Osteoporosis is a mineral bone disease arising from the predominance of osteoclastic bone resorption. Bisphosphonates which inhibit osteoclasts are commonly used in osteoporosis treatment, but are not without severe adverse effects like osteonecrosis of the jaw. The mechanisms behind the development of such phenomena is not well understood. Bone homeostasis is achieved through an intimate cross-talk between osteoclasts and osteoblasts. Thus, it is important to visualise activities of these cells simultaneously in situ. Currently, there are means to visualise osteoclast shape and numbers with tartrate-resistant alkaline phosphatase (TRAP) staining but no practical and accurate methods to quantify osteoclast activity in situ. This investigation aims to establish the use of ELF97, a substrate of TRAP, to visualise and quantify osteoclast activity. This provides vital clues to mechanisms of various bone disorders. TRAP dephosphorylation of ELF97 results in a detectable fluorescent product at areas of osteoclast activity. Osteoclastic activity was initiated in zebrafish by inducing crush injuries in tail fin rays. Colocalisation of ELF97 fluorescence with osteoclast-specific DsRed in transgenic zebrafish, visualised under confocal microscopy, is used to further establish the specificity of ELF97 to sites of osteoclastic activity. Quantification is established by comparing fluorescence between wild type, osteoclast-deficient mutants and bisphosphonate-treated zebrafish. The utility of ELF97 will also be investigated in terms of the stability of the florescent product. The investigation revealed that ELF97 and DsRed fluorescence were found commonly at crush sites with osteoclastic activity. Wild type zebrafish had greater fluorescence compared to osteoclast-deficient (p<0.0001) and bisphosphonate-treated zebrafish (p<0.0001) after 7 and 14 days post-crush, revealing that fluorescence from ELF97 corresponds to expected osteoclastic activity. Fluorescence of tail fins treated with ELF97 did not diminish over a period of 21 days of storage, demonstrating its stability. ELF97 is thus a useful means to visualise osteoclast activity, potentially crucial in more advanced investigations to understand bone disorders. It could be used in combination with other cellular markers in whole biological samples to study and experimentally manipulate bone remodelling

Summary. Objective assessment of tendon histomorphology, particularly in the context of tissue repair, requires comprehensive analyses of both cellular distribution and matrix architecture. Fourier Transform analyses of histological images collected with second harmonic generation (SHG-FT) technique provide objective, quantitative assessment of collagen fiber organization with high specificity. Concurrent nuclear staining allows simultaneous analyses of cell morphology and distribution. Introduction. Tendon injuries can be career-limiting in human and equine athletes, since the architectural organization of the tissues are lost in the course of fibrotic repair. Objective assessment of tendon repair is problematical, particularly in research addressing potential therapies. Fourier Transform analyses of histological images collected with second harmonic generation (SHG-FT) technique can provide objective, quantitative assessments of collagen fiber organization with high specificity. This study describes the use of SHG-FT with fluorescently-labelled tendon-derived cells (TDC) in an in-vivo model of equine tendinitis to assess the temporal and spatial effects of cell delivery on collagen fiber organization. Materials and methods. Collagenase-induced tendinitis was created in the mid-metatarsal region of one hindlimb superficial digital flexor tendons (SDFT) in two horses. SDFTs from two clinically normal adult horses and were also used as controls. Autogenous TDCs were isolated from the lateral digital extensor tendon of the contralateral hind limb. Four weeks post-collagenase injection, 10×10. 6. DiI-labeled TDCs were injected into the tendon lesions. Tendon samples were obtained for histologic evaluation following euthanasia, 2-weeks after cell injections. Tendon samples were cryo-sectioned to 25–30μ exposed to nuclear counter stains (DAPI and PI) and imaged immediately through a confocal microscope (Zeiss LSM 710) with a 2-photon laser source, to obtain backward SHG (bSHG) and forward SHG (fSHG) images. In addition, images with DiI and DAPI fluorescence were acquired using 500–550 nm (green) or 565–615 nm (orange) emission filters, respectively. Fourier analysis of the SHG images was carried out using imageJ software. Results. DiI-labeled TDCs could be imaged successfully under two-photon fluorescence concurrently with SHG imaging. This was possible because the excitation wavelength of the two-photon laser (780nm) and detection of emissions above 565nm do not interfere with the bSHG band (380–400nm). Images collected with bSHG included signals from DAPI-stained nuclei. In contrast, emissions from PI-labeled nuclei were acquired independently of SHG signals. The contrast generated by individual collagen fibers was higher in images collected with fSHG than bSHG. SHG-FT of fSHG images provided accurate assessment of collagen fiber orientation in repair tissue and normal tendon. Discussion/Conclusions. Objective assessment of collagen orientation, along with spatial distribution of cells within healing tendon serves as useful indices of healing. Injected DiI-labeled TDCs could be imaged successfully under two-photon fluorescence concurrently with SHG imaging. However, DiI fluorescence is susceptible to photo-bleaching during SHG acquisition. Use of an alternative nuclear counter stains, such as PI, that do not emit along with SHG signal should be considered to optimise data acquisition and support simultaneous analyses of collagen structure, cellular morphology and cell distribution. SHG-FT histologic analysis along with biochemical and biomechanical indices collectively provide comprehensive assessment of therapies for tendon repair

Aim

Adequate debridement of necrotic bone is of paramount importance for eradication of infection in chronic osteomyelitis. Currently, no tools are available to detect the exact amount of necrotic bone in order to optimize surgical resection. The aim of the present study was to evaluate the feasibility of an intraoperative illumination method (VELscope^®) and the correlation between intraoperative and pathohistological findings in surgically treated chronic fracture related infection patients.

Osteoarthritis (OA) is a degenerative joint disease affecting millions worldwide. Early detection of OA and monitoring its progression is essential for effective treatment and for preventing irreversible damage. Although sensors have emerged as a promising tool for monitoring analytes in patients, their application for monitoring the state of pathology is currently restricted to specific fields (such as diabetes). In this study, we present the development of an optical sensor system for real-time monitoring of inflammation based on the measurement of nitric oxide (NO), a molecule highly produced in tissues during inflammation. Single-walled carbon nanotubes (SWCNT) were functionalized with a single-stranded DNA (ssDNA) wrapping designed using an artificial intelligence approach and tested using S-nitroso-N-acetyl penicillamine (SNAP) as a standard released-NO marker. An optical SWIR reader with LED excitation at 650 nm, 730 nm and detecting emission above 1000 nm was developed to read the fluorescence signal from the SWCNTs. Finally, the SWCNT was embedded in GelMa to prove the feasibility of monitoring the release of NO in bovine chondrocyte and osteochondral inflamed cultures (1–10 ng/ml IL1β) monitored over 48 hours. The stability of the inflammation model and NO release was indirectly validated using the Griess and DAF-FM methods. A microfabricated sensor tag was developed to explore the possibility of using ssDNA-SWCNT in an ex vivo anatomic set-up for surgical feasibility, the limit of detection, and the stability under dynamic flexion. The SWCNT sensor was sensitive to NO in both in silico and in vitro conditions during the inflammatory response from chondrocyte and osteochondral plug cultures. The fluorescence signal decreased in the inflamed group compared to control, indicating increased NO concentration. The micro-tag was suitable and stable in joints showing a readable signal at a depth of up to 6 mm under the skin. The ssDNA-SWCNT technology showed the possibility of monitoring inflammation continuously in an in vitro set-up and good stability inside the joint. However, further studies in vivo are needed to prove the possibility of monitoring disease progression and treatment efficacy in vivo. Acknowledgments: The project was co-financed by Innosuisse (grant nr. 56034.1 IP-LS)

Aim. Prosthetic joint infections pose a major clinical challenge. Developing novel material surface technologies for orthopedic implants that prevent bacterial adhesion and biofilm formation is essential. Antimicrobial coatings applicable to articulating implant surfaces are limited, due to the articulation mechanics inducing wear, coating degradation, and toxic particle release. Noble metals are known for their antimicrobial activity and high mechanical strength and could be a viable coating alternative for orthopaedic implants [1]. In this study, the potential of thin platinum-based metal alloy coatings was developed, characterized, and tested on cytotoxicity and antibacterial properties. Method. Three platinum-based metal alloy coatings were sputter-coated on medical-grade polished titanium discs. The coatings were characterized using optical topography and scanning electron microscopy with energy dispersive spectroscopy (SEM/EDS). Ion release was measured using inductively coupled plasma optical emission spectrometry (ICP-OES). Cytotoxicity was tested according to ISO10993-5 using mouse fibroblasts (cell lines L929 and 3T3). Antibacterial surface activity, bacterial adhesion, bacterial proliferation, and biofilm formation were tested with gram-positive Staphylococcus aureus ATCC 25923 and gram-negative Escherichia coli ATCC 25922. Colony forming unit (CFU) counts, live-dead fluorescence staining, and SEM-EDS images were used to assess antibacterial activity. Results. Three different platinum-based metal alloys consisting of platinum-iridium, platinum-copper, and platinum-zirconium. The coatings were found 80 nm thick, smooth (roughness average < 60 nm), and non-toxic. The platinum-copper coating showed a CFU reduction larger than one logarithm in adherent bacteria compared to uncoated titanium. The other coatings showed a smaller reduction. This data was confirmed by SEM and live-dead fluorescence images, and accordingly, ICP-OES measurements showed low levels of metal ion release from the coatings. Conclusions. The platinum-copper coating showed low anti-adhesion properties, even with extremely low metal ions released. These platinum-based metal alloy coatings cannot be classified as antimicrobial yet. Further optimization of the coating composition to induce a higher ion release based on the galvanic principle is required and copper looks most promising as the antimicrobial compound of choice. Acknowledgments. This publication is supported by the DARTBAC project (with project number NWA.1292.19.354) of the research program NWA-ORC which is (partly) financed by the Dutch Research Council (NWO); and the AMBITION project (with project number NSP20–1-302), co-funded by the PPP Allowance made available by Health-Holland, Top Sector Life Sciences & Health to ReumaNederland

3D Printed polyether-ether-ketone (PEEK) has gained widespread use in clinical practice due to its excellent biocompatibility, biomechanical compatibility, and personalization. However, pre-printed PEEK implants are not without their flaws, including bioinert, optimization distortion of 3D printing digital model and prosthetic mismatching. Recent advancements in mechanical processing technology have made it possible to print bone implants with PEEK fused deposition, allowing for the construction of mechanically adaptable implants. In this study, we aimed to synthesize silanized polycitrate (PCS) via thermal polymerization and in situ graft it to PEEK surface to construct an elastomer coating for 3D printed PEEK implants (PEEK-PCS). This incorporation of PCS allows the implant to exhibit adaptive space filling ability and stress dispersal. In vivo and in vitro results, PEEK-PCS exhibited exceptional osseointegration and osteogenesis properties along with macrophage M2 phenotypic polarization, inflammatory factors reducing, promotion of osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). Additionally, PEEK-PCS displays good autofluorescence properties in vitro and in vivo, with stable fluorescence for 14 days, suggesting potential bioimaging applications. The study confirms that PEEK in situ grafting with thermo-polymerized PCS elastomers is a viable approach for creating multifunctional (bone defect adaptation, bioimaging, immune regulation, and osseointegration) implants for bone tissue engineering

While high-performance ceramics like alumina and zirconia exhibit excellent wear resistance, they provide poor osseointegration capacity. As osseointegration is crucial for non-cemented joint prostheses, new techniques have been successfully developed for biofunctionalizing high-performance ceramic surfaces. Stable cell adhesion can be achieved by covalently bound specific peptides. In this study we investigate the effect of sterilization processes on organo-chemically functionalized surfaces. To enhance the performance of alumina-toughened zirconia ceramics (ATZ), a 3-aminopropyldiisopropylethoxysilane (APDS) monolayer was applied and coupled with cyclo-RGD peptides (cRGD) by using bifunctional crosslinker bis(sulfosuccinimidyl)suberat (BS³). The samples were sterilized using e-beam or gamma-sterilization at 25 kGy, either before or after biofunctionalization with cRGD. Functionalization stability was investigated by contact angle measurements. The functionality of cRGD after sterilization was demonstrated using proliferation tests and cytotoxicity assays. Immunofluorescence staining (pFAK, Actin, DAPI) was conducted to evaluate the adhesion potential between the samples and human mesenchymal stem cells (hMSCs). Functionalized samples before and after sterilization showed no significant difference regarding their contact angles. A proliferation test demonstrated that the cells on functionalized samples proliferate significantly more than on untreated samples before and after sterilization. hMSCs showed a significant higher proliferation on gamma sterilized samples compared to all other groups after 14 days. It was confirmed that the samples did not exhibit cytotoxic behavior before or after sterilization. Fluorescence microscopy demonstrated that both, cells on sterilized and on non-sterilized samples, expressed high levels of pFAK-Y397. The investigated functionalization enables improved adhesion and proliferation of hMSCs and is stable against the investigated sterilization processes. This is of importance as the option of having a sterile product enables the start of the translation of this biofunctional coating towards preclinical and subsequently first-in-man applications. Acknowledgments: We acknowledge the financial support of the Federal Ministry of Education and Research, BMBF (13GW0452A-C)

The development of cytoplasmic processes from in situ chondrocytes is a characteristic feature of early osteoarthritis in human cartilage. The processes involve cytoskeletal elements and are distinct from the short primary cilia described in human chondrocytes. Vimentin is an intermediate filament playing an essential structural and signal-transduction role. We determined cellular levels and distribution of vimentin in chondrocytes of different morphologies in non-degenerate and mildly osteoarthritic cartilage. Femoral heads were obtained after consent from patients undergoing hip arthroplasty following femoral neck fracture. Cartilage explants were graded as non-degenerate (grade 0;G0) or mildly osteoarthritic (grade 1;G1) and labelled with the cytoplasmic dye CMFDA (5-chloromethylfluorescein-diacetate) for cell shape. Explants were cryosectioned and labelled for vimentin by fluorescence immunohistochemistry. In situ chondrocyte morphology was identified by confocal microscopy as either normal (rounded/elliptical) or abnormal (with one or more cytoplasmic process of ≥2µm) and vimentin levels and distribution determined semi-quantitatively and related to chondrocyte morphology. When all cells in G0 and G1 cartilage were compared, there was no difference between average levels of vimentin per cell (P=0.144)[6(261)];femoral heads:cells). When cells were separated on the basis of morphology, there was no difference between vimentin levels in cells with one or more cytoplasmic process compared to those of normal morphology (P>0.05;[6(261)]). However vimentin levels were much greater at the base of cytoplasmic processes compared to distant areas of the same cells (P=0.021)[5(29)]). Although overall levels of chondrocyte vimentin do not change in these early stages of osteoarthritis, the formation and structure of these substantial chondrocyte cytoplasmic processes involves changes to its distribution. These morphological changes are similar to those occurring during chondrocyte de-differentiation to fibroblasts reported in osteoarthritis which results in the formation of mechanically-inferior fibro-cartilage. Alterations to chondrocyte vimentin distribution either directly or indirectly may play a role in cartilage degeneration

To investigate temporal changes in synovial lymphatic system (SLS) drainage function after Anterior cruciate ligament (ACL) injury, a non-invasive ACL rupture model was used to induce the PTOA phenotype without altering the SLS structure. We have created a non-invasive ACL rupture model in the right knee (single overload impact) of 12- week-old C57bl/6 male mice to mimic the ACL rupture-induced PTOA development. 70 kDa-TxRedDextran were injected into the right knee of the mice at 0, 1, 2, and 4 wks post modeling (n=5/group), and the fluorescence signal distribution and intensity were measured by the IVIS system at 1 and 6 hrs post-injection. After 24 hrs, the drainage lymph nodes and whole knee joint were harvested and subjected to ex vivo IVIS imaging and immunofluorescence detection respectively. Manual ACL rupture was induced by 12N overloaded force and validated by a front drawer test. Intraarticular clearance of TxRed-Dextran detected by the IVIS was significantly reduced at 1, and 2 wks at a level of 43% and 55% respectively but was not significantly different from baseline levels at 4 wks (89%). TxRed-Dextran signal in draining lymph nodes was significantly reduced at 1 week at the level of but not for 2 and 4 wks compared to baseline levels (week 1–29%, week 2–50%, week 4–94%). TxRed-Dextran particle was significantly enriched in the synovium at 1, 2 wks but was not significantly different from baseline levels at 4 wks rupture-post ACL rupture (Particle numbers: Sham Ctrl-34 ±14, week 1, 113 ± 17; week 2, 89 ± 13; week 4, 46 ± 18; mean ± SD). We observed the drainage function of SLS significantly decreased at 1 and 2 wks after the ACL rupture, and was slowly restored at 4 wks post-injury in a non-invasive ACL rupture model. Early impairment of SLS drainage function may lead to accumulation of inflammatory factors and promote PTOA progression

Early changes within articular cartilage during human idiopathic osteoarthritis are poorly understood. However alterations to chondrocyte morphology occur with the development of fine cytoplasmic processes and cell clusters, potentially playing a role in cartilage degeneration. The aggrecanase ADAMTS-4 (A disintegrin and metalloproteinase with thrombospondin motifs-4) has been implicated as an important factor in cartilage degradation, so we investigated the relationship between chondrocyte morphology and levels of ADAMTS-4 in both non-degenerate and mildly osteoarthritic human cartilage. Human femoral heads were obtained following consent from patients undergoing hip arthroplasty following femoral neck fracture. Cartilage explants of normal (grade 0; G0) and mildly osteoarthritic (grade 1; G1) cartilage were labelled with the cytoplasmic dye CMFDA (5-chloromethylfluorescein-diacetate). Explants were cryosectioned (30μm sections), and labelled for ADAMTS-4 by fluorescence immunohistochemistry. Sections were imaged with confocal microscopy, allowing the semi-quantitative analysis of ADAMTS-4 and 3D visualisation of in situ cell morphology. With cartilage degeneration from G0 to G1, there was a decrease in the proportion of chondrocytes with normal rounded morphology (P<0.001) but an increase in the proportion of cells with processes (P<0.01) and those in clusters (P<0.001;[4(1653)]; femoral heads:cells). Although average levels of ADAMTS-4 for all cells was the same between G0 and G1 (P>0.05), a change was evident in the distribution curves for cell-specific ADAMTS-4 labelling. Cell-by-cell analysis showed that ADAMTS-4 levels were higher in chondrocytes with cytoplasmic processes compared to normal cells (P=0.044) however cells in clusters had lower levels than normal cells (P=0.003;[3(436)]). Preliminary data suggested that ADAMTS-4 levels increased with larger chondrocyte clusters. These results suggest complex heterogeneous changes to levels of cell-associated ADAMTS-4 with early cartilage degeneration – increasing in cells with processes and initially decreasing in clusters. Increased levels of ADAMTS-4 are likely to produce focal areas of matrix weakness potentially leading to early cartilage degeneration

Joint tissues release extracellular vesicles (EVs) that potentially sustain joint homeostasis and contribute to osteoarthritis (OA) pathogenesis. EVs are putative novel therapeutics for OA, and transport biologically active molecules (including small non-coding RNAs (SNCRNAs)) between cells. This study identified altering SNCRNA cargo in EVs in OA which may act as early diagnostic markers and treatment targets. OA was surgically induced in four skeletally mature Standardbred horses using an osteochondral fragment model in the left middle carpal joint. The right joint underwent sham surgery. Synovial fluid (SF) and plasma were obtained weekly throughout the 70-day study. EVs were isolated using size exclusion chromatography and characterised using nanoparticle tracking (Nanosight), and exosome fluorescence detection and tetraspanin phenotyping (Exoview). RNA was extracted from EVs derived from SF (sham and OA joints) and plasma collected at days 10, 35, 42, 49, 56, 63, and subjected to small RNA sequencing on a NovaSeq SP100 flow cell (Illumina). Nanosight-derived EV characteristics of size and concentration were not significantly different following disease induction. The diameter of the temporal population of plasma and SF-derived exosomes changed significantly for CD9 and CD81 following OA induction with significant temporal, and disease-related changes in CD63 and CD81 protein expressin in plasma and SF. In SF and plasma-derived EVs snoRNAs, snRNAs, tRNAs, lncRNA, y-RNA, piRNAs and scRNA were found. Following pairwise analysis of all-time points we identified 27 miRs DE in plasma and 45 DE miRs in SF. Seven were DE in plasma and SF; miR-451, miR-25, miR-215, miR-92a, miR-let-7c, miR-486-5p, miR-23a. In plasma and SF 35 and 21 snoRNAs were DE with four DE in plasma and SF; U3, snord15, snord46, snord58. This work has identified alterations to OA EV sncRNAs in plasma and SF providing a greater understanding of the role of EVs in early OA

Preventing infections in joint replacements is a major ongoing challenge, with limited effective clinical technologies currently available for uncemented knee and hip prostheses. This research aims to develop a coating for titanium implants, consisting of a supported lipid bilayer (SLB) encapsulating an antimicrobial agent. The SLB will be robustly tethered to the titanium using self-assembled monolayers (SAMs) of octadecylphosphonic acid (ODPA). The chosen antimicrobial is Novobiocin, a coumarin-derived antibiotic known to be effective against resistant strains of Staphylococcus aureus. ODPA SAMs were deposited on TiO. 2. -coated quartz crystal microbalance (QCM) sensors using two environmentally friendly non-polar solvents (anisole and cyclopentyl methyl ether, CPME), two concentrations of ODPA (0.5mM and 1mM) and two processing temperatures (21°C and 60°C). QCM, water contact angle measurements, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and temperature-programmed desorption mass spectrometry (TPD-MS) were used to characterise the ODPA SAM. A SLB with encapsulated Novobiocin was subsequently developed on the surface of the ODPA SAM using fluorescent lipids and a solvent assisted method. The prototype implant surface was tested for antimicrobial activity against S. aureus. A well-ordered, uniform ODPA SAM was rapidly formed using 0.5 mM ODPA in CPME at 21°C during 10 min, as confirmed by high Sauerbrey mass (≍285-290 ng/cm. 2. ), high atomic percentage phosphorus (detected using XPS) and high water contact angles (117.6±2.5°). QCM measurements combined with fluorescence microscopy provided evidence of complete planar lipid bilayer formation on the titanium surface using a solvent assisted method. Incorporation of Novobiocin into the SLB resulted in reduced attachment and viability of S. aureus. Key parameters were established for the rapid, robust and uniform formation of an ODPA SAM on titanium (solvent, temperature and concentration). This allowed the successful formation of an antimicrobial SLB, which demonstrated potential for reducing attachment and viability of pathogens associated with joint replacement infections

Stem cells are known to have low levels of intracellular reactive oxygen species (ROS) and high levels of glutathione. ROS are thought to interact with several pathways that affect the transcription machinery required for stem cell differentiation, and are critical for maintaining stem cell function. In this study, we are developing a new fluorescent probe that rapidly and reversibly reacts with glutathione (GSH), the most abundant non-protein thiol in living cells that acts as an antioxidant and redox regulator. Multipotent perivascular progenitor cells derived from human ESCs (hESC-PVPCs): Differentiated ESCs as embryoid bodies in the presence of BMP4 to induce mesoderm differentiation followed by a simple cell selection strategy using attachment of single cells onto collagen-coated dishes. Differential gene expression profiling was performed among H9 hESCs, EBs induced by BMP4 and naturally selected CD140B+CD44+ population at Day 7 (PVPCs). Colony-forming assay: GSHhigh and GSHlow PVPCs were plated on 10-cm tissue culture-treated polystyrene dishes in triplicate in growth medium and cultured for 14 days. Transwell migration assay: GSHhigh and GSHlow PVPCs at passage 4 were resuspended at 1 × 10. 6. /mL in the migration medium and seeded in the upper chamber. The following human recombinant SDF-1 and PDGF-AA proteins were used as chemoattractants in the lower compartment. Probe-GSH conjugate shows shifts in fluorescence excitation and emission spectra that enables ratiometric measurement of GSH levels. Using these properties, stem cells can be purified by FACS-based technology according to intracellular GSH level. We are developing a protocol both for comparing GSH level in stem cell from different culture conditions and for preparing stem cells with high-GSH level . Our results reveal that GSHhigh PVPC purified by FACS show increased colony forming ability compared with that GSHlow PVPC, indicating that intracellular GSH contributes to the maintenance of stemness. Moreover, transplantation of GSHlow PVPC is more effective than that of GSHlow PVPC for cartilage regeneration in osteochondral defect. This technique enable FACS-based sorting of stem cells according to intracellular GSH levels and thus investigation of functional role of GSH (high antioxidant capacity) in the stem cell maintenance and chondrogenic differentiation

For soft tissue sarcoma patients receiving preoperative radiation therapy, wound complications are common and potentially devastating; they may result in multiple subsequent surgeries and significant patient morbidity. The purpose of this study was to assess the feasibility of intraoperative indocyanine green fluorescent angiography (ICGA) as a predictor of wound complications in resections of irradiated soft tissue sarcoma of the extremities. A consecutive series of patients of patients with soft tissue sarcoma of the extremities or pelvis who received neoadjuvant radiation and a subsequent radical resection received intraoperative ICGA with the SPY PHI device (Stryker Inc, Kalamazoo MI) at the time of closure. Three fellowship trained Orthopaedic Oncologic Surgeons were asked to prospectively predict likelihood of wound complications based on fluorescence. Retrospective analysis of fluorescence signal along multiple points of the wound length was performed and quantified. The primary endpoint was wound complication, defined as delayed wound healing or wound dehiscence, within 3 months of surgery. An a priori power analysis demonstrated that 5 patients were necessary to achieve statistical significance. Univariate and multivariate statistical analyses were performed to identify predictors of wound complications. 14 patients were consecutively imaged. The diagnosis was undifferentiated pleomorphic sarcoma in 9 (64.3%) of patients; 11 (78.6%) tumors were high grade. There were 6 patients with wound complications classified as “aseptic” in 5 cases and secondary to hematoma in 1 case. Using the ICGA, blinded surgeons correctly predicted wound complications in 75% of cases. In the area of wound complication, the mean % of maximal signal for wound complications was 49% during the inflow phase and 48% during the peak phase. The mean % maximal signal for peri-incisional tissue without wound complications was 77% during the inflow phase and 83% during the peak phase (p=0.003 and p<0.001). During the inflow phase, a mean ratio of normal of 0.62 maximized the area under the curve (AUC=0.90) for predicting wound complications with a sensitivity of 100% and specificity of 77.4%. During the peak phase, a mean ratio of normal of 0.55 maximized the area under the curve (AUC=0.95) for predicting wound complications with a sensitivity of 88.9% and a specificity 100%. Intraoperative use of indocyanine green fluorescent angiography may help to predict wound complications in patients undergoing resection of preoperatively irradiated soft tissue sarcomas of the extremities and pelvis. Future studies are necessary to validate this technology in a prospective manner and to determine if interventions can be instituted to prevent predicted wound complications

Aim. There is growing evidence that bacteria encountered in periprosthetic joint infections (PJI) form surface-attached biofilms on prostheses, as well as biofilm aggregates embedded in synovial fluid and tissues. However, models allowing the investigation of these biofilms and the assessment of their antimicrobial susceptibility in physiologically relevant conditions are currently lacking. To address this, we developed a synthetic synovial fluid (SSF) model and we validated this model in terms of growth, aggregate formation and antimicrobial susceptibility testing, using multiple PJI isolates. Methods. 17 PJI isolates were included, belonging to Staphylococcus aureus, coagulase negative staphylococci, Cutibacterium acnes, Pseudomonas aeruginosa, enterococci, streptococci, Candida species and Enterobacterales. Growth and aggregate formation in SSF, under microaerophilic or anaerobic conditions, were evaluated using light microscopy. The biofilm preventing concentration (BPC) and minimum biofilm inhibitory concentration (MBIC) of relevant antibiotics (doxycyclin, rifampicin and oxacillin) were determined for the staphylococcal strains (n=8). To this end, a high throughput approach was developed, using a fluorescent viability resazurin staining. BPC and MBIC values were compared to the minimum inhibitory concentration (MIC) obtained with conventional methods. Results. The SSF model allowed all isolates to grow well under microaerophilic or anaerobic conditions. When cultured in SSF, all isolates formed biofilm aggregates, varying in size and shape along different species. A susceptibility testing method based on measuring resazurin-derived fluorescence was successfully developed, allowing high throughput determination of the BPC and the MBIC in SSF. For all staphylococci cultured in SSF a reduced susceptibility to the tested antibiotics was observed when compared to susceptibility data obtained in general medium. For rifampicin and doxycyclin the BPC was consistently higher than the MIC (two- to fourfold dilution difference for rifampicin and four- to sixfold dilution difference for doxycyclin). For oxacillin the MIC equaled the BPC for two isolates, while for the other isolates the BPC was higher than the MIC (two- to fourfold dilution difference). Expectedly, the MBIC was higher than the BPC and differences with the MIC were even more pronounced for all antibiotics tested (differences of six- to fourteenfold dilutions were observed). Conclusion. Our data indicate that the in vitro SSF model could provide more insight in how PJI-related pathogens form biofilms in physiologically relevant conditions. The BPC and MBIC were consistently and substantially higher than MIC. This model could be a valuable addition to evaluate the antimicrobial susceptibility in biofilms in a PJI context. Sources of funding: FWO-Vlaanderen (grant G066523N)

A substantial body of evidence supports the use of extracorporeal shock wave therapy (ESWT) for fracture non-unions in human medicine. However, the success rate (i.e., radiographic union at six months after ESWT) is only approximately 75%. Detailed knowledge regarding the underlying mechanisms that induce bio-calcification after ESWT is limited. The aim of the present study was to analyse the biological response within mineralized tissue of a new invertebrate model organism, the zebra mussel Dreissena polymorpha, after exposure with extracorporeal shock waves (ESWs). Mussels were exposed to ESWs with positive energy density of 0.4 mJ/mm. 2. or were sham exposed. Detection of newly calcified tissue was performed by concomitantly exposing the mussels to fluorescent markers. Two weeks later, the fluorescence signal intensity of the valves was measured. Mussels exposed to ESWs showed a statistically significantly higher mean fluorescence signal intensity within the shell zone than mussels that were sham exposed. Additional acoustic measurements revealed that the increased mean fluorescence signal intensity within the shell of those mussels that were exposed to ESWs was independent of the size and position of the focal point of the ESWs. These data demonstrate that induction of bio-calcification after ESWT may not be restricted to the region of direct energy transfer of ESWs into calcified tissue. The results of the present study are of relevance for better understanding of the molecular and cellular mechanisms that induce formation of new mineralized tissue after ESWT. Specifically, bio-calcification following ESWT may extend beyond the direct area of treatment

Summary. A novel in vivo animal model to establish new surgical interventions for patients with ACL insufficiency. Introduction. After ACL reconstruction, recruited cells from surrounding tissues play crucial roles in ligamentization to obtain adequate structural properties. To allow athletes to return sports activity sooner, these remodeling processes should be elucidated and be accelerated. However, in conventional animal models, it has been difficult to differentiate donor and recipient cells. Here we introduce the transgenic Kusabira-Orange pigs, in which cells produce fluorescence systemically, as in vivo model to trace cell recruitment after ACL reconstruction. Methods. After the approval by the Institutional Animal Care and Used Committee, a transgenic pig that carries and produces red fluorescent Kusabira-Orange (KO) was established. Skeletally immature transgenic pigs (n=12) (20 wks old, 76.0 ± 17.5 kg) and wild type (WT) pigs were used as recipient and donor, respectively. For validation of the pigs as in vivo model, the ACL histological structure, cell shape, mitogenic activity, and migration activity were assessed and were compared to those of wild type pigs. The sensitivity and specificity of KO fluorescence under microscopy were analyzed. Histological analyses were conducted with HE, Masson trichrome (MT), and DAPI staining. The length change pattern in our ACL reconstruction was evaluated to validate the surgical procedure. After allograft ACL reconstruction with fresh-frozen flexor digital tendon of WT pigs, pigs were euthanised at 3, 6, 12, and 24 weeks postoperatively (3 pigs each) for the histological analyses. Results. The histological analyses, and mitogenic/migration assays did not show any apparent differences between KO and WT pigs. The sensitivity and specificity of KO fluorescence revealed to be 98%. Maximal length change of the reconstructed ACL was less than 3.5 mm. Three weeks postoperatively, host cells producing KO fluorescence repopulated mainly at the peripheral part of the graft, while, interestingly, cells also located in inter-territorial space of collagen fascicles. More cells migrated towards the mid of the graft in 6–12 week. Cell distribution became homogeneous in parallel to matrix remodeling in 12–24 week. Discussion. As far as we know, this is the first study to apply the genetically engineered pig producing a fluorescent protein as in vivo model to analyze biological remodeling processes after ACL reconstruction. ACL fibroblasts in KO pigs could be detected under fluorescence with high sensitivity and specificity. In addition, structural organization, mitogenic and migration activity were not different from those in WT. As for histological experiments, the recipient cells could be easily and effectively differentiated from donor cells especially 3 weeks postoperatively. Cells migrated in the inter-territorial region among collagen fascicles earlier than we expected. We are going to investigate angiogenesis, matrix remodeling, and structural properties in parallel to the cell migration

Introduction. It has been shown in vitro that human monocytes can phagocytose submicron polyethylene wear particles generated from total hip arthroplasties (THA) with highly cross-linked polyethylene inlays. The aim of our study was to detect the presence and possible phagocytosis of such particles in peripheral blood monocytes of patients with respective THA. Patients and methods. All patients were operated using the same implant, the cementless SL Plus stem; Bicon cup and a cross-linked polyethylene insert Rexpol (Smith and Nephew). Besides clinical and radiographic check-up, blood samples were collected at follow-up and analyzed by flow cytometry. Polyethylene can be identified by its auto fluorescence when stimulated by a laser with the wavelength of fluorescein isothiocyanate (FITC). Presence of wear particles in monocytes was identified by determination of their size and granularity. Some samples were scrutinized by confocal laser scanning microscopy to correlate the intracellular position of the particles. Blood samples of patients without total joint replacement served as controls. Results. 18 samples of patients with THA were compared to 18 controls. Flow cytometry didn't show any difference of size, granularity and auto fluorescence of the investigated cells between the two groups. Furthermore confocal laser scanning microscopy was unable to establish the intracellular position of the auto fluorescence. There were 11 female and 7 male patients with a mean age of 70,4 years at the time of surgery and an average body mass index of 32 (23 – 41). Average follow-up time was 6,5 years (6 – 8 years). 2 patients had been revised, one for a periprosthetic fracture postoperatively, the other for cup loosening at 5 years. Radiographically there were no signs of loosening. Conclusion. Flow cytometry and confocal laser scanning microscopy were unable to detect submicron polyethylene wear particles in human monocytes in vivo following THA. This could be due to a lack of sensitivity or/and specificity although the in vitro study showing phagocytosis of submicron particles in vitro applied the same methods. The analysis could be too early if the number of wear particles hasn't possibly reached a critical mass at 6.5 years. Potentially the conclusion of the in vitro study is inapplicable and human monocytes are unable to phagocytose polyethylene wear particles. In any case further research in this field seems necessary

Recapitulating tissue elasticity can direct mesenchymal stromal cell (MSC) differentiation; however, it is unclear how substrate elasticity affects MSC metabolism. It is hypothesized MSCs subjected to stiffnesses, atypical of standard tissue culture plastic, display altered metabolic phenotypes during differentiation. In this study, such alterations in MSC metabolic profiles, based on the fluorescence lifetime of NAD(P)H, a critical co-factor in energy production, were monitored using Fluorescence lifetime imaging microscopy (FLIM) as an evaluation tool. Polyacrylamide substrates with varying stiffnesses were fabricated to model the native elasticity of cartilage and bone. MSCs cultured on these substrates exhibited potent alterations in their metabolic status over a 14-day period that were detectable as early as day 3 using FLIM. Overall, soft substrates induced a more glycolytic response after 10 days of culture that persisted at day 14 (as measured by protein-bound NAD(P)H contributions to the lifetime decay). Similarly, by day 10; MSCs on intermediate-stiffness substrates favoured glycolysis. MSCs on stiffer substrates initially displayed a glycolytic phenotype followed by a transition to oxidative phosphorylation by day 10. Staining for mineralised nodules and glycosaminoglycans verified MSCs on stiffer substrates differentiating towards an osteogenic lineage, while MSCs on intermediate substrates showed similarities with differentiated chondrocytes. Overall, it can be concluded that matrix stiffness can induce metabolic perturbations in MSCs for up to 14 days. From this research, ideal culture conditions in which the metabolics of MSCs could be manipulated to promote maximum potency could potentially be defined in the future

Introduction and Objective. Individuals with type 2 diabetes (T2D) have a 3-fold increased risk of bone fracture compared to non-diabetics, with the majority of fractures occurring in the hip, vertebrae and wrists. However, unlike osteoporosis, in T2D, increased bone fragility is generally not accompanied by a reduction in bone mineral density (BMD). This implies that T2D is explained by poorer bone quality, whereby the intrinsic properties of the bone tissue itself are impaired, rather than bone mass. Yet, the mechanics remain unclear. The objective of this study is to (1) assess the fracture mechanics of bone at the structural and tissue level; and (2) investigate for changes in the composition of bone tissue along with measuring total fluorescent advanced glycation end products (fAGEs) from the skin, as T2D progresses with age in Zucker diabetic fatty (ZDF (fa/fa)) and lean Zucker (ZL (fa/+)) rats. Materials and Methods. Right ulnae and skin sections were harvested from ZDF (fa/fa) (T2D) and ZL (fa/+) (Control) rats at 12 and 46 weeks (wks) of age (n = 8, per strain and age) and frozen. Right ulnae were thawed for 12 hrs before micro-CT (μCT) scanning to assess the microstructure and measure BMD. After scanning, ulnae were loaded until failure via three-point bending. Fourier transform-infrared microspectroscopy (FTIR) was used to measure various bone mineral- and collagen-related parameters such as, mineral-to-matrix ratio and nonenzymatic cross-link ratio. Finally, fAGEs were measured from skin sections using fluorescence spectrometry and an absorbance assay, reported in units of ng quinine/ mg collagen. Results. At 12 and 46 wks bone size was significantly smaller in length (p < 0.01), cortical area (p < 0.001) and cross-sectional moment of inertia (p < 0.001) in T2D rats compared to age-matched controls. A slight reduction in BMD was observed in T2D rats compared to controls at both ages, however, this was not significant. Structural properties of T2D bone were significantly altered at 12 and 46 wks, with bending rigidity increasing approximately 2.5-fold and 1.5-fold in control and T2D rats with age, respectively (p < 0.0001). Similarly, yield and ultimate moment significantly reduced in T2D rats with age in comparison to controls (p < 0.0001). Energy absorbed to failure was significantly reduced in T2D rats at 46 weeks of age compared to controls (p < 0.01). The amount of energy absorbed to failure increased approximately 1.4-fold from 12 to 46 wks in control rats, however, in T2D rats a reduction was seen with age, although not significant. At 12 wks, there was no significant deficits in tissue material properties, whereas, at 46 wks a significant reduction in yield stress, yield strain and ultimate stress was observed for T2D rats in comparison to controls (p < 0.05). Conclusions. These findings show that longitudinal growth is impaired as early as 12 wks of age and by 46 wks bone size is significantly reduced in T2D rats compared to controls. The reduction in T2D structural properties is likely attributed to the bone geometry deficits. At 12 wks of age, the tissue material properties are not altered in T2D bone versus controls. However, at 46 wks, bone strength is reduced in T2D, leading to the conclusion that tissue properties are altered as the disease progresses