Modification of macroporous titanium tracheal implants with biodegradable structures: tracking in vivo integration for determination of optimal in situ epithelialization conditions

Previously, we showed that macroporous titanium implants, colonized in vivo together with an epithelial graft, are viable options for tracheal replacement in sheep. To decrease the number of operating steps, biomaterial-based replacements for epithelial graft and intramuscular implantation were developed in the present study. Hybrid microporous PLLA/titanium tracheal implants were designed to decrease initial stenosis and provide a surface for epithelialization. They have been implanted in New Zealand white rabbits as tracheal substitutes and compared to intramuscular implantation samples. Moreover, a basement membrane like coating of the implant surface was also designed by Layer-by-Layer (LbL) method with collagen and alginate. The results showed that the commencement of stenosis can be prevented by the microporous PLLA. For determination of the optimum time point of epithelialization after implantation, HPLC analysis of blood samples, C-reactive protein (CRP), and Chromogranin A (CGA) analyses and histology were carried out. Following 3 weeks the implant would be ready for epithelialization with respect to the amount of tissue integration. Calcein-AM labeled epithelial cell seeding showed that after 3 weeks implant surfaces were suitable for their attachment. CRP readings were steady after an initial rise in the first week. Cross-linked collagen/alginate structures show nanofibrillarity and they form uniform films over the implant surfaces without damaging the microporosity of the PLLA body. Human respiratory epithelial cells proliferated and migrated on these surfaces which provided a better alternative to PLLA film surface. In conclusion, collagen/alginate LbL coated hybrid PLLA/titanium implants are viable options for tracheal replacement, together with in situ epithelialization.     

Cell cultivation on porous titanium implants with various structures

The paper presents data on the cultivation of human dermal fibroblasts and rabbit mesenchymal stromal cells on two types of porous titanium implants, i.e., those with irregular pores formed by pressed titanium particles and those with regular pores formed by the cohesion of one-size titanium particles inside the implant. The goal of this study was to determine what type of titanium implant porosity ensured its strongest interaction with cells. Cells were cultivated on implants for 7 days. During this period, they formed a confluent monolayer on the implant surface. Cells grown on titanium implants were monitored by scanning electron microscopy. Fibroblasts interaction with implants depended on the implant porosity structure. On implants with irregular pores cells were more spread. On implants with regular pores fibroblasts enveloped particles and were only occasionally bound with neighboring particles by small outgrowths. There was no tight interaction of particles inside the implant. In implants formed by pressed particles, cells grow not only on surface, but also in the depth of the implant. Thus, we suppose that a tighter interaction of cells with the titanium implant and, supposedly, tissues with the implant in the organism will take place in the variant when the implant structure is formed by pressed titanium particles, i.e., cellular interaction was observed inside the implant. In implants with irregular pores, cells grew both on the surface and in the depth. Thus, cells exhibited more adequate interactions with irregular pore titanium implants in vitro and hopefully the same interaction will be true in tissues after the implantation of the prosthesis into the organism.     

The cultivation of cells on the porous titanium implants with the different structure

The data on human dermal fibroblasts and rabbit mesenchymal stromal cells cultivation on porous titanium implants are presented in given paper. Two types of implants were used: type 1--with irregular pores formed by pressed titanium particles and type 2--with regular pores formed by coalescence of one-size titanium particles into implant. The goal of this study was to choose the type of titanium implant porosity which ensures the tightest interaction of titanium implant with surrounding tissue cells after implantation in the body. Cells were cultivated on implants for 7 days and in both cases they formed confluent monolayer on the implants surfaces. That indicated adhesion, migration and proliferation of cells on such implants. Condition of cells cultured on titanium implants was controlled by scanning electron microscopy. The character of fibroblasts interaction with given implants was different depending on porosity type of implants. On implants with irregular pores, the cells were more spread and overlapped the pores spreading over neighbored particles. On implants with regular pores that formed by one-size particles into implant, the fibroblasts covered these particles not overlapping the pores and seldom interacted with neighbored particles by small outgrowths. There was no tight interaction of particles into implant. In implants formed by pressed particles, the cells grew not only on the surface but also in the depth of implant. Thereby, we suppose that more tight interaction of cells with titanium implant and, supposedly, tissues with implant in an organism will take place in the case of implant structure formed by pressed titanium particles.     

The connective tissue response to Ti, NiCr and AgPd alloys

The aim of the study was to compare the connective tissue response of Lewis rats to Ti, NiCr and AgPd alloys. It was found that implants were covered by collagen-rich, well vascularized capsules. Titanium was covered by the thinnest capsule (57 ± 20 μm) and AgPd alloy was covered by the thickest capsule (239 ± 50 μm). The PCNA+ cell prevalence in the capsules was lower for titanium than for AgPd and NiCr. Mast cells formed a gradient to a depth of 1200 μm only for titanium implants. Cells with brown to black silver granules in the cytoplasm were observed close to AgPd implants. The results suggest that titanium implants induce a weaker connective tissue response than implants made from NiCr and AgPd alloys.     

Does titanium in ionic form display a tissue-specific distribution?

Most studies have focused on the biodistribution of titanium(IV) oxide as nanoparticles or crystals in organism. But several reports suggested that titanium is released from implant in ionic form. Therefore, gaining insight into toxicokinetics of Ti ions will give valuable information, which may be useful when assessing the health risks of long-term exposure to titanium alloy implants in patients. A micro synchrotron radiation-induced X-ray fluorescence (µ-SRXRF) was utilized to investigate the titanium distribution in the liver, spleen and kidneys of rats following single intravenous or 30-days oral administration of metal (6 mg Ti/b.w.) in ionic form. Titanium was mainly retained in kidneys after both intravenous and oral dosing, and also its compartmentalization in this organ was observed. Titanium in the liver was non-uniformly distributed—metal accumulated in single aggregates, and some of them were also enriched in calcium. Correlation analysis showed that metal did not displace essential elements, and in liver titanium strongly correlated with calcium. Two-dimensional maps of Ti distribution show that the location of the element is characteristic for the route of administration and time of exposure. We demonstrated that µ-SRXRF can provide information on the distribution of titanium in internal structures of whole organs, which helps in enhancing our understanding of the mechanism of ionic titanium accumulation in the body. This is significant due to the popularity of titanium implants and the potential release of metal ions from them to the organism.     

Effects of titanium(iv) ions on human monocyte-derived dendritic cells

Orthopaedic metal implants composed of titanium are routinely used in bone fracture repair and for joint replacement therapies. A considerable fraction of implant recipients are unable to benefit due to implant failure resulting from aseptic loosening, while others may experience cutaneous sensitivity to titanium after implantation. An adaptive immune reactivity towards titanium ions, originating from the biocorrosion of the implants, could play a role. As an initiator of the adaptive immune response, dendritic cells (DC) were studied for uptake and characteristics after titanium exposure. Energy filtered transmission electron microscopy showed uptake of titanium(iv) (Ti(iv)) ions by DCs in vitro and co-localisation with phosphorus-rich cell structures of the DC membranes (phospholipids), cytoplasm (ribosomes and phosphorylated proteins) and the nucleus (DNA). DC maturation and function were investigated by measuring cell surface marker expression by flow cytometry. After exposure, DCs showed a decrease in MHC class II (HLA-DR), co-stimulatory molecules (CD40, CD80 & CD86) and chemokine receptors (CCR) 6 and CCR7 but an increase in CCR4 after Ti(iv) treatment. However, Ti(iv) treated DCs had an increased stimulatory capacity towards allogenic lymphocytes. A Ti(iv) concentration dependant increase of IL-12p70 was observed amidst decrease of the other measured cytokines (TGF-β1 and TGF-β2). Hence, Ti(iv) alters DC properties, resulting in an enhanced T lymphocyte reactivity and deviation towards a Th1 type immune response. This effect may be responsible for the inflammatory side effects of titanium implants seen in patients.     

Multi-Scale Modification of Metallic Implants With Pore Gradients,Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.     

Effect of heparin and alendronate coating on titanium surfaces on inhibition of osteoclast and enhancement of osteoblast function

The failure of orthopedic and dental implants has been attributed mainly to loosening of the implant from host bone, which may be due to weak bonding of the implant material to bone tissue. Titanium (Ti) is used in the field of orthopedic and dental implants because of its excellent biocompatibility and outstanding mechanical properties. Therefore, in the field of materials science and tissue engineering, there has been extensive research to immobilize bioactive molecules on the surface of implant materials in order to provide the implants with improved adhesion to the host bone tissue.In this study, chemically active functional groups were introduced on the surface of Ti by a grafting reaction with heparin and then the Ti was functionalized by immobilizing alendronate onto the heparin-grafted surface. In the MC3T3-E1 cell osteogenic differentiation study, the alendronate-immobilized Ti substrates significantly enhanced alkaline phosphatase activity (ALP) and calcium content. Additionally, nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation of RAW264.7 cells was inhibited with the alendronate-immobilized Ti as confirmed by TRAP analysis. Real time PCR analysis showed that mRNA expressions of osteocalcin and osteopontin, which are markers for osteogenesis, were upregulated in MC3T3-E1 cells cultured on alendronate-immobilized Ti. The mRNA expressions of TRAP and Cathepsin K, markers for osteoclastogenesis, in RAW264.7 cells cultured on alendronate-immobilized Ti were down-regulated. Our study suggests that alendronate-immobilized Ti may be a bioactive implant with dual functions to enhance osteoblast differentiation and to inhibit osteoclast differentiation simultaneously.     

Preliminary fabrication and characterization of electron beam melted Ti–6Al–4V customized dental implant

The current study was aimed to fabricate customized root form dental implant using additive manufacturing technique for the replacement of missing teeth. The root form dental implant was designed using Geomagic™ and Magics™, the designed implant was directly manufactured by layering technique using ARCAM A2™ electron beam melting system by employing medical grade Ti–6Al–4V alloy powder. Furthermore, the fabricated implant was characterized in terms of certain clinically important parameters such as surface microstructure, surface topography, chemical purity and internal porosity. Results confirmed that, fabrication of customized dental implants using additive rapid manufacturing technology offers an attractive method to produce extremely pure form of customized titanium dental implants, the rough and porous surface texture obtained is expected to provide better initial implant stabilization and superior osseointegration.     

Spark plasma sintering synthesis of porous nanocrystalline titanium alloys for biomedical applications

The reason for the extended use of titanium and its alloys as implant biomaterials stems from their lower elastic modulus, their superior biocompatibility and improved corrosion resistance compared to the more conventional stainless steel and cobalt-based alloys [Niinomi, M., Hattori, T., Niwa, S., 2004. Material characteristics and biocompatibility of low rigidity titanium alloys for biomedical applications. In: Jaszemski, M.J., Trantolo, D.J., Lewandrowski, K.U., Hasirci, V., Altobelli, D.E., Wise, D.L. (Eds.), Biomaterials in Orthopedics. Marcel Dekker Inc., New York, pp. 41-62]. Nanostructured titanium-based biomaterials with tailored porosity are important for cell-adhesion, viability, differentiation and growth. Newer technologies like foaming or low-density core processing were recently used for the surface modification of titanium alloy implant bodies to stimulate bone in-growth and improve osseointegration and cell-adhesion, which in turn play a key role in the acceptance of the implants. We here report preliminary results concerning the synthesis of mesoporous titanium alloy bodies by spark plasma sintering. Nanocrystalline cp Ti, Ti-6Al-4V, Ti-Al-V-Cr and Ti-Mn-V-Cr-Al alloy powders were prepared by high-energy wet-milling and sintered to either full-density (cp Ti, Ti-Al-V) or uniform porous (Ti-Al-V-Cr, Ti-Mn-V-Cr-Al) bulk specimens by field-assisted spark plasma sintering (FAST/SPS). Cellular interactions with the porous titanium alloy surfaces were tested with osteoblast-like human MG-63 cells. Cell morphology was investigated by scanning electron microscopy (SEM). The SEM analysis results were correlated with the alloy chemistry and the topographic features of the surface, namely porosity and roughness.     

钛及钛合金种植体表面处理的研究进展

钛的优良特性早已被公认为首选牙用种植材料,其表面氧化膜是形成骨结合的关建。为了进一步增加其耐腐蚀性和生物相容性,人们采取了多种方式进行表面处理,最终达到在植入体的不同部位产生不同的粗糙表面,以利于不同细胞在不同地方的附着,增加其生物活性。同时,达到避免各种污染物对纯钛种植体影响的目的。近年来钛种植体材料表面处理已经成为研究热点,本文就表面处理的方法进行综述。     

Soft and hard tissue response to zirconium dioxide dental implants--a clinical study in man

Titanium dental implants have been used successfully in implantology for more than 40 years. Recent research, however, suggests that titanium might have more side effects than previously believed. Zirconia ceramics have been employed in orthopaedic surgery for approximately 30 years and were recently introduced into dentistry as a metal replacement for crown and bridge work as well as implant abutments. Zirconium dioxide has been shown in both in vitro and in vivo studies to have desirable osseointegrative properties. This clinical study shows that dental implants made from zirconia are a feasible alternative to titanium dental implants. In addition to excellent cosmetic results, zirconia implants allow a degree of osseointegration and soft tissue response that is superior to titanium dental implants.     

PHB/PLLA组织工程前交叉韧带支架材料改性的实验研究

目的:探索体外构建组织工程前交叉韧带(anterior cruciate ligament,ACL)的三维支架材料。方法:以聚羟基丁酸已酯/聚左旋乳酸(PHB/PLLA1:1)制备"三明治"样结构共聚物并测量其孔隙率等指标。以I型胶原对制备的PHB/PLLA支架进行杂化,获得PHB/PLLA胶原杂化支架。扫描电镜观察其表面结构。将兔皮肤成纤维细胞(SF)接种于PHB/PLLA支架与PHB/PLLA胶原杂化支架,观察其在材料上生长情况。结果:PHB/PLLA支架杂化后胶原填充于纤维空隙,分布比较均匀。体外培养的胶原杂化支架材料上要比PHB/PLLA支架有更多的皮肤成纤维细胞生长。结论:胶原杂化有利于细胞种植和生长,PHB/PLLA胶原杂化支架具有良好的三维构型和生物相容性,有望为前交叉韧带损伤的修复提供了一种新型的支架材料。     

Modulation of bone ingrowth of rabbit femur titanium implants by in vivo axial micromechanical loading.

Titanium implants commonly used in orthopedics and dentistry integrate into host bone by a complex and coordinated process. Despite increasingly well illustrated molecular healing processes, mechanical modulation of implant bone ingrowth is poorly understood. The objective of the present study was to determine whether micromechanical forces applied axially to titanium implants modulate bone ingrowth surrounding intraosseous titanium implants. We hypothesized that small doses of micromechanical forces delivered daily to the bone-implant interface enhance implant bone ingrowth. Small titanium implants were placed transcortically in the lateral aspect of the proximal femur in 15 New Zealand White rabbits under general anesthesia and allowed to integrate with the surrounding bone for 6 wk. Micromechanical forces at 200 mN and 1 Hz were delivered axially to the right femur implants for 10 min/day over 12 consecutive days, whereas the left femur implants served as controls. The average bone volume 1 mm from mechanically loaded implants (n = 15) was 73 +/- 12%, which was significantly greater than the average bone volume (52 +/- 21%) of the contralateral controls (n = 15) (P < 0.01). The average number of osteoblast-like cells per endocortical bone surface was 55 +/- 8 cells/mm(2) for mechanically loaded implants, which was significantly greater than the contralateral controls (35 +/- 6 cells/mm(2)) (P < 0.01). Dynamic histomorphometry showed a significant increase in mineral apposition rate and bone-formation rate of mechanically stressed implants (3.8 +/- 1.2 microm/day and 2.4 +/- 1.0 microm(3).microm(-2).day(-1), respectively) than contralateral controls (2.2 +/- 0.92 microm/day and 1.2 +/- 0.60 microm(3).microm(-2).day(-1), respectively; P < 0.01). Collectively, these data suggest that micromechanical forces delivered axially on intraosseous titanium implants may have anabolic effects on implant bone ingrowth.     

Dual Action of Lysophosphatidate-Functionalised Titanium: Interactions with Human (MG63) Osteoblasts and Methicillin Resistant Staphylococcus aureus

Titanium (Ti) is a widely used material for surgical implants; total joint replacements (TJRs), screws and plates for fixing bones and dental implants are forged from Ti. Whilst Ti integrates well into host tissue approximately 10% of TJRs will fail in the lifetime of the patient through a process known as aseptic loosening. These failures necessitate revision arthroplasties which are more complicated and costly than the initial procedure. Finding ways of enhancing early (osseo)integration of TJRs is therefore highly desirable and continues to represent a research priority in current biomaterial design. One way of realising improvements in implant quality is to coat the Ti surface with small biological agents known to support human osteoblast formation and maturation at Ti surfaces. Lysophosphatidic acid (LPA) and certain LPA analogues offer potential solutions as Ti coatings in reducing aseptic loosening. Herein we present evidence for the successful bio-functionalisation of Ti using LPA. This modified Ti surface heightened the maturation of human osteoblasts, as supported by increased expression of alkaline phosphatase. These functionalised surfaces also deterred the attachment and growth of Staphylococcus aureus, a bacterium often associated with implant failures through sepsis. Collectively we provide evidence for the fabrication of a dual-action Ti surface finish, a highly desirable feature towards the development of next-generation implantable devices.     

Correlation between vertical misfits and stresses transmitted to implants from metal frameworks

An inappropriate prosthetic fit could cause stress over the interface implant/bone. The objective of this study was to compare stresses transmitted to implants from frameworks cast using different materials and to investigate a possible correlation between vertical misfits and these stresses. Fifteen one-piece cast frameworks simulating bars for fixed prosthesis in a model with five implants were fabricated and arranged into three different groups according to the material used for casting: CP Ti (commercially pure titanium), Co-Cr (cobalt-chromium) or Ni-Cr-Ti (nickel-chromium-titanium) alloys. Each framework was installed over the metal model with all screws tightened to a 10 N cm torque and then, vertical misfits were measured using an optical microscope. The stresses transmitted to implants were measured using quantitative photoelastic analysis in values of maximum shear stress (τ), when each framework was tightened to the photoelastic model to a 10 N cm standardized torque. Stress data were statistically analyzed using one-way ANOVA and Tukey's test and correlation tests were performed using Pearson's rank correlation (α = 0.05). Mean and standard deviation values of vertical misfit are presented for CP Ti (22.40 ± 9.05 μm), Co-Cr (66.41 ± 35.47 μm) and Ni-Cr-Ti (32.20 ± 24.47 μm). Stresses generated by Co-Cr alloy (τ = 7.70 ± 2.16 kPa) were significantly higher than those generated by CP Ti (τ = 5.86 ± 1.55 kPa, p = 0.018) and Ni-Cr-Ti alloy (τ = 5.74 ± 3.05 kPa, p = 0.011), which were similar (p = 0.982). Correlations between vertical misfits and stresses around the implants were not significant as for any evaluated materials.     

Shear stability of an elastomeric disk spacer within an intervertebral joint: a parametric study.

A parametric study was conducted to compare the resistance to shear force provided by three surgical implantation techniques and three endplate designs for use with a polymeric lumbar intervertebral disk spacer. While under an axial load, the implant was pushed out laterally through the surgical window created for implantation. Force at 0.3 mm displacement, slope of the initial portion of the force-displacement graph, and maximum force were measured. The results indicate that implants with pegs, inclined planes, or domes on the surfaces of the disk will add significantly to the shear stability of the implant while maintaining the simplicity of a single part device.     

Interruption of Electrical Conductivity of Titanium Dental Implants Suggests a Path Towards Elimination Of Corrosion

Peri-implantitis is an inflammatory disease that results in the destruction of soft tissue and bone around the implant. Titanium implant corrosion has been attributed to the implant failure and cytotoxic effects to the alveolar bone. We have documented the extent of titanium release into surrounding plaque in patients with and without peri-implantitis. An in vitro model was designed to represent the actual environment of an implant in a patient’s mouth. The model uses actual oral microbiota from a volunteer, allows monitoring electrochemical processes generated by biofilms growing on implants and permits control of biocorrosion electrical current. As determined by next generation DNA sequencing, microbial compositions in experiments with the in vitro model were comparable with the compositions found in patients with implants. It was determined that the electrical conductivity of titanium implants was the key factor responsible for the biocorrosion process. The interruption of the biocorrosion current resulted in a 4–5 fold reduction of corrosion. We propose a new design of dental implant that combines titanium in zero oxidation state for osseointegration and strength, interlaid with a nonconductive ceramic. In addition, we propose electrotherapy for manipulation of microbial biofilms and to induce bone healing in peri-implantitis patients.     

Development and Evaluation of Sustained-Release Etoposide-Loaded Poly(ε-Caprolactone) Implants

Poly(ε-caprolactone) implants containing etoposide, an important chemotherapeutic agent and topoisomerase II inhibitor, were fabricated by a melt method and characterized in terms of content uniformity, morphology, drug physical state, and sterility. In vitro and in vivo drug release from the implants was also evaluated. The cytotoxic activity of implants against HeLa cells was studied. The short-term tolerance of the implants was investigated after subcutaneous implantation in mice. The original chemical structure of etoposide was preserved after incorporation into the polymeric matrix, in which the drug was dispersed uniformly. Etoposide was present in crystalline form in the polymeric implant. In vitro release study showed prolonged and controlled release of etoposide, which showed cytotoxicity activity against HeLa cells. After implantation, good correlation between in vitro and in vivo drug release was found. The implants demonstrated good short-term tolerance in mice. These results tend to show that etoposide-loaded implants could be potentially applied as a local etoposide delivery system.     

A paradigm for the development and evaluation of novel implant topologies for bone fixation: Implant design and fabrication

The future development of bio-integrated devices will improve the functionality of robotic prosthetic limbs. A critical step in the advancement of bio-integrated prostheses will be establishing long-term, secure fixation to the remnant bone. To overcome limitations associated with contemporary bone-anchored prosthetic limbs, we established a paradigm for developing and fabricating novel orthopedic implants undergoing specified loading. A topology optimization scheme was utilized to generate optimal implant macrostructures that minimize deformations near the bone-implant interface. Variations in implant characteristics and interfacial connectivity were investigated to examine how these variables influence the layout of the optimized implant. For enhanced tissue integration, the optimally designed macroscopic geometry of a titanium (Ti)-alloy implant was further modified by introducing optimized microstructures. The complex geometries of selected implants were successfully fabricated using selective laser sintering (SLS) technology. Fabrication accuracy was assessed by comparing volumes and cross-sectional areas of fabricated implants to CAD data. The error of fabricated volume to CAD design volume was less than 8% and differences in cross sectional areas between SEM images of fabricated implants and corresponding cross sections from CAD design were on average less than 9%. We have demonstrated that this computational design method, combined with solid freeform fabrication techniques, provides a versatile way to develop novel orthopedic implants.