载BMP-2多肽P24的巯基化壳聚糖水凝胶诱导异位成骨的实验研究
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摘要
目的探讨载BMP-2多肽P24的巯基化壳聚糖(chitosan-4-thio-butylamidine,CS-TBA)水凝胶的异位成骨能力及体内生物相容性。方法采用壳聚糖、2-亚氨基硫烷盐酸盐、羟基磷灰石(hydroxyapatite,HA)制备CS-TBA/HA溶液,进一步加入P24多肽制备CS-TBA/5%P24/HA、CS-TBA/10%P24/HA溶液;取上述3种溶液,加入β-甘油磷酸二钠(β-glycerophosphate disodium,β-GP),获得CS-TBA/HA/β-GP、CS-TBA/5%P24/HA/β-GP、CS-TBA/10%P24/HA/β-GP水凝胶。取雌性SD大鼠18只,随机分为A、B、C 3组(n=6),制备竖脊肌肌袋后,分别植入CS-TBA/HA/β-GP水凝胶(A组)、CS-TBA/5%P24/HA/β-GP水凝胶(B组)、CS-TBA/10%P24/HA/β-GP水凝胶(C组)。术后观察大鼠存活情况,4、8周取材采用micro-CT检测标本骨小梁厚度(trabecular thickness,Tb.Th)、骨小梁数量(trabecular number,Tb.N)、皮质骨骨密度(bone mineral density,BMD),组织学观察(HE、Masson染色)分析材料的生物降解性及成骨作用。结果术后各组大鼠均存活至取材时间点。micro-CT显示,术后随时间延长,各组新生骨逐渐增多;同一时间点B、C组较A组明显,且随着P24浓度的增加,C组新生骨形成更多。术后各组Tb.Th、Tb.N、BMD逐渐增加,除A组Tb.Th外,其余各指标4周与8周间比较差异均有统计学意义(P<0.05)。各时间点,B、C组Tb.Th、Tb.N、BMD明显高于A组,C组高于B组,比较差异均有统计学意义(P<0.05)。组织学染色观察示,B、C组材料具有良好的生物降解性,且成骨效果随P24浓度升高而增强。结论 P24多肽有助于提升CS-TBA水凝胶的异位成骨作用,且10%浓度效果更强。
Objective To study the ectopic osteogenesis and biocompatibility of bone morphogenetic protein 2(BMP-2)-derived peptide P24 loaded chitosan-4-thio-butylamidine(CS-TBA) hydrogel. Methods First, the CSTBA/hydroxyapatite(HA) solution was prepared by using chitosan, 2-iminothiolane hydrochloride, and HA. Then, the different amount of P24 peptides were added to the CS-TBA/HA to prepare the CS-TBA/5%P24/HA and CSTBA/10%P24/HA solutions. Finally, β-glycerophosphate disodium(β-GP) was added to the CS-TBA/HA, CSTBA/5%P24/HA, and CS-TBA/10%P24/HA to prepare the CS-TBA/HA/β-GP, CS-TBA/5%P24/HA/β-GP, and CSTBA/10%P24/HA/β-GP hydrogels, respectively. Eighteen Sprague Dawley female rats were randomly divided into 3 groups(n=6), which were injected into the back muscle pouches with equal volume CS-TBA/HA/β-GP hydrogel(group A), CS-TBA/5%P24/HA/β-GP hydrogel(group B), and CS-TBA/10%P24/HA/β-GP hydrogel(group C). The animals were sacrificed at 4 and 8 weeks and conducted micro-CT. The ability of biodegradation and osteogenesis of hydrogl wasdetected by trabecular thickness(Tb.Th), trabecular number(Tb.N), bone mineral density(BMD), and histological staining(HE and Masson). Results All the rats survived to the time point of the harvest. Micro-CT results showed that the new bones gradually increased in each group after operation. At the same time, the new bone formation was more obvious in groups B and C than in group A, and with the increase of P24 concentration, new bone formation in group C was much more than that in group B. The Tb.Th, Tb.N, and BMD increased gradually in 3 groups, and the differences between 4 and 8 weeks were significant(P<0.05) except the Tb.Th in group A. At different time points, the Tb.Th, Tb.N,and BMD were significantly higher in groups B and C than in group A(P<0.05), and in group C was higher than in group B(P<0.05), showing significant differences between groups. Histological staining showed that the materials of groups B and C were biodegradable, and the osteogenic effect was increased with the increase of P24 concentration. Conclusion P24 peptide can improve the ectopic osteogenesis of CS-TBA hydrogel, and the 10% concentration is more effective.
Objective To study the ectopic osteogenesis and biocompatibility of bone morphogenetic protein 2(BMP-2)-derived peptide P24 loaded chitosan-4-thio-butylamidine(CS-TBA) hydrogel. Methods First, the CSTBA/hydroxyapatite(HA) solution was prepared by using chitosan, 2-iminothiolane hydrochloride, and HA. Then, the different amount of P24 peptides were added to the CS-TBA/HA to prepare the CS-TBA/5%P24/HA and CSTBA/10%P24/HA solutions. Finally, β-glycerophosphate disodium(β-GP) was added to the CS-TBA/HA, CSTBA/5%P24/HA, and CS-TBA/10%P24/HA to prepare the CS-TBA/HA/β-GP, CS-TBA/5%P24/HA/β-GP, and CSTBA/10%P24/HA/β-GP hydrogels, respectively. Eighteen Sprague Dawley female rats were randomly divided into 3 groups(n=6), which were injected into the back muscle pouches with equal volume CS-TBA/HA/β-GP hydrogel(group A), CS-TBA/5%P24/HA/β-GP hydrogel(group B), and CS-TBA/10%P24/HA/β-GP hydrogel(group C). The animals were sacrificed at 4 and 8 weeks and conducted micro-CT. The ability of biodegradation and osteogenesis of hydrogl wasdetected by trabecular thickness(Tb.Th), trabecular number(Tb.N), bone mineral density(BMD), and histological staining(HE and Masson). Results All the rats survived to the time point of the harvest. Micro-CT results showed that the new bones gradually increased in each group after operation. At the same time, the new bone formation was more obvious in groups B and C than in group A, and with the increase of P24 concentration, new bone formation in group C was much more than that in group B. The Tb.Th, Tb.N, and BMD increased gradually in 3 groups, and the differences between 4 and 8 weeks were significant(P<0.05) except the Tb.Th in group A. At different time points, the Tb.Th, Tb.N,and BMD were significantly higher in groups B and C than in group A(P<0.05), and in group C was higher than in group B(P<0.05), showing significant differences between groups. Histological staining showed that the materials of groups B and C were biodegradable, and the osteogenic effect was increased with the increase of P24 concentration. Conclusion P24 peptide can improve the ectopic osteogenesis of CS-TBA hydrogel, and the 10% concentration is more effective.
引文
1 Venkatesan J,Anil S,Kim SK,et al.Chitosan as a vehicle for growth factor delivery:Various preparations and their applications in bone tissue regeneration.Int J Biol Macromol,2017,104(Pt B):1383-1397.
2 Wang J,Wang L,Yu H,et al.Recent progress on synthesis,property and application of modified chitosan:An overview.Int J Biol Macromol,2016,88:333-344.
3 Liu X,Chen Y,Huang Q,et al.A novel thermo-sensitive hydrogel based on thiolated chitosan/hydroxyapatite/beta-glycerophosphate.Carbohydr Polym,2014,110:62-69.
4 Sarti F,Bernkop-Schniirch A.Chitosan and thiolated chitosan.Advances in Polymer Science,2011,243:93-110.
5 Park K.Chitosan-gelatin-platelet gel composite scaffold for bone regeneration.J Control Release,2017,254:137-144.
6 Fiedler J,Roderer G,Gunther KP,et al.BMP-2,BMP-4,and PDGF-bb stimulate chemotactic migration of primary human mesenchymal progenitor cells.J Cell Biochem,2002,87(3):305-312.
7 Inai K,Norris RA,Hoffman S,et al.BMP-2 induces cell migration and periostin expression during atrioventricular valvulogenesis.Dev Biol,2008,315(2):383-396.
8 Niu X,Feng Q,Wang M,et al.Preparation and characterization of chitosan microspheres for controlled release of synthetic oligopeptide derived from BMP-2.J Microencapsul,2009,26(4):297-305.
9 Lin ZY,Duan ZX,Guo XD,et al.Bone induction by biomimetic PLGA-(PEG-ASP)n copolymer loaded with a novel synthetic BMP-2-related peptide in vitro and in vivo.J Control Release,2010,144(2):190-195.
10 Chen Y,Liu X,Liu R,et al.Zero-order controlled release of BMP2-derived peptide P24 from the chitosan scaffold by chemical grafting modification technique for promotion of osteogenesis in vitro and enhancement of bone repair in vivo.Theranostics,2017,7(5):1072-1087.
11 Ohba S.Tissue regenerative therapies based on regulatory mechanisms underlying bone and cartilage development.Clin Calcium,2016,26(12):1765-1771.
12 Fan L,Wu H,Cao M,et al.Enzymatic synthesis of collagen peptide-carboxymethylated chitosan copolymer and its characterization.React Funct Polym,2014,76(1):26-31.
13 Fan L,Wu H,Zhou X,et al.Transglutaminase-catalyzed grafting collagen on chitosan and its characterization.Carbohydr Polym,2014,105:253-259.
14 Xu B,Zheng P,Gao F,et al.A mineralized high strength and tough hydrogel for skull bone regeneration.Adv Funct Mater,2017,27(4):1604327.
15 Baker RM,Tseng LF,Iannolo MT,et al.Self-deploying shapememory polymer scaffolds for grafting and stabilizing complex bone defects:A mouse femoral segmental defect study.Biomaterials,2016,76:388-398.
16 Shi L,Wang F,Zhu W,et al.Self-healing silk fibroin-based hydrogel for bone regeneration:Dynamic metal-ligand selfassembly approach.Adv Funct Mater,2017,27(37):1700591.
17 Kim S,Kang Y,Mercado-Pagan AE,et al.In vitro evaluation of photo-crosslinkable chitosan-lactide hydrogels for bone tissue engineering.J Biomed Mater Res B Appl Biomater,2014,102(7):1393-1406.
18 Nandi SK,Kundu B,Basu D,et al.Protein growth factors loaded highly porous chitosan scaffold:a comparison of bone healing properties.Mater Sci Eng C Mater Biol Appl,2013,33(3):1267-1275.
19 Saito A,Suzuki Y,Ogata S,et al.Activation of osteo-progenitor cells by a novel synthetic peptide derived from the bone morphogenetic protein-2 knuckle epitope.Biochim Biophys Acta,2003,1651(1-2):60-67.
20 Li J,Hong J,Zheng Q,et al.Repair of rat cranial bone defects with nHAC/PLLA and BMP-2-related peptide or rhBMP-2.J Orthop Res,2011,29(11):1745-1752.
21 Liu X,Yu B,Huang Q,Liu R,et al.In vitro BMP-2 peptide release from thiolated chitosan based hydrogel.Int J Biol Macromol,2016,93(Pt A):314-321.
2 Wang J,Wang L,Yu H,et al.Recent progress on synthesis,property and application of modified chitosan:An overview.Int J Biol Macromol,2016,88:333-344.
3 Liu X,Chen Y,Huang Q,et al.A novel thermo-sensitive hydrogel based on thiolated chitosan/hydroxyapatite/beta-glycerophosphate.Carbohydr Polym,2014,110:62-69.
4 Sarti F,Bernkop-Schniirch A.Chitosan and thiolated chitosan.Advances in Polymer Science,2011,243:93-110.
5 Park K.Chitosan-gelatin-platelet gel composite scaffold for bone regeneration.J Control Release,2017,254:137-144.
6 Fiedler J,Roderer G,Gunther KP,et al.BMP-2,BMP-4,and PDGF-bb stimulate chemotactic migration of primary human mesenchymal progenitor cells.J Cell Biochem,2002,87(3):305-312.
7 Inai K,Norris RA,Hoffman S,et al.BMP-2 induces cell migration and periostin expression during atrioventricular valvulogenesis.Dev Biol,2008,315(2):383-396.
8 Niu X,Feng Q,Wang M,et al.Preparation and characterization of chitosan microspheres for controlled release of synthetic oligopeptide derived from BMP-2.J Microencapsul,2009,26(4):297-305.
9 Lin ZY,Duan ZX,Guo XD,et al.Bone induction by biomimetic PLGA-(PEG-ASP)n copolymer loaded with a novel synthetic BMP-2-related peptide in vitro and in vivo.J Control Release,2010,144(2):190-195.
10 Chen Y,Liu X,Liu R,et al.Zero-order controlled release of BMP2-derived peptide P24 from the chitosan scaffold by chemical grafting modification technique for promotion of osteogenesis in vitro and enhancement of bone repair in vivo.Theranostics,2017,7(5):1072-1087.
11 Ohba S.Tissue regenerative therapies based on regulatory mechanisms underlying bone and cartilage development.Clin Calcium,2016,26(12):1765-1771.
12 Fan L,Wu H,Cao M,et al.Enzymatic synthesis of collagen peptide-carboxymethylated chitosan copolymer and its characterization.React Funct Polym,2014,76(1):26-31.
13 Fan L,Wu H,Zhou X,et al.Transglutaminase-catalyzed grafting collagen on chitosan and its characterization.Carbohydr Polym,2014,105:253-259.
14 Xu B,Zheng P,Gao F,et al.A mineralized high strength and tough hydrogel for skull bone regeneration.Adv Funct Mater,2017,27(4):1604327.
15 Baker RM,Tseng LF,Iannolo MT,et al.Self-deploying shapememory polymer scaffolds for grafting and stabilizing complex bone defects:A mouse femoral segmental defect study.Biomaterials,2016,76:388-398.
16 Shi L,Wang F,Zhu W,et al.Self-healing silk fibroin-based hydrogel for bone regeneration:Dynamic metal-ligand selfassembly approach.Adv Funct Mater,2017,27(37):1700591.
17 Kim S,Kang Y,Mercado-Pagan AE,et al.In vitro evaluation of photo-crosslinkable chitosan-lactide hydrogels for bone tissue engineering.J Biomed Mater Res B Appl Biomater,2014,102(7):1393-1406.
18 Nandi SK,Kundu B,Basu D,et al.Protein growth factors loaded highly porous chitosan scaffold:a comparison of bone healing properties.Mater Sci Eng C Mater Biol Appl,2013,33(3):1267-1275.
19 Saito A,Suzuki Y,Ogata S,et al.Activation of osteo-progenitor cells by a novel synthetic peptide derived from the bone morphogenetic protein-2 knuckle epitope.Biochim Biophys Acta,2003,1651(1-2):60-67.
20 Li J,Hong J,Zheng Q,et al.Repair of rat cranial bone defects with nHAC/PLLA and BMP-2-related peptide or rhBMP-2.J Orthop Res,2011,29(11):1745-1752.
21 Liu X,Yu B,Huang Q,Liu R,et al.In vitro BMP-2 peptide release from thiolated chitosan based hydrogel.Int J Biol Macromol,2016,93(Pt A):314-321.