A Complex Evaluation of the In-Vivo Biocompatibility and Degradation of an Extruded ZnMgSr Absorbable Alloy Implanted into Rabbit Bones for 360 Days

The increasing incidence of trauma in medicine brings with it new demands on the materials used for the surgical treatment of bone fractures. Titanium, its alloys, and steel are used worldwide in the treatment of skeletal injuries. These metallic materials, although inert, are often removed after the injured bone has healed. The second-stage procedure—the removal of the plates and screws—can overwhelm patients and overload healthcare systems. The development of suitable absorbable metallic materials would help us to overcome these issues. In this experimental study, we analyzed an extruded Zn-0.8Mg-0.2Sr (wt.%) alloy on a rabbit model. From this alloy we developed screws which were implanted into the rabbit tibia. After 120, 240, and 360 days, we tested the toxicity at the site of implantation and also within the vital organs: the liver, kidneys, and brain. The results were compared with a control group, implanted with a Ti-based screw and sacrificed after 360 days. The samples were analyzed using X-ray, micro-CT, and a scanning electron microscope. Chemical analysis revealed only small concentrations of zinc, strontium, and magnesium in the liver, kidneys, and brain. Histologically, the alloy was verified to possess very good biocompatibility after 360 days, without any signs of toxicity at the site of implantation. We did not observe raised levels of Sr, Zn, or Mg in any of the vital organs when compared with the Ti group at 360 days. The material was found to slowly degrade in vivo, forming solid corrosion products on its surface.

Effect of the Acid-Etching on Grit-Blasted Dental Implants to Improve Osseointegration: Histomorphometric Analysis of the Bone-Implant Contact in the Rabbit Tibia Model

Previous studies have shown that the most reliable way to evaluate the success of an implant is by bone-to-implant contact (BIC). Recent techniques allow modifications to the implant surface that improve mechanical and biological characteristics, and also upgrade osseointegration. Objective: The aim was to evaluate the osseointegration in rabbit tibia of two different titanium dental implant surfaces: shot-blasted with Al2O3 (SB) and the same treatment with an acid-etching by immersion for 15 s in HCl/H2SO4 (SB + AE). Material and methods: Roughness parameters (Ra, Rt, and Rz) were determined by white light interferometer microscopy. Surface wettability was evaluated with a contact angle video-based system using water, di-iodomethane, and formamide. Surface free energy was determined by means of Owens and Wendt equations. Scanning electron microscopy equipped with X-ray microanalysis was used to study the morphology and determine the chemical composition of the surfaces. Twenty-four grade 4 titanium dental implants (Essential Klockner®) were implanted in the rabbit’s tibia, 12 for each surface treatment, using six rabbits. Six weeks later the rabbits were sacrificed and the implants were sent for histologic analysis. Resonance frequency analysis (RFA) was recorded both at the time of surgery and the end of the research with each device (Osstell Mentor and Osstell ISQ). Results: The roughness measurements between the two treatments did not show statistically significant differences. However, the effect of the acid etching made the surface slightly more hydrophilic (decreasing contact angle from 74.7 for SB to 64.3 for SB + AE) and it presented a higher surface energy. The bone-to-implant contact ratio (BIC %) showed a similar tendency, with 55.18 ± 15.67 and 59.9 ± 13.15 for SB and SB + AE implants, respectively. After 6 weeks of healing, the SB + AE showed an implant stability quotient (ISQ) value of 76 ± 4.47 and the shot-blasted one an ISQ value of 75.83 ± 8.44 (no statistically significant difference). Implants with different surface properties had distinctive forms of behavior regarding osseointegration. Furthermore, the Osstell system was an invasive and reliable method to measure implant stability. Conclusion: Both surfaces of implants studied showed high osseointegration. The SB and SB + AE implants used in our study had similar behavior both in terms of BIC values and RFA. The RFA systems in Osstell Mentor and Osstell ISQ confirmed nearly perfect reproducibility and repeatability.

3D-printed Poly-Lactic Co-Glycolic Acid (PLGA) scaffolds in non-critical bone defects impede bone regeneration in rabbit tibia bone

BACKGROUND: An increasing number of bone graft materials are commercially available and vary in their composition, mechanism of action, costs, and indications. OBJECTIVE: A commercially available PLGA scaffold produced using 3D printing technology has been used to promote the preservation of the alveolar socket after tooth extraction. We examined its influence on bone regeneration in long bones of New Zealand White rabbits. METHODS: 5.0-mm-diameter circular defects were created on the tibia bones of eight rabbits. Two groups were studied: (1) control group, in which the bone defects were left empty; (2) scaffold group, in which the PLGA scaffolds were implanted into the bone defect. Radiography was performed every two weeks postoperatively. After sacrifice, bone specimens were isolated and examined by micro-computed tomography and histology. RESULTS: Scaffolds were not degraded by eight weeks after surgery. Micro-computed tomography and histology showed that in the region of bone defects that was occupied by scaffolds, bone regeneration was compromised and the total bone volume/total volume ratio (BV/TV) was significantly lower. CONCLUSION: The implantation of this scaffold impedes bone regeneration in a non-critical bone defect. Implantation of bone scaffolds, if unnecessary, lead to a slower rate of fracture healing.

Evaluation of Vertical Guided Bone Regeneration Using a Particulate Form of Experimental Bioactive Glass in a Rabbit: A Case Report with Literature Review

Background: There are a large number of different types of bone-grafting materials that are used for the regeneration of atrophied alveolar ridges in order to make dental implantation possible. However, available surgical techniques and materials for bone augmentation do not contribute to the achievement of the desired reliable results and require a search for new solutions to an existing problem. A group of synthetic osteoplastic materials based on bioactive glass (BAG) may become a matter of choice in bone tissue regeneration because of special osteogenic properties. The aim of this study was to visually and histologically evaluate the behavior of an experimental BAG in rabbit tibia bone samples, which were collected from the animal 6 weeks after filling the bone defects. Methods and Results: The observation was carried out on one outbred rabbit whose tibia bone defects were filled with an experimental osteoplastic material based on the BAG. The chemical composition of the experimental osteoplastic material included SiO2 (41%), Na2O (21%), CaO (28.5%), P2O5 (6%), CaF2 (1.5%), MgO (1%), Al2O3 (1%). For histological analysis, H&E staining of paraffin-embedded tissues was performed according to the standard technique. Light microscopy of tissue samples was performed using a Leitz HM-LUX microscope (Germany). Six weeks after filling the bone defects, a strong bond between the augmented hard tissue and rabbit tibia was recognized. Also, a dense fusion of adjacent soft tissues with a newly formed bone without signs of chronic inflammation or graft particles in granular tissue was noted. Microscopic examination of the stained sections showed the presence of mature viable BT with a uniform distribution of osteocytes. Also, residual fragments of the degraded biomaterial surrounded by the fibers of a woven bone were revealed in several slices. Conclusion: In accordance with the results of this experiment, it can be concluded that the usage of BAG related to the system SiO2(41%)-Na2O(21%)-CaO(28.5%)-P2O5(6%)-CaF2(1.5%)-MgO(1%)-Al2O3(1%) may increase the volume of bone without application of barrier membrane. However, further research involving more animals needs to be done to estimate the scientific significance of the obtained data and to evaluate the mechanical properties of augmented bone.

Octacalcium Phosphate Bone Substitute (Bontree®): From Basic Research to Clinical Case Study

Bone grafts used in alveolar bone regeneration can be categorized into autografts, allografts, xenografts, and synthetic bones, depending on their origin. The purpose of this study was to evaluate the effect of a commercialized octacalcium phosphate (OCP)-based synthetic bone substitute material (Bontree®) in vitro, in vivo, and in clinical cases. Material characterization of Bontree® granules (0.5 mm and 1.0 mm) using scanning electron microscopy and X-ray diffraction showed that both 0.5 mm and 1.0 mm Bontree® granules were uniformly composed mainly of OCP. The receptor activator of NF-κB ligand (RANKL) and alkaline phosphatase (ALP) activities of MG63 cells were assessed and used to compare Bontree® with a commercial biphasic calcium phosphate ceramic (MBCP+TM). Compared with MBCP+TM, Bontree® suppressed RANKL and increased ALP activity. A rabbit tibia model used to examine the effects of granule size of Bontree® grafts showed that 1.0 mm Bontree® granules had a higher new bone formation ability than 0.5 mm Bontree® granules. Three clinical cases using Bontree® for ridge or sinus augmentation are described. All eight implants in the three patients showed a 100% success rate after 1 year of functional loading. This basic research and clinical application demonstrated the safety and efficacy of Bontree® for bone regeneration.

Assessment of seawater bacterial infection in rabbit tibia by Illumina MiSeq sequencing and bacterial culture

Objectives We aimed to explore the bacterial community composition following ocean bacterial infection using an animal model. Methods This animal-based experiment was conducted from September 2019 to November 2019. Eighteen seawater filter membranes were collected from Changle City, Fujiian Province, China, on September 8, 2019. Ten filter membranes were used for implantation. Eight filter membranes that were used in the bacterial culture for the exploration of seawater bacteria were assigned to the seawater group (SG). Fourteen healthy adult New Zealand rabbits were randomly divided into the experimental group (EG) and control group (CG). Seawater filter membranes and asepsis membranes were implanted into the tibia in the EG and CG, respectively. One week after surgery, tibial bone pathology tissues were collected and assessed using light microscopy and scanning electron microscopy (SEM). Medullary cavity tissues were collected for the performance of Illumina MiSeq sequencing and bacterial culture. The differences between EG and CG were assessed by pathological observation under light microscopy and SEM, high-throughput bacterial sequencing, and bacterial culture. Results Compared with the CG, the infection rate was 100%, and the mortality value was 20% after the implantation of the filter membranes in the EG. Both light microscopy and SEM showed that a large number of bacteria were distributed in the bone marrow cavity after ocean bacterial infection. No bacterial growth was found in the CG. Illumina MiSeq sequencing found that Firmicutes, Proteobacteria, Thermotogae, Fusobacteria, Bacteroidetes, and Actinobacteria were the dominant bacteria at the phylum level and Clostridium_sensu_stricto_7, Haloimpatiens, Clostridium_sensu_stricto_15, Clostridiaceae_1, Clostridium_sensu_stricto_18, and Oceanotoga were the dominant bacteria in genus level among the EG. In the bacterial culture of the medullary cavity tissues, Klebsiella pneumoniae, Shewanella algae, Staphylococcus aureus, Escherichia coli, Enterobacter cloacae, and Vibrio vulnificus were the predominant infective species. Moreover, compared with the SG, the EG showed a higher detection rate of E. coli and S. aureus (P = 0.008 and P = 0.001, respectively). The detection rates of V. alginolyticus, V. parahaemolyticus, and V. fluvialis were higher in the SG than the EG (P = 0.007, P = 0.03, and P = 0.03, respectively). Conclusions Our model, which was comprehensively evaluated using four techniques: histopathology and SEM observation, gene detection, and bacteria culture, provides a scientific basis for the clinical diagnosis and treatment of patients in such settings.

Porous matrix nanometric scaffold repairs rabbit tibia injury by promoting bone marrow mesenchymal stem cell osteogenic differentiation and up-regulating type I and II collagen content

To explore the effect of porous matrix nanometer scaffold on Bone marrow mesenchymal stem cell osteogenic differentiation and type I and II collagen content in rabbit tibial injury. 22 rabbits, weighing 2.20–3.10 kg, were equally divided into model and treatment groups. Von Kossa was used to detect osteogenic differentiation, while Western blot detected type I and type II collagen. The bone density meter measurement software was used to assess tibial bone density. Universal material testing machine was used to detect biomechanical indicators level, while HE staining detected bone morphology. Adherent Cells growth were observed in a week; where cells clusters appeared and continued to grow 2 weeks later, and the number of osteoblasts and mineralized nodules increased significantly 3 weeks later. The collagen level in the treatment group was significantly higher than that in model group (P < 0.05). The tibia bone mineral density in model group was significantly lower at the sixth and twelfth weeks after surgery (P < 0.05). The maximum bending load and bending stiffness of model group were significantly lower than those of treatment group with higher damage degree (P < 0.05). Treatment increased the bone tissue and osteoblasts, leading to production of trabecular bone structure. No clustered regenerated bone-like tissues in model group were observed. The porous matrix nanometer scaffold can repair rabbit tibia injury possibly by promoting Bone marrow mesenchymal stem cell osteogenic differentiation and increasing type I and type II collagen level.

The impact of surface treatment in 3-dimensional printed implants for early osseointegration: a comparison study of three different surfaces

Abstract3D printing technology has been gradually applied to various areas. In the present study, 3D-printed implants were fabricated with direct metal laser sintering technique for a dental single root with titanium. The 3D implants were allocated into following groups: not treated (3D-None), sandblasted with a large grit and acid-etched (3D-SLA), and target-ion-induced plasma-sputtered surface (3D-TIPS). Two holes were drilled in each tibia of rabbit, and the three groups of implants were randomly placed with a mallet. Rabbits were sacrificed at two, four, and twelve weeks after the surgery. Histologic and histomorphometric analyses were performed for the evaluation of mineralized bone-to-implant contact (mBIC), osteoid-to-implant contact (OIC), total bone-to-implant contact (tBIC), mineralized bone area fraction occupancy (mBAFO), osteoid area fraction occupancy (OAFO), and total bone area fraction occupancy (tBAFO) in the inner and outer areas of lattice structure. At two weeks, 3D-TIPS showed significantly higher inner and outer tBIC and inner tBAFO compared with other groups. At four weeks, 3D-TIPS showed significantly higher outer OIC than 3D-SLA, but there were no significant differences in other variables. At twelve weeks, there were no significant differences. The surface treatment with TIPS in 3D-printed implants could enhance the osseointegration process in the rabbit tibia model, meaning that earlier osseointegration could be achieved.

Correlation between two-dimensional micro-CT and histomorphometry for assessment of the implant osseointegration in rabbit tibia model

Background Histology is considered as a gold standard for analyzing bone architecture. However, histomorphometry is a destructive method and only offers the bone information of a limited location. Micro-computed tomography (μCT) is a non-destructive technology and provides a slice at any site. The aim of this study was to compare the correlation of the Bone-to-Implant Contact ratio (BIC) between 2D micro-CT (μCT) and histomorphometry and to investigate a method for assessing the osseointegration of the implant by 2D μCT. Methods A total of 18 implants were divided into three groups (6 implants per group), and inserted into the rabbit tibia defects as follow: implant only (Implant group), implant with β-TCP/hydrogel (TCP group), implant with rhBMP-2 loaded β-TCP/hydrogel composite (BMP-2 group). After 4 weeks of implantation, the specimens were collected to take the micro-CT scan with an aluminum filter and performed H&E staining on the undecalcified sections. The 2D μCT slices were chosen at an angle of 0°, 45°, 90° and 135° with the representative histological section to measure BIC. And the correlations between BICs of 2D μCT and BICs of histology were evaluated. Results In each group, BICs at the same sites measured by histomorphometry and corresponding 2D μCT presented the same trend and shown no significant difference between the two methods (P > 0.05). BICs of histological sections and BICs of corresponding 2D μCT slices presented a strong correlation in the implant group (γ = 0.74, P = 0.09), a moderate correlation in the TCP group (γ = 0.46, P = 0.35), a weak correlation in the BMP-2 group (γ = 0.30, P = 0.56). In the implant group, the relationship between BIC-Mean-μCTs and BICs-Histology has presented a significant linear correlation (γ = 0.84, P = 0.04). Conclusions Integrating bone information of several 2D μCT slices in different sites to measure BIC is a feasible method for assessing the implant osseointegration.

Effect of mesoporous silica and its combination with hydroxyapatite on the regeneration of rabbit’s bone defects: A pilot study

BACKGROUND: Bone volume augmentation is a routine technique used in oral implantology and periodontology. Advances in the surgical techniques and the biomaterials field have allowed a greater accessibility to these treatments. Nevertheless, dehiscence and fenestrations incidence during dental implant procedures are still common in patients with bone loss. OBJECTIVE: The main objective is to evaluate in a pilot experimental study the biological response to mesoporous silica (MS) hybrid scaffolds and its regenerative capacity in different formulations. METHODS: Two defects per rabbit tibia were performed (one for control and other for test) and the biomaterials tested in this study have been used to fill the bone defects, prepared in two different formulations (3D hybrid scaffolds or powdered material, in 100% pure MS form, or 50% MS with 50% hydroxyapatite (HA). Euthanasia was performed 4 months after surgery for bone histopathological study and radiographic images were acquired by computerized microtomography. RESULTS: Results showed that radiographically and histopathologically pure MS formulations lead to a lower biological response, e.g when formulated with HA, the osteogenic response in terms of osteoconduction was greater. CONCLUSIONS: We observed tolerance and lack of toxicity of the MS and HA, without registering any type of local or systemic allergic reaction.