磷酸钙/壳聚糖/小分子腺苷复合材料修复大鼠颅骨极限骨缺损
|
摘要
目的观察新型生物复合材料在SD大鼠颅骨极限骨缺损中的修复效果,为骨组织工程的相关的基础研究提供实验证据。方法取18只8周龄的雄性SD大鼠,制作颅骨缺损直径达8 mm的圆形极限骨缺损模型,并随机分入不同组,观察时间为12周。分组情况如下:在颅骨缺损中植入单纯使用蒸馏水作为成形剂的8 mm直径的磷酸钙骨修复替代材料(CPC)支架材料(CPC对照组);在颅骨缺损中植入使用10%壳聚糖溶液作为成形剂的8 mm直径的CPC支架材料(CPC/CN组);在颅骨缺损中植入用10%壳聚糖液体作为成形剂并每个含有300μg小分子腺苷的8 mm直径的CPC支架材料(CPC/CN/AD组)。对标本进行X线、CT、苏木素-伊红染色(HE染色)及相关的定量分析。结果 X线影像提示各组骨缺损均有不同程度的修复,骨折线逐渐模糊,CPC/CN/AD组的骨缺损基本修复。CT结果也是如此。HE染色切片3组均未见急慢性炎症细胞,缺损中可见新生骨组织,新生骨内及周围可见散在的新生血管,新生骨面积百分数提示CPC/CN/AD组新生骨面积百分数显著大于CPC/CN组和CPC对照组(P<0.05),新生血管密度定量提示CPC/CN/AD组的新生血管密度均显著大于另外两组(P<0.05),但CPC/CN组与CPC对照组之间差异无统计学意义(P>0.05)。结论磷酸钙/壳聚糖/小分子腺苷复合材料与传统单纯磷酸钙骨修复替代材料相比具有更好的促成骨作用,是一种相对理想的新型骨再生修复替代材料之一。
Objective To evaluate the effect of a novel biomaterial in repairing large cranial defects in rats. Methods Eighteen SD rats were used to establish rat modes of large cranial defect(8 mm in diameter). The rat models were randomized into 3 groups and the cranial defects were repaired using different scaffold materials, namely CPC paste prepared with distilled water(CPC control group), CPC paste mixed with 10% chitosan(CPC/CN group), or CPC paste with 10% chitosan and 300 mg adenosine(CPC/CN/AD group). The defects were examined 12 weeks after the surgery with X-ray, CT, HE staining and quantitative assessments. Results X-ray showed that the defect was repaired in all the groups. The fracture line became obscure and the defects were almost fully repaired by regenerated bone tissues in CPC/CN/AD group, which was consistent with CT findings. In all the 3 groups, HE staining revealed the presence of new bones in the defects and new vessels in and around the new bones without inflammatory cells. The new bone area was significantly greater in CPC/CN/AD group than in CPC/CN group and CPC control group(P<0.05). The new vessel density was the highest in CPC/CN/AD group(P>0.05) but similar between CPC/CN group and CPC control group(P>0.05). Conclusion This novel calcium phosphate cement preloaded with chitosan and small molecule adenosine can better promote bone regeneration than calcium phosphate cement for repairing large bone defects to serve as a good replacement material for bone regeneration.
Objective To evaluate the effect of a novel biomaterial in repairing large cranial defects in rats. Methods Eighteen SD rats were used to establish rat modes of large cranial defect(8 mm in diameter). The rat models were randomized into 3 groups and the cranial defects were repaired using different scaffold materials, namely CPC paste prepared with distilled water(CPC control group), CPC paste mixed with 10% chitosan(CPC/CN group), or CPC paste with 10% chitosan and 300 mg adenosine(CPC/CN/AD group). The defects were examined 12 weeks after the surgery with X-ray, CT, HE staining and quantitative assessments. Results X-ray showed that the defect was repaired in all the groups. The fracture line became obscure and the defects were almost fully repaired by regenerated bone tissues in CPC/CN/AD group, which was consistent with CT findings. In all the 3 groups, HE staining revealed the presence of new bones in the defects and new vessels in and around the new bones without inflammatory cells. The new bone area was significantly greater in CPC/CN/AD group than in CPC/CN group and CPC control group(P<0.05). The new vessel density was the highest in CPC/CN/AD group(P>0.05) but similar between CPC/CN group and CPC control group(P>0.05). Conclusion This novel calcium phosphate cement preloaded with chitosan and small molecule adenosine can better promote bone regeneration than calcium phosphate cement for repairing large bone defects to serve as a good replacement material for bone regeneration.
引文
[1]Langer R,Vacanti JP.Tissue engineering[J].Science,1993,260(5110):920-6.
[2]Petite H,Viateau V,Bensa?d W,et al.Tissue-engineered bone regeneration[J].Nat Biotechnol,2000,18(9):959-63.
[3]Salgado AJ,Coutinho OP,Reis RL.Bone tissue engineering:state of the art and future trends[J].Macromol Biosci,2004,4(8):743-65.
[4]Lode A,Heiss C,Knapp G,et al.Strontium-modified premixed Calcium phosphate cements for the therapy of osteoporotic bone defects[J].Acta Biomater,2018,65(9):475-85.
[5]Ródenas-Rochina J,Ribelles JL,Lebourg M.Comparative study of PCL-HAp and PCL-bioglass composite scaffolds for bone tissue engineering[J].J Mater Sci Mater Med,2013,24(5):1293-308.
[6]Chow BA.A new calcium phosphate[C].Watersetting Cement,1987:352-79.
[7]Frankenburg EP,Goldstein SA,Bauer TW,et al.Biomechanical and histological evaluation of a calcium phosphate cement[J].J Bone Joint Surg Am,1998,80(8):1112-24.
[8]Ambard AJ,Mueninghoff L.Calcium phosphate cement:review of mechanical and biological properties[J].J Prosthodont,2006,15(5):321-8.
[9]Xu HH,Quinn JB,Takagi S,et al.Synergistic reinforcement of in situ hardening calcium phosphate composite scaffold for bone tissue engineering[J].Biomaterials,2004,25(6):1029-37.
[10]Zhao L,Tang M,Weir MD,et al.Osteogenic media and rh BMP-2-induced differentiation of umbilical cord mesenchymal stem cells encapsulated in alginate microbeads and integrated in an injectable calcium phosphate-chitosan fibrous scaffold[J].Tissue Eng Part A,2011,17(7/8):969-79.
[11]Weir MD,Xu HH.Osteoblastic induction on Calcium phosphate cement-chitosan constructs for bone tissue engineering[J].J Biomed Mater Res A,2010,94(1):223-33.
[12]Ramazzotti G,Bavelloni A,Blalock W,et al.BMP-2 induced expression of PLCβ1 that is a positive regulator of osteoblast differentiation[J].J Cell Physiol,2016,231(3):623-9.
[13]Abreu FA,Ferreira CL,Silva GA,et al.Effect of PDGF-BB,IGF-I growth factors and their combination carried by liposomes in tooth socket healing[J].Braz Dent J,2013,24(4):299-307.
[14]M?ller-Siegert J,Parmentier J,Laquerrière P,et al.Physicochemical regulation of TGF and VEGF delivery from mesoporous calcium phosphate bone substitutes[J].Nanomedicine(Lond),2017,12(15):1835-50.
[15]Discher DE,Mooney DJ,Zandstra PW.Growth factors,matrices,and forces combine and control stem cells[J].Science,2009,324(5935):1673-7.
[16]Poudel SB,Bhattarai G,Kook SH,et al.Recombinant human IGF-1produced by transgenic plant cell suspension culture enhances new bone formation in calvarial defects[J].Growth Horm IGF Res,2017,36(6):1-10.
[17]Mediero A,Cronstein BN.Adenosine and bone metabolism[J].Trends Endocrinol Metab,2013,24(6):290-300.
[18]Kang H,Shih YR,Nakasaki M,et al.Small molecule-driven direct conversion of human pluripotent stem cells into functional osteoblasts[J].Sci Adv,2016,2(8):e1600691.
[19]Lee CH,Cook JL,Mendelson A,et al.Regeneration of the articular surface of the rabbit synovial joint by cell homing:a proof of concept study[J].Lancet,2010,376(9739):440-8.
[20]Weir MD,Xu HH,Simon CG.Strong Calcium phosphate cementchitosan-mesh construct containing cell-encapsulating hydrogel beads for bone tissue engineering[J].J Biomed Mater Res A,2006,77(3):487-96.
[21]Samavedi S,Whittington AR,Goldstein AS.Calcium phosphate ceramics in bone tissue engineering:a review of properties and their influence on cell behavior[J].Acta Biomater,2013,9(9):8037-45.
[22]Costantino PD,Friedman CD,Jones K,et al.Experimental hydroxyapatite cement cranioplasty[J].Plast Reconstr Surg,1992,90(2):174-85;discussion 186-91.
[23]Bohner M,Gbureck U,Barralet JE.Technological issues for the development of more efficient calcium phosphate bone cements:a critical assessment[J].Biomaterials,2005,26(33):6423-9.
[24]Chow LC.Next Generation calcium phosphate-based biomaterials[J].Dent Mater J,2009,28(1):1-10.
[25]Ginebra MP,Espanol M,Montufar EB,et al.New processing approaches in calcium phosphate cements and their applications in regenerative medicine[J].Acta Biomater,2010,6(8):2863-73.
[26]Younes RM.Chitosan preparation from marine sources,structure,properties and applications[J].Mar Drugs,2015,13(3):1133-74.
[27]Rochet N,Balaguer T,Boukhechba F,et al.Differentiation and activity of human preosteoclasts on chitosan enriched calcium phosphate cement[J].Biomaterials,2009,30(26):4260-7.
[28]Mediero A,Wilder T,Perez-Aso M,et al.Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein-2[J].FASEB J,2015,29(4):1577-90.
[29]Bradaschia-Correa V,Josephson AM,Egol AJ,et al.Ecto-5'-nucleotidase(CD73)regulates bone formation and remodeling during intramembranous bone repair in aging mice[J].Tissue Cell,2017,49(5):545-51.
[30]Mediero A,Wilder T,Reddy VS,et al.Ticagrelor regulates osteoblast and osteoclast function and promotes bone formation in vivo via an adenosine-dependent mechanism[J].FASEB J,2016,30(11):3887-900.
[2]Petite H,Viateau V,Bensa?d W,et al.Tissue-engineered bone regeneration[J].Nat Biotechnol,2000,18(9):959-63.
[3]Salgado AJ,Coutinho OP,Reis RL.Bone tissue engineering:state of the art and future trends[J].Macromol Biosci,2004,4(8):743-65.
[4]Lode A,Heiss C,Knapp G,et al.Strontium-modified premixed Calcium phosphate cements for the therapy of osteoporotic bone defects[J].Acta Biomater,2018,65(9):475-85.
[5]Ródenas-Rochina J,Ribelles JL,Lebourg M.Comparative study of PCL-HAp and PCL-bioglass composite scaffolds for bone tissue engineering[J].J Mater Sci Mater Med,2013,24(5):1293-308.
[6]Chow BA.A new calcium phosphate[C].Watersetting Cement,1987:352-79.
[7]Frankenburg EP,Goldstein SA,Bauer TW,et al.Biomechanical and histological evaluation of a calcium phosphate cement[J].J Bone Joint Surg Am,1998,80(8):1112-24.
[8]Ambard AJ,Mueninghoff L.Calcium phosphate cement:review of mechanical and biological properties[J].J Prosthodont,2006,15(5):321-8.
[9]Xu HH,Quinn JB,Takagi S,et al.Synergistic reinforcement of in situ hardening calcium phosphate composite scaffold for bone tissue engineering[J].Biomaterials,2004,25(6):1029-37.
[10]Zhao L,Tang M,Weir MD,et al.Osteogenic media and rh BMP-2-induced differentiation of umbilical cord mesenchymal stem cells encapsulated in alginate microbeads and integrated in an injectable calcium phosphate-chitosan fibrous scaffold[J].Tissue Eng Part A,2011,17(7/8):969-79.
[11]Weir MD,Xu HH.Osteoblastic induction on Calcium phosphate cement-chitosan constructs for bone tissue engineering[J].J Biomed Mater Res A,2010,94(1):223-33.
[12]Ramazzotti G,Bavelloni A,Blalock W,et al.BMP-2 induced expression of PLCβ1 that is a positive regulator of osteoblast differentiation[J].J Cell Physiol,2016,231(3):623-9.
[13]Abreu FA,Ferreira CL,Silva GA,et al.Effect of PDGF-BB,IGF-I growth factors and their combination carried by liposomes in tooth socket healing[J].Braz Dent J,2013,24(4):299-307.
[14]M?ller-Siegert J,Parmentier J,Laquerrière P,et al.Physicochemical regulation of TGF and VEGF delivery from mesoporous calcium phosphate bone substitutes[J].Nanomedicine(Lond),2017,12(15):1835-50.
[15]Discher DE,Mooney DJ,Zandstra PW.Growth factors,matrices,and forces combine and control stem cells[J].Science,2009,324(5935):1673-7.
[16]Poudel SB,Bhattarai G,Kook SH,et al.Recombinant human IGF-1produced by transgenic plant cell suspension culture enhances new bone formation in calvarial defects[J].Growth Horm IGF Res,2017,36(6):1-10.
[17]Mediero A,Cronstein BN.Adenosine and bone metabolism[J].Trends Endocrinol Metab,2013,24(6):290-300.
[18]Kang H,Shih YR,Nakasaki M,et al.Small molecule-driven direct conversion of human pluripotent stem cells into functional osteoblasts[J].Sci Adv,2016,2(8):e1600691.
[19]Lee CH,Cook JL,Mendelson A,et al.Regeneration of the articular surface of the rabbit synovial joint by cell homing:a proof of concept study[J].Lancet,2010,376(9739):440-8.
[20]Weir MD,Xu HH,Simon CG.Strong Calcium phosphate cementchitosan-mesh construct containing cell-encapsulating hydrogel beads for bone tissue engineering[J].J Biomed Mater Res A,2006,77(3):487-96.
[21]Samavedi S,Whittington AR,Goldstein AS.Calcium phosphate ceramics in bone tissue engineering:a review of properties and their influence on cell behavior[J].Acta Biomater,2013,9(9):8037-45.
[22]Costantino PD,Friedman CD,Jones K,et al.Experimental hydroxyapatite cement cranioplasty[J].Plast Reconstr Surg,1992,90(2):174-85;discussion 186-91.
[23]Bohner M,Gbureck U,Barralet JE.Technological issues for the development of more efficient calcium phosphate bone cements:a critical assessment[J].Biomaterials,2005,26(33):6423-9.
[24]Chow LC.Next Generation calcium phosphate-based biomaterials[J].Dent Mater J,2009,28(1):1-10.
[25]Ginebra MP,Espanol M,Montufar EB,et al.New processing approaches in calcium phosphate cements and their applications in regenerative medicine[J].Acta Biomater,2010,6(8):2863-73.
[26]Younes RM.Chitosan preparation from marine sources,structure,properties and applications[J].Mar Drugs,2015,13(3):1133-74.
[27]Rochet N,Balaguer T,Boukhechba F,et al.Differentiation and activity of human preosteoclasts on chitosan enriched calcium phosphate cement[J].Biomaterials,2009,30(26):4260-7.
[28]Mediero A,Wilder T,Perez-Aso M,et al.Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein-2[J].FASEB J,2015,29(4):1577-90.
[29]Bradaschia-Correa V,Josephson AM,Egol AJ,et al.Ecto-5'-nucleotidase(CD73)regulates bone formation and remodeling during intramembranous bone repair in aging mice[J].Tissue Cell,2017,49(5):545-51.
[30]Mediero A,Wilder T,Reddy VS,et al.Ticagrelor regulates osteoblast and osteoclast function and promotes bone formation in vivo via an adenosine-dependent mechanism[J].FASEB J,2016,30(11):3887-900.