Secondary ion mass spectrometry for bone research.

The purpose of this Tutorial is to highlight the suitability of time-of-flight secondary ion mass spectrometry (ToF-SIMS) and OrbiTrap™ SIMS (Orbi-SIMS) in bone research by introducing fundamentals and best practices of bone analysis with these mass spectrometric imaging (MSI) techniques. The Tutorial includes sample preparation, determination of best-suited measurement settings, data acquisition, and data evaluation, as well as a brief overview of SIMS applications in bone research in the current literature. SIMS is a powerful analytical technique that allows simultaneous analysis and visualization of mineralized and nonmineralized bone tissue, bone marrow as well as implanted biomaterials, and interfaces between bone and implants. Compared to histological staining, which is the standard analytical procedure in bone research, SIMS provides chemical imaging of nonstained bone sections that offers insights beyond what is conventionally obtained. The Tutorial highlights the versatility of ToF- and Orbi-SIMS in addressing important questions in bone research. By illustrating the value of these MSI techniques, it demonstrates how they can contribute to advance progress in bone research.

Identification of Pseudomonas aeruginosa exopolysaccharide Psl in biofilms using 3D OrbiSIMS.

Secondary ion mass spectrometry (SIMS) offers advantages over both liquid extraction mass spectrometry and matrix assisted laser desorption mass spectrometry in that it provides the direct in situ analysis of molecules and has the potential to preserve the 3D location of an analyte in a sample. Polysaccharides are recognized as challenging analytes in the mass spectrometry of liquids and are also difficult to identify and assign using SIMS. Psl is an exopolysaccharide produced by Pseudomonas aeruginosa, which plays a key role in biofilm formation and maturation. In this Letter, we describe the use of the OrbiTrap analyzer with SIMS (3D OrbiSIMS) for the label-free mass spectrometry of Psl, taking advantage of its high mass resolving power for accurate secondary ion assignment. We study a P. aeruginosa biofilm and compare it with purified Psl to enable the assignment of secondary ions specific to the Psl structure. This resulted in the identification of 17 peaks that could confidently be ascribed to Psl fragments within the biofilm matrix. The complementary approach of the following neutral loss sequences is also shown to identify multiple oligosaccharide fragments without the requirement of a biological reference sample.

Identifying new biomarkers of aggressive Group 3 and SHH medulloblastoma using 3D hydrogel models, single cell RNA sequencing and 3D OrbiSIMS imaging.

The most common malignant brain tumour in children, medulloblastoma (MB), is subdivided into four clinically relevant molecular subgroups, although targeted therapy options informed by understanding of different cellular features are lacking. Here, by comparing the most aggressive subgroup (Group 3) with the intermediate (SHH) subgroup, we identify crucial differences in tumour heterogeneity, including unique metabolism-driven subpopulations in Group 3 and matrix-producing subpopulations in SHH. To analyse tumour heterogeneity, we profiled individual tumour nodules at the cellular level in 3D MB hydrogel models, which recapitulate subgroup specific phenotypes, by single cell RNA sequencing (scRNAseq) and 3D OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) imaging. In addition to identifying known metabolites characteristic of MB, we observed intra- and internodular heterogeneity and identified subgroup-specific tumour subpopulations. We showed that extracellular matrix factors and adhesion pathways defined unique SHH subpopulations, and made up a distinct shell-like structure of sulphur-containing species, comprising a combination of small leucine-rich proteoglycans (SLRPs) including the collagen organiser lumican. In contrast, the Group 3 tumour model was characterized by multiple subpopulations with greatly enhanced oxidative phosphorylation and tricarboxylic acid (TCA) cycle activity. Extensive TCA cycle metabolite measurements revealed very high levels of succinate and fumarate with malate levels almost undetectable particularly in Group 3 tumour models. In patients, high fumarate levels (NMR spectroscopy) alongside activated stress response pathways and high Nuclear Factor Erythroid 2-Related Factor 2 (NRF2; gene expression analyses) were associated with poorer survival. Based on these findings we predicted and confirmed that NRF2 inhibition increased sensitivity to vincristine in a long-term 3D drug treatment assay of Group 3 MB. Thus, by combining scRNAseq and 3D OrbiSIMS in a relevant model system we were able to define MB subgroup heterogeneity at the single cell level and elucidate new druggable biomarkers for aggressive Group 3 and low-risk SHH MB.

Development of nanoparticle loaded microneedles for drug delivery to a brain tumour resection site.

Systemic drug delivery to the central nervous system (CNS) has been historically impeded by the presence of the blood brain barrier rendering many therapies inefficacious to any cancer cells residing within the brain. Therefore, local drug delivery systems are being developed to overcome this shortfall. Here we have manufactured polymeric microneedle (MN) patches, which can be anchored within a resection cavity site following surgical removal of a tumour such as isocitrate dehydrogenase wild type glioblastoma (GBM). These MN patches have been loaded with polymer coated nanoparticles (NPs) containing cannabidiol (CBD) or olaparib (OLA) and applied to an in vitro brain simulant and ex vivo rat brain tissue to assess drug release and distance of penetration. MN patches loaded with methylene blue dye were placed into a cavity of 0.6 % agarose to simulate brain tissue. The results showed that clear channels were generated by the MNs and the dye spread laterally throughout the agarose. When loaded with CBD-NPs, the agarose showed a CBD concentration of 12.5 µg/g at 0.5 cm from the MN insertion site. Furthermore, high performance liquid chromatography of ex vivo brain tissue following CBD-NP/MN patch insertion showed successful delivery of 59.6 µg/g into the brain tissue. Similarly, OLA-NP loaded MN patches showed delivery of 5.2 µg/g OLA into agarose gel at 0.5 cm distance from the insertion site. Orbitrap secondary ion mass spectrometry (OrbiSIMS) analysis confirmed the presence of OLA and the MN patch at up to 6 mm away from the insertion site following its application to a rat brain hemisphere. This data has provided insight into the capabilities and versatility of MN patches for use in local brain drug delivery, giving promise for future research.

Elucidating the molecular landscape of the stratum corneum.

SignificanceSkin is recognized as an intricate assembly of molecular components, which facilitate cell signaling, metabolism, and protein synthesis mechanisms in order to offer protection, regulation, and sensation to the body. Our study takes significant steps to characterize in more detail the complex chemistry of the skin, in particular by generating a better understanding of the uppermost layer, the stratum corneum. Using a state-of-the-art 3D OrbiSIMS technique, we were able to observe the depth distribution, in situ, for a wide range of molecular species. This unprecedented molecular characterization of skin provides information that has the potential to benefit research into fundamental processes, such as those associated with skin aging and disease, and the development and delivery of effective topical formulations.

Surgical treatment outcome after serial debridement of infected nonunion-A retrospective cohort study.

PURPOSE: Reported outcome after multiple staged surgical treatment of infected nonunion is scarce. We, therefore, asked: (1) What is the clinical outcome in infected nonunion patients after multiple staged revision surgery? (2) Are different pathogens evidenced after surgical treatment in patients who have undergone more or less surgeries? METHODS: All enrolled patients were surgically treated for long bone-infected nonunion between January 2010 and March 2018. Besides patients´ demographics outcome in terms of bony consolidation and major complications defined as death during inward treatment, amputation and recurrence of infection during follow-up of at least 12 months were assessed. Microbiological findings were assessed and compared between two groups with less than five versus five or more surgical revisions. RESULTS: Bone consolidation was achieved in 86% of the patients while complications such as femoral or transtibial amputation, recurrence of infection or even death during inpatient treatment could be evidenced in six patients (14%). In patients who underwent multiple-stage surgery for five or more times, germ changes and repeated germ detection was more common than in patients with less surgeries. CONCLUSIONS: Surgical treatment of infected nonunions poses a high burden on the patients with major complications occurring in about 14% of the patients using a multiple staged treatment concept. Future prospective studies comparing outcomes after limited with multiple staged revision surgeries are necessary.

Polymicrobial infections and microbial patterns in infected nonunions - a descriptive analysis of 42 cases.

BACKGROUND: Frequencies of polymicrobial infection and pathogens evidenced in course of infected nonunion treatment are largely unknown. Therefore, this study aims at investigating microbial patterns in infected nonunions. METHODS: Surgically treated patients with long bone infected nonunion admitted between January 2010 and March 2018 were included in the study. Microbiological culture and polymerase-chain-reaction results of tissue samples of initial and follow-up revision surgeries were assessed and compared with patient and treatment characteristics. RESULTS: Forty two patients with a mean age of 53.9 ± 17.7 years were included. In six patients (14.3%) polymicrobial infection was evident. A change of pathogens evidenced in course of the treatment occurred in 21 patients (50%). In 16 patients (38.1%) previously detected bacteria could be determined by microbial testing after further revision surgery. Staphylococcus aureus was most often detected (n = 34, 30.6%), followed by Enterococcus spp. (n = 25, 22.5%) and Staphylococcus epidermidis (n = 18, 16.2%). Five Staphylococcus aureus were resistant to methicillin (MRSA). In patients without polymicrobial infection or further germ detection in course of the treatment, 86.4% of the infections were due to Staphylococcus spp.. Infections due to Streptococcus spp. and gram-negative bacteria were only present in patients with polymicrobial infection and germ-change in course of the treatment. CONCLUSION: A low rate of polymicrobial infections was evidenced in the present study. Germ-change often occurs in course of revision surgeries. Prospective studies with more sensitive diagnostic tools are necessary to elucidate the therapeutical relevance of microbiological testing results for surgical as well as medical treatment in infected nonunions.

Computed tomography for managing periprosthetic femoral fractures. A retrospective analysis.

BACKGROUND: Periprosthetic fractures (PPF) present a common cause for revision surgery after arthroplasty. The choice of performing either an osteosynthesis or revision arthroplasty depends on the orthopedic implant anchored and loosening. Standard diagnostics include x-ray imaging. CT is usually performed to confirm implant loosening in case of ambiguous diagnosis on standard x-ray imaging. This study aimed to examine the role of CT as a diagnostic modality and its implications for treatment planning and outcome. METHODS: Patients treated for PPF from January 2010 to February 2018 were included. X-ray and CT reports were analyzed to assess implant loosening. The planning for surgery and the final surgical treatment were evaluated. In addition, patient characteristics were analyzed and compared between patients with and without additional CT as a preoperative diagnostic procedure. RESULTS: Seventy-five patients were eligible for the study. X-ray imaging was performed in 90.7% of cases. CT was performed in 60% of the cases as part of the preoperative diagnostic. A clear statement on implant stability or loosening could not be made in 69.1% after X-ray imaging and in 84.4% following CT imaging. Revision arthroplasty for loosened femoral prosthesis components was necessary in 40% of cases. No difference could be determined comparing patients with X-ray imaging to those with X-ray and additional CT. In both groups, operative treatment did not deviate from the preoperative planning. DISCUSSION: In two thirds of the conventional radiographic findings, no reliable evaluation of implant loosening was possible in femoral PPFs. Intriguingly, additional CT did not improve the evaluation of implant loosening. Nonetheless, CT scans are often performed if loosening assessment is unclear on regular radiographs. This fact can explain the bias CT results in comparison to regular radiography. However, software-supported CT diagnosis could help to adequately answer the question of loosened implants in PPF in the near future. Since the diagnosis of fracture and their morphology assessment is currently adequately performed using X-rays, CT shall not be considered as the gold standard.

Do Systemic Factors Influence the Fate of Nonunions to Become Atrophic? A Retrospective Analysis of 162 Cases.

INTRODUCTION: Nonunions are a challenge for orthopedic surgeons. In hypertrophic nonunions, improvement of mechanical stability usually is the satisfactory treatment, whereas in atrophic nonunions improvement of the biological environment is most important. However, scientific evidence revealed that "avital" nonunions are not avascular and fibrous tissue contains cells with osteogenic potential. To find out if systemic factors suppress this intrinsic potential in atrophic nonunions, this study compares characteristics of hypertrophic with atrophic nonunion patients. METHODS: We analyzed medical records of 162 surgically treated patients suffering from aseptic long bone nonunions. Atrophic and hypertrophic nonunions were distinguished by absence or presence of callus and calcification in the fracture gap. Mechanical implant loosening and patient characteristics such as age, gender, and body mass index were assessed. Fracture classification according to AO/OTA, open and closed fractures, and osteosynthesis were recorded. In addition, comorbidities and allergies between both groups were compared. RESULTS: A higher number of hypertrophic nonunion patients were male with often allergies. Hypertrophic nonunion occurred more often after intramedullary nailing compared to atrophic nonunions. Atrophic nonunion patients being nonallergic were significantly older than nonallergic patients suffering from hypertrophic nonunions. In both atrophic and hypertrophic nonunion patients, age was lower in patients with accompanying injuries compared with age of patients with isolated fractures. CONCLUSION: Systemic factors influence development of nonunion types. In nonallergic patients, atrophic nonunions occur more often in the elderly. This manuscript is a first step to identify different factors which might influence the nature of nonunion. To enable nonunion treatment which is tailored to individual patient characteristics, further prospective studies with more sophisticated research methods are necessary.

Quantitative analysis of individual cell-free DNA concentration before and after penetrating trauma.

INTRODUCTION: Cell-free DNA (cfDNA) elevations were remarked in the blood of trauma patients. Published increases refer to comparative values of a healthy control group, ignoring thereby inter- and intra-individual differences under normal conditions. The aim of this study was to quantify cfDNA in patients in the time course of a planned orthopedic surgery, which constitutes the advantage of obtaining individual pre- and post-trauma values for each patient. By this approach, a basis should be established for the potential future application of cfDNA as biomarker for the detection of mild injuries related to volunteer experiments in forensic biomechanics. METHODS: Plasma samples of ten patients obtaining knee or hip arthroplasty were analyzed quantitatively for cfDNA by real-time qPCR the day prior operation (Prior), immediately afterwards (Day0), and the day after the surgery (Day1). RESULTS: Prior values exhibited a broad range, indicating pronounced inter-individual differences in the basic level of cfDNA. After surgery, levels were significantly elevated on both days (Wilcoxon test p = 0.002). In nine patients, highest values were measured on Day0, whereby a fold change of 19 was remarked once. After Day0, values decreased, though they did not reach Prior values until Day1 in nine patients. CONCLUSION: Endoprosthesis surgery represents a well-defined trauma scenario for the measurement of individual cfDNA elevations. The analysis of pre- to post-trauma alterations lay the groundwork for the application of cfDNA as biomarker for the detection of minor injuries in the field of forensic biomechanics.

Computational segmentation of collagen fibers in bone matrix indicates bone quality in ovariectomized rat spine.

Bone loss varies according to disease and age and these variations affect bone cells and extracellular matrix. Osteoporosis rat models are widely investigated to assess mechanical and structural properties of bone; however, bone matrix proteins and their discrepant regulation of diseased and aged bone are often overlooked. The current study considered the spine matrix properties of ovariectomized rats (OVX) against control rats (Sham) at 16 months of age. Diseased bone showed less compact structure with inhomogeneous distribution of type 1 collagen (Col1) and changes in osteocyte morphology. Intriguingly, demineralization patches were noticed in the vicinity of blood vessels in the OVX spine. The organic matrix structure was investigated using computational segmentation of collagen fibril properties. In contrast to the aged bone, diseased bone showed longer fibrils and smaller orientation angles. The study shows the potential of quantifying transmission electron microscopy images to predict the mechanical properties of bone tissue.

A New Clinically Relevant T-Score Standard to Interpret Bone Status in a Sheep Model.

BACKGROUND Osteoporosis is diagnosed by bone loss using a radiological parameter called T-score. Preclinical studies use DXA to evaluate bone status were the T-score is referenced on bone mineral density (BMD) values of the same animals before treatment. Clinically, the reference BMD represents values of an independent group of healthy patients around 30 years old. The present study established a clinically similar T-score standard to diagnose osteoporosis in a sheep model. MATERIAL AND METHODS We used 31 female merino land sheep (average 5.5 years old) to study osteoporosis. The following groups were compared using DXA measurement: 1) control; 2) ovariectomized (OVX); 3) OVX combined with a deficient diet (OVXD); and 4) OVXD combined with methylprednisolone administration (OVXDS). Further, an independent group of 32 healthy sheep (4-6 years old) were measured as an independent baseline. BMD was measured at 0 months, 3 months, and 8 months after treatment. RESULTS The same significance pattern between the treated groups and either baseline groups was seen. However, using an independent baseline changed the "clinical" interpretation of the data from an osteoporotic bone status (T-score <-2.5) after 3 months of OXDS treatment into an osteopenic bone status (T-score <-1.5 to -2.4). CONCLUSIONS Using an independent baseline enhanced the statistical significance and showed the clinical relevance. Furthermore, an independent baseline is a reliable alternative to use of a new control group for future experiments and thus reduces the number of animals needed by eliminating the need for a control and corresponding to clinical practice.

Osteocyte Regulation of Receptor Activator of NF-κB Ligand/Osteoprotegerin in a Sheep Model of Osteoporosis.

Osteoporosis induction in a sheep model by steroid administration combined with ovariectomy recapitulates decreased bone formation and substandard matrix mineralization in patients. Recently, the role of osteocytes has been frequently addressed, with focus on their role in osteoclastogenesis. However, the quantification of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) signaling in osteocytes was not studied in sheep. The current study reproduced the sheep model of osteoporosis to study the RANKL/OPG ratio correlation to the method of osteoporosis induction. We investigated the induction of osteoporosis after 8 months using 31 female merino land sheep divided into four groups: control, ovariectomy, ovariectomy with dietary limitation, and ovariectomy with dietary limitation and steroid injection. In accordance to previous reports, the present study showed trabecular thinning, higher numbers of apoptotic osteocytes, and imbalanced metabolism, leading to defective mineralization. The global RANKL/OPG ratio in the spine after 8 months of steroid and dietary treatment was not different from that of the control. Interestingly, assessment of the osteocyte-specific RANKL/OPG ratio showed that the steroid-induced osteoporosis in its late progressive phase stimulates RANKL expression in osteocytes. Sclerostin is suggested to induce RANKL expression in osteocytes. The findings of this study can contribute to further explain the success of sclerostin antibodies in treating osteoporotic patients despite increased osteocyte-expressed RANKL.

Postembedding Decalcification of Mineralized Tissue Sections Preserves the Integrity of Implanted Biomaterials and Minimizes Number of Experimental Animals.

Bone histology of decalcified or undecalcified samples depends on the investigation. However, in research each method provides different information to answer the scientific question. Decalcification is the first step after sample fixation and governs what analysis is later feasible on the sections. Besides, decalcification is favored for immunostaining and in situ hybridization. Otherwise, sample decalcification can be damaging to bone biomaterials implants that contains calcium or strontium. On the other hand, after decalcification mineralization cannot be assessed using histology or imaging mass spectrometry. The current study provides a solution to the hardship caused by material presence within the bone tissue. The protocol presents a possibility of gaining sequential and alternating decalcified and undecalcified sections from the same bone sample. In this manner, investigations using histology, protein signaling, in situ hybridization, and mass spectrometry on the same sample can better answer the intended research question. Indeed, decalcification of sections and grindings resulted in well-preserved sample and biomaterials integrity. Immunostaining was comparable to that of classically decalcified samples. The study offers a novel approach that incites correlative analysis on the same sample and reduces the number of processed samples whether clinical biopsies or experimental animals.

Inhibitor-mediated stabilization of the conformational structure of a histone deacetylase-like amidohydrolase.

Histone deacetylases are major regulators of eukaryotic gene expression. Not unexpectedly, histone deacetylases are among the most promising targets in cancer therapy. However, despite huge efforts in histone deacetylase inhibitor design, very little is known about the impact of histone deacetylase inhibitors on enzyme stability. In this study, the conformational stability of a well-established histone deacetylase homolog with high structural similarity (histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes species FB188) was investigated using denaturation titrations and stopped-flow kinetics. Based on the results of these complementary approaches, we conclude that the interconversion of native histone deacetylase-like amidohydrolase into its denatured form involves several intermediates possessing different enzyme activities and conformational structures. The refolding kinetics has shown to be strongly dependent on Zn(2+) and to a lesser extent on K(+), which underlines their importance not only for catalytic function but also for maintaining the correct conformational structure of the enzyme. Two main unfolding processes of histone deacetylase-like amidohydrolase were differentiated. The unfolding occurring at submolar concentrations of the denaturant guanidine hydrochloride was not affected by inhibitor binding, whereas the unfolding at higher concentrations of guanidine hydrochloride was strongly affected. It was shown that the known inhibitors suberoylanilide hydroxamic acid and cyclopentylpropionyl hydroxamate are capable of stabilizing the conformational structure of histone deacetylase-like amidrohydrolase. Judging from the free energies of unfolding, the protein stability was increased by 9.4 and 5.4 kJ.mol(-1) upon binding of suberoylanilide hydroxamic acid and cyclopentylpropionyl hydroxamate, respectively.