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.

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.

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.

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.

Elastic-plastic fracture toughness and rising JR-curve behavior of cortical bone is partially protected from irradiation-sterilization-induced degradation by ribose protectant.

OBJECTIVE: This study tested the hypothesis that pre-treating cortical bone with ribose would protect the rising fracture resistance curve behavior and crack initiation fracture toughness of both bovine and human cortical bone from the degrading effects of γ-irradiation sterilization. MATERIALS AND METHODS: A ribose pre-treatment (1.8 M for bovine, and 1.2 M for human, in PBS at 60 °C for 24 h) was applied to single-edge notched bending fracture specimens prior to sterilization with a 33 kGy dose of γ-irradiation. Fracture resistance curves were generated with a single specimen method using an optical crack length measurement technique. The effect of the treatment on overall fracture resistance behavior, crack initiation fracture toughness, and tearing modulus was compared with non-irradiated and conventionally irradiation sterilized controls. Hydrothermal isometric tension testing was used to examine collagen network connectivity and thermal stability to explore relationships between collagen network quality and fracture resistance. RESULTS: The ribose pre-treatment successfully protected the crack growth initiation fracture toughness of bovine and human bone by 32% and 63%, respectively. The rising JR-curve behavior was also partially protected. Furthermore, collagen connectivity and thermal stability followed similar patterns to those displayed by fracture toughness. CONCLUSIONS: This paper demonstrates that the fracture toughness of irradiation-sterilized bone tissue can be partially protected with a ribose pre-treatment. This new approach shows potential for the production and clinical application of sterilized allografts with improved mechanical performance and durability.

Osteolytic and mixed cancer metastasis modulates collagen and mineral parameters within rat vertebral bone matrix.

Metastatic involvement in vertebral bone diminishes the mechanical integrity of the spine; however minimal data exist on the potential impact of metastases on the intrinsic material characteristics of the bone matrix. Thirty-four (34) female athymic rats were inoculated with HeLa (N = 17) or Ace-1 (N = 17) cancer cells lines producing osteolytic or mixed (osteolytic and osteoblastic) metastases, respectively. A maximum of 21 days was allowed between inoculation and rat sacrifice for vertebrae extraction. High performance liquid chromatography (HPLC) was utilized to determine modifications in collagen-I parameters such as proline hydroxylation and the formation of specific enzymatic and non-enzymatic (pentosidine) cross-links. Raman spectroscopy was used to determine relative changes in mineral crystallinity, mineral carbonation, mineral/collagen matrix ratio, collagen quality ratio, and proline hydroxylation. HPLC results showed significant increase in the formation of pentosidine and decrease in the formation of the enzymatic cross-link deoxy-pryridinoline within osteolytic bone compared to mixed bone. Raman results showed decreased crystallinity, increased carbonation, and collagen quality (aka 1660/1690 sub-band) ratio with osteolytic bone compared to mixed bone and healthy controls along with an observed increase in proline hydroxylation with metastatic involvement. The mineral/matrix ratio decreased in both osteolytic and mixed bone compared to healthy controls. Quantifying modifications within the intrinsic characteristics of bone tissue will provide a foundation to assess the impact of current therapies on the material behavior of bone tissue in the metastatic spine and highlight targets for the development of new therapeutics and approaches for treatment. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2126-2136, 2016.

Biomechanical comparison of the human cadaveric pelvis with a fourth generation composite model.

The use of cadavers for orthopaedic biomechanics research is well established, but presents difficulties to researchers in terms of cost, biosafety, availability, and ease of use. High fidelity composite models of human bone have been developed for use in biomechanical studies. While several studies have utilized composite models of the human pelvis for testing orthopaedic reconstruction techniques, few biomechanical comparisons of the properties of cadaveric and composite pelves exist. The aim of this study was to compare the mechanical properties of cadaveric pelves to those of the 4th generation composite model. An Instron ElectroPuls E10000 mechanical testing machine was used to load specimens with orientation, boundary conditions and degrees of freedom that approximated those occurring during the single legged phase of walking, including hip abductor force. Each specimen was instrumented with strain gauge rosettes. Overall specimen stiffness and principal strains were calculated from the test data. Composite specimens showed significantly higher overall stiffness and slightly less overall variability between specimens (composite K=1448±54N/m, cadaver K=832±62N/m; p<0.0001). Strains measured at specific sites in the composite models and cadavers were similar (but did differ) only when the applied load was scaled to overall construct stiffness. This finding regarding strain distribution and the difference in overall stiffness must be accounted for when using these composite models for biomechanics research. Altering the cortical wall thickness or tuning the elastic moduli of the composite material may improve future generations of the composite model.

Maximum load to failure and tensile displacement of an all-suture glenoid anchor compared with a screw-in glenoid anchor.

PURPOSE: The purpose of this study was to evaluate the biomechanical behavior of an all-suture glenoid anchor in comparison with a more conventional screw-in glenoid anchor, with regard to maximum load to failure and tensile displacement. METHODS: All mechanical testing was performed using an Instron ElectroPuls E1000 mechanical machine, with a 10 N pre-load and displacement rate of 10 mm/min. Force-displacement curves were generated, with calculation of maximum load, maximum displacement, displacement at 50 N and stiffness. Pretesting of handset Y-Knots in bone analog models revealed low force displacement below 60 N of force. Subsequently, three groups of anchors were tested for pull out strength in bovine bone and cadaver glenoid bone: a bioabsorbable screw-in anchor (Bio Mini-Revo, ConMed Linvatec), a handset all-suture anchor (Y-Knot, ConMed Linvatec) and a 60 N pre-tensioned all-suture anchor (Y-Knot). A total of 8 anchors from each group was tested in proximal tibia of bovine bone and human glenoids (age range 50-90). RESULTS: In bovine bone, the Bio Mini-Revo displayed greater maximum load to failure (206 ± 77 N) than both the handset (140 ± 51 N; P = 0.01) and the pre-tensioned Y-Knot (135 ± 46 N; P = 0.001); no significant difference was seen between the three anchor groups in glenoid bone. Compared to the screw-in anchors, the handset all-suture anchor displayed inferior fixation, early displacement and greater laxity in the bovine bone and cadaveric bone (P < 0.05). Pre-tensioning the all-suture anchor to 60 N eliminated this behavior in all bone models. CONCLUSIONS: Handset Y-Knots display low force anchor displacement, which is likely due to slippage in the pilot hole. Pre-tensioning the Y-Knot to 60 N eliminates this behavior. LEVEL OF EVIDENCE: I.

γ-Irradiation sterilized bone strengthened and toughened by ribose pre-treatment.

OBJECTIVE: This study tested the hypothesis that a ribose-based pre-treatment would protect the strength, ductility and toughness of γ-irradiation sterilized cortical bone. METHODS: Experiment 1: The effects of ribose pre-treatment (1.8M in PBS at 60°C for 24h) prior to 33 kGy of irradiation on strength, ductility and toughness (beams in three-point bending) and fracture toughness (J-integral at instability in single edge notched (bending)) were tested against matched non-irradiated and irradiated controls from bovine tibiae. Experiment 2: Three-point bending tests were conducted using beams from human femora (males, 59-67 years). Bone collagen thermal stability and network connectivity were examined using hydrothermal isometric tension testing. RESULTS: Ribose pre-treatment protected the strength, ductility and toughness of irradiation sterilized bovine and human specimens to differing degrees. Their ultimate strength was not detectably different from non-irradiated control levels; toughness in bovine and human specimens was protected by 57 and 76%, respectively. Untreated human bone was less affected by irradiation and ribose pre-treatment was more effective in human bone than bovine bone. CONCLUSIONS: This paper presents the first proof-of-principle that irradiation-sterilized bone with improved mechanical properties can be produced through the application of a ribose pre-irradiation treatment, which provides a more stable and connected collagen network than found in conventionally irradiated controls.

Collagen modifications in postmenopausal osteoporosis: advanced glycation endproducts may affect bone volume, structure and quality.

The classic model of postmenopausal osteoporosis (PM-OP) starts with the depletion of estrogen, which in turn stimulates imbalanced bone remodeling, resulting in loss of bone mass/volume. Clinically, this leads to fractures because of structural weakness. Recent work has begun to provide a more complete picture of the mechanisms of PM-OP involving oxidative stress and collagen modifications known as advanced glycation endproducts (AGEs). On one hand, AGEs may drive imbalanced bone remodeling through signaling mediated by the receptor for AGEs (RAGE), stimulating resorption and inhibiting formation. On the other hand, AGEs are associated with degraded bone material quality. Oxidative stress promotes the formation of AGEs, inhibits normal enzymatically derived crosslinking and can degrade collagen structure, thereby reducing fracture resistance. Notably, there are multiple positive feedback loops that can exacerbate the mechanisms of PM-OP associated with oxidative stress and AGEs. Anti-oxidant therapies may have the potential to inhibit the oxidative stress based mechanisms of this disease.

Bone embrittlement and collagen modifications due to high-dose gamma-irradiation sterilization.

Bone allografts are often used in orthopedic reconstruction of skeletal defects resulting from trauma, bone cancer or revision of joint arthroplasty. γ-Irradiation sterilization is a widely-used biological safety measure; however it is known to embrittle bone. Irradiation has been shown to affect the post-yield properties, which are attributed to the collagen component of bone. In order to find a solution to the loss of toughness in irradiated bone allografts, it is important to fully understand the effects of irradiation on bone collagen. The objective of this study was to evaluate changes in the structure and integrity of bone collagen as a result of γ-irradiation, with the hypothesis that irradiation fragments collagen molecules leading to a loss of collagen network connectivity and therefore loss of toughness. Using cortical bone from bovine tibiae, sample beams irradiated at 33kGy on dry ice were compared to native bone beams (paired controls). All beams were subjected to three-point bend testing to failure followed by characterization of the decalcified bone collagen, using differential scanning calorimetry (DSC), hydrothermal isometric tension testing (HIT), high performance liquid chromatography (HPLC) and gel electrophoresis (SDS-PAGE). The carbonyl content of demineralized bone collagen was also measured chemically to assess oxidative damage. Barium sulfate staining after single edge notch bending (SEN(B)) fracture testing was also performed on bovine tibia bone beams with a machined and sharpened notch to evaluate the fracture toughness and ability of irradiated bone to form micro-damage during fracture. Irradiation resulted in a 62% loss of work-to-fracture (p≤0.001). There was significantly less micro-damage formed during fracture propagation in the irradiated bone. HPLC showed no significant effect on pentosidine, pyridinoline, or hydroxypyridinoline levels suggesting that the loss of toughness is not due to changes in these stable crosslinks. For DSC, there was a 20% decrease in thermal stability (p<0.001) with a 100% increase (p<0.001) in enthalpy of denaturation (melting). HIT testing also showed a decrease in thermal stability (20% lower denaturation temperature, p<0.001) and greatly reduced measures of collagen network connectivity (p<0.001). Interestingly, the increase in enthalpy of denaturation suggests that irradiated collagen requires more energy to denature (melt), perhaps a result of alterations in the hydrogen bonding sites (increased carbonyl content detected in the insoluble collagen) on the irradiated bone collagen. Altogether, this new data strongly indicates that a large loss of overall collagen connectivity due to collagen fragmentation resulting from γ-irradiation sterilization leads to inferior cortical bone toughness. In addition, notable changes in the thermal denaturation of the bone collagen along with chemical indicators of oxidative modification of the bone collagen indicate that the embrittlement may be a function not only of collagen fragmentation but also of changes in bonding.

3BP2-deficient mice are osteoporotic with impaired osteoblast and osteoclast functions.

A fine balance between bone resorption by osteoclasts and bone formation by osteoblasts maintains bone homeostasis. In patients with cherubism, gain-of-function mutations in 3BP2, which is encoded by SH3-domain binding protein 2 (SH3BP2), cause cystic lesions with activated osteoclasts that lead to craniofacial abnormalities. However, little is known about the function of wild-type 3BP2 in regulating bone homeostasis. Here we have shown that 3BP2 is required for the normal function of both osteoblasts and osteoclasts. Initial analysis showed that Sh3bp2-/-mice developed osteoporosis as a result of reduced bone formation despite the fact that bone resorption was impaired. We demonstrated using reciprocal bone marrow chimeras, a cell-intrinsic defect of the osteoblast and osteoclast compartments in vivo. Further, Sh3bp2-/- osteoblasts failed to mature and form mineralized nodules in vitro, while Sh3bp2-/- osteoclasts spread poorly and were unable to effectively degrade dentine matrix in vitro. Finally, we showed that 3BP2 was required for Abl activation in osteoblasts and Src activation in osteoclasts, and demonstrated that the in vitro defect of each cell type was restored by the respective expression of activated forms of these kinases. These findings reveal an unanticipated role for the 3BP2 adapter protein in osteoblast function and in coordinating bone homeostatic signals in both osteoclast and osteoblast lineages.

Phenotypic variation of fluoride responses between inbred strains of mice.

Excessive systemic exposure to fluoride (F) can lead to disturbances in bone homeostasis and dental enamel development. We have previously shown strain-specific responses to F in the development of dental fluorosis (DF) and in bone formation/mineralization. The current study was undertaken to further investigate F responsive variations in bone metabolism and to determine possible relationships with DF susceptibility. Seven-week-old male mice from FVB/NJ, C57BL/6J, C3H/HeJ, A/J, 129S1/SvImJ, AKR/J, DBA/2J, and BALB/cByJ inbred strains were exposed to NaF (0 or 50 ppm as F(-)) in drinking water for 60 days. Sera were collected for F, Ca, Mg, PO(4), iPTH, sRANKL, and ALP levels. Bone marrow cells were subjected to ex vivo cell culture for osteoclast potential and CFU colony assays (CFU-fibroblast, CFU-osteoblast, CFU-erythrocyte/granulocyte/macrophage/megakaryocyte, CFU-granulocyte/macrophage, CFU-macrophage, and CFU-granulocyte). Femurs and vertebrae were subjected to micro-CT analyses, biomechanical testing, and F, Mg, and Ca content assays. DF was evaluated using quantitative fluorescence and clinical criteria. Strain-specific responses to F were observed for DF, serum studies, ex vivo cell culture studies, and bone quality. Among the strains, there were no patterns or significant correlations between DF severity and the actions of F on bone homeostasis (serum studies, ex vivo assays, or bone quality parameters). The genetic background continues to play a role in the actions of F on tooth enamel development and bone homeostasis. F exposure led to variable phenotypic responses between strains involving dental enamel development and bone metabolism.

Differential effects of PPAR-{gamma} activation versus chemical or genetic reduction of DPP-4 activity on bone quality in mice.

Patients with type 2 diabetes mellitus have an increased risk of fracture that can be further exacerbated by thiazolidinediones. A new class of antidiabetic agents control glucose through reduction of dipeptidyl peptidase-4 (DPP-4) activity; however the importance of DPP-4 for the control of bone quality has not been extensively characterized. We compared the effects of the thiazolidinedione pioglitazone and the DPP-4 inhibitor sitagliptin on bone quality in high-fat diet (HFD)-fed wild-type mice. In complementary studies, we examined bone quality in Dpp4(+/+) vs. Dpp4(-/-) mice. Pioglitazone produced yellow bones with greater bone marrow adiposity and significantly reduced vertebral bone mechanics in male, female, and ovariectomized (OVX) HFD fed female mice. Pioglitazone negatively affected vertebral volumetric bone mineral density, trabecular architecture, and mineral apposition rate in male mice. Sitagliptin treatment of HFD-fed wild-type mice significantly improved vertebral volumetric bone mineral density and trabecular architecture in female mice, but these improvements were lost in females after OVX. Genetic inactivation of Dpp4 did not produce a major bone phenotype in male and female Dpp4(-/-) mice; however, OVX Dpp4(-/-) mice exhibited significantly reduced femoral size and mechanics. These findings delineate the skeletal consequences of pharmacological and genetic reduction of DPP-4 activity and reveal significant differences in the effects of pioglitazone vs. sitagliptin vs. genetic Dpp4 inactivation on bone mechanics in mice.