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Xenogeneic bone grafting materials
异种骨移植材料
Author: Dr Mike Barbeck, Dr Ronald Unger, Prof. Dr Frank Witte, Prof. Dr Sabine Wenisch & Prof. Dr Dr Reiner Schnettler, Germany
翻译者：西瓜柚子
Nowadays, a variety of bone substitutes are available for the clinical user. Interestingly, these materials significantly differ regarding their raw materials or manufacturing processes. As an alternative to autologous bone tissue (autograft), which is still applied as “gold standard” due to its extensive regenerative properties, bone substitutes from other natural sources become more and more relevant in regenerative dentistry. These bone substitute materials are either derived from human (allograft) or animal origin (xenograft).
如今，临床用户可以选择多种骨替代材料。有趣的是，这些材料在原料或制造过程方面有很大的不同。作为自体骨组织（自体移植）的替代品，自体骨组织由于其广泛的再生特性仍然被应用为“金标准”，来自其他天然来源的骨替代材料在再生牙科中变得越来越重要。这些骨替代材料要么来自人类（同种异体移植），要么来自动物（异种移植）。
图1 cerabone®颗粒保留有松质骨结构
图2,3 cerabone®颗粒的表面微结构，显示了天然微结构的保留和基于无细胞成骨细胞小室的纯化状态。cerabone®颗粒的横截面(微CT)，显示了异种骨移植材料完成纯化后保留有层状天然结构。
In case of these materials, the obtained bony extracellular matrix based on calcium phosphates should finally serve as bone substitute (Figs. 1–3). Based on the physicochemical similarity of this class of bone substitutes to the autologous bone tissue, it can be assumed that these materials are the ideal choice for osseous regeneration. Preferentially, bovine bone is used as source tissue in the daily dental practice, as in case of the two primarily applied bone substitute materials Bio-Oss™ and cerabone®.
在这些材料的情况下，所得到的基于磷酸钙的骨质细胞外基质最终应该作为骨替代材料（图1-3）。基于这类骨替代材料与自体骨组织在物理化学上的相似性，可以假设这些材料是骨再生的理想选择。在日常牙科实践中，牛骨是作为源组织的首选，就像两种主要应用的骨替代材料Bio-Oss™和cerabone®一样。
Safety aspects and purification processes
安全性方面和纯化过程
For the clinical application of bone substitutes from natural sources it is inalienable to purify the donor tissue from immunogens to guarantee a regeneration process without complications such as rejections or disease transmissions. To ensure the safe application of such bone substitute materials, different purification steps of the donor tissue are applied.
对于来自天然来源的骨替代材料的临床应用，必须将供体组织从免疫原中纯化，以保证一个没有并发症（如排斥或疾病传播）的再生过程。为了确保这类骨替代材料的安全应用，对供体组织进行了不同的纯化步骤。
The first step is the suitable selection of donor animals before the initiation of the purification process. Hence, for the production of Bio-Oss™ and cerabone® bovine femoral heads from registered suppliers located in Australia and New Zealand are processed as both countries are recognised to have a negligible BSE risk according to the World Organisation for Animal Health (OIE). Afterwards, complex purification steps including both chemical and physical methods are applied for a complete purification. However, those methods are occasionally discussed because of possible rejection reactions or a transfer of pathogens while applying bone grafting materials. In this context, the temperature treatment for the purification plays a major role. Bio-Oss™ is processed at temperatures of approximately 300 °C, while the bone substitute material cerabone® is purified by notably higher temperatures of up to 1,250 °C.This difference in temperature seems to be of significant importance for the safe application of xenogeneic bone substitutes.
在纯化过程的启动之前，第一步是合适的选择供体动物。因此，为了生产Bio-Oss™和cerabone®，来自澳大利亚和新西兰的注册供应商提供的牛股骨头被用于加工，因为这两个国家被世界动物卫生组织(OIE)认定为具有可忽略的BSE风险。之后，应用复杂的纯化步骤，包括化学和物理方法，以实现完全纯化。然而，这些方法有时会引起争议，因为在应用骨移植材料时可能会发生排斥反应或病原体的转移。在这方面，纯化过程中的温度处理起着重要作用。Bio-Oss™是在约300°C的温度下加工的，而骨替代材料cerabone®则是在高达1,250°C的温度下纯化的。这种温度差似乎对异种骨替代物的安全应用非常重要。
The purification process of bovine bone tissue was evaluated in a recent review by Kim et al. 3 Interestingly, the authors concluded that the inactivation of prions in Bio-Oss™ is rather based on the applied temperature than a result of the treatment with highly concentrated sodium hydroxide (NaOH). While this chemical process was described as efficient by Wenz et al., the reliability and sensitivity of the used tests were questioned by Kim et al. In this review, the authors describe that prions will only be effectively destroyed by heating up to 1,000 °C for five minutes. Furthermore, the according EU-guidelines for medical devices utilising animal tissues and their derivatives (Part 1: Application of risk management, EN ISO 22442-1), point out that a treatment at temperatures above 800 °C is reducing the risk of the transmission of Transmissible Spongiform Encephalopathies (TSEs) to an acceptable minimum.
Kim等人对牛骨组织的纯化过程进行了评价。有趣的是，作者得出结论，Bio-Oss™中朊病毒的灭活更多地是基于应用的温度，而不是高浓度氢氧化钠(NaOH)的处理结果。虽然Wenz等人描述了这种化学过程是有效的，但Kim等人质疑了所使用的测试的可靠性和敏感性。在这篇综述中，作者描述了朊病毒只有在加热到1,000°C五分钟后才能有效地被破坏。此外，关于利用动物组织及其衍生物(第1部分：风险管理的应用，EN ISO 22442-1)的医疗器械的欧盟指南指出，在800°C以上的温度下处理可以将传播可传染性海绵状脑病(TSEs)的风险降低到可接受的最低水平。
To assure a maximum level of safety, cerabone® is heated to temperatures above 1,200 °C during processing. Thus, organic parts like cells and proteins are removed and even potentially contained prions and other pathogens are destroyed. Despite the treatment at high temperatures, the natural bone structure is preserved (Figs. 1–3) making cerabone® a safe and reliable product for bone regeneration applications.
为了确保最高水平的安全性，在加工过程中，cerabone®被加热到超过1,200°C的温度。因此，有机部分如细胞和蛋白质被去除，甚至可能含有的朊病毒和其他病原体也被破坏。尽管经过高温处理，天然骨结构仍然保持不变(图1-3)，使cerabone®成为骨再生应用中安全可靠的产品。
Figure 4一个示意图，说明了大多数应用的骨移植材料引起的细胞和炎症过程，植入床血管化过程和骨组织再生过程之间的相关性
Inflammation and bone regeneration
炎症和骨再生
Data from preclinical and clinical studies show comparable values for new bone formation, remaining bone grafting material and connective tissue for both xenogeneic bone substitutes mentioned above (Tab. 1). These results refer to similar biological activities of Bio-Oss™ and cerabone®. However, in case of cerabone® higher numbers of multinucleated giant cells (MNGCs) were found within the first days after its implantation. Furthermore, the comparison to different other studies shows that the initial number of MNGCs in case of cerabone® is significantly lower as found in the implant bed of fast degradable synthetic materials based on tricalcium phosphates. These results confirm several other studies claiming the long-term stability of xenogeneic bone substitutes as it was shown that MNGCs are involved in the biodegradation of bone-grafting materials by phagocytosis.6, 7
来自临床前和临床研究的数据显示，对于上述两种异种骨替代物，新骨形成、残留骨移植材料和结缔组织的值相当(表1)。这些结果表明Bio-Oss™和cerabone®具有类似的生物活性。然而，在cerabone®植入后的最初几天内，发现多核巨细胞(MNGCs)的数量较高。此外，与不同其他研究的比较显示，在cerabone®植入床中发现MNGCs初始数量明显低于磷酸三钙这种快速可降解的合成材料。这些结果证实了其他几项研究的说法，即异种骨替代物的长期稳定性，因为已经证明MNGCs通过吞噬作用参与骨移植材料的生物降解。
Interestingly, the MNGCs were identified as foreign body giant cells (FBGCs) based on their molecule expression. 8 However, more information is still needed to get further conclusion regarding their differentiation. 8, 9 Interestingly, the degradation process of bone substitutes and the process of bone tissue regeneration are closely connected via the relevant cell types such as macrophages and MNGCs (Fig. 4). In this context, it was shown that both macrophages and MNGCs on the one side express pro-inflammatory molecules that are relevant for the degradation process, but also secrete antiinflammatory substances needed for tissue regeneration. 9 One of the most important signaling molecules is the vascular endothelial growth factor (VEGF), which has direct and indirect impact onto different processes important for successful tissue regeneration. 8, 9 Thus, VEGF induces angiogenesis at the implant site, which has indirectly a positive influence on bone tissue growth, and also direct influence on the development and activity of osteoblasts.8,10
有趣的是，根据它们的分子表达，MNGCs被鉴定为异物巨细胞(FBGCs)。然而，还需要更多的信息才能得出更进一步的结论，关于它们的分化。有趣的是，骨替代材料的降解过程和骨组织再生过程通过相关细胞类型如巨噬细胞和MNGCs紧密联系在一起(图4)。在这方面，已经证明，巨噬细胞和MNGCs一方面表达对降解过程有关的促炎分子，但另一方面也分泌对组织再生有必要的抗炎物质。最重要的信号分子之一是血管内皮生长因子(VEGF)，它对成功组织再生的不同过程有直接和间接的影响。因此，VEGF在植入部位诱导血管生成，这对骨组织生长有间接的积极影响，也对成骨细胞的发育和活性有直接影响。
Figure 5 Cerabone ® 产品家族
Tab .1 Bio-Oss™和cerabone®在组织形态学方面的结果，显示了新形成的骨、残留的骨移植材料和结缔组织的相当值
In case of the xenogeneic bone substitute material cerabone®, it can be assumed that the observed higher numbers of MNGCs might have a positive effect on bone regeneration. Interestingly, an initially improved bioactivity for cerabone® combined with a higher vascularisation at the implant site was demonstrated, which might be based on the increased number of MNGCs compared to Bio-Oss™. 2 Thus, an improving effect on bone regeneration could be concluded after the application of cerabone®. In combination with the hydrophilic nature of this material, 1 which has been shown to significantly support the regeneration process by promoting the growth of osteoblasts, cerabone® can be considered as a reliable bone grafting material with an assured safety for both clinical user and patient.
就cerabone®这种异种骨替代材料而言，可以推测观察到的MNGCs数量较高可能对骨再生有积极作用。有趣的是，cerabone®与Bio-Oss™相比，显示出初始改善的生物活性和植入部位更高的血管化，这可能是基于MNGCs数量增加的原因。因此，在应用cerabone®后可以得出对骨再生有改善作用的结论。结合这种材料的亲水性质，1 它已经被证明能够通过促进成骨细胞的生长显著地支持再生过程，cerabone®可以被认为是一种可靠的骨移植材料，为临床用户和患者确保最高可能的安全性。
Summary
总结
Altogether, it can be concluded that the xenogeneic bone substitute material cerabone® is able to ensure the highest possible safety from disease transmission due to the high temperature treatment. Furthermore, it is assumable that the relatively high numbers of multinucleated giant cells express high amounts of antiinflammatory molecules and support a fast and high implant bed vascularisation and therefore, might favour the bone regeneration process.
总之，可以得出结论，异种骨替代材料cerabone®由于高温处理能够确保高安全性，降低疾病传播。此外，MNGCs相对较高的数量表达大量抗炎分子，并支持快速和高效植入部位血管化，因此可能有利于骨再生过程。
附录： 对比异种骨移植物与天然骨成分和结构对比
Note:
1.两种骨移植材料，均不是完全的化学配比的羟基磷灰石材料，钙磷比( Ca/P ratio )在1.75 和1.33之间，钙占主要成分
2.比较低温烧结的Bio-Oss，具有纤维质纹理（Fibrillar texture）[是指细胞或组织中纤维状结构，如细胞骨架或细胞外基质的排列和取向];而两次高温烧结的Cerabone,是致密光滑的表面，并且伴有小颗粒，这个和羟基磷灰石陶瓷一样。
3.晶体尺寸也随着温度的增加，而增加，溶解度降低。很多物理特征随着温度的增加而改变：比表面积，结晶度，晶体粒度，物相组成成分等。
参考文献：
Barbeck M, Unger R, Witte F, et al. Xenogeneic bone grafting materials[J]. Int Mag Oral Implant, 2017, 3: 34-36.
Perić Kačarević Z, Kavehei F, Houshmand A, et al. Purification processes of xenogeneic bone substitutes and their impact on tissue reactions and regeneration[J]. The International journal of artificial organs, 2018, 41(11): 789-800.
