Autologous mesenchymal stem cells loaded in Gelfoam<Superscript>®</Superscript> for structural bone allograft healing in rabbits

This study was designed to evaluate the effect of autologous bone marrow mesenchymal stem cells (MSCs) seeded into Gelfoam^® on structural bone allograft healing. Thirty New Zealand white rabbits were divided into two groups. Segmental bone defect was created on diaphysis of the femur, and the defect was reconstructed with structural bone allograft. In experimental group, structural allograft was wrapped around by Gelfoam^® containing autologous MSCs, whereas cells were not included in control group. At 4, 8, 12 weeks, the femur of rabbits underwent radiographic and histologic evaluation for bony union. Bone morphogenic protein-2 (BMP-2), BMP-4, BMP-7, vascular endothelial growth factor (VEGF), and receptor activator of nuclear factor-kappa B ligand (RANKL) were measured within the grafted periosteal tissue. Bony union was not achieved in both groups at 4 and 8 weeks. At 12 weeks, three out of five femurs in experimental group were united, but one out of five in control group was united. Mean Taira scores were significantly different between two groups. The expression of BMP-2 was significantly higher at 4, 8 weeks, the expressions of BMP-4 and BMP-7 were significantly higher at 8 and 12 weeks, and the expression of VEGF and RANKL were significantly higher at all time points in experimental group. Incorporation of the structural bone allograft could be enhanced if allograft is covered with Gelfoam^® containing autologous MSCs. MSCs have influence on not only bone formation, but neo-angiogenesis, and bone resorption.     

Direct perfusion measurements of cancellous bone anisotropic permeability

More extensive characterization of trabecular connectivity and intertrabecular space will be instrumental in understanding disease states and designing engineered bone. This project presents an experimental protocol to define the directional dependence of transport properties as measured from healthy cancellous bone when considered as a biologic, porous medium. In the initial design phases, mature bovine bone was harvested from the femoral neck (n=6 cylinders) and distal condyle (n=4 cubes) regions and used for "proof of concept" experimentation. A power study on those results led to the presented work on 20 cubic samples (mean volume=4.09cm(3)) harvested from a single bovine distal femur. Anisotropic intrinsic permeabilities (k(i)) were quantified along the orthogonal anatomic axes (i=medial-lateral, anterior-posterior, and superior-inferior) from each individual cubic bone sample. Using direct perfusion measurements, permeability was calculated based upon Darcy's Law describing flow through porous media. The maximum mean value was associated with the superior-inferior orientation (4.65x10(-10)m(2)) in comparison with the mean anterior-posterior (4.52x10(-10)m(2)) and medial-lateral (2.33x10(-10)m(2)) direction values. The results demonstrate the anisotropic (p=0.0143) and heterogeneous (p=0.0002) nature of the tissue and encourage the ongoing quantification of parameters within the established poroelastic models.     

An automatic approach for calibrating dielectric bone properties by combining finite-element and optimization software tools

The dielectric properties of human bone are one of the most essential inputs required by electromagnetic stimulation for improved bone regeneration. Measuring the electric properties of bone is a difficult task because of the complexity of the bone structure. Therefore, an automatic approach is presented to calibrate the electric properties of bone. The numerical method consists of three steps: generating input from experimental data, performing the numerical simulation, and calibrating the bone dielectric properties. As an example, the dielectric properties at 20 Hz of a rabbit distal femur were calibrated. The calibration process was considered as an optimization process with the aim of finding the optimum dielectric bone properties that match most of the numerically calculated simulation and experimentally measured data sets. The optimization was carried out automatically by the optimization software tool iSIGHT in combination with the finite-element solver COMSOL Multiphysics. As a result, the optimum conductivity and relative permittivity of the rabbit distal femur at 20 Hz were found to be 0.09615 S/m and 19522 for cortical bone and 0.14913 S/m and 1561507 for cancellous bone, respectively. The proposed method is a potential tool for the identification of realistic dielectric properties of the entire bone volume. The presented approach combining iSIGHT with COMSOL is applicable to, amongst others, designing implantable electro-stimulative devices or the optimization of electrical stimulation parameters for improved bone regeneration.     

The use of deep frozen and irradiated bone allografts in the reconstruction of tibial plateau fractures

To investigate the clinical behavior of deep frozen and irradiated bone allografts in the treatment of depressed tibial plateau fractures. Twenty-two patients with a tibial plateau fracture were treated with cancellous bone allografts. The bone allograft preparation process included fresh-freezing at ?70 °C for 4 weeks and gamma-irradiation at 25 kGy. All of the patients were followed for 1–2 years. The clinical effects were assessed using the Rasmussen score for tibial head fractures and X-rays. Postoperatively, the average excellent and fair Rasmussen scores were 88.9 %. Only one patient developed an infection, with no integration between allograft and recipient bone observed. All of the other bone allografts were incorporated successfully, and no osteoporosis or sclerosis was observed. The frozen and gamma-irradiated bone allograft is a good alternative in the treatment of tibial plateau fractures, which we have shown can integrate with the surrounding host bone.     

Deformation of the distal femur: a contribution towards the pathogenesis of osteochondrosis dissecans in the knee joint.

Osteochondrosis dissecans (OD) is a process of subchondral bone necrosis occurring predominantly in young individuals at specific sites. The aetiology of this disease remains controversial with mechanical processes due to trauma and/or ischaemic factors being proposed. This study aims at explaining the aetiology of OD in the knee joint as a result of the particular deformation of the condyles. A finite element analysis of the distal third of the femur was performed. A three-dimensional model was developed based on computed tomography scans of a normal femur, consisting of cortical bone, cancellous bone and articular cartilage. This model was subjected to physiological loads at 0, 30, 60 and 90 degrees of knee flexion. A complex deformation was found within each condyle as well as between the two condyles. Both medial and lateral condyles are deformed in the medio-lateral direction and at the same time compressed between the patella and the tibia in the antero-posterior direction. This effect is highest at 60 degrees of knee flexion. In both planes, the medial condyle is distorted more than the lateral one. Strain concentration in the subchondral bone facing the patella varies with flexion, especially for angles exceeding 60 degrees. The deformation of the femur in the predominant locus of OD in the medial condyle exceeds that of the lateral condyle considerably. The analysis shows that repeated vigorous exercise including extreme knee flexion may produce rapidly changing strains which in turn could ultimately be responsible for local subchondral bone collapse.     

The effectiveness of bone banking in Central Serbia: audit of the first seven years

We analyzed the incidence and predisposing factors for overall discard rate after retrieval of 295 femoral head allografts. The aim of the present study was to evaluate the quality system of institutional bone banking and to ensure that we can provide high standard allografts with low infection rate. Audit of bone banking was conducted on 295 donors and 180 recipients. Of the 295 donated femoral heads 77 were discarded, giving an overall discard rate of 26.1 %. At retrieval, 37 allografts were positive, giving an overall contamination rate of 12.54 %. The organism most commonly identified was Staphylococcus species. Seven (2.37 %) of the 295 allografts failed the blood screening tests. Twelve allografts (4.06 %) were discarded because of suspected damage of the packaging or disuse during surgery. Due to donor death or inability to perform serology retests, 21 (7.11 %) allografts were discarded. In the postoperative survey an infection rate of 2.22 % was found. After 7 years of bone banking, our results show that overall discard rate and allograft related infection rate are in accordance with the international standards. The leading cause of allograft discarding was bacterial contamination influenced by the surgical team. We suggest stringent aseptic allograft handling during harvesting and thawing within highly concentrated antibiotic solution to reduce a possibility of its contamination.     

Towards automatic measurement of anteversion and neck–shaft angles in human femurs using CT images

Automatic assessment of human femur morphology may provide useful clinical information with regard to hip and knee surgery, prosthesis design and management of hip instability. To this end, neck–shaft and anteversion angles are usually used. We propose a full automatic method to estimate these angles in human femurs. Multislice CT images from 18 dried bones were analysed. The algorithm fits 3D cylinders to different regions of the bone to estimate the angles. A manual segmentation and a conventional angle assessment were used for validation. We found anteversion angle as 20 ± 7° and neck–shaft angle as 130 ± 9°. Mean distances from femur surface to cylinders were 5.5 ± 0.6, 3.5 ± 0.6 and 2.4 ± 0.4 mm for condyles, diaphysis and neck regions, respectively. Automatic and conventional angles were positively correlated (r²>0.85). Manual and automatic segmentations did not differ. The method was fast and 100% reproducible. A robust in vivo segmentation algorithm should be integrated to advance towards a clinically compliant methodology.     

Automatic allograft bone selection through band registration and its application to distal femur

Clinical reports suggest that large bone defects could be effectively restored by allograft bone transplantation, where allograft bone selection acts an important role. Besides, there is a huge demand for developing the automatic allograft bone selection methods, as the automatic methods could greatly improve the management efficiency of the large bone banks. Although several automatic methods have been presented to select the most suitable allograft bone from the massive allograft bone bank, these methods still suffer from inaccuracy. In this paper, we propose an effective allograft bone selection method without using the contralateral bones. Firstly, the allograft bone is globally aligned to the recipient bone by surface registration. Then, the global alignment is further refined through band registration. The band, defined as the recipient points within the lifted and lowered cutting planes, could involve more local structure of the defected segment. Therefore, our method could achieve robust alignment and high registration accuracy of the allograft and recipient. Moreover, the existing contour method and surface method could be unified into one framework under our method by adjusting the lift and lower distances of the cutting planes. Finally, our method has been validated on the database of distal femurs. The experimental results indicate that our method outperforms the surface method and contour method.     

Effect of storage temperature on gentamicin release from antibiotic-coated bone chips

Freezing is the most common method for storing bones until use in skeletal reconstruction. However, the effect of freezing on antibiotic delivery from antibiotic-coated bone has not been evaluated. In this study, we compared antibiotic delivery in vitro from gentamicin-coated human bone stored at different temperatures. Bone chips obtained from human femur heads were chemically cleaned and mixed with gentamicin sulfate. Samples were stored for 4 months at ?20 °C, 4 months at ?80 °C, or evaluated immediately without freezing. Antibiotic release from the bone chips was measured using Bacillus subtilis as an indicator strain. Zones of inhibition and rates of gentamicin release were similar in all three groups. Storage at ?20 and ?80 °C for bone allografts has no effect on gentamicin release from chemically cleaned bone chips.     

External-internal relationships in humeri of modern human females

With data from an early twentieth century human skeletal collection, this exploratory study investigates associations between inner cortical and medullary cavity structures and outer shaft and epiphyseal features of a long bone. Humeri are measured directly in both whole bone and transverse section contexts; data along 2 axes at 2 sites are obtained. Twenty-two probable females, with an age range concentrated in middle adulthood, comprise the sample. Correlations between multiple external and internal bone measurements are analyzed, with the aim of yielding information on the physical nature of bone and on the effects of different measurement types, locations, and orientations for bone relationships. The study also examines whether prediction of inner humeral dimensions from outer measurements would be appropriate. Results indicate biepicondylar width and maximum length as the strongest external correlates of cortical dimensions. Contrasting with studies on the proximal femur, the humeral head shows external size changeability, mostly in the transverse plane, in response to modeling forces shared with the shaft. Epiphyseal measures are more highly associated with absolute rather than percent, and areal rather than linear, cortical variables. Medullary cavity dimensions are not significantly correlated with whole bone measures. Most associations demonstrate shape or proportion influences rather than a shared effect of linear body size. Regarding location and orientation, the distal site and medial-lateral axis display the strongest correlations among external and internal bone dimensions. In light of the demonstrated correlation patterns, prediction of humeral cortical quantity from external bone measures in living and skeletal populations would benefit from utilizing (1) biepicondylar width and maximum length; (2) an absolute, areal cortical measure; (3) a distal location; and (4) a medial-lateral orientation.     

Articular-surface-based automatic anatomical coordinate systems for the knee bones

Increasing use of patient-specific surgical procedures in orthopaedics means that patient-specific anatomical coordinate systems (ACSs) need to be determined. For knee bones, automatic algorithms constructing ACSs exist and are assumed to be more reliable than manual methods, although both approaches are based on non-unique numerical reconstructions of true bone geometries. Furthermore, determining the best algorithms is difficult, as algorithms are evaluated on different datasets. Thus, in this study, we developed 3 algorithms, each with 3 variants, and compared them with 5 from the literature on a dataset comprising 24 lower-limb CT-scans. To evaluate algorithms’ sensitivity to the operator-dependent reconstruction procedure, the tibia, patella and femur of each CT-scan were each reconstructed once by three different operators.Our algorithms use principal inertia axis (PIA), cross-sectional area, surface normal orientations and curvature data to identify the bone region underneath articular surfaces (ASs). Then geometric primitives are fitted to ASs, and the ACSs are constructed from the geometric primitive points and/or axes. For each bone type, the algorithm displaying the least inter-operator variability is identified. The best femur algorithm fits a cylinder to posterior condyle ASs and a sphere to the femoral head, average axis deviations: 0.12°, position differences: 0.20 mm. The best patella algorithm identifies the AS PIAs, average axis deviations: 0.91°, position differences: 0.19 mm. The best tibia algorithm finds the ankle AS center and the 1st PIA of a layer around a plane fitted to condyle ASs, average axis deviations: 0.38°, position differences: 0.27 mm.     

The mechanical behaviour of intracondylar cancellous bone of the femur at different loading rates

The compressive properties of human cancellous bone of the distal intracondylar femur in its wet condition were determined. Specimens were obtained from six cadaveric femora and were tested at a strain rate of 0.002, 0.10 and 9.16 sec⁻¹. It was found that the compressive strength decreases with an increasing vertical distance from the joint. The highest compressive strength level was recorded in the posterior medial condyle. Correlations among the mechanical properties, the bulk specimen density and the bone mineral content yield (i) highly significant correlations between the compressive strength and the elastic modulus (ii) highly significant correlations between the compressive strength or the modulus of elasticity and the bulk specimen density (iii) a doubtful correlation between the compressive strength and the bone mineral content. All recorded graphs of the impact loaded specimens displayed several well defined stress peaks, unlike the graphs recorded at low loading rates. It can be concluded that upon impact loading the localized trabecular failure which is associated with each peak, does not affect the spongy bone's stress capacity in a detrimental way.     

The effect of temperature exposure during shipment on a commercially available demineralized bone matrix putty

During August and September of 2013, temperature data loggers were shipped to and from an AATB accredited and FDA registered allograft tissue processing facility in Belgrade, MT (Bacterin International, Inc.) to five warm climate cities (Dallas, TX, El Paso, TX, New Orleans, LA, Phoenix, AZ, and Tampa, FL). Shipping data acquired from 72 independent shipments were analyzed to generate an assessment of temperature exposure, shipment times, and shipping event durations experienced during routine distribution. Overall the packages experienced an average temperature of 26.2 ± 2.3 °C which mirrored the average external ambient temperature of 25.8 ± 3.0 °C. However, temperature spikes above 40 °C were frequently observed. The data from the model shipments were extrapolated to provide a worst-case high temperature spike of 52.9 °C for 12 h and 14 min. Multiple lots of a commercially available demineralized bone matrix (DBM) putty (OsteoSelect^® DBM Putty) were subjected to continuous heating at 50 °C, to multiple worst-case temperature spikes, and to multiple freeze–thaw cycles to assess the effects of these temperature extremes on the handling and osteoinductivity of the allograft tissue. Five weeks of continuous exposure to 50 °C and 12 simulated worst-case one-way shipments did not adversely affect the handling characteristics or the in vivo osteoinductivity of the product.     

Assessment of the bilateral asymmetry of human femurs based on physical,densitometric, and structural rigidity characteristics

The purpose of this study was to perform a comprehensive geometric, densitometric, biomechanical, and statistical analysis of paired femurs for an adult population over a wide age range using three imaging modalities to quantify the departure from symmetry in size, bone mineral density, and cross-sectional structural rigidities.Femur measurements were obtained from 20 pairs of cadaveric femurs. Dimensions of these anatomic sites were measured using calipers directly on the bone and plain radiographs. Dual energy X-ray absorptiometry was used to measure bone mineral density. Bone mineral content and axial and bending rigidities were determined from the CT imaging.No differences were observed between the geometric measurements, DXA based bone mineral density and axial and bending rigidities of left and right femurs (P>0.05 for all cases). Left and right proximal femurs are not significantly different based on geometric, densitometric, and structural rigidity measurements. However, absolute left–right differences for individual patients can be substantial. When using the contralateral femur as a control, the number of femur pairs required to assess significant changes in anatomic dimensions and structural properties induced by a tumor, infection, fracture, or implanted device can range from 3 to 165 pairs depending on the desired effect size or sensitivity (5% or 10% difference).This information is important both for femoral arthroplasty implant design and the use of the contralateral femur as an intra-subject control for clinical assessment and research studies. In addition, our statistical analysis provides sample size estimates for planning future orthopedic research studies.     

The effect of ribose pre-treatment of cortical bone on γ-irradiation sterilization effectiveness

Reconstruction of large skeletal defects is a significant and challenging issue. Tissue banks often use γ-irradiation (15–35 kGy) to sterilize bone allografts, which, however, drastically impairs the post-yield mechanical properties. In previous studies, we reported the development of a method that protects human bone collagen connectivity through ribose crosslinking while still undergoing γ-irradiation. Given these promising results, the next step was to determine if the presence of ribose within the bone tissue would interfere with the effectiveness of the γ-irradiation sterilization against bacteria. This study had two stages. The aim of the first stage was to assess the protective effect of ribose in solution using a Bacillus pumilus spore strip model. The aim of the second stage was to assess the protective effect of ribose (R) on spores within a human cortical bone model in comparison to conventionally irradiated bone (I). Treatment of B. pumilus spore strips with ribose in solution led to temperature-dependent effects on spore viability versus spore strips treated with PBS alone. Ribose solution at 60 °C led to a notable two logs decrease in spore count relative to PBS at 60 °C. In the human bone model, the number of spores in the I and R groups were greatly decreased in comparison to the non-irradiated N group. No spore colonies were detected in the R group (n = 4) whereas two of the four plates of group I formed colonies. This study provides evidence that the method of pre-treating bone with ribose crosslinking prior to irradiation sterilization, while improving irradiation sterilized bone allograft quality, also may improve the effectiveness of the sterilization process.     

Effect of thermodisinfection on mechanic parameters of cancellous bone

Revision surgery of joint replacements is increasing and raises the demand for allograft bone since restoration of bone stock is crucial for longevity of implants. Proceedings of bone grafts influence the biological and mechanic properties differently. This study examines the effect of thermodisinfection on mechanic properties of cancellous bone. Bone cylinders from both femoral heads with length 45 mm were taken from twenty-three 6–8 months-old piglets, thermodisinfected at 82.5 °C according to bone bank guidelines and control remained native. The specimens were stored at ?20 °C immediately and were put into 21 °C Ringer’s solution for 3 h before testing. Shear and pressure modulus were tested since three point bending force was examined until destruction. Statistical analysis was done with non-parametric Wilcoxon, t test and SPSS since p < 0.05 was significant. Shear modulus was significantly reduced by thermodisinfection to 1.02 ± 0.31 GPa from 1.28 ± 0.68 GPa for unprocessed cancellous bone (p = 0.029) since thermodisinfection reduced pressure modulus not significantly from 6.30 ± 4.72 GPa for native specimens to 4.97 ± 2.23 GPa and maximum bending force was 270.03 ± 116.68 N for native and 228.80 ± 70.49 N for thermodisinfected cancellous bone. Shear and pressure modulus were reduced by thermodisinfection around 20 % and maximum bending force was impaired by about 15 % compared with native cancellous bone since only the reduction of shear modulus reached significance. The results suggest that thermodisinfection similarly affects different mechanic properties of cancellous bone and the reduction of mechanic properties should not relevantly impair clinical use of thermodisinfected cancellous bone.     

Accuracy of femur reconstruction from sparse geometric data using a statistical shape model

Sparse geometric information from limited field-of-view medical images is often used to reconstruct the femur in biomechanical models of the hip and knee. However, the full femur geometry is needed to establish boundary conditions such as muscle attachment sites and joint axes which define the orientation of joint loads. Statistical shape models have been used to estimate the geometry of the full femur from varying amounts of sparse geometric information. However, the effect that different amounts of sparse data have on reconstruction accuracy has not been systematically assessed. In this study, we compared shape model and linear scaling reconstruction of the full femur surface from varying proportions of proximal and distal partial femur geometry in combination with morphometric and landmark data. We quantified reconstruction error in terms of surface-to-surface error as well as deviations in the reconstructed femur’s anatomical coordinate system which is important for biomechanical models. Using a partial proximal femur surface, mean shape model-based reconstruction surface error was 1.8 mm with 0.15° or less anatomic axis error, compared to 19.1 mm and 2.7–5.6° for linear scaling. Similar results were found when using a partial distal surface. However, varying amounts of proximal or distal partial surface data had a negligible effect on reconstruction accuracy. Our results show that given an appropriate set of sparse geometric data, a shape model can reconstruct full femur geometry with far greater accuracy than simple scaling.     

KRT8, FAF1 and PTH1R gene polymorphisms are associated with leg weakness traits in pigs

The liability to lesions of dysfunctions of bone and joints in pigs, summarized as leg weakness and mostly expressed as osteochondrosis, is an animal welfare and economic issue in pig production. The objective of this study was to identify polymorphisms in the functional and positional candidate genes keratin 8 (KRT8), Fas-associated factor 1 (FAF1) and parathyroid hormone type I receptor (PTH1R) and to evaluate their association with leg weakness traits. Therefore, osteochondrosis lesions were scored in animals of a Duroc × Pietrain F2 population (DuPi; n = 310) and commercial herds of the breed Large White (n = 298). In addition, bone mineralization traits were observed in DuPi population. SNPs were identified in genes KRT8 (g.8,039G > A), FAF1 (g.380,914T > C) and PTH1R (c.1,672C > T). KRT8 showed significant association with bone mineral density and content (P ≤ 0.05). FAF1 was association with OC lesions score of all joints inspected (P ≤ 0.05). PTH1R showed significant dominance effects on OC lesion scores of the distal femur articular cartilage (P = 0.01) and epiphysis of the distal ulna (P = 0.05) as well as sums of scores of all joints (OCsum, P = 0.04) and assignment to groups of either severely or gently affected animals (OCcat, P = 0.01). This study reveals clear genetic-statistical evidence for a link of KRT8, FAF1 and PTH1R with some of leg weakness related traits in pigs.     

Osteoprotective effect of green tea polyphenols and annatto-extracted tocotrienol in obese mice is associated with enhanced microbiome vitamin K2 biosynthetic pathways

The role of the gut microbiome in bone health has received significant attention in the past decade. We investigated the effects of green tea polyphenols (GTP) and annatto-extracted tocotrienols (AT) on bone properties and gut microbiome in obese mice. Male mice were assigned to a two (no AT vs. 400 mg/kg diet AT) × two (no GTP vs. 0.5% w/v GTP) factorial design, namely control, G, T, and G+T group respectively, for 14 weeks. The 4th lumbar vertebra (LV-4) and femur were harvested for bone microstructural analysis using μ-CT. Microbiome analysis using 16S rRNA gene sequencing of cecal feces was performed. AT increased bone volume at distal femur. GTP increased serum procollagen type 1 N-terminal propeptide concentration, bone volume at the distal femur and the LV-4, and trabecular number at distal femur; whereas GTP decreased trabecular separation at distal femur. Interactions between GTP and AT were observed in serum C-terminal telopeptide of type I collagen level (control>G=T=G+T) as well as the cortical bone area (control<G=T=G+T) and thickness (T≥G+T≥G≥control) at femur mid-diaphysis. Redundancy analysis showed a significant difference in the gut microbiome profile among different groups and the relative abundance of Akkermansia muciniphila, Clostridum saccharogumia, and Subdoligranulum variabile was increased in the GTP- and AT-supplemented groups. Functional profiling of the gut microbiome showed the combination of GTP and AT induced biosynthetic pathways for vitamin K₂. Our results suggest that GTP and AT supplementation benefits bone properties in obese mice through modifying gut microbiome composition and function.