Bone Tissue Samples Research Articles

MiR‑483‑3p promotes the osteogenesis of human osteoblasts bytargeting Dikkopf 2 (DKK2) and the Wnt signaling pathway.

Osteoporosis is a systemic metabolic bone disease during which bone mass decreases and bone quality is reduced. Maintaining the bone formation capacity of osteoblasts is crucial for the treatment of osteoporosis. In the present study, bioinformatics analysis was performed on online microarray expression profiles to identify miRNA(s) related to osteoblast proliferation and bone marrow-derived mesenchymal stem cell (BMSC) osteogenic differentiation. The specific effects of candidate miRNAs on cell proliferation, osteogenic differentiation and Wnt signaling-related factors were examined. As regards the downstream mechanisms, online tools were employed to predict the downstream targets of candidate miRNAs and the predicted miRNA-mRNA binding was verified. Finally, the dynamic effects of miRNAs and mRNAs were examined. The results revealed that miR-483-3p expression was decreased in bone tissue samples from patients with osteoporosis. In miR-483-3p-overexpressing human osteoblasts, cell viability, DNA synthesis capacity and osteogenesis were promoted, and the protein levels of Wnt1, β-catenin and cyclin D1 were increased. However, the protein receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio and cell apoptotic rate were decreased. The Wnt signaling, antagonist Dikkopf 2 (DKK2), was targeted and negatively regulated by miR-483-3p. DKK2 knockdown exerted similar effects as miR-483-3p overexpression, while DKK2 overexpression inhibited cell viability, DNA synthesis capacity and osteogenesis. DKK2 overexpression also decreased the Wnt1, β-catenin, and cyclin D1 protein levels, whereas it promoted the the RANKL/OPG ratio and the apoptosis of human osteoblasts. DKK2 overexpression reversed the functions of miR-483-3p overexpression. On the whole, the findings of the present study demonstrate that the miR-483-3p/DKK2 axis modulates the bone formation process by affecting osteoblast proliferation, pre-osteoblast differentiation into mature osteoblasts and new bone matrix formation.

Comparison between Conventional Decalcification and a Microwave-Assisted Method in Bone Tissue Affected with Mycetoma.

Mycetoma is a lifelong granulomatous disease of subcutaneous tissues and bones. Histopathology is a substantiated indicative method based on the assumption of a definitive diagnosis of mycetoma. It requires efficient processing of tissues including bone decalcification. The decalcification process must ensure complete removal of calcium and also a proper preservation of tissue and microorganisms' staining ability. Objectives. To compare the conventional method used in decalcification with the microwave method using different decalcification solutions. Different characteristics were tested, including the speed of decalcification and morphological and fungal preservation in bone tissue affected with mycetoma. Materials and Methods. Three decalcification solutions were employed to remove calcium from 50 bone tissue samples affected with mycetoma, including 10% neutral buffered EDTA (pH 7.4), 5% nitric acid, and 5% hydrochloric acid. Conventional and microwave methods were used. Haematoxylin-eosin (HE) stain, Gridley's stain, and Grocott hexamine-silver stain were employed to evaluate the bone and fungi morphologies. Results. The decalcification time of the conventional method compared with the microwave method with 10% EDTA (pH 7.4) took 120 hours and 29 hours, while 5% hydrochloric acid and 5% nitric acid took 8 hours and 3 hours, separately. Also, 10% EDTA is the best decalcifying agent for HE staining and fungal stains. 5% hydrochloric acid and 5% nitric acid can be used for fungal staining. Conclusion. The current study investigated the effects of different decalcifying agents as well as two decalcification procedures on the preservation of the bone structure and fungal staining, which will help to develop suitable protocols for the analyses of the bone tissue affected with mycetoma infection.

Pathological and microbiological impact of a gentamicin-loaded biocomposite following limited or extensive debridement in a porcine model of osteomyelitis.

AimsCERAMENT|G is an absorbable gentamicin-loaded biocomposite used as an on-site vehicle of antimicrobials for the treatment of chronic osteomyelitis. The purpose of the present study was to investigate the sole effect of CERAMENT|G, i.e. without additional systemic antimicrobial therapy, in relation to a limited or extensive debridement of osteomyelitis lesions in a porcine model.MethodsOsteomyelitis was induced in nine pigs by inoculation of 104 colony-forming units (CFUs) of Staphylococcus aureus into a drill hole in the right tibia. After one week, the pigs were allocated into three groups. Group A (n = 3) received no treatment during the study period (19 days). Groups B (n = 3) and C (n = 3) received limited or extensive debridement seven days postinoculation, respectively, followed by injection of CERAMENT|G into the bone voids. The pigs were euthanized ten (Group C) and 12 (Group B) days after the intervention.ResultsAll animals presented confirmatory signs of bone infection post-mortem. The estimated amount of inflammation was substantially greater in Groups A and B compared to Group C. In both Groups B and C, peptide nucleic acid fluorescence in situ hybridization (PNA FISH) of CERAMENT|G and surrounding bone tissue revealed bacteria embedded in an opaque matrix, i.e. within biofilm. In addition, in Group C, the maximal measured post-mortem gentamicin concentrations in CERAMENT|G and surrounding bone tissue samples were 16.6 μg/ml and 6.2 μg/ml, respectively.ConclusionThe present study demonstrates that CERAMENT|G cannot be used as a standalone alternative to extensive debridement or be used without the addition of systemic antimicrobials.Cite this article: Bone Joint Res 2020;9(7):394–401.

Distribution of Young's modulus at various sampling points in a human lumbar spine vertebral body

BACKGROUND CONTEXTMathematical modeling for creating computer spine models is one of the basic methods underlying many scientific publications. The accuracy of strength parameters of tissues introduced into such models translates directly into the reliability of obtained results. Experimental determination of Young's modulus (E) in various areas of spongy bone tissue seems to be crucial for creating a reliable spine model without excessive simplifications in the form of a single E value for the whole vertebral body. PURPOSEThe aim of the study was to determine Young's modulus in different parts of the lumbar vertebral column for samples subjected to compression and bending. STUDY DESIGNCylindrical spongy bone tissue samples were subjected to bending and compression strength tests. METHODSThe study included 975 pathologically unchanged samples of spongy bone tissue harvested from the lumbar vertebrae of 15 male donors. The samples were subjected to compression or bending strength tests and then Young's modulus was determined for each sample depending on its location in the vertebral body. The samples were tested differently between given locations within one vertebra as well as between vertebrae. RESULTSCompressed specimens are characterized by highly significantly different Young's modulus values depending on the location in the vertebral body. Samples No. 7 and No. 9 in the anterior part of the vertebral body have highly significantly higher Young's modulus values compared to those in the posterior part of the vertebral body for all lumbar vertebrae. Samples subjected to bending showed significant differences (p<.05) between samples located closer to the vertebral canal (No.16, No.17) and samples located further away (No.14, No.15) with higher values for the samples located in the posterior part of the vertebral body. CONCLUSIONSAccommodating the anisotropic structure of spongy bone in computer models and the application of different Young's module values for areas within one vertebral body will allow one to obtain realistic results of computer simulations used. CLINICAL SIGNIFICANCEDetermining the exact strength parameters of spongy bone tissue within one vertebra and changes in these properties in subsequent vertebrae will allow to create more accurate computer models of the lumbar spine and the whole spine. This, in turn, will translate into more reliable computer simulations used, among others, to determine the risk of fractures or osteoporotic changes, or simulation of the procedure of spinal fusion.

Clonal hematopoiesis in angioimmunoblastic T-cell lymphoma with divergent evolution to myeloid neoplasms

AbstractTET2 and DNMT3A mutations are frequently identified in T-cell lymphomas of T follicular helper cell origin (TCL-TFH), clonal hematopoiesis (CH), and myeloid neoplasms (MNs). The relationships among these 3 entities, however, are not well understood. We performed comprehensive genomic studies on paired bone marrow and tissue samples as well as on flow cytometry–sorted bone marrow and peripheral blood subpopulations from a cohort of 22 patients with TCL-TFH to identify shared CH-type mutations in various hematopoietic cell compartments. Identical mutations were detected in the neoplastic T-cell and myeloid compartments of 15 out of 22 patients (68%), including TET2 (14/15) and DNMT3A (10/15). Four patients developed MNs, all of which shared CH-type mutations with their TCL-TFH; additional unique genetic alterations were also detected in each patient's TCL-TFH and MN. These data demonstrate that CH is prevalent in patients with TCL-TFH and that divergent evolution of a CH clone may give rise to both TCL-TFH and MNs.

Microbiological analysis of autologous bone particles obtained by low-speed drilling and treated with different decontamination agents

The aim of this study was to compare the effectiveness of three agents – two antibiotics (amoxicillin and clindamycin) and an antiseptic (chlorhexidine) – to decontaminate bone grafts obtained by low-speed drilling. The study included 248 bone tissue samples harvested from 62 patients by low-speed drilling before dental implant placement. Each of four samples obtained from every patient was dropped, using a sterile instrument, into a sterile tube containing a 500-μl solution of 400μg/mL amoxicillin, 150μg/mL clindamycin, 0.12% chlorhexidine, or physiological saline for 1min. The number of colony-forming units (CFU) was determined at 48h of culture. The use of clindamycin, amoxicillin, or chlorhexidine as decontaminant for 1min significantly reduced the CFU count when compared to physiological saline (control agent). In both anaerobic and CO2-rich atmospheres, significant differences in CFU/mL were found between the control and chlorhexidine groups (P<0.001), control and amoxicillin groups (P<0.001), control and clindamycin groups (P<0.001), chlorhexidine and amoxicillin groups (P<0.0001), and chlorhexidine and clindamycin groups (P<0.0001). In conclusion, clindamycin had the highest decontaminating effect on bone particles obtained by low-speed drilling, followed by chlorhexidine and amoxicillin. Clindamycin may therefore be a valid alternative option for the routine decontamination of intraoral bone grafts.

Dynamics of hematological parameters, macromorphological and radiological results of reparative osteogenesis in rabbits for the use of platelet concentrates and hydroxyapatite ceramics

The results of the dynamics of the morphological parameters of the blood of a rabbit with model defects of the opening of the spongy and compact bone tissue at diff erent periods of reparative osteogenesis are presented. Formed 4 groups of rabbits. Bone lesions in the fi rst experimental group were fi lled with injectable platelet-rich fi brin, in the second - platelet-rich fi brin, in the third - a combination of platelet-rich fi brin, and hydroxyapatite with β-tricalcium phosphate.In the control group, the defects remained healed under a blood clot. All animals were in the same conditions of feeding and keeping, had unlimited access to water. During the study, rabbits were clinically observed. Blood was taken for morphological examination before surgery and on the 3rd, 7th, 14th, 21st, 42nd day. Animals were taken from the experiment on the 14th, 21st and 42nd days, an X-ray examination was carried out, bone tissue samples were taken. It was established that trauma of bone tissue leads to a number of reactions of the body aimed at restoring the damaged area. Against the background of the general picture of the obtained morphological results, the level of platelets signifi cantly changes compared to the physiological norm, while the number of red blood cells and white blood cells does not go beyond it. There is a slight increase in hemoglobin levels, especially in the experimental groups on the twenty-fi rst and forty-second day. Signifi cant changes in the number of red blood cells, white blood cells, platelets and hemoglobin concentration in the experimental groups were noted compared with the control group at diff erent periods of reparative osteogenesis.The use of various types of platelet concentrates aff ects the overall reaction of the body. On radiographs of the radial bones (compact bone tissue) on the twenty-fi rst day in the second and third experimental groups, where fi brin enriched with platelets and its combination with hydroxyapatite materials with β-tricalcium phosphate were used to replace bone defects, the infl ammatory reaction was manifested to a lesser extent. There was no signifi cant formation of bone callus compared with the fi rst, in which model defects were fi lled with injectable platelet-rich fi brin and the control group. Each of the types of platelet concentrates and their combination with hydroxyapatite ceramics have a diff erent eff ect on the restoration of bone defects and is accompanied by the appearance of a number of reactions, both local and general. According to the degree of intensity of this process, they can be placed in the following sequence: i-PRF ˂ PRF ˂ PRF+GT. Macromorphologically and radiologically signifi cant diff erences were not detected for diff erent types of bone tissue due to the use of each of the substances that concentrates platelets. The combination of hydroxyapatite granules with β-tricalcium phosphate and platelet-rich fi brin proved to be the best option for repairing the damaged area. Its use provides a signifi cantly smaller manifestation of the local infl ammatory reaction and causes the formation of optimal bone marrow. Key words: PRF, i-PRF, granules, centrifuges, growth factors, bone marrow.

Radiographic, macromorphological and hematological evaluation of hydroxyapatite ceramics with diff erent physicochemical properties

Treatments of fragmentary fractures in case of loss regenerative potential of bone tissue require usingdiff erent composite materials. Among all of them, ceramics based on synthetic hydroxyapatite and tricalcium phosphate are considered promising. The reparative osteogenesis in spongy and compact rabbit’s bone tissueusing 3 composite materials with diff erent physicochemical propertieswas studied. A reparative osteogenesiscontrolled by monitoring clinical, radiologic and macromorphologic parameters. Composite materials infl uence on the organism was studied by determining the dynamics of hematological parameters throughout the study period. To realize this scheme was formed a 3 experimental groups and one control group of rabbits. The created defects of animals of fi rst experimental group were fi lled out with GT + α-TKF-500, thedefects of second oneanimals group fi lled out with GTlKl-2, and defects ofthird oneanimals group fi lled out with GTlKg-700. The GTlKl-700materials were doped with silicon. In control animals, bone defects were allowed to heal under a blood clot. During the studies, all animals were located in the same environment conditions. A blood samples for hematological investigation was taken from the external jugular vein before anesthesia and at the 3rd, 7th, 14th, 21st and 42nd day after surgery. X-ray examination was performed on 14th, 21st, and 42nddays.The animals were removed from the experiment on 21stand 42nd days. The samples were examined by macromorphologic method. By radiologic examination was found that composite materials had osteoconductive properties except the silicondoped example which hadosteoinductive properties. The samples of compact bone tissue of 1st and 3rd experimental groups characterized by formation of punctate osteosclerosis with a compacted contour of the periosteumon the 42nd day. But the bone samples of 2nd experimental group had compacted contours of the periosteum only. However, in the cancellous bone tissue on the 42nd day in the animals of the 1st experimental group developed point osteosclerosis, which visualized as individual granules of the composite.But in the animals of 2nd group thewhite spot which was outlined shape and homogeneous structure at the defect was found. The bone samples of 3rd animals group shoved a clearly limited point osteosclerosisforming were was found ceramic granules. In case using ofGT + α-TKF-500, a strong connection of granules from the formed bone tissue and without any periosteal growthswas noted. Using GTlKl-2 places of defects were identical to not injured sites of radial bones. Using GTlKg-700 shows that bone defect is fi lled to the level of the plane of the bone surface and covered with a periosteum without visible growths. The granules of the composite are evenly distributed in the regenerate and associated with bone tissue. The analysis of hematological parameters did not reveal any fundamental diff erences, but the use of hydroxyapatite implants is not accompanied, in contrast to spontaneous reparative osteogenesis, the development of leukocytosis, which indicates a moderate course of its infl ammatory reaction. But the latter is accompanied by a platelet reaction, the most pronounced when using implants, which is probably due to the infl uence of platelet factors and is indirect evidence of early osteoblastic reaction. Key words: fractures, rabbits, erythrocytes, leukocytes, platelets, hemoglobin.

In-vivo histocompatibility and osteogenic potential of biodegradable PLDLA composites containing silica-based bioactive glass fiber.

The purpose of this two-year study was to evaluate the histocompatibility and osteogenic properties of a composite material consisting of poly(l-co-d,l lactide) (PLDLA) and silica-based bioactive glass fibers in vivo. PLDLA and PLDLA/silica-based bioactive glass fibers pins were implanted into the erector spinae muscles and femurs of beagles. Muscle and bone tissue samples were harvested 6, 12, 16, 26, 52, 78, and 104 weeks after implantation. Histology analysis was used to assess the histocompatibility, angiogenesis, and bone-implant contact. Micro-computed tomography was used to evaluate bone formation around the pins. Immunohistochemistry and western blotting revealed the expression level of the osteogenesis-related proteins. Addition of bioactive glass was demonstrated to possess better histocompatibility and reduce the inflammatory reactions in vivo. Moreover, PLDLA/silica-based bioactive glass fibers pins were demonstrated to promote angiogenesis and increase osteogenesis-related proteins expression, and thus played a positive role in osteogenesis and osseointegration after implantation. Our findings indicated that a composite of PLDLA and silica-based bioactive glass fiber is a promising biodegradable material for clinical use.

Is the bone tissue of the femoral neck demineralised in patients with hip fracture?

The aim of this study is to establish the falsifiability of the "osteoporotic hypothesis" for hip fracture, according to which the bone density and mineral composition of bone tissue in patients with hip fracture is poorer than when no such fracture is present, and that this circumstance is relevant to the occurrence of a fracture. The study population consisted of forty patients treated with arthroplasty. Twenty patients with femoral neck fracture and another twenty with hip osteoarthritis received the same diagnostic protocol and the same antibiotic, anaesthetic, surgical and antithrombotic prophylaxis. Levels of calcium (Ca), phosphorus (P) and vitamin D in blood, amongst other values, were determined, and five samples of bone tissue from the proximal femoral metaphysis were obtained and characterised by optical microscopy and microanalytical analysis. No statistically significant differences were observed between the two groups with respect to the trabecular number, area or thickness, or inter-trabecular distance. However, there were differences in the length of the trabeculae, which was greater in the patients with hip osteoarthritis (p = 0.002), but not when the groups were compared by gender. When compared by age, a greater inter-trabecular distance was observed in the patients aged over 75 years (p = 0.036) but there were no differences in the remaining parameters. Serum levels of Ca (p = 0.03), P (p < 0.01) and vitamin D (p < 0.01) were lower in the fracture group. In the quantitative microanalytical analysis, no significant differences were observed in bone levels of Ca or P or in the Ca/P index, nor was there any correlation between serum and levels of bone Ca or P (Ca-0.197:p = 0.314;P-0.274:p = 0.158).Multiple linear regression revealed no correlation between the diagnoses, vitamin D and bone levels of Ca or P. Despite the reduced serum levels of Ca and P in the patients with hip fracture, no correlation was observed with bone levels of Ca and P,which were similar in both groups. There were differences in the organic bone structure, in terms of length and inter-trabecular distance. For patients with osteoporosis, treatment should be aimed at increasing the synthesis of bone trabeculae to reinforce their structure. Nevertheless, no such treatment can prevent falls, and therefore no reduction in hip fractures amongst this population can be assured.

Concordance of intraoperative frozen section diagnosis with routine histopathological diagnosis

Introduction: Intra-operative frozen section plays an important role in the management of surgical patients and yet it must be used prudently to avoid the indiscriminate usage of this important technique as it is subject to many limitations in comparison to the routine paraffin embedded tissue sections. In this study we have attempted to look at the concordance between the intraoperative frozen section diagnosis versus the regular histopathological diagnosis in our patients. Aim of the study: To correlate the diagnosis given on Frozen section with Histopathological diagnosis.Materials and Methods: This was a prospective study carried over a period of six months, from January 2019 to June 2019. Intraoperative Frozen sections were compared with routine regular paraffin embedded histopathology diagnosis in 51 patients. Detailed clinical history like age, gender, site, duration of lesions were noted. Results: Various specimen types were received for Frozen sections during the time frame of this study, which included bone, soft tissue, genitourinary, skin, brain, and gastrointestinal tract tissue samples. Frozen section requests included asking for a diagnosis of a lesion (72.5%), margin status of a resected specimen containing malignancy (3.92%), lymph node status for the presence of metastatic tumor (23.5%). All the 51 cases (100%) of frozen section impressions were concordant with routine histopathological diagnosis.Conclusion: The Intra-operative consultation using Frozen section is a very useful tool and guides the surgeon to intraoperatively modify the surgical approach. However, regular histopathological analysis is the gold standard for diagnosis.

Efficacy of NEMO-binding domain peptide used to treat experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus: an in-vivo study

PurposeTreatment of chronic osteomyelitis (bone infection) remains a clinical challenge. Our previous study had demonstrated that NEMO-binding domain (NBD) peptide effectively ameliorates the inhibition of osteoblast differentiation by TNF-α in vitro. In this work, NBD peptide was evaluated in vivo for treating chronic osteomyelitis induced by methicillin-resistant Staphylococcus aureus (MRSA) in a rabbit model.MethodsTibial osteomyelitis was induced in 50 New Zealand white rabbits by tibial canal inoculation of MRSA strain. After 3 weeks, 45 rabbits with osteomyelitis were randomly divided into four groups that correspondingly received the following interventions: 1) Control group (9 rabbits, no treatment); 2) Van group (12 rabbits, debridement and parenteral treatment with vancomycin); 3) NBD + Van group (12 rabbits, debridement and local NBD peptide injection, plus parenteral treatment with vancomycin); 4) NBD group (12 rabbits, debridement and local NBD peptide injection). Blood samples were collected weekly for the measurement of leucocyte count, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels. The rabbits in all four groups were sacrificed 6 weeks after debridement; the anti-infective efficacy was evaluated by radiological, histological, and microbiological examination, and promotion of bone remodeling was quantified by micro-CT using the newly formed bone.ResultsExcept two rabbits in the Control group and one in the NBD group that died from severe infection before the end point, the remaining 42 animals (7, 12, 12, 11 in the Control, Van, NBD + Van, and NBD group respectively) were sacrificed 6 weeks after debridement. In general, there was no significant difference in the leucocyte count, and ESR and CRP levels, although there were fluctuations throughout the follow-up period after debridement. MRSA was still detectable in bone tissue samples of all animals. Interestingly, treatment with NBD peptide plus vancomycin significantly reduced radiological and histological severity scores compared to that in other groups. The best therapeutic efficacy in bone defect repair was observed in the NBD peptide + Van group.ConclusionsIn a model of osteomyelitis induced by MRSA, despite the failure in demonstrating antibacterial effectiveness of NBD peptide in vivo, the results suggest antibiotics in conjunction with NBD peptide to possibly have promising therapeutic potential in osteomyelitis.

Differentially expressed microRNA and target gene analysis in patients with postmenopausal osteoporosis

Objective To analyze the differentially expressed microRNA (miRNA) and their target genes in peripheral blood and bone tissue of postmenopausal osteoporosis (PMOP), and provide basis for the study of pathogenesis as well as biomarkers identification of PMOP. Methods Two miRNA datasets of PMOP from the public platform NCBI-GEO DataSets were obtained, including GSE64433 (the miRNA expression profile of peripheral blood samples, including 23 PMOP patients and 25 controls) and GSE74209 (the miRNA expression profile of the femoral neck bone tissue sample, including six PMOP patients and six controls). R/Bioconductor was performed for data analysis and differentially expressed miRNA screening, and miRNAs with fold change>2 & P<0.05 between osteoporosis and controls were selected as differentially expressed miRNA. The miRNA target gene prediction was performed by combining TargetScan, miRDB and miRTarBase databases. The miRNA-target gene regulatory network was constructed and analyzed by Cytoscape. Results There were 224 differentially expressed miRNAs (75 up-regulated miRNAs and 149 down-regulated miRNAs) in the peripheral blood samples of PMOP group (GSE64433 dataset) and 132 differentially expressed miRNAs (58 up-regulated miRNAs and 74 down-regulated miRNAs) in the femoral neck bone tissue of PMOP group (GSE74209 dataset). We combined the results from the two datasets and obtained 8 miRNAs down-regulated in both datasets, and the 8 miRNAs regulated a total of 327 target genes, and 10 of these target genes were co-regulated by two miRNAs. Conclusions The core miRNAs and the target genes regulated by multiple miRNAs in the regulatory network may play important roles in the pathogenesis of PMOP and have potential application values as molecular biomarkers of disease. These findings are meaningful for the studies of PMOP pathogenesis, biomarkers screening and prevention of osteoporotic fractures. Key words: Osteoporosis, postmenopausal; MicroRNAs; Regulatory network

High Frequencies of Mutated EZH2 and IRF8 and Other Epigenetic Genes in Primary Bone Lymphomas Are Indicative of GCB-Phenotype

Introduction Primary bone diffuse large B-cell lymphoma (PB-DLBCL) is a rare extranodal lymphoma comprising 1-2% of all malignant lymphomas. This study aims to elucidate the genetic background of a homogeneous cohort of PB-DLBCL. Methods This retrospective study consists of primary DLBCL-patients with bone localization(s) of which pretreatment fresh frozen or formalin-fixed paraffin-embedded bone tissue samples were available. Patients were diagnosed (2003-2019) at Leiden University Medical Center (LUMC), a center of expertise for bone tumors, Amsterdam University Medical Center (AUMC), Erasmus MC and affiliated Dutch hospitals. Based on strict definitions regarding radiological assessment of anatomical disease localizations at diagnosis three subgroups were categorized: solely osseous involvement (single or multiple bone lesions; PB-DLBCL), osseous involvement and locoregional lymphadenopathy (locoregional disease), and osseous and (multiple) extra-osseous localizations (disseminated disease). Cell-of-origin (COO) was determined by immunohistochemistry (BCL6, CD10, and MUM1) and classified according to the Hans' algorithm. Additionally, COO was confirmed with NanoString and the Lymph2Cx assay (Scott et al., Blood 2014), in a subset of patients. With similar procedures (Vermaat et al., Haematologica 2019), molecular profiles were determined with an in-house developed and validated targeted next-generation sequencing (tNGS) panel, comprising 52 DLBCL-specific genes, for sequencing with the Ion S5TM System. Obtained results were compared to sequencing data of (1) an independent 'in-house' cohort of 23 primary GCB (Germinal Center B-Cell)-DLBCL patients without bone localization ('non-osseous') and (2) pooled data of 651 GCB-DLBCL patients from literature (Chapuy et al., Nature Medicine 2018, Karube et al., Leukemia 2018, Reddy et al., Cell 2017, Schmitz et al., NEJM 2018). Results Our cohort contained 56 patients (males, N=33, (59%)) with a median age at diagnosis of 62 years (range 13-92). Twenty-four patients had PB-DLBCL (45%), 8 had locoregional disease (14%), and 23 had disseminated disease (41%). In general, immunohistochemistry and Lymph2Cx identified a GCB subtype for the majority of all DLBCL with bone localizations (Figure-1A) and these results for the hitherto unperformed cases will follow shortly. tNGS identified 48 genes with 'pathogenic' mutations, with on average four mutated genes per patients (range 0-10; Figure-1A). Overall, high mutation frequencies were observed in TNFRSF14 (33%), KMT2D (27%), EZH2 (25%), CREBBP (22%), B2M (22%), and TP53 (20%) in DLBCL with bone localizations and mainly genes involved in epigenetic machinery. In PB-DLBCLs, high frequency of mutated EZH2 (38%) and IRF8 (25%) were identified. Both are epigenetic genes that regulates tumor suppression and type I interferon, respectively. In four PB-DLBCLs EZH2 and IRF8 were concomitantly mutated. Locoregional disease showed a similar molecular profile as PB-DLBCL. Association with clinical characteristics will be performed shortly. Compared to our cohort of non-osseous GCB-DLBCL (Figure-1B) and pooled data of GCB-DLBCL in large sequencing studies (Figure-1C), EZH2 (Chi-square; P=0.046 and P=0.005, respectively) was significantly more frequently mutated in PB-DLBCL, though IRF8 did not attain this significance (Chi-square; P=0.121 and P=0.111, respectively; Figure-1D). Conclusion This study is the first that provides integrative analyses of immunohistochemistry, Lymph2Cx, and tNGS of a homogeneous cohort of PB-DLBCL, demonstrating the importance of epigenetic genes in lymphomagenesis. In contrast to (non-osseous) GCB-DLBCLs, the molecular profile of PB-DLBCL is characterized by significantly frequent mutations in EZH2 and frequent mutations in IRF8 and other epigenetic genes, which is indicative for a GCB phenotype (Scherer F. et al., Sci Transl Med 2016) and supported by immunohistochemistry and Lymph2Cx data. These results suggest that PB-DLBCL is a specific DLBCL-entity with a unique molecular profile and provide a rationale for exploration of novel targeted treatment with EZH2 (and IRF8) inhibitors for PB-DLBCL patients. Disclosures Lugtenburg: Genmab: Consultancy, Honoraria; Servier: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy; Celgene: Consultancy, Honoraria; Janssen Cilag: Honoraria; Takeda: Consultancy, Honoraria, Research Funding. Kersten:Gilead: Honoraria; Mundipharma: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Takeda Oncology: Research Funding; Miltenyi: Honoraria; Kite Pharma: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria.

Adjacent Level Tuberculous Spondylodiscitis Leading to Proximal Junctional Kyphosis: Rare and Unusual Presentation

Vertebral osteomyelitis manifesting as a compression fracture misdiagnosed in the setting of steroid-induced or senile osteoporosis is very rare, although such patients are prone to infection or reactivation, as their immune system is exhausted. Spondylodiscitis occurring at adjacent levels following instrumented spinal fusion leading to pathologic fracture and proximal junctional failure, especially caused by tuberculosis, to our knowledge, has not been discussed in the literature. In case 1, a 61-year-old woman with osteoporotic T12 collapse was treated with corpectomy, anterior reconstruction, and posterior fixation from T9-L2. Initial biopsy and culture were normal. She presented 4 months later with compression fracture of T8; T8 corpectomy with anterior reconstruction and proximal extension of the construct was performed. In case 2, a 65-year-old woman with multiple comorbidities and osteoporotic L1 compression fracture was treated with L1 corpectomy, anterior reconstruction, and posterior instrumentation from T11-L3. She presented 4 months later with T10 vertebral body acute collapse; 2-stage anterior corpectomy and reconstruction was performed. In both cases, probing the affected vertebral body yielded pus. Pus and bone tissue samples sent for culture and histopathologic examination were positive for tuberculosis suggesting tuberculous spondylitis in both cases. In both patients, tuberculous spondylodiscitis at the proximal adjacent level was diagnosed <1 year after the initial spinal surgery. Neither patient had a previous history of pulmonary or extrapulmonary tuberculosis. They were successfully treated with antituberculous therapy and proximal extension of the construct with anterior reconstruction. Adjacent segment spondylodiscitis should be suspected and intraoperative biopsy must be considered for histopathologic and microbiologic examination to rule out subclinical infection in immunosuppressed patients with multiple comorbidities. Management should be individualized, considering the context of infection, causative organism, extent of bone destruction, and neurologic involvement.

2019 J. Leonard Goldner Award Winner: A Novel Intraoperative Technique Seeding Morselized Bone Tissue into Pediatric Blood Culture Bottles Improves Microbiological Diagnosis in Patients with Foot and Ankle Osteomyelitis

Category:Basic Sciences/Biologics, DiabetesIntroduction/Purpose:Bone infection in the foot is a challenging pathology that frequently leads to amputation and major disability. Agent identification and antimicrobial susceptibility-guided therapy is the key for a successful treatment. Standard culture methods for bone tissue have demonstrated poor sensitivity. It has been reported that synovial fluid culture sensitivity is enhanced when seeded into pediatric blood culture bottles (PBCB), however, its performance for bone infection has yet to be determined. The aim of this study was to evaluate a novel method to obtain and process infected bone tissue using PBCB and comparing it with the standard technique.Methods:Patients admitted at a single university hospital with foot osteomyelitis between 2008 and 2017 were recruited prospectively. The diagnosis was made by clinical, laboratory and imaging findings. A representative sample of infected bone tissue was obtained for each patient and simultaneously processed using the standard technique for solid tissue cultures (SCM) and a morselized bone sample that was seeded into a PBCB (BacT/ALERT® PF). Culture sensitivity and number of agents detected for each technique were compared using the McNemar and the Mann Whitney tests respectively.Results:Over the period studied, 107 patients fitted the inclusion criteria. Of these, 63 patients had a diabetic-related infection. The causative agent was identified in 60.7% using the SCM and in 97.2% using PBCB (p < 0.001). We detected a mean of 1.05 bacteria species using SCM and 1.67 using PBCB (p<0.01). PBCB retrieved 1 bacterial species in 50.5% of the samples and 2 or more bacterial species in 46.7%. The SCM detected 1 bacterial species in 29% of the samples and 2 or more bacterial species in 31.7%.Conclusion:In this cohort, culture technique using morselized bone tissue seeded in PBCB identified the causative agent in a significantly larger percentage than SCM. Additionally, this method identified a larger number of agents. These findings demonstrate that this novel, simple and reproducible technique for culturing bone tissue samples allows a better microbiological diagnosis, without additional intraoperative risks, compared with standard methods.

Simple Defocused Fiber Optic Volume Probe for Subsurface Raman Spectroscopy in Turbid Media.

We investigated the ability to perform deep subsurface Raman spectroscopy in turbid media using a simple fiber optic volume probe. Being able to collect Raman signals from regions deep within a biological sample provides the ability to noninvasively study underlying living tissue and tissue engineered constructs with high chemical specificity. Spatially offset Raman spectroscopy has shown great potential for obtaining subsurface Raman signals in biological samples. The applicability of the method for invivo studies depends on the system complexity and small size probes are often desirable. Most real-time studies on human patients utilizing Raman spectroscopy have been performed with easy-to-handle miniaturized probes. Here we show both experimentally and theoretically that the sampling depth from a simple volume probe can be controlled by changing the probe to sample distance effectively suppressing Raman and fluorescence contributions from shallow sample layers while favoring the collection of signals from deeper layers. Relative spectral intensities as function of probe to sample distance were investigated for layered phantoms of poly(methyl methacrylate) and trans-stilbene and compared with theory. The volume probe was then utilized for the collection of spectra from phantoms mimicking invivo transcutaneous measurement configurations of bone and engineered scaffold as well as from an ex vivo sample of bone and soft tissue. Together the results show that Raman fiber optic volume probes can be utilized for subsurface Raman spectroscopy in turbid media, providing a simple alternative to spatially offset Raman systems for, e.g., noninvasive monitoring and studying mineralized tissue and implanted scaffolds invivo.

Rapid Repair of Goat Large Segmental Loading Bone Defect and Functional Reconstruction by Interventional Micro-Circulation System

Objective: This study aimed to explore the efficacy of interventional micro-circulation system (IMCS), combining the stromal cell-derived factor 1 (SDF-1)/type I collagen/chitosan composite membrane into TEB in treating with the model of goat tibial LSBD. Methods: The model of goat tibial LSBD was constructed by introducing the IMCS and SDF-1 guiding membrane. Perfusion treatment of CXCR4 + -BMSCs and BMP-2 was performed on the animal model, general conditions of the goats were observed, and changes in the goat's tibia was observed at different time points through imaging techniques. Using RNA extraction and reverse transcription, cDNA was acquired. Alkaline phosphatase (ALP), core binding factor-α1 (Cbfα1) and osteocalcin (OC) content, as well as the mRNA of transformation growth factor-β1 (TGF-β1) in fixed bone tissue samples of the goat, were detected by real-time quantitative PCR (RT-qPCR). Results: The model of goat tibial LSBD repaired by IMCS was successfully constructed. The standing, walking and running functions of the goat recovered well after two months; and imaging revealed that the callus was closely connected and most of the scaffold materials of TEB were absorbed. The detection of the ALP gene in bone tissue samples failed, and the amplified product was non-specific. Furthermore, the levels of Cbfα1, OC and mRNA of TGF-β1 were significantly higher than in normal bones (P &lt; 0.05). Conclusion: The combination treatment of IMCS with slow controlled drug delivery systems and TEB functioned well in repairing goat tibial LSBD. This is a novel method for repairing bone defects.

Hemostasis and Bone Regeneration Using Chitosan/Gelatin-BCP Bi-layer Composite Material.

The aim of the study was to determine the hemostatic activity of a composite bi-layered topical hemostat composed of electrospun gelatin loaded with bi-phasic calcium phosphate and chitosan layer and its effect on bone formation. Morphology of the composite hemostat and its individual components were observed using scanning electron microscopy. In vitro biocompatibility of the topical hemostat tested using preosteoblasts cells (MC3t3-E1) showed no adverse toxicity. Confocal microscopy of seeded cells showed good cell adhesion while 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated 15% increased cell proliferation at the end of 1 week culture period. The material's efficiency as a hemostatic agent was tested by testing blood adsorption capacity and in vivo bone bleeding models. Blood absorption indicates that test sample measuring 14.14 mm becomes fully saturated within 5 minutes of blood contact. Bone bleeding was induced on the frontal plates of rat skulls and samples were applied as either removable topical hemostat or actual degradable hemostat. The effect of the bi-layered hemostat on bone formation was determined through analysis of micro-computed tomography (microCT) and observation of histological sections of extracted bone tissue samples. Results indicate that the bi-layer hemostat was able to halt bleeding within 3 minutes of application on the bleeding site and significantly enhanced bone regeneration. Using the bi-layer material as a degradable hemostat also drastically improved bone regeneration of the 3 mm defect.

Abstract 2036: Osteoblasts use multiple mechanisms of cellular communication to regulate breast cancer cell proliferation in bone

Abstract Breast cancer (BC) preferentially metastasizes to bone. While the mechanism for preferred metastasis is unknown, bone likely provides a hospitable environment that attracts BC cells (BCCs) and allows them to colonize and grow. Bone metastases perturb the balance of normal bone remodeling. Emerging evidence suggests that OBs may be altered and respond differently to cancer cells during metastatic BC progression. According to one study, OBs suppress cancer cell growth in early metastasis. Our work suggests OBs reduce BCC growth in bone prior to macrometastatic colonization. Thus, our data suggest OBs have tumor-inhibitory properties. We have new evidence to suggest that communication between OBs and BCCs ‘educates’ OBs to produce factors that suppress BCC proliferation in bone. We have in-vitro and in-vivo mouse-model evidence that ‘educated’ osteoblasts (EOs) have a unique secretory protein profile compared to ‘uneducated’ OBs. We also identified EOs as being present in the bone tissue samples of human patients with bone metastatic BC via multi-plex immunofluorescence. When we treated BCCs with EO conditioned media (CM), BCC proliferation was reduced in both triple negative and ER+ metastatic BCCs, while CM from ‘uneducated’ OBs did not affect BCC proliferation. This effect was mediated through alterations in EO production of decorin and NOV. We identified EO CM as a rich source of exosomes (exo) and confirmed the presence of an exo population via iodixanol density gradient and western blotting for specific exo protein markers. We found that EO-derived exo, but not ‘uneducated’ OB-derived exo, decreased proliferation of ER+ and triple negative BCC. Also, exposure to EO-derived exo 1) decreased the number of Ki67 positive metastatic BCC, and 2) decreased the number of metastatic BCC in the S phase of cell cycle as measured by EdU incorporation. Moreover, we labeled EO exo with RFP-conjugated CD63 to visually confirm exo transfer from EO cells to BCCs using confocal microscopy. And, co-culture with EOs increased triple negative and ER+ metastatic BC expression of p21 compared to co-cultures with ‘uneducated’ OBs. Our data suggest that EOs use multiple mechanisms of cellular communication to regulate BCC proliferation in bone. Impact: Our data suggest OBs produce factors that suppress metastatic BCC growth. Much less attention has been given to OB interactions with tumor cells at sites of bone metastasis due to observations that OB populations are reduced at sites of advanced osteolysis. However, we propose that OBs may be valuable endogenous targets to aid in suppression of metastatic BC growth in the niche in concert with therapeutic drugs to kill the cancer cells. Our data suggest there is a population of OBs that demonstrate a functional role in suppressing metastatic BCC growth; a property capable of exploitation. For these reasons, OBs and EOs are strong candidates for therapeutic targeting. Citation Format: Alexus D. Kolb, Alison B. Shupp, Dimpi Mukhopdhyay, Karen M. Bussard. Osteoblasts use multiple mechanisms of cellular communication to regulate breast cancer cell proliferation in bone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2036.