Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density.

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease

Abrogation of fluid suppression in intracranial postcontrast fluid‐attenuated inversion recovery magnetic resonance imaging: A clinical and phantom study

Postcontrast, fluid-attenuated inversion recovery (FLAIR) sequences are reported to be of variable value in veterinary and human neuroimaging. The source of hyperintensity in postcontrast-T2 FLAIR images is inconsistently reported and has implications for the significance of imaging findings. We hypothesized that the main source of increased signal intensity in postcontrast-T2 FLAIR images would be due to gadolinium leakage into adjacent fluid, and that the resulting gadolinium-induced T1 shortening causes reappearance of fluid hyperintensity, previously nulled on precontrast FLAIR images. A retrospective, descriptive study was carried out comparing T2 weighted, pre- and postcontrast T1 weighted and pre- and postcontrast weighted T2 FLAIR images in a variety of intracranial diseases in dogs and cats. A prospective, experimental, phantom, in vitro study was also done to compare the relative effects of gadolinium concentration on T2 weighted, T1 weighted, and FLAIR images. A majority of hyperintensities on postcontrast-T2 FLAIR images that were not present on precontrast FLAIR images were also present on precontrast T2 weighted images, and were consistent with normal or pathological fluid filled structures. Phantom imaging demonstrated increased sensitivity of FLAIR sequences to low concentrations of gadolinium compared to T1 weighted sequences. Apparent contrast enhancement on postcontrast-T2 FLAIR images often reflects leakage of gadolinium across normal or pathology specific barriers into fluid-filled structures, and hyperintensity may therefore represent normal fluid structures as well as pathological tissues. Findings indicated that postcontrast-T2 FLAIR images may provide insight into integrity of biological structures such as the ependymal and subarachnoid barriers that may be relevant to progression of disease

Temporal development of near-native functional properties and correlations with qMRI in self-assembling fibrocartilage treated with exogenous lysyl oxidase homolog 2.

Advances in cartilage tissue engineering have led to constructs with mechanical integrity and biochemical composition increasingly resembling that of native tissues. In particular, collagen cross-linking with lysyl oxidase has been used to significantly enhance the mechanical properties of engineered neotissues. In this study, development of collagen cross-links over time, and correlations with tensile properties, were examined in self-assembling neotissues. Additionally, quantitative MRI metrics were examined in relation to construct mechanical properties as well as pyridinoline cross-link content and other engineered tissue components. Scaffold-free meniscus fibrocartilage was cultured in the presence of exogenous lysyl oxidase, and assessed at multiple time points over 8weeks starting from the first week of culture. Engineered constructs demonstrated a 9.9-fold increase in pyridinoline content, reaching 77% of native tissue values, after 8weeks of culture. Additionally, engineered tissues reached 66% of the Young's modulus in the radial direction of native tissues. Further, collagen cross-links were found to correlate with tensile properties, contributing 67% of the tensile strength of engineered neocartilages. Finally, examination of quantitative MRI metrics revealed several correlations with mechanical and biochemical properties of engineered constructs. This study displays the importance of culture duration for collagen cross-link formation, and demonstrates the potential of quantitative MRI in investigating properties of engineered cartilages.Statement of significanceThis is the first study to demonstrate near-native cross-link content in an engineered tissue, and the first study to quantify pyridinoline cross-link development over time in a self-assembling tissue. Additionally, this work shows the relative contributions of collagen and pyridinoline to the tensile properties of collagenous tissue for the first time. Furthermore, this is the first investigation to identify a relationship between qMRI metrics and the pyridinoline cross-link content of an engineered collagenous tissue

Temporal development of near-native functional properties and correlations with qMRI in self-assembling fibrocartilage treated with exogenous lysyl oxidase homolog 2.

Advances in cartilage tissue engineering have led to constructs with mechanical integrity and biochemical composition increasingly resembling that of native tissues. In particular, collagen cross-linking with lysyl oxidase has been used to significantly enhance the mechanical properties of engineered neotissues. In this study, development of collagen cross-links over time, and correlations with tensile properties, were examined in self-assembling neotissues. Additionally, quantitative MRI metrics were examined in relation to construct mechanical properties as well as pyridinoline cross-link content and other engineered tissue components. Scaffold-free meniscus fibrocartilage was cultured in the presence of exogenous lysyl oxidase, and assessed at multiple time points over 8weeks starting from the first week of culture. Engineered constructs demonstrated a 9.9-fold increase in pyridinoline content, reaching 77% of native tissue values, after 8weeks of culture. Additionally, engineered tissues reached 66% of the Young's modulus in the radial direction of native tissues. Further, collagen cross-links were found to correlate with tensile properties, contributing 67% of the tensile strength of engineered neocartilages. Finally, examination of quantitative MRI metrics revealed several correlations with mechanical and biochemical properties of engineered constructs. This study displays the importance of culture duration for collagen cross-link formation, and demonstrates the potential of quantitative MRI in investigating properties of engineered cartilages.Statement of significanceThis is the first study to demonstrate near-native cross-link content in an engineered tissue, and the first study to quantify pyridinoline cross-link development over time in a self-assembling tissue. Additionally, this work shows the relative contributions of collagen and pyridinoline to the tensile properties of collagenous tissue for the first time. Furthermore, this is the first investigation to identify a relationship between qMRI metrics and the pyridinoline cross-link content of an engineered collagenous tissue

A Modified Hydroxyproline Assay Based on Hydrochloric Acid in Ehrlich's Solution Accurately Measures Tissue Collagen Content.

Collagen quantification has long been relevant to biomedical research and clinical practice to characterize tissues and determine disease states. The hydroxyproline assay, while a broadly employed method of quantifying collagen, uses perchloric acid to dissolve Ehrlich's reagent. Since perchloric acid poses occupational safety hazards and high costs, in this study, a new hydroxyproline assay was developed that replaces perchloric acid with a relatively safer and cheaper alternative, hydrochloric acid (HCl). To validate this biochemical technique, first, using either acid to dissolve Ehrlich's reagent, the assays were completed for native and engineered collagenous tissues. No statistical differences were identified between the assays (p = 0.32). Subsequently, both biochemical techniques were compared to amino acid analysis, considered a proteomics gold standard. Interestingly, utilizing HCl in lieu of perchloric acid yielded greater concordance with amino acid analysis (ρc = 0.980) than did the traditional assay (ρc = 0.947); that is, the HCl-based assay more closely estimates hydroxyproline content, and, consequently, true collagen content. Thus, using Ehrlich's reagent containing HCl in the hydroxyproline assay represents an advance in both mitigating laboratory safety hazards and improving biochemical collagen quantification

A Modified Hydroxyproline Assay Based on Hydrochloric Acid in Ehrlich's Solution Accurately Measures Tissue Collagen Content.

Collagen quantification has long been relevant to biomedical research and clinical practice to characterize tissues and determine disease states. The hydroxyproline assay, while a broadly employed method of quantifying collagen, uses perchloric acid to dissolve Ehrlich's reagent. Since perchloric acid poses occupational safety hazards and high costs, in this study, a new hydroxyproline assay was developed that replaces perchloric acid with a relatively safer and cheaper alternative, hydrochloric acid (HCl). To validate this biochemical technique, first, using either acid to dissolve Ehrlich's reagent, the assays were completed for native and engineered collagenous tissues. No statistical differences were identified between the assays (p = 0.32). Subsequently, both biochemical techniques were compared to amino acid analysis, considered a proteomics gold standard. Interestingly, utilizing HCl in lieu of perchloric acid yielded greater concordance with amino acid analysis (ρc = 0.980) than did the traditional assay (ρc = 0.947); that is, the HCl-based assay more closely estimates hydroxyproline content, and, consequently, true collagen content. Thus, using Ehrlich's reagent containing HCl in the hydroxyproline assay represents an advance in both mitigating laboratory safety hazards and improving biochemical collagen quantification

Antigen removal for the production of biomechanically functional, xenogeneic tissue grafts.

Xenogeneic tissues are derived from other animal species and provide a source of material for engineering mechanically functional tissue grafts, such as heart valves, tendons, ligaments, and cartilage. Xenogeneic tissues, however, contain molecules, known as antigens, which invoke an immune reaction following implantation into a patient. Therefore, it is necessary to remove the antigens from a xenogeneic tissue to prevent immune rejection of the graft. Antigen removal can be accomplished by treating a tissue with solutions and/or physical processes that disrupt cells and solubilize, degrade, or mask antigens. However, processes used for cell and antigen removal from tissues often have deleterious effects on the extracellular matrix (ECM) of the tissue, rendering the tissue unsuitable for implantation due to poor mechanical properties. Thus, the goal of an antigen removal process should be to reduce the antigen content of a xenogeneic tissue while preserving its mechanical functionality. To expand the clinical use of antigen-removed xenogeneic tissues as biomechanically functional grafts, it is essential that researchers examine tissue antigen content, ECM composition and architecture, and mechanical properties as new antigen removal processes are developed

Regenerating Mandibular Bone Using rhBMP-2: Part 1-Immediate Reconstruction of Segmental Mandibulectomies.

ObjectiveTo describe a surgical technique using a regenerative approach and internal fixation for immediate reconstruction of critical size bone defects after segmental mandibulectomy in dogs.Study designProspective case series.AnimalsDogs (n = 4) that had reconstruction after segmental mandibulectomy for treatment of malignant or benign tumors.MethodsUsing a combination of extraoral and intraoral approaches, a locking titanium plate was contoured to match the native mandible. After segmental mandibulectomy, the plate was secured and a compression resistant matrix (CRM) infused with rhBMP-2, implanted in the defect. The implant was then covered with a soft tissue envelope followed by intraoral and extraoral closure.ResultsAll dogs that had mandibular reconstruction healed with intact gingival covering over the mandibular defect and had immediate return to normal function and occlusion. Mineralized tissue formation was observed clinically within 2 weeks and solid cortical bone formation within 3 months. CT findings at 3 months showed that the newly regenerated mandibular bone had ∼50% of the bone density and porosity compared to the contralateral side. No significant complications occurred.ConclusionMandibular reconstruction using internal fixation and CRM infused with rhBMP-2 is an excellent solution for immediate reconstruction of segmental mandibulectomy defects in dogs

Regenerating Mandibular Bone Using rhBMP-2: Part 1-Immediate Reconstruction of Segmental Mandibulectomies.

ObjectiveTo describe a surgical technique using a regenerative approach and internal fixation for immediate reconstruction of critical size bone defects after segmental mandibulectomy in dogs.Study designProspective case series.AnimalsDogs (n = 4) that had reconstruction after segmental mandibulectomy for treatment of malignant or benign tumors.MethodsUsing a combination of extraoral and intraoral approaches, a locking titanium plate was contoured to match the native mandible. After segmental mandibulectomy, the plate was secured and a compression resistant matrix (CRM) infused with rhBMP-2, implanted in the defect. The implant was then covered with a soft tissue envelope followed by intraoral and extraoral closure.ResultsAll dogs that had mandibular reconstruction healed with intact gingival covering over the mandibular defect and had immediate return to normal function and occlusion. Mineralized tissue formation was observed clinically within 2 weeks and solid cortical bone formation within 3 months. CT findings at 3 months showed that the newly regenerated mandibular bone had ∼50% of the bone density and porosity compared to the contralateral side. No significant complications occurred.ConclusionMandibular reconstruction using internal fixation and CRM infused with rhBMP-2 is an excellent solution for immediate reconstruction of segmental mandibulectomy defects in dogs