An improved linear systems model of hydrothermal isometric tension testing to aid in assessing bone collagen quality: Effects of ribation and type-2 diabetes.

This study sought to further develop and validate a previously proposed physics-based model that maps denaturation kinetics from differential scanning calorimetry (DSC) to the isometric tension generated during hydrothermal isometric tension (HIT) testing of collagenous tissues. The primary objectives of this study were to verify and validate two physics-based model parameters: α, which indicates the amount of instantaneous isometric tension developed per unit of collagen denaturation, and ß, which captures the proportionality between temperature and the generated isometric tension post denaturation initiation. These parameters were used as measures of bone collagen quality, employing data from HIT and DSC testing of human bone collagen from two previous studies. Additionally, given the physical basis of the model, the study aimed to further validate Max.Slope, the rate of change in isometric tensile stress with change in temperature, as an independent measure of collagen network connectivity. Max.Slope has previously been positively correlated with measures of cortical bone fracture resistance. Towards this verification and validation, the hypotheses were a) that α would correlate strongly with HIT denaturation temperature, Td, and the enthalpy of melting (ΔH) from DSC, and b) that ß would correlate positively and strongly with Max.Slope. The model was employed in the analysis of HIT-DSC data from the testing of demineralized bone collagen isolated from cadaveric human femurs in two prior studies. In one study, data were collected from HIT-DSC testing of cortical bone collagen from 74 donors. Among them, 38 had a history of type 2 diabetes +/- chronic kidney disease, while the remaining 36 had no history of T2D again with or without CKD. Cortical bone specimens were extracted from the lateral mid-shaft. The second study involved 15 donor femora, with four cortical bone specimens extracted from each. Of these four, two specimens underwent a 4-week incubation in 0.1 M ribose at 37 °C to induce non-enzymatic ribation and advanced glycation endproducts, while the other two served as non-ribated controls. The examination involved investigating correlations between the model parameters α and ß and various measures, such as Max.Slope, Td, ΔH, age, and duration of type 2 diabetes. The results revealed positive correlations between the model parameter ß and Max.Slope (r = 0.55-0.58). The parameter α was found to be associated with Td, but also sensitive to the shape of the HIT curve around Td resulting in difficulties with variability and interpretation. As a result, while both hypotheses are confirmed, Max.Slope and ß are better indicators of bone collagen quality because they are measures of the connectivity or, more generally, the integrity of the bone collagen network.

In vitro evaluation of bone cell response to novel 3D-printable nanocomposite biomaterials for bone reconstruction.

Critically-sized segmental bone defects represent significant challenges requiring grafts for reconstruction. 3D-printed synthetic bone grafts are viable alternatives to structural allografts if engineered to provide appropriate mechanical performance and osteoblast/osteoclast cell responses. Novel 3D-printable nanocomposites containing acrylated epoxidized soybean oil (AESO) or methacrylated AESO (mAESO), polyethylene glycol diacrylate, and nanohydroxyapatite (nHA) were produced using masked stereolithography. The effects of volume fraction of nHA and methacrylation of AESO on interactions of differentiated MC3T3-E1 osteoblast (dMC3T3-OB) and differentiated RAW264.7 osteoclast cells with 3D-printed nanocomposites were evaluated in vitro and compared with a control biomaterial, hydroxyapatite (HA). Higher nHA content and methacrylation significantly improved the mechanical properties. All nanocomposites supported dMC3T3-OB cells' adhesion and proliferation. Higher amounts of nHA enhanced cell adhesion and proliferation. mAESO in the nanocomposites resulted in greater adhesion, proliferation, and activity at day 7 compared with AESO nanocomposites. Excellent osteoclast-like cells survival, defined actin rings, and large multinucleated cells were only observed on the high nHA fraction (30%) mAESO nanocomposite and the HA control. Thus, mAESO-based nanocomposites containing higher amounts of nHA have better interactions with osteoblast-like and osteoclast-like cells, comparable with HA controls, making them a potential future alternative graft material for bone defect repair.

The effect of triglycerol diacrylate on the printability and properties of UV curable, bio-based nanohydroxyapatite composites.

3D printable biopolymer nanocomposites composed of hydroxyapatite nanoparticles and functionalized plant-based monomers demonstrate potential as sustainable and structural biomaterials. To increase this potential, their printability and performance must be improved. For extrusion-based 3D printing, such as Direct Ink Writing (DIW), printability is important for print fidelity. In this work, triglycerol diacrylate (TGDA) was added to an acrylated epoxidized soybean oil:polyethylene glycol diacrylate resin to increase hydrogen bonding. Greater hydrogen bonding was hypothesized to improve printability by increasing the ink's shear yield strength, and therefore shape holding after deposition. The effects of this additive on material and mechanical properties were quantified. Increased hydrogen bonding due to TGDA content increased the ink's shear yield stress and viscosity by 916% and 27.6%, respectively. This resulted in improved printability, with best performance at 3 vol% TGDA. This composition achieved an ultimate tensile strength (UTS) of 32.4 ± 2.1 MPa and elastic modulus of 1.15 ± 0.21 GPa. These were increased from the 0 vol% TGDA composite, which had an UTS of 24.8 ± 1.8 MPa and a modulus of 0.88 ± 0.06 GPa. This study demonstrates the development of bio-based additive manufacturing feedstocks for potential uses in sustainable manufacturing, rapid prototyping, and biomaterial applications.

Fracture Toughness: Bridging the Gap Between Hip Fracture and Fracture Risk Assessment.

PURPOSE OF REVIEW: This review surveys recent literature related to cortical bone fracture mechanics and its application towards understanding bone fragility and hip fractures. RECENT FINDINGS: Current clinical tools for hip fracture risk assessment have been shown to be insensitive in some cases of elevated fracture risk leading to the question of what other factors account for fracture risk. The emergence of cortical bone fracture mechanics has thrown light on other factors at the tissue level that are important to bone fracture resistance and therefore assessment of fracture risk. Recent cortical bone fracture toughness studies have shown contributions from the microstructure and composition towards cortical bone fracture resistance. A key component currently overlooked in the clinical evaluation of fracture risk is the importance of the organic phase and water to irreversible deformation mechanisms that enhance the fracture resistance of cortical bone. Despite recent findings, there is an incomplete understanding of which mechanisms lead to the diminished contribution of the organic phase and water to the fracture toughness in aging and bone-degrading diseases. Notably, studies of the fracture resistance of cortical bone from the hip (specifically the femoral neck) are few, and those that exist are mostly consistent with studies of bone tissue from the femoral diaphysis. Cortical bone fracture mechanics highlights that there are multiple determinants of bone quality and therefore fracture risk and its assessment. There is still much more to learn concerning the tissue-level mechanisms of bone fragility. An improved understanding of these mechanisms will allow for the development of better diagnostic tools and therapeutic measures for bone fragility and fracture.

Effect of ribose incubation on physical, chemical, and mechanical properties of human cortical bone.

Raman spectroscopy (RS) is sensitive to the accumulation of advanced glycation end-products (AGEs), and it measures matrix-sensitive properties that correlate with the fracture toughness of human cortical bone. However, it is unclear whether sugar-mediated accumulation of AGEs affects the fracture toughness of human cortical bone in a manner that is consistent with the negative correlations between amide I sub-peak ratios and fracture toughness. Upon machining 64 single-edge notched beam (SENB) specimens from cadaveric femurs (8 male and 7 female donors between 46 years and 61 years of age), pairs of SENB specimens were incubated in 15 mL of phosphate buffered saline with or without 0.1 M ribose for 4 weeks at 37 °C. After acquiring 10 Raman spectra per bone specimen (n = 32 per incubation group), paired SENB specimens were loaded in three-point bending at a quasi-static or a high loading rate approximating 10-4 s-1 or 10-2 s-1, respectively (n = 16 per incubation group per loading rate). While 2 amide I sub-peak ratios, I1670/I1640 and I1670/I1610, decreased by 3-5% with a 100% increase in AGE content, as confirmed by fluorescence measurements, the ribose incubation to accumulate AGEs in bone did not affect linear elastic (KIc) nor non-linear elastic (KJc) measurements of bone's ability to resist crack growth. Moreover, AGE accumulation did not affect the change in these properties when the loading rate changed. Increasing the loading rate increased KIc but decreased KJc. Ribose incubation did not affect mineral-related RS properties such as mineral-to-matrix ratios, Type B carbonate substitutions, and crystallinity. It did however increase the thermal stability of demineralized bone (differential scanning calorimetry), without affecting the network connectivity of the organic matrix (i.e., maximum slope during a hydrothermal isometric tension test of demineralized bone). In conclusion, RS is sensitive to AGE accumulation via the amide I band (plus the hydroxyproline-to-proline ratio), but the increase in AGE content due to ribose incubation was not sufficient to affect the fracture toughness of human cortical bone.

Enhanced mechanical performance of mSLA-printed biopolymer nanocomposites due to phase functionalization.

Functionalized phases can effectively increase the mechanical properties of nanocomposites through interfacial bonding. This work demonstrates masked stereolithography (mSLA) of biopolymer-based nanocomposites and the improvement of their mechanical properties by the functionalization of both polymer matrix and nanoparticles with methacrylate groups. 3D printable nanocomposite inks were prepared from plant-derived acrylated epoxidized soybean oil (AESO), polyethylene glycol diacrylate (PEGDA), and nano-hydroxyapatite (nHA). Both AESO and nHA were further functionalized with additional methacrylate groups. We hypothesized that the additional functionalization of AESO and surface functionalization of nHA would improve the tensile strength and fracture toughness of these nanocomposites by increasing the degree of crosslinking and the strength of the interface between the matrix and nanoparticles. Curing efficiency, rheology, and print-fidelity of the nanocomposites were evaluated. Mechanical test specimens were prepared by mSLA-based 3D printing. Tensile mechanical properties, Poisson's ratio, and Mode-I fracture toughness were measured by following ASTM standards. Fracture surfaces of the tested specimens were studied using scanning electron microscopy. Thermomechanical behavior, especially glass transition temperature (Tg), was studied using dynamic mechanical analysis (DMA). Functionalized AESO (mAESO) improved rheological, tensile, and fracture mechanical properties. For instance, by replacing AESO with mAESO, tensile strength, Young's modulus, fracture toughness (K1c), and Tg increased by 33%, 53%, 40%, and 38% respectively. In addition, the combination of both functionalized nHA and mAESO improved the fracture toughness of the 10% volume fraction nHA nanocomposites but made them less extensible presumably due to reduced chain mobility due to greater crosslinking.

A critical evaluation of cortical bone fracture toughness testing methods.

Cortical bone fracture mechanics which quantifies the tissue's resistance to fracture is widely regarded as important to finding key determinants of bone fragility and fracture. Currently, the most widely used fracture mechanics approach is the J-integral resistance (J-R) curve as defined in ASTM E1820 standard. This standard employs an unloading compliance (UC) method to estimate crack extension, necessary for fracture toughness and resistance curve (R-curve) quantification. Further, this UC method requires a series of unload-reload cycles to be conducted during the fracture test. However, cortical bone violates some assumptions on which the UC method is based, which are: no energy loss during the unload-reload cycles and any change in unloading compliance is only due to crack extension. Consequently, the aim of this study was to examine the impact of the UC method on the accuracy of fracture toughness measurement for bovine cortical bone. Ten pairs of single edged notched bend specimens were prepared from the posterior diaphysis of bovine tibiae and underwent three-point bending fracture tests. The paired specimens were divided into two groups: a cyclic loaded group and a monotonic loaded group. Further, crack extension was determined by the UC method for the cyclic group and by an optical method for both the cyclic and monotonic groups. From these, three different approaches were used to generate J-R curves from which three fracture toughness parameters were computed and compared between the three approaches. This comparison allowed the impact of crack extension estimation by the UC method as well as the unload-reload cycles on the accuracy of the fracture toughness measures to be assessed. Results show that the UC method underestimates crack extension by an average error of 73%. In addition, the combined effects from crack extension estimation using the UC method and the unload-reload cycles lead to a significant overestimation of the specimen's fracture toughness measures. This highlights the need for more studies to establish a standardized approach to cortical bone fracture testing.

The impact of fall-related loading rate on the formation of micro-damage in human cortical bone fracture.

The quest for better predictive tools as well as new preventative and therapeutic measures for bone fragility and fracture has highlighted the need for greater mechanistic understanding of the bone fracture process. Cortical bone, the major load bearing part of the bone, employs different toughening mechanisms to either inhibit or slow down crack growth which leads to fracture. Among these toughening mechanisms, is the formation of a micro-damage process zone (MDPZ) around the region of the propagating crack. Investigations into the MDPZ to date have primarily been based on quasi-static or cyclic loading rate experiments which do not necessarily replicate physiological fracture rates. Consequently, the impact of fall-related loading rates on the formation of the micro-damage process zone was investigated comparing these to quasi-static loading rate equivalents. The size of MDPZ was found to be 42% smaller in the high-rate group compared to the quasi-static rate group. The smaller MDPZ size was associated with a brittle, unstable fracture behaviour and an overall smaller fracture resistance measure (Jmax). This result points to the possibility of a strain rate hardening mechanism at the heart of micro-damage formation, which is hampered under high loading rates, resulting in lower overall fracture resistance.

Causative or associative: A critical review of the role of advanced glycation end-products in bone fragility.

The accumulation of advanced glycation end-products (AGEs) in the organic matrix of bone with aging and chronic disease such as diabetes is thought to increase fracture risk independently of bone mass. However, to date, there has not been a clinical trial to determine whether inhibiting the accumulation of AGEs is effective in preventing low-energy, fragility fractures. Moreover, unlike with cardiovascular or kidney disease, there are also no pre-clinical studies demonstrating that AGE inhibitors or breakers can prevent the age- or diabetes-related decrease in the ability of bone to resist fracture. In this review, we critically examine the case for a long-standing hypothesis that AGE accumulation in bone tissue degrades the toughening mechanisms by which bone resists fracture. Prior research into the role of AGEs in bone has primarily measured pentosidine, an AGE crosslink, or bulk fluorescence of hydrolysates of bone. While significant correlations exist between these measurements and mechanical properties of bone, multiple AGEs are both non-fluorescent and non-crosslinking. Since clinical studies are equivocal on whether circulating pentosidine is an indicator of elevated fracture risk, there needs to be a more complete understanding of the different types of AGEs including non-crosslinking adducts and multiple non-enzymatic crosslinks in bone extracellular matrix and their specific contributions to hindering fracture resistance (biophysical and biological). By doing so, effective strategies to target AGE accumulation in bone with minimal side effects could be investigated in pre-clinical and clinical studies that aim to prevent fragility fractures in conditions that bone mass is not the underlying culprit.

Empirical evidence that bone collagen molecules denature as a result of bone fracture.

Cortical bone tissue, primarily composed of collagen, hydroxyapatite, and water, is a strong and tough natural, structural biomaterial. The integrity of the collagenous phase (native triple helix vs. damaged/denatured coil) has previously been correlated via various means, including hydrothermal isometric tension testing and FTIR and Raman spectroscopy, with the capability of cortical bone to undergo stable fracture. Collagen is a relatively stable protein, requiring around 70 J/g to thermally denature its native triple helix structure, through the melting of hydrogen bonds. It is widely thought that bone collagen molecules denature (unravel) during fracture, acting as a molecular-scale mechanical toughening mechanism, but this has not been empirically demonstrated to date. A new technology, fluorescently-labeled collagen hybridizing peptides (F-CHP), enables imaging that specifically detects denatured collagen. This provides an opportunity to empirically test whether bone collagen molecules do denature during bone fracture. Here, F-CHP was used to stain fracture surfaces produced by transverse Mode-I fracture of chevron-notched bovine and human cortical bone beams. The fracture surfaces demonstrated increased staining, above the level of rigorous paired controls, and the staining directly correlated with the work-to-fracture (WFx) of bovine bone beams. This increased denaturation signal was also constrained to a rough textured region visible on the fracture surface, which is known to correspond with stable tearing. Similar staining was also detected on the fracture surfaces of human cortical bone. Increased staining was not detected on the fracture surfaces of specimens that were dehydrated prior to fracture, suggesting a role for water in the denaturation process. This study provides the first empirical evidence of bone collagen denaturation resulting from cortical bone fracture and extends our understanding of this mechanism towards the mechanical performance of cortical bone.

A linear systems model of the hydrothermal isometric tension test for assessing collagenous tissue quality.

Collagen is the most abundant structural protein in the animal kingdom. Its thermal and thermomechanical properties are often measured using differential scanning calorimetry (DSC) and hydrothermal isometric tension (HIT) tests, respectively. In living tissues, not all collagenous structures (molecules, fibrils, etc.) have the same "quality," and the heterogeneity among these structures in specific tissues increases with remodeling, aging, and/or disease states. In this paper, first, a peak-fitting analysis is carried out to separate and distinguish the sequential denaturation events in a DSC endotherm, which presumably stem from heterogeneity in the collagen fibrils. The fitting analysis uses one of two functions: a Gaussian function or a function proposed by Miles. The individual endotherms were then convolved with a physics-based parametric function, J(T), proposed by the authors, to model the development of the isometric tension in two stages: 1) tension development due to a sudden increase in conformational entropy as each collagen packet denatures, and 2) additional isometric tension development due to increasing temperature, consistent with rubber thermo-elasticity. The proposed function parameters were then found by fitting to actual HIT curves using a global optimization technique. This model provides a decoupling of the effects of denaturation kinetics and collagen network connectivity and therefore an improved interpretation of HIT test results during the temperature ramp from ambient temperature to 90 °C. The simple model outputs are two parameters, α and ß, that have physical meaning and aid in assessing collagenous tissue quality in terms of connectivity and integrity.

mSLA-based 3D printing of acrylated epoxidized soybean oil - nano-hydroxyapatite composites for bone repair.

Structural bone allografts are used to treat critically sized segmental bone defects (CSBDs) as such defects are too large to heal naturally. Development of biomaterials with competent mechanical properties that can also facilitate new bone formation is a major challenge for CSBD repair. 3D printed synthetic bone grafts are a possible alternative to structural allografts if engineered to provide appropriate structure with sufficient mechanical properties. In this work, we fabricated a set of novel nanocomposite biomaterials consisting of acrylated epoxidized soybean oil (AESO), polyethylene glycol diacrylate (PEGDA) and nanohydroxyapatite (nHA) by using masked stereolithography (mSLA)-based 3D printing. The nanocomposite inks possess suitable rheological properties and good printability to print complex, anatomically-precise, 'by design' grafts. The addition of nHA to the AESO/PEGDA resin improved the tensile strength and fracture toughness of the mSLA printed nanocomposites, presumably due to small-scale reinforcement. By adding 10 vol% nHA, tensile strength, modulus and fracture toughness (KIc) were increased to 30.8 ± 1.2 MPa (58% increase), 1984.4 ± 126.7 MPa (144% increase) and 0.6 ± 0.1 MPa·m1/2 (42% increase), respectively (relative to the pure resin). The nanocomposites did not demonstrate significant hydrolytic, enzymatic or oxidative degradation when incubated for 28 days, assuring chemical and mechanical stability at early stages of implantation. Apatite nucleated and covered the nanocomposite surfaces within 7 days of incubation in simulated body fluid. Good viability and proliferation of differentiated MC3T3-E1 osteoblasts were also observed on the nanocomposites. Taken all together, our nanocomposites demonstrate excellent bone-bioactivity and potential for bone defect repair.

Acrylated epoxidized soybean oil/hydroxyapatite-based nanocomposite scaffolds prepared by additive manufacturing for bone tissue engineering.

The mechanical properties and biocompatibility of nanocomposites composed of Acrylated Epoxidized Soybean Oil (AESO), nano-Hydroxyapatite (nHA) rods and either 2-Hydroxyethyl Acrylate (HEA) or Polyethylene Glycol Diacrylate (PEGDA) and 3D printed using extrusion-based additive manufacturing methods were investigated. The effects of addition of HEA or PEGDA on the rheological, mechanical properties and cell-biomaterial interactions were studied. AESO, PEGDA (or HEA), and nHA were composited using an ultrasonic homogenizer and scaffolds were 3D printed using a metal syringe on an extrusion-based 3D printer while simultaneously UV cured during layer-by-layer deposition. Nanocomposite inks were characterized for their viscosity before curing, and dispersion of the nHA particles and tensile mechanical properties after curing. Proliferation and differentiation of human bone marrow-derived mesenchymal stem cells (BM-MSCs) were studied by seeding cells onto the scaffolds and culturing in osteogenic differentiation medium for 7, 14 and 21 days. Overall, each of the scaffolds types demonstrated controlled morphology resulting from the printability of nanocomposite inks, well-dispersed nHA particles within the polymer matrices, and were shown to support cell proliferation and osteogenic differentiation after 14 and 21 days of culture. However, the nature of the functional groups present in each ink detectably affected the mechanical properties and cytocompatibility of the scaffolds. For example, while the incorporation of HEA reduced nHA dispersion and tensile strength of the final nanocomposite, it successfully enhanced shear yield strength, and printability, as well as cell adhesion, proliferation and osteogenic differentiation, establishing a positive effect perhaps due to additional hydrogen bonding.

Effect of sterilization treatment on mechanical properties, biodegradation, bioactivity and printability of GelMA hydrogels.

Gelatin methacryloyl (GelMA) hydrogel scaffolds and GelMA-based bioinks are widely used in tissue engineering and bioprinting due to their ability to support cellular functions and new tissue development. Unfortunately, while terminal sterilization of the GelMA is a critical step for translational tissue engineering applications, it can potentially cause thermal or chemical modifications of GelMA. Thus, understanding the effect of terminal sterilization on GelMA properties is an important, though often overlooked, aspect of material design for translational tissue engineering applications. To this end, we characterized the effects of FDA-approved terminal sterilization methods (autoclaving, ethylene oxide treatment, and gamma (γ)-irradiation) on GelMA prepolymer (bioink) and GelMA hydrogels in terms of the relevant properties for biomedical applications, including mechanical strength, biodegradation rate, cell culture in 2D and 3D, and printability. Autoclaving and ethylene oxide treatment of the GelMA decreased the stiffness of the hydrogel, but the treatments did not modify the biodegradation rate of the hydrogel; meanwhile, γ-irradiation increased the stiffness, reduced the pore size and significantly slowed the biodegradation rate. None of the terminal sterilization methods changed the 2D fibroblast or endothelial cell adhesion and spreading. However, ethylene oxide treatment significantly lowered the fibroblast viability in 3D cell culture. Strikingly, γ-irradiation led to significantly reduced ability of the GelMA prepolymer to undergo sol-gel transition. Furthermore, printability studies showed that the bioinks prepared from γ-irradiated GelMA had significantly reduced printability as compared to the GelMA bioinks prepared from autoclaved or ethylene oxide treated GelMA. These results reveal that the choice of the terminal sterilization method can strongly influence important properties of GelMA bioink and hydrogel. Overall, this study provides further insight into GelMA-based material design with consideration of the effect of terminal sterilization.

Mechanical properties of nanocomposite biomaterials improved by extrusion during direct ink writing.

In this study, single filaments of acrylated epoxidized soybean oil (AESO)/polyethylene glycol diacrylate (PEGDA)/nanohydroxyapatite (nHA)-based nanocomposites intended for bone defect repair have displayed significant improvement of their mechanical properties when extruded through smaller needle gauges before UV curing. These nanocomposite inks can be deposited layer-by-layer during direct ink writing (DIW) - a form of additive manufacturing. Single filaments were prepared by extruding the nanocomposite ink through needles with varying diameters from 0.21 mm to 0.84 mm and then UV cured. Filaments and cast specimens were tensile tested to determine elastic modulus, strength and toughness. The cured nanocomposite filaments were further characterized using thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM). SEM confirmed that the hydroxyapatite nanoparticles were well dispersed in the polymer matrices. The ultimate tensile strength and moduli increased as the diameter of the extrusion needle was decreased. These correlated with increased matrix crystallinity and fewer defects. For instance, filaments extruded through 0.84 mm diameter needles had ultimate tensile stress and modulus of 26.3 ± 2.8 MPa and 885 ± 100 MPa, respectively, whereas, filaments extruded through 0.21 mm needles had ultimate tensile stress and modulus of 48.9 ± 4.0 MPa and 1696 ± 172 MPa, respectively. This study has demonstrated enhanced mechanical properties resulting from extrusion-based direct ink writing of a new AESO-PEGDA-nHA nanocomposite biomaterial intended for biomedical applications. These enhanced properties are the result of fewer defects and increased crystallinity. A means of achieving mechanical properties suitable for repairing bone defects is apparent.

Ribose pre-treatment can protect the fatigue life of γ-irradiation sterilized bone.

Structural bone allografts are often sterilized with γ-irradiation to decrease infection risk, which unfortunately degrades the bone collagen connectivity, making the bone weak and brittle. In previous studies, we successfully protected the quasi-static mechanical properties of human cortical bone by pre-treating with ribose, prior to irradiation. This study focused on the quasi-static and fatigue tensile properties of ribose treated irradiated sterilized bone allografts. Seventy-five samples were cut from the mid-shaft diaphysis of human femurs into standardized dog-bone shape geometries for quasi-static and fatigue tensile testing. Specimens were prepared in sets of three adjacent specimens. Each set was made of a normal (N), irradiated (I) and ribose pre-treated + irradiation (R) group. The R group was incubated in a 1.2 M ribose solution before γ-irradiation. The quasi-static tensile and decalcified tests were conducted to failure under displacement control. The fatigue samples were tested under cyclic loading (10 Hz, peak stress of 45MP, minimum-to-maximum stress ratio of 0.1) until failure or reaching 10 million cycles. Ribose pre-treatment significantly improved significantly the mechanical properties of irradiation sterilized human bone in the quasi-static tensile and decalcified tests. The fatigue life of the irradiated group was impaired by 99% in comparison to the normal control. Surprisingly, the R-group has significantly superior properties over the I-group and N-group (p < 0.01, p < 0.05) (> 100%). This study shows that incubating human cortical bone in a ribose solution prior to irradiation can indeed improve the fatigue life of irradiation-sterilized cortical bone allografts.

Pre-clinical evaluation of bone allograft toughened with a novel sterilization method: An in vivo rabbit study.

Bone allografts often undergo γ-irradiation sterilization to decrease infection risk. However this consequently degrades bone collagen and makes the allograft brittle. Our laboratory has previously found that pre-treatment with ribose ex vivo protects the bone. However, it remains unclear whether or not ribose-treated γ-irradiated allografts are able to unite and remodel in vivo. Using New Zealand White rabbits (NZWr), we aimed to evaluate if ribose-treated allografts can unite with host bone (compared to untreated (fresh-frozen) and conventionally-irradiated allografts). A critically-sized defect was created in the radii of NZWr and reconstructed with allografts fixed with an intramedullary Kirschner wire. Healing and union were assessed at 2, 6, and 12 weeks post operation, with radiographs, µCT, static and dynamic histomorphometry, backscatter electron microscopy, and torsion testing. Intramedullary fixation achieved stable reconstructions and bony union in all groups and no differences were found in the radiographic and biomechanical parameters tested. Interestingly, γ-irradiated allografts had significantly less bone volume due to evident resorption of the grafts. In contrast, ribose pre-treatment protected γ-irradiated allografts from this bone loss, with results similar to the fresh frozen controls. In conclusion, ribose-pretreated γ-irradiated allografts were able to unite in vivo. In addition to achieving bony union with host bone, ribose pre-treatment may protect against allograft resorption. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Bone collagen network integrity and transverse fracture toughness of human cortical bone.

Greater understanding of the determinants of skeletal fragility is highly sought due to the great burden that bone affecting diseases and fractures have on economies, societies and health care systems. Being a complex, hierarchical composite of collagen type-I and non-stoichiometric substituted hydroxyapatite, bone derives toughness from its organic phase. In this study, we tested whether early observations that a strong correlation between bone collagen integrity measured by thermomechanical methods and work to fracture exist in a more general and heterogeneous sampling of the population. Neighboring uniform specimens from an established, highly characterized and previously published collection of human cortical bone samples (femur mid-shaft) were decalcified in EDTA. Fifty-four of the original 62 donors were included (26 male and 28 females; ages 21-101 years; aging, osteoporosis, diabetes and cancer). Following decalcification, bone collagen was tested using hydrothermal isometric tension (HIT) testing in order to measure the collagen's thermal stability (denaturation temperature, Td) and network connectivity (maximum rate of isometric tension generation; Max.Slope). We used linear regression and general linear models (GLMs) with several explanatory variables to determine whether relationships between HIT parameters and generally accepted bone quality factors (e.g., cortical porosity, pentosidine content [pen], pyridinoline content [pyd]), age, and measures of fracture toughness (crack initiation fracture toughness, Kinit, and total energy release/dissipation rate evaluated at the point of unstable fast fracture, J-int) were significant. Bone collagen connectivity (Max.Slope) correlated well with the measures of fracture toughness (R2 = 24-35%), and to a lesser degree with bound water fraction (BW; R2 = 7.9%) and pore water fraction (PW; R2 = 9.1%). Significant correlations with age, apparent volumetric bone mineral density (vBMD), and mature enzymatic [pyd] and non-enzymatic collagen crosslinks [pen] were not detected. GLMs found that Max.Slope and vBMD (or BW), with or without age as additional covariate, all significantly explained the variance in Kinit (adjusted-R2 = 36.7-49.0%). Also, the best-fit model for J-int (adjusted-R2 = 35.7%) included only age and Max.Slope as explanatory variables with Max.Slope contributing twice as much as age. Max.Slope and BW without age were also significant predictors of J-int (adjusted-R2 = 35.5%). In conclusion, bone collagen integrity as measured by thermomechanical methods is a key factor in cortical bone fracture toughness. This study further demonstrates that greater attention should be paid to degradation of the overall organic phase, rather than a specific biomarker (e.g. [pen]), when seeking to understand elevated fracture rates in aging and disease.

Impact of Side Chain Polarity on Non-Stoichiometric Nano-Hydroxyapatite Surface Functionalization with Amino Acids.

In this study the affinity of three amino acids for the surface of non-stoichiometric hydroxyapatite nanoparticles (ns-nHA) was investigated under different reaction conditions. The amino acids investigated were chosen based on their differences in side chain polarity and potential impact on this surface affinity. While calcium pre-saturation of the calcium-deficient ns-nHA was not found to improve attachment of any of the amino acids studied, the polarity and fraction of ionized functional side groups was found to have a significant impact on this attachment. Overall, amino acid attachment to ns-nHA was not solely reliant on carboxyl groups. In fact, it seems that amine groups also notably interacted with the negative ns-nHA surface and increased the degree of surface binding achieved. As a result, glycine and lysine had greater attachment to ns-nHA than aspartic acid under the reaction conditions studied. Lastly, our results suggest that a layer of each amino acid forms at the surface of ns-nHA, with aspartic acid attachment the most stable and its surface coverage the least of the three amino acids studied.

Mechanical behavior of metastatic vertebrae are influenced by tissue architecture, mineral content, and organic feature alterations.

Diminished vertebral mechanical behavior with metastatic involvement is typically attributed to modified architecture and trabecular bone content. Previous work has identified organic and mineral phase bone quality changes in the presence of metastases, yet limited work exists on the potential influence of such tissue level modifications on vertebral mechanical characteristics. This work seeks to determine correlations between features of bone (structural and tissue level) and mechanical behavior in metastatically involved vertebral bone. It is hypothesized that tissue level properties (mineral and organic) will improve these correlations beyond architectural properties and BMD alone. Twenty-four female athymic rats were inoculated with HeLa or Ace-1 cancer cells lines producing osteolytic (N = 8) or mixed (osteolytic/osteoblastic, N = 7) metastases, respectively. Twenty-one days post-inoculation L1-L3 pathologic vertebral motion segments were excised and µCT imaged. 3D morphometric parameters and axial rigidity of the L2 vertebrae were quantified. Sequential loading and µCT imaging measured progression of failure, stiffness and peak force. Relationships between mechanical testing (whole bone and tissue-level) and tissue-level material property modifications with metastatic involvement were evaluated utilizing linear regression models. Osteolytic involvement reduced vertebral trabecular bone volume, structure, CT-derived axial rigidity, stiffness and failure force compared to healthy controls (N = 9). Mixed metastases demonstrated similar trends. Previously assessed collagen cross-linking and proline-based residues were correlated to mechanical behavior and improved the predictive ability of the regression models. Similarly, collagen organization improved predictive regression models for metastatic bone hardness. This work highlights the importance of both bone content/architecture and organic tissue-level features in characterizing metastatic vertebral mechanics. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3013-3022, 2018.