Repopulation of decellularised articular cartilage by laser-based matrix engraving.

BACKGROUND: In spite of advances in the treatment of cartilage defects using cell and scaffold-based therapeutic strategies, the long-term outcome is still not satisfying since clinical scores decline years after treatment. Scaffold materials currently used in clinical settings have shown limitations in providing suitable biomechanical properties and an authentic and protective environment for regenerative cells. To tackle this problem, we developed a scaffold material based on decellularised human articular cartilage. METHODS: Human articular cartilage matrix was engraved using a CO2 laser and treated for decellularisation and glycosaminoglycan removal. Characterisation of the resulting scaffold was performed via mechanical testing, DNA and GAG quantification and in vitro cultivation with adipose-derived stromal cells (ASC). Cell vitality, adhesion and chondrogenic differentiation were assessed. An ectopic, unloaded mouse model was used for the assessment of the in vivo performance of the scaffold in combination with ASC and human as well as bovine chondrocytes. The novel scaffold was compared to a commercial collagen type I/III scaffold. FINDINGS: Crossed line engravings of the matrix allowed for a most regular and ubiquitous distribution of cells and chemical as well as enzymatic matrix treatment was performed to increase cell adhesion. The biomechanical characteristics of this novel scaffold that we term CartiScaff were found to be superior to those of commercially available materials. Neo-tissue was integrated excellently into the scaffold matrix and new collagen fibres were guided by the laser incisions towards a vertical alignment, a typical feature of native cartilage important for nutrition and biomechanics. In an ectopic, unloaded in vivo model, chondrocytes and mesenchymal stromal cells differentiated within the incisions despite the lack of growth factors and load, indicating a strong chondrogenic microenvironment within the scaffold incisions. Cells, most noticeably bone marrow-derived cells, were able to repopulate the empty chondrocyte lacunae inside the scaffold matrix. INTERPRETATION: Due to the better load-bearing, its chondrogenic effect and the ability to guide matrix-deposition, CartiScaff is a promising biomaterial to accelerate rehabilitation and to improve long term clinical success of cartilage defect treatment. FUNDING: Austrian Research Promotion Agency FFG ("CartiScaff" #842455), Lorenz Böhler Fonds (16/13), City of Vienna Competence Team Project Signaltissue (MA23, #18-08).

Regional diversity in the murine cortical vascular network is revealed by synchrotron X-ray tomography and is amplified with age.

Cortical bone is permeated by a system of pores, occupied by the blood supply and osteocytes. With ageing, bone mass reduction and disruption of the microstructure are associated with reduced vascular supply. Insight into the regulation of the blood supply to the bone could enhance the understanding of bone strength determinants and fracture healing. Using synchrotron radiation-based computed tomography, the distribution of vascular canals and osteocyte lacunae was assessed in murine cortical bone and the influence of age on these parameters was investigated. The tibiofibular junction from 15-week- and 10-month-old female C57BL/6J mice were imaged post-mortem. Vascular canals and three-dimensional spatial relationships between osteocyte lacunae and bone surfaces were computed for both age groups. At 15 weeks, the posterior region of the tibiofibular junction had a higher vascular canal volume density than the anterior, lateral and medial regions. Intracortical vascular networks in anterior and posterior regions were also different, with connectedness in the posterior higher than the anterior at 15 weeks. By 10 months, cortices were thinner, with cortical area fraction and vascular density reduced, but only in the posterior cortex. This provided the first evidence of age-related effects on murine bone porosity due to the location of the intracortical vasculature. Targeting the vasculature to modulate bone porosity could provide an effective way to treat degenerative bone diseases, such as osteoporosis.

The inferomedial femoral neck is compromised by age but not disease: Fracture toughness and the multifactorial mechanisms comprising reference point microindentation.

The influence of ageing on the fracture mechanics of cortical bone tissue is well documented, though little is known about if and how related material properties are further affected in two of the most prominent musculoskeletal diseases, osteoporosis and osteoarthritis (OA). The femoral neck, in close proximity to the most pertinent osteoporotic fracture site and near the hip joint affected by osteoarthritis, is a site of particular interest for investigation. We have recently shown that Reference Point micro-Indentation (RPI) detects differences between cortical bone from the femoral neck of healthy, osteoporotic fractured and osteoarthritic hip replacement patients. RPI is a new technique with potential for in vivo bone quality assessment. However, interpretation of RPI results is limited because the specific changes in bone properties with pathology are not well understood and, further, because it is not conclusive what properties are being assessed by RPI. Here, we investigate whether the differences previously detected between healthy and diseased cortical bone from the femoral neck might reflect changes in fracture toughness. Together with this, we investigate which additional properties are reflected in RPI measures. RPI (using the Biodent device) and fracture toughness tests were conducted on samples from the inferomedial neck of bone resected from donors with: OA (41 samples from 15 donors), osteoporosis (48 samples from 14 donors) and non age-matched cadaveric controls (37 samples from 10 donoros) with no history of bone disease. Further, a subset of indented samples were imaged using micro-computed tomography (3 osteoporotic and 4 control samples each from different donors) as well as fluorescence microscopy in combination with serial sectioning after basic fuchsin staining (7 osteoporotic and 5 control samples from 5 osteoporotic and 5 control donors). In this study, the bulk indentation and fracture resistance properties of the inferomedial femoral neck in osteoporotic fracture, severe OA and control bone were comparable (p > 0.05 for fracture properties and <10% difference for indentation) but fracture toughness reduced with advancing age (7.0% per decade, r = -0.36, p = 0.029). Further, RPI properties (in particular, the indentation distance increase, IDI) showed partial correlation with fracture toughness (r = -0.40, p = 0.023) or derived elastic modulus (r = -0.40, p = 0.023). Multimodal indent imaging revealed evidence of toughening mechanisms (i.e. crack deflection, bridging and microcracking), elastoplastic response (in terms of the non-conical imprint shape and presence of pile-up) and correlation of RPI with damage extent (up to r = 0.79, p = 0.034) and indent size (up to r = 0.82, p < 0.001). Therefore, crack resistance, deformation resistance and, additionally, micro-structure (porosity: r = 0.93, p = 0.002 as well as pore proximity: r = -0.55, p = 0.027 for correlation with IDI) are all contributory to RPI. Consequently, it becomes clear that RPI measures represent a multitude of properties, various aspects of bone quality, but are not necessarily strongly correlated to a single mechanical property. In addition, osteoporosis or osteoarthritis do not seem to further influence fracture toughness of the inferomedial femoral neck beyond natural ageing. Since bone is highly heterogeneous, whether this finding can be extended to the whole femoral neck or whether it also holds true for other femoral neck quadrants or other material properties remains to be shown.

Structure-mechanics relationships of collagen fibrils in the osteogenesis imperfecta mouse model.

The collagen molecule, which is the building block of collagen fibrils, is a triple helix of two α1(I) chains and one α2(I) chain. However, in the severe mouse model of osteogenesis imperfecta (OIM), deletion of the COL1A2 gene results in the substitution of the α2(I) chain by one α1(I) chain. As this substitution severely impairs the structure and mechanics of collagen-rich tissues at the tissue and organ level, the main aim of this study was to investigate how the structure and mechanics are altered in OIM collagen fibrils. Comparing results from atomic force microscopy imaging and cantilever-based nanoindentation on collagen fibrils from OIM and wild-type (WT) animals, we found a 33% lower indentation modulus in OIM when air-dried (bound water present) and an almost fivefold higher indentation modulus in OIM collagen fibrils when fully hydrated (bound and unbound water present) in phosphate-buffered saline solution (PBS) compared with WT collagen fibrils. These mechanical changes were accompanied by an impaired swelling upon hydration within PBS. Our experimental and atomistic simulation results show how the structure and mechanics are altered at the individual collagen fibril level as a result of collagen gene mutation in OIM. We envisage that the combination of experimental and modelling approaches could allow mechanical phenotyping at the collagen fibril level of virtually any alteration of collagen structure or chemistry.

Variability in reference point microindentation and recommendations for testing cortical bone: location, thickness and orientation heterogeneity.

Reference Point Indentation (RPI) has been proposed as a new clinical tool to aid the diagnosis of Osteoporosis. This study has examined the performance of the tool within entire femurs to improve the understanding of the mechanical properties of bone and also to guide future RPI testing to optimize repeatability of results obtained using the technique. Human, bovine, porcine and rat femurs were indented along three longitudinal axes: anterior and posterior: medial and lateral as well as around the circumference of the femoral head and neck. Cortical and subchondral bone thickness was measured using CT and radiography. The study shows that in some samples, bone is too thin to support the high loads applied with the technique and in these cases, RPI values are highly influenced by thickness. The technique will be useful in the mid-shaft region where cortical thickness is greatest, providing previously established guidelines are followed to optimize measurement repeatability, including performing multiple measurements per sample and investigating multiple samples. The study has also provided evidence that RPI values vary significantly with test site, hence mechanical properties should not be inferred from RPI findings alone away from the test site, even within the same bone. In conclusion, RPI appears to be a useful tool for scientific investigation; however further work is required to examine the feasibility of using RPI for assessing differences between healthy and diseased bone in a clinical setting.

An experimental and computational study of the hydrodynamics of high-velocity water microdrops for interproximal tooth cleaning.

The flow field and local hydrodynamics of high-velocity water microdrops impacting the interproximal (IP) space of typodont teeth were studied experimentally and computationally. Fourteen-day old Streptococcus mutans biofilms in the IP space were treated by a prototype AirFloss delivering 115 µL of water at a maximum exit-velocity of 60 ms(-1) in a 33-ms burst. Using high-speed imaging, footage was generated showing the details of the burst, and demonstrating the removal mechanism of the biofilms. Footage was also generated to characterize the viscoelastic behavior of the biofilms when impacted by an air-only burst, which was compared to the water burst. Image analysis demonstrated the importance of fluid forces on the removal pattern of interdental biofilms. X-ray micro-Computed Tomography (µ-CT) was used to obtain 3D images of the typodont and the IP spaces. Computational Fluid Dynamics (CFD) simulations were performed to study the effect of changing the nozzle position and design on the hydrodynamics within the IP space. Results confirmed our previous data regarding the wall shear stress generated by high-velocity water drops which dictated the efficacy of biofilm detachment. Finally, we showed how CFD models could be used to optimize water drop or burst design towards a more effective biofilm removal performance.

Variability in reference point microindentation and recommendations for testing cortical bone: maximum load, sample orientation, mode of use, sample preparation and measurement spacing.

Reference Point Indentation (RPI) is a novel microindentation tool that has emerging clinical potential for the assessment of fracture risk as well as use as a laboratory tool for straight-forward mechanical characterisation of bone. Despite increasing use of the tool, little research is available to advise the set-up of testing protocols or optimisation of testing parameters. Here we consider five such parameters: maximum load, sample orientation, mode of use, sample preparation and measurement spacing, to investigate how they affect the Indentation Distance Increase (IDI), the most published measurement parameter associated with the RPI device. The RPI tool was applied to bovine bone; indenting in the proximal midshaft of five femora and human bone; indenting five femoral heads and five femoral neck samples. Based on the findings of these studies we recommend the following as the best practice. (1) Repeat measurements should be utilised to reduce the coefficient of variation (e.g. 8-15 repeats to achieve a 5-10% error, however the 3-5 measurements used here gives a 15-20% error). (2) IDI is dependent on maximum load (r=0.45 on the periosteal surface and r=0.94 on the machined surface, p<0.05), mode of use (i.e. comparing the device held freehand compared to fixed in its stand, p=0.04) and surface preparation (p=0.004) so these should be kept consistent throughout testing. Though sample orientation appears to have minimal influence on IDI (p>0.05), care should also be taken in combining measurements from different orientations. (3) The coefficient of variation is higher (p=0.04) when holding the device freehand, so it should ideally be kept supported in its stand. (4) Removing the periosteum (p=0.04) and machining the surface of the bone (p=0.08) reduces the coefficient of variation, so should be performed where practical. (5) There is a hyperbolic relationship between thickness and IDI (p<0.001) with a sample thickness 10 fold greater than the maximum indentation depth recommended, to ensure a representative measurement. (6) Measurement spacing does not appear to influence the IDI (p>0.05), so it can be as low as 500 µm. By following these recommendations, RPI users can minimise the potential confounding effects associated with the variables investigated here and reduce the coefficient of variation, hence achieving more consistent testing. This optimisation of the technique enhances both the clinical and laboratory potential of the tool.

Anisotropy of bovine cortical bone tissue damage properties.

Bone is a heterogeneous, anisotropic natural composite material. Several studies have measured human cortical bone elastic properties in different anatomical directions and found that the Young's modulus was highest in the longitudinal, followed by the tangential and then by the radial direction. This study compared the Young's modulus, the accumulated microdamage and local strains related to the failure process in these three anatomical directions. Cortical bone samples (≈360 µm×360 µm) were mechanically tested in three-point bending and concomitantly imaged to assess local strains using digital image correlation technique. The bone whitening effect was used to detect microdamage formation and propagation. No statistically significant difference was found between the Young's modulus of longitudinal (9.4±2.0 GPa) and tangential (9.9±1.8 GPa) bovine bone samples, as opposed to previous findings on human bone samples. The same similarity was found for the whitening values (5000±1900 pix/mm(2) for longitudinal, 5800±2600 pix/mm(2) for tangential) and failure strains (16.8±7.0% for longitudinal, 19.1±3.2% for tangential) as well. However, significantly lower values were observed in the radial samples for Young's modulus (5.92±0.77 GPa), whitening (none or minimal) and failure strain (10.8±3.8%). For strains at whitening onset, no statistically significant difference was seen for the longitudinal (5.1±1.6%) and radial groups (4.2±2.0%), however, the tangential values were significantly greater (7.0±2.4%). The data implies that bovine cortical bone tissue in long bones is designed to withstand higher loads in the longitudinal and tangential directions than in the radial one. A possible explanation of the anisotropy in the mechanical parameters derived here might be the structure of the tissues in the three directions tested.

Similar damage initiation but different failure behavior in trabecular and cortical bone tissue.

The mechanical properties of bone tissue are reflected in its micro- and nanostructure as well as in its composition. Numerous studies have compared the elastic mechanical properties of cortical and trabecular bone tissue and concluded that cortical bone tissue is stiffer than trabecular bone tissue. This study compared the progression of microdamage leading to fracture and the related local strains during this process in trabecular and cortical bone tissue. Unmachined single bovine trabeculae and similarly-sized cortical bovine bone samples were mechanically tested in three-point bending and concomitantly imaged to assess local strains using a digital image correlation technique. The bone whitening effect was used to detect microdamage formation and propagation. This study found that cortical bone tissue exhibits significantly lower maximum strains (trabecular 36.6%±14% vs. cortical 22.9%±7.4%) and less accumulated damage (trabecular 16100±8800 pix/mm2 vs. cortical 8000±3400 pix/mm2) at failure. However, no difference was detected for the maximum local strain at whitening onset (trabecular 5.8%±2.6% vs. cortical 7.2%±3.1%). The differences in elastic modulus and mineral distribution in the two tissues were investigated, using nanoindentation and micro-Raman imaging, to explain the different mechanical properties found. While cortical bone was found to be overall stiffer and more highly mineralized, no apparent differences were noted in the distribution of modulus values or mineral density along the specimen diameter. Therefore, differences in the mechanical behavior of trabecular and cortical bone tissue are likely to be in large part due to microstructural (i.e. orientation and distribution of cement lines) and collagen related compositional differences.

Site specific increase in heterogeneity of trabecular bone tissue mineral during oestrogen deficiency.

Although osteoporosis reduces overall bone mass causing bone fragility, recent studies report that the remaining bone tissue is significantly stiffer. Preliminary studies indicate that alterations in bone tissue mineral content might explain these changes, albeit that other studies report conflicting observations. The objective of this study is to quantify whether the distribution of bone tissue mineral is altered during oestrogen deficiency. Individual trabeculae were harvested from the proximal femur of 7 ovariectomised sheep (OVX), sacrificed 12 months post-surgery, and 5 age-matched controls. Mineral content (wt% Ca) was determined using a quantitative backscattered scanning electron microscopy imaging approach. Mineral heterogeneity within individual trabeculae was compared by calculating the full width at half maximum (FWHM) of mineral density distributions. Mean calcium content, the spatial distribution of mineral within trabeculae and the inter-trabecular variation between regions of proximal femora were also compared. Oestrogen deficiency increased mineral heterogeneity within individual trabeculae compared to healthy controls, as measured by FWHM (3.57 ± 0.68 vs. 3.17 ± 0.36 wt% Ca, p = 0.04). In particular mineral variability increased between superficial and deep regions of trabeculae of OVX animals (p = 0.04). Interestingly, mineralisation variability between greater and lesser trochanters (i.e. intertrochanteric fracture line) was increased in OVX compared to CON, as indicated by a greater % difference in the standard deviation of trabecular mineral content (77.11 ± 11.70 vs. 45.64 ± 23.70 %, p = 0.03). Such changes are undetectable by evaluating the mean mineral content of bone tissue, but may contribute to changes in bone mechanical strength following osteoporotic bone loss.

Local strain and damage mapping in single trabeculae during three-point bending tests.

The use of bone mineral density as a surrogate to diagnose bone fracture risk in individuals is of limited value. However, there is growing evidence that information on trabecular microarchitecture can improve the assessment of fracture risk. One current strategy is to exploit finite element analysis (FEA) applied to 3D image data of several mm-sized trabecular bone structures obtained from non-invasive imaging modalities for the prediction of apparent mechanical properties. However, there is a lack of FE damage models, based on solid experimental facts, which are needed to validate such approaches and to provide criteria marking elastic-plastic deformation transitions as well as microdamage initiation and accumulation. In this communication, we present a strategy that could elegantly lead to future damage models for FEA: direct measurements of local strains involved in microdamage initiation and plastic deformation in single trabeculae. We use digital image correlation to link stress whitening in bone, reported to be correlated to microdamage, to quantitative local strain values. Our results show that the whitening zones, i.e. damage formation, in the presented loading case of a three-point bending test correlate best with areas of elevated tensile strains oriented parallel to the long axis of the samples. The average local strains along this axis were determined to be (1.6±0.9)% at whitening onset and (12±4)% just prior to failure. Overall, our data suggest that damage initiation in trabecular bone is asymmetric in tension and compression, with failure originating and propagating over a large range of tensile strains.

Mechanical properties of single bovine trabeculae are unaffected by strain rate.

For a better understanding of traumatic bone fractures, knowledge of the mechanical behavior of bone at high strain rates is required. Importantly, it needs to be clarified how quasistatic mechanical testing experiments relate to real bone fracture. This merits investigating the mechanical behavior of bone with an increase in strain rate. Various studies examined how cortical and trabecular bone behave at varying strain rates, but no one has yet looked at this question using individual trabeculae. In this study, three-point bending tests were carried out on bovine single trabeculae excised from a proximal femur to test the trabecular material's strain rate sensitivity. An experimental setup was designed, capable of measuring local strains at the surface of such small specimens, using digital image correlation. Microdamage was detected using the bone whitening effect. Samples were tested through two orders of magnitude, at strain rates varying between 0.01 and 3.39 s(-1). No linear relationship was observed between the strain rate and the Young's modulus (1.13-16.46 GPa), the amount of microdamage, the maximum tensile strain at failure (14.22-61.65%) and at microdamage initiation (1.95-12.29%). The results obtained in this study conflict with previous studies reporting various trends for macroscopic cortical and trabecular bone samples with an increase in strain rate. This discrepancy might be explained by the bone type, the small sample geometry, the limited range of strain rates tested here, the type of loading and the method of microdamage detection. Based on the results of this study, the strain rate can be ignored when modeling trabecular bone.

In situ observation of fluoride-ion-induced hydroxyapatite-collagen detachment on bone fracture surfaces by atomic force microscopy.

The topography of freshly fractured bovine and human bone surfaces was determined by the use of atomic force microscopy (AFM). Fracture surfaces from both kinds of samples exhibited complex landscapes formed by hydroxyapatite mineral platelets with lateral dimensions ranging from ∼90 nm × 60 nm to ∼20 nm × 20 nm. Novel AFM techniques were used to study these fracture surfaces during various chemical treatments. Significant topographical changes were observed following exposure to aqueous solutions of ethylenediaminetetraacetic acid (EDTA) or highly concentrated sodium fluoride (NaF). Both treatments resulted in the apparent loss of the hydroxyapatite mineral platelets on a timescale of a few seconds. Collagen fibrils situated beneath the overlying mineral platelets were clearly exposed and could be resolved with high spatial resolution in the acquired AFM images. Time-dependent mass loss experiments revealed that the applied agents (NaF or EDTA) had very different resulting effects. Despite the fact that the two treatments exhibited nearly identical results following examination by AFM, bulk bone samples treated with EDTA exhibited a ∼70% mass loss after 72 h, whereas for the NaF-treated samples, the mass loss was only of the order of ∼10%. These results support those obtained from previous mechanical testing experiments, suggesting that enhanced formation of superficial fluoroapatite dramatically weakens the protein-hydroxyapatite interfaces. Additionally, we discovered that treatment with aqueous solutions of NaF resulted in the effective extraction of noncollagenous proteins from bone powder.

Time-lapsed investigation of three-dimensional failure and damage accumulation in trabecular bone using synchrotron light.

Synchrotron radiation micro-computed tomography (SRmicroCT) is a very useful technique when it comes to three-dimensional (3D) imaging of complex internal and external geometries. Being a fully non-destructive technique, SRmicroCT can be combined with other experiments in situ for functional imaging. We are especially interested in the combination of SRmicroCT with mechanical testing in order to gain new insights in the failure mechanism of trabecular bone. This interest is motivated by the immense costs in health care due to patients suffering from osteoporosis, a systemic skeletal disease resulting in decreased bone stability and increased fracture risk. To better investigate the different failure mechanisms on the microlevel, we have developed a novel in situ mechanical compression device, capable of exerting both static and dynamic displacements on experimental samples. The device was calibrated for mechanical testing using solid aluminum and bovine trabecular bone samples. To study different failure mechanisms in trabecular bone, we compared a fatigued and a non-fatigued bovine bone sample with respect to failure initiation and propagation. The fatigued sample failed in a burst-like fashion in contrast to the non-fatigued sample, which exhibited a distinct localized failure band. Moreover, microscopic cracks - microcracks and microfractures - were uncovered in a 3D fashion illustrating the failure process in great detail. The majority of these cracks were connected to a bone surface. The data also showed that the classification of microcracks and -fractures from 2D section can sometimes be ambiguous, which is also true for the distinction of diffuse and distinct microdamage. Detailed investigation of the failure mechanism in these samples illustrated that trabecular bone often fails in delamination, providing a mechanism for energy dissipation while conserving trabecular bone architecture. In the future, this will allow an even better understanding of bone mechanics related to its hierarchical structural organization.