Electron Beam Melting Manufacturing Technology for Individually Manufactured Jaw Prosthesis: A Case Report.

In the field of maxillofacial reconstruction, additive manufacturing technologies, specifically electron beam melting (EBM), offer clinicians the potential for patient-customized design of jaw prostheses, which match both load-bearing and esthetic demands. The technique allows an innovative, functional design, combining integrated porous regions for bone ingrowth and secondary biological fixation with solid load-bearing regions ensuring the biomechanical performance. A patient-specific mandibular prosthesis manufactured using EBM was successfully used to reconstruct a patient's mandibular defect after en bloc resection. Over a 9-month follow-up period, the patient had no complications. A short operating time, good esthetic outcome, and high level of patient satisfaction as measured by quality-of-life questionnaires-the European Organisation for Research and Treatment of Cancer QLQ-C30 (30-item quality-of-life core questionnaire) and H&N35 (head and neck cancer module)-were reported for this case. Individually planned and designed EBM-produced prostheses may be suggested as a possible future alternative to fibular grafts or other reconstructive methods. However, the role of porosity, the role of geometry, and the optimal combination of solid and porous parts, as well as surface properties in relation to soft tissues, should be carefully evaluated in long-term clinical trials.

Osseointegration of titanium with an antimicrobial nanostructured noble metal coating.

Nanometer scale surface features on implants and prostheses can potentially be used to enhance osseointegration and may also add further functionalities, such as infection resistance, to the implant. In this study, a nanostructured noble metal coating consisting of palladium, gold and silver, never previously used in bone applications, was applied to machined titanium screws to evaluate osseointegration after 6 and 12 weeks in rabbit tibiae and femurs. Infection resistance was confirmed by in vitro adhesion test. A qualitatively and quantitatively similar in vivo bone response was observed for the coated and uncoated control screws, using histology, histomorphometry and electron microscopy. The bone-implant interface analysis revealed an extensive bone formation and direct bone-implant contact. These results demonstrate that the nanostructured noble metal coating with antimicrobial properties promotes osseointegration and may therefore be used to add extra implant functionality in the form of increased resistance to infection without the use of antibiotics. FROM THE CLINICAL EDITOR: The authors of this paper demonstrate that nanostructured noble metal coating of implants and prostheses used in orthopedic procedures promotes osseointegration and may be used to add extra implant functionality in the form of increased resistance to infection without the use of antibiotics.

Integrin and chemokine receptor gene expression in implant-adherent cells during early osseointegration.

The mechanisms of early cellular recruitment and interaction to titanium implants are not well understood. The aim of this study was to investigate the expression of pro-inflammatory cytokines, chemokines and adhesion markers during the first 24 h of implantation. Anodically oxidized and machined titanium implants were inserted in rat tibia. After 3, 12, and 24 h the implants were unscrewed and analyzed with quantitative polymerase chain reaction. Immunohistochemistry and scanning electron microscopy revealed different cell types, morphology and adhesion at the two implant surfaces. A greater amount of cells, as indicated by higher expression of small subunit ribosomal RNA (18S), was detected on the oxidized surface. Higher expression of CXC chemokine receptor-4 (at 12 h) and integrins, alphav (at 12 h), beta1 (at 24 h) and beta2 (at 12 and 24 h) was detected at the oxidized surfaces. Significantly higher tumor necrosis factor-alpha (at 3 h) and interleukin-1beta (at 24 h) expression was demonstrated for the machined surface. It is concluded that material surface properties rapidly modulate the expression of receptors important for the recruitment and adhesion of cells which are crucial for the inflammatory and regenerative processes at implant surfaces in vivo.

A technique for evaluating bone ingrowth into 3D printed, porous Ti6Al4V implants accurately using X-ray micro-computed tomography and histomorphometry.

This paper investigates the application of X-ray micro-computed tomography (micro-CT) to accurately evaluate bone formation within 3D printed, porous Ti6Al4V implants manufactured using Electron Beam Melting (EBM), retrieved after six months of healing in sheep femur and tibia. All samples were scanned twice (i.e., before and after resin embedding), using fast, low-resolution scans (Skyscan 1172; Bruker micro-CT, Kontich, Belgium), and were analysed by 2D and 3D morphometry. The main questions posed were: (i) Can low resolution, fast scans provide morphometric data of bone formed inside (and around) metal implants with a complex, open-pore architecture?, (ii) Can micro-CT be used to accurately quantify both the bone area (BA) and bone-implant contact (BIC)?, (iii) What degree of error is introduced in the quantitative data by varying the threshold values?, and (iv) Does resin embedding influence the accuracy of the analysis? To validate the accuracy of micro-CT measurements, each data set was correlated with a corresponding centrally cut histological section. The results show that quantitative histomorphometry corresponds strongly with 3D measurements made by micro-CT, where a high correlation exists between the two techniques for bone area/volume measurements around and inside the porous network. On the contrary, the direct bone-implant contact is challenging to estimate accurately or reproducibly. Large errors may be introduced in micro-CT measurements when segmentation is performed without calibrating the data set against a corresponding histological section. Generally, the bone area measurement is strongly influenced by the lower threshold limit, while the upper threshold limit has little or no effect. Resin embedding does not compromise the accuracy of micro-CT measurements, although there is a change in the contrast distributions and optimisation of the threshold ranges is required.

Bioactive Spheres: The Way of Treating Dentin Hypersensitivity.

Sealing exposed dental tubules is the most effective and long-term way to relieve the pain induced by dental sensitivity. A bioactive hollow sphere (strontium substituted calcium phosphate) was synthesized and added in toothpaste to study its effect on dental hypersensitivity via tooth tubules occlusion and mineralization. The size of spheres is perfect for penetrating into dental tubules, reaching to 20 µm into the tubules. The exposed dental tubules were occluded by spheres and new apatite layer after 3 days brushing. The spheres attached to the surface of dentin and the mineralized surface contained two layers, a porous layer followed by a dense apatite layer. The porous layer can be dissolved in an acidic solution, but the following dense layer could be kept even after soaking in an acid solution. In conclusion, Sr-substituted calcium phosphate spheres could be a good candidate for at-home treatment of dental hypersensitivity.

Long-term osseointegration of 3D printed CoCr constructs with an interconnected open-pore architecture prepared by electron beam melting.

UNLABELLED: In orthopaedic surgery, cobalt chromium (CoCr) based alloys are used extensively for their high strength and wear properties, but with concerns over stress shielding and bone resorption due to the high stiffness of CoCr. The structural stiffness, principally related to the bulk and the elastic modulus of the material, may be lowered by appropriate design modifications, to reduce the stiffness mismatch between metal/alloy implants and the adjacent bone. Here, 3D printed CoCr and Ti6Al4V implants of similar macro-geometry and interconnected open-pore architecture prepared by electron beam melting (EBM) were evaluated following 26week implantation in adult sheep femora. Despite higher total bone-implant contact for Ti6Al4V (39±4%) than CoCr (27±4%), bone formation patterns were similar, e.g., densification around the implant, and gradual ingrowth into the porous network, with more bone in the outer half (periphery) than the inner half (centre). Raman spectroscopy revealed no major differences in mineral crystallinity, the apatite-to-collagen ratio, or the carbonate-to-phosphate ratio. Energy dispersive X-ray spectroscopy showed similar Ca/P ratio of the interfacial tissue adjacent to both materials. Osteocytes made direct contact with CoCr and Ti6Al4V. While osteocyte density and distribution in the new-formed bone were largely similar for the two alloys, higher osteocyte density was observed at the periphery of the porous network for CoCr, attributable to slower remodelling and a different biomechanical environment. The results demonstrate the possibility to achieve bone ingrowth into open-pore CoCr constructs, and attest to the potential for fabricating customised osseointegrated CoCr implants for load-bearing applications. STATEMENT OF SIGNIFICANCE: Although cobalt chromium (CoCr) based alloys are used extensively in orthopaedic surgery, stress shielding due to the high stiffness of CoCr is of concern. To reduce the stiffness mismatch between CoCr and bone, CoCr and Ti6Al4V implants having an interconnected open-pore architecture were prepared by electron beam melting (EBM). After six months of submerged healing in sheep, both alloys showed similar patterns of bone formation, with densification around the implant and gradual ingrowth into the porous network. The molecular and elemental composition of the interfacial tissue was similar for both alloys. Osteocytes made direct contact with both alloys, with similar overall osteocyte density and distribution. The work attests to the potential for achieving osseointegration of EBM manufactured porous CoCr implants.

Monocyte viability on titanium and copper coated titanium.

The role of apoptosis/cell death in the inflammatory response at the implanted materials is unexplored. Two surfaces with different cytotoxic potential and in vivo outcomes, titanium (Ti) and copper (Cu) were incubated in vitro with human monocytes and studied using a method to discriminate apoptotic and necrotic cells (Annexin V/PI staining). Further, staurosporine, a potent inducer of apoptosis, was added to the surface adherent monocytes. Lactate dehydrogenase (a marker of cell membrane injury) and TNF-alpha and IL-10, cytokines, previously suggested to play a major role in the monocyte apoptosis, were assayed in the culture medium. The results demonstrated that Ti surfaces displayed enhanced monocyte survival and production of IL-10 and TNF-alpha. Cu adherent cells exhibited apoptotic signs as early as 1h after incubation. In contrast to Ti, after 48 h the predominance of apoptotic cells switched to apoptotic/necrotic cells on Cu surfaces. Staurosporine treatment of Ti adherent cells mediated similar type of cell death. LDH and cytokine contents were low around Cu surfaces, partly explained by interference between Cu ions and LDH and cytokines. This study suggests that material properties rapidly influence the onset of human monocyte apoptosis and progression to late apoptosis/necrosis. Early detection of apoptosis and cell death may be important for the understanding of the biological response to implanted materials.

In vivo cytokine secretion and NF-kappaB activation around titanium and copper implants.

The early biological response at titanium (Ti), copper (Cu)-coated Ti and sham sites was evaluated in an in vivo rat model. Material surface chemical and topographical properties were characterized using Auger electron spectroscopy, energy dispersive X-ray spectroscopy and interferometry, respectively. The number of leukocytes, cell types and cell viability (release of lactate dehydrogenase) were determined in the implant-interface exudate. The contents of activated nuclear transcription factor NF-kappaB, interleukin-6 (IL-6) and interleukin-10 (IL-10) were determined by enzyme linked immunosorbent assay. An increase in the number of leukocytes, in particular, polymorphonuclear leukocytes, was observed between 12 and 48 h around Cu. A marked decrease of exudate cell viability was found around Cu after 48 h. The total amounts of activated NF-kappaB after 12 h was highest in Ti exudates whereas after 48 h the highest amount of NF-kappaB was detected around Cu. The levels of cytokine IL-6 were consistently high around Cu at both time periods. No differences in IL-10 contents were detected, irrespective of material/sham and time. The results show that materials with different toxicity grades (titanium with low and copper with high toxicity) exhibit early differences in the activation of NF-kappaB, extracellular expression and secretion of mediators, causing major differences in inflammatory cell accumulation and death in vivo.

A novel soft tissue model for biomaterial-associated infection and inflammation - bacteriological, morphological and molecular observations.

Infection constitutes a major risk for implant failure, but the reasons why biomaterial sites are more vulnerable than normal tissue are not fully elucidated. In this study, a soft tissue infection model was developed, allowing the analysis of cellular and molecular responses in each of the sub-compartments of the implant-tissue interface (on the implant surface, in the surrounding exudate and in the tissue). Smooth and nanostructured titanium disks with or without noble metal chemistry (silver, gold, palladium), and sham sites, were inoculated with Staphylococcus epidermidis and analysed with respect to number of viable bacteria, number, viability and gene expression of host cells, and using different morphological techniques after 4 h, 24 h and 72 h. Non-infected rats were controls. Results showed a transient inflammatory response at control sites, whereas bacterial administration resulted in higher recruitment of inflammatory cells (mainly polymorphonuclear), higher, continuous cell death and higher gene expression of tumour necrosis factor-alpha, interleukin-6, interleukin-8, Toll-like receptor 2 and elastase. At all time points, S. epidermidis was predominantly located in the interface zone, extra- and intracellularly, and lower levels were detected on the implants compared with surrounding exudate. This model allows detailed analysis of early events in inflammation and infection associated to biomaterials in vivo leading to insights into host defence mechanisms in biomaterial-associated infections.

Enhancement of CRF-PEEK osseointegration by plasma-sprayed hydroxyapatite: A rabbit model.

Carbon-fibre-reinforced polyether ether ketone (CFR-PEEK) exhibits excellent biomechanical properties as it has an elastic modulus similar to bone. However, CFR-PEEK displays inferior biocompatibility compared with titanium alloy and coating techniques are therefore of interest in order to improve integration. In this paper, the early biological response to CFR-PEEK implants, with and without hydroxyapatite coating, was investigated. Furthermore, a hydroxyapatite-coated titanium alloy reference served as a clinically relevant control. The study was conducted in a rabbit model, both in femur trabecular bone as well as in tibia cortical bone. The results demonstrated that an hydroxyapatite coating significantly enhances the bone response to PEEK implants in vivo. Moreover, in cortical bone, hydroxyapatite-coated PEEK implants induced superior bone response compared with hydroxyapatite-coated Ti ones. These results suggest that hydroxyapatite-coated CFR-PEEK is a suitable material for in vivo implantation.

Stability after maxillary segmentation for correction of anterior open bite: a cohort study of 33 cases.

The aim of this cohort study was to evaluate the stability after multi-segmentation of the maxilla for correction of anterior open bite deformities. A total of 33 patients who underwent segmented maxillary osteotomy between 1994 and 2006 were included in the study. Rigid fixation with plates and postsurgical intermaxillary fixation for 6 weeks was applied to each patient. All patients were then followed in a standardized examination procedure at months 6, 12, 18 and up to 30 months postoperatively. Vertical and horizontal relation of the incisors was measured both clinically and on cast models. The main finding was that statistically significant relapse was found vertically, whereas the horizontal relationship to the mandible was unchanged. The vertical relapses were predominantly seen in patients with severe open bite evident preoperatively.

Acute inflammatory response to laser-induced micro- and nano-sized titanium surface features.

BACKGROUND: The inflammatory process induced by implant surfaces is an important component of the tissue response, where limited knowledge is available regarding the role of surface topography. With laser ablation, a combined micro- and nanoscale surface modification could be created, which have been shown to enhance bone growth and biomechanical stability in vivo. PURPOSE: The aim of this article was to evaluate the early in vivo inflammatory response to laser-modified titanium disks, with machined titanium disks and sham operation sites serving as controls. MATERIALS AND METHODS: Circular disks were installed in a subcutaneous rat model for 24 and 72 hours, where the cell number, cell types, and cytokine levels were evaluated. RESULTS: The results revealed that significantly fewer inflammatory cells (mononuclear and polymorphonuclear) were attracted to the sites with the laser-modified implants compared with the machined titanium implants. Similar concentrations of pro-inflammatory cytokines (TNF-a and MCP-1), together with slightly higher cell viability, were observed around the laser-modified surface compared with the machined surface. CONCLUSIONS: The results in the present study suggest that the combination of surface micro and nano features of the laser-treated surface contributes to the downregulation of early inflammatory events.

Gene expression of inflammation and bone healing in peri-implant crevicular fluid after placement and loading of dental implants. A kinetic clinical pilot study using quantitative real-time PCR.

PURPOSE: Early detection of healing complications after placement of dental implants is a pressing but elusive goal. This paper proposes a non-invasive diagnostic tool for monitoring healing- and peri-implant disease specific genes, complementary to clinical evaluations. MATERIAL AND METHODS: Eighteen partially edentulous patients were recruited to this pilot study. Three Brånemark TiUnite® implants/patient (Nobel Biocare) were placed in a one-stage procedure. Abutments with smooth or rough (TiUnite®) surface were placed. The test group (n = 9) received fixed bridges (immediate loading), whereas the control group (n = 9) implants were loaded 3 months after surgery. In addition to clinical measurements, crevicular fluid was collected using paper strips at the implant abutments 2, 14, 28, and 90 days postoperative. mRNA was extracted, purified, and converted to cDNA. Quantitative PCR assays for IL-1ß, TNF-α, Osteocalcin (OC), Alkaline Phosphatase (ALP), Cathepsin K, Tartrate Resistant Acid Phosphatase, and 18S ribosomal RNA were designed and validated. Relative gene expression levels were calculated. RESULTS: One implant was lost in the control group and three in the test group. In one test patient, one implant showed lowered stability after 2 to 4 weeks and was unloaded. Later implant stability improved which allowed for loading after 3 to 4 months. TNF-α and ALP most commonly showed correlation with clinical parameters followed by IL-1ß and OC. The strongest correlation was found for TNF-α with clinical complications at 2 and 14 days (p = .01/r = -048, and p = .0004/r = -0.56, respectively; test and control groups together). In some cases, gene expression predicted clinical complications (TNF-α, ALP, CK). CONCLUSION: This study is based on samples from few individuals; still, some genes showed correlation with clinical findings. Further studies are needed to refine and optimize the sampling process, to find the appropriate panel, and to validate gene expression for monitoring implant healing.

The correlation between gene expression of proinflammatory markers and bone formation during osseointegration with titanium implants.

An in vivo interfacial gene expression model combined with biomechanical analysis was used in order to determine the relationship between the molecular events taking place during osseointegration and the biomechanical stability of the implant. Anodically oxidized and machined, threaded titanium implants were characterized topographically, chemically and ultrastructurally. The implants were inserted in rat tibiae and the implant bone torsion stability was evaluated. After measurements, the implants were removed and analyzed with qPCR. Results showed an increase in the breakpoint torque of 140%, 170% and 190%, after 6, 14, and 28 days, respectively, at the oxidized implants as compared to the machined. Gene expression analysis revealed higher expression of runt related transcription factor-2 (Runx2) (after 28 d), osteocalcin (OC) and tartrate resistant acid phosphatase (TRAP) (after 6, 14 and 28 d) and cathepsin K (CATK) (after 6 and 14 d) at the oxidized implants. On the other hand, machined implants were associated with higher expression of tumor necrosis factor-α (TNF-α) (after 6 and 28 d) and interleukin-1ß (IL-1ß) (after 6, 14 and 28 d) compared to the oxidized implants. In conclusion, the favorable cellular and molecular events at the oxidized implants were in parallel with significantly stronger bone anchorage during osseointegration.

The influence of bone type on the gene expression in normal bone and at the bone-implant interface: experiments in animal model.

BACKGROUND: Studies on the biological processes in different bone types and the reaction of different bone types to biomaterials are often hindered because of the difficulties in sampling procedures and lack of sensitive techniques. PURPOSE: The purpose was to assess the suitability of quantitative polymerase chain reaction (qPCR) for investigation of the biological differences between cortical and trabecular bone types and their responses to biomaterials. MATERIALS AND METHODS: Gene expression of selected markers in rat bone samples from different locations was evaluated. Samples were harvested by trephines from the trabecular femoral epiphysis, cortico-trabecular proximal tibial metaphysic, and the cortical distal tibial metaphysis. Gene expression was also evaluated at the surfaces of anodically oxidized implants retrieved from cortical and trabecular sites after 3 days of implantation. mRNA in the bone samples and in the tissue associated with the implant surfaces was extracted and quantified using qPCR. Tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), alkaline phosphatase (ALP), osteocalcin (OC), tartrate-resistant acid phosphatase (TRAP), cathepsin K (CATK), and 18S ribosomal subunits (18S) were analyzed. RESULTS: In the bone samples, higher expression of ALP, OC, TRAP, and CATK was found in femoral epiphysis compared to proximal or distal tibial metaphysis, indicating a higher turnover in the trabecular bone. On the other hand, TNF-α and IL-1ß showed higher expression in both tibia sites compared with the femur site, which suggests higher inflammatory potential in the cortical bone. In response to the oxidized implants trabecular bone expressed a higher level of IL-1ß, whereas the implants in cortical bone were associated with higher expression of ALP and OC. CONCLUSION: There are biological differences between cortical and trabecular bone types, both in the normal steady-state condition and in response to biomaterials. Such differences can be characterized and discriminated quantitatively using a sensitive technique such as qPCR.

In vivo evaluation of noble metal coatings.

A nanotopographic noble metal (Ag, Au, Pd) coating has been applied on commercial urinary catheters and used in more than 80,000 patients, with good clinical results. We have previously evaluated the biocompatibility of different variations of this coating, showing high cellular viability and function in vitro. However, the reasons for good clinical and preclinical behavior are not known. This in vivo study aimed to investigate the soft tissue peri-implant reaction to five coatings with systematically altered noble metal ratios after 1, 3, and 21 days of implantation in rats. The results show that coatings of silver only, or silver with medium amounts of gold and low-medium palladium content were superior to other tested coatings. Such surfaces were during the first days after implantation associated with a decreased recruitment of inflammatory cells to implant close exudates, a lower percentage of neutrophils, higher cell viability, and lower production of monocyte chemoattractant protein-1 (MCP-1), compared to the other coatings and uncoated silicone (PDMS) control. In contrast, the addition of higher concentrations of gold and palladium to silver induced a thicker soft tissue capsule. Coatings with high concentration of palladium induced the thickest fibrouscapsule after 21 days of implantation. The study demonstrates that by varying the noble metal ratio at implant surfaces it is possible to modulate inflammation and fibrosis in soft tissue.

In vivo gene expression in response to anodically oxidized versus machined titanium implants.

A quantitative polymerase chain reaction technique (qPCR) in combination with scanning electron microscopy was applied for the evaluation of early gene expression response and cellular reactions close to titanium implants. Anodically oxidized and machined titanium miniscrews were inserted in rat tibiae. After 1, 3, and 6 days the implants were unscrewed and the surrounding bone was retrieved using trephines. Both the implants and bone were analyzed with qPCR. A greater amount of cells, as indicated with higher expression of 18S, was detected on the oxidized surface after 1 and 6 days. Significantly higher osteocalcin (at day 6), alkaline phosphatase (at days 3 and 6), and cathepsin K (at day 3) expression was demonstrated for the oxidized surface. Higher expression of tumor necrosis factor-alpha (at day 1) and interleukin-1beta (at days 1 and 6) was detected on the machined surfaces. SEM revealed a higher amount of mesenchymal-like cells on the oxidized surface. The results show that the rapid recruitment of mesenchymal cells, the rapid triggering of gene expression crucial for bone remodeling and the transient nature of inflammation, constitute biological mechanisms for osseointegration, and high implant stability associated with anodically oxidized implants.

Fibrous capsule formation around titanium and copper.

Previous studies suggest that implant material properties influence the quality and quantity of fibrous capsule around the implant. However, the precise relation between material surface chemistry, early inflammatory response, and fibrous subsequent repair outcome is still unknown. Titanium (Ti) and copper (Cu), surfaces with different inflammatory potential, were implanted subcutaneously in rats and retrieved fibrous capsules were analyzed after 28 and 56 days. Histological examinations show pronounced differences in capsule morphology. The fibrous capsule around Ti was thinner than that around Cu, with less number of the inflammatory cells in the layer close to the implant surface, and less and smaller blood vessels. The capsule around Cu was thick, with a large number of the inflammatory cells, particularly macrophages and giant cells, and increased number of blood vessels. Our study suggests that material surface properties, which initiate early, multiple cellular inflammatory events, are also associated with increased fibrosis and angiogenesis during repair phase.