Chondroitin/dermatan sulfate glycosyltransferase genes are essential for craniofacial development.

Chondroitin/dermatan sulfate (CS/DS) proteoglycans are indispensable for animal development and homeostasis but the large number of enzymes involved in their biosynthesis have made CS/DS function a challenging problem to study genetically. In our study, we generated loss-of-function alleles in zebrafish genes encoding CS/DS biosynthetic enzymes and characterized the effect on development in single and double mutants. Homozygous mutants in chsy1, csgalnact1a, csgalnat2, chpfa, ust and chst7, respectively, develop to adults. However, csgalnact1a-/- fish develop distinct craniofacial defects while the chsy1-/- skeletal phenotype is milder and the remaining mutants display no gross morphological abnormalities. These results suggest a high redundancy for the CS/DS biosynthetic enzymes and to further reduce CS/DS biosynthesis we combined mutant alleles. The craniofacial phenotype is further enhanced in csgalnact1a-/-;chsy1-/- adults and csgalnact1a-/-;csgalnact2-/- larvae. While csgalnact1a-/-;csgalnact2-/- was the most affected allele combination in our study, CS/DS is still not completely abolished. Transcriptome analysis of chsy1-/-, csgalnact1a-/- and csgalnact1a-/-;csgalnact2-/- larvae revealed that the expression had changed in a similar way in the three mutant lines but no differential expression was found in any of fifty GAG biosynthesis enzymes identified. Thus, zebrafish larvae do not increase transcription of GAG biosynthesis genes as a consequence of decreased CS/DS biosynthesis. The new zebrafish lines develop phenotypes similar to clinical characteristics of several human congenital disorders making the mutants potentially useful to study disease mechanisms and treatment.

The role of Gdf5 in the development of the zebrafish fin endoskeleton.

BACKGROUND: The development of the vertebrate limb skeleton requires a complex interaction of multiple factors to facilitate the correct shaping and positioning of bones and joints. Growth and differentiation factor 5 (Gdf5) is involved in patterning appendicular skeletal elements including joints. Expression of gdf5 in zebrafish has been detected in fin mesenchyme condensations and segmentation zones as well as the jaw joint, however, little is known about the functional role of Gdf5 outside of Amniota. RESULTS: We generated CRISPR/Cas9 knockout of gdf5 in zebrafish and analyzed the resulting phenotype at different developmental stages. Homozygous gdf5 mutant zebrafish displayed changes in segmentation of the endoskeletal disc and, as a consequence, loss of posterior radials in the pectoral fins. Mutant fish also displayed disorganization and reduced length of endoskeletal elements in the median fins, while joints and mineralization seemed unaffected. CONCLUSIONS: Our study demonstrates the importance of Gdf5 in the development of the zebrafish pectoral and median fin endoskeleton and reveals that the severity of the effect increases from anterior to posterior elements. Our findings are consistent with phenotypes observed in the human and mouse appendicular skeleton in response to Gdf5 knockout, suggesting a broadly conserved role for Gdf5 in Osteichthyes.

Developmental low-dose exposure to bisphenol A induces chronic inflammation, bone marrow fibrosis and reduces bone stiffness in female rat offspring only.

BACKGROUND: Developmental exposure to low doses of the endocrine disruptor bisphenol A (BPA) is known to alter bone tissue in young rodents, although how bone tissue is affected in aged animals is not well known. We have recently shown that low-dose developmental exposure to BPA increases procollagen type I N-terminal propeptide (P1NP) levels, a peptide formed during type 1 collagen synthesis, in plasma of 5-week-old female rat offspring while male offspring showed reduced bone size. OBJECTIVE: To analyze offspring bone phenotype at 52 weeks of age and clarify whether the BPA-induced increase in P1NP levels at 5 weeks is an early sign of bone marrow fibrosis development. METHODS: As in our 5-week study, pregnant Fischer 344 rats were exposed to BPA via drinking water corresponding to 0.5 µg/kg BW/day (BPA0.5), which is in the range of human daily exposure, or 50 µg/kg BW/day (BPA50) from gestational day 3.5 until postnatal day 22. Controls were given only vehicle. The offspring were sacrificed at 52 weeks of age. Bone effects were analyzed using peripheral quantitative and micro-computed tomography (microCT), 3-point bending test, plasma markers and histological examination. RESULTS: Compared to a smaller bone size at 5 weeks, at the age of 52 weeks, femur size in male offspring had been normalized in developmentally BPA-exposed rats. The 52-week-old female offspring showed, like the 5-week-old siblings, higher plasma P1NP levels compared to controls but no general increasing bone growth or strength. However, 2 out of 14 BPA-exposed female offspring bones developed extremely thick cortices later in life, discovered by systematic in vivo microCT scanning during the study. This was not observed in male offspring or in female controls. Biomechanical testing revealed that both doses of developmental BPA exposure reduced femur stiffness only in female offspring. In addition, histological analysis showed an increased number of fibrotic lesions only in the bone marrow of female rat offspring developmentally exposed to BPA. In line with this, plasma markers of inflammation, Tnf (in BPA0.5) and Timp1 (in BPA50) were increased exclusively in female offspring. CONCLUSIONS: Developmental BPA exposure at an environmentally relevant concentration resulted in female-specific effects on bone as well as on plasma biomarkers of collagen synthesis and inflammation. Even a dose approximately eight times lower than the current temporary EFSA human tolerable daily intake of 4 µg/kg BW/day, appeared to induce bone stiffness reduction, bone marrow fibrosis and chronic inflammation in female rat offspring later in life.

Compressive fatigue properties of an acidic calcium phosphate cement-effect of phase composition.

Calcium phosphate cements (CPCs) are synthetic bone grafting materials that can be used in fracture stabilization and to fill bone voids after, e.g., bone tumour excision. Currently there are several calcium phosphate-based formulations available, but their use is partly limited by a lack of knowledge of their mechanical properties, in particular their resistance to mechanical loading over longer periods of time. Furthermore, depending on, e.g., setting conditions, the end product of acidic CPCs may be mainly brushite or monetite, which have been found to behave differently under quasi-static loading. The objectives of this study were to evaluate the compressive fatigue properties of acidic CPCs, as well as the effect of phase composition on these properties. Hence, brushite cements stored for different lengths of time and with different amounts of monetite were investigated under quasi-static and dynamic compression. Both storage and brushite-to-monetite phase transformation was found to have a pronounced effect both on quasi-static compressive strength and fatigue performance of the cements, whereby a substantial phase transformation gave rise to a lower mechanical resistance. The brushite cements investigated in this study had the potential to survive 5 million cycles at a maximum compressive stress of 13 MPa. Given the limited amount of published data on fatigue properties of CPCs, this study provides an important insight into the compressive fatigue behaviour of such materials.

Novel Calcium Phosphate Promotes Interbody Bony Fusion in a Porcine Anterior Cervical Discectomy and Fusion Model.

STUDY DESIGN: Experimental porcine anterior cervical discectomy and fusion (ACDF) model: a proof-of-concept study. OBJECTIVE: The effect of monetite synthetic bone graft containing calcium pyrophosphate (Ca-PP) and ß-tricalcium phosphate (ß-TCP) on cervical spinal fusion in a non-instrumented two-level large animal model. SUMMARY OF BACKGROUND DATA: ACDF is the gold standard surgical technique for the treatment of degenerative cervical spinal diseases. However, pseudarthrosis associated with increased patient morbidity occurs in approximately 2,6% of the surgeries. Synthetic bone graft (SBG) may enhance bony fusion and subsequently decrease the risk of pseudarthrosis. Recent studies on monetite-based synthetic bone grafts for use in large cranial defects in humans have shown promising bone healing results, necessitating further investigation of their use in cervical spinal fusion. METHODS: Four adult female Danish Göttingen mini-pigs received partial cervical anterior discectomy and intervertebral defects at an upper and lower level. One defect was filled with SBG and the other was left empty. Bony fusion was evaluated using computed tomography (CT) at three-month intervals for 12 months. Fifteen months post-surgery, the animals were euthanized for further ex vivo qualitative histopathological and micro-CT evaluations. Fusion rates were compared using Fisher´s exact test at each time point. RESULTS: Increased interbody bony fusion rates were observed at synthetic bone graft levels (4/4) compared with control levels (0/4) evaluated by CT at 6- and 9-months post-surgery ( P = 0.029). Fusion was observed at all synthetic bone graft levels 12 months post-surgery and at only one control level. Histopathological evaluation confirmed high-quality interbody bony fusion at all synthetic bone graft levels, and fusion by spondylosis at one control level. CONCLUSION: This proof-of-concept study provides preliminary evidence of a novel, Ca-PP -and ß-TCP-containing monetite SBG that promotes bony fusion compared to a negative control in a clinically relevant porcine model of ACDF. LEVEL OF EVIDENCE: N/A.

Quantification of Bone Height and Bone Volume Around Dental Implants After Open Maxillary Sinus Elevation Surgery Using CBCT.

Purpose: To assess, using CBCT, the volume and height of bone formation after open maxillary sinus elevation without the use of grafts. Materials and Methods: The study was retrospective and included 24 patients with a total of 67 implants. CBCT examinations were conducted at baseline (0 to 43 days postsurgery) and after an average healing period of 6.2 months (range: 5.1 to 7.8 months). The image analysis included metal artifact reduction, registration, and a standardized protocol for segmenting the anatomical structures of the maxillary sinus, including calculating the 3D volumetric changes after bone formation. Conventional manual 2D measurement of vertical bone formation was executed for comparison. Clinical factors assumed to be relevant for bone formation were obtained from patient medical records. Results: One implant was lost before prosthetic loading, representing an early implant loss rate of 1.5%. Differences in intra- and interexaminer reproducibility were registered for the conventional 2D method (P < .05). The average vertical bone formation measured with the 2D method was 4.8 mm (4.6 to 5.0 mm), covering 60.2% of the implant height within the sinus. The average volumetric bone formation measured with the developed 3D image-analysis method was 801 mm3 in total and 195 mm3 in a restricted region around each implant. Bone formation was registered in 62% of the volume of the restricted region. A correlation regarding bone formation was found between the two methods (R2 = 0.705). Clinical factors such as age, smoking, general health, and postoperative complications did not correlate with the amount of bone formed. Conclusion: CBCT image analysis is a promising method for objective 3D evaluation of bone formation after sinus elevation. A correlation was seen between the manually measured bone height (2D) and the bone volume in a restricted region around each implant using the developed method (3D). Reducing visual interpretation minimizes errors related to examiner reliability. Clinical factors did not significantly affect the volumetric bone formation.

Long-term mechanical properties of a novel low-modulus bone cement for the treatment of osteoporotic vertebral compression fractures.

In spite of the success of vertebroplasty (VP) and balloon kyphoplasty (BKP), which are widely used for stabilizing painful vertebral compression fractures, concerns have been raised about use of poly(methyl methacrylate) (PMMA) bone cements for these procedures since the high compressive modulus of elasticity (E) of the cement is thought to be one of the causes of the higher number of adjacent-level vertebral fractures. Therefore, bone cements with E comparable to that of cancellous bone have been proposed. While the quasi-static compressive properties of these so-called "low-modulus" cements have been widely studied, their fatigue performance remains underassessed. The purpose of the present study was to critically compare a commercial bone cement (control cement) and its low-modulus counterpart on the basis of quasi-static compressive strength (CS), E, fatigue limit under compression-compression loading, and release of methyl methacrylate (MMA). At 24 h, mean CS and E of the low-modulus material were 72% and 77% lower than those of the control cement, whereas, at 4 weeks, mean CS and E were 60% and 54% lower, respectively. The fatigue limit of the control cement was estimated to be 43-45 MPa compared to 3-5 MPa for the low-modulus cement. The low-modulus cement showed an initial burst release of MMA after 24 h followed by a plateau, similar to many other commercially available cements, whereas the control cement showed a much lower, stable release from day 1 and up to 1 week. The low-modulus cement may be a promising alternative to currently available PMMA bone cements, with the potential for reducing the incidence of adjacent fractures following VP/BKP.

Density and mechanical properties of vertebral trabecular bone-A review.

Being able to predict the mechanical properties of vertebrae in patients with osteoporosis and other relevant pathologies is essential to prevent fractures and to develop the most favorable fracture treatments. Furthermore, a reliable prediction is important for developing more patient- and pathology-specific biomaterials. A plethora of studies correlating bone density to mechanical properties has been reported; however, the results are variable, due to a variety of factors, including anatomical site and methodological differences. The aim of this study was to provide a comprehensive literature review on density and mechanical properties of human vertebral trabecular bone as well as relationships found between these properties. A literature search was performed to include studies, which investigated mechanical properties and bone density of trabecular bone. Only studies on vertebral trabecular bone tissue, reporting bone density or mechanical properties, were kept. A large variation in reported vertebral trabecular bone densities, mechanical properties, and relationships between the two was found, as exemplified by values varying between 0.09 and 0.35 g/cm3 for the wet apparent density and from 0.1 to 976 MPa for the elastic modulus. The differences were found to reflect variations in experimental and analytical processes that had been used, including testing protocol and specimen geometry. The variability in the data decreased in studies where bone tissue testing occurred in a standardized manner (eg, the reported differences in average elastic modulus decreased from 400% to 10%). It is important to take this variability into account when analyzing the predictions found in the literature, for example, to calculate fracture risk, and it is recommended to use the models suggested in the present review to reduce data variability.

The broad role of Nkx3.2 in the development of the zebrafish axial skeleton.

The transcription factor Nkx3.2 (Bapx1) is an important chondrocyte maturation inhibitor. Previous Nkx3.2 knockdown and overexpression studies in non-mammalian gnathostomes have focused on its role in primary jaw joint development, while the function of this gene in broader skeletal development is not fully described. We generated a mutant allele of nkx3.2 in zebrafish with CRISPR/Cas9 and applied a range of techniques to characterize skeletal phenotypes at developmental stages from larva to adult, revealing loss of the jaw joint, fusions in bones of the occiput, morphological changes in the Weberian apparatus, and the loss or deformation of bony elements derived from basiventral cartilages of the vertebrae. Axial phenotypes are reminiscent of Nkx3.2 knockout in mammals, suggesting that the function of this gene in axial skeletal development is ancestral to osteichthyans. Our results highlight the broad role of nkx3.2 in zebrafish skeletal development and its context-specific functions in different skeletal elements.

Functional Properties of Low-Modulus PMMA Bone Cements Containing Linoleic Acid.

Acrylic bone cements modified with linoleic acid are a promising low-modulus alternative to traditional high-modulus bone cements. However, several key properties remain unexplored, including the effect of autoclave sterilization and the potential use of low-modulus cements in other applications than vertebral augmentation. In this work, we evaluate the effect of sterilization on the structure and stability of linoleic acid, as well as in the handling properties, glass transition temperature, mechanical properties, and screw augmentation potential of low-modulus cement containing the fatty acid. Neither 1H NMR nor SFC-MS/MS analysis showed any detectable differences in autoclaved linoleic acid compared to fresh one. The peak polymerization temperature of the low-modulus cement was much lower (28-30 °C) than that of the high-modulus cement (67 °C), whereas the setting time remained comparable (20-25 min). The Tg of the low-modulus cement was lower (75-78 °C) than that of the high-stiffness cement (103 °C). It was shown that sterilization of linoleic acid by autoclaving did not significantly affect the functional properties of low-modulus PMMA bone cement, making the component suitable for sterile production. Ultimately, the low-modulus cement exhibited handling and mechanical properties that more closely match those of osteoporotic vertebral bone with a screw holding capacity of under 2000 N, making it a promising alternative for use in combination with orthopedic hardware in applications where high-stiffness augmentation materials can result in undesired effects.

Monetite-based composite cranial implants demonstrate long-term clinical volumetric balance by concomitant bone formation and degradation.

The use of calcium phosphates (CaPs) as synthetic bone substitutes should ideally result in a volumetric balance with concomitant bone formation and degradation. Clinical data on such properties is nevertheless lacking, especially for monetite-based CaPs. However, a monetite-based composite implant has recently shown promising cranial reconstructions, with both CaP degradation and bone formation. In this study, the volumetric change at the implant site was quantified longitudinally by clinical computed tomography (CT). The retrospective CT datasets had been acquired postoperatively (n = 10), in 1-year (n = 9) and 3-year (n = 5) follow-ups. In the 1-year follow-up, the total volumetric change at the implant site was -8 ±â€¯8%. A volumetric increase (bone formation) was found in the implant-bone interface, and a volumetric decrease was observed in the central region (CaP degradation). In the subjects with 2- or 3-year follow-ups, the rate of volumetric decrease slowed down or plateaued. The reported degradation rate is lower than previous clinical studies on monetite, likely due to the presence of pyrophosphate in the monetite-based CaP-formulation. A 31-months retrieval specimen analysis demonstrated that parts of the CaP had been remodeled into bone. The CaP phase composition remained stable, with 6% transformation into hydroxyapatite. In conclusion, this study demonstrates successful bone-bonding between the CaP-material and the recipient bone, as well as a long-term volumetric balance in cranial defects repaired with the monetite-based composite implant, which motivates further clinical use. The developed methods could be used in future studies for correlating spatiotemporal information regarding bone regeneration and CaP degradation to e.g. patient demographics. STATEMENT OF SIGNIFICANCE: In bone defect reconstructions, the use of calcium phosphate (CaP) bioceramics ideally results in a volumetric balance between bone formation and CaP degradation. Clinical data on the volumetric balance is nevertheless lacking, especially for monetite-based CaPs. Here, this concept is investigated for a composite cranial implant. The implant volumes were quantified from clinical CT-data: postoperatively, one year and three years postoperatively. In total, -8 ±â€¯8% (n = 9) volumetric change was observed after one year. But the change plateaued, with only 2% additional decrease at the 3-year follow-up (n = 5), indicating a lower CaP degradation rate. Osseointegration was seen at the bone-implant interface, with a 9 ±â€¯7% volumetric change after one year. This study presented the first quantitative spatiotemporal CT analysis of monetite-based CaPs.

The effect of two types of resorbable augmentation materials - a cement and an adhesive - on the screw pullout pullout resistance in human trabecular bone.

Augmentation materials, such as ceramic and polymeric bone cements, have been frequently used to improve the physical engagement of screws inserted into bone. While ceramic, degradable cements may ultimately improve fixation stability, reports regarding their effect on early fixation stability have been inconsistent. On the other hand, a newly developed degradable ceramic adhesive that can bond with tissues surrounding the screw, may improve the pullout performance, ensure early stability, and subsequent bony integration. The aim of this study was to investigate failure mechanisms of screw/trabecular bone constructs by comparing non-augmented screws with screws augmented with a calcium phosphate cement or an adhesive, i.e. a phosphoserine-modified calcium phosphate. Pullout tests were performed on screws inserted into trabecular cylinders extracted from human femoral bone. Continuous and stepwise pullout loading was applied with and without real-time imaging in a synchrotron radiation micro-computed tomograph, respectively. Statistical analysis that took the bone morphology into account confirmed that augmentation with the adhesive supported significantly higher pullout loads compared to cement-augmented, or non-augmented screws. However, the adhesive also allowed for a higher injection volume compared to the cement. In-situ imaging showed cracks in the vicinity of the screw threads in all groups, and detachment of the augmentation materials from the trabecular bone in the augmented specimens. Additional cracks at the periphery of the augmentation and the bone-material interfaces were only observed in the adhesive-augmented specimen, indicating a contribution of surface bonding to the pullout resistance. An adhesive that has potential for bonding with tissues, displayed superior pullout resistance, compared to a brushite cement, and may be a promising material for cementation or augmentation of implants.

A biomimetic engineered bone platform for advanced testing of prosthetic implants.

Existing methods for testing prosthetic implants suffer from critical limitations, creating an urgent need for new strategies that facilitate research and development of implants with enhanced osseointegration potential. Herein, we describe a novel, biomimetic, human bone platform for advanced testing of implants in vitro, and demonstrate the scientific validity and predictive value of this approach using an assortment of complementary evaluation methods. We anchored titanium (Ti) and stainless steel (SS) implants into biomimetic scaffolds, seeded with human induced mesenchymal stem cells, to recapitulate the osseointegration process in vitro. We show distinct patterns of gene expression, matrix deposition, and mineralization in response to the two materials, with Ti implants ultimately resulting in stronger integration strength, as seen in other preclinical and clinical studies. Interestingly, RNAseq analysis reveals that the TGF-beta and the FGF2 pathways are overexpressed in response to Ti implants, while the Wnt, BMP, and IGF pathways are overexpressed in response to SS implants. High-resolution imaging shows significantly increased tissue mineralization and calcium deposition at the tissue-implant interface in response to Ti implants, contributing to a twofold increase in pullout strength compared to SS implants. Our technology creates unprecedented research opportunities towards the design of implants and biomaterials that can be personalized, and exhibit enhanced osseointegration potential, with reduced need for animal testing.

Implicit and explicit finite element models predict the mechanical response of calcium phosphate-titanium cranial implants.

The structural integrity of cranial implants is of great clinical importance, as they aim to provide cerebral protection after neurosurgery or trauma. With the increased use of patient-specific implants, the mechanical response of each implant cannot be characterized experimentally in a practical way. However, computational models provide an excellent possibility for efficiently predicting the mechanical response of patient-specific implants. This study developed finite element models (FEMs) of titanium-reinforced calcium phosphate (CaP-Ti) implants. The models were validated with previously obtained experimental data for two different CaP-Ti implant designs (D1 and D2), in which generically shaped implant specimens were loaded in compression at either quasi-static (1 mm/min) or impact (5 kg, 1.52 m/s) loading rates. The FEMs showed agreement with experimental data in the force-displacement response for both implant designs. The implicit FEMs predicted the peak load with an underestimation for D1 (9%) and an overestimation for D2 (11%). Furthermore, the shape of the force-displacement curves were well predicted. In the explicit FEMs, the first part of the force-displacement response showed 5% difference for D1 and 2% difference for D2, with respect to the experimentally derived peak loads. The explicit FEMs efficiently predicted the maximum displacements with 1% and 4% difference for D1 and D2, respectively. Compared to the CaP-Ti implant, an average parietal cranial bone FEM showed a stiffer response, greater energy absorption and less deformation under the same impact conditions. The framework developed for modelling the CaP-Ti implants has a potential for modelling CaP materials in other composite implants in future studies since it only used literature based input and matched boundary conditions. Furthermore, the developed FEMs make an important contribution to future evaluations of patient-specific CaP-Ti cranial implant designs in various loading scenarios.

Mechanical behaviour of composite calcium phosphate-titanium cranial implants: Effects of loading rate and design.

Cranial implants are used to repair bone defects following neurosurgery or trauma. At present, there is a lack of data on their mechanical response, particularly in impact loading. The aim of the present study was to assess the mechanical response of a recently developed composite calcium phosphate-titanium (CaP-Ti) implant at quasi-static and impact loading rates. Two different designs were tested, referred to as Design 1 (D1) and Design 2 (D2). The titanium structures in the implant specimens were additively manufactured by a powder-bed fusion process and subsequently embedded in a self-setting CaP material. D1 was conceptually representative of the clinically used implants. In D2, the titanium structure was simplified in terms of geometry in order to facilitate the manufacturing. The mechanical response of the implants was evaluated in quasi-static compression, and in impact using a drop-tower. Similar peak loads were obtained for the two designs, at the two loading rates: 808 ± 29 N and 852 ± 34 for D1, and 840 ± 40 N and 814 ± 13 for D2. A strain rate dependency was demonstrated for both designs, with a higher stiffness in the impact test. Furthermore, the titanium in the implant fractured in the quasi-static test (to failure) but not in the impact test (to 5.75 J) for D1. For D2, the displacement at peak load was significantly lower in the impact test than in the quasi-static test. The main difference between the designs was seen in the quasi-static test results where the deformation zones, i.e. notches in the titanium structure between the CaP tiles, in D1 likely resulted in a localization of the deformation, compared to in D2 (which did not have deformation zones). In the impact test, the only significant difference between the designs was a higher maximum displacement of D2 than of D1. In comparison with other reported mechanical tests on osteoconductive ceramic-based cranial implants, the CaP-Ti implant demonstrates the highest reported strength in quasi-static compression. In conclusion, the titanium structure seems to make the CaP-Ti implant capable of cerebral protection in impact situations like the one tested in this study.

The effect of housing environment on bone healing in a critical radius defect in New Zealand White rabbits.

In animal studies on bone healing, the effect of housing space and physical activity are seldom taken into account. Bone formation was evaluated in New Zealand White rabbits (mean ± SEM BW: 3.9 ± 0.11 kg) with a critical bone defect after 12 weeks of rehabilitation in pair-housing in 3 m2 large floor pens (Floor, n = 10) or standard single housing in 0.43 m2 cages (Cage, n = 10). In the randomised full-factorial study, a bone replica of calcium phosphate cement (CPC, n = 10) or autologous bone (AB, n = 10) was implanted in the unilateral 20 mm radius defect. Post-mortem, the oxidative capacity was measured by citrate synthase (CS) activity in M. quadriceps and the defect filling volume and density evaluated by microcomputer tomography (µ-CT). Histology sections were evaluated by subjective scoring and histomorphometry. Fourteen rabbits remained until the end of the study. Group Floor (n = 7; 3 CPC + 4 AB) had a higher CS activity and a larger bone defect filling volume and lower density by µ-CT measurements than group Cage (n = 7; 3 CPC + 4 AB). Three out of four rabbits in AB-Floor presented fusion of the defect with reorganisation of trabecular bone, whereas three of four in AB-Cage showed areas of incomplete healing. Floor rabbits had a higher score of bony fusion between the radius and ulna than Cage rabbits. There were no differences between groups in histomorphometry. The study found that a larger housing space increased physical activity and promoted bone formation.

Effect of Copper Ion Concentration on Bacteria and Cells.

In the oral cavity, dental implants-most often made of commercially pure titanium-come in contact with bacteria, and antibacterial management has been researched extensively to improve patient care. With antibiotic resistance becoming increasingly prevalent, this has resulted in copper being investigated as an antibacterial element in alloys. In this study, the objective was to investigate the copper ion concentrations at which cyto-toxicity is avoided while bacterial inhibition is ensured, by comparing Cu ion effects on selected eukaryotes and prokaryotes. To determine relevant copper ion concentrations, ion release rates from copper and a 10 wt. % Cu Ti-alloy were investigated. Survival studies were performed on MC3T3 cells and Staphylococcus epidermidis bacteria, after exposure to Cu ions concentrations ranging from 9 × 10-3 to 9 × 10-12 g/mL. Cell survival increased from <10% to >90% after 24 h of exposure, by reducing Cu concentrations from 9 × 10-5 to 9 × 10-6 g/mL. Survival of bacteria also increased in the same range of Cu concentrations. The maximum bacteria growth was found at 9 × 10-7 g/mL, probably due to stress response. In conclusion, the minimum inhibitory concentrations of Cu ions for these prokaryotes and eukaryotes were found in the range from 9 × 10-5 to 9 × 10-6 g/mL. Interestingly, the Cu ion concentration correlating to the release rate of the 10 wt. % Cu alloy (9 × 10-8 g/mL) did not kill the bacteria, although this alloy has previously been found to be antibacterial. Further studies should investigate in depth the bacteria-killing mechanism of copper.

Bone Volume Assessment Around Dental Implants After Open Maxillary Sinus Elevation Surgery: A Quantitative Approach to CBCT Images.

PURPOSE: Cone beam computed tomography (CBCT) is an important imaging technique in maxillofacial evaluations. However, application-specific image analysis methods aimed at extracting quantitative information from these images need to be further developed. The aim of this study was to provide a robust and objective method that could assess radiologic changes around dental implants after sinus elevation surgery with simultaneous implant placement. MATERIALS AND METHODS: The study was performed retrospectively on patients fulfilling the inclusion criteria. The included patients had been CBCT scanned preoperatively, at baseline (early after surgery), and 6 months postoperatively. In order to quantify the radiologic changes, an image analysis workflow was developed based on the postoperative baseline and 6-month scans. The workflow included metal artefact reduction, registration, and a standardized protocol for semiautomatic segmentation. Validation of different steps of the method was conducted by comparing scans from all time points. Comparison of constant volumes (eg, screws and bony parts not subjected to change) was used. Additionally, the Dice similarity coefficient (DSC) was used to measure the overlap of the segmentations. RESULTS: The study included nine maxillary sinuses from six patients. The bone formation was quantified and visualized in 3D. In the validation, no significant differences were found for the constant volumes at the different scanning time points. The DSC showed accurate results with values > 0.92. CONCLUSION: The method presented in this study provides an objective and robust evaluation of bone formation around dental implants. The same methodologies can be applied in other studies of dental CBCT images, eg, for comparison of grafting materials or surgical strategies.

Antibacterial investigation of titanium-copper alloys using luminescent Staphylococcus epidermidis in a direct contact test.

Commercially pure titanium (CP-Ti), used as oral implants, is often populated by various bacterial colonies in the oral cavity. These bacteria can cause Peri-implantitis, leading to loss of bone tissue and failure of implants. With the increased awareness of antibiotic resistance, research has been directed towards alternative solutions and recent findings have indicated titanium­copper (Ti-Cu) alloys as a promising antibacterial material. The aim of this study was to produce homogeneous Ti-Cu alloys, with various concentrations of copper, and to characterise their antibacterial properties through direct contact tests, using luminescent bacteria, in addition to traditional materials characterisation techniques. Samples of CP-Ti and four different Ti-Cu alloys (1, 2.5, 3 and 10 wt%Cu) were produced in an arc-furnace, heated treated and rapidly quenched. X-ray diffraction revealed that Ti2Cu, was present only in the 10 wt%Cu alloy, however, scanning electron microscopy (SEM) indicated precipitates at the grain boundaries of the 3 wt%Cu alloy, which were confirmed to be of a copper rich phase by energy dispersive x-ray spectroscopy (EDS) analysis. EDS line scans confirmed that the alloys were homogenous. After 6 h, a trend between copper content and antibacterial rate could be observed, with the 10 wt%Cu alloy having the highest rate. SEM confirmed fewer bacteria on the 3 wt%Cu and especially the 10 wt%Cu samples. Although the 10 wt%Cu alloy gave the best antibacterial results, it is desired that the Cu concentration is below ~3 wt%Cu to maintain similar mechanical and corrosive performance as CP-Ti. Therefore, it is proposed that future work focuses on the 3 wt%Cu alloy.

Crack nucleation and propagation in microcrystalline-cellulose based granules subject to uniaxial and triaxial load.

Cracking patterns in four kinds of granules, based on the common pharmaceutical excipient microcrystalline cellulose (MCC) and subject to compressive load, were examined. The initial pore structure and the location of initial failure under uniaxial compression were assessed using X-ray micro-computed tomography, whereas contact force development and onset of cracking under more complex compressive load were examined using a triaxial testing apparatus. Smoothed particle hydrodynamics (SPH) simulations were employed for numerical analysis of the stress distributions prior to cracking. For granules subject to uniaxial compression, initial cracking always occurred along the meridian and the precise location of the crack depended on the pore structure. Likewise, for granules subject to triaxial compression, the fracture plane of the primary crack was generally parallel to the dominant loading direction. The occurrence of cracking was highly dependent on the triaxiality ratio, i.e. the ratio between the punch displacements in the secondary and dominant loading directions. Compressive stresses in the lateral directions, induced by triaxial compression, prevented crack opening and fragmentation of the granule, something that could be verified by simulations. These results provide corroboration as well as further insights into previously observed differences between confined and unconfined compression of granular media.