골 결합 개선을 위한 티타늄의 구리 도핑 미세 다공성 코팅에 대한 조골 세포 반응

토론

금속 티타늄 및 그 합금은 우수한 기계적 특성과 생체 적합성으로 인해 치과, 성형 외과 및 기타 분야에서 널리 사용됩니다. 그러나 임플란트로서의 티타늄은 뼈 조직과 수동적으로만 결합할 수 있습니다. 이 조합은 종종 기계적 조합으로 임플란트가 느슨해지거나 가라앉기 쉬워 임플란트 실패로 이어집니다. 현재 임플란트 표면 개질 방법은 골유착 능력을 향상시키기 위해 주로 사용된다[19]. 이상적인 임플란트의 표면은 골전도성과 골유도성이 모두 있어야 하고 생체적합성이 좋아야 하며 임플란트와 골조직 사이의 골유착 형성이 촉진되어야 한다[20]. 이 연구에서 우리는 혁신적인 미세 다공성 Cu–TiO₂를 준비했습니다. 티타늄 표면에 코팅하여 티타늄의 생물학적 활성과 생체 적합성을 향상시키고 현재 임상 응용 분야에서 티타늄 임플란트의 단점을 극복하기를 희망합니다.

구리 이온과 이산화티타늄은 생물학적 활성이 좋은 것으로 입증되었습니다[21]. 이 연구에서 미세다공성 Cu–TiO₂ 티타늄 표면의 마이크로아크 산화에 의해 제조된 코팅은 코팅과 티타늄 기판 사이의 긴밀한 결합의 가장 큰 장점을 보여 문헌에서 확인된 바 있다[22]. 마이크로아크 산화 피막의 우수한 결합력은 형성 과정과 밀접한 관련이 있습니다. 마이크로아크 산화 과정에서 화학적 산화, 전기 화학적 산화 및 플라즈마 산화가 공존합니다. 아크 방전에 의해 발생하는 순간적인 고온 고압의 작용하에 티타늄 표면은 기판 표면에서 "성장" 방식으로 기판 산화물을 중심으로 성장합니다. 세라믹 코팅, 코팅 및 기질 송곳니는 엇갈리고 결합력이 좋습니다[23].

생물학적 물질의 표면 특성은 물질의 생물학적 특성에 직접적인 영향을 미칩니다. 마이크로아크 산화 코팅은 전자현미경에서 거칠고 다공성인 표면 형태를 나타냅니다. 형태는 주로 서로 침투하는 다양한 크기의 미세 기공으로 구성됩니다. 이러한 작은 기공은 마이크로아크 산화 과정에서 형성되고, 고전압에서 금속 표면이 파괴되며, 마이크로아크 영역에서의 순간적인 고온 소결은 티타늄 매트릭스를 결정질 세라믹 상 구조를 갖는 세라믹 필름으로 직접 산화 및 소결시킨다. , 전기 고장이 발생하는 곳. 전자현미경으로 관찰된 미세기공이 형성된다. These rough and porous structures can not only increase the attachment area of tissue cells, but these interpenetrating micropores are also equivalent to a three-dimensional scaffold structure, which can induce bone tissue to grow into the pores and promote cell adhesion and extension. Pan et al. [24] prepared micro/nanohierarchical structured TiO₂ coatings on polished titanium by micro-arc oxidation and found that the coatings were favorable for the adhesion and extension of MG63 cells. Zhang et al. [25] prepared a Si–TiO₂ coating by micro-arc oxidation, and further study showed that the adhesion of MC3T3-E1 cells on this silicon-containing TiO₂ coating was significantly higher than that on a Si-free TiO₂ coating and pure Ti.

The greatest advantage of the microarc oxidation coating is that the ions in the electrolyte solution can be introduced into the coating during the microarc oxidation process. In this study, the EDS analysis results of the coating surface showed that the microporous Cu–TiO₂ coating is mainly composed of Cu, Ca, P, O and Ti elements, of which titanium comes from the matrix, calcium and phosphorus come from the basic electrolyte solution, and the copper ions in the electrolyte are deposited in the coating along with the formation of the ceramic film. The calcium and phosphorus components on the surface of the implant can not only improve the surface properties of the material but also induce bone formation. In addition to calcium and phosphorus, the copper ions in the microporous Cu–TiO₂ coating have good biocompatibility and biological activity. Copper-doped coatings on the surface of implants have also been reported in the literature. Astasov-Frauenhoffer et al. [26] deposited copper on Ti via a spark-assisted anodization method and confirmed that the viability of the bacterial cells was strongly inhibited. Zong et al. [27] combined anodization and magnetron sputtering to combine copper into TiO₂ nanotubes and prepare copper (Cu) into TiO₂ NTAs (Cu–Ti–O NTAs), and further study showed that Cu–Ti–O NTAs have excellent long-term antibacterial ability and favorable angiogenic activity.

Biocompatibility is the minimum requirement for measuring implants and is also the basic guarantee for implant safety. In this study, biologically active copper was introduced into the surface of titanium implants through microarc oxidation; however, copper ions, as heavy metal ions, have potential toxicity. Therefore, we must consider whether the microporous Cu–TiO₂ coating is cytotoxic. In this study, live/dead cell staining was used to evaluate the microporous Cu–TiO₂ coating. The results showed no obvious cytotoxicity on the surface of the microporous Cu–TiO₂ coating on the titanium surface, and good cell compatibility was observed. We speculate that this finding may be related to the low copper content of the coating. Huang et al. [28] fabricated gap-bridging chitosan–gelatin nanocomposite coatings incorporated with different amounts of copper (Cu; 0.01, 0.1, 1, and 10 mM for Cu I, II, III, and IV groups, respectively) on Ti and demonstrated that the activities of bone marrow stromal cells were not impaired on Cu-doped coatings except for the Cu IV group.

Cell adhesion and proliferation are the basis of implant osseointegration in the later stage. The more cells that adhere and proliferate on the surface of the implant, the better the effect of implant-bone interface osseointegration. The results of this study showed that on the first day after the material surface was inoculated, the amount of cell adhesion on the surface of the samples of each group differed, and the amount of adhesion on the surface of the microporous Cu–TiO₂ coating group increased significantly. The number of cells that adhered to the sample surface gradually increased, but the number of cells that adhered to the group with microporous Cu–TiO₂ coating was significantly greater than that of the other two groups. The difference was statistically significant, indicating that the microporous Cu–TiO₂ coating was doped with copper ions. A porous, rough surface is most conducive to cell adhesion. Similar to cell adhesion, cell proliferation on the surface of each group of materials also showed similar results. Our research results are similar to previous reports [29].

In addition to adhesion and proliferation, the degree of cell differentiation on the surface of the material can further reflect the performance of the implant's osseointegration. The osteogenic differentiation marker genes ALP , BMP , RUNX2 , OCN and COL-I can reflect cell differentiation. In this study, as time went by, the expression of BMP, OCN, ALP and COL-I on the surface of each group of samples increased, but the expression of the microporous Cu–TiO₂ coating group was significantly higher than that of the control group. This finding is closely related to the promotion of osteogenic differentiation by copper ions. Komarova et al. [30] prepared Zn- and Cu-containing CaP-based coatings by microarc oxidation on Ti and showed that low amounts of Cu and Zn in the coatings promoted high motility of human adipose-derived multipotent mesenchymal stromal cells and subsequent ability to differentiate into osteoblasts.

Osseointegration is the key to the success or failure of the implant. This means that there is no fibrous tissue between the implant and the bone tissue. There is direct contact between the implant and the bone tissue, and it can directly bear stress to realize the relationship between the implant and the bone tissue, establishing a functional connection. The osseointegration between orthopedic implants and bone tissue is affected by many factors, such as the initial stability of the implant and the mechanical properties of the implant material, implant surface properties, biocompatibility, biological activity and the condition of the surrounding bone tissue [31].

An ideal implant position and a stable biomechanical environment are the prerequisite and basis for the osseointegration of the implant-bone interface. In this study, the femoral condyle was chosen to be implanted with a copper-doped microporous coating because the abundant blood supply and sufficient bone volume at the femoral condyle can provide a good anatomical basis and a relatively stable mechanical environment for the implant. Moreover, the femoral condyle is mostly cancellous bone. After implantation, bone formation and the effect of implant-bone interface osseointegration can be more intuitively evaluated.

Micro-CT is currently a common method for observing the osteogenesis performance of implants and is also an effective method for evaluating the osseointegration between implants and bone tissue. Bone microstructure is visualized through three-dimensional reconstruction and the region of interest (ROI) analysis with the assistance of related software to obtain the relevant parameters of new bone tissue. Among all the parameters, BV/TV represents the total amount of bone formation and is an important indicator reflecting the osseointegration of the implant. In this study, we chose BV/TV as the detection index. Four weeks after implantation, the BV/TV of the microporous Cu–TiO₂ coating group was higher than that of the control group. Eight weeks after implantation, the BV/TV values of the microporous Cu–TiO₂ coating group and the control group were higher than those at 4 weeks, and the BV/TV of the microporous Cu–TiO₂ coating group was higher than that of the control group. On the basis of micro-CT detection, we performed histological observation and quantitative analysis of the bone tissue around the implant through hard tissue slices. The results of VG staining showed that the microporous Cu–TiO₂ coating group formed more new bone than the control group, and the new bone that formed around it was in direct contact with the internal implant without fibrous tissue infiltration. These results indicate that microporous Cu–TiO₂ coating on the titanium surface can promote the osseointegration of titanium implants. This finding is similar to previous in vitro studies. The rough, porous structure produced by microarc oxidation mimics the micro/nanostructure of normal bone tissue. More importantly, biologically active copper ions promote bone tissue regeneration. Under the action of these common factors, bone tissue regeneration on the surface of the implant is promoted. Our research results are consistent with literature reports. Milan et al. [32] designed a multifunctional Cu/a-C:H thin coating deposited on Ti–6Al–4 V alloy (TC4) via magnetron sputtering and found that the coating composition can stimulate angiogenesis and osteogenesis and control the host response, thereby increasing the success rate of implants.