Zywicka Bogusława, Czarny Anna, Zaczyńska Ewa, Karaś Joanna
Zakład Chirurgii Eksperymentalnej, i Badania Biomateriałów AM, Wrocławiu.
Polim Med. 2006;36(3):23-35.
Ceramic materials based on calcium carbonate have been prepared in response for the demand for resorbable materials for use in bone surgery. Calcium carbonate in the form of crystalline aragonite or calcite with various amount of lithium fluoride was the raw material. Material CC-1FA from crystalline aragonite (99% CaCO3 and 1% LiF), CC-5FA - from crystalline aragonite (95% CaCO3 and 5% LiF) and CC-1FK -material from crystalline calcite (99% CaCO3 and 1% LiF) was studied. To evaluate their biocompatibility and inflammatory effect we investigated the activity of nuclear factor kappaB (NF-kappaB) and proinflammatory cytokine concentrations: tumor necrosis factor TNF-alpha, interleukins 11-6 and IL-8 in peripheral human leukocytes (PBL) after stimulation in vitro with tested calcite materials. Evaluation of local soft tissue reaction after implantation was also the aim of our study. Proinflammatory cytokines take part in inflammatory reaction caused by biomaterials. Expression of these proteins is controlled by proinflammatory regulatory transcription factors including the commonly appearing NF-kappaB (Nuclear Factor kappaB). In a quiescent cell NF-kappaB resides in the cytosol in an inactive form which its activated under the influence of kinases. The activated NF-kappaB protein translocates from cytosol to nucleus of cell and binding to specific DNA sequence it initiates transcriptions. Thanks to the quick regulation of immunological response it ensures the survival of cells in unfavorable reactions of environmental factors. It regulates the expression of many genes mainly connected with the course of the inflammatory process (of some cytokines, proteins of acute phase, collagenasis, stromilozine other enzymes decomposing the elements of matrix) and with proliferation and differentiating of cells. However its excessive activity can lead to unfavorable reactions, for example uncontrollable division of cells, appearance of giant cells of foreign body type. In our study protein expression of NF-kappaB in PBL were assessed using anti-c-Rel-antibody (PBL expressing c-Rel in the nucleus = labelled NF-kappaB (+) cells). The NF-kappaB activation in PBL was expressed as: the percentage of NF-kappaB (+) cells. The level of cytokines: IL-6, IL-8 and TNF-alpha level in the supernatants from leukocytes culture with tested materials was determined by an enzyme-linked immunoabsorbent assay (ELISA) after 24 and 72 hours incubation. The local tissue reaction in vivo was evaluated 1 and 3 months after implantation calcite devices into dorsal muscles of rats. The achieved results showed that the tested calcite material incubated 24 and 72 hours with PBL culture didn't synthesized higher amounts of the cytokines IL-6 and IL-8 in comparison with the untreated cells. All tested materials stimulated after 24 and 72 hours PBL culture to produce a significant level of TNF-alpha (p < 0.05) (higher level after CC-5FA and CC-1FA materials and lower after CC-1FK). Activation of transcription factor nuclear factor NF-kappaB in leukocytes after 24 hours incubation with CC-5FA, CC-1FA was higher than CC-1FK but significantly was only after CC-5FA stimulating. The expression of NF-kappaB after 72 hours of incubation decreased and was on the level comparable with control group. Histological observation 1 month after implantation showed that the carbonate ceramic devices were surrounded with connective tissue surrounding implants capsule with presence of macrophages. Additionally after 3 month in the dense connective tissue cartilaginous and calcification focus was visible. Soft tissue reaction showed chronic inflammatory reaction connected with resorbtion of biomaterials, the most increased after implantation of CC-5FA, than CC-1FA and CC-1FK.
Transcription nuclear factor NF-kappaB was activated after in vitro stimulation in short time by the tested calcite material CC-5FA. This activity was correlated with induction in vitro of TNF-alpha in short and long time. The most increased soft tissue reaction 1 and 3 month after implantation of CC-5FA material was found. Our study showed that calcite materials can activate NF-kappaB and demonstrated differences in biocompatibility of tested materials.
基于碳酸钙的陶瓷材料是为满足骨外科可吸收材料的需求而制备的。以结晶文石或方解石形式存在的碳酸钙与不同量的氟化锂作为原料。研究了来自结晶文石的材料CC - 1FA（99% CaCO₃和1% LiF）、CC - 5FA（95% CaCO₃和5% LiF）以及来自结晶方解石的材料CC - 1FK（99% CaCO₃和1% LiF）。为评估它们的生物相容性和炎症效应，我们在体外用人外周血白细胞（PBL）与测试的方解石材料刺激后，研究了核因子κB（NF - κB）的活性以及促炎细胞因子浓度：肿瘤坏死因子TNF - α、白细胞介素11 - 6和IL - 8。本研究的目的还包括评估植入后局部软组织反应。促炎细胞因子参与生物材料引起的炎症反应。这些蛋白质的表达受促炎调节转录因子控制，包括常见的NF - κB（核因子κB）。在静止细胞中，NF - κB以无活性形式存在于细胞质中，在激酶的影响下被激活。激活的NF - κB蛋白从细胞质转移到细胞核，并与特定DNA序列结合，启动转录。由于免疫反应的快速调节，它确保细胞在环境因素的不利反应中存活。它调节许多基因的表达，主要与炎症过程（一些细胞因子、急性期蛋白、胶原酶、基质金属蛋白酶及其他分解基质成分的酶）以及细胞增殖和分化有关。然而，其过度活性可能导致不良反应，例如细胞的不可控分裂、异物型巨细胞的出现。在我们的研究中，使用抗c - Rel抗体评估PBL中NF - κB的蛋白表达（细胞核中表达c - Rel的PBL = 标记的NF - κB(+)细胞）。PBL中NF - κB的激活表示为：NF - κB(+)细胞的百分比。用测试材料培养白细胞24小时和72小时后，通过酶联免疫吸附测定（ELISA）测定上清液中细胞因子IL - 6、IL - 8和TNF - α的水平。将方解石装置植入大鼠背部肌肉1个月和3个月后评估体内局部组织反应。所获得的结果表明，与未处理细胞相比，测试的方解石材料与PBL培养物孵育24小时和72小时后，未合成更高量的细胞因子IL - 6和IL - 8。所有测试材料在培养PBL 24小时和72小时后均刺激产生显著水平的TNF - α（p < 0.05）（CC - 5FA和CC - 1FA材料刺激后水平较高，CC - 1FK刺激后水平较低）。与CC - 1FK相比，CC - 5FA、CC - 1FA与白细胞孵育24小时后转录因子核因子NF - κB的激活更高，但仅在CC - 5FA刺激后显著。孵育72小时后NF - κB的表达下降，与对照组水平相当。植入1个月后的组织学观察表明，碳酸盐陶瓷装置被结缔组织包围，植入物周围有巨噬细胞。此外，3个月后在致密结缔组织中可见软骨和钙化灶。软组织反应显示与生物材料吸收相关的慢性炎症反应，CC - 5FA植入后反应增加最多，其次是CC - 1FA和CC - 1FK。
测试的方解石材料CC - 5FA在体外短时间刺激后激活了转录核因子NF - κB。这种活性与短时间和长时间体外诱导TNF - α相关。发现CC - 5FA材料植入1个月和3个月后软组织反应增加最多。我们的研究表明方解石材料可激活NF - κB，并证明了测试材料在生物相容性方面的差异。
