兔骨髓间充质干细胞片（MSCs）包裹新型rhBMP-2-硫酸钙支架修复兔大段尺骨缺损的实验研究
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摘要
研究背景：
随着我国经济和交通的高速发展,高能量的各种车祸伤创伤等所导致的骨缺损越来越多；以及骨组织感染、肿瘤切除和发育异常等疾病等导致的骨缺损发病率也在增高。这不仅给患者和社会带来沉重的经济负担,而且也严重影响患者的生活质量,是临床治疗的难题。目前,临床多采用新鲜自体骨移植治疗,但由于骨来源的限制、二次损伤及治疗相关并发症等问题,限制了该方法的广泛应用。因此,研究新的治疗策略已十分必要。最近,组织工程技术的发展为治疗大段骨缺损提出了新的思路,即将活细胞体外培养扩增后接种于某些特殊支架材料上,以构建生物人工组织,植入受体后在局部微环境的作用下发挥细胞的成骨效应。
目的：
研究复合重组人骨形态发生蛋白-2(rhBMP-2)硫酸钙支架体外对骨髓间充质干细胞分化的影响；其次,在体内,研究骨髓间充质干细胞(MSCs)包裹的复合重组人骨形态发生蛋白-2(rhBMP-2)硫酸钙人工骨支架修复兔长骨骨缺损的作用与效果。
方法：
1.兔骨髓间充质干细胞的分离与培养：8周龄雄性新西兰大白兔15只,每只动物均行双侧髂骨骨髓穿刺抽取骨髓,体外密度梯度离心法联合细胞贴壁法分离培养扩增新西兰兔MSCs。2.支架对MSCs的成骨分化：将材料做成直径6mm,厚度2mm的圆盘状,放在培养板中,种上密度为5000个/cm2的细胞,常规进行贴壁培养7天、14天后对细胞的碱性磷酸酶含量进行测定。3.干细胞片的制作：将密度为50000个/cm2的细胞悬液用培养基连续培养2-3周,直至形成细胞薄片。将细胞片包裹在硫酸钙支架上备有。4.兔骨缺损修复：选用20只新西兰大白兔,制成单侧前肢15mm的尺骨骨缺损模型,随机分为四组：A组植入单纯硫酸钙支架；B组植入复合重组人骨形态发生蛋白-2(rhBMP-2)硫酸钙支架；C组植入经骨髓间充质干细胞(MSCs)包裹的硫酸钙支架；D组植入骨髓间充质干细胞(MSCs)包裹的复合重组人骨形态发生蛋白-2(rhBMP-2)硫酸钙支架。在术后4周随机处死动物,作下列检测：大体观察、X线检查、microCT、组织学检测、X线片的计算机图像分析、microCT的骨量分析、组织切片的计算机图像分析并对以上结果进行统计学分析。
结果：
1.体外7天测得碱性磷酸酶含量(nmol/s/mg蛋白)为：rhBMP-2-硫酸钙支架组含量为0.51±0.08,比单纯的硫酸钙组(含量为：0.30±0.03)明显升高(p<0.05)；在14天时,碱性磷酸酶含量rhBMP-2-硫酸钙支架组(1.22±0.21)明显高于单纯硫酸钙组(0.424±0.04),P<0.05；但是,在两个时间点上无论是rhBMP-2-硫酸钙支架组还是单纯硫酸钙支架组的碱性磷酸酶含量都明显高于单纯培养瓶组(P<0.05)。2.标本大体观察：A组缺损大部分由纤维组织连接；B组与c组大体上区别不大,两组的缺损外缘和中间有很多纤维组织,紧靠挠骨处和两个断端周围都有一定量的骨组织存在；D组缺损几乎都被大量骨组织替代；3.X线检查示：B组与C组无明显差别,但是都优于A组(p<0.05)；D组优于B组和C组(P<0.05)。X线评分所示：D组>B组≈C组>A组。4.micro CT与骨量分析：在四周后,各组硫酸钙大部分都已经吸收,A组骨组织非常有限；而B组和C组CT扫描显示有大量骨痂生成,但是在缺损中心以及远离挠骨一侧的缺损处骨量很少,并且髓腔结构不明显；在D组,大量骨痂形成,并且骨缺损已经成骨性愈合,可见大部分髓腔阴影。CT骨量分析示：B组与C组无明显差别,但是都优于A组(p<0.05)；D组优于B组和C组(P<0.05)。5.组织学切片与组织学半定量：组织切片结果与microCT相似,组织半定量也显示B组与C组无明显差别,但是都优于A组(p<0.05)。
结论：
1.证明硫酸钙支架是生长因子rhBMP-2的良好载体,并可以作为间充质干细胞片附着的良好支架。2.骨髓间充质干细胞片的移植可以有效地促进新骨的生成及骨缺损的愈合。3.rhBMP-2复合硫酸钙诱导成骨的作用也非常明显。4.结论也说明MSCs片包裹的rhBMP-2-硫酸钙支架由于具有成骨的三要素细胞、诱导因子以及支架结构,可以有效地促进新骨的生成,对于长段骨缺损的修复将发挥很大的作用。
Background:
Bone defects induced by high energy-wounds were more and mere popular with the economic development of our contry, and the incidence of the bone defects after the treatment of the bone infection, bone tumor and some anamorphosis deseases, which would make the patents or their family endure large burdens. Autograft is considered the gold standard for filling bone defects, however, it has the shortcomings such as donor site morbidity and donor shortage. As to allograft, immunologic response and endemic risk can not be ignored. These difficulties have resulted in the search for alternative bone graft substitutes. In recent years, the regenerative medicine or tissue engineering provide the possibility of successfully repairing and restoring the function in damaged or diseased tissues, which is based on three factors:scaffolds; cells and growth factors.
Objective:
The aim of this research is to assessing the efficency of the rhBMP-2-calcium sulfate coated by the MSCs cell sheets in the long bone defects models.
Methods:
Firstly, MSCs was cultured from the 8 weeks-male-rabbits; secondly, the osteogenic differentiation of MSCs cultured on rhBMP-2 loaded CS was investigated; thirdly, the defects were treated with CS (group A), rhBMP-2 loaded CS (group B), MSC sheet wrapped CS (group C) and MSC sheet wrapped rhBMP-2 loaded CS (group D).
Results:
The ALP of MSCs cultured on rhBMP-2-loaded CS was significantly higher than on CS both at 7 and 14 days (p<0.05). The defects treated with MSC sheet wrapped rhBMP-2-loaded-CS showed significantly higher scores by X-ray analysis and more bone formation determined by both histology and micro-computer tomography than other three groups after 4 and 8 weeks (p<0.05). There was no significant difference in X-ray score and bone formation between group B and C, both significantly higher than group A (p<0.05).
Conclusions:
MSC sheet wrapped rhBMP-2 loaded CS may be an effective approach to promote repair of segmental bone defects and have great potential for repairing large segmental bone defects in clinic.
随着我国经济和交通的高速发展,高能量的各种车祸伤创伤等所导致的骨缺损越来越多；以及骨组织感染、肿瘤切除和发育异常等疾病等导致的骨缺损发病率也在增高。这不仅给患者和社会带来沉重的经济负担,而且也严重影响患者的生活质量,是临床治疗的难题。目前,临床多采用新鲜自体骨移植治疗,但由于骨来源的限制、二次损伤及治疗相关并发症等问题,限制了该方法的广泛应用。因此,研究新的治疗策略已十分必要。最近,组织工程技术的发展为治疗大段骨缺损提出了新的思路,即将活细胞体外培养扩增后接种于某些特殊支架材料上,以构建生物人工组织,植入受体后在局部微环境的作用下发挥细胞的成骨效应。
目的：
研究复合重组人骨形态发生蛋白-2(rhBMP-2)硫酸钙支架体外对骨髓间充质干细胞分化的影响；其次,在体内,研究骨髓间充质干细胞(MSCs)包裹的复合重组人骨形态发生蛋白-2(rhBMP-2)硫酸钙人工骨支架修复兔长骨骨缺损的作用与效果。
方法：
1.兔骨髓间充质干细胞的分离与培养：8周龄雄性新西兰大白兔15只,每只动物均行双侧髂骨骨髓穿刺抽取骨髓,体外密度梯度离心法联合细胞贴壁法分离培养扩增新西兰兔MSCs。2.支架对MSCs的成骨分化：将材料做成直径6mm,厚度2mm的圆盘状,放在培养板中,种上密度为5000个/cm2的细胞,常规进行贴壁培养7天、14天后对细胞的碱性磷酸酶含量进行测定。3.干细胞片的制作：将密度为50000个/cm2的细胞悬液用培养基连续培养2-3周,直至形成细胞薄片。将细胞片包裹在硫酸钙支架上备有。4.兔骨缺损修复：选用20只新西兰大白兔,制成单侧前肢15mm的尺骨骨缺损模型,随机分为四组：A组植入单纯硫酸钙支架；B组植入复合重组人骨形态发生蛋白-2(rhBMP-2)硫酸钙支架；C组植入经骨髓间充质干细胞(MSCs)包裹的硫酸钙支架；D组植入骨髓间充质干细胞(MSCs)包裹的复合重组人骨形态发生蛋白-2(rhBMP-2)硫酸钙支架。在术后4周随机处死动物,作下列检测：大体观察、X线检查、microCT、组织学检测、X线片的计算机图像分析、microCT的骨量分析、组织切片的计算机图像分析并对以上结果进行统计学分析。
结果：
1.体外7天测得碱性磷酸酶含量(nmol/s/mg蛋白)为：rhBMP-2-硫酸钙支架组含量为0.51±0.08,比单纯的硫酸钙组(含量为：0.30±0.03)明显升高(p<0.05)；在14天时,碱性磷酸酶含量rhBMP-2-硫酸钙支架组(1.22±0.21)明显高于单纯硫酸钙组(0.424±0.04),P<0.05；但是,在两个时间点上无论是rhBMP-2-硫酸钙支架组还是单纯硫酸钙支架组的碱性磷酸酶含量都明显高于单纯培养瓶组(P<0.05)。2.标本大体观察：A组缺损大部分由纤维组织连接；B组与c组大体上区别不大,两组的缺损外缘和中间有很多纤维组织,紧靠挠骨处和两个断端周围都有一定量的骨组织存在；D组缺损几乎都被大量骨组织替代；3.X线检查示：B组与C组无明显差别,但是都优于A组(p<0.05)；D组优于B组和C组(P<0.05)。X线评分所示：D组>B组≈C组>A组。4.micro CT与骨量分析：在四周后,各组硫酸钙大部分都已经吸收,A组骨组织非常有限；而B组和C组CT扫描显示有大量骨痂生成,但是在缺损中心以及远离挠骨一侧的缺损处骨量很少,并且髓腔结构不明显；在D组,大量骨痂形成,并且骨缺损已经成骨性愈合,可见大部分髓腔阴影。CT骨量分析示：B组与C组无明显差别,但是都优于A组(p<0.05)；D组优于B组和C组(P<0.05)。5.组织学切片与组织学半定量：组织切片结果与microCT相似,组织半定量也显示B组与C组无明显差别,但是都优于A组(p<0.05)。
结论：
1.证明硫酸钙支架是生长因子rhBMP-2的良好载体,并可以作为间充质干细胞片附着的良好支架。2.骨髓间充质干细胞片的移植可以有效地促进新骨的生成及骨缺损的愈合。3.rhBMP-2复合硫酸钙诱导成骨的作用也非常明显。4.结论也说明MSCs片包裹的rhBMP-2-硫酸钙支架由于具有成骨的三要素细胞、诱导因子以及支架结构,可以有效地促进新骨的生成,对于长段骨缺损的修复将发挥很大的作用。
Background:
Bone defects induced by high energy-wounds were more and mere popular with the economic development of our contry, and the incidence of the bone defects after the treatment of the bone infection, bone tumor and some anamorphosis deseases, which would make the patents or their family endure large burdens. Autograft is considered the gold standard for filling bone defects, however, it has the shortcomings such as donor site morbidity and donor shortage. As to allograft, immunologic response and endemic risk can not be ignored. These difficulties have resulted in the search for alternative bone graft substitutes. In recent years, the regenerative medicine or tissue engineering provide the possibility of successfully repairing and restoring the function in damaged or diseased tissues, which is based on three factors:scaffolds; cells and growth factors.
Objective:
The aim of this research is to assessing the efficency of the rhBMP-2-calcium sulfate coated by the MSCs cell sheets in the long bone defects models.
Methods:
Firstly, MSCs was cultured from the 8 weeks-male-rabbits; secondly, the osteogenic differentiation of MSCs cultured on rhBMP-2 loaded CS was investigated; thirdly, the defects were treated with CS (group A), rhBMP-2 loaded CS (group B), MSC sheet wrapped CS (group C) and MSC sheet wrapped rhBMP-2 loaded CS (group D).
Results:
The ALP of MSCs cultured on rhBMP-2-loaded CS was significantly higher than on CS both at 7 and 14 days (p<0.05). The defects treated with MSC sheet wrapped rhBMP-2-loaded-CS showed significantly higher scores by X-ray analysis and more bone formation determined by both histology and micro-computer tomography than other three groups after 4 and 8 weeks (p<0.05). There was no significant difference in X-ray score and bone formation between group B and C, both significantly higher than group A (p<0.05).
Conclusions:
MSC sheet wrapped rhBMP-2 loaded CS may be an effective approach to promote repair of segmental bone defects and have great potential for repairing large segmental bone defects in clinic.
引文
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48. Scaglione, S., et al., Engineering of osteoinductive grafts by isolation and expansion of ovine bone marrow stromal cells directly on 3D ceramic scaffolds. Biotechnol Bioeng,2006.93(1):p.181-7.
49. Mankani, M.H., et al., In vivo bone formation by human bone marrow stromal cells: effect of carrier particle size and shape. Biotechnol Bioeng,2001.72(1):p. 96-107.
50. Yang, J., et al., Cell sheet engineering:recreating tissues without biodegradable scaffolds. Biomaterials,2005.26(33):p.6415-22.
51. Ouyang, H.W., et al., Assembly of bone marrow stromal cell sheets with knitted poly (L-lactide) scaffold for engineering ligament analogs. J Biomed Mater Res B Appl Biomater,2005.75(2):p.264-71.
52. Cooper, M.L., et al., Direct comparison of a cultured composite skin substitute containing human keratinocytes and fibroblasts to an epidermal sheet graft containing human keratinocytes on athymic mice. J Invest Dermatol,1993.101(6): p.811-9.
53. Nishida, K., et al., Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med,2004.351(12):p. 1187-96.
54. Shimizu, T., et al., Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues. FASEB J,2006.20(6):p.708-10.
55. Goldstein, A.S., Effect of seeding osteoprogenitor cells as dense clusters on cell growth and differentiation. Tissue Eng,2001.7(6):p.817-27.
56. Hoshino, M., et al., Repair of long intercalated rib defects in dogs using recombinant human bone morphogenetic protein-2 delivered by a synthetic polymer and beta-tricalcium phosphate. J Biomed Mater Res A,2009,90(2):p. 514-21.
57. Bruder, S.P., et al., The effect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects. J Bone Joint Surg Am, 1998.80(7):p.985-96.
58. Roldan, J.C., et al., Bone formation in the presence of platelet-rich plasma vs. bone morphogenetic protein-7. Bone,2004.34(1):p.80-90.
59. Rasmusson, I., Immune modulation by mesenchymal stem cells. Exp Cell Res, 2006.312(12):p.2169-79.
60. Aggarwal, S. and M.F. Pittenger, Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood,2005.105(4):p.1815-22.
1. J.L. Ricci, H.A., R Nadkarni, M. Hawkins,J. Turner, S. Rosenblum, Biological Mechanisms of Calcium-Sulfate. Replacement by Bone. Bone Engineering. Toronto, 2000. pp(332):p.334.
2. Anusavice, K., ed. Phillips'Science of Dental Materials Vol. pp 255-281.2003. 832.
3. Gao, C., et al., Characteristics of calcium sulfate/gelatin composite biomaterials for bone repair. J Biomater Sci Polym Ed,2007.18(7):p.799-824.
4. Neira-Carrillo, A., et al., Selective crystallization of calcium salts by poly(acrylate)-grafted chitosan. J Colloid Interface Sci,2005.286(1):p.134-41.
5. Thomas, M.V. and D.A. Puleo, Calcium sulfate:Properties and clinical applications. J Biomed Mater Res B Appl Biomater,2009.88(2):p.597-610.
6. Guarnieri, R., et al., Maxillary sinus augmentation using granular calcium sulfate (surgiplaster sinus):radiographic and histologic study at 2 years. Int J Periodontics Restorative Dent,2006.26(1):p.79-85.
7. Guarnieri, R. and M. Bovi, Maxillary sinus augmentation using prehardened calcium sulfate:a case report. Int J Periodontics Restorative Dent,2002.22(5):p. 503-8.
8. Orsini, G, et al., Bone-defect healing with calcium-sulfate particles and cement:an experimental study in rabbit. J Biomed Mater Res B Appl Biomater,2004.68(2):p. 199-208.
9. Lioliou, M.G., et al., Calcium sulfate precipitation in the presence of water-soluble polymers. J Colloid Interface Sci,2006.303(1):p.164-70.
10. Payne, J.M., et al., Migration of human gingival fibroblasts over guided tissue regeneration barrier materials. J Periodontol,1996.67(3):p.236-44.
11. Lillo, R. and L.F. Peltier, The substitution of plaster of Paris rods for portions of the diaphysis of the radius in dogs. Surg Forum,1956.6:p.556-8.
12. Alderman, N.E., Sterile plaster of paris as an implant in the infrabony environment: a preliminary study. J Periodontol,1969.40(1):p.11-3.
13. Bahn, S.L., Plaster:a bone substitute. Oral Surg Oral Med Oral Pathol,1966. 21(5):p.672-81.
14. Shaffer, C.D. and G.R. App, The use of plaster of paris in treating infrabony periodontal defects in humans. J Periodontol,1971.42(11):p.685-90.
15. Bell, W.H., Resorption Characteristics of Bone and Bone Substitutes. Oral Surg Oral Med Oral Pathol,1964.17:p.650-7.
16. Peltier, L.F., The use of plaster of Paris to fill large defects in bone:a preliminary report.1959. Clin Orthop Relat Res,2001(382):p.3-5.
17. Peltier, L.F., et al., The use of plaster of paris to fill defects in bone. Ann Surg, 1957.146(1):p.61-9.
18. de Wet, I.S. and C. Jansen, The use of plaster of Paris to fill large defects in bone. An experimental study to determine the ultimate fate of a measured amount of plaster of Paris inserted into the bone of a living animal. S Afr J Surg,1973.11(1): p.1-8.
19. Gourley, I.M. and J.P. Arnold, The experimental replacement of segmental defects in bone with a plaster of Paris-epoxy resin mixture. Am J Vet Res,1960.21:p. 1119-22.
20. Peltier, L.F., The use of plaster of Paris to fill defects in bone. Clin Orthop,1961. 21:p.1-31.
21. Elkins, A.D. and L.P. Jones, The effects of plaster of Paris and autogenous cancellous bone on the healing of cortical defects in the femurs of dogs. Vet Surg, 1988.17(2):p.71-6.
22. Kim, S.G., et al., Use of particulate dentin-plaster of Paris combination with/without platelet-rich plasma in the treatment of bone defects around implants. Int J Oral Maxillofac Implants,2002.17(1):p.86-94.
23. Shi, B., et al., Alveolar ridge preservation prior to implant placement with surgical-grade calcium sulfate and platelet-rich plasma:a pilot study in a canine model. Int J Oral Maxillofac Implants,2007.22(4):p.656-65.
24. Kelly, C.M., et al., The use of a surgical grade calcium sulfate as a bone graft substitute:results of a multicenter trial. Clin Orthop Relat Res,2001(382):p. 42-50.
25. Scarano, A., et al., Peri-implant bone regeneration with calcium sulfate:a light and transmission electron microscopy case report. Implant Dent,2007.16(2):p. 195-203.
26. Wang, M.L., et al., Altered bioreactivity and limited osteoconductivity of calcium sulfate-based bone cements in the osteoporotic rat spine. Spine J,2008.8(2):p. 340-50.
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44. Wang, M.L., et al., Altered bioreactivity and limited osteoconductivity of calcium sulfate-based bone cements in the osteoporotic rat spine. Spine J,2008.8(2):p. 340-50.
45. Saito, N., et al., Local bone formation by injection of recombinant human bone morphogenetic protein-2 contained in polymer carriers. Bone,2003.32(4):p. 381-6.
46. Bessho, K., et al., Experimental studies on bone induction using low-molecular-weight poly (DL-lactide-co-glycolide) as a carrier for recombinant human bone morphogenetic protein-2. J Biomed Mater Res,2002.61(1):p.61-5.
47. Trojani, C., et al., Ectopic bone formation using an injectable biphasic calcium phosphate/Si-HPMC hydrogel composite loaded with undifferentiated bone marrow stromal cells. Biomaterials,2006.27(17):p.3256-64.
48. Scaglione, S., et al., Engineering of osteoinductive grafts by isolation and expansion of ovine bone marrow stromal cells directly on 3D ceramic scaffolds. Biotechnol Bioeng,2006.93(1):p.181-7.
49. Mankani, M.H., et al., In vivo bone formation by human bone marrow stromal cells: effect of carrier particle size and shape. Biotechnol Bioeng,2001.72(1):p. 96-107.
50. Yang, J., et al., Cell sheet engineering:recreating tissues without biodegradable scaffolds. Biomaterials,2005.26(33):p.6415-22.
51. Ouyang, H.W., et al., Assembly of bone marrow stromal cell sheets with knitted poly (L-lactide) scaffold for engineering ligament analogs. J Biomed Mater Res B Appl Biomater,2005.75(2):p.264-71.
52. Cooper, M.L., et al., Direct comparison of a cultured composite skin substitute containing human keratinocytes and fibroblasts to an epidermal sheet graft containing human keratinocytes on athymic mice. J Invest Dermatol,1993.101(6): p.811-9.
53. Nishida, K., et al., Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med,2004.351(12):p. 1187-96.
54. Shimizu, T., et al., Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues. FASEB J,2006.20(6):p.708-10.
55. Goldstein, A.S., Effect of seeding osteoprogenitor cells as dense clusters on cell growth and differentiation. Tissue Eng,2001.7(6):p.817-27.
56. Hoshino, M., et al., Repair of long intercalated rib defects in dogs using recombinant human bone morphogenetic protein-2 delivered by a synthetic polymer and beta-tricalcium phosphate. J Biomed Mater Res A,2009,90(2):p. 514-21.
57. Bruder, S.P., et al., The effect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects. J Bone Joint Surg Am, 1998.80(7):p.985-96.
58. Roldan, J.C., et al., Bone formation in the presence of platelet-rich plasma vs. bone morphogenetic protein-7. Bone,2004.34(1):p.80-90.
59. Rasmusson, I., Immune modulation by mesenchymal stem cells. Exp Cell Res, 2006.312(12):p.2169-79.
60. Aggarwal, S. and M.F. Pittenger, Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood,2005.105(4):p.1815-22.
1. J.L. Ricci, H.A., R Nadkarni, M. Hawkins,J. Turner, S. Rosenblum, Biological Mechanisms of Calcium-Sulfate. Replacement by Bone. Bone Engineering. Toronto, 2000. pp(332):p.334.
2. Anusavice, K., ed. Phillips'Science of Dental Materials Vol. pp 255-281.2003. 832.
3. Gao, C., et al., Characteristics of calcium sulfate/gelatin composite biomaterials for bone repair. J Biomater Sci Polym Ed,2007.18(7):p.799-824.
4. Neira-Carrillo, A., et al., Selective crystallization of calcium salts by poly(acrylate)-grafted chitosan. J Colloid Interface Sci,2005.286(1):p.134-41.
5. Thomas, M.V. and D.A. Puleo, Calcium sulfate:Properties and clinical applications. J Biomed Mater Res B Appl Biomater,2009.88(2):p.597-610.
6. Guarnieri, R., et al., Maxillary sinus augmentation using granular calcium sulfate (surgiplaster sinus):radiographic and histologic study at 2 years. Int J Periodontics Restorative Dent,2006.26(1):p.79-85.
7. Guarnieri, R. and M. Bovi, Maxillary sinus augmentation using prehardened calcium sulfate:a case report. Int J Periodontics Restorative Dent,2002.22(5):p. 503-8.
8. Orsini, G, et al., Bone-defect healing with calcium-sulfate particles and cement:an experimental study in rabbit. J Biomed Mater Res B Appl Biomater,2004.68(2):p. 199-208.
9. Lioliou, M.G., et al., Calcium sulfate precipitation in the presence of water-soluble polymers. J Colloid Interface Sci,2006.303(1):p.164-70.
10. Payne, J.M., et al., Migration of human gingival fibroblasts over guided tissue regeneration barrier materials. J Periodontol,1996.67(3):p.236-44.
11. Lillo, R. and L.F. Peltier, The substitution of plaster of Paris rods for portions of the diaphysis of the radius in dogs. Surg Forum,1956.6:p.556-8.
12. Alderman, N.E., Sterile plaster of paris as an implant in the infrabony environment: a preliminary study. J Periodontol,1969.40(1):p.11-3.
13. Bahn, S.L., Plaster:a bone substitute. Oral Surg Oral Med Oral Pathol,1966. 21(5):p.672-81.
14. Shaffer, C.D. and G.R. App, The use of plaster of paris in treating infrabony periodontal defects in humans. J Periodontol,1971.42(11):p.685-90.
15. Bell, W.H., Resorption Characteristics of Bone and Bone Substitutes. Oral Surg Oral Med Oral Pathol,1964.17:p.650-7.
16. Peltier, L.F., The use of plaster of Paris to fill large defects in bone:a preliminary report.1959. Clin Orthop Relat Res,2001(382):p.3-5.
17. Peltier, L.F., et al., The use of plaster of paris to fill defects in bone. Ann Surg, 1957.146(1):p.61-9.
18. de Wet, I.S. and C. Jansen, The use of plaster of Paris to fill large defects in bone. An experimental study to determine the ultimate fate of a measured amount of plaster of Paris inserted into the bone of a living animal. S Afr J Surg,1973.11(1): p.1-8.
19. Gourley, I.M. and J.P. Arnold, The experimental replacement of segmental defects in bone with a plaster of Paris-epoxy resin mixture. Am J Vet Res,1960.21:p. 1119-22.
20. Peltier, L.F., The use of plaster of Paris to fill defects in bone. Clin Orthop,1961. 21:p.1-31.
21. Elkins, A.D. and L.P. Jones, The effects of plaster of Paris and autogenous cancellous bone on the healing of cortical defects in the femurs of dogs. Vet Surg, 1988.17(2):p.71-6.
22. Kim, S.G., et al., Use of particulate dentin-plaster of Paris combination with/without platelet-rich plasma in the treatment of bone defects around implants. Int J Oral Maxillofac Implants,2002.17(1):p.86-94.
23. Shi, B., et al., Alveolar ridge preservation prior to implant placement with surgical-grade calcium sulfate and platelet-rich plasma:a pilot study in a canine model. Int J Oral Maxillofac Implants,2007.22(4):p.656-65.
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