Method for assessing coating uniformity of angioplasty balloons coated with poly(lactic-co-glycolic acid) nanoparticles loaded with quercetin.

In this study, we aim to quantify coating uniformity and correlate fluorescence intensity to drug loading for drug-coated angioplasty balloons (DCB) coated with 5, 10, 15, or 20 layers of poly(lactic-co-glycolic acid) nanoparticles (NPs) entrapped with quercetin. Uniformity was quantified from histograms and horizontal line profiles of microscopic fluorescent images acquired with sample specific parameters, and cracks in the coating were measured and counted. The fluorescence of images acquired with global parameters was correlated with quercetin loading measured via gravimetric/HPLC analysis. More layers on DCBs may be associated with less uniform coatings, as indicated by differences in histogram standard deviations. The line profile percent deviation from average for each sample was <20%. Cracks were present on all balloons, but their length was not significantly different between samples. The 5-layer DCBs had the fewest cracks, whereas the 15-layer DCBs had the most cracks. A strong positive correlation (R = 0.896) was identified between fluorescence intensity and drug loading. A relationship between the number of layers and coating uniformity seems to exist, but further investigations are required for confirmation. Fluorescence intensity appears to strongly predict drug loading, demonstrating that fluorescent imaging may be a viable alternative to drug release studies.

Method for assessing coating uniformity of angioplasty balloons coated with poly(lactic-co-glycolic acid) nanoparticles loaded with quercetin.

Significance: Drug-coated angioplasty balloons (DCBs) are used to treat peripheral artery disease, and proper dosage depends on coating characteristics like uniformity and number of layers. Aim: Quantify coating uniformity and correlate fluorescence intensity to drug loading for DCBs coated with 5, 10, 15, or 20 layers of poly(lactic-co-glycolic acid) nanoparticles (NPs) entrapped with quercetin. Approach: Images of DCBs were acquired using fluorescence microscopy. Coating uniformity was quantified from histograms and horizontal line profiles, and cracks on the balloons were measured and counted. Fluorescence intensity was correlated with the drug loading of quercetin found from gravimetric analysis coupled with high-performance liquid chromatography (HPLC). Results: Higher numbers of coating layers on DCBs may be associated with less uniform coatings. Cracks in the coating were present on all balloons, and the length of cracks was not significantly different between balloons coated with different numbers of layers or balloons coated with the same number of layers. A strong positive correlation was identified between fluorescence intensity and drug loading. Conclusion: There may be a relationship between the number of NP layers and the uniformity of the coating, but further investigation is needed to confirm this. Fluorescence intensity appears to be a strong predictor of drug loading on DCBs coated with quercetin-entrapped NPs, demonstrating that fluorescent imaging may be a viable alternative to drug release studies.

Evaluating differences in Young's Modulus of regenerated and uninjured mouse digit bone through microCT density-based calculation and nanoindentation testing.

The mouse digit tip amputation model is an excellent model of bone regeneration, but its size and shape present an obstacle for biomechanical testing. As a result, assessing the structural quality of the regenerated bone in this model has focused on mineral density and bone architecture analysis. Here we describe an image-processing based method for assessment of mechanical properties in the regenerated digit by using micro-computed tomography mineral density data to calculate spatially discrete Young's modulus values throughout the entire distal third phalange. Further, we validate this method through comparison to nanoindentation-measured values for Young's modulus. Application to a set of regenerated and unamputated digits shows that regenerated bone has a lower Young's modulus compared to the uninjured digit, with a similar trend for experimental hardness values. Importantly, this method heightens the utility of the digit regeneration model, allows for more impactful treatment evaluation using the model, and introduces an analysis platform that can be used for other bones that do not conform to a standard long-bone model.

Spatial transcriptomics reveals metabolic changes underly age-dependent declines in digit regeneration.

De novo limb regeneration after amputation is restricted in mammals to the distal digit tip. Central to this regenerative process is the blastema, a heterogeneous population of lineage-restricted, dedifferentiated cells that ultimately orchestrates regeneration of the amputated bone and surrounding soft tissue. To investigate skeletal regeneration, we made use of spatial transcriptomics to characterize the transcriptional profile specifically within the blastema. Using this technique, we generated a gene signature with high specificity for the blastema in both our spatial data, as well as other previously published single-cell RNA-sequencing transcriptomic studies. To elucidate potential mechanisms distinguishing regenerative from non-regenerative healing, we applied spatial transcriptomics to an aging model. Consistent with other forms of repair, our digit amputation mouse model showed a significant impairment in regeneration in aged mice. Contrasting young and aged mice, spatial analysis revealed a metabolic shift in aged blastema associated with an increased bioenergetic requirement. This enhanced metabolic turnover was associated with increased hypoxia and angiogenic signaling, leading to excessive vascularization and altered regenerated bone architecture in aged mice. Administration of the metabolite oxaloacetate decreased the oxygen consumption rate of the aged blastema and increased WNT signaling, leading to enhanced in vivo bone regeneration. Thus, targeting cell metabolism may be a promising strategy to mitigate aging-induced declines in tissue regeneration.

Age-Dependent Changes in Bone Architecture, Patterning, and Biomechanics During Skeletal Regeneration.

Mouse digit amputation provides a useful model of bone growth after injury, in that the injury promotes intramembranous bone formation in an adult animal. The digit tip is composed of skin, nerves, blood vessels, bones, and tendons, all of which regenerate after digit tip amputation, making it a powerful model for multi-tissue regeneration. Bone integrity relies upon a balanced remodeling between bone resorption and formation, which, when disrupted, results in changes to bone architecture and biomechanics, particularly during aging. In this study, we used recently developed techniques to evaluate bone patterning differences between young and aged regenerated bone. This analysis suggests that aged mice have altered trabecular spacing and patterning and increased mineral density of the regenerated bone. To further characterize the biomechanics of regenerated bone, we measured elasticity using a micro-computed tomography image-processing method combined with nanoindentation. This analysis suggests that the regenerated bone demonstrates decreased elasticity compared with the uninjured bone, but there is no significant difference in elasticity between aged and young regenerated bone. These data highlight distinct architectural and biomechanical differences in regenerated bone in both young and aged mice and provide a new analysis tool for the digit amputation model to aid in evaluating the outcomes for potential therapeutic treatments to promote regeneration.

A new approach to analyzing regenerated bone quality in the mouse digit amputation model using semi-automatic processing of microCT data.

Bone regeneration is a critical area of research impacting treatment of diseases such as osteoporosis, age-related decline, and orthopaedic implants. A crucial question in bone regeneration is that of bone architectural quality, or how "good" is the regenerated bone tissue structurally? Current methods address typical long bone architecture, however there exists a need for improved ability to quantify structurally relevant parameters of bone in non-standard bone shapes. Here we present a new analysis approach based on open-source semi-automatic methods combining image processing, solid modeling, and numerical calculations to analyze bone tissue at a more granular level using µCT image data from a mouse digit model of bone regeneration. Examining interior architecture, growth patterning, spatial mineral content, and mineral density distribution, these methods are then applied to two types of 6-month old mouse digits - 1) those prior to amputation injury (unamputated) and 2) those 42 days after amputation when bone has regenerated. Results show regenerated digits exhibit increased inner void fraction, decreased patterning, different patterns of spatial mineral distribution, and increased mineral density values when compared to unamputated bone. Our approach demonstrates the utility of this new analysis technique in assessment of non-standard bone models, such as the regenerated bone of the digit, and aims to bring a deeper level of analysis with an open-source, integrative platform to the greater bone community.

Significant correlation of bone material strength index as measured by the OsteoProbe with Vickers and Rockwell hardness.

The bone material strength index (BMSi), as measured by the OsteoProbe, is significantly correlated with Vickers hardness and Rockwell (RW) hardness measurements on conventional materials. The Vickers and RW measurements were carried out according to American Society for Testing and Materials standard test methods, and OsteoProbe measurements followed published standardized testing methods. The correlations between the BMSi and RW hardness, r = 0.93, and between the BMSi and Vickers hardness, r = 0.94, are comparable with the correlation between RW and Vickers hardness, r = 0.87. The correlation between the BMSi and RW is significant at p < 0.01, and the correlation between the BMSi and Vickers hardness is significant at p < 0.01. These results show that the indentation measurement performed by the OsteoProbe may be considered as a type of hardness measurement comparable to widely used conventional methods, with specific applications targeted by its portable and narrow design.

Analyzing the effect of hydration on the wedge indentation fracture behavior of cortical bone.

Hydration directly affects the mechanical properties of bone. An initial and basic procedure shows both wedge indentation fracture experiments under plane strain conditions in cortical bone and numerical simulation with finite elements agree that dry bone fractures much more easily than fully hydrated bone submerged in an aqueous environment, such as in the body of an animal. The wedge indentation experiments were performed with high speed video microscopy, under dry and fully hydrated (submerged) conditions. The numerical simulation, specifically finite element analysis using cohesive elements to simulate fracture, was utilized to capture plasticity, fracture initiation and propagation, and to study the applicability of brittle material based indentation fracture theory. Experiment and theory give similar results for the dependence of depth of fracture initiation, and size of plastic zone, on hydration state. Comparison of fracture propagation characteristics between wet and dry bone are examined and discussed. This research demonstrates the ability to quantitatively assess the effect of hydration on the fracture initiation, propagation, and plastic zone size of cortical bone, through an approach using simple wedge indentation, with important implications for efforts in developing methods to understand clinical diagnostic testing and general fracture behavior of living bone in the ultimate interest of health care purposes.

Effect of various testing conditions on results for a handheld reference point indentation instrument in horses.

OBJECTIVE: To compare results obtained with a handheld reference point indentation instrument for bone material strength index (BMSi) measurements in the equine third metacarpal bone for various testing conditions. SAMPLE: 24 third metacarpal bones. PROCEDURES: Third metacarpal bones from both forelimbs of 12 horses were obtained. The dorsal surface of each bone was divided into 6 testing regions. In vivo and ex vivo measurements of BMSi were obtained through the skin and on exposed bone, respectively, to determine effects of each testing condition. Difference plots were used to assess agreement between BMSi obtained for various conditions. Linear regression analysis was used to assess effects of age, sex, and body weight on BMSi. A mixed-model ANOVA was used to assess effects of age, sex, limb, bone region, and testing condition on BMSi values. RESULTS: Indentation measurements were performed on standing sedated and recumbent anesthetized horses and on cadaveric bone. Regional differences in BMSi values were detected in adult horses. A significant linear relationship (r(2) = 0.71) was found between body weight and BMSi values. There was no difference between in vivo and ex vivo BMSi values. A small constant bias was detected between BMSi obtained through the skin, compared with values obtained directly on bone. CONCLUSIONS AND CLINICAL RELEVANCE: Reference point indentation can be used for in vivo assessment of the resistance of bone tissue to microfracture in horses. Testing through the skin should account for a small constant bias, compared with results for testing directly on exposed bone.

Study of indentation of a sample equine bone using finite element simulation and single cycle reference point indentation.

In an attempt to study the mechanical behavior of bone under indentation, methods of analyses and experimental validations have been developed, with a selected test material. The test material chosen is from an equine cortical bone. Stress-strain relationships are first obtained from conventional mechanical property tests. A finite element simulation procedure is developed for indentation analyses. The simulation results are experimentally validated by determining (1) the maximum depth of indentation with a single cycle type of reference point indentation, and (2) the profile and depth of the unloaded, permanent indentation with atomic force microscopy. The advantage of incorporating in the simulation a yield criterion calibrated by tested mechanical properties, with different values in tension and compression, is demonstrated. In addition, the benefit of including damage through a reduction in Young's modulus is shown in predicting the permanent indentation after unloading and recovery. The expected differences in response between two indenter tips with different sharpness are predicted and experimentally observed. Results show predicted indentation depths agree with experimental data. Thus, finite element simulation methods with experimental validation, and with damage approximation by a reduction of Young's modulus, may provide a good approach for analysis of indentation of cortical bone. These methods reveal that multiple factors affect measured indentation depth and that the shape of the permanent indentation contains useful information about bone material properties. Only further work can determine if these methods or extensions to these methods can give useful insights into bone pathology, for example the bone fragility of thoroughbred racehorses.

Applications of a New Handheld Reference Point Indentation Instrument Measuring Bone Material Strength.

A novel, hand-held Reference Point Indentation (RPI) instrument, measures how well the bone of living patients and large animals resists indentation. The results presented here are reported in terms of Bone Material Strength, which is a normalized measure of how well the bone resists indentation, and is inversely related to the indentation distance into the bone. We present examples of the instrument's use in: (1) laboratory experiments on bone, including experiments through a layer of soft tissue, (2) three human clinical trials, two ongoing in Barcelona and at the Mayo Clinic, and one completed in Portland, OR, and (3) two ongoing horse clinical trials, one at Purdue University and another at Alamo Pintado Stables in California. The instrument is capable of measuring consistent values when testing through soft tissue such as skin and periosteum, and does so handheld, an improvement over previous Reference Point Indentation instruments. Measurements conducted on horses showed reproducible results when testing the horse through tissue or on bare bone. In the human clinical trials, reasonable and consistent values were obtained, suggesting the Osteoprobe® is capable of measuring Bone Material Strength in vivo, but larger studies are needed to determine the efficacy of the instrument's use in medical diagnosis.