P15蛋白促进骨形成及其作用机制的实验研究
|
摘要
一、研究背景
细胞-基质的相互作用对于细胞骨架结构、生长和分化的调节具有重要的意义。细胞的存活依赖于细胞和细胞外基质（extracellular matrix，ECM）、其它细胞以及血清中的生长因子之间的相互作用。如果这些相互作用被阻遏，正常细胞会经历凋亡，这是一种细胞程序性死亡的生理过程。细胞凋亡在维持细胞数目和组织结构方面起着至关重要的作用，其调控机制十分复杂。其中某些细胞-基质的作用是通过整合素蛋白进行调节。这是一种细胞表面受体家族。大多数整合素蛋白通过基质蛋白调节细胞的贴附，这些基质蛋白包括胶原、纤连蛋白和玻连蛋白。整合素可以产生细胞外信号来调节细胞的贴附、形态、分化、增殖和凋亡。
整合素可以和数种细胞基质外分子相互作用，包括纤维连接蛋白，骨桥蛋白，骨唾液蛋白，I型， II型和X型胶原。由于这些蛋白包含着特定的氨基酸序列，多种类型的整联蛋白α和β亚基形成非共价的异二聚体以适应每个不同的结合位点来调控细胞应答。P15蛋白（序列GTPGPQGIAGQRGVV）是I型胶原α1链中15个连续的氨基酸。在本研究中，我们用P15蛋白共价结合钛合金表面，并研究P15蛋白是否能够增强成骨细胞的贴附，增殖和分化。此外，我们还探索了整联蛋白的激活是否也参与其中，从而评价P15蛋白作为一种有效的生物活性分子应用于骨组织再生工程可行性。
骨形成是细胞和基质进行三维组合的复杂过程。正常骨的形成包括以下两种机制：膜内成骨和软骨内成骨。膜内成骨主要是通过内在的骨膜成骨板成骨，并且不经过软骨形成的阶段。软骨内成骨是在骨骺和骺板软骨形成并且延长以成骨之后，在矿化的软骨支架上进行骨合成，其机制依赖于修复中应力环境。这些机制同样在骨折和截骨后修复发挥作用。通过膜内成骨，间充质干细胞从成骨前体细胞分化为成骨细胞，而软骨内成骨的特征是软骨的合成在先，而之后骨形成是按照软骨内成骨的顺序进行的。本研究探索了外源添加的P15蛋白对于软骨细胞形态，碱性磷酸酶水平，软骨基因的改变以及蛋白表达的作用来研究软骨内成骨是否参与了P15蛋白促进骨形成的作用机制。
自体骨移植被认为是骨移植材料中的“金标准”，能够增强骨再生和修复骨缺损，但是取自体骨会带来相关的供体区并发症，进而限制了这项技术的应用。此外，应用异体骨同样也存在不足之处，包括高成本，消毒和存储的问题，并且最重要的是潜在的宿主对外来组织的免疫源性反应和传染病输入的风险。总体上，骨移植材料的需求最近几年一直在增加，原因主要是因为需要骨移植的手术数量增加了，例如脊柱融合手术，关节置换翻修，继发于创伤的保肢手术，肿瘤或者骨畸形。未来的研究主要是克服前述提到的自体骨和异体骨移植物的缺点，进而发展出新的可以作为传统骨移植物的替代品或辅助品的“原生物”材料，从而满足临床治疗对于骨再生的要求。
无机牛源性羟基磷灰石（Anorganic bovine-derived hydroxyapatite, ABM)是一种天然的微孔羟基磷灰石复合物（HA），ABM已经用于修补牙周缺损。HA具有很好地生物相容性。其多孔的结构可以用于制作支架和促进骨的生长。然而，HA具有惰性并且骨传导性较差。为了增强骨的生长和诱导新骨的形成，我们将P15蛋白和HA相结合。P15蛋白主要参与成纤维细胞和成骨细胞的贴附。P15蛋白被证明可以促进细胞间贴附，并且可以支持细胞在HA表面的贴附。在本研究中，我们通过小鼠皮下模型和山羊脊柱缺损模型分析评价了ABM/P15作为骨移植替代物的有效性和安全性。
二、研究目的
（一）探索外源添加的P15蛋白对于软骨细胞的形态、碱性磷酸酶水平以及软骨基因的改变和蛋白表达水平的影响。
（二）研究P15蛋白对于特定整联蛋白的激活作用。
（三）通过体外小鼠皮下软骨内成骨模型评价P15蛋白对于MSC诱导，软骨细胞分化、和之后的骨化和矿化作用的影响。
（四）探索P15蛋白共价结合的钛合金表面对于成骨细胞和间充质干细胞的贴附、增殖和成熟的影响。
（五）P15/ABM复合物骨修复效果的评价及P15蛋白的细胞毒性分析。
三、内容和方法
（一）成软骨细胞微团培养和染色用于观察P15蛋白对于细胞生长分化的影响。
（二）双免疫荧光法检测P15蛋白和α5整联蛋白共表达。蛋白免疫印迹实验和免疫组织化学染色检测含有P15蛋白培养基培养的鼠间充质干细胞软骨分化基因的表达。
（三）将含有P15蛋白的生物基质胶注射于小鼠皮下，之后通过微型CT扫描、苏木精伊红/阿辛蓝染色和免疫组织化学染色分析P15蛋白对于骨形成作用的影响。
（四）乳酸脱氢酶和MTT实验检测P15蛋白共价连接的钛合金表面的细胞毒性。实时定量qPCR、蛋白免疫印迹实验和免疫组织化学荧光检测被用于评价培养细胞的成骨基因表达和分化。
（五） P15蛋白在骨形成中的作用通过山羊脊柱缺损模型进行评价。此外，蛋白免疫印迹实验和免疫组织化学荧光检测被用于评价P15蛋白对于人关节软骨细胞和成纤维细胞成软骨分化的影响。LDH和MTT实验被用于研究P15蛋白的细胞毒性。
四、研究结果
（一）P15蛋白组细胞增殖、生长和分化较对照组明显加快。
（二）P15蛋白和α5整联蛋白在P15蛋白组细胞中共表达，P15蛋白组的软骨分化基因表达显著高于对照组。
（三）小鼠皮下软骨骨化模型表明P15蛋白组的骨形成体积显著高于对照组，有更多的软骨生成，并且成软骨基因表达高于对照组。
（四）接种于P15蛋白共价连接的钛合金表面的细胞LDH和MTT结果与对照组无显著统计学差异（P＞0.05），且可以增强培养细胞的成骨基因表达水平。
（五）P15/ABM组山羊脊柱缺损区可以观察到大量的新骨形成，并且未观察到炎性反应的发生。P15蛋白组的成纤维细胞LDH和MTT实验结果与对照组无统计学差异（P＞0.05），且细胞凋亡基因表达水平与对照组无统计学差异（P＞0.05）。
五、实验结论
（一）P15蛋白可以促进MSC细胞的增殖、生长和成软骨分化。
（二）P15蛋白可以通过软骨内成骨促进骨的生长。
（三）P15蛋白共价连接的钛金属表面对于MSC细胞无毒性，并且可以使细胞成骨分化的基因表达升高。
（四）P15/ABM能够促进脊柱骨缺损的修复，并且不会产生免疫应答。P15蛋白对于成纤维细胞无毒性并且不会诱导细胞凋亡。
Back ground
Cell–matrix interactions are crucial for the regulation of cytoskeletal structure, growthand differentiation. Cell survival depends on cellular interaction with extracellular matrix(ECM), with other cells and with soluble growth factors in the serum. If these interactionsare prevented, normal cells may undergo apoptosis, a physiological form of programmedcell death. Apoptosis provides a vital mechanism for maintaining cell number and tissuestructure with complex mechanisms of regulation. Certain cell–ECM interactions aremediated by integrins, a major family of cell surface receptors. Most integrins mediate celladhesion to matrix proteins, including collagen, fibronectin, and vitronectin. Integrins cangenerate intracellular signals that regulate cell adhesion, morphology, differentiation,proliferation and apoptosis.
Integrins can interact with several ECM molecules including; fibronectin, osteopontin,bone sialoprotien, collagen I, II and X. As each of these proteins contain specific aminoacid sequences, multiple types of integrin alpha and beta subunits exist and formnoncovalent heterodimers to conform to each different binding site thereby modulatingcellular response. P15is the common name of a synthetic15amino acid peptide(GTPGPQGIAGQRGVV) whose sequence is taken from the cell-binding domain ofhuman collagen type I protein. In this study, we explore covalently attaching the P15peptide to a titanium surface to evaluate whether it will enhance osteoblastic adhesion,proliferation and differentiation. Additionally, we determine if integrin activation isinvolved in this process to evaluate the feasibility of P15peptide as an effectively bioactivemolecule in the use of bone tissue regeneration.
Bone formation is complex but the three-dimensional positioning of cells andmatrices is straightforward. Normal bone develops using only2mechanisms:Intramembranous bone formation and endochondral bone formation. Intramembranousbone formation is mediated by the inner periosteal osteogenic layer with bone synthesizedinitially without the mediation of a cartilage phase. Endochondral bone formation describesthe synthesis of bone on a mineralized cartilage scaffold after epiphyseal and physealcartilage have shaped and elongated the developing organ. These mechanisms are alsoused in fracture and osteotomy repair with the specific mechanism dependent on the mechanical environment provided during repair. With intramembranous bone repair,mesenchymal cells differentiate along a preosteoblast to osteoblast line while endochondralbone repair is characterized by the initial synthesis of cartilage followed by theendochondral sequence of bone formation. This study investigate the effect ofexogenously added P-15peptide on chondrocyte morphology, alkaline phosphatase levels,and alteration of chondrocytic gene and protein expression to analyze whether theendochondral bone formation is involed in the mechanism of P15peptide in the promotionof bone formation.
The autologous bone graft is considered to be the “gold standard” bone graftingmaterial to enhance bone regeneration and repair bone defects, its harvesting is associatedwith donor-site morbidity and restricted availability. Furthermore, the alternative use ofallografts has also certain drawbacks, including high cost, issues of processing, sterilizationand storage, and most importantly potential immunogenic response by the host to theforeign tissue and disease transmission. Overall, the requirements for bone graftingmaterial have increased significantly within the recent years, mainly due to the increasingnumber of procedures requiring bone augmentation, such as spinal fusion procedures,revision arthroplasties and limb salvage procedures secondary to trauma, tumour orskeletal abnormalities. Ongoing research to overcome the aforementioned limitations ofautologous and allogeneic bone grafts led to development of new “orthobiologic” materialseither as adjuncts or as alternatives to the “traditional” grafting materials for themanagement of any clinical condition-requiring enhancement of bone regeneration.
Anorganic bovine-derived hydroxyapatite （ABM） is a natural microporous HAcompound that is approved for the filling of periodontal defects. HA has excellentbiocompatibility and its porous structure serves as a scaffold and allows bone ingrowth.However, HA is rather inert and has a weak osteoinductive activity. In order to enhancebone ingrowth and to induce new bone formation, we added the cell-binding peptide (P-15)to HA. P-15is a synthetic clone of15-amino acid sequence of type I collagen, which ismainly involved in the adhesion/junction of cells, primarily fibroblasts and osteoblasts.P-15has been shown to have the ability to support the intracellular adhesion; moreover itsupports the adhesion of cells on HA-surface. In this study, we analyze and evaluate theefficacy and safety of the ABM/P15as the bone graft substitute in the mouse subdermalmodel and sheep vertebral defect model.
Objective
1. To examine the effect of exogenously added P-15peptide on chondrocyte morphology,alkaline phosphatase levels, and alteration of chondrocytic gene and protein expression.
2. To investigate the role of P-15peptide on activation of specific integrins.
3. An in vivo murine subdermal endochondral ossification model was utilized to evaluateeffect of P-15peptide on MSC attraction, chondrocytic differentiation, and subsequentossification and mineralization.
4. To investigated osteoblast and mesenchymal cell (MSC) adhesion, proliferation andmaturation on covalently attached P15-titanium (Ti-P15) surfaces.
5. The evaluation of the efficacy of the P15/ABM in the bone repair and the cellularcytotoxicity of P-15peptide.
Methods
1. The chondrogenic micromass culture and staining were used to observe the effects ofP15peptide on the cellular growth and differentiation.
2. The double immunofluorescence was used to test the coexpression of P15peptide andα5integrin. The western blot analysis and immunohistochemistry were performed to testthe expression of chondrogenic genes in mouse stem cells cultured with P15peptide.
3. The biological matrigel containing P15peptide was injected subcutaneously to themouse. Then, the microCT, HE and alcian blue and immunohistochemical staining wereused to analyze the effects of P15peptide on bone formation.
4. P15peptide was covalently bonded to titanium alloy surfaces. LDH and MTT assaywere used to evaluate the cellular cytotoxity of titanium alloy surface covalently bondedwith P15peptide. The real-time qPCR, western blot and fluorescent immunohistochemistrywere performed to measure osteoblast gene expression and differentiation of the culturedcells.
5. The role of the P-15peptide in bone formation was investigated using in vivo sheepvertebral defect and murine subdermal endochondral ossification models. In addition,western blot and fluorescent immunohistochemistry were performed to evaluate the effects of P-15peptide on the human articular chondrocytes and fibroblasts. LDH and MTT assaywere also used to investigate the cellular cytotoxicity of P-15peptide.
Results
1. The proliferation, growth and differentiation of the cells in P15group are significantlyenhanced compared with control group.
2. The coexpression of the P15peptide and α5integrin was observed in P15group. Theexpression of chondrogenic genes in P15group was significantly higher than the controlgroup.
3. The mouse subdermal endochondral ossification model showed that the volume of thenewly formed bone in P15group was significantly higher than the control group. The P15group has more newly formed cartilage and higher expression of the chondrogenic genescompared with control group.
4. The LDH and MTT results showed no statistical difference between the cells growed onthe P15-titanium surface and those on the titanium surface (P＞0.05), and the P15peptidecan enhance the expression of the osteogenic genes in cells grown on the P15-titaniumsurface.
5. Large amount of newly formed bone was observed in the defect area of the sheep spinein P15/ABM group without the presence of inflammatory response. The LDH and MTTtests showed no statistical difference between the fibroblasts in P15group and those incontrol group (P＞0.05). No statistical difference of the expression of apoptosis genes wasfound between the P15and control group (P＞0.05).
Conclusion
1. P15peptide can enhance the proliferation, growth and chondrogenic differentiation ofMSCs.
2. P15peptide can promote the bone growth by the endochondral bone formation.3. The P15-titanium has no cellular cytotoxicity to the MSCs and can elevate the
expression of osteogenic genes of MSCs.
4. P15/ABM can facilitate the bone repair in the spinal defect area without theinflammatory response. P15peptide has no cellular cytotoxicity to the fibroblasts andcannot induce the apoptosis.
细胞-基质的相互作用对于细胞骨架结构、生长和分化的调节具有重要的意义。细胞的存活依赖于细胞和细胞外基质（extracellular matrix，ECM）、其它细胞以及血清中的生长因子之间的相互作用。如果这些相互作用被阻遏，正常细胞会经历凋亡，这是一种细胞程序性死亡的生理过程。细胞凋亡在维持细胞数目和组织结构方面起着至关重要的作用，其调控机制十分复杂。其中某些细胞-基质的作用是通过整合素蛋白进行调节。这是一种细胞表面受体家族。大多数整合素蛋白通过基质蛋白调节细胞的贴附，这些基质蛋白包括胶原、纤连蛋白和玻连蛋白。整合素可以产生细胞外信号来调节细胞的贴附、形态、分化、增殖和凋亡。
整合素可以和数种细胞基质外分子相互作用，包括纤维连接蛋白，骨桥蛋白，骨唾液蛋白，I型， II型和X型胶原。由于这些蛋白包含着特定的氨基酸序列，多种类型的整联蛋白α和β亚基形成非共价的异二聚体以适应每个不同的结合位点来调控细胞应答。P15蛋白（序列GTPGPQGIAGQRGVV）是I型胶原α1链中15个连续的氨基酸。在本研究中，我们用P15蛋白共价结合钛合金表面，并研究P15蛋白是否能够增强成骨细胞的贴附，增殖和分化。此外，我们还探索了整联蛋白的激活是否也参与其中，从而评价P15蛋白作为一种有效的生物活性分子应用于骨组织再生工程可行性。
骨形成是细胞和基质进行三维组合的复杂过程。正常骨的形成包括以下两种机制：膜内成骨和软骨内成骨。膜内成骨主要是通过内在的骨膜成骨板成骨，并且不经过软骨形成的阶段。软骨内成骨是在骨骺和骺板软骨形成并且延长以成骨之后，在矿化的软骨支架上进行骨合成，其机制依赖于修复中应力环境。这些机制同样在骨折和截骨后修复发挥作用。通过膜内成骨，间充质干细胞从成骨前体细胞分化为成骨细胞，而软骨内成骨的特征是软骨的合成在先，而之后骨形成是按照软骨内成骨的顺序进行的。本研究探索了外源添加的P15蛋白对于软骨细胞形态，碱性磷酸酶水平，软骨基因的改变以及蛋白表达的作用来研究软骨内成骨是否参与了P15蛋白促进骨形成的作用机制。
自体骨移植被认为是骨移植材料中的“金标准”，能够增强骨再生和修复骨缺损，但是取自体骨会带来相关的供体区并发症，进而限制了这项技术的应用。此外，应用异体骨同样也存在不足之处，包括高成本，消毒和存储的问题，并且最重要的是潜在的宿主对外来组织的免疫源性反应和传染病输入的风险。总体上，骨移植材料的需求最近几年一直在增加，原因主要是因为需要骨移植的手术数量增加了，例如脊柱融合手术，关节置换翻修，继发于创伤的保肢手术，肿瘤或者骨畸形。未来的研究主要是克服前述提到的自体骨和异体骨移植物的缺点，进而发展出新的可以作为传统骨移植物的替代品或辅助品的“原生物”材料，从而满足临床治疗对于骨再生的要求。
无机牛源性羟基磷灰石（Anorganic bovine-derived hydroxyapatite, ABM)是一种天然的微孔羟基磷灰石复合物（HA），ABM已经用于修补牙周缺损。HA具有很好地生物相容性。其多孔的结构可以用于制作支架和促进骨的生长。然而，HA具有惰性并且骨传导性较差。为了增强骨的生长和诱导新骨的形成，我们将P15蛋白和HA相结合。P15蛋白主要参与成纤维细胞和成骨细胞的贴附。P15蛋白被证明可以促进细胞间贴附，并且可以支持细胞在HA表面的贴附。在本研究中，我们通过小鼠皮下模型和山羊脊柱缺损模型分析评价了ABM/P15作为骨移植替代物的有效性和安全性。
二、研究目的
（一）探索外源添加的P15蛋白对于软骨细胞的形态、碱性磷酸酶水平以及软骨基因的改变和蛋白表达水平的影响。
（二）研究P15蛋白对于特定整联蛋白的激活作用。
（三）通过体外小鼠皮下软骨内成骨模型评价P15蛋白对于MSC诱导，软骨细胞分化、和之后的骨化和矿化作用的影响。
（四）探索P15蛋白共价结合的钛合金表面对于成骨细胞和间充质干细胞的贴附、增殖和成熟的影响。
（五）P15/ABM复合物骨修复效果的评价及P15蛋白的细胞毒性分析。
三、内容和方法
（一）成软骨细胞微团培养和染色用于观察P15蛋白对于细胞生长分化的影响。
（二）双免疫荧光法检测P15蛋白和α5整联蛋白共表达。蛋白免疫印迹实验和免疫组织化学染色检测含有P15蛋白培养基培养的鼠间充质干细胞软骨分化基因的表达。
（三）将含有P15蛋白的生物基质胶注射于小鼠皮下，之后通过微型CT扫描、苏木精伊红/阿辛蓝染色和免疫组织化学染色分析P15蛋白对于骨形成作用的影响。
（四）乳酸脱氢酶和MTT实验检测P15蛋白共价连接的钛合金表面的细胞毒性。实时定量qPCR、蛋白免疫印迹实验和免疫组织化学荧光检测被用于评价培养细胞的成骨基因表达和分化。
（五） P15蛋白在骨形成中的作用通过山羊脊柱缺损模型进行评价。此外，蛋白免疫印迹实验和免疫组织化学荧光检测被用于评价P15蛋白对于人关节软骨细胞和成纤维细胞成软骨分化的影响。LDH和MTT实验被用于研究P15蛋白的细胞毒性。
四、研究结果
（一）P15蛋白组细胞增殖、生长和分化较对照组明显加快。
（二）P15蛋白和α5整联蛋白在P15蛋白组细胞中共表达，P15蛋白组的软骨分化基因表达显著高于对照组。
（三）小鼠皮下软骨骨化模型表明P15蛋白组的骨形成体积显著高于对照组，有更多的软骨生成，并且成软骨基因表达高于对照组。
（四）接种于P15蛋白共价连接的钛合金表面的细胞LDH和MTT结果与对照组无显著统计学差异（P＞0.05），且可以增强培养细胞的成骨基因表达水平。
（五）P15/ABM组山羊脊柱缺损区可以观察到大量的新骨形成，并且未观察到炎性反应的发生。P15蛋白组的成纤维细胞LDH和MTT实验结果与对照组无统计学差异（P＞0.05），且细胞凋亡基因表达水平与对照组无统计学差异（P＞0.05）。
五、实验结论
（一）P15蛋白可以促进MSC细胞的增殖、生长和成软骨分化。
（二）P15蛋白可以通过软骨内成骨促进骨的生长。
（三）P15蛋白共价连接的钛金属表面对于MSC细胞无毒性，并且可以使细胞成骨分化的基因表达升高。
（四）P15/ABM能够促进脊柱骨缺损的修复，并且不会产生免疫应答。P15蛋白对于成纤维细胞无毒性并且不会诱导细胞凋亡。
Back ground
Cell–matrix interactions are crucial for the regulation of cytoskeletal structure, growthand differentiation. Cell survival depends on cellular interaction with extracellular matrix(ECM), with other cells and with soluble growth factors in the serum. If these interactionsare prevented, normal cells may undergo apoptosis, a physiological form of programmedcell death. Apoptosis provides a vital mechanism for maintaining cell number and tissuestructure with complex mechanisms of regulation. Certain cell–ECM interactions aremediated by integrins, a major family of cell surface receptors. Most integrins mediate celladhesion to matrix proteins, including collagen, fibronectin, and vitronectin. Integrins cangenerate intracellular signals that regulate cell adhesion, morphology, differentiation,proliferation and apoptosis.
Integrins can interact with several ECM molecules including; fibronectin, osteopontin,bone sialoprotien, collagen I, II and X. As each of these proteins contain specific aminoacid sequences, multiple types of integrin alpha and beta subunits exist and formnoncovalent heterodimers to conform to each different binding site thereby modulatingcellular response. P15is the common name of a synthetic15amino acid peptide(GTPGPQGIAGQRGVV) whose sequence is taken from the cell-binding domain ofhuman collagen type I protein. In this study, we explore covalently attaching the P15peptide to a titanium surface to evaluate whether it will enhance osteoblastic adhesion,proliferation and differentiation. Additionally, we determine if integrin activation isinvolved in this process to evaluate the feasibility of P15peptide as an effectively bioactivemolecule in the use of bone tissue regeneration.
Bone formation is complex but the three-dimensional positioning of cells andmatrices is straightforward. Normal bone develops using only2mechanisms:Intramembranous bone formation and endochondral bone formation. Intramembranousbone formation is mediated by the inner periosteal osteogenic layer with bone synthesizedinitially without the mediation of a cartilage phase. Endochondral bone formation describesthe synthesis of bone on a mineralized cartilage scaffold after epiphyseal and physealcartilage have shaped and elongated the developing organ. These mechanisms are alsoused in fracture and osteotomy repair with the specific mechanism dependent on the mechanical environment provided during repair. With intramembranous bone repair,mesenchymal cells differentiate along a preosteoblast to osteoblast line while endochondralbone repair is characterized by the initial synthesis of cartilage followed by theendochondral sequence of bone formation. This study investigate the effect ofexogenously added P-15peptide on chondrocyte morphology, alkaline phosphatase levels,and alteration of chondrocytic gene and protein expression to analyze whether theendochondral bone formation is involed in the mechanism of P15peptide in the promotionof bone formation.
The autologous bone graft is considered to be the “gold standard” bone graftingmaterial to enhance bone regeneration and repair bone defects, its harvesting is associatedwith donor-site morbidity and restricted availability. Furthermore, the alternative use ofallografts has also certain drawbacks, including high cost, issues of processing, sterilizationand storage, and most importantly potential immunogenic response by the host to theforeign tissue and disease transmission. Overall, the requirements for bone graftingmaterial have increased significantly within the recent years, mainly due to the increasingnumber of procedures requiring bone augmentation, such as spinal fusion procedures,revision arthroplasties and limb salvage procedures secondary to trauma, tumour orskeletal abnormalities. Ongoing research to overcome the aforementioned limitations ofautologous and allogeneic bone grafts led to development of new “orthobiologic” materialseither as adjuncts or as alternatives to the “traditional” grafting materials for themanagement of any clinical condition-requiring enhancement of bone regeneration.
Anorganic bovine-derived hydroxyapatite （ABM） is a natural microporous HAcompound that is approved for the filling of periodontal defects. HA has excellentbiocompatibility and its porous structure serves as a scaffold and allows bone ingrowth.However, HA is rather inert and has a weak osteoinductive activity. In order to enhancebone ingrowth and to induce new bone formation, we added the cell-binding peptide (P-15)to HA. P-15is a synthetic clone of15-amino acid sequence of type I collagen, which ismainly involved in the adhesion/junction of cells, primarily fibroblasts and osteoblasts.P-15has been shown to have the ability to support the intracellular adhesion; moreover itsupports the adhesion of cells on HA-surface. In this study, we analyze and evaluate theefficacy and safety of the ABM/P15as the bone graft substitute in the mouse subdermalmodel and sheep vertebral defect model.
Objective
1. To examine the effect of exogenously added P-15peptide on chondrocyte morphology,alkaline phosphatase levels, and alteration of chondrocytic gene and protein expression.
2. To investigate the role of P-15peptide on activation of specific integrins.
3. An in vivo murine subdermal endochondral ossification model was utilized to evaluateeffect of P-15peptide on MSC attraction, chondrocytic differentiation, and subsequentossification and mineralization.
4. To investigated osteoblast and mesenchymal cell (MSC) adhesion, proliferation andmaturation on covalently attached P15-titanium (Ti-P15) surfaces.
5. The evaluation of the efficacy of the P15/ABM in the bone repair and the cellularcytotoxicity of P-15peptide.
Methods
1. The chondrogenic micromass culture and staining were used to observe the effects ofP15peptide on the cellular growth and differentiation.
2. The double immunofluorescence was used to test the coexpression of P15peptide andα5integrin. The western blot analysis and immunohistochemistry were performed to testthe expression of chondrogenic genes in mouse stem cells cultured with P15peptide.
3. The biological matrigel containing P15peptide was injected subcutaneously to themouse. Then, the microCT, HE and alcian blue and immunohistochemical staining wereused to analyze the effects of P15peptide on bone formation.
4. P15peptide was covalently bonded to titanium alloy surfaces. LDH and MTT assaywere used to evaluate the cellular cytotoxity of titanium alloy surface covalently bondedwith P15peptide. The real-time qPCR, western blot and fluorescent immunohistochemistrywere performed to measure osteoblast gene expression and differentiation of the culturedcells.
5. The role of the P-15peptide in bone formation was investigated using in vivo sheepvertebral defect and murine subdermal endochondral ossification models. In addition,western blot and fluorescent immunohistochemistry were performed to evaluate the effects of P-15peptide on the human articular chondrocytes and fibroblasts. LDH and MTT assaywere also used to investigate the cellular cytotoxicity of P-15peptide.
Results
1. The proliferation, growth and differentiation of the cells in P15group are significantlyenhanced compared with control group.
2. The coexpression of the P15peptide and α5integrin was observed in P15group. Theexpression of chondrogenic genes in P15group was significantly higher than the controlgroup.
3. The mouse subdermal endochondral ossification model showed that the volume of thenewly formed bone in P15group was significantly higher than the control group. The P15group has more newly formed cartilage and higher expression of the chondrogenic genescompared with control group.
4. The LDH and MTT results showed no statistical difference between the cells growed onthe P15-titanium surface and those on the titanium surface (P＞0.05), and the P15peptidecan enhance the expression of the osteogenic genes in cells grown on the P15-titaniumsurface.
5. Large amount of newly formed bone was observed in the defect area of the sheep spinein P15/ABM group without the presence of inflammatory response. The LDH and MTTtests showed no statistical difference between the fibroblasts in P15group and those incontrol group (P＞0.05). No statistical difference of the expression of apoptosis genes wasfound between the P15and control group (P＞0.05).
Conclusion
1. P15peptide can enhance the proliferation, growth and chondrogenic differentiation ofMSCs.
2. P15peptide can promote the bone growth by the endochondral bone formation.3. The P15-titanium has no cellular cytotoxicity to the MSCs and can elevate the
expression of osteogenic genes of MSCs.
4. P15/ABM can facilitate the bone repair in the spinal defect area without theinflammatory response. P15peptide has no cellular cytotoxicity to the fibroblasts andcannot induce the apoptosis.
引文
[1] Shapiro F. Cortical bone repair. The relationship of the lacunar-canalicular system andintercellular gap junctions to the repair process. J Bone Joint Surg [Am],1988,70:1067-1081.
[2] Forriol F, Shapiro F. Bone development. Interaction of molecular components and biophysicalforces. Clin Orthop Rel Res,2005,432:14-33.
[3] Rivas R, Shapiro F. Structural stages in the development of the long bones and epiphyses: astudy in the New Zealand rabbit. J Bone Joint Surg [Am]2002,84:85-100.
[4] Shapiro F, Holtrop ME, Glimcher MJ. Organization and cellular biology of the perichondrialossification groove of Ranvier. J Bone Joint Surg [Am],1977,59:703-723.
[5] Rhinelander FW. The normal microcirculation of diaphyseal cortex and its response to fracture.J Bone Joint Surg [Am],1968,50:784-800.
[6] Trueta J, Morgan JD. The vascular contribution to osteogenesis. I. Studies by the injectionmethod. J Bone Joint Surg [Br],1960,42:97-109.
[7] Haines RW. Cartilage canals. J Anat,1933,68:45-64.
[8] Shapiro F. Epiphyseal and physeal cartilage vascularization: a light microscopic andautoradiographic study of cartilage canals in newborn and young postnatal rabbit bone. Anat Rec,1998,252:140-148.
[9] Dale GG, Harris WR. Prognosis of epiphyseal separations. An experimental study. J Bone JointSurg [Br],1958,40:116-122.
[10] Burger EH, Klein-Nuland J. Mechanotransduction in bone–role of lacuno-canalicularnetwork. FASEB J,1999,13(Suppl): S101-S112.
[11] Palumbo C, Palazinni S, Marotti G. Morphological study of intercellular junctions duringosteocyte differentiation. Bone,1990a,11:401-406.
[12] Palumbo C, Palazinni S, Zaffe D, Marotti G. Osteocyte differentiation in the tibia of newbornrabbit: an ultrastructural study of the formation of cytoplasmic processes. Acta Anat,1990b,137:350-358.
[13] Rubinacci A, Covini M, Bisogni C, Villa I, Galli M, Palumbo C, Ferretti M, Muglia MA,Marotti G. Bone as an ion exchange system: evidence for a link between mechanotransduction andmetabolic needs. Am J Physiol Endocrinol Metab,2002,282:851-864.
[14] Shapiro F. Cortical bone repair. The relationship of the lacunar-canalicular system andintercellular gap junctions to the repair process. J Bone Joint Surg [Am],1988,70:1067-1081.
[15] Shapiro F. Variable conformation of gap junctions linking bone cells: a transmission electronmicroscopic study of linear, stacked linear, curvilinear, oval and annular junctions. Calcif TissueInt,1997,61:285-293.
[16] Shapiro F, Cahill C, Malatantis G, Nayak RC. Transmission electron microscopicdemonstration of vimentin in rat osteoblast and osteocyte cell bodies and processes using theimmunogold technique. Anat Rec,1995,241:39-48.
[17] Borgens RB. Endogenous ionic currents traverse intact and damaged bone. Science,1984,225:478-482.
[18] Duncan RL, Turner CH. Mechanotransduction and the functional response of bone tomechanical strains. Calcif Tissue Int,1995,57:344-358.
[19] Henderson JH, Carter DR. Mechanical induction in limb morphogenesis. The role ofgrowth-generated strains and pressures. Bone,2002,31:645-653.
[20] Forriol F, Shapiro F. Bone development. Interaction of molecular components and biophysicalforces. Clin Orthop Rel Res,2005,432:14-33.
[21] Wolff J. Das Gesetz der Transformation der Knochen. Berlin: A Hirschwald.[Englishtranslation: The Law of Bone Remodelling.(P Maquet, R. Furlong, transl). Berlin, Heidelberg,New York,1976.
[22] Roux W. Anpassungslehre, Histomechanik, Histochemie. Mit Bemerkunngen uber dieEntwicklung und Formgestaltung der Gelenke [Adaptation, Histomechanics, Histochemistry. WithComments on the Development and Morphology of the Joints]. Virchows Arch,1912,207:168-209.
[23] Krompecher S. Die Knochenbildung [Bone Formation]. Gustav Fischer, Jena,1937.
[24] Krompecher S. Die Entwicklung der Knochenzellen und die Bildung derKnochengrundsubstanz bei der knorpelig und bindegewbig vorgebildeten sowie der primarenreinen Knochenbildung [The development of bone cells and the formation of the ground substanceof bone during bone preformed by cartilage or connective tissue as well as during primary purebone formation]. Verh Anat Ges,1934,42:34-53.
[25] Krompecher S. Die Beeinflussbarkeit der Gewebsdifferenzierung der granulierendenKnochenoberflachen insbesondere die der Callusbildung [The possibility to influence tissuedifferentiation of the granulating bone surfaces, especially those of callus formation]. LangenbecksArch Klin Chir,1956,281:472-512.
[26] Pauwels F. Biomechanics of the Normal and Diseased Hip. Theoretical foundation, techniqueand results of treatment. An atlas.(RJ Furlong, P Maquet, transl.). Springer-Verlag, Berlin,Heidelberg, New York,1976.
[27] Weinans H, Prendergast PJ. Tissue adaptation as a dynamical process far from equilibrium.Bone,1996,19:143-149.
[28] Carter DR, Beaupre GS, Giori NJ, Helms JA. Mechanobiology of skeletal regeneration. ClinOrthop Rel Res,1998,355S: S41-S55.
[29] Claes LE, Heigele CA. Magnitudes of local stress and strain along bony surfaces predict thecourse and type of fracture healing. J Biomech,1999,32:255-266.
[30] Owan I, Burr DB, Turner CH, Qui J, Tu Y, Onga JE, Duncan RL. Mechanotransduction inbone: osteoblasts are more responsive to fluid flow than mechanical strain. Am J Physiol,1997,273: C810-815.
[31] Prendergast PJ, van der Meulen MCH. Mechanics of bone regeneration. In: Bone MechanicsHandbook, second edition (SJ Cowin, ed),2001,32.1-32.13.
[32] Bassett CAL, Becker RO. Generation of electric potentials by bone in response to mechanicalstress. Science,1962,137:1063-1064.
[33] Friedenberg ZB, Brighton CT. Bioelectric potentials in bone. J Bone Joint Surg [Am],1966,48:915-923.
[34] Basset CAL, Pawluk RJ, Becker RO. Effects of electric currents on bone in vivo. Nature,1964,204:652-654.
[35] Bassett CAL, Mitchell SN, Gaston SR. Pulsing electromagnetic field treatment in ununitedfractures and failed arthrodeses. J Am Med Assoc,1982,247:623-628.
[36] Brighton CT. The treatment of non-unions with electricity. J Bone Joint Surg [Am],1981,63:847-851.
[37] Chao E, Inoue N. Biophysical stimulation of bone fracture repair, regeneration andremodelling. Eur Cell Mater,2003,6:72-85.
[38] Barnes GL, Kostenuik PJ, Gerstenfeld LC, Einhorn TA. Growth factor regulation of fracturerepair. J Bone Miner Res,1999,14:1805-1815.
[39] Einhorn TA. The cell and molecular biology of fracture healing. Clin Orthop Rel Res,1998,355S: S7-S21.
[40] Ferguson C, Alpern E, Miclau T, Helms JA. Does adult fracture repair recapitulate embryonicskeletal formation? Mech Dev,1999,87:57-66.
[41] Gerstenfeld LC, Cullinane DM, Barnes GL, Graves DT, Einhorn TA. Fracture healing as apost-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J CellBiochem,2003,88:873-884.
[42] Vortkamp A, Pathi S, Peretti GM, Caruso EM, Zaleske DJ, Tabin CJ. Recapitulation of signalsregulating embryonic bone formation during postnatal growth and in fracture repair. Mech Dev,1998,71:65-76.
[43] Foote JS. A contribution to the comparative histology of the femur. Smithsonian Contributionsto Knowledge,1916,35:1-230.
[44] Modis L. Organization of the Extracellular Matrix: A Polarization Microscopic Approach.CRC Press, Boca Raton, FL, USA,1991.
[45] Gebhardt W. Uber funktionell wichtige Anordnungsweisen der feineren und groberenBauelemente des Wirbeltierknochens. II. Spezieller Teil. I. Der Bau der HaversschenLamellensysteme und seine funktionelle Bedeutung [On functionally important structures of thefiner and coarser building blocks of the vertebrate bone. II. Special part. The structure of theHaversian lamellar system and its functional significance],1905.
[46] Petersen H. Die Organe der Skelettystems. In: Handbuch der Mikroskopischen Anatomie desMenschen [The organs of the skeletal system. In. The Manual of Human Microscopic Anatomy](von Mollendorff W, ed).2:1930,520-678.
[47] Giraud-Guille MM. Twisted plywood architecture of collagen fibrils in human compact boneosteons. Calcif Tissue Int,1988,42:167-180.
[48] Weidenreich F. Das Knochengewebe. In: Handbuch der Mikroskopischen Anatomie desMenschen [Bone Tissue. In: Manual of Human Microscopic Anatomy](von Mollendorff W, ed).2:1930,391-520.
[49] Weiner S, Arad T, Sabanay I, Traub W. Rotated plywood structure of primary lamellar bone inthe rat: orientations of the collagen fibril array. Bone,1997,20:509-514.
[51] Bouligand Y, Denefle J-P, Lechaire J-P, Maillard M. Twisted architectures in cell-freeassembled collagen gels: study of collagen substrates used for cultures. Biol Cell,1985,54:143-162.
[52] Cisneros DA, Hung C, Franz CM, Muller DJ. Observing growth steps of collagenself-assembly by timelapse high-resolution atomic force microscopy. J Struc Biol,2006,154:232-245.
[53] Kadler KE, Holmes DF, Trotter JA, Chapman JA. Collagen fibril formation. Biochem J,1996,316:1-11.
[54] Dupuytren G. On the formation of callus; and on the means of remedying its faulty ormisshapen deposit. In: On the Injuries and Diseases of Bones (Le Gros Clark F, ed and transl)Sydenham Society, London,1847,40-72.
[55] Cornil V, Coudray P. Du cal. Au point de vue experimental et histologique [The callus. Fromthe experimental and histological point of view]. J Anat Physiol,1904,40:113-179.
[56] Koch H. Experimentelle studien uber Knochenregeneration und knochencallusbildung
[Experimental studies on bone regeneration and callus formation]. Bruns Beitr Klin Chir,1924,132:364-440.
[57] McKibbin B. The biology of fracture healing in long bones. J Bone Joint Surg [Br],1978,60:150-162.
[58] Wehner E. Experimentelle Studie über die Knochencallusentwicklung unter dem Einflusse desfunktionellen Reizes ohne und mit besonderer Sch digung von Periost und Knochenmark
[Experimental study on the development of bone callus under the influence of the functionalstimulation without and with special damage to periosteum and bone marrow]. Bruns Beitr KlinChir,1921,123:541-583.
[59] Urist MB, Johnson RW Jr. Calcification and ossification. IV. The healing of fractures in manunder clinical conditions. J Bone Joint Surg,1943,25:375-426.
[60] Ham AW, Harris WR. Repair and transplantation of bone. In: The Biochemistry andPhysiology of Bone,2nd ed (Bourne GH, ed) Academic Press, New York,1971,337-399.
[61] Ham AW. The healing of a simple fracture of a long bone. In: Histology,8th ed (Ham AW,Crmack DH, eds) Lippincott, Philadelphia.1979,450-455.
[62] Perren SM, Russenberger M, Steinemann S, Muller ME, Allgower M. The reaction of corticalbone to compression. Acta Orthop Scand. Supp,1969,125:19-30.
[63] Perren SM. Physical and biological aspects of fracture healing with special reference tointernal fixation. Clin Orthop Rel Res,1979,138:175-196.
[64] Schenk R, Willenegger H. Zur histologie der primaren knochenheilung [The histology ofprimary bone repair]. Langenbecks Arch Klin Chir,1964,308:440-452.
[65] Muller ME. Bases experimentales et principes de1’osteosynthese par compression
[Experimental basis and principles of osteosynthesis by compression]. Internat Orthop,1978,2:115-125.
[66] Schenk R, Willenegger H. Morphological findings in primary fracture healing. Symp BiolHung,1967,7:75-86.
[67] Olerud S, Danckwardt-Lilliestr m G. Fracture healing in compression osteosynthesis in thedog. J Bone Joint Surg [Br],1968,50:844-851.
[68] Wieder H. Regeneration of bone. An essay, illustrated by photographs and photomicrographs,dealing with the repair of simple fractures and the regeneration of bone after partial injury andsubperiosteal resection. Univ Pennsylvania Med Bull,1907,20:109-138.
[69] Schenk RK, Hunziker EB. Histologic and ultrastructural features of fracture healing. In: BoneFormation and Repair (Brighton CT, Friedlaender GE, Lane JM, eds) American Academy ofOrthopaedic Surgeons, Rosemont, IL.1994,117-146.
[70] Glimcher MJ, Shapiro F, Ellis RD, Eyre DR. Changes in tissue morphology and collagencomposition during the repair of cortical bone in the adult chicken. J Bone Joint Surg [Am],1980,62:964-973.
[71] Draenert Y, Draenert K. Gap healing of compact bone. Scan Elec Microsc1980,4:103-111.
[72] Johner R. Zur Knochenheilung in Abh ngigkeit von der Defektgrosse [On the healing of bonein relation to the magnitude of the defect]. Helv Chir Acta,1972,39:409-411.
[73] Schenk R, Willenegger H. Zur histologie der primaren knochenheilung [The histology ofprimary bone repair]. Unfallheilkunde,1977,80:155-160.
[74] Claes L, Augat P, Suger G, Wilke, H-J. Influence of size and stability of the osteotomy gap onthe success of fracture healing. J Orthop Res,1997,15:577-584.
[75] Claes L, Eckert-Hubner K, Augat P. The effect of mechanical stability on local vascularizationand tissue differentiation in callus healing. J Orthop Res,2002,20:1099-1105.
[76] Aronson J, Harrison BH, Stewart CL, Harp JH Jr. The histology of distraction osteogenesisusing different external fixators. Clin Orthop Rel Res,1989,241:106-116.
[77] Barnes GL, Kostenuik PJ, Gerstenfeld LC, Einhorn TA. Growth factor regulation of fracturerepair. J Bone Miner Res,1999,14:1805-1815.
[78] Delloye C, Delefortrie G, Coutelier L, Vincent A. Bone regenerate formation in cortical boneduring distraction lengthening. Clin Orthop Rel Res,1990,250:34-42.
[79] Ilizarov GA. The tension-stress effect on the genesis and growth of tissues. Part1. Theinfluence of stability of fixation and soft-tissue preservation. Clin Orthop Rel Res,1989,238:249-281.
[80] Kojimoto H, Yasui N, Goto T, Matsuda S, Shimomura Y. Bone lengthening in rabbits bycallus distraction. The role of periosteum and endosteum. J Bone Joint Surg [Br],1988,70:543-549.
[81] Wiedemann M. Callus distraction: a new method? Clin Orthop Rel Res,1996,327:291-304.
[82] Peltonen JI, Kahri AI, Lindberg L-A, Heikkila PS, Karaharju EO, Aalto KA. Bone formationafter distraction osteotomy of the radius in the sheep. Acta Orthop Scand,1992,63:599-603.
[83] Yasui N, Sato M, Ochi T, Kimura T, Kawahata H, Kitamura Y, Nomura S. Three modes ofossification during distraction osteogenesis in the rat. J Bone Joint Surg [Br],1997,79:824-830.
[84] Galotto M, Campanile G, Robino G, Descalzi Cancedda F, Bianco P, Cancedda R.Hypertrophic chondrocytes undergo further differentiation to osteoblastlike cells and participate inthe initial bone formation in developing chick embryo. J Bone Miner Res,1994,9:1239-1249.
[85] Kusuzaki K, Kageyama N, Shinjo H, Takeshita H, Murata H, Hashiguchi S, Ashihara T,Hirasawa Y. Development of bone canaliculi during bone repair. Bone,2000,27:655-659.
[86] Charnley J, Baker SL. Compression arthrodesis of the knee. A clinical and histological study.J Bone Joint Surg [Br],1952,34:187-199.
[87] Uhthoff HK, Rahn BA. Healing patterns of metaphyseal fractures. Clin Orthop Rel Res,1981,160:295-303.
[88] Perren SM. Evolution of the internal fixation of long bone fractures. The scientific basis ofbiological internal fixation: Choosing a balance between stability and biology. J Bone Joint Surg[Br],2002,84:1093-1110.
[89] Zimmermann R, Jakubietz R, Jakubietz M, Strasser E, Schlegel A, Wiltfang J, Eckstein R.Different preparation methods to obtain platelet components as a source of growth factors for localapplication. Transfusion2001,41(10):1217–24.
[90] Schlegel KA, Donath K. Bio-Oss-a resorbable bone substitute? Int J Biomater Med Implants1998,8(3–4):201–9.
[91] Wiltfang J, Schlegel KA, Schultze-Mosgau S, Nkenke E, Zimmermann R, Kessler P. Sinusfloor augmentation with betatricalciumphosphate (beta-TCP): does platelet-rich plasma promote itsosseous integration and degradation? Clin Oral Implants Res2003,14(2):213–8.
[92] Ishikawa K, Asaoka K. Estimation of ideal mechanical strength and critical porosity ofcalcium phosphate cement. J Biomed Mater Res1995,29(12):1537–43.
[93] Nkenke E, Schultze-Mosgau S, Radespiel-Troger M, Kloss F, Neukam FW. Morbidity ofharvesting of chin grafts: a prospective study. Clin Oral Implants Res2001,12(5):495–502.
[94] Nkenke E, Radespiel-Troger M, Wiltfang J, Schultze-Mosgau S, Winkler G, Neukam FW.Morbidity of harvesting of retromolar bone grafts: a prospective study. Clin Oral Implants Res2002,13(5):514–21.
[95] Nkenke E, Weisbach V, Winckler E, Kessler P, Schultze-Mosgau S, Wiltfang J, Neukam FW.Morbidity of harvesting of bone grafts from the iliac crest for preprosthetic augmentationprocedures: a prospective study. Int J Oral Maxillofac Surg2004,33(2):157–63.
[96] Khairoun I, Driessens FC, Boltong MG, Planell JA, Wenz R. Addition of cohesion promotorsto calcium phosphate cements. Biomaterials1999,20(4):393–8.
[97] McAllister B, Margolin M, Cogan A, Buck D, Hollinger J, Lynch S. Eighteen-monthradiographic and histologic evaluation of sinus grafting with anorganic bovine bone in thechimpanzee. Int J Oral Maxillofac Implants1999,14:56–73.
[98] Schlegel KA, Fichtner G, Schultze-Mosgau S, Wiltfang J. Histologic findings in sinusaugmentation with autogenous bone chips versus a bovine bone substitute. Int J Oral MaxillofacImplants2003,18(1):53–8.
[99] Sanan A, Haines SJ. Repairing holes in the head: a history of cranioplasty. Neurosurgery1997,40(3):588–603.
[100] Parikh SN. Bone graft substitutes: past, present, future. J Postgrad Med2002,48(2):142–8.
[101] Aichelmann-Reidy ME, Yukna RA. Bone replacement grafts. The bone substitutes. DentClin North Am1998,42(3):491–503.
[102] Albrektsson T, Johansson C. Osteoinduction, osteoconduction and osseointegration. EurSpine J2001,10(Suppl.2): S96–S101.
[103] Neukam FW, Esser E. Implantology. Mund Kiefer Gesichtschir2000,4(Suppl.1): S249–56.
[104] Nkenke E, Kloss F, Wiltfang J, Schultze-Mosgau S, Radespiel-Troger M, Loos K, NeukamFW. Histomorphometric and fluorescence microscopic analysis of bone remodelling afterinstallation of implants using an osteotome technique. Clin Oral Implants Res2002,13(6):595–602.
[105] Park J, Ries J, Gelse K, Kloss F, von der MK, Wiltfang J, Neukam FW, Schneider H. Boneregeneration in critical size defects by cell-mediated BMP-2gene transfer: a comparison ofadenoviral vectors and liposomes. Gene Ther2003,10(13):1089–98.
[106] Thorwarth M, Schlegel KA, Wiltfang J, Rupprecht S, Park JH. Experimental pilot study onsurface activation of implants with liposomal vectors. Mund Kiefer Gesichtschir2004,8(4):250–5.
[107] Nkenke E, Weisbach V, Winckler E, Kessler P, Schultze-Mosgau S, Wiltfang J, Neukam FW.Morbidity of harvesting of bone grafts from the iliac crest for preprosthetic augmentationprocedures: a prospective study. Int J Oral Maxillofac Surg2004,33(2):157–63.
[108] Oreffo RO. Bone tissue engineering-biomimetic scaffolds and engineered human progenitors.Eur Cells Mater2003,5(Suppl.2):28.
[109] Nguyen H, Qian JJ, Bhatnagar RS, Li S. Enhanced cell attachment and osteoblastic activityby P-15peptide-coated matrix in hydrogels. Biochem Biophys Res Commun2003,311(1):179–86.
[110] Bhatnagar RS, Qian JJ, Wedrychowska A, Sadeghi M, Wu YM, Smith N. Design ofbiomimetic habitats for tissue engineering with P-15, a synthetic peptide analogue of collagen.Tissue Eng1999,5(1):53–65.
[111] Qian JJ, Bhatnagar RS. Enhanced cell attachment to anorganic bone mineral in the presenceof a synthetic peptide related to collagen. J Biomed Mater Res1996,31(4):545–54.
[112] Bhatnagar RS, Qian JJ, Wedrychowska A, Sadeghi M, Wu YM, Smith N. Design ofbiomimetic habitats for tissue engineering with P-15, a synthetic peptide analogue of collagen.Tissue Eng1999,5(1):53–65.
[113] Nguyen H, Qian JJ, Bhatnagar RS, Li S. Enhanced cell attachment and osteoblastic activityby P-15peptide-coated matrix in hydrogels. Biochem Biophys Res Commun2003,311(1):179–86.
[114] Qian JJ, Bhatnagar RS. Enhanced cell attachment to anorganic bone mineral in the presenceof a synthetic peptide related to collagen. J Biomed Mater Res1996,31(4):545–54.
[115] Hanks T, Atkinson BL. Comparison of cell viability on anorganic bone matrix with orwithout P-15cell binding peptide. Biomaterials2004,25(19):4831-6.
[116] Barboza EP, de Souza RO, Caula AL, Neto LG, Caula FO, Duarte ME. Bone regeneration oflocalized chronic alveolar defects utilizing cell binding peptide associated with anorganicbovine-derived bone mineral: a clinical and histological study. J Periodontol2002,73(10):1153-9.
[117] Tehemar S, Hanes P, Sharawy M. Enhancement of osseointegration of implants placed intoextraction sockets of healthy and periodontally diseased teeth by using graft material, an ePTFEmembrane, or a combination. Clin Implant Dent Relat Res2003,5(3):193-211.
[118] Scarano A, Iezzi G, Petrone G, Orsini G, Degidi M, Strocchi R, Piattelli A. Cortical boneregeneration with a synthetic cellbinding peptide: a histologic and histomorphometric pilot study.Implant Dent2003,12(4):318-24..
[1] Ferguson C, Alpern E, Miclau T, Helms JA. Does adult fracture repair recapitulateembryonic skeletal formation? Mech Dev,1999,87:57-66.
[2] Gerstenfeld LC, Cullinane DM, Barnes GL, Graves DT, Einhorn TA. Fracture healingas a post-natal developmental process: molecular, spatial, and temporal aspects of itsregulation. J Cell Biochem,2003,88:873-884.
[3] Vortkamp A, Pathi S, Peretti GM, Caruso EM, Zaleske DJ, Tabin CJ. Recapitulation ofsignals regulating embryonic bone formation during postnatal growth and in fracture repair.Mech Dev,1998,71:65-76.
[4] Sanan A, Haines SJ. Repairing holes in the head: a history of cranioplasty.Neurosurgery1997,40(3):588–603.
[5] Parikh SN. Bone graft substitutes: past, present, future. J Postgrad Med2002,48(2):142–8.
[1] Long M, Rack HJ. Titanium alloys in total joint replacement--a materials scienceperspective. Biomaterials.1998,19(18):1621-39.
[2] Niinomi M. Metallic biomaterials. J Artif Organs.2008,11(3):105-10.
[3] Esposito M, Hirsch JM, Lekholm U, Thomsen P. Biological factors contributing tofailures of osseointegrated oral implants.(II). etiopathogenesis. Eur J Oral Sci.1998,106(3):721-64.
[4] Glassman SD, Anagnost SC, Parker A, Burke D, Johnson JR, Dimar JR. The effect ofcigarette smoking and smoking cessation on spinal fusion. Spine (Phila Pa1976).2000,25(20):2608-15.
[5] Tsao AK, Jones LC, Lewallen DG. Implant Wear Symposium2007Clinical WorkGroup. What patient and surgical factors contribute to implant wear and osteolysis in totaljoint arthroplasty? J Am Acad Orthop Surg.2008,16Suppl1:S7-13.
[6] Van Steenberghe D, Jacobs R, Desnyder M, Maffei G, Quirynen M. The relativeimpact of local and endogenous patient-related factors on implant failure up to theabutment stage. Clin Oral Implants Res.2002,13(6):617-22.
[7] Hynes RO. Integrins: Versatility, modulation, and signaling in cell adhesion. Cell.1992,69(1):11-25.
[8] Bhatnagar RS, Qian JJ, Wedrychowska A, Sadeghi M, Wu YM, Smith N. Design ofbiomimetic habitats for tissue engineering with P-15, a synthetic peptide analogue ofcollagen. Tissue Eng.1999,5(1):53-65.
[9] Bhatnagar RS, Qian JJ, Gough CA. The role in cell binding of a beta-bend within thetriple helical region in collagen alpha1(I) chain: Structural and biological evidence forconformational tautomerism on fiber surface. J Biomol Struct Dyn.1997,14(5):547-60.
[10] Hanks T, Atkinson BL. Comparison of cell viability on anorganic bone matrix with orwithout P-15cell binding peptide. Biomaterials.2004,25(19):4831-6.
[11] Gomar F, Orozco R, Villar JL, Arrizabalaga F. P-15small peptide bone graftsubstitute in the treatment of non-unions and delayed union. A pilot clinical trial. IntOrthop.2007,31(1):93-9.
[12] Sherman BP, Lindley EM, Turner AS, Seim HB,3rd, Benedict J, Burger EL, et al.Evaluation of ABM/P-15versus autogenous bone in an ovine lumbar interbody fusionmodel. Eur Spine J.2010,19(12):2156-63.
[13] De Long WG,Jr, Einhorn TA, Koval K, McKee M, Smith W, Sanders R, et al. Bonegrafts and bone graft substitutes in orthopaedic trauma surgery. A critical analysis. J BoneJoint Surg Am.2007,89(3):649-58.
[14] Kim KH, Ramaswamy N. Electrochemical surface modification of titanium indentistry. Dent Mater J.2009,28(1):20-36.
[15] Yao C, Webster TJ. Anodization: A promising nano-modification technique oftitanium implants for orthopedic applications. J Nanosci Nanotechnol.2006,6:2682-92.
[16] Schliephake H, Scharnweber D, Dard M, Sewing A, Aref A, Roessler S.Functionalization of dental implant surfaces using adhesion molecules. J Biomed MaterRes B Appl Biomater.2005,73(1):88-96.
[17] Kantlehner M, Schaffner P, Finsinger D, Meyer J, Jonczyk A, Diefenbach B, et al.Surface coating with cyclic RGD peptides stimulates osteoblast adhesion and proliferationas well as bone formation. Chembiochem.2000,18;1(2):107-14.
[18] Hennessy KM, Clem WC, Phipps MC, Sawyer AA, Shaikh FM, Bellis SL. The effectof RGD peptides on osseointegration of hydroxyapatite biomaterials. Biomaterials.2008,29(21):3075-83.
[19] Secchi AG, Grigoriou V, Shapiro IM, Cavalcanti-Adam EA, Composto RJ, DucheyneP, et al. RGDS peptides immobilized on titanium alloy stimulate bone cell attachment,differentiation and confer resistance to apoptosis. J Biomed Mater Res A.2007,83(3):577-84.
[20] Kroese-Deutman HC, van den Dolder J, Spauwen PH, Jansen JA. Influence ofRGDloaded titanium implants on bone formation in vivo. Tissue Eng.2005,11:1867-75.
[21] Ketonis C, Parvizi J, Adams CS, Shapiro IM, Hickok NJ. Topographic featuresretained after antibiotic modification of ti alloy surfaces: Retention of topography withattachment of antibiotics. Clin Orthop Relat Res.2009,467(7):1678-87.
[22] Antoci V,Jr, King SB, Jose B, Parvizi J, Zeiger AR, Wickstrom E, et al. Vancomycincovalently bonded to titanium alloy prevents bacterial colonization. J Orthop Res.2007,25(7):858-66.
[23] Bhatnagar R, Li S. Biomimetic scaffolds for tissue engineering. Conf Proc IEEE EngMed Biol Soc.2004,7:5021-3.
[24] Canalis E. Effect of growth factors on bone cell replication and differentiation. ClinOrthop Relat Res.1985,193:246-63.
[25] Xiao G, Wang D, Benson MD, Karsenty G, Franceschi RT. Role of thealpha2-integrin in osteoblast-specific gene expression and activation of the Osf2transcription factor. J Biol Chem.1998,273(49):32988-94.
[26] Salasznyk RM, Klees RF, Williams WA, Boskey A, Plopper GE. Focal adhesionkinase signaling pathways regulate the osteogenic differentiation of human mesenchymalstem cells. Exp Cell Res.2007,313(1):22-37.
[27] Salasznyk RM, Klees RF, Hughlock MK, Plopper GE. ERK signaling pathwaysregulate the osteogenic differentiation of human mesenchymal stem cells on collagen I andvitronectin. Cell Commun Adhes.2004,11(5-6):137-53.
[28] Kubler A, Neugebauer J, Oh JH, Scheer M, Zoller JE. Growth and proliferation ofhuman osteoblasts on different bone graft substitutes: An in vitro study. Implant Dent.2004,13(2):171-9.
[29] Reddi AH. Role of morphogenetic proteins in skeletal tissue engineering andregeneration. Nat Biotechnol.1998,16(3):247-52.
[30] Ripamonti U, Reddi AH. Growth and morphogenetic factors in bone induction: Roleof osteogenin and related bone morphogenetic proteins in craniofacial and periodontalbone repair. Crit Rev Oral Biol Med.1992,3(1-2):1-14.
[1] Burwell R. Studies in the transplantation of bone: VII. The fresh compositehomograft-autograft of cancellous bone: an analysis of factors leading to osteogenesis inmarrow transplants and in marrow containing bone grafts. J Bone Joint Surg [Br],1964，46:110–140.
[2] Younger EM, Chapman MW. Morbidity at bone graft donor site. J Orthop Trauma，1989，3:192–195.
[3] R. S. Bhatnagar, Synthetic compounds and compositions with enhanced cell binding,US Patent5354736,1994.
[4] Yang XB, Bhatnagar RS, Li S, Oreffo RO. Biomimetic collagen scaffolds for humanbone cell growth and differentiation. Tissue Eng，2004，10:1148–1159.
[5] Bhatnagar RS, Qian JJ, Wedrychowska A, Sadeghi M, Wu YM, Smith N. Design ofbiomimetic habitats for tissue engineering with P-15, a synthetic peptide analogue ofcollagen. Tissue Eng,1999,5(1):53–65.
[6] Nguyen H, Qian JJ, Bhatnagar RS, Li S. Enhanced cell attachment and osteoblasticactivity by P-15peptide-coated matrix in hydrogels. Biochem Biophys Res Commun,2003,311(1):179–86.
[7] Qian JJ, Bhatnagar RS. Enhanced cell attachment to anorganic bone mineral in thepresence of a synthetic peptide related to collagen. J Biomed Mater Res,1996,31(4):545–54.
[8] Hanks T, Atkinson BL. Comparison of cell viability on anorganic bone matrix with orwithout P-15cell binding peptide. Biomaterials,2004,25(19):4831–6.
[9] Kubler A, Neugebauer J, Oh JH, Scheer M, Zoller JE. Growth and proliferation ofhuman osteoblasts on different bone graft substitutes: an in-vitro study. Implant Dent,2004,13:171–179.
[10] Qian JJ, Bhatnagar RS. Enhanced cell attachment to anorganic bone mineral in thepresence of a synthetic peptide related to collagen. J Biomed Mater Res,1996,31:545–554.
[11] Barboza EP, de Souza RO, Caula AL, Neto LG, Caula FO, Duarte ME. Boneregeneration of localized chronic alveolar defects utilizing cell binding peptide associatedwith anorganic bovine-derived bone mineral: a clinical and histological study. JPeriodontol,2002,73(10):1153–9.
[12] Tehemar S, Hanes P, Sharawy M. Enhancement of osseointegration of implantsplaced into extraction sockets of healthy and periodontally diseased teeth by using graftmaterial, an ePTFE membrane, or a combination. Clin Implant Dent Relat Res,2003,5(3):193–211.
[13] Scarano A, Iezzi G, Petrone G, Orsini G, Degidi M, Strocchi R, Piattelli A. Corticalbone regeneration with a synthetic cell binding peptide: a histologic andhistomorphometric pilot study. Implant Dent,2003,12(4):318–24.
[14] Frederic Shapiro. Bone development and its relation to fracture repair. The role ofmesenchymal osteoblasts and surface osteoblasts. European Cells and Materials,2008,15:53-76.
[15] Nguyen H, Qian JJ, Bhatnagar RS, Li S. Enhanced cell attachment and osteoblasticactivity by P-15peptide-coated matrix in hydrogels. Biochem Biophys Res Commun.2003,311(1):179-86.
[16] Gomar F, Orozco R, Villar JL, Arrizabalaga F. P-15small peptide bone graftsubstitute in the treatment of non-unions and delayed union. A pilot clinical trial. IntOrthop,2007,31:93–99.
[17] Hanks T, Atkinson BL. Comparison of cell viability on anorganic bone matrix with orwithout P-15cell binding peptide. Biomaterials,2004,25:4831–4836.
[18] Kubler A, Neugebauer J, Oh JH, Scheer M, Zoller JE. Growth and proliferation ofhuman osteoblasts on different bone graft substitutes: an in vitro study. Implant Dent,2004,13:171–179.
[19] Cheung SS, Tofe AJ. Mechanism of cell growth on calcium phosphate particles: roleof cell-mediated dissolution of calcium phosphate matrix. STP Pharm Sci,1993,3:51–55.
[20] Callan DP, Rohrer MD. Use of bovine-derived hydroxyapatite in the treatment ofedentulous ridge defects: a human clinical and histologic case report. J Periodontol,1993,64:575–582.
[21] Zaner DJ, Yukna RA. Particle size of periodontal bone grafting materials. JPeriodontol,1984,55:406–409.
[22] Thorwarth M, Schultze-Mosgau S, Wehrhan F, Srour S, Wiltfang J, Neukam FW,Schlegel KA Enhanced bone regeneration with a synthetic cell-binding peptide–in vivoresults. Biochem Biophys Res Commun,2005,329:789–795.
[23] Scarano A, Iezzi G, Petrone G, Orsini G, Degidi M, Strocchi R, Piattelli A. Corticalbone regeneration with a synthetic cell-binding peptide: a histologic andhistomorphometric pilot study. Implant Dent.2003,12(4):318-24.
[24] DeLustro F, Dasch J, Keefe J, Ellingsworth L. Immune responses to allogeneic andxenogeneic implants of collagen and collagen derivatives. Clin Orthop Relat Res,1990,260:263–279.
[1] De Long Jr WG, Einhorn TA, Koval K, McKee M, Smith W, Sanders R, et al. Bonegrafts and bone graft substitutes in orthopaedic trauma surgery. A critical analysis. J BoneJt Surg Am2007,89(3):649-58.
[2] Finkemeier CG. Bone-grafting and bone-graft substitutes. J Bone Jt Surg Am,2002,84(3):454-64.
[3] Ahlmann E, Patzakis M, Roidis N, Shepherd L, Holtom P. Comparison of anterior andposterior iliac crest bone graft in terms of harvest-site morbidity and functional outcomes. JBone Jt Surg Am,2002,84(5):716-20.
[4] St John TA, Vaccaro AR, Sah AP, Schaefer M, Berta SC, Albert T, et al. Physical andmonetary costs associated with autogenous bone graft harvesting. Am J Orthop,2003,32(1):18-23.
[5] Greenwald AS, Boden SD, Goldberg VM, Khan Y, Laurencin CT, Rosier RN,American Academy of Orthopaedic Surgeons. The committee on biological implants.Bone-graft substitutes: facts, fictions, and applications. Bone Jt Surg Am,2001,83(Suppl.2Pt2):98-103.
[6] Jahangir AA, Nunley RM, Mehta S, Sharan A, the Washington Health Policy Fellows.Bone-graft substitutes in orthopaedic surgery. AAOS Now,2008.
[7] Desai BM. Osteobiologics. Am J Orthop (Belle Mead NJ),2007,36(4Suppl.):8-11.
[8] Giannoudis PV, Dinopoulos H, Tsiridis E. Bone substitutes: an update. Injury,2005,36:S20-7.
[9] Einhorn TA. Enhancement of fracture healing. J Bone Jt Surg Am,1995,77:940-56.
[10] Dimitriou R, Tsiridis E, Giannoudis PV. Current concepts of molecular aspects ofbone healing. Injury,2005,36(12):1392-404.
[11] Keramaris NC, Calori GM, Nikolaou VS, Schemitsch EH, Giannoudis PV. Fracturevascularity and bone healing: a systematic review of the role of VEGF. Injury,2008,39(Suppl.2):S45-57.
[12] Bielby R, Jones E, McGonagle D. The role of mesenchymal stem cells in maintenanceand repair of bone. Injury,2007,38(Suppl.1): S26-32.
[13] Laurencin C, Khan Y, El-Amin SF. Bone graft substitutes. Expert Rev Med Devices2006,3(1):49-57.
[14] Bauer TW, Muschler GF. Bone graft materials: an overview of the basic science. ClinOrthop Relat Res,2000,371:10-27.
[15] Khan SN, Cammisa Jr FP, Sandhu HS, Diwan AD, Girardi FP, Lane JM. The biologyof bone grafting. J Am Acad Orthop Surg,2005,13:77-86.
[16] Sen MK, Miclau T. Autologous iliac crest bone graft: should it still be the goldstandard for treating nonunions? Injury,2007,38(Suppl.1): S75-80.
[17] Horne LT, Murray PM, Saha S, Sidhar K. Effects of distal radius bone graft harveston the axial compressive strength of the radius. J Hand Surg Am,2010,35(2):262-6.
[18] Krause JO, Perry CR. Distal femur as a donor site of autogenous cancellous bone graft.J Orthop Trauma,1995,9(2):145-51.
[19] Mazock JB, Schow SR, Triplett RG. Proximal tibia bone harvest: review of technique,complications, and use in maxillofacial surgery. Int J Oral Maxillofac Implants2004;19(4):586-93.
[20] Raikin SM, Brislin K. Local bone graft harvested from the distal tibia or calcaneus forsurgery of the foot and ankle. Foot Ankle Int,2005,26(6):449-53.
[21] Cox G, Jones E, McGonagle D, Giannoudis PV. Reamerirrigator-aspirator indicationsand clinical results: a systematic review. Int Orthop2011[Epub ahead of print].
[22] Dell PC, Burchardt H, Glowczewskie Jr FP. A roentgenographic, biomechanical, andhistological evaluation of vascularized and non-vascularized segmental fibular canineautografts. J Bone Jt Surg Am,1985,67(1):105-12.
[23] Connolly JF. Injectable bone marrow preparations to stimulate osteogenic repair. ClinOrthop Relat Res,1995,313:8-18.
[24] Curylo LJ, Johnstone B, Petersilge CA, Janicki JA, Yoo JU. Augmentation of spinalarthrodesis with autologous bone marrow in a rabbit po-sterolateral spine fusion model.Spine,1999,24:434-8.
[25] Pountos I, Georgouli T, Kontakis G, Giannoudis PV. Efficacy of minimally invasivetechniques for enhancement of fracture healing: evidence today. Int Orthop,2010,34(1):3-12.
[26] Hernigou P, Poignard A, Beaujean F, Rouard H. Percutaneous autologousbone-marrow grafting for nonunions. Influence of the number and concentration ofprogenitor cells. J Bone Jt Surg Am,2005,87(7):1430-7.
[27] Ja¨ ger M, Herten M, Fochtmann U, Fischer J, Hernigou P, Zilkens C, et al. Bridgingthe gap: bone marrow aspiration concentrate reduces autologous bone grafting in osseousdefects. J Orthop Res,2011,29(2):173-80.
[28] Tiedeman JJ, Garvin KL, Kile TA, Connolly JF. The role of a composite, DBM andbone marrow in the treatment of osseous defects. Orthopedics,1995,18:1153-8.
[29] D’Ippolito G, Schiller PC, Ricordi C, Roos BA, Howard GA. Agerelated osteogenicpotential of mesenchymal stromal stem cells from human vertebral bone marrow. J BoneMiner Res,1999,14(7):1115-22.
[30] Huibregtse BA, Johnstone B, Goldberg VM, Caplan AI. Effect of age and samplingsite on the chondro-osteogenic potential of rabbit marrow-derived mesenchymal progenitorcells. J Orthop Res,2000,18(1):18-24.
[31] Farrington M, Matthews I, Foreman J, Richardson KM, Caffrey E. Microbiologicalmonitoring of bone grafts: two the surgeon10(2012)230-239237237years’ experience ata tissue bank. J Hosp Infect,1998,38:261-71.
[32] Kappe T, Cakir B, Mattes T, Reichel H, Flo¨ren M. Infections after bone allograftsurgery: a prospective study by a hospital bone bank using frozen femoral heads fromliving donors. Cell Tissue Bank,2010,11(3):253-9.
[33] Mankin HJ, Hornicek FJ, Raskin KA. Infection in massive bone allografts. ClinOrthop Relat Res,2005,432:210-6.
[34] Buck BE, Malinin TI, Brown MD. Bone transplantation and humanimmunodeficiency virus. An estimate of risk of acquired immunodeficiency syndrome(AIDS). Clin Orthop Relat Res,1989,240:129-36.
[35] Centers for Disease Control and Prevention. Hepatitis C virus transmission from anantibody-negative organ and tissue donor, United States,2000-2002. Morb Mortal WklyRep,2003,52:273-6.
[36] Katz JM, Nataraj C, Jaw R, Deigl E, Bursac P. Demineralized bone matrix as anosteoinductive biomaterial and in vitro predictors of its biological potential. J BiomedMater Res B, Appl Biomater,2009,89:127-34.
[37] Peterson B, Whang PG, Iglesias R, Wang JC, Lieberman JR. Osteoinductivity ofcommercially available demineralized bone matrix. Preparations in a spine fusion model. JBone Jt Surg Am,2004,86:2243-50.
[38] Wang JC, Alanay A, Mark D, Kanim LEA, Campbell PA, Dawson EG, et al. Acomparison of commercially available demineralized bone matrix for spinal fusion. EurSpine J,2007,16:1233-40.
[39] Docquier PL, Delloye C. Treatment of aneurysmal bone cysts by introduction ofdemineralized bone and autogenous bone marrow. J Bone Jt Surg Am,2005,87:2253-8.
[40] Tynan JR, Schachar NS, Marshall GB, Gray RR. Pathologic fracture through aunicameral bone cyst of the pelvis: CTguided percutaneous curettage, biopsy, and bonematrix injection. J Vasc Interv Radiol,2005,16:293-6.
[41] Dinopoulos HT, Giannoudis PV. Safety and efficacy of use of demineralised bonematrix in orthopaedic and trauma surgery. Expert Opin Drug Saf,2006,5(6):847-66.
[42] Urist MR. Bone: formation by autoinduction. Science,1965,150(698):893-9.
[43] Giannoudis PV, Einhorn TA. Bone morphogenetic proteins in musculoskeletalmedicine. Injury2009;40(Suppl.3):S1-3.
[44] Nauth A, Giannoudis PV, Einhorn TA, Hankenson KD, Friedlaender GE, Li R, et al.Growth factors: beyond bone morphogenetic proteins. J Orthop Trauma,2010,24(9):543-6.
[45] Simpson AH, Mills L, Noble B. The role of growth factors and related agents inaccelerating fracture healing. J Bone Jt Surg Br,2006,88(6):701-5.
[46] Dallari D, Savarino L, Stagni C, Cenni E, Cenacchi A, Fornasari PM, et al. Enhancedtibial osteotomy healing with use of bone grafts supplemented with platelet gel or plateletgel and bone marrow stromal cells. J Bone Jt Surg Am,2007,89:2413-20.
[47] Tang CH, Yang RS, Huang TH, Liu SH, Fu WM. Enhancement of fibronectinfibrillogenesis and bone formation by basic fibroblast growth factor via protein kinasec-dependent pathway in rat osteoblasts. Mol Pharmacol,2004,66:440-9.
[48] Geiger F, Lorenz H, Xu W, Szalay K, Kasten P, Claes L, et al. VEGF producing bonemarrow stromal cells (BMSC) enhance vascularization and resorption of a natural coralbone substitute. Bone,2007,41:516-22.
[49] Argintar E, Edwards S, Delahay J. Bone morphogenetic proteins in orthopaedictrauma surgery. Injury,2011,42(8):730-4.
[50] Blokhuis TJ. Formulations and delivery vehicles for bone morphogenetic proteins:latest advances and future directions. Injury,2009,40(Suppl.3):S8-11.
[51] Alsousou J, Thompson M, Hulley P, Noble A, Willett K. The biology of platelet-richplasma and its application in trauma and orthopaedic surgery: a review of the literature. JBone Jt Surg Br,2009,91(8):987-96.
[52] Lowery GL, Kulkarni S, Pennisi AE. Use of autologous growth factors in lumbarspinal fusion. Bone,1999,25(2Suppl.):47-50.
[53] Jenis LG, Banco RJ, Kwon B. A prospective study of Autologous Growth Factors(AGF) in lumbar interbody fusion. Spine J,2006,6(1):14-20.
[54] Bi L, Cheng W, Fan H, Pei G. Reconstruction of goat tibial defects using an injectabletricalcium phosphate/chitosan in combination with autologous platelet-rich plasma.Biomaterials,2010,31:3201-11.
[55] Hartmann EK, Heintel T, Morrison RH, Weckbach A. Infiuence of platelet-richplasma on the anterior fusion in spinal injuries: a qualitative and quantitative analysis usingcomputer tomography. Arch Orthop Trauma Surg,2010,130:909-14.
[56] Cenni E, Avnet S, Fotia C, Salerno M, Baldini N. Platelet-rich plasma impairsosteoclast generation from human precursors of peripheral blood. J Orthop Res,2010,28:792-7.
[57] Forriol F, Longo UG, Concejo C, Ripalda P, Maffulli N, Denaro V. Platelet-richplasma, rhOP-1(rhBMP-7) and frozen rib allograft for the reconstruction of bonymandibular defects in sheep. A pilot experimental study. Injury,2009,40(Suppl.3): S44-9.
[58] McGonagle D, English A, Jones EA. The relevance of mesenchymal stem cells invivo for future orthopaedic strategies aimed at fracture repair. Curr Orthop2007;21(4):262-7.
[59] Robey PG. Stem cells near the century mark. J Clin Invest,2000,105:1489-91.
[60] Watt FM, Hogan BL. Out of Eden: stem cells and their niches. Science,2000,287:1427-30.
[61] Papathanasopoulos A, Giannoudis PV. Biological considerations of mesenchymalstem cells and endothelial progenitor cells. Injury,2008,39(Suppl.2): S21-32.
[62] Sempuku T, Ohgushi H, Okumura M, Tamai S. Osteogenic potential of allogeneic ratmarrow cells in porous hydroxyapatite ceramics: a histological study. J Orthop Res,2005,14:907-13.
[63] Arinzeh TL, Peter SJ, Archambault MP, van den Bos C, Gordon S, Kraus K, et al.Allogeneic mesenchymal stem cells regenerate bone in a critical-sized canine segmentaldefect. J Bone Jt Surg Am,2003,85:1927-35.
[64] Patterson TE, Kumagai K, Griffith L, Muschler GF. Cellular strategies forenhancement of fracture repair. J Bone Jt Surg Am,2008,90(Suppl.1):111-9.
[65] Cornell CN. Osteoconductive materials and their role as substitutes for autogenousbone grafts. Orthop Clin North Am,1999,30(4):591-8.
[66] Nandi SK, Roy S, Mukherjee P, Kundu B, De DK, Basu D. Orthopaedic applicationsof bone graft&graft substitutes: a review. Indian J Med Res,2010,132:15-30.
[67] Fleming Jr JE, Cornell CN, Muschler GF. Bone cells and matrices in orthopaedictissue engineering. Orthop Clin North Am,2000,31:357-74.
[68] Chen Y. Orthopaedic application of gene therapy. J Orthop Sci,2001,6:199-207.
[69] Lind M, Bu¨ nger C. Orthopaedic applications of gene therapy. Int Orthop,2005,29:205-9.
[70] Calori GM, Donati D, Di Bella C, Tagliabue L. Bone morphogenetic proteins andtissue engineering: future directions. Injury,2009,40(Suppl.3): S67-76.
[71] Bohner M. Calcium orthophosphates in medicine: from ceramics to calciumphosphate cements. Injury,2000,31(Suppl.4):37-47.
[72] Hak DJ. The use of osteoconductive bone graft substitutes in orthopaedic trauma. JAm Acad Orthop Surg,2007,15:525-36.
[73] Balc ik C, Tokdemir T, Senko¨ ylu¨ A, Koc N, Timuc in M, Akin S, et al. Earlyweight bearing of porous HA/TCP (60/40) ceramics in vivo: a longitudinal study in asegmental bone defect model of rabbit. Acta Biomater,2007,3:985-96.
[74] Ghosh SK, Nandi SK, Kundu B, Datta S, De DK, Roy SK, et al. In vivo response ofporous HA and-TCP prepared by aqueous solution combustion method and comparisonwith bioglass scaffolds. J Biomed Mater Res B Appl Biomater,2008,86:217-27.
[75] Noshi T, Yoshikawa T, Ikeuchi M, Dohi Y, Ohgushi H, Horiuchi K, et al.Enhancement of the in vivo osteogenic potential of marrow/hydroxyapatite composites bybovine bone morphogenetic protein. J Biomed Mater Res,2000,52(4):621-30.
[76] Daculsi G. Biphasic calcium phosphate concept applied to artificial bone, implantcoating and injectable bone substitute. Biomaterials,1998,19(16):1473-8.
[77] Moroni A, Pegreffi F, Cadossi M, Hoang-Kim A, Lio V, Giannini S.Hydroxyapatite-coated external fixation pins. Expert Rev Med Devices,2005,2:465-71.
[78] Nguyen HQ, Deporter DA, Pilliar RM, Valiquette N, Yakubovich R. The effect ofsol-gel-formed calcium phosphate coatings on bone ingrowth and osteoconductivity ofporous-surfaced Ti alloy implants. Biomaterials,2004,25:865-76.
[79] Kopylov P, Runnqvist K, Jonsson K, Aspenberg P. Norian SRS versus externalfixation in redisplaced distal radial fractures. A randomized study in40patients. ActaOrthop Scand,1999,70:1-5.
[80] Sanchez-Sotelo J, Munuera L, Madero R. Treatment of fractures of the distal radiuswith remodellable bone cement: a prospective, randomised study using Norian SRS. JBone Jt Surg Br,2000,82:856-63.
[81] Gross V, Brandes J. The ultrastructure of the interface between a glass ceramic andbone. J Biomed Mater Res,1981,15:291-305.
[82] Kinnunen I, Aitasalo K, Pollonen M, Varpula M. Reconstruction of orbital fioorfractures using bioactive glass. J Craniomaxillofac Surg,2000,28:22-34.
[83] Peltola M, Suonpaa J, Aitasalo K, Ma¨a¨ tta¨nen H, Andersson O, Yli-Urpo A, et al.Experimental follow-up model for clinical frontal sinus obliteration with bioactive glass.Acta Otolaryngol Suppl,2000,543:167-9.
[84] Cao W, Hench LL. Bioactive materials. Ceramics Int,1996,22:493-507.
[85] Peters CL, Hines JL, Bachus KN, Craig MA, Bloebaum RD. Biological effects ofcalcium sulphate as a bone graft substitute in ovine metaphyseal defects. J Biomed MaterRes A,2006,76:456-62.
[86] Maeda ST, Bramane CM, Taga R, Garcia RB, de Moraes IG, Bernadineli N.Evaluation of surgical cavities filled with three types of calcium sulphate. J Appl Oral Sci,2007,15:416-9.
[87] Mirzayan R, Panossian V, Avedian R, Forrester DM, Menendez LR. The use ofcalcium sulphate in the treatment of benign bone lesions. A preliminary report. J Bone JtSurg Am,2001,83:355-8.
[88] Chiroff RT, White EW, Weber KN, Roy DM. Tissue ingrowth of replamineformimplants. J Biomed Mater Res,1975,9:29-45.
[89] Coughlin MJ, Grimes JS, Kennedy MP. Coralline hydroxyapatite bone graft substitutein hindfoot surgery. Foot Ankle Int,2006,27(1):19-22.
[90] Giannoudis PV, Einhorn TA, Marsh D. Fracture healing: the diamond concept. Injury,2007,38(Suppl.4): S3-6.
[91] Dimitriou R, Jones E, McGonagle D, Giannoudis PV. Bone regeneration: currentconcepts and future directions. BMC Med,2011,31;9:66.
[92] Brydone AS, Meek D, Maclaine S. Bone grafting, orthopaedic biomaterials, and theclinical need for bone engineering. Proc Inst Mech Eng H,2010,224(12):1329-43.
[93] Aloy-Pro′sper A, Maestre-Ferrin L, Pen arrocha-Oltra D, Pen arrocha-Diago M.Bone regeneration using particulate grafts: an update. Med Oral Patol Oral Cir Bucal,2011,16(2). E210-4.
[2] Forriol F, Shapiro F. Bone development. Interaction of molecular components and biophysicalforces. Clin Orthop Rel Res,2005,432:14-33.
[3] Rivas R, Shapiro F. Structural stages in the development of the long bones and epiphyses: astudy in the New Zealand rabbit. J Bone Joint Surg [Am]2002,84:85-100.
[4] Shapiro F, Holtrop ME, Glimcher MJ. Organization and cellular biology of the perichondrialossification groove of Ranvier. J Bone Joint Surg [Am],1977,59:703-723.
[5] Rhinelander FW. The normal microcirculation of diaphyseal cortex and its response to fracture.J Bone Joint Surg [Am],1968,50:784-800.
[6] Trueta J, Morgan JD. The vascular contribution to osteogenesis. I. Studies by the injectionmethod. J Bone Joint Surg [Br],1960,42:97-109.
[7] Haines RW. Cartilage canals. J Anat,1933,68:45-64.
[8] Shapiro F. Epiphyseal and physeal cartilage vascularization: a light microscopic andautoradiographic study of cartilage canals in newborn and young postnatal rabbit bone. Anat Rec,1998,252:140-148.
[9] Dale GG, Harris WR. Prognosis of epiphyseal separations. An experimental study. J Bone JointSurg [Br],1958,40:116-122.
[10] Burger EH, Klein-Nuland J. Mechanotransduction in bone–role of lacuno-canalicularnetwork. FASEB J,1999,13(Suppl): S101-S112.
[11] Palumbo C, Palazinni S, Marotti G. Morphological study of intercellular junctions duringosteocyte differentiation. Bone,1990a,11:401-406.
[12] Palumbo C, Palazinni S, Zaffe D, Marotti G. Osteocyte differentiation in the tibia of newbornrabbit: an ultrastructural study of the formation of cytoplasmic processes. Acta Anat,1990b,137:350-358.
[13] Rubinacci A, Covini M, Bisogni C, Villa I, Galli M, Palumbo C, Ferretti M, Muglia MA,Marotti G. Bone as an ion exchange system: evidence for a link between mechanotransduction andmetabolic needs. Am J Physiol Endocrinol Metab,2002,282:851-864.
[14] Shapiro F. Cortical bone repair. The relationship of the lacunar-canalicular system andintercellular gap junctions to the repair process. J Bone Joint Surg [Am],1988,70:1067-1081.
[15] Shapiro F. Variable conformation of gap junctions linking bone cells: a transmission electronmicroscopic study of linear, stacked linear, curvilinear, oval and annular junctions. Calcif TissueInt,1997,61:285-293.
[16] Shapiro F, Cahill C, Malatantis G, Nayak RC. Transmission electron microscopicdemonstration of vimentin in rat osteoblast and osteocyte cell bodies and processes using theimmunogold technique. Anat Rec,1995,241:39-48.
[17] Borgens RB. Endogenous ionic currents traverse intact and damaged bone. Science,1984,225:478-482.
[18] Duncan RL, Turner CH. Mechanotransduction and the functional response of bone tomechanical strains. Calcif Tissue Int,1995,57:344-358.
[19] Henderson JH, Carter DR. Mechanical induction in limb morphogenesis. The role ofgrowth-generated strains and pressures. Bone,2002,31:645-653.
[20] Forriol F, Shapiro F. Bone development. Interaction of molecular components and biophysicalforces. Clin Orthop Rel Res,2005,432:14-33.
[21] Wolff J. Das Gesetz der Transformation der Knochen. Berlin: A Hirschwald.[Englishtranslation: The Law of Bone Remodelling.(P Maquet, R. Furlong, transl). Berlin, Heidelberg,New York,1976.
[22] Roux W. Anpassungslehre, Histomechanik, Histochemie. Mit Bemerkunngen uber dieEntwicklung und Formgestaltung der Gelenke [Adaptation, Histomechanics, Histochemistry. WithComments on the Development and Morphology of the Joints]. Virchows Arch,1912,207:168-209.
[23] Krompecher S. Die Knochenbildung [Bone Formation]. Gustav Fischer, Jena,1937.
[24] Krompecher S. Die Entwicklung der Knochenzellen und die Bildung derKnochengrundsubstanz bei der knorpelig und bindegewbig vorgebildeten sowie der primarenreinen Knochenbildung [The development of bone cells and the formation of the ground substanceof bone during bone preformed by cartilage or connective tissue as well as during primary purebone formation]. Verh Anat Ges,1934,42:34-53.
[25] Krompecher S. Die Beeinflussbarkeit der Gewebsdifferenzierung der granulierendenKnochenoberflachen insbesondere die der Callusbildung [The possibility to influence tissuedifferentiation of the granulating bone surfaces, especially those of callus formation]. LangenbecksArch Klin Chir,1956,281:472-512.
[26] Pauwels F. Biomechanics of the Normal and Diseased Hip. Theoretical foundation, techniqueand results of treatment. An atlas.(RJ Furlong, P Maquet, transl.). Springer-Verlag, Berlin,Heidelberg, New York,1976.
[27] Weinans H, Prendergast PJ. Tissue adaptation as a dynamical process far from equilibrium.Bone,1996,19:143-149.
[28] Carter DR, Beaupre GS, Giori NJ, Helms JA. Mechanobiology of skeletal regeneration. ClinOrthop Rel Res,1998,355S: S41-S55.
[29] Claes LE, Heigele CA. Magnitudes of local stress and strain along bony surfaces predict thecourse and type of fracture healing. J Biomech,1999,32:255-266.
[30] Owan I, Burr DB, Turner CH, Qui J, Tu Y, Onga JE, Duncan RL. Mechanotransduction inbone: osteoblasts are more responsive to fluid flow than mechanical strain. Am J Physiol,1997,273: C810-815.
[31] Prendergast PJ, van der Meulen MCH. Mechanics of bone regeneration. In: Bone MechanicsHandbook, second edition (SJ Cowin, ed),2001,32.1-32.13.
[32] Bassett CAL, Becker RO. Generation of electric potentials by bone in response to mechanicalstress. Science,1962,137:1063-1064.
[33] Friedenberg ZB, Brighton CT. Bioelectric potentials in bone. J Bone Joint Surg [Am],1966,48:915-923.
[34] Basset CAL, Pawluk RJ, Becker RO. Effects of electric currents on bone in vivo. Nature,1964,204:652-654.
[35] Bassett CAL, Mitchell SN, Gaston SR. Pulsing electromagnetic field treatment in ununitedfractures and failed arthrodeses. J Am Med Assoc,1982,247:623-628.
[36] Brighton CT. The treatment of non-unions with electricity. J Bone Joint Surg [Am],1981,63:847-851.
[37] Chao E, Inoue N. Biophysical stimulation of bone fracture repair, regeneration andremodelling. Eur Cell Mater,2003,6:72-85.
[38] Barnes GL, Kostenuik PJ, Gerstenfeld LC, Einhorn TA. Growth factor regulation of fracturerepair. J Bone Miner Res,1999,14:1805-1815.
[39] Einhorn TA. The cell and molecular biology of fracture healing. Clin Orthop Rel Res,1998,355S: S7-S21.
[40] Ferguson C, Alpern E, Miclau T, Helms JA. Does adult fracture repair recapitulate embryonicskeletal formation? Mech Dev,1999,87:57-66.
[41] Gerstenfeld LC, Cullinane DM, Barnes GL, Graves DT, Einhorn TA. Fracture healing as apost-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J CellBiochem,2003,88:873-884.
[42] Vortkamp A, Pathi S, Peretti GM, Caruso EM, Zaleske DJ, Tabin CJ. Recapitulation of signalsregulating embryonic bone formation during postnatal growth and in fracture repair. Mech Dev,1998,71:65-76.
[43] Foote JS. A contribution to the comparative histology of the femur. Smithsonian Contributionsto Knowledge,1916,35:1-230.
[44] Modis L. Organization of the Extracellular Matrix: A Polarization Microscopic Approach.CRC Press, Boca Raton, FL, USA,1991.
[45] Gebhardt W. Uber funktionell wichtige Anordnungsweisen der feineren und groberenBauelemente des Wirbeltierknochens. II. Spezieller Teil. I. Der Bau der HaversschenLamellensysteme und seine funktionelle Bedeutung [On functionally important structures of thefiner and coarser building blocks of the vertebrate bone. II. Special part. The structure of theHaversian lamellar system and its functional significance],1905.
[46] Petersen H. Die Organe der Skelettystems. In: Handbuch der Mikroskopischen Anatomie desMenschen [The organs of the skeletal system. In. The Manual of Human Microscopic Anatomy](von Mollendorff W, ed).2:1930,520-678.
[47] Giraud-Guille MM. Twisted plywood architecture of collagen fibrils in human compact boneosteons. Calcif Tissue Int,1988,42:167-180.
[48] Weidenreich F. Das Knochengewebe. In: Handbuch der Mikroskopischen Anatomie desMenschen [Bone Tissue. In: Manual of Human Microscopic Anatomy](von Mollendorff W, ed).2:1930,391-520.
[49] Weiner S, Arad T, Sabanay I, Traub W. Rotated plywood structure of primary lamellar bone inthe rat: orientations of the collagen fibril array. Bone,1997,20:509-514.
[51] Bouligand Y, Denefle J-P, Lechaire J-P, Maillard M. Twisted architectures in cell-freeassembled collagen gels: study of collagen substrates used for cultures. Biol Cell,1985,54:143-162.
[52] Cisneros DA, Hung C, Franz CM, Muller DJ. Observing growth steps of collagenself-assembly by timelapse high-resolution atomic force microscopy. J Struc Biol,2006,154:232-245.
[53] Kadler KE, Holmes DF, Trotter JA, Chapman JA. Collagen fibril formation. Biochem J,1996,316:1-11.
[54] Dupuytren G. On the formation of callus; and on the means of remedying its faulty ormisshapen deposit. In: On the Injuries and Diseases of Bones (Le Gros Clark F, ed and transl)Sydenham Society, London,1847,40-72.
[55] Cornil V, Coudray P. Du cal. Au point de vue experimental et histologique [The callus. Fromthe experimental and histological point of view]. J Anat Physiol,1904,40:113-179.
[56] Koch H. Experimentelle studien uber Knochenregeneration und knochencallusbildung
[Experimental studies on bone regeneration and callus formation]. Bruns Beitr Klin Chir,1924,132:364-440.
[57] McKibbin B. The biology of fracture healing in long bones. J Bone Joint Surg [Br],1978,60:150-162.
[58] Wehner E. Experimentelle Studie über die Knochencallusentwicklung unter dem Einflusse desfunktionellen Reizes ohne und mit besonderer Sch digung von Periost und Knochenmark
[Experimental study on the development of bone callus under the influence of the functionalstimulation without and with special damage to periosteum and bone marrow]. Bruns Beitr KlinChir,1921,123:541-583.
[59] Urist MB, Johnson RW Jr. Calcification and ossification. IV. The healing of fractures in manunder clinical conditions. J Bone Joint Surg,1943,25:375-426.
[60] Ham AW, Harris WR. Repair and transplantation of bone. In: The Biochemistry andPhysiology of Bone,2nd ed (Bourne GH, ed) Academic Press, New York,1971,337-399.
[61] Ham AW. The healing of a simple fracture of a long bone. In: Histology,8th ed (Ham AW,Crmack DH, eds) Lippincott, Philadelphia.1979,450-455.
[62] Perren SM, Russenberger M, Steinemann S, Muller ME, Allgower M. The reaction of corticalbone to compression. Acta Orthop Scand. Supp,1969,125:19-30.
[63] Perren SM. Physical and biological aspects of fracture healing with special reference tointernal fixation. Clin Orthop Rel Res,1979,138:175-196.
[64] Schenk R, Willenegger H. Zur histologie der primaren knochenheilung [The histology ofprimary bone repair]. Langenbecks Arch Klin Chir,1964,308:440-452.
[65] Muller ME. Bases experimentales et principes de1’osteosynthese par compression
[Experimental basis and principles of osteosynthesis by compression]. Internat Orthop,1978,2:115-125.
[66] Schenk R, Willenegger H. Morphological findings in primary fracture healing. Symp BiolHung,1967,7:75-86.
[67] Olerud S, Danckwardt-Lilliestr m G. Fracture healing in compression osteosynthesis in thedog. J Bone Joint Surg [Br],1968,50:844-851.
[68] Wieder H. Regeneration of bone. An essay, illustrated by photographs and photomicrographs,dealing with the repair of simple fractures and the regeneration of bone after partial injury andsubperiosteal resection. Univ Pennsylvania Med Bull,1907,20:109-138.
[69] Schenk RK, Hunziker EB. Histologic and ultrastructural features of fracture healing. In: BoneFormation and Repair (Brighton CT, Friedlaender GE, Lane JM, eds) American Academy ofOrthopaedic Surgeons, Rosemont, IL.1994,117-146.
[70] Glimcher MJ, Shapiro F, Ellis RD, Eyre DR. Changes in tissue morphology and collagencomposition during the repair of cortical bone in the adult chicken. J Bone Joint Surg [Am],1980,62:964-973.
[71] Draenert Y, Draenert K. Gap healing of compact bone. Scan Elec Microsc1980,4:103-111.
[72] Johner R. Zur Knochenheilung in Abh ngigkeit von der Defektgrosse [On the healing of bonein relation to the magnitude of the defect]. Helv Chir Acta,1972,39:409-411.
[73] Schenk R, Willenegger H. Zur histologie der primaren knochenheilung [The histology ofprimary bone repair]. Unfallheilkunde,1977,80:155-160.
[74] Claes L, Augat P, Suger G, Wilke, H-J. Influence of size and stability of the osteotomy gap onthe success of fracture healing. J Orthop Res,1997,15:577-584.
[75] Claes L, Eckert-Hubner K, Augat P. The effect of mechanical stability on local vascularizationand tissue differentiation in callus healing. J Orthop Res,2002,20:1099-1105.
[76] Aronson J, Harrison BH, Stewart CL, Harp JH Jr. The histology of distraction osteogenesisusing different external fixators. Clin Orthop Rel Res,1989,241:106-116.
[77] Barnes GL, Kostenuik PJ, Gerstenfeld LC, Einhorn TA. Growth factor regulation of fracturerepair. J Bone Miner Res,1999,14:1805-1815.
[78] Delloye C, Delefortrie G, Coutelier L, Vincent A. Bone regenerate formation in cortical boneduring distraction lengthening. Clin Orthop Rel Res,1990,250:34-42.
[79] Ilizarov GA. The tension-stress effect on the genesis and growth of tissues. Part1. Theinfluence of stability of fixation and soft-tissue preservation. Clin Orthop Rel Res,1989,238:249-281.
[80] Kojimoto H, Yasui N, Goto T, Matsuda S, Shimomura Y. Bone lengthening in rabbits bycallus distraction. The role of periosteum and endosteum. J Bone Joint Surg [Br],1988,70:543-549.
[81] Wiedemann M. Callus distraction: a new method? Clin Orthop Rel Res,1996,327:291-304.
[82] Peltonen JI, Kahri AI, Lindberg L-A, Heikkila PS, Karaharju EO, Aalto KA. Bone formationafter distraction osteotomy of the radius in the sheep. Acta Orthop Scand,1992,63:599-603.
[83] Yasui N, Sato M, Ochi T, Kimura T, Kawahata H, Kitamura Y, Nomura S. Three modes ofossification during distraction osteogenesis in the rat. J Bone Joint Surg [Br],1997,79:824-830.
[84] Galotto M, Campanile G, Robino G, Descalzi Cancedda F, Bianco P, Cancedda R.Hypertrophic chondrocytes undergo further differentiation to osteoblastlike cells and participate inthe initial bone formation in developing chick embryo. J Bone Miner Res,1994,9:1239-1249.
[85] Kusuzaki K, Kageyama N, Shinjo H, Takeshita H, Murata H, Hashiguchi S, Ashihara T,Hirasawa Y. Development of bone canaliculi during bone repair. Bone,2000,27:655-659.
[86] Charnley J, Baker SL. Compression arthrodesis of the knee. A clinical and histological study.J Bone Joint Surg [Br],1952,34:187-199.
[87] Uhthoff HK, Rahn BA. Healing patterns of metaphyseal fractures. Clin Orthop Rel Res,1981,160:295-303.
[88] Perren SM. Evolution of the internal fixation of long bone fractures. The scientific basis ofbiological internal fixation: Choosing a balance between stability and biology. J Bone Joint Surg[Br],2002,84:1093-1110.
[89] Zimmermann R, Jakubietz R, Jakubietz M, Strasser E, Schlegel A, Wiltfang J, Eckstein R.Different preparation methods to obtain platelet components as a source of growth factors for localapplication. Transfusion2001,41(10):1217–24.
[90] Schlegel KA, Donath K. Bio-Oss-a resorbable bone substitute? Int J Biomater Med Implants1998,8(3–4):201–9.
[91] Wiltfang J, Schlegel KA, Schultze-Mosgau S, Nkenke E, Zimmermann R, Kessler P. Sinusfloor augmentation with betatricalciumphosphate (beta-TCP): does platelet-rich plasma promote itsosseous integration and degradation? Clin Oral Implants Res2003,14(2):213–8.
[92] Ishikawa K, Asaoka K. Estimation of ideal mechanical strength and critical porosity ofcalcium phosphate cement. J Biomed Mater Res1995,29(12):1537–43.
[93] Nkenke E, Schultze-Mosgau S, Radespiel-Troger M, Kloss F, Neukam FW. Morbidity ofharvesting of chin grafts: a prospective study. Clin Oral Implants Res2001,12(5):495–502.
[94] Nkenke E, Radespiel-Troger M, Wiltfang J, Schultze-Mosgau S, Winkler G, Neukam FW.Morbidity of harvesting of retromolar bone grafts: a prospective study. Clin Oral Implants Res2002,13(5):514–21.
[95] Nkenke E, Weisbach V, Winckler E, Kessler P, Schultze-Mosgau S, Wiltfang J, Neukam FW.Morbidity of harvesting of bone grafts from the iliac crest for preprosthetic augmentationprocedures: a prospective study. Int J Oral Maxillofac Surg2004,33(2):157–63.
[96] Khairoun I, Driessens FC, Boltong MG, Planell JA, Wenz R. Addition of cohesion promotorsto calcium phosphate cements. Biomaterials1999,20(4):393–8.
[97] McAllister B, Margolin M, Cogan A, Buck D, Hollinger J, Lynch S. Eighteen-monthradiographic and histologic evaluation of sinus grafting with anorganic bovine bone in thechimpanzee. Int J Oral Maxillofac Implants1999,14:56–73.
[98] Schlegel KA, Fichtner G, Schultze-Mosgau S, Wiltfang J. Histologic findings in sinusaugmentation with autogenous bone chips versus a bovine bone substitute. Int J Oral MaxillofacImplants2003,18(1):53–8.
[99] Sanan A, Haines SJ. Repairing holes in the head: a history of cranioplasty. Neurosurgery1997,40(3):588–603.
[100] Parikh SN. Bone graft substitutes: past, present, future. J Postgrad Med2002,48(2):142–8.
[101] Aichelmann-Reidy ME, Yukna RA. Bone replacement grafts. The bone substitutes. DentClin North Am1998,42(3):491–503.
[102] Albrektsson T, Johansson C. Osteoinduction, osteoconduction and osseointegration. EurSpine J2001,10(Suppl.2): S96–S101.
[103] Neukam FW, Esser E. Implantology. Mund Kiefer Gesichtschir2000,4(Suppl.1): S249–56.
[104] Nkenke E, Kloss F, Wiltfang J, Schultze-Mosgau S, Radespiel-Troger M, Loos K, NeukamFW. Histomorphometric and fluorescence microscopic analysis of bone remodelling afterinstallation of implants using an osteotome technique. Clin Oral Implants Res2002,13(6):595–602.
[105] Park J, Ries J, Gelse K, Kloss F, von der MK, Wiltfang J, Neukam FW, Schneider H. Boneregeneration in critical size defects by cell-mediated BMP-2gene transfer: a comparison ofadenoviral vectors and liposomes. Gene Ther2003,10(13):1089–98.
[106] Thorwarth M, Schlegel KA, Wiltfang J, Rupprecht S, Park JH. Experimental pilot study onsurface activation of implants with liposomal vectors. Mund Kiefer Gesichtschir2004,8(4):250–5.
[107] Nkenke E, Weisbach V, Winckler E, Kessler P, Schultze-Mosgau S, Wiltfang J, Neukam FW.Morbidity of harvesting of bone grafts from the iliac crest for preprosthetic augmentationprocedures: a prospective study. Int J Oral Maxillofac Surg2004,33(2):157–63.
[108] Oreffo RO. Bone tissue engineering-biomimetic scaffolds and engineered human progenitors.Eur Cells Mater2003,5(Suppl.2):28.
[109] Nguyen H, Qian JJ, Bhatnagar RS, Li S. Enhanced cell attachment and osteoblastic activityby P-15peptide-coated matrix in hydrogels. Biochem Biophys Res Commun2003,311(1):179–86.
[110] Bhatnagar RS, Qian JJ, Wedrychowska A, Sadeghi M, Wu YM, Smith N. Design ofbiomimetic habitats for tissue engineering with P-15, a synthetic peptide analogue of collagen.Tissue Eng1999,5(1):53–65.
[111] Qian JJ, Bhatnagar RS. Enhanced cell attachment to anorganic bone mineral in the presenceof a synthetic peptide related to collagen. J Biomed Mater Res1996,31(4):545–54.
[112] Bhatnagar RS, Qian JJ, Wedrychowska A, Sadeghi M, Wu YM, Smith N. Design ofbiomimetic habitats for tissue engineering with P-15, a synthetic peptide analogue of collagen.Tissue Eng1999,5(1):53–65.
[113] Nguyen H, Qian JJ, Bhatnagar RS, Li S. Enhanced cell attachment and osteoblastic activityby P-15peptide-coated matrix in hydrogels. Biochem Biophys Res Commun2003,311(1):179–86.
[114] Qian JJ, Bhatnagar RS. Enhanced cell attachment to anorganic bone mineral in the presenceof a synthetic peptide related to collagen. J Biomed Mater Res1996,31(4):545–54.
[115] Hanks T, Atkinson BL. Comparison of cell viability on anorganic bone matrix with orwithout P-15cell binding peptide. Biomaterials2004,25(19):4831-6.
[116] Barboza EP, de Souza RO, Caula AL, Neto LG, Caula FO, Duarte ME. Bone regeneration oflocalized chronic alveolar defects utilizing cell binding peptide associated with anorganicbovine-derived bone mineral: a clinical and histological study. J Periodontol2002,73(10):1153-9.
[117] Tehemar S, Hanes P, Sharawy M. Enhancement of osseointegration of implants placed intoextraction sockets of healthy and periodontally diseased teeth by using graft material, an ePTFEmembrane, or a combination. Clin Implant Dent Relat Res2003,5(3):193-211.
[118] Scarano A, Iezzi G, Petrone G, Orsini G, Degidi M, Strocchi R, Piattelli A. Cortical boneregeneration with a synthetic cellbinding peptide: a histologic and histomorphometric pilot study.Implant Dent2003,12(4):318-24..
[1] Ferguson C, Alpern E, Miclau T, Helms JA. Does adult fracture repair recapitulateembryonic skeletal formation? Mech Dev,1999,87:57-66.
[2] Gerstenfeld LC, Cullinane DM, Barnes GL, Graves DT, Einhorn TA. Fracture healingas a post-natal developmental process: molecular, spatial, and temporal aspects of itsregulation. J Cell Biochem,2003,88:873-884.
[3] Vortkamp A, Pathi S, Peretti GM, Caruso EM, Zaleske DJ, Tabin CJ. Recapitulation ofsignals regulating embryonic bone formation during postnatal growth and in fracture repair.Mech Dev,1998,71:65-76.
[4] Sanan A, Haines SJ. Repairing holes in the head: a history of cranioplasty.Neurosurgery1997,40(3):588–603.
[5] Parikh SN. Bone graft substitutes: past, present, future. J Postgrad Med2002,48(2):142–8.
[1] Long M, Rack HJ. Titanium alloys in total joint replacement--a materials scienceperspective. Biomaterials.1998,19(18):1621-39.
[2] Niinomi M. Metallic biomaterials. J Artif Organs.2008,11(3):105-10.
[3] Esposito M, Hirsch JM, Lekholm U, Thomsen P. Biological factors contributing tofailures of osseointegrated oral implants.(II). etiopathogenesis. Eur J Oral Sci.1998,106(3):721-64.
[4] Glassman SD, Anagnost SC, Parker A, Burke D, Johnson JR, Dimar JR. The effect ofcigarette smoking and smoking cessation on spinal fusion. Spine (Phila Pa1976).2000,25(20):2608-15.
[5] Tsao AK, Jones LC, Lewallen DG. Implant Wear Symposium2007Clinical WorkGroup. What patient and surgical factors contribute to implant wear and osteolysis in totaljoint arthroplasty? J Am Acad Orthop Surg.2008,16Suppl1:S7-13.
[6] Van Steenberghe D, Jacobs R, Desnyder M, Maffei G, Quirynen M. The relativeimpact of local and endogenous patient-related factors on implant failure up to theabutment stage. Clin Oral Implants Res.2002,13(6):617-22.
[7] Hynes RO. Integrins: Versatility, modulation, and signaling in cell adhesion. Cell.1992,69(1):11-25.
[8] Bhatnagar RS, Qian JJ, Wedrychowska A, Sadeghi M, Wu YM, Smith N. Design ofbiomimetic habitats for tissue engineering with P-15, a synthetic peptide analogue ofcollagen. Tissue Eng.1999,5(1):53-65.
[9] Bhatnagar RS, Qian JJ, Gough CA. The role in cell binding of a beta-bend within thetriple helical region in collagen alpha1(I) chain: Structural and biological evidence forconformational tautomerism on fiber surface. J Biomol Struct Dyn.1997,14(5):547-60.
[10] Hanks T, Atkinson BL. Comparison of cell viability on anorganic bone matrix with orwithout P-15cell binding peptide. Biomaterials.2004,25(19):4831-6.
[11] Gomar F, Orozco R, Villar JL, Arrizabalaga F. P-15small peptide bone graftsubstitute in the treatment of non-unions and delayed union. A pilot clinical trial. IntOrthop.2007,31(1):93-9.
[12] Sherman BP, Lindley EM, Turner AS, Seim HB,3rd, Benedict J, Burger EL, et al.Evaluation of ABM/P-15versus autogenous bone in an ovine lumbar interbody fusionmodel. Eur Spine J.2010,19(12):2156-63.
[13] De Long WG,Jr, Einhorn TA, Koval K, McKee M, Smith W, Sanders R, et al. Bonegrafts and bone graft substitutes in orthopaedic trauma surgery. A critical analysis. J BoneJoint Surg Am.2007,89(3):649-58.
[14] Kim KH, Ramaswamy N. Electrochemical surface modification of titanium indentistry. Dent Mater J.2009,28(1):20-36.
[15] Yao C, Webster TJ. Anodization: A promising nano-modification technique oftitanium implants for orthopedic applications. J Nanosci Nanotechnol.2006,6:2682-92.
[16] Schliephake H, Scharnweber D, Dard M, Sewing A, Aref A, Roessler S.Functionalization of dental implant surfaces using adhesion molecules. J Biomed MaterRes B Appl Biomater.2005,73(1):88-96.
[17] Kantlehner M, Schaffner P, Finsinger D, Meyer J, Jonczyk A, Diefenbach B, et al.Surface coating with cyclic RGD peptides stimulates osteoblast adhesion and proliferationas well as bone formation. Chembiochem.2000,18;1(2):107-14.
[18] Hennessy KM, Clem WC, Phipps MC, Sawyer AA, Shaikh FM, Bellis SL. The effectof RGD peptides on osseointegration of hydroxyapatite biomaterials. Biomaterials.2008,29(21):3075-83.
[19] Secchi AG, Grigoriou V, Shapiro IM, Cavalcanti-Adam EA, Composto RJ, DucheyneP, et al. RGDS peptides immobilized on titanium alloy stimulate bone cell attachment,differentiation and confer resistance to apoptosis. J Biomed Mater Res A.2007,83(3):577-84.
[20] Kroese-Deutman HC, van den Dolder J, Spauwen PH, Jansen JA. Influence ofRGDloaded titanium implants on bone formation in vivo. Tissue Eng.2005,11:1867-75.
[21] Ketonis C, Parvizi J, Adams CS, Shapiro IM, Hickok NJ. Topographic featuresretained after antibiotic modification of ti alloy surfaces: Retention of topography withattachment of antibiotics. Clin Orthop Relat Res.2009,467(7):1678-87.
[22] Antoci V,Jr, King SB, Jose B, Parvizi J, Zeiger AR, Wickstrom E, et al. Vancomycincovalently bonded to titanium alloy prevents bacterial colonization. J Orthop Res.2007,25(7):858-66.
[23] Bhatnagar R, Li S. Biomimetic scaffolds for tissue engineering. Conf Proc IEEE EngMed Biol Soc.2004,7:5021-3.
[24] Canalis E. Effect of growth factors on bone cell replication and differentiation. ClinOrthop Relat Res.1985,193:246-63.
[25] Xiao G, Wang D, Benson MD, Karsenty G, Franceschi RT. Role of thealpha2-integrin in osteoblast-specific gene expression and activation of the Osf2transcription factor. J Biol Chem.1998,273(49):32988-94.
[26] Salasznyk RM, Klees RF, Williams WA, Boskey A, Plopper GE. Focal adhesionkinase signaling pathways regulate the osteogenic differentiation of human mesenchymalstem cells. Exp Cell Res.2007,313(1):22-37.
[27] Salasznyk RM, Klees RF, Hughlock MK, Plopper GE. ERK signaling pathwaysregulate the osteogenic differentiation of human mesenchymal stem cells on collagen I andvitronectin. Cell Commun Adhes.2004,11(5-6):137-53.
[28] Kubler A, Neugebauer J, Oh JH, Scheer M, Zoller JE. Growth and proliferation ofhuman osteoblasts on different bone graft substitutes: An in vitro study. Implant Dent.2004,13(2):171-9.
[29] Reddi AH. Role of morphogenetic proteins in skeletal tissue engineering andregeneration. Nat Biotechnol.1998,16(3):247-52.
[30] Ripamonti U, Reddi AH. Growth and morphogenetic factors in bone induction: Roleof osteogenin and related bone morphogenetic proteins in craniofacial and periodontalbone repair. Crit Rev Oral Biol Med.1992,3(1-2):1-14.
[1] Burwell R. Studies in the transplantation of bone: VII. The fresh compositehomograft-autograft of cancellous bone: an analysis of factors leading to osteogenesis inmarrow transplants and in marrow containing bone grafts. J Bone Joint Surg [Br],1964，46:110–140.
[2] Younger EM, Chapman MW. Morbidity at bone graft donor site. J Orthop Trauma，1989，3:192–195.
[3] R. S. Bhatnagar, Synthetic compounds and compositions with enhanced cell binding,US Patent5354736,1994.
[4] Yang XB, Bhatnagar RS, Li S, Oreffo RO. Biomimetic collagen scaffolds for humanbone cell growth and differentiation. Tissue Eng，2004，10:1148–1159.
[5] Bhatnagar RS, Qian JJ, Wedrychowska A, Sadeghi M, Wu YM, Smith N. Design ofbiomimetic habitats for tissue engineering with P-15, a synthetic peptide analogue ofcollagen. Tissue Eng,1999,5(1):53–65.
[6] Nguyen H, Qian JJ, Bhatnagar RS, Li S. Enhanced cell attachment and osteoblasticactivity by P-15peptide-coated matrix in hydrogels. Biochem Biophys Res Commun,2003,311(1):179–86.
[7] Qian JJ, Bhatnagar RS. Enhanced cell attachment to anorganic bone mineral in thepresence of a synthetic peptide related to collagen. J Biomed Mater Res,1996,31(4):545–54.
[8] Hanks T, Atkinson BL. Comparison of cell viability on anorganic bone matrix with orwithout P-15cell binding peptide. Biomaterials,2004,25(19):4831–6.
[9] Kubler A, Neugebauer J, Oh JH, Scheer M, Zoller JE. Growth and proliferation ofhuman osteoblasts on different bone graft substitutes: an in-vitro study. Implant Dent,2004,13:171–179.
[10] Qian JJ, Bhatnagar RS. Enhanced cell attachment to anorganic bone mineral in thepresence of a synthetic peptide related to collagen. J Biomed Mater Res,1996,31:545–554.
[11] Barboza EP, de Souza RO, Caula AL, Neto LG, Caula FO, Duarte ME. Boneregeneration of localized chronic alveolar defects utilizing cell binding peptide associatedwith anorganic bovine-derived bone mineral: a clinical and histological study. JPeriodontol,2002,73(10):1153–9.
[12] Tehemar S, Hanes P, Sharawy M. Enhancement of osseointegration of implantsplaced into extraction sockets of healthy and periodontally diseased teeth by using graftmaterial, an ePTFE membrane, or a combination. Clin Implant Dent Relat Res,2003,5(3):193–211.
[13] Scarano A, Iezzi G, Petrone G, Orsini G, Degidi M, Strocchi R, Piattelli A. Corticalbone regeneration with a synthetic cell binding peptide: a histologic andhistomorphometric pilot study. Implant Dent,2003,12(4):318–24.
[14] Frederic Shapiro. Bone development and its relation to fracture repair. The role ofmesenchymal osteoblasts and surface osteoblasts. European Cells and Materials,2008,15:53-76.
[15] Nguyen H, Qian JJ, Bhatnagar RS, Li S. Enhanced cell attachment and osteoblasticactivity by P-15peptide-coated matrix in hydrogels. Biochem Biophys Res Commun.2003,311(1):179-86.
[16] Gomar F, Orozco R, Villar JL, Arrizabalaga F. P-15small peptide bone graftsubstitute in the treatment of non-unions and delayed union. A pilot clinical trial. IntOrthop,2007,31:93–99.
[17] Hanks T, Atkinson BL. Comparison of cell viability on anorganic bone matrix with orwithout P-15cell binding peptide. Biomaterials,2004,25:4831–4836.
[18] Kubler A, Neugebauer J, Oh JH, Scheer M, Zoller JE. Growth and proliferation ofhuman osteoblasts on different bone graft substitutes: an in vitro study. Implant Dent,2004,13:171–179.
[19] Cheung SS, Tofe AJ. Mechanism of cell growth on calcium phosphate particles: roleof cell-mediated dissolution of calcium phosphate matrix. STP Pharm Sci,1993,3:51–55.
[20] Callan DP, Rohrer MD. Use of bovine-derived hydroxyapatite in the treatment ofedentulous ridge defects: a human clinical and histologic case report. J Periodontol,1993,64:575–582.
[21] Zaner DJ, Yukna RA. Particle size of periodontal bone grafting materials. JPeriodontol,1984,55:406–409.
[22] Thorwarth M, Schultze-Mosgau S, Wehrhan F, Srour S, Wiltfang J, Neukam FW,Schlegel KA Enhanced bone regeneration with a synthetic cell-binding peptide–in vivoresults. Biochem Biophys Res Commun,2005,329:789–795.
[23] Scarano A, Iezzi G, Petrone G, Orsini G, Degidi M, Strocchi R, Piattelli A. Corticalbone regeneration with a synthetic cell-binding peptide: a histologic andhistomorphometric pilot study. Implant Dent.2003,12(4):318-24.
[24] DeLustro F, Dasch J, Keefe J, Ellingsworth L. Immune responses to allogeneic andxenogeneic implants of collagen and collagen derivatives. Clin Orthop Relat Res,1990,260:263–279.
[1] De Long Jr WG, Einhorn TA, Koval K, McKee M, Smith W, Sanders R, et al. Bonegrafts and bone graft substitutes in orthopaedic trauma surgery. A critical analysis. J BoneJt Surg Am2007,89(3):649-58.
[2] Finkemeier CG. Bone-grafting and bone-graft substitutes. J Bone Jt Surg Am,2002,84(3):454-64.
[3] Ahlmann E, Patzakis M, Roidis N, Shepherd L, Holtom P. Comparison of anterior andposterior iliac crest bone graft in terms of harvest-site morbidity and functional outcomes. JBone Jt Surg Am,2002,84(5):716-20.
[4] St John TA, Vaccaro AR, Sah AP, Schaefer M, Berta SC, Albert T, et al. Physical andmonetary costs associated with autogenous bone graft harvesting. Am J Orthop,2003,32(1):18-23.
[5] Greenwald AS, Boden SD, Goldberg VM, Khan Y, Laurencin CT, Rosier RN,American Academy of Orthopaedic Surgeons. The committee on biological implants.Bone-graft substitutes: facts, fictions, and applications. Bone Jt Surg Am,2001,83(Suppl.2Pt2):98-103.
[6] Jahangir AA, Nunley RM, Mehta S, Sharan A, the Washington Health Policy Fellows.Bone-graft substitutes in orthopaedic surgery. AAOS Now,2008.
[7] Desai BM. Osteobiologics. Am J Orthop (Belle Mead NJ),2007,36(4Suppl.):8-11.
[8] Giannoudis PV, Dinopoulos H, Tsiridis E. Bone substitutes: an update. Injury,2005,36:S20-7.
[9] Einhorn TA. Enhancement of fracture healing. J Bone Jt Surg Am,1995,77:940-56.
[10] Dimitriou R, Tsiridis E, Giannoudis PV. Current concepts of molecular aspects ofbone healing. Injury,2005,36(12):1392-404.
[11] Keramaris NC, Calori GM, Nikolaou VS, Schemitsch EH, Giannoudis PV. Fracturevascularity and bone healing: a systematic review of the role of VEGF. Injury,2008,39(Suppl.2):S45-57.
[12] Bielby R, Jones E, McGonagle D. The role of mesenchymal stem cells in maintenanceand repair of bone. Injury,2007,38(Suppl.1): S26-32.
[13] Laurencin C, Khan Y, El-Amin SF. Bone graft substitutes. Expert Rev Med Devices2006,3(1):49-57.
[14] Bauer TW, Muschler GF. Bone graft materials: an overview of the basic science. ClinOrthop Relat Res,2000,371:10-27.
[15] Khan SN, Cammisa Jr FP, Sandhu HS, Diwan AD, Girardi FP, Lane JM. The biologyof bone grafting. J Am Acad Orthop Surg,2005,13:77-86.
[16] Sen MK, Miclau T. Autologous iliac crest bone graft: should it still be the goldstandard for treating nonunions? Injury,2007,38(Suppl.1): S75-80.
[17] Horne LT, Murray PM, Saha S, Sidhar K. Effects of distal radius bone graft harveston the axial compressive strength of the radius. J Hand Surg Am,2010,35(2):262-6.
[18] Krause JO, Perry CR. Distal femur as a donor site of autogenous cancellous bone graft.J Orthop Trauma,1995,9(2):145-51.
[19] Mazock JB, Schow SR, Triplett RG. Proximal tibia bone harvest: review of technique,complications, and use in maxillofacial surgery. Int J Oral Maxillofac Implants2004;19(4):586-93.
[20] Raikin SM, Brislin K. Local bone graft harvested from the distal tibia or calcaneus forsurgery of the foot and ankle. Foot Ankle Int,2005,26(6):449-53.
[21] Cox G, Jones E, McGonagle D, Giannoudis PV. Reamerirrigator-aspirator indicationsand clinical results: a systematic review. Int Orthop2011[Epub ahead of print].
[22] Dell PC, Burchardt H, Glowczewskie Jr FP. A roentgenographic, biomechanical, andhistological evaluation of vascularized and non-vascularized segmental fibular canineautografts. J Bone Jt Surg Am,1985,67(1):105-12.
[23] Connolly JF. Injectable bone marrow preparations to stimulate osteogenic repair. ClinOrthop Relat Res,1995,313:8-18.
[24] Curylo LJ, Johnstone B, Petersilge CA, Janicki JA, Yoo JU. Augmentation of spinalarthrodesis with autologous bone marrow in a rabbit po-sterolateral spine fusion model.Spine,1999,24:434-8.
[25] Pountos I, Georgouli T, Kontakis G, Giannoudis PV. Efficacy of minimally invasivetechniques for enhancement of fracture healing: evidence today. Int Orthop,2010,34(1):3-12.
[26] Hernigou P, Poignard A, Beaujean F, Rouard H. Percutaneous autologousbone-marrow grafting for nonunions. Influence of the number and concentration ofprogenitor cells. J Bone Jt Surg Am,2005,87(7):1430-7.
[27] Ja¨ ger M, Herten M, Fochtmann U, Fischer J, Hernigou P, Zilkens C, et al. Bridgingthe gap: bone marrow aspiration concentrate reduces autologous bone grafting in osseousdefects. J Orthop Res,2011,29(2):173-80.
[28] Tiedeman JJ, Garvin KL, Kile TA, Connolly JF. The role of a composite, DBM andbone marrow in the treatment of osseous defects. Orthopedics,1995,18:1153-8.
[29] D’Ippolito G, Schiller PC, Ricordi C, Roos BA, Howard GA. Agerelated osteogenicpotential of mesenchymal stromal stem cells from human vertebral bone marrow. J BoneMiner Res,1999,14(7):1115-22.
[30] Huibregtse BA, Johnstone B, Goldberg VM, Caplan AI. Effect of age and samplingsite on the chondro-osteogenic potential of rabbit marrow-derived mesenchymal progenitorcells. J Orthop Res,2000,18(1):18-24.
[31] Farrington M, Matthews I, Foreman J, Richardson KM, Caffrey E. Microbiologicalmonitoring of bone grafts: two the surgeon10(2012)230-239237237years’ experience ata tissue bank. J Hosp Infect,1998,38:261-71.
[32] Kappe T, Cakir B, Mattes T, Reichel H, Flo¨ren M. Infections after bone allograftsurgery: a prospective study by a hospital bone bank using frozen femoral heads fromliving donors. Cell Tissue Bank,2010,11(3):253-9.
[33] Mankin HJ, Hornicek FJ, Raskin KA. Infection in massive bone allografts. ClinOrthop Relat Res,2005,432:210-6.
[34] Buck BE, Malinin TI, Brown MD. Bone transplantation and humanimmunodeficiency virus. An estimate of risk of acquired immunodeficiency syndrome(AIDS). Clin Orthop Relat Res,1989,240:129-36.
[35] Centers for Disease Control and Prevention. Hepatitis C virus transmission from anantibody-negative organ and tissue donor, United States,2000-2002. Morb Mortal WklyRep,2003,52:273-6.
[36] Katz JM, Nataraj C, Jaw R, Deigl E, Bursac P. Demineralized bone matrix as anosteoinductive biomaterial and in vitro predictors of its biological potential. J BiomedMater Res B, Appl Biomater,2009,89:127-34.
[37] Peterson B, Whang PG, Iglesias R, Wang JC, Lieberman JR. Osteoinductivity ofcommercially available demineralized bone matrix. Preparations in a spine fusion model. JBone Jt Surg Am,2004,86:2243-50.
[38] Wang JC, Alanay A, Mark D, Kanim LEA, Campbell PA, Dawson EG, et al. Acomparison of commercially available demineralized bone matrix for spinal fusion. EurSpine J,2007,16:1233-40.
[39] Docquier PL, Delloye C. Treatment of aneurysmal bone cysts by introduction ofdemineralized bone and autogenous bone marrow. J Bone Jt Surg Am,2005,87:2253-8.
[40] Tynan JR, Schachar NS, Marshall GB, Gray RR. Pathologic fracture through aunicameral bone cyst of the pelvis: CTguided percutaneous curettage, biopsy, and bonematrix injection. J Vasc Interv Radiol,2005,16:293-6.
[41] Dinopoulos HT, Giannoudis PV. Safety and efficacy of use of demineralised bonematrix in orthopaedic and trauma surgery. Expert Opin Drug Saf,2006,5(6):847-66.
[42] Urist MR. Bone: formation by autoinduction. Science,1965,150(698):893-9.
[43] Giannoudis PV, Einhorn TA. Bone morphogenetic proteins in musculoskeletalmedicine. Injury2009;40(Suppl.3):S1-3.
[44] Nauth A, Giannoudis PV, Einhorn TA, Hankenson KD, Friedlaender GE, Li R, et al.Growth factors: beyond bone morphogenetic proteins. J Orthop Trauma,2010,24(9):543-6.
[45] Simpson AH, Mills L, Noble B. The role of growth factors and related agents inaccelerating fracture healing. J Bone Jt Surg Br,2006,88(6):701-5.
[46] Dallari D, Savarino L, Stagni C, Cenni E, Cenacchi A, Fornasari PM, et al. Enhancedtibial osteotomy healing with use of bone grafts supplemented with platelet gel or plateletgel and bone marrow stromal cells. J Bone Jt Surg Am,2007,89:2413-20.
[47] Tang CH, Yang RS, Huang TH, Liu SH, Fu WM. Enhancement of fibronectinfibrillogenesis and bone formation by basic fibroblast growth factor via protein kinasec-dependent pathway in rat osteoblasts. Mol Pharmacol,2004,66:440-9.
[48] Geiger F, Lorenz H, Xu W, Szalay K, Kasten P, Claes L, et al. VEGF producing bonemarrow stromal cells (BMSC) enhance vascularization and resorption of a natural coralbone substitute. Bone,2007,41:516-22.
[49] Argintar E, Edwards S, Delahay J. Bone morphogenetic proteins in orthopaedictrauma surgery. Injury,2011,42(8):730-4.
[50] Blokhuis TJ. Formulations and delivery vehicles for bone morphogenetic proteins:latest advances and future directions. Injury,2009,40(Suppl.3):S8-11.
[51] Alsousou J, Thompson M, Hulley P, Noble A, Willett K. The biology of platelet-richplasma and its application in trauma and orthopaedic surgery: a review of the literature. JBone Jt Surg Br,2009,91(8):987-96.
[52] Lowery GL, Kulkarni S, Pennisi AE. Use of autologous growth factors in lumbarspinal fusion. Bone,1999,25(2Suppl.):47-50.
[53] Jenis LG, Banco RJ, Kwon B. A prospective study of Autologous Growth Factors(AGF) in lumbar interbody fusion. Spine J,2006,6(1):14-20.
[54] Bi L, Cheng W, Fan H, Pei G. Reconstruction of goat tibial defects using an injectabletricalcium phosphate/chitosan in combination with autologous platelet-rich plasma.Biomaterials,2010,31:3201-11.
[55] Hartmann EK, Heintel T, Morrison RH, Weckbach A. Infiuence of platelet-richplasma on the anterior fusion in spinal injuries: a qualitative and quantitative analysis usingcomputer tomography. Arch Orthop Trauma Surg,2010,130:909-14.
[56] Cenni E, Avnet S, Fotia C, Salerno M, Baldini N. Platelet-rich plasma impairsosteoclast generation from human precursors of peripheral blood. J Orthop Res,2010,28:792-7.
[57] Forriol F, Longo UG, Concejo C, Ripalda P, Maffulli N, Denaro V. Platelet-richplasma, rhOP-1(rhBMP-7) and frozen rib allograft for the reconstruction of bonymandibular defects in sheep. A pilot experimental study. Injury,2009,40(Suppl.3): S44-9.
[58] McGonagle D, English A, Jones EA. The relevance of mesenchymal stem cells invivo for future orthopaedic strategies aimed at fracture repair. Curr Orthop2007;21(4):262-7.
[59] Robey PG. Stem cells near the century mark. J Clin Invest,2000,105:1489-91.
[60] Watt FM, Hogan BL. Out of Eden: stem cells and their niches. Science,2000,287:1427-30.
[61] Papathanasopoulos A, Giannoudis PV. Biological considerations of mesenchymalstem cells and endothelial progenitor cells. Injury,2008,39(Suppl.2): S21-32.
[62] Sempuku T, Ohgushi H, Okumura M, Tamai S. Osteogenic potential of allogeneic ratmarrow cells in porous hydroxyapatite ceramics: a histological study. J Orthop Res,2005,14:907-13.
[63] Arinzeh TL, Peter SJ, Archambault MP, van den Bos C, Gordon S, Kraus K, et al.Allogeneic mesenchymal stem cells regenerate bone in a critical-sized canine segmentaldefect. J Bone Jt Surg Am,2003,85:1927-35.
[64] Patterson TE, Kumagai K, Griffith L, Muschler GF. Cellular strategies forenhancement of fracture repair. J Bone Jt Surg Am,2008,90(Suppl.1):111-9.
[65] Cornell CN. Osteoconductive materials and their role as substitutes for autogenousbone grafts. Orthop Clin North Am,1999,30(4):591-8.
[66] Nandi SK, Roy S, Mukherjee P, Kundu B, De DK, Basu D. Orthopaedic applicationsof bone graft&graft substitutes: a review. Indian J Med Res,2010,132:15-30.
[67] Fleming Jr JE, Cornell CN, Muschler GF. Bone cells and matrices in orthopaedictissue engineering. Orthop Clin North Am,2000,31:357-74.
[68] Chen Y. Orthopaedic application of gene therapy. J Orthop Sci,2001,6:199-207.
[69] Lind M, Bu¨ nger C. Orthopaedic applications of gene therapy. Int Orthop,2005,29:205-9.
[70] Calori GM, Donati D, Di Bella C, Tagliabue L. Bone morphogenetic proteins andtissue engineering: future directions. Injury,2009,40(Suppl.3): S67-76.
[71] Bohner M. Calcium orthophosphates in medicine: from ceramics to calciumphosphate cements. Injury,2000,31(Suppl.4):37-47.
[72] Hak DJ. The use of osteoconductive bone graft substitutes in orthopaedic trauma. JAm Acad Orthop Surg,2007,15:525-36.
[73] Balc ik C, Tokdemir T, Senko¨ ylu¨ A, Koc N, Timuc in M, Akin S, et al. Earlyweight bearing of porous HA/TCP (60/40) ceramics in vivo: a longitudinal study in asegmental bone defect model of rabbit. Acta Biomater,2007,3:985-96.
[74] Ghosh SK, Nandi SK, Kundu B, Datta S, De DK, Roy SK, et al. In vivo response ofporous HA and-TCP prepared by aqueous solution combustion method and comparisonwith bioglass scaffolds. J Biomed Mater Res B Appl Biomater,2008,86:217-27.
[75] Noshi T, Yoshikawa T, Ikeuchi M, Dohi Y, Ohgushi H, Horiuchi K, et al.Enhancement of the in vivo osteogenic potential of marrow/hydroxyapatite composites bybovine bone morphogenetic protein. J Biomed Mater Res,2000,52(4):621-30.
[76] Daculsi G. Biphasic calcium phosphate concept applied to artificial bone, implantcoating and injectable bone substitute. Biomaterials,1998,19(16):1473-8.
[77] Moroni A, Pegreffi F, Cadossi M, Hoang-Kim A, Lio V, Giannini S.Hydroxyapatite-coated external fixation pins. Expert Rev Med Devices,2005,2:465-71.
[78] Nguyen HQ, Deporter DA, Pilliar RM, Valiquette N, Yakubovich R. The effect ofsol-gel-formed calcium phosphate coatings on bone ingrowth and osteoconductivity ofporous-surfaced Ti alloy implants. Biomaterials,2004,25:865-76.
[79] Kopylov P, Runnqvist K, Jonsson K, Aspenberg P. Norian SRS versus externalfixation in redisplaced distal radial fractures. A randomized study in40patients. ActaOrthop Scand,1999,70:1-5.
[80] Sanchez-Sotelo J, Munuera L, Madero R. Treatment of fractures of the distal radiuswith remodellable bone cement: a prospective, randomised study using Norian SRS. JBone Jt Surg Br,2000,82:856-63.
[81] Gross V, Brandes J. The ultrastructure of the interface between a glass ceramic andbone. J Biomed Mater Res,1981,15:291-305.
[82] Kinnunen I, Aitasalo K, Pollonen M, Varpula M. Reconstruction of orbital fioorfractures using bioactive glass. J Craniomaxillofac Surg,2000,28:22-34.
[83] Peltola M, Suonpaa J, Aitasalo K, Ma¨a¨ tta¨nen H, Andersson O, Yli-Urpo A, et al.Experimental follow-up model for clinical frontal sinus obliteration with bioactive glass.Acta Otolaryngol Suppl,2000,543:167-9.
[84] Cao W, Hench LL. Bioactive materials. Ceramics Int,1996,22:493-507.
[85] Peters CL, Hines JL, Bachus KN, Craig MA, Bloebaum RD. Biological effects ofcalcium sulphate as a bone graft substitute in ovine metaphyseal defects. J Biomed MaterRes A,2006,76:456-62.
[86] Maeda ST, Bramane CM, Taga R, Garcia RB, de Moraes IG, Bernadineli N.Evaluation of surgical cavities filled with three types of calcium sulphate. J Appl Oral Sci,2007,15:416-9.
[87] Mirzayan R, Panossian V, Avedian R, Forrester DM, Menendez LR. The use ofcalcium sulphate in the treatment of benign bone lesions. A preliminary report. J Bone JtSurg Am,2001,83:355-8.
[88] Chiroff RT, White EW, Weber KN, Roy DM. Tissue ingrowth of replamineformimplants. J Biomed Mater Res,1975,9:29-45.
[89] Coughlin MJ, Grimes JS, Kennedy MP. Coralline hydroxyapatite bone graft substitutein hindfoot surgery. Foot Ankle Int,2006,27(1):19-22.
[90] Giannoudis PV, Einhorn TA, Marsh D. Fracture healing: the diamond concept. Injury,2007,38(Suppl.4): S3-6.
[91] Dimitriou R, Jones E, McGonagle D, Giannoudis PV. Bone regeneration: currentconcepts and future directions. BMC Med,2011,31;9:66.
[92] Brydone AS, Meek D, Maclaine S. Bone grafting, orthopaedic biomaterials, and theclinical need for bone engineering. Proc Inst Mech Eng H,2010,224(12):1329-43.
[93] Aloy-Pro′sper A, Maestre-Ferrin L, Pen arrocha-Oltra D, Pen arrocha-Diago M.Bone regeneration using particulate grafts: an update. Med Oral Patol Oral Cir Bucal,2011,16(2). E210-4.