation and tissue healing. As materials for tissue engineering, good biocompatibility and suitable mechanical property are required, while in some case, the biodegradable property is to be considered [11]. We found the cell adhesion of PLGA is not better than gelatin,
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while the cell adhesion of gelatin is very good but its mechanical property is too poor. Therefore, in this study, we fabricated the PLGA–gelatin compound scaffolds consisting of different mass ratio of PLGA and gelatin, and investigated their mechanical property, degradation behavior in vitro, 3D microstructure and the ability of Schwann cell adhesion, with an purpose to illustrate the potentiality of the complex as artificial nerve scaffold. 2. Materials and methods 2.1. Fabrication of PLGA–gelatin compound scaffolds At first, gelatin (purchased from Sigma) was prepared into gelatin microballoons according to our previous report [12], and then the gelatin microballoons was sieved out with 400-screen grit to obtain gelatin microballoons with a diameter smaller than 10 m for later use. Then PLGA and gelatin microballoons were respectively added to dichlormethane (DCM) according to the mass ratio of PLGA:gelatin = 9:1, 8:2, 7:3, 6:4, 5:5 and 3:7, respectively. When PLGA dissolved into DCM completely ultrasonic wave was applied for the uniform dispersion of gelatin microballoons in the PLGA-DCM solution (gelatin microballoons does not dissolve in DCM), thus made a suspension. Then the suspension was transferred into a special tooting for shaping, following the removal of solvent DCM under natural condition, thus made the hollow scaffolds. Finally, the scaffolds were vacuum dried for 48 h to remove solvent DCM completely, thus made the scaffold as shown in Fig. 1. 2.2. Mechanical property of PLGA–gelatin compound scaffolds The scaffolds were cut into lamellar test pieces (5 mm in width, 50 mm in length) following elongation test by mechanical test machine for materials (INSTRON 1011, USA). The elongation speed was 10 mm/min; the measure parameters were “Broken Strength” and “Broken extensibility”.
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2.3. 3D microstructure of PLGA–gelatin compound scaffolds The scaffolds were made into SEM samples, and then underwent SEM scan (AMRAY 1000B, USA) to observe their 3D microstructure. 2.4. In vitro degradation of PLGA–gelatin compound scaffolds First, the scaffolds were weighed (0.01 mg precisely) respectively to get the initial mass (m0 ), and then cut them into two pieces, respectively. The two pieces was immersed in an ampoule with 10 ml PBS, then the ampoule was placed on a rocking bed with 50 rpm, finally the rocking bed was placed in a constant temperature incubator at 37 ± 0.5 ◦ C for 1 month. During the 1month experiment period, the pieces were taken out to get their wet weight and dry weight as well as observe their appearance at each sampling time point, while the PBS solution was updated weekly, and the content of hydroxyproline degraded from the PLGA–gelatin into the solution was assayed with ProOH-Kits (purchased from Nanking Jiancheng Company, China) at each updating time point. 2.5. Cell adhesion test of PLGA–gelatin compound scaffolds The scaffold was cut into pieces (≈20 mm2 ) following ETOX-sterilization. Then the pieces were co culturing with cells (obtained from mouse’s sciatic nerve) in DMEM (purchased from Sigma). After the pieces were co cultured with Schwann cells for 3 days, the pieces were taken out and then fixed with glutaraldehyde for 24 h, finally the fixed pieces were made into SEM samples, following SEM scan to observe the cell adhesiveness by scanning electron microscope (AMRAY 1000B, USA). 3. Results 3.1. The mechanical property of PLGA–gelatin compound scaffolds The result of “Broken Strength” of the PLGA–gelatin compound scaffolds was shown in Fig. 2a, while the result of “Broken extensibility” was shown in Fig. 2b. Generally speaking, from Fig. 2 it could be seen the friability of the scaffolds increased with the increase of gelatin in the scaffold, while the tenacity became poor with the increase of gelatin in the scaffold. 3.2. 3D microstructure of PLGA–gelatin compound scaffolds
Fig. 1. PLGA–gelatin scaffold.
Their 3D microstructure measured by SEM was shown in Fig. 3. Generally speaking, from Fig. 3 it was observed the homogeneity of the compound scaffolds became poor with the increase of gelatin in it.
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Fig. 2. Elongation mechanical property of the scaffolds. The size of all the lamellar test pieces were 5 mm (width) × 50 mm (length), elongation test by mechanical test machine for materials (INSTRON 1011, USA). The elongation speed was 10 mm/min. (a) Broken strength and (b) broken extensibility.
3.3. Schwann cell adhesion test of PLGA–gelatin compound scaffolds
3.4. In vitro degradation behavior of PLGA–gelatin compound scaffolds
found the more the gelatin in the scaffold the greater the scaffold swells. Meanwhile, it was also found the time for the scaffolds keeping their original shape will shorten with the increase of gelatin in the scaffold, especially for the “3:7” scaffold, its original shape could not be kept any more after 6 h, while the others could mainly keep their original shape after 8 weeks. And the more the PLGA in the scaffold, the better the scaffold kept its shape, thus suggested the importance of mechanical property provided by PLGA for the scaffold.
3.4.1. Gross observation It could be seen the scaffolds would adsorb water and swell with time elapsing during the degradation period, and it was also
3.4.2. The max water uptake The max water uptake of scaffolds in PBS with different mass ratio during the degradation period in vitro was shown in Fig. 5,
The result was shown in Fig. 4. It was obvious that Schwann cells can adhere to all the surface of the scaffolds with different mass ratio.
Fig. 3. 3D microstructure of the scaffolds with different mass ratio of PLGA and gelatin, SEM, Scanned by AMRAY 1000B. The mass ratio of PLGA and gelatin was 9:1, 8:2, 7:3, 6:4, 5:5 and 3:7, respectively.
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Fig. 4. Cells adhering to the scaffolds with different mass ratio of PLGA and gelatin after co-culturing for 3 days. SEM, Scanned by AMRAY 1000B. The mass ratio of PLGA and gelatin was 9:1, 8:2, 7:3, 6:4, 5:5 and 3:7, respectively.
and the formula for the max water uptake was as follow: max water uptake (%) = (((max wet weight) − (initial dried weight))/ (initial dried weight)) × 100%. 3.4.3. Mass loss rate The mass loss rate of the scaffolds varying with degradation time was shown in Fig. 6, and the formula for the mass loss rate was as follow: mass loss rate (%) = (((initial dried weight) − (dried weight at sampling time point))/
Generally speaking, from Fig. 5 it could be seen that the max water uptake increased with the increment of gelatin; and it could be seen from Fig. 6 that the mass loss rate became faster with the increase of gelatin in scaffolds. 3.4.4. The accumulative releasing ratio of hydroxyproline from the scaffolds into the solutions varying with degradation time The result of the accumulative releasing ratio of hydroxyproline from the scaffolds into the solutions varying with degradation time was shown in Fig. 7. Generally speaking, from Fig. 7 it could be considered that all the scaffolds with different mass ratio had sustained release performance during the degradation period. Obviously, the sus-
(initial dried weight)) × 100%.
Fig. 5. The max water uptake of the scaffolds. The mass ratio of PLGA and gelatin was 9:1, 8:2, 7:3, 6:4, 5:5 and 3:7, respectively. The scaffolds were immersed in PBS at 37 ◦ C for 8 weeks.
Fig. 6. The mass loss rate of the scaffolds varying with time. The mass ratio of PLGA and gelatin was 9:1, 8:2, 7:3, 6:4, 5:5 and 3:7, respectively. The scaffolds were immersed in PBS at 37 ◦ C for 8 weeks.
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Fig. 7. The accumulative releasing ratio of hydroxyproline varying with degradation time. The mass ratio of PLGA and gelatin was 9:1, 8:2, 7:3, 6:4, 5:5 and 3:7, respectively. The scaffolds were immersed in PBS at 37 ◦ C for 8 weeks.
tained release performance would become poor with the increase of gelatin in scaffold. 4. Discussion Generally, from Fig. 2, it could be seen the mechanical property of scaffolds became poor with the increase of gelatin in the scaffolds. As scaffold for tissue engineering, suitable mechanical property is required except for good biocompatibility and/or biodegradation [11]. It is known to all that suitable mechanical property required for tissue engineering scaffold is to meet the surgical operation and bear the load produced by the motion of the host’s body. PLGA is a polymer and has elasticity, tenacity and rigidity, while in body fluid gelatin has no elasticity, tenacity and rigidity, therefore, theoretically the fragility and tenacity of the scaffold would become poor with the increase of gelatin in the scaffold, and the result of mechanical test also proved the hypothesis. And from the result, it could be considered that the addition of gelatin could adjust the mechanical property of PLGA polymer. It was also seen from Fig. 3 that the homogeneity of scaffolds became poor with the increase of gelatin in the scaffolds. Obviously, It is certain that the greater the homogeneity of scaffolds the better the consistency of the whole and the local property of the scaffolds. The reason for the change of homogeneity may be that dried gelatin microballoons is non-sticky, therefore, its capability to disperse in the PLGA solution was provided by the viscosity of PLGA solution, increasing the content of gelatin will accordingly result in the decrease of PLGA, while the decrease of PLGA will accordingly reduce the viscosity of PLGA solution, thus will cause the poor stability of PLGA–(gelatin microballoons) solution. During the fabricating process, the poor stability of PLGA–(gelatin microballoons) solution will lead to the poor homogeneity of finished scaffolds. Good biocompatibility is a required property for tissue engineering scaffold [11]. The biocompatibility of PLGA and gelatin has been proved [9,10,13–15]. From Fig. 4, we can see all the scaffolds have good property for cell adhesion, thus illustrates
the good biocompatibility of the scaffolds fabricated in the study. Meanwhile, from the result of cell adhesion test, we found the addition of gelatin could improve the biocompatibility of PLGA. Generally speaking, it could be seen from Fig. 5 that the max water uptake of scaffolds increased with the increase of gelatin in them, and the more the content of gelatin in scaffold the shorter the time for the scaffold gets to the max water uptake varying from several hours to days. PLGA is a polymer while gelatin is a macromolecule, however, the hydrophilicity of gelatin macromolecule is far better than that of PLGA polymer; thus gelatin can easily dissolve in water while PLGA only gradually swells by the permeation of water molecules [15]. Therefore, the addition of gelatin can enhance the hydroscopicity of PLGA, obviously, the more the addition of gelatin the greater the water uptake of the scaffold. However, the enhancement of hydroscopicity of the scaffold may lead to the easier disaggregation and the decrease of strength of the scaffold, as well as excessive swell of scaffold in dimension thus may cause the greater compression response at the host’s implanting site. Therefore, the content of gelatin in the scaffold is restricted to a proper extent. It could be seen from Fig. 6 that the mass loss rate of scaffolds quickened with the increase of gelatin in them. It is obvious that gelatin is easily dissolvable in 37 ◦ C water, therefore, the more the content of gelatin in scaffold the more and the easier gelatin dissolves out, thus resulted in the increase of mass loss rate. Taking “3:7” scaffold for example, because of the higher content of gelatin, its mass loss rate is the fastest and it got to the max mass loss ratio in several hours. Generally speaking, from Fig. 7 it could be seen the accumulative releasing ratio of hydroxyproline increased with the degradation time prolonged, and the releasing rate quickened with the increase of gelatin. Obviously, hydroxyproline came from gelatin; therefore, the releasing rate/ratio of hydroxyproline also represents the releasing rate/ratio of gelatin liberating from the scaffold. The result also suggests that the content of gelatin can affect the sustained-releasing property of the compound scaffold. Additionally, especially for the “3:7” sample, at 2 weeks, there was almost no hydroxyproline left in the scaffold, which indicated gelatin had almost been dissolved out from the scaffold into artificial degradation solution. The reason may be the very poor homogeneity in “3:7”, which resulted in the outburst of gelatin from the scaffold and complete release before the start of PLGA degradation. The successful repair of disrupted peripheral nerve will be affected by many factors, such as scaffold providing proper route and microenvironment to guide its regeneration, drug or nerve growth factors promoting its regeneration, therefore, active compound nerve scaffold should function better than that of simple and inactive one. Studies have shown addition of nerve growth factor into scaffold or seeding Schwann cell upon scaffold can promote the regeneration of disrupted nerve [16–18]. However, nerve growth factors bear shorter half-life time in vivo [19], which requires sustained release of them for better performance in vivo; similarly, seeding Schwann cell upon scaffold requires good cell adhesion property of artificial nerve scaf-
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fold. To achieve the purpose of sustained release and prevention of activity loss of nerve growth factor, it could be encapsulated into gelatin-microballoons as was reported in our previous work [12], then scaffold designed by this method should be of good biocompatibility and proper mechanical property, as well as could load and sustained release nerve growth factor. From Fig. 7, it could be seen the scaffold could sustained release hydroxyproline with time, if nerve growth factor were to be encapsulated into the gelatin-microballoons it could be believed the scaffold should control the release of nerve growth factor. 5. Conclusion The scaffold designed in the study is of good biocompatibility, sustained release property and proper mechanical property. Taking the comprehensive parameters into consideration, to meet the requirement of nerve scaffold, the compound scaffold should consist mainly of PLGA with the addition of gelatin as adjusting reagent. Therefore, the scaffold fabricated by this method could be a kind of promising nerve scaffold, and its in vivo investigation is onging. Acknowledgements This work was supported by The National Basic Research Program of China (973 Grant No. 2005CB522603), the Program of New Century Excellent Talents in University (Li xiaokun), ZheJiang province key Natural Science Foundation (Grant No. Z205755) and “111-Plan” (College of Bioengineering, Chongqing University, PR China).
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Characteristics of PLGA–gelatin complex as potential artificial nerve scaffold Xiao-kun Li a,d , Shao-xi Cai b , Bin Liu b,c , Zhi-ling Xu b,e , Xiao-zhen Dai b , Kai-wang Ma b,∗ , Shao-qiang Li d , Li Yang b,e , K.L. Paul Sung e,f , Xiao-bing Fu g a
Engineering Research Center of Bioreactor and Pharmaceutical Development, Ministry of Education, Jilin Agricultural University, Changchun, 130118, PR China b College of Bioengineering, Chongqing University, Chongqing, 400044, PR China c College of Life sciences, Southwest University, Chongqing, 400716, PR China d School of Pharmacy, Wenzhou Medical College, Wenzhou, 325027, PR China e China-USA International Collaborative Lab, Chongqing University and UCSD, College of Bioengineering, Chongqing University, Chongqing, 400044, PR China f Departments of Orthopaedics and Bioengineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0412, USA g Wound Healing and Cell Biology Laboratory Institute of Basic Medical Science, PLA General Hospital, Postgraduate Medical College, Beijing, 100853, PR China Received 21 December 2006; received in revised form 31 January 2007; accepted 2 February 2007 Available online 13 February 2007
Abstract The segmentation lesion of peripheral nerve will seriously impair the motion and sensation of the patients, and the satisfactory recovery of segmented peripheral nerve by autograft or allograft is still a great challenge posing to the neurosurgery. Apart from autograft for nerve repair, different allograft has been studying. In this study, a scaffold fabricated with polylactic acid-co-glycolic acid (PLGA) copolymer and gelatin was evaluated to be a potential artificial nerve scaffold in vitro. The effect of different mass ratio between PLGA and gelatin upon the characteristics of PLGA–gelatin scaffolds such as microstructure, mechanical property, degradation behavior in PBS, cell adhesion property were investigated. The results showed the homogeneity and mechanical property of the scaffolds became poor with the increase of gelatin, and the rate of max water-uptake and the mass loss of scaffolds increases with the increase of gelatin, and the cells could adhere to the scaffolds. Those indicated the scaffolds fabricated by the PLGA–gelatin complex had excellent biocompatibility, suitable mechanical property and sustained-release characteristics, which would meet the requirements for artificial nerve scaffold. © 2007 Elsevier B.V. All rights reserved. Keywords: Nerve scaffold; Nerve repair; PLGA; Gelatin
1. Introduction The human being peripheral nerve possesses a strong regeneration potentiality. The disrupted peripheral nerve could successfully regenerate [1] if a proper environment and route were provided. At present, to recover the disrupted nerve is clinically achieved with autograft [2], but there still exist certain limits and disadvantages: the donator source limitation for
∗
Corresponding author at: College of Bioengineering, Chongqing University, Chongqing, 400044, PR China. Tel.: +86 23 65102507; fax: +86 23 65112097. E-mail address: [email protected] (K.-w. Ma). 0927-7765/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2007.02.010
autograft [3] and the accompanying risks in harvesting autograft. To avoid those disadvantages, artificial nerve scaffold has been developed and shown to regenerate the disrupted nerve successfully [1,4]. Poly (lactic acid and glycolic acid) copolymer (PLGA), due to its excellent biocompatibility, biodegradable and suitable mechanical properties, has been extensively used in tissue engineering [5–8]. Gelatin, a kind of excellent natural biomaterial, has also been used in nerve repair [9,10] for it was considered to promote cell proliferation and tissue healing. As materials for tissue engineering, good biocompatibility and suitable mechanical property are required, while in some case, the biodegradable property is to be considered [11]. We found the cell adhesion of PLGA is not better than gelatin,
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while the cell adhesion of gelatin is very good but its mechanical property is too poor. Therefore, in this study, we fabricated the PLGA–gelatin compound scaffolds consisting of different mass ratio of PLGA and gelatin, and investigated their mechanical property, degradation behavior in vitro, 3D microstructure and the ability of Schwann cell adhesion, with an purpose to illustrate the potentiality of the complex as artificial nerve scaffold. 2. Materials and methods 2.1. Fabrication of PLGA–gelatin compound scaffolds At first, gelatin (purchased from Sigma) was prepared into gelatin microballoons according to our previous report [12], and then the gelatin microballoons was sieved out with 400-screen grit to obtain gelatin microballoons with a diameter smaller than 10 m for later use. Then PLGA and gelatin microballoons were respectively added to dichlormethane (DCM) according to the mass ratio of PLGA:gelatin = 9:1, 8:2, 7:3, 6:4, 5:5 and 3:7, respectively. When PLGA dissolved into DCM completely ultrasonic wave was applied for the uniform dispersion of gelatin microballoons in the PLGA-DCM solution (gelatin microballoons does not dissolve in DCM), thus made a suspension. Then the suspension was transferred into a special tooting for shaping, following the removal of solvent DCM under natural condition, thus made the hollow scaffolds. Finally, the scaffolds were vacuum dried for 48 h to remove solvent DCM completely, thus made the scaffold as shown in Fig. 1. 2.2. Mechanical property of PLGA–gelatin compound scaffolds The scaffolds were cut into lamellar test pieces (5 mm in width, 50 mm in length) following elongation test by mechanical test machine for materials (INSTRON 1011, USA). The elongation speed was 10 mm/min; the measure parameters were “Broken Strength” and “Broken extensibility”.
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2.3. 3D microstructure of PLGA–gelatin compound scaffolds The scaffolds were made into SEM samples, and then underwent SEM scan (AMRAY 1000B, USA) to observe their 3D microstructure. 2.4. In vitro degradation of PLGA–gelatin compound scaffolds First, the scaffolds were weighed (0.01 mg precisely) respectively to get the initial mass (m0 ), and then cut them into two pieces, respectively. The two pieces was immersed in an ampoule with 10 ml PBS, then the ampoule was placed on a rocking bed with 50 rpm, finally the rocking bed was placed in a constant temperature incubator at 37 ± 0.5 ◦ C for 1 month. During the 1month experiment period, the pieces were taken out to get their wet weight and dry weight as well as observe their appearance at each sampling time point, while the PBS solution was updated weekly, and the content of hydroxyproline degraded from the PLGA–gelatin into the solution was assayed with ProOH-Kits (purchased from Nanking Jiancheng Company, China) at each updating time point. 2.5. Cell adhesion test of PLGA–gelatin compound scaffolds The scaffold was cut into pieces (≈20 mm2 ) following ETOX-sterilization. Then the pieces were co culturing with cells (obtained from mouse’s sciatic nerve) in DMEM (purchased from Sigma). After the pieces were co cultured with Schwann cells for 3 days, the pieces were taken out and then fixed with glutaraldehyde for 24 h, finally the fixed pieces were made into SEM samples, following SEM scan to observe the cell adhesiveness by scanning electron microscope (AMRAY 1000B, USA). 3. Results 3.1. The mechanical property of PLGA–gelatin compound scaffolds The result of “Broken Strength” of the PLGA–gelatin compound scaffolds was shown in Fig. 2a, while the result of “Broken extensibility” was shown in Fig. 2b. Generally speaking, from Fig. 2 it could be seen the friability of the scaffolds increased with the increase of gelatin in the scaffold, while the tenacity became poor with the increase of gelatin in the scaffold. 3.2. 3D microstructure of PLGA–gelatin compound scaffolds
Fig. 1. PLGA–gelatin scaffold.
Their 3D microstructure measured by SEM was shown in Fig. 3. Generally speaking, from Fig. 3 it was observed the homogeneity of the compound scaffolds became poor with the increase of gelatin in it.
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Fig. 2. Elongation mechanical property of the scaffolds. The size of all the lamellar test pieces were 5 mm (width) × 50 mm (length), elongation test by mechanical test machine for materials (INSTRON 1011, USA). The elongation speed was 10 mm/min. (a) Broken strength and (b) broken extensibility.
3.3. Schwann cell adhesion test of PLGA–gelatin compound scaffolds
3.4. In vitro degradation behavior of PLGA–gelatin compound scaffolds
found the more the gelatin in the scaffold the greater the scaffold swells. Meanwhile, it was also found the time for the scaffolds keeping their original shape will shorten with the increase of gelatin in the scaffold, especially for the “3:7” scaffold, its original shape could not be kept any more after 6 h, while the others could mainly keep their original shape after 8 weeks. And the more the PLGA in the scaffold, the better the scaffold kept its shape, thus suggested the importance of mechanical property provided by PLGA for the scaffold.
3.4.1. Gross observation It could be seen the scaffolds would adsorb water and swell with time elapsing during the degradation period, and it was also
3.4.2. The max water uptake The max water uptake of scaffolds in PBS with different mass ratio during the degradation period in vitro was shown in Fig. 5,
The result was shown in Fig. 4. It was obvious that Schwann cells can adhere to all the surface of the scaffolds with different mass ratio.
Fig. 3. 3D microstructure of the scaffolds with different mass ratio of PLGA and gelatin, SEM, Scanned by AMRAY 1000B. The mass ratio of PLGA and gelatin was 9:1, 8:2, 7:3, 6:4, 5:5 and 3:7, respectively.
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Fig. 4. Cells adhering to the scaffolds with different mass ratio of PLGA and gelatin after co-culturing for 3 days. SEM, Scanned by AMRAY 1000B. The mass ratio of PLGA and gelatin was 9:1, 8:2, 7:3, 6:4, 5:5 and 3:7, respectively.
and the formula for the max water uptake was as follow: max water uptake (%) = (((max wet weight) − (initial dried weight))/ (initial dried weight)) × 100%. 3.4.3. Mass loss rate The mass loss rate of the scaffolds varying with degradation time was shown in Fig. 6, and the formula for the mass loss rate was as follow: mass loss rate (%) = (((initial dried weight) − (dried weight at sampling time point))/
Generally speaking, from Fig. 5 it could be seen that the max water uptake increased with the increment of gelatin; and it could be seen from Fig. 6 that the mass loss rate became faster with the increase of gelatin in scaffolds. 3.4.4. The accumulative releasing ratio of hydroxyproline from the scaffolds into the solutions varying with degradation time The result of the accumulative releasing ratio of hydroxyproline from the scaffolds into the solutions varying with degradation time was shown in Fig. 7. Generally speaking, from Fig. 7 it could be considered that all the scaffolds with different mass ratio had sustained release performance during the degradation period. Obviously, the sus-
(initial dried weight)) × 100%.
Fig. 5. The max water uptake of the scaffolds. The mass ratio of PLGA and gelatin was 9:1, 8:2, 7:3, 6:4, 5:5 and 3:7, respectively. The scaffolds were immersed in PBS at 37 ◦ C for 8 weeks.
Fig. 6. The mass loss rate of the scaffolds varying with time. The mass ratio of PLGA and gelatin was 9:1, 8:2, 7:3, 6:4, 5:5 and 3:7, respectively. The scaffolds were immersed in PBS at 37 ◦ C for 8 weeks.
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Fig. 7. The accumulative releasing ratio of hydroxyproline varying with degradation time. The mass ratio of PLGA and gelatin was 9:1, 8:2, 7:3, 6:4, 5:5 and 3:7, respectively. The scaffolds were immersed in PBS at 37 ◦ C for 8 weeks.
tained release performance would become poor with the increase of gelatin in scaffold. 4. Discussion Generally, from Fig. 2, it could be seen the mechanical property of scaffolds became poor with the increase of gelatin in the scaffolds. As scaffold for tissue engineering, suitable mechanical property is required except for good biocompatibility and/or biodegradation [11]. It is known to all that suitable mechanical property required for tissue engineering scaffold is to meet the surgical operation and bear the load produced by the motion of the host’s body. PLGA is a polymer and has elasticity, tenacity and rigidity, while in body fluid gelatin has no elasticity, tenacity and rigidity, therefore, theoretically the fragility and tenacity of the scaffold would become poor with the increase of gelatin in the scaffold, and the result of mechanical test also proved the hypothesis. And from the result, it could be considered that the addition of gelatin could adjust the mechanical property of PLGA polymer. It was also seen from Fig. 3 that the homogeneity of scaffolds became poor with the increase of gelatin in the scaffolds. Obviously, It is certain that the greater the homogeneity of scaffolds the better the consistency of the whole and the local property of the scaffolds. The reason for the change of homogeneity may be that dried gelatin microballoons is non-sticky, therefore, its capability to disperse in the PLGA solution was provided by the viscosity of PLGA solution, increasing the content of gelatin will accordingly result in the decrease of PLGA, while the decrease of PLGA will accordingly reduce the viscosity of PLGA solution, thus will cause the poor stability of PLGA–(gelatin microballoons) solution. During the fabricating process, the poor stability of PLGA–(gelatin microballoons) solution will lead to the poor homogeneity of finished scaffolds. Good biocompatibility is a required property for tissue engineering scaffold [11]. The biocompatibility of PLGA and gelatin has been proved [9,10,13–15]. From Fig. 4, we can see all the scaffolds have good property for cell adhesion, thus illustrates
the good biocompatibility of the scaffolds fabricated in the study. Meanwhile, from the result of cell adhesion test, we found the addition of gelatin could improve the biocompatibility of PLGA. Generally speaking, it could be seen from Fig. 5 that the max water uptake of scaffolds increased with the increase of gelatin in them, and the more the content of gelatin in scaffold the shorter the time for the scaffold gets to the max water uptake varying from several hours to days. PLGA is a polymer while gelatin is a macromolecule, however, the hydrophilicity of gelatin macromolecule is far better than that of PLGA polymer; thus gelatin can easily dissolve in water while PLGA only gradually swells by the permeation of water molecules [15]. Therefore, the addition of gelatin can enhance the hydroscopicity of PLGA, obviously, the more the addition of gelatin the greater the water uptake of the scaffold. However, the enhancement of hydroscopicity of the scaffold may lead to the easier disaggregation and the decrease of strength of the scaffold, as well as excessive swell of scaffold in dimension thus may cause the greater compression response at the host’s implanting site. Therefore, the content of gelatin in the scaffold is restricted to a proper extent. It could be seen from Fig. 6 that the mass loss rate of scaffolds quickened with the increase of gelatin in them. It is obvious that gelatin is easily dissolvable in 37 ◦ C water, therefore, the more the content of gelatin in scaffold the more and the easier gelatin dissolves out, thus resulted in the increase of mass loss rate. Taking “3:7” scaffold for example, because of the higher content of gelatin, its mass loss rate is the fastest and it got to the max mass loss ratio in several hours. Generally speaking, from Fig. 7 it could be seen the accumulative releasing ratio of hydroxyproline increased with the degradation time prolonged, and the releasing rate quickened with the increase of gelatin. Obviously, hydroxyproline came from gelatin; therefore, the releasing rate/ratio of hydroxyproline also represents the releasing rate/ratio of gelatin liberating from the scaffold. The result also suggests that the content of gelatin can affect the sustained-releasing property of the compound scaffold. Additionally, especially for the “3:7” sample, at 2 weeks, there was almost no hydroxyproline left in the scaffold, which indicated gelatin had almost been dissolved out from the scaffold into artificial degradation solution. The reason may be the very poor homogeneity in “3:7”, which resulted in the outburst of gelatin from the scaffold and complete release before the start of PLGA degradation. The successful repair of disrupted peripheral nerve will be affected by many factors, such as scaffold providing proper route and microenvironment to guide its regeneration, drug or nerve growth factors promoting its regeneration, therefore, active compound nerve scaffold should function better than that of simple and inactive one. Studies have shown addition of nerve growth factor into scaffold or seeding Schwann cell upon scaffold can promote the regeneration of disrupted nerve [16–18]. However, nerve growth factors bear shorter half-life time in vivo [19], which requires sustained release of them for better performance in vivo; similarly, seeding Schwann cell upon scaffold requires good cell adhesion property of artificial nerve scaf-
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fold. To achieve the purpose of sustained release and prevention of activity loss of nerve growth factor, it could be encapsulated into gelatin-microballoons as was reported in our previous work [12], then scaffold designed by this method should be of good biocompatibility and proper mechanical property, as well as could load and sustained release nerve growth factor. From Fig. 7, it could be seen the scaffold could sustained release hydroxyproline with time, if nerve growth factor were to be encapsulated into the gelatin-microballoons it could be believed the scaffold should control the release of nerve growth factor. 5. Conclusion The scaffold designed in the study is of good biocompatibility, sustained release property and proper mechanical property. Taking the comprehensive parameters into consideration, to meet the requirement of nerve scaffold, the compound scaffold should consist mainly of PLGA with the addition of gelatin as adjusting reagent. Therefore, the scaffold fabricated by this method could be a kind of promising nerve scaffold, and its in vivo investigation is onging. Acknowledgements This work was supported by The National Basic Research Program of China (973 Grant No. 2005CB522603), the Program of New Century Excellent Talents in University (Li xiaokun), ZheJiang province key Natural Science Foundation (Grant No. Z205755) and “111-Plan” (College of Bioengineering, Chongqing University, PR China).
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