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Injectable photo-crosslinking cartilage decellularized extracellular matrix for cartilage tissue regeneration
Materials Letters 268 (2020) 127609
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/mlblue
Injectable photo-crosslinking cartilage decellularized extracellular matrix for cartilage tissue regeneration Yong Xu b,1, Litao Jia d,1, Zongxin Wang d,1, Gening Jiang b, Guangdong Zhou d,⇑, Weiming Chen c,⇑, Ru Chen a,⇑ a
Department of Breast Surgery, Hainan General Hospital, Hainan Medical University, Hainan, China Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China c Shanghai Yapeng Biotechnology Co., Ltd, Shanghai, China d Research Institute of Plastic Surgery, Wei Fang Medical College, Shandong, China b
a r t i c l e
i n f o
Article history: Received 25 July 2019 Received in revised form 22 February 2020 Accepted 4 March 2020 Available online 5 March 2020 Keywords: Decellularized extracellular matrix Biomaterials Sol-gel preparation Photo-crosslinking
a b s t r a c t Cartilage decellularized extracellular matrix (CDEM) is a promising biomaterial for preparing tissue engineering scaffolds, and CDEM-based hydrogels constitute an ideal choice for repairing cartilage damage. In this study, CDEM powders and photo-responsive hyaluronic acid (pHA) were successfully fabricated into injectable and photo-crosslinkable hydrogel (CDEM-pHA). The rheological properties of CDEM-pHA indicated that CDEM-pHA exhibited shear-thinning behavior, and the storage modulus of the hydrogel increased with increasing UV irradiation. Chondrocytes were loaded into CDEM-pHA and implanted into the subcutaneous tissue of nude mice for eight weeks; the results indicated that CDEM-pHA combined with chondrocytes successfully regenerated mature cartilage in vivo. This study established a novel strategy for preparing CDEM-based hydrogel for cartilage regeneration. Ó 2020 Elsevier B.V. All rights reserved.
1. Introduction CDEM contains complex tissue components and simulates a native microenvironment, which provides attractive bioactivity and exhibits great advantages for cartilage tissue remodeling [1]. However, traditional CDEM-based scaffolds have a dense structure, which is not beneficial for cell infiltration and tissue regeneration [2]. Processing solid CDEM into injectable and in situ formation of hydrogels is a promising approach for cartilage defect repair. Injectable CDEM-based hydrogels possess tremendous advantages for cartilage regeneration [1,3,4]; for example, cells or drugs can be encapsulated into scaffolds uniformly, hydrogels can form diverse shapes for filling irregular defect, in situ formation of hydrogels will be beneficial for the integration of scaffolds with tissue. Photocrosslinking hydrogels allow the injection of hydrogel precursor and subsequent rapid gelation at target tissue position, which shows tremendous promise in clinics [5,6]. However, there still exists challenges in preparing injectable photo-crosslinking CDEM-based hydrogels, which require rapid gelation at irregular defect position, efficient tissue integration, and excellent cartilage regeneration ability [7]. In this study, CDEM-based hydrogels (CDEM-pHA) were successfully prepared by combining CDEM ⇑ Corresponding authors. E-mail addresses: [email protected] (G. Zhou), weimingchen07@163. com (W. Chen), [email protected] (R. Chen). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.matlet.2020.127609 0167-577X/Ó 2020 Elsevier B.V. All rights reserved.
powders and pHA. The CDEM-pHA is injectable and photocrosslinkable. Most importantly, CDEM-pHA and chondrocytes constructs achieved excellent cartilage regeneration in vivo, indicating their potential application value for cartilage regeneration. 2. Experimental 2.1. Preparation of CDEM-pHA CDEM derived from crow scapular cartilage was processed into powders; this was performed according to the previous study [8]. Briefly, cartilage was cut into slices and decellularized in a 1 M sodium hydroxide solution for 4 h. The decellularized matrix was crushed with a grinder and homogenized with a high-speed homogenizer. pHA was provided by East China University of Science and Technology as previous literature described [6]; 0.08 g of pHA was dissolved in 1 mL PBS at 37 in water bath and 0.16 g CDEM powders were blended with pHA solution to form uniformed hydrogel with concentration of 24%. Then, the hydrogel was irradiated for 90 s with 365 nm light (10 mW cm2) until the hydrogel reached gelation (Fig. 1). 2.2. Characterization Attenuated total reflection fourier transform infrared spectroscopy (ATR-FTIR) of CDEM-pHA hydrogels before and after UV
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Y. Xu et al. / Materials Letters 268 (2020) 127609
Fig. 1. The synthesis process of injectable photo-crosslinking CDEM-pHA.
crosslinking was performed with a Nicolet-670 FTIR spectrometer. The rheological properties of CDEM-pHA hydrogels were tested by an Anton Paar MCR302 rheometer with parallel-plate (25 mm diameter) geometry at room temperature. Dynamic rheology tests were performed to measure the storage modulus (G0 ) and the loss modulus (G00 ) of hydrogels under UV irradiation (365 nm, 30 mW cm2) for 400 s.
2.3. Cartilage regeneration in vivo For this test, 0.08 g of pHA was completely dissolved in 1 mL DMEM (containing 10% FBS, 100 mg/mL streptomycin, and100 U/mL penicillin); 0.16 g CDEM powder was blended with the pHA solution to form hydrogel; and 100 mL chondrocytes derived from auricular cartilage of New Zealand white rabbits weighing 1.5–2.0 kg with a concentration of 5.0 108 cells/mL were placed into 900 mL hydrogel to form cells loaded hydrogel (cell-containing group). 200 mL cells loaded hydrogel was placed into a cylindrical mold (8 mm in dimeter). Then, the hydrogel was irradiated for 90 s under 365 nm light (10 mW cm2) until the hydrogel reached gelation. These cell-hydrogel constructs were cultured in DMEM for 7 days in vitro, then implanted into the subcutaneous tissue of nude mice (n = 4) for 4 weeks and 8 weeks. Finally, these cellhydrogel constructs were evaluated by histological and quantitative analysis. The same hydrogel without cell seeding (cell-less group) was parallelly treated as a control. For histological evaluation, the paraffin sections were stained with hematoxylin and eosin (HE) and safranin O, type-II collagen was also detected by immunohistochemical staining of the tissue sections to evaluate the cartilage-specific phenotypes. The content of DNA, total collagen, and sulfated glycosaminoglycan (GAG), and
Young’s modulus of the cell-hydrogel constructs were tested and fresh auricular cartilage (native) from New Zealand white rabbits was used as a positive control, which were performed according to a previously reported study [2].
3. Results and discussion As shown in Fig. 1, mixtures of CDEM and pHA were made into sol before crosslinking, but gelation formed after UV irradiation. In a previous study [7], it was demonstrated that pHA and polymers containing an amino group can cause photo-triggered imine reaction. The ATR-FTIR spectra of hydrogels further proved that the chemical group of the CDEM-pHA hydrogel changed after crosslinking. As shown in Fig. 2a, the –NO2 stretching vibration at 1332 cm1 and –OH stretching vibration at 11451 cm1 a disappeared after the imine reaction. From the rheological results, the CDEM-pHA hydrogel exhibited shear-thinning behavior (Fig. 2b), which is necessary for an injectable hydrogel to flow continuously. The storage modulus (G0 ) of the hydrogel increased with increasing UV irradiation (Fig. 2c and Fig. 2d), indicating that gelation was formed after photo-triggered crosslinking and the G0 of hydrogel was adjustable by controlling the irradiation time. Chondrocytes loaded hydrogel showed excellent moldability after UV crosslinking (Fig. 3a). The cell-hydrogel construct retained their original shape and gradually formed a white cartilage-like tissue with increased in vivo culture time (Fig. 3b and c). Histological examination showed that chondrocytes were evenly distributed in the hydrogel and a typical lacunae structure and homogeneous cartilage-specific ECM deposition was observed (Fig. 3d-f, j-l), while the cell-less group showed no cartilage feature with negative staining of safranin-O and type II collagen (Fig. 3g-i, m-o).
Y. Xu et al. / Materials Letters 268 (2020) 127609
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Fig. 2. ATR-FTIR and rheological analysis of CDEM-pHA hydrogel. (a) ATR-FTIR spectra of hydrogels before and after UV crosslinking. (b) Viscosity of the hydrogel at 25 . (c) Dynamic modulus (G0 and G00 ) of hydrogel with UV light at varying time. (d) Storage modulus of hydrogels under UV irradiation at 10, 100, and 200 s (*p < 0.05, n = 3).
Fig. 3. Cartilage regeneration in vivo. (a) Cell-hydrogel construct after UV crosslinking. Gross view of cell-containing group after four (b) and eight (c) weeks of implantation. HE (d, g, j, and m), Safranin-O (e, h, k, and n), and type-II collagen (f, h, l, and o) staining of cell-containing and cell-less groups after four and eight weeks of implantation.
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Y. Xu et al. / Materials Letters 268 (2020) 127609
Fig. 4. Quantitative analysis of engineered cartilage after four and eight weeks of implantation. (a) DNA content, (b) total collagen, (c) GAG content, and (d) Young’s modulus of engineered cartilage. (*p < 0.05, n = 3).
Compared with four weeks, more typical lacunae structure and strong positive staining of Safranin-O and immunohistochemical type II collagen formed after 8 weeks of subcutaneous implantation, indicating that cell-hydrogel construct formed mature cartilage tissue. Quantitative analysis indicated that the DNA content of the cellhydrogel construct no longer increased after four weeks of implantation (Fig. 4a). However, the total collagen content, GAG content, and Young’s modulus of the cell-hydrogel construct increase with the implantation time (Fig. 4b-d), and were even closed to those of native cartilage after eight weeks of implantation. Collectively, all these results indicated that CDEM-pHA combined with chondrocytes achieved excellent cartilage regeneration in vivo. CDEMpHA provides a native microenvironment for chondrocytes growth, which is the main reason that CDEM-pHA combined chondrocytes achieved excellent cartilage regeneration in vivo.
CRediT authorship contribution statement Yong Xu: Investigation, Methodology, Writing - review & editing. Litao Jia: Software, Resources. Zongxin Wang: Data curation, Writing - original draft. Gening Jiang: Visualization. Guangdong Zhou: Supervision, Formal analysis. Weiming Chen: Conceptualization, Project administration. Ru Chen: Funding acquisition, Validation. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements
4. Conclusions In the current study, an injectable photo-crosslinking CDEMbased hydrogel is provided for cartilage tissue regeneration. For the first time, CDEM was processed into UV-crosslinkable gelation, which exhibited many advantages for cartilage regeneration. CDEM-pHA was injectable and photo-crosslinkable, which is suitable for filling and repairing irregularly shaped defects. Moreover, it is convenient to add the cells into hydrogel to form a cell-scaffold construct. Importantly, the hydrogel provides the physicochemical features of cell’s native microenvironment, which is beneficial for cartilage regeneration.
This work was supported by The National Key Research and Development Program of China (2017YFC1103900) and National Natural Science Foundation of China (81970014). References [1] J. Wu, Q. Ding, A. Dutta, Y. Wang, Y.H. Huang, H. Weng, L. Tang, Y. Hong, An injectable extracellular matrix derived hydrogel for meniscus repair and regeneration, Acta Biomater. 16 (2015) 49–59. [2] Y. Xu, D. Li, Z. Yin, A. He, M. Lin, G. Jiang, X. Song, X. Hu, Y. Liu, J. Wang, Tissueengineered trachea regeneration using decellularized trachea matrix treated with laser micropore technique, Acta Biomater. 58 (2017) 113–121.
Y. Xu et al. / Materials Letters 268 (2020) 127609 [3] J. Zhang, Y. Yang, Y. Chen, X. Liu, S. Guo, L. Zhu, Y. Wang, An in situ phototriggered-imine-crosslink composite hydrogel for bone defect repair, J. Mater. Chem. B 4 (2016) 973–981. [4] H.G. Yi, Y.J. Choi, J.W. Jung, J. Jang, T.H. Song, S. Chae, M. Ahn, T.H. Choi, J.W. Rhie, D.W. Cho, Three-dimensional printing of a patient-specific engineered nasal cartilage for augmentative rhinoplasty, J. Tissue Eng. 10 (2019). [5] B. Li, L. Wang, F. Xu, X. Gang, U. Demirci, D. Wei, Y. Li, Y. Feng, D. Jia, Y. Zhou, Hydrosoluble, UV-crosslinkable and injectable chitosan for patterned cell-laden microgel and rapid transdermal curing hydrogel in vivo, Acta Biomater. 22 (2015) 59–69.
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[6] H. Lin, A.M. Beck, K. Shimomura, J. Sohn, M.R. Fritch, Y. Deng, E.J. Kilroy, Y. Tang, P.G. Alexander, R.S. Tuan, Optimization of photocrosslinked gelatin/hyaluronic acid hybrid scaffold for the repair of cartilage defect, J. Tissue Eng. Regen. Med. 13 (8) (2019) 1418–1429. [7] Y. Yang, J. Zhang, Z. Liu, Q. Lin, X. Liu, C. Bao, Y. Wang, L. Zhu, Tissue-integratable and biocompatible photogelation by the imine crosslinking reaction, Adv. Mater. 28 (14) (2016) 2724–2730. [8] X. Zhang, Z. Deng, H.Z. Yang, W. Guo, Y. Li, D. Ma, C. Yu, Y. Zhang, Y. Jin, Expansion and delivery of human fibroblasts on micronized acellular dermal matrix for skin regeneration, Biomaterials 30 (14) (2009) 2666–2674.
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/mlblue
Injectable photo-crosslinking cartilage decellularized extracellular matrix for cartilage tissue regeneration Yong Xu b,1, Litao Jia d,1, Zongxin Wang d,1, Gening Jiang b, Guangdong Zhou d,⇑, Weiming Chen c,⇑, Ru Chen a,⇑ a
Department of Breast Surgery, Hainan General Hospital, Hainan Medical University, Hainan, China Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China c Shanghai Yapeng Biotechnology Co., Ltd, Shanghai, China d Research Institute of Plastic Surgery, Wei Fang Medical College, Shandong, China b
a r t i c l e
i n f o
Article history: Received 25 July 2019 Received in revised form 22 February 2020 Accepted 4 March 2020 Available online 5 March 2020 Keywords: Decellularized extracellular matrix Biomaterials Sol-gel preparation Photo-crosslinking
a b s t r a c t Cartilage decellularized extracellular matrix (CDEM) is a promising biomaterial for preparing tissue engineering scaffolds, and CDEM-based hydrogels constitute an ideal choice for repairing cartilage damage. In this study, CDEM powders and photo-responsive hyaluronic acid (pHA) were successfully fabricated into injectable and photo-crosslinkable hydrogel (CDEM-pHA). The rheological properties of CDEM-pHA indicated that CDEM-pHA exhibited shear-thinning behavior, and the storage modulus of the hydrogel increased with increasing UV irradiation. Chondrocytes were loaded into CDEM-pHA and implanted into the subcutaneous tissue of nude mice for eight weeks; the results indicated that CDEM-pHA combined with chondrocytes successfully regenerated mature cartilage in vivo. This study established a novel strategy for preparing CDEM-based hydrogel for cartilage regeneration. Ó 2020 Elsevier B.V. All rights reserved.
1. Introduction CDEM contains complex tissue components and simulates a native microenvironment, which provides attractive bioactivity and exhibits great advantages for cartilage tissue remodeling [1]. However, traditional CDEM-based scaffolds have a dense structure, which is not beneficial for cell infiltration and tissue regeneration [2]. Processing solid CDEM into injectable and in situ formation of hydrogels is a promising approach for cartilage defect repair. Injectable CDEM-based hydrogels possess tremendous advantages for cartilage regeneration [1,3,4]; for example, cells or drugs can be encapsulated into scaffolds uniformly, hydrogels can form diverse shapes for filling irregular defect, in situ formation of hydrogels will be beneficial for the integration of scaffolds with tissue. Photocrosslinking hydrogels allow the injection of hydrogel precursor and subsequent rapid gelation at target tissue position, which shows tremendous promise in clinics [5,6]. However, there still exists challenges in preparing injectable photo-crosslinking CDEM-based hydrogels, which require rapid gelation at irregular defect position, efficient tissue integration, and excellent cartilage regeneration ability [7]. In this study, CDEM-based hydrogels (CDEM-pHA) were successfully prepared by combining CDEM ⇑ Corresponding authors. E-mail addresses: [email protected] (G. Zhou), weimingchen07@163. com (W. Chen), [email protected] (R. Chen). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.matlet.2020.127609 0167-577X/Ó 2020 Elsevier B.V. All rights reserved.
powders and pHA. The CDEM-pHA is injectable and photocrosslinkable. Most importantly, CDEM-pHA and chondrocytes constructs achieved excellent cartilage regeneration in vivo, indicating their potential application value for cartilage regeneration. 2. Experimental 2.1. Preparation of CDEM-pHA CDEM derived from crow scapular cartilage was processed into powders; this was performed according to the previous study [8]. Briefly, cartilage was cut into slices and decellularized in a 1 M sodium hydroxide solution for 4 h. The decellularized matrix was crushed with a grinder and homogenized with a high-speed homogenizer. pHA was provided by East China University of Science and Technology as previous literature described [6]; 0.08 g of pHA was dissolved in 1 mL PBS at 37 in water bath and 0.16 g CDEM powders were blended with pHA solution to form uniformed hydrogel with concentration of 24%. Then, the hydrogel was irradiated for 90 s with 365 nm light (10 mW cm2) until the hydrogel reached gelation (Fig. 1). 2.2. Characterization Attenuated total reflection fourier transform infrared spectroscopy (ATR-FTIR) of CDEM-pHA hydrogels before and after UV
2
Y. Xu et al. / Materials Letters 268 (2020) 127609
Fig. 1. The synthesis process of injectable photo-crosslinking CDEM-pHA.
crosslinking was performed with a Nicolet-670 FTIR spectrometer. The rheological properties of CDEM-pHA hydrogels were tested by an Anton Paar MCR302 rheometer with parallel-plate (25 mm diameter) geometry at room temperature. Dynamic rheology tests were performed to measure the storage modulus (G0 ) and the loss modulus (G00 ) of hydrogels under UV irradiation (365 nm, 30 mW cm2) for 400 s.
2.3. Cartilage regeneration in vivo For this test, 0.08 g of pHA was completely dissolved in 1 mL DMEM (containing 10% FBS, 100 mg/mL streptomycin, and100 U/mL penicillin); 0.16 g CDEM powder was blended with the pHA solution to form hydrogel; and 100 mL chondrocytes derived from auricular cartilage of New Zealand white rabbits weighing 1.5–2.0 kg with a concentration of 5.0 108 cells/mL were placed into 900 mL hydrogel to form cells loaded hydrogel (cell-containing group). 200 mL cells loaded hydrogel was placed into a cylindrical mold (8 mm in dimeter). Then, the hydrogel was irradiated for 90 s under 365 nm light (10 mW cm2) until the hydrogel reached gelation. These cell-hydrogel constructs were cultured in DMEM for 7 days in vitro, then implanted into the subcutaneous tissue of nude mice (n = 4) for 4 weeks and 8 weeks. Finally, these cellhydrogel constructs were evaluated by histological and quantitative analysis. The same hydrogel without cell seeding (cell-less group) was parallelly treated as a control. For histological evaluation, the paraffin sections were stained with hematoxylin and eosin (HE) and safranin O, type-II collagen was also detected by immunohistochemical staining of the tissue sections to evaluate the cartilage-specific phenotypes. The content of DNA, total collagen, and sulfated glycosaminoglycan (GAG), and
Young’s modulus of the cell-hydrogel constructs were tested and fresh auricular cartilage (native) from New Zealand white rabbits was used as a positive control, which were performed according to a previously reported study [2].
3. Results and discussion As shown in Fig. 1, mixtures of CDEM and pHA were made into sol before crosslinking, but gelation formed after UV irradiation. In a previous study [7], it was demonstrated that pHA and polymers containing an amino group can cause photo-triggered imine reaction. The ATR-FTIR spectra of hydrogels further proved that the chemical group of the CDEM-pHA hydrogel changed after crosslinking. As shown in Fig. 2a, the –NO2 stretching vibration at 1332 cm1 and –OH stretching vibration at 11451 cm1 a disappeared after the imine reaction. From the rheological results, the CDEM-pHA hydrogel exhibited shear-thinning behavior (Fig. 2b), which is necessary for an injectable hydrogel to flow continuously. The storage modulus (G0 ) of the hydrogel increased with increasing UV irradiation (Fig. 2c and Fig. 2d), indicating that gelation was formed after photo-triggered crosslinking and the G0 of hydrogel was adjustable by controlling the irradiation time. Chondrocytes loaded hydrogel showed excellent moldability after UV crosslinking (Fig. 3a). The cell-hydrogel construct retained their original shape and gradually formed a white cartilage-like tissue with increased in vivo culture time (Fig. 3b and c). Histological examination showed that chondrocytes were evenly distributed in the hydrogel and a typical lacunae structure and homogeneous cartilage-specific ECM deposition was observed (Fig. 3d-f, j-l), while the cell-less group showed no cartilage feature with negative staining of safranin-O and type II collagen (Fig. 3g-i, m-o).
Y. Xu et al. / Materials Letters 268 (2020) 127609
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Fig. 2. ATR-FTIR and rheological analysis of CDEM-pHA hydrogel. (a) ATR-FTIR spectra of hydrogels before and after UV crosslinking. (b) Viscosity of the hydrogel at 25 . (c) Dynamic modulus (G0 and G00 ) of hydrogel with UV light at varying time. (d) Storage modulus of hydrogels under UV irradiation at 10, 100, and 200 s (*p < 0.05, n = 3).
Fig. 3. Cartilage regeneration in vivo. (a) Cell-hydrogel construct after UV crosslinking. Gross view of cell-containing group after four (b) and eight (c) weeks of implantation. HE (d, g, j, and m), Safranin-O (e, h, k, and n), and type-II collagen (f, h, l, and o) staining of cell-containing and cell-less groups after four and eight weeks of implantation.
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Y. Xu et al. / Materials Letters 268 (2020) 127609
Fig. 4. Quantitative analysis of engineered cartilage after four and eight weeks of implantation. (a) DNA content, (b) total collagen, (c) GAG content, and (d) Young’s modulus of engineered cartilage. (*p < 0.05, n = 3).
Compared with four weeks, more typical lacunae structure and strong positive staining of Safranin-O and immunohistochemical type II collagen formed after 8 weeks of subcutaneous implantation, indicating that cell-hydrogel construct formed mature cartilage tissue. Quantitative analysis indicated that the DNA content of the cellhydrogel construct no longer increased after four weeks of implantation (Fig. 4a). However, the total collagen content, GAG content, and Young’s modulus of the cell-hydrogel construct increase with the implantation time (Fig. 4b-d), and were even closed to those of native cartilage after eight weeks of implantation. Collectively, all these results indicated that CDEM-pHA combined with chondrocytes achieved excellent cartilage regeneration in vivo. CDEMpHA provides a native microenvironment for chondrocytes growth, which is the main reason that CDEM-pHA combined chondrocytes achieved excellent cartilage regeneration in vivo.
CRediT authorship contribution statement Yong Xu: Investigation, Methodology, Writing - review & editing. Litao Jia: Software, Resources. Zongxin Wang: Data curation, Writing - original draft. Gening Jiang: Visualization. Guangdong Zhou: Supervision, Formal analysis. Weiming Chen: Conceptualization, Project administration. Ru Chen: Funding acquisition, Validation. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements
4. Conclusions In the current study, an injectable photo-crosslinking CDEMbased hydrogel is provided for cartilage tissue regeneration. For the first time, CDEM was processed into UV-crosslinkable gelation, which exhibited many advantages for cartilage regeneration. CDEM-pHA was injectable and photo-crosslinkable, which is suitable for filling and repairing irregularly shaped defects. Moreover, it is convenient to add the cells into hydrogel to form a cell-scaffold construct. Importantly, the hydrogel provides the physicochemical features of cell’s native microenvironment, which is beneficial for cartilage regeneration.
This work was supported by The National Key Research and Development Program of China (2017YFC1103900) and National Natural Science Foundation of China (81970014). References [1] J. Wu, Q. Ding, A. Dutta, Y. Wang, Y.H. Huang, H. Weng, L. Tang, Y. Hong, An injectable extracellular matrix derived hydrogel for meniscus repair and regeneration, Acta Biomater. 16 (2015) 49–59. [2] Y. Xu, D. Li, Z. Yin, A. He, M. Lin, G. Jiang, X. Song, X. Hu, Y. Liu, J. Wang, Tissueengineered trachea regeneration using decellularized trachea matrix treated with laser micropore technique, Acta Biomater. 58 (2017) 113–121.
Y. Xu et al. / Materials Letters 268 (2020) 127609 [3] J. Zhang, Y. Yang, Y. Chen, X. Liu, S. Guo, L. Zhu, Y. Wang, An in situ phototriggered-imine-crosslink composite hydrogel for bone defect repair, J. Mater. Chem. B 4 (2016) 973–981. [4] H.G. Yi, Y.J. Choi, J.W. Jung, J. Jang, T.H. Song, S. Chae, M. Ahn, T.H. Choi, J.W. Rhie, D.W. Cho, Three-dimensional printing of a patient-specific engineered nasal cartilage for augmentative rhinoplasty, J. Tissue Eng. 10 (2019). [5] B. Li, L. Wang, F. Xu, X. Gang, U. Demirci, D. Wei, Y. Li, Y. Feng, D. Jia, Y. Zhou, Hydrosoluble, UV-crosslinkable and injectable chitosan for patterned cell-laden microgel and rapid transdermal curing hydrogel in vivo, Acta Biomater. 22 (2015) 59–69.
5
[6] H. Lin, A.M. Beck, K. Shimomura, J. Sohn, M.R. Fritch, Y. Deng, E.J. Kilroy, Y. Tang, P.G. Alexander, R.S. Tuan, Optimization of photocrosslinked gelatin/hyaluronic acid hybrid scaffold for the repair of cartilage defect, J. Tissue Eng. Regen. Med. 13 (8) (2019) 1418–1429. [7] Y. Yang, J. Zhang, Z. Liu, Q. Lin, X. Liu, C. Bao, Y. Wang, L. Zhu, Tissue-integratable and biocompatible photogelation by the imine crosslinking reaction, Adv. Mater. 28 (14) (2016) 2724–2730. [8] X. Zhang, Z. Deng, H.Z. Yang, W. Guo, Y. Li, D. Ma, C. Yu, Y. Zhang, Y. Jin, Expansion and delivery of human fibroblasts on micronized acellular dermal matrix for skin regeneration, Biomaterials 30 (14) (2009) 2666–2674.