Preparation and <i>in vivo</i> osteogenesis of acellular dermal matrix/dicalcium phosphate composite scaffold for bone repair_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LAN Yinan ^1,2 ^# , ZHANG Jingyi ³ , RAN Yongfeng ³ , LI Bo ³ , CAI Xiaobin ² ^# , JIANG Tao ³ ,  XUE Deting ¹

1. Department of Orthopedics, the Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou Zhejiang, 310009, P. R. China;
2. Department of Orthopedics, Lishui Hospital of Zhejiang University, Lishui Zhejiang, 323000, P. R. China;
3. Hangzhou Huamai Medical Technology Co. Ltd., Hangzhou Zhejiang, 310052, P. R. China;

Corresponding author：

XUE Deting, Email: blueskine@zju.edu.cn

Keywords：

Bone tissue engineering; acellular dermal matrix; dicalcium phosphate; composite scaffold; bone repair

DOI：

10.7507/1002-1892.202403059

Video：

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Objective To investigate the physicochemical properties, osteogenic properties, and osteogenic ability in rabbit model of femoral condylar defect of acellular dermal matrix (ADM)/dicalcium phosphate (DCP) composite scaffold. Methods ADM/DCP composite scaffolds were prepared by microfibril technique, and the acellular effect of ADM/DCP composite scaffolds was detected by DNA residue, fat content, and α-1, 3-galactosyle (α-Gal) epitopes; the microstructure of scaffolds was characterized by field emission scanning electron microscopy and mercury porosimetry; X-ray diffraction was used to analyze the change of crystal form of scaffold; the solubility of scaffolds was used to detect the pH value and calcium ion content of the solution; the mineralization experiment in vitro was used to observe the surface mineralization. Twelve healthy male New Zealand white rabbits were selected to prepare the femoral condylar defect models, and the left and right defects were implanted with ADM/DCP composite scaffold (experimental group) and skeletal gold^® artificial bone repair material (control group), respectively. Gross observation was performed at 6 and 12 weeks after operation; Micro-CT was used to detect and quantitatively analyze the related indicators [bone volume (BV), bone volume/tissue volume (BV/TV), bone surface/bone volume (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), bone mineral density (BMD)], and HE staining and Masson staining were performed to observe the repair of bone defects and the maturation of bone matrix. Results Gross observation showed that the ADM/DCP composite scaffold was a white spongy solid. Compared with ADM, ADM/DCP composite scaffolds showed a significant decrease in DNA residue, fat content, and α-Gal antigen content (P<0.05). Field emission scanning electron microscopy showed that the ADM/DCP composite scaffold had a porous structure, and DCP particles were attached to the porcine dermal fibers. The porosity of the ADM/DCP composite scaffold was 76.32%±1.63% measured by mercury porosimetry. X-ray diffraction analysis showed that the crystalline phase of DCP in the ADM/DCP composite scaffolds remained intact. Mineralization results in vitro showed that the hydroxyapatite layer of ADM/DCP composite scaffolds was basically mature. The repair experiment of rabbit femoral condyle defect showed that the incision healed completely after operation without callus or osteophyte. Micro-CT showed that bone healing was complete and a large amount of new bone tissue was generated in the defect site of the two groups, and there was no difference in density between the defect site and the surrounding bone tissue, and the osteogenic properties of the two groups were equivalent. There was no significant difference in BV, BV/TV, BS/BV, Tb.Th, Tb.N, and BMD between the two groups (P>0.05), except that the Tb.Sp in the experimental group was significantly higher than that in the control group (P<0.05). At 6 and 12 weeks after operation, HE staining and Masson staining showed that the new bone and autogenous bone fused well in both groups, and the bone tissue tended to be mature. Conclusion The ADM/DCP composite scaffold has good biocompatibility and osteogenic ability similar to the artificial bone material in repairing rabbit femoral condylar defects. It is a new scaffold material with potential in the field of bone repair.

Citation： LAN Yinan, ZHANG Jingyi, RAN Yongfeng, LI Bo, CAI Xiaobin, JIANG Tao, XUE Deting. Preparation and in vivo osteogenesis of acellular dermal matrix/dicalcium phosphate composite scaffold for bone repair. Chinese Journal of Reparative and Reconstructive Surgery, 2024, 38(6): 755-762. doi: 10.7507/1002-1892.202403059 Copy

1.	Busch A, Wegner A, Haversath M, et al. Bone substitutes in orthopaedic surgery: Current status and future perspectives. Z Orthop Unfall, 2021, 159(3): 304-313.
2.	Busch A, Jäger M. Synthetic bone replacement substances. Die Orthopädie, 2022, 51(12): 1023-1032.
3.	Ferraz MP. Bone grafts in dental medicine: An overview of autografts, allografts and synthetic materials. Materials (Basel), 2023, 16(11): 4117. doi: 10.3390/ma16114117.
4.	Wang J, Liu Q, Guo Z, et al. Progress on biomimetic mineralization and materials for hard tissue regeneration. ACS Biomater Sci Eng, 2023, 9(4): 1757-1773.
5.	van der Rest M, Garrone R. Collagen family of proteins. FASEB J, 1991, 5(13): 2814-2823.
6.	Roth KE, Maier GS, Schmidtmann I, et al. Release of antibiotics out of a moldable collagen-β-tricalciumphosphate-composite compared to two calcium phosphate granules. Materials (Basel), 2019, 12(24): 4056. doi: 10.3390/ma12244056.
7.	Qiu ZY, Cui Y, Tao CS, et al. Mineralized collagen: Rationale, current status, and clinical applications. Materials (Basel), 2015, 8(8): 4733-4750.
8.	Sheehy EJ. Collagen-based biomaterials for tissue regeneration and repair. Peptides and Proteins as Biomaterials for Tissue Regeneration and Repair, 2018. doi: 10.1016/B978-0-08-100803-4.00005-X.
9.	Bose S, Li S, Mele E, et al. Dry vs. wet: Properties and performance of collagen films. Part Ⅱ. Cyclic and time-dependent behaviours. J Mech Behav Biomed Mater, 2020, 112: 104040. doi: 10.1016/j.jmbbm.2020.104040.
10.	Brown B, Lindberg K, Reing J, et al. The basement membrane component of biologic scaffolds derived from extracellular matrix. Tissue Eng, 2006, 12(3): 519-526.
11.	Xie H, Wang Z, Zhang L, et al. Extracellular vesicle-functionalized decalcified bone matrix scaffolds with enhanced pro-angiogenic and pro-bone regeneration activities. Sci Rep, 2017, 7: 45622. doi: 10.1038/srep45622.
12.	Hussey GS, Dziki JL, Badylak SF. Extracellular matrix-based materials for regenerative medicine. Nature Reviews Materials, 2018, 3: 159-173.
13.	Van Orden K, Santos J, Stanfield B, et al. Bovine versus porcine acellular dermal matrix for abdominal wall herniorrhaphy or bridging. Eur J Trauma Emerg Surg, 2022, 48(3): 1993-2001.
14.	Tan YH, Helms HR, Nakayama KH. Decellularization strategies for regenerating cardiac and skeletal muscle tissues. Front Bioeng Biotechnol, 2022, 10: 831300. doi: 10.3389/fbioe.2022.831300.
15.	Fu XY, Jiang ZY, Zhang CY, et al. New hope for esophageal stricture prevention: A prospective single-center trial on acellular dermal matrix. World J Gastrointest Endosc, 2023, 15(12): 725-734.
16.	Cole W, Samsell B, Moore MA. Achilles tendon augmented repair using human acellular dermal matrix: A case series. J Foot Ankle Surg, 2018, 57(6): 1225-1229.
17.	Das P, Singh YP, Mandal BB, et al. Tissue-derived decellularized extracellular matrices toward cartilage repair and regeneration. Methods Cell Biol, 2020, 157: 185-221.
18.	Ko CL, Chen JC, Tien YC, et al. Osteoregenerative capacities of dicalcium phosphate-rich calcium phosphate bone cement. J Biomed Mater Res A, 2015, 103(1): 203-210.
19.	国家食品药品监督管理总局. YY/T 0606. 25-2014 组织工程医疗产品第25部分: 动物源性生物材料DNA残留量测定法: 荧光染色法. 2015-07-01.
20.	中华人民共和国国家卫生和计划生育委员会, 国家食品药品监督管理总局. GB 5009.6-2016 食品安全国家标准食品中脂肪的测定. 2017-06-23.
21.	国家食品药品监督管理总局. YY/T 1561-2017 组织工程医疗器械产品动物源性支架材料残留α-Gal抗原检测. 2018-04-01.
22.	国家食品药品监督管理总局. YY/T 1465.5-2016 医疗器械免疫原性评价方法第5部分: 用M86抗体测定动物源性医疗器械中α-Gal抗原清除率. 2017-06-01.
23.	国家食品药品监督管理总局. YY/T 1558.3-2017/ISO 13175-3: 2012 外科植入物磷酸钙第3部分: 羟基磷灰石和β-磷酸三钙骨替代物. 2018-10-01.
24.	Kokubo T, Takadama H. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials, 2006, 27(15): 2907-2915.
25.	Gilbert TW. Strategies for tissue and organ decellularization. J Cell Biochem, 2012, 113(7): 2217-2222.
26.	Ye C, Chen J, Qu Y, et al. Naringin in the repair of knee cartilage injury via the TGF-β/ALK5/Smad2/3 signal transduction pathway combined with an acellular dermal matrix. J Orthop Translat, 2021, 32: 1-11.
27.	Melkonyan KI, Popandopulo KI, Bazlov SB, et al. Results of experimental hernioplasty with acellular dermal matrix. Bull Exp Biol Med, 2023, 174(4): 514-517.
28.	Xia W, Lin C, Tu Z, et al. Preparation of laser microporous porcine acellular dermal matrix and observation of wound transplantation. Cell Tissue Bank, 2023, 24(1): 191-202.

1. Busch A, Wegner A, Haversath M, et al. Bone substitutes in orthopaedic surgery: Current status and future perspectives. Z Orthop Unfall, 2021, 159(3): 304-313.
2. Busch A, Jäger M. Synthetic bone replacement substances. Die Orthopädie, 2022, 51(12): 1023-1032.
3. Ferraz MP. Bone grafts in dental medicine: An overview of autografts, allografts and synthetic materials. Materials (Basel), 2023, 16(11): 4117. doi: 10.3390/ma16114117.
4. Wang J, Liu Q, Guo Z, et al. Progress on biomimetic mineralization and materials for hard tissue regeneration. ACS Biomater Sci Eng, 2023, 9(4): 1757-1773.
5. van der Rest M, Garrone R. Collagen family of proteins. FASEB J, 1991, 5(13): 2814-2823.
6. Roth KE, Maier GS, Schmidtmann I, et al. Release of antibiotics out of a moldable collagen-β-tricalciumphosphate-composite compared to two calcium phosphate granules. Materials (Basel), 2019, 12(24): 4056. doi: 10.3390/ma12244056.
7. Qiu ZY, Cui Y, Tao CS, et al. Mineralized collagen: Rationale, current status, and clinical applications. Materials (Basel), 2015, 8(8): 4733-4750.
8. Sheehy EJ. Collagen-based biomaterials for tissue regeneration and repair. Peptides and Proteins as Biomaterials for Tissue Regeneration and Repair, 2018. doi: 10.1016/B978-0-08-100803-4.00005-X.
9. Bose S, Li S, Mele E, et al. Dry vs. wet: Properties and performance of collagen films. Part Ⅱ. Cyclic and time-dependent behaviours. J Mech Behav Biomed Mater, 2020, 112: 104040. doi: 10.1016/j.jmbbm.2020.104040.
10. Brown B, Lindberg K, Reing J, et al. The basement membrane component of biologic scaffolds derived from extracellular matrix. Tissue Eng, 2006, 12(3): 519-526.
11. Xie H, Wang Z, Zhang L, et al. Extracellular vesicle-functionalized decalcified bone matrix scaffolds with enhanced pro-angiogenic and pro-bone regeneration activities. Sci Rep, 2017, 7: 45622. doi: 10.1038/srep45622.
12. Hussey GS, Dziki JL, Badylak SF. Extracellular matrix-based materials for regenerative medicine. Nature Reviews Materials, 2018, 3: 159-173.
13. Van Orden K, Santos J, Stanfield B, et al. Bovine versus porcine acellular dermal matrix for abdominal wall herniorrhaphy or bridging. Eur J Trauma Emerg Surg, 2022, 48(3): 1993-2001.
14. Tan YH, Helms HR, Nakayama KH. Decellularization strategies for regenerating cardiac and skeletal muscle tissues. Front Bioeng Biotechnol, 2022, 10: 831300. doi: 10.3389/fbioe.2022.831300.
15. Fu XY, Jiang ZY, Zhang CY, et al. New hope for esophageal stricture prevention: A prospective single-center trial on acellular dermal matrix. World J Gastrointest Endosc, 2023, 15(12): 725-734.
16. Cole W, Samsell B, Moore MA. Achilles tendon augmented repair using human acellular dermal matrix: A case series. J Foot Ankle Surg, 2018, 57(6): 1225-1229.
17. Das P, Singh YP, Mandal BB, et al. Tissue-derived decellularized extracellular matrices toward cartilage repair and regeneration. Methods Cell Biol, 2020, 157: 185-221.
18. Ko CL, Chen JC, Tien YC, et al. Osteoregenerative capacities of dicalcium phosphate-rich calcium phosphate bone cement. J Biomed Mater Res A, 2015, 103(1): 203-210.
19. 国家食品药品监督管理总局. YY/T 0606. 25-2014 组织工程医疗产品第25部分: 动物源性生物材料DNA残留量测定法: 荧光染色法. 2015-07-01.
20. 中华人民共和国国家卫生和计划生育委员会, 国家食品药品监督管理总局. GB 5009.6-2016 食品安全国家标准食品中脂肪的测定. 2017-06-23.
21. 国家食品药品监督管理总局. YY/T 1561-2017 组织工程医疗器械产品动物源性支架材料残留α-Gal抗原检测. 2018-04-01.
22. 国家食品药品监督管理总局. YY/T 1465.5-2016 医疗器械免疫原性评价方法第5部分: 用M86抗体测定动物源性医疗器械中α-Gal抗原清除率. 2017-06-01.
23. 国家食品药品监督管理总局. YY/T 1558.3-2017/ISO 13175-3: 2012 外科植入物磷酸钙第3部分: 羟基磷灰石和β-磷酸三钙骨替代物. 2018-10-01.
24. Kokubo T, Takadama H. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials, 2006, 27(15): 2907-2915.
25. Gilbert TW. Strategies for tissue and organ decellularization. J Cell Biochem, 2012, 113(7): 2217-2222.
26. Ye C, Chen J, Qu Y, et al. Naringin in the repair of knee cartilage injury via the TGF-β/ALK5/Smad2/3 signal transduction pathway combined with an acellular dermal matrix. J Orthop Translat, 2021, 32: 1-11.
27. Melkonyan KI, Popandopulo KI, Bazlov SB, et al. Results of experimental hernioplasty with acellular dermal matrix. Bull Exp Biol Med, 2023, 174(4): 514-517.
28. Xia W, Lin C, Tu Z, et al. Preparation of laser microporous porcine acellular dermal matrix and observation of wound transplantation. Cell Tissue Bank, 2023, 24(1): 191-202.

Chinese Journal of Reparative and Reconstructive Surgery

Preparation and in vivo osteogenesis of acellular dermal matrix/dicalcium phosphate composite scaffold for bone repair

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