Surgical site infections (SSIs) after spinal fusion surgery increase healthcare costs, morbidity and mortality. Routine measures of obesity fail to consider site specific fat distribution. We aimed to assess the association between the spine adipose index and deep surgical site infection and determine a threshold value for spine adipose index that can assist in preoperative risk stratification in patients undergoing posterior instrumented lumbar fusion (PILF). A multicentre retrospective case-control study was completed. We reviewed patients who underwent PILF from January 1, 2010 to December 31, 2018. All patients developing a deep primary incisional or organ-space SSI within 90 days of surgery as per US Centre for Disease Control and Prevention criteria were identified. We gathered potential pre-operative and intra-operative deep infection risk factors for each patient. Spine adipose index was measured on pre-operative mid-sagittal cuts of T2 weighted MRI scans. Each measurement was repeated twice by three authors in a blinded fashion, with each series of measurement separated by a period of at least six weeks. Forty-two patients were included in final analysis, with twenty-one cases and twenty-one matched controls. The spine adipose index was significantly greater in patients developing deep SSI (p =0.029), and this relationship was maintained after adjusting for confounders (p=0.046). Risk of developing deep SSI following PILF surgery was increased 2.0-fold when the spine adipose index was ≥0.51. The spine adipose index had excellent (ICC >0.9; p <0.001) inter- and intra-observer reliabilities. The spine adipose index is a novel radiographic measure and an independent risk factor for developing deep SSI, with 0.51 being the ideal threshold value for pre-operative risk stratification in patients undergoing PILF surgery

Introduction. Autologous fat grafting has favourable potential as a regenerative strategy and is the current gold-standard to repair large contour defects, as needed in breast reconstruction after mastectomy and traumatic soft tissue reconstruction. Clinically, there is a limit on the volume of lipoaspirate which can be utilised to repair a soft-tissue defect. Surgical complications are the result of poor structural fidelity of lipoaspirate and graft resorption as a filling material and are hindered further by poor graft vascularisation. This study aims to develop injectable lipoaspirate-derived adipose tissue grafts with enhanced biologically and clinically-admissible structural and functional properties adopting light photocrosslinking of unmodified lipoaspirate. Methods. Patient-derived lipoaspirate was harvested and crosslinked using novel photoinitiator and exposure to visible light (wavelength 450nm) in surgery, establishing bonds between extracellular matrix (ECM) proteins within the material. The degree of crosslinking was tuned (photoinitiator concentration, light exposure, light intensity) and covalent bond formation measured using mass spectrometry. To predict patient response, SWATH-MS was used to identify differences in patient ECM and crosslinked grafts were implanted in vivo using a subcutaneous mouse model. Functional vessel formation and resorption were quantified using micro-CT and tissue-remodelling was assessed via histology. Results. There was an increase in the relative abundance of covalent bonds present with increasing degree of crosslinking. When injected, crosslinked lipoaspirate had better shape fidelity compared with native lipoaspirate – demonstrated by a smaller fibre diameter. Crosslinked lipoaspirate remained viable over long term culture and resulted in more predictable resorption profiles when implanted in vivo. Conclusions. The crosslinking approach described here is tunable and functional across different patient samples. Improving the structural properties of lipoaspirate through minimal manipulation has clinical utility for the delivery of grafts with higher shape fidelity and therefore increased graft survival when implanted

PURPOSE. Recently, in tissue engineering several methods using stem cells have been developed to repair chondral and osteochondral defects. Most of these methods rely on the use of scaffolds. Studies in the literature have demonstrated, first in animals and then in humans, that the use of mesenchymal stem cells withdrawn by several methods from adipose tissue allows to regenerate hyaline articular cartilage. In fact, it has been cleared that adipose-derived cells have multipotentiality equivalent to bone marrow-derived stem cells and that they can very easily and very quickly be isolated in large amounts enabling their immediate use in operating room for one-step cartilage repair techniques. The purpose of this study is to evaluate the therapeutic effect of adipose-derived stem cells on cartilage repair and present our experience in the treatment of knee cartilage defects by the novel AMIC REPAIR TECHNIQUE AUGMENTED by immersing the collagen scaffold with mesenchymal stem cells withdrawn from adipose tissue of the abdomen. MATERIALS AND METHODS. Fat tissue processing involves mechanical forces and does not mandatorily require any enzymatic or chemical treatment in order to obtain the regenerative cells from the lipoaspirate. In our study, mesenchymal adipose stem cells were obtained by non-enzymatic filtration or microfragmentation of lipoaspirates of the abdomen adipose tissue that enabled the separation of the stromal vascular fraction and were used in one-step reconstruction of knee cartilage defects by means of this new AUGMENTED AMIC TECHNIQUE. The focal defects underwent bone marrow stimulation microfractures, followed by coverage with collagen double layer resorbable membrane (Chondro-gide. TM. -Geistlich Pharma AG, Wolhusen, Switzerland) soaked in the cells obtained from fat in 18 patients, aged between 31 and 58 years, at the level of the left knee in 10 cases and in the right in eight, with follow-up ranging between 12 and 36 months. RESULTS: Surgical procedures have been completed without technical problems neither intraoperative or early postoperative complications. The evaluation scores (IKDC, KOOS and VAS) showed a significant improvement, more than 30%, at the initial 6 months follow-up and furtherly improved in the subsequent follow-ups. Also the control MRIs showed a progressive filling and maturation of the repair tissue of the defects. CONCLUSIONS. Since we are reporting a short and medium-term experience, it is not, of course, possible to provide conclusive assessment considerations on this technique, as the experience has to mature along with the progression of follow-ups. The simplicity together with the absence of intraoperative difficulties or immediate complications and the experience gained by other authors, first in animals and then in early clinical cases, makes it, however, possible to say that this can be considered one of the techniques to which resort for one-step treatment of cartilage defects in the knee because it improves patient's conditions and has the potential to regenerate hyaline-like cartilage. Future follow-up works will confirm the results

Purpose. Adipose derived stem cells have been shown to enhance both wound and bone healing. The stem cells are harvested, purified, cultured and the viability assessed in order to provide adequate cellular yield. The isolation process requires trained laboratory staff, intensive procedures utilizing multiple purification solutions and expensive equipment for culturing and interpretation of viability of the isolated stem cells. The aim of the study was to investigate the effect of simple lipo-aspirate on wound and bone healing. Methodology. This is a prospective, interventional study to investigate the effect of adipocyte extract on wound and bone healing. 9 Young, healthy, large white female pigs were used in the study. Fat was harvested using standard liposuction technique and injected around the defects created. Skin defects were evaluated for secondary wound healing macroscopically and histologically. 3 pigs were used in a pilot study to evaluate the possibility of investigating the effects of lipo-aspirate in bone defects. Results. Histological evaluation shows accelerated secondary wound healing with the treatment of adipose tissue compared to control groups. The thickness of regenerated epidermis, the number of new vascular nests was increased and the wound surface area was decreased in adipose treated wounds. Bacteriology results showed no significant differences. Conclusion. Results indicate a potential benefit in the treatment of wounds with the use of lipo-aspirated extract. The procedure allows for a cost effective method to enhance wound healing in a developing country. Due to the encouraging results in wound healing and osteogenic potential of lipo-aspirate, a pilot study to evaluate lipo-aspirate effects on bone healing has been drawn up. NO DISCLOSURES

Aim. This study evaluated target tissue concentrations of double dose cefuroxime administered intravenously as either one 15 min infusion of 3,000 mg (Group 1) or two single 15 min infusions of 1,500 mg administered 4 h apart (Group 2). Method. Sixteen pigs were randomised into two groups of eight. Cortical and cancellous bone, synovial fluid of the knee joint and subcutaneous adipose tissue concentrations were measured based on sampling via microdialysis. Plasma samples were collected as a reference. Comparison of the groups was based on time with concentrations above relevant minimal inhibitory concentrations (fT>MIC) of 4 μg/mL. Results. The mean time fT>MIC (4 μg/mL) across compartments was longer for Group 2 (280–394 min) than for Group 1 (207–253 min) (p<0.01). Cortical bone showed a tendency towards longer fT>MIC (4 μg/mL) in Group 2 (280 min) than in Group 1 (207 min) (p=0.053). Within 50 min after administration, the mean concentration of 4 μg/mL was reached in all compartments for both groups. The mean concentrations decreased below 4 μg/mL after approximately 4 h (Group 1) and 3 h (Group 2) from initiation of administration (time zero). Conclusions. During an 8 h interval, double-dose cefuroxime administered as 2 × 1,500 mg with a 4 h interval provides longer time above MIC breakpoint for Staphylococcus aureus (4 μg/mL) than a single bolus of 3,000 mg cefuroxime. To maintain sufficient tissue concentrations during longer surgeries, re-administration of cefuroxime (1,500 mg) should be considered 3 h after the first administration

Aberrant infrapatellar fat metabolism is a notable feature provoking inflammation and fibrosis in the progression of osteoarthritis (OA). Irisin, a secretory subunit of fibronectin type III domain containing 5 (FNDC5) regulate adipose morphogenesis, energy expenditure, skeletal muscle, and bone metabolism. This study aims to characterize the biological roles of Irisin signaling in an infrapatellar fat formation and OA development. Injured articular specimens were harvested from 19 patients with end-stage knee OA and 11 patients with the femoral neck fracture. Knee joints in mice that overexpressed Irisin were subjected to intra-articular injection of collagenase to provoke OA. Expressions of Irisin, adipokines, and MMPs probed with RT-quantitative PCR. Infrapatellar adiposity, articular cartilage damage, and synovial integrity verified with histomorphometry and immunohistochemistry. Infrapatellar adipose and synovial tissues instead of articular cartilage exhibited Irisin immunostaining. Human OA specimens showed 40% decline in Irisin expression than the non-OA group. In vitro, the gain of Irisin function enabled synovial fibroblasts but not chondrocytes to display minor responses to the IL-1β provocation of MMP3 and MMP9 expression. Of note, Irisin signaling reduced adipogenic gene expression and adipocyte formation of mesenchymal progenitor cells. In collagenase-mediated OA knee pathogenesis, forced FNDC5 expression in articular compromised the collagenase-induced infrapatellar adipose hypertrophy, synovial hypercellularity, and membrane hyperplasia. These adipose-regulatory actions warded off the affected knees from cartilage destruction and gait aberrance. Likewise, intra-articular injection of Irisin recombinant protein mitigated the development of infrapatellar adiposity and synovitis slowing down the progression of cartilage erosion and walking profile irregularity. Affected joints and adipocytes responded to the Irisin recombinant protein treatment by reducing the expressions of cartilage-deleterious adipokines IL-6, leptin, and adiponectin through regulating PPAR&gamma, function. Irisin dysfunction is relevant to the existence of end-stage knee OA. Irisin signaling protects from excessive adipogenesis of mesenchymal precursor cells and diminished inflammation and cartilage catabolism actions aggravated by adipocytes and synovial cells. This study sheds emerging new light on the Irisin signaling stabilization of infrapatellar adipose homeostasis and the perspective of the therapeutic potential of Irisin recombinant protein for deescalating knee OA development

Aim. Pyogenic spondylodiscitis remains a therapeutic challenge, as demonstrated by divergent treatment guidelines. The combination of moxifloxacin and rifampicin may be an attractive treatment option for cases caused by staphylococci; however, previous studies have reported a reduction in plasma concentrations of moxifloxacin when co-administered with rifampicin. The magnitude of this reduction in spinal tissues is not known. We aimed to investigate the interaction of rifampicin on moxifloxacin tissue concentrations in vertebral cancellous bone, intervertebral disc and subcutaneous adipose tissue in steady-state conditions using microdialysis in a porcine model. Method. Twenty female pigs were randomized into two groups of ten pigs: Group A received moxifloxacin 400 mg orally once daily for three days preoperatively. Group B received moxifloxacin 400 mg orally for three days preoperatively combined with rifampicin 450 mg twice daily for seven days preoperatively. Measurements were obtained from plasma, vertebral cancellous bone, intervertebral disc and subcutaneous adipose tissue for 24 h. Microdialysis was applied for sampling in solid tissues. Results. Co-administration of moxifloxacin and rifampicin demonstrated a reduction of free moxifloxacin concentrations in spinal tissues. The peak drug concentration (C. max. ) and the area under the concentration-time curve (AUC. 0–24. ) in all tissue compartments decreased in the range of 66–79% and 65–76%, respectively. Conclusions. Using microdialysis, we demonstrated a significant reduction of moxifloxacin C. max. and AUC. 0–24. in the spinal tissues when co-administered with rifampicin. Further studies are warranted to understand the clinical implications of this finding for the treatment of pyogenic spondylodiscitis

In Denmark the most common postoperative pathogen is S. aureus (1), sensitive to dicloxacillin. These bacteria can cause a postoperative infection despite using prophylactic antibiotics. Whether the tissue concentration reached is above the minimal inhibitory concentration (MIC) for the pathogens is unknown, and if lower than expected could result in a postoperative infection. Thus a trial was conducted, measuring the actual tissue concentration of dicloxacillin in human muscle and adipose tissue and compared these to the plasma concentration. MIC for dicloxacillin against S. aureus was determined using the broth macrodilution method. Six healthy male volunteers aging 25 to 27 years (body-mass-index; 20–28), were recruited. A CMA63 (Mdialysis, Stockholm, Sweden) catheter was placed in the subcutaneous tissue of the abdomen and in the rectus muscle of the thigh and the volunteers given 2 g dicloxacillin intravenously over 5 minutes. In 10 min intervals for the following 6 hours, samples from blood and Microdialysis fluid (flowrate 5 ml/min) were collected. Recovery was determined in vitro. Plasma was isolated from blood samples. The unbound dicloxacillin was isolated from plasma using filter plates (AcroPrep 30K Omega, Pall Corporation, US) centrifuged for 30 minutes at 1000 × g and 37°C. All samples were analyzed with High Performance Liquid Chromatography. MIC was determined to be 0.125 µg/ml. Average recovery was 73,7 % Maximum concentrations were reached in muscle tissue after a median of 0.5 hours and adipose tissue after 0.8 hours. The geometric mean ration (GMR) of AUC0-6h for adipose tissue compared to plasma was 0.32 [0.15–0.71]. GMR of AUC0-6h for muscle tissue compared to plasma and adipose tissue compared to muscle showed no statistically significant differences. The tissue concentrations were above MIC for 3.4 hours for adipose tissue and 4.1 hours for muscle tissue. The administration of prophylactic dicloxacillin should be given at least 30 minutes prior to incision to ensure maximum tissue concentrations at the onset of surgery. A second dose should be given after 3.4 hours in case of long surgery time. Since the dicloxacillin concentration reached in the adipose tissue is lower than in plasma, it should be investigated whether this difference is more prominent in adipose patients or patients with impaired peripheral circulation, since these patients are at a greater risk of postoperative infections

Aim. Prompt and sufficient broad spectrum empirical antibiotic treatment is key to prevent infection following open tibial fractures. Succeeding co-administration, we dynamically assessed the time for which vancomycin and meropenem concentrations were above relevant epidemiological cut-off minimal inhibitory concentrations (T>MIC) in tibial compartments for the bacteria most frequently encountered in open fractures. Low and high MIC-targets were applied: 1 and 4 µg/mL for vancomycin and 0.125 and 2 µg/mL for meropenem. Materials and methods. 8 pigs received a single dose of 1000 mg vancomycin and 1000 mg meropenem simultaneously over 100 min and 10 min, respectively. Microdialysis catheters were placed for sampling over 8 h in tibial cancellous bone, cortical bone, and adjacent subcutaneous adipose tissue. Venous blood samples were collected as references. Results. Across the targeted epidemiological cut-off values, vancomycin displayed longer T>MIC in all the investigated compartments in comparison to meropenem. For both drugs, cortical bone exhibited the shortest T>MIC. For the low MIC targets and across compartments, T>MIC ranged between 208–499 min (46–100%) for vancomycin and 189–406 min (42–90%) for meropenem. For the high MIC targets, T>MIC ranged between 30–446 min (7–99%) for vancomycin and 45–181 min (10–40%) for meropenem. Conclusion. The differences in the T>MIC between the low and high targets illustrates how the interpretation of these results is highly susceptible to the defined MIC target. To encompass any trauma, contaminating or individual tissue differences, a more aggressive dosing approach may be considered to achieve longer T>MIC in all the exposed tissues and thereby lowering the risk of acquiring an infection after open tibial fractures

Aims. Vancomycin may be an important drug for intravenous perioperative antimicrobial prophylaxis in spine surgery. We assessed single-dose vancomycin intervertebral disc, vertebral cancellous bone, and subcutaneous adipose tissue concentrations using microdialysis in a pig model. Methods. 8 female pigs received 1,000 mg of vancomycin intravenously as a single dose over 100 minutes. Microdialysis probes were placed in the C3-C4 intervertebral disc, C3 vertebral cancellous bone, and subcutaneous adipose tissue, and vancomycin concentrations were obtained over 8 hours. Venous blood samples were obtained as reference. Results. Ranging from 0.24 to 0.60, vancomycin tissue penetration, expressed as the ratio of tissue to plasma area under the concentration-time curve from 0 to the last measured value, was incomplete for all compartments. The lowest penetration was found in the intervertebral disc. The time to a mean clinically relevant minimal inhibitory concentration (MIC) of 4 μg/mL were 3, 17, 25, and 156 min for plasma, subcutaneous adipose tissue, vertebral cancellous bone and the intervertebral disc, respectively. In contrast to the other compartments, a mean MIC of 8 μg/mL was not reached in the intervertebral disc. An approximately 3-time longer elimination rate was observed in the intervertebral disc in comparison to all the other compartments (p < 0.001), and the time to peak drug concentration was higher for all tissues compared with plasma. Conclusions. Preoperative administration of 1,000 mg of vancomycin may provide adequate vancomycin tissue concentrations with a considerable delay, though tissue penetration was incomplete. However, in order also to achieve adequate intervertebral disc concentrations in all individuals and accommodating a potentially higher MIC target, supplemental application of vancomycin may be necessary

Numerous investigators have described osteogenic differentiation of bone marrow stromal cells obtained from both murine and human sources over the past decade. The ease of access and large available quantity of adipose tissue, however, makes Adipose-Derived Stem Cells (ADSC) a far more practical alternative for clinical applications, such as operative treatment of non-unions and regeneration of critical bone defects. Therefore, the primary goal of this research endeavor is to achieve osteogenic differentiation of ADSC. Previous work has already demonstrated that bone morphogenetic protein receptor 1A (BMP receptor 1A) signaling is required for healing critical bone defects. Based on this evidence, we used a lentiviral vector to increase expression of BMP receptor 1A by our stem cell population in order to direct their differentiation into the osteoblastic lineage. We harvested subcutaneous adipose tissue intraoperatively from consenting patients undergoing elective lipoplasty and panniculectomy procedures. The stromal vascular fraction was isolated from this tissue and further refined by passaging in selective media to yield a stable population of ADSC in primary culture. Both the identity and homogeneity of this stem cell population was confirmed using adipogenic induction media and differentiation cocktails. In addition, we subcloned an expression plasmid containing the BMP receptor 1A locus in tandem with green fluorescent protein (GFP) under the transcriptional control of a single promoter. This plasmid was packaged into a lentiviral vector to provide a reliable method of achieving both genomic integration and long-term expression of the BMP receptor 1A gene. Hence, transduction of ADSC using this vector resulted in overexpression of BMP receptor 1A by these multipotent cells. The GFP was then utilized as a reporter gene to screen and enrich the ADSC population for only those stem cells with a robust expression of BMP receptor 1A. The ADSC that overexpressed BMP receptor 1A were found to achieve osteogenic differentiation after 18 to 20 days of in vitro culture, as revealed by immunohistochemistry assays for osteocalcin. Osteogenic differentiation was further confirmed by alizarin red staining and quantitative PCR for alkaline phosphatase gene expression as a biomarker for the osteoblastic lineage. Our results demonstrate that stem cells derived from the adipose tissue of a patient represent a viable means of culturing autologous osteoblasts in vitro for future implantation at the site of critical bone defects. This method of attaining osseous regeneration is intuitively appealing, given the minimal donor site morbidity associated with removing subcutaneous fat. By transducing the ADSC with a lentiviral vector, we have also collected further evidence implicating the critical importance of signaling mediated by the BMP receptor 1A during osteogenesis. Further tissue engineering studies are now in progress to evaluate the osteogenic differentiation potential of these stem cells under hydrostatic and fluid flow shearing mechanical loads

Tissue engineering by self-assembly is a technique that consists of growing cells on surfaces made of thermoresponsive polymers, that allow the production of contiguous cell sheets by simply lowering the temperature below the polymer's low critical solution temperature. In this approach cell-cell junctions and deposited extracellular matrix (ECM) remain intact, which provides a better cell localisation at the site of injury. However, these systems lack the possibility to fabricate multi-layered and three-dimensional cell sheets that would better recapitulate native tissues. Moreover, the fabrication of ECM-rich cell sheets would be highly desirable. This limitation could be overcome by inducing macromolecular crowding (MMC) conditions. Herein we venture to fabricate electrospun thermoresponsive nanofibres to sustain the growth and detachment of ECM-rich tissue substitutes in the presence of a MMC microenvironment. A copolymer of 85% poly-N-isopropylacrylamide and 15% N-tert-butylacrylamide (pNIPAAm/NTBA) were used for all experiments. To create aligned nanofibers, the polymer was electrospun and collected on a mandrel rotating at 2000 rpm. Human adipose derived stem cells (hADSC) were treated with media containing macromolecular crowders to enhance matrix deposition. Cell viability and morphology were assessed, and immunocytochemistry was conducted in order to estimate matrix deposition and composition. Adipogenic, osteogenic and chondrogenic assays were performed both with and without the presence of MMC. Non-invasive cell detachment was enabled by decreasing the temperature of culture to 10 °C for 20 minutes. The electrospinning process resulted in the production of pNIPAm/NTBA fibres in the diameter range from 1 to 2 µm and an overall alignment of 80%. Cell viability, proliferation and metabolic activity revealed that hADSCs were able to grow on the thermoresponsive scaffold. The cells were able to detach as an intact cell sheet in presence of MMC. Moreover, it was demonstated that MMC, by a volume extrusion effect, enhances Collagen type I deposition, which is one of the main components of the ECM. Histological analysis revealed that in the presence of MMC the cells were able to self-assembled into three dimensional multi-layers. The cells were able to differentiate towards the osteogenic and adipogenic lineage in the presence of MMC. Interestingly we were able to fabricate three-dimensional chondrogenic cell sheet both with and without MMC. Collectively the pNIPAm/NTBA thermoresponsive fibres were able to sustain the growth and the detachment of ECM-rich multi-layered cell sheets. The pNIPAm/NTBA fibres were able to successfully sustain growth and detachment of ECM-rich tissue equivalents. We believe that replacement, repair and restoration of tissue function can be accomplished best using cells that create their own tissue-specific extracellular matrix with a precision and stoichiometric efficiency still unmatched by man-made devices

Osteogenesis Imperfecta (OI) is a heritable bone disorder characterized by bone fragility and often caused by mutations in the Type I collagen-encoding genes COL1A1 and COL1A2. The pathophysiology of OI, particularly at the cellular level, is still not well understood. This contributes to the lack of a cure for this disorder as well as an effective preventive or management options of its complications. In the bone environment, mesenchymal stem cells (MSCs) and osteoblasts (Ob) exert their function, at least partially, through the secretion of extracellular vesicles (EV). EV is a heterogeneous group of nanosized membrane-enclosed vesicles that carry/transfer a cargo of proteins, lipid and nucleic acids from the secreting cell to its target cells. Our objective is to characterize EVs secreted by human control (HC)- and OI-MSCs and their derived Obs, with focus on their protein content. We hypothesize that there will be differences in the protein content of EVs secreted by OI-Obs compared to HC-Ob, which may indicate a deviation from healthy Ob behavior and, thus, a role in OI pathophysiology. MSCs were harvested from the adipose tissue of four COL1A1-OI and two HC patients. They were proliferated in an EV-depleted media, then induced to differentiate to extracellular matrix (ECM)-producing osteoblasts, which then gets mineralized. EVs secreted by MSCs (MSC-EV) and Obs (Ob-EV) were then purified and concentrated. Using liquid chromatography- tandem mass spectrometry, proteomic analysis of the EV groups was done. A total of 384 unique proteins were identified in all EVs, 373 were found in Vesiclepedia indicating a good enrichment of our samples with EV proteins. 67 proteins of the total 384 were exclusively or significantly upregulated (p-value < 0 .05) in OI-Ob-EV and 28 proteins in the HC-Ob-EVs, relative to each other. These two groups of differentially expressed proteins were compared by Gene Ontology (GO) analysis of their cellular compartment, molecular functions and biological processes. We observed that there were differences in the cellular origin of EV-proteins, which may indicate heterogeneity of the isolated EVs. Molecular function and biological process analyses of the HC-Ob-EV proteins showed, as expected, predominantly calcium-related activities such as extracellular matrix (ECM) mineralization. OI-Ob-EV proteins were still predominantly exhibiting ECM organization and formation functions. Annexins A1,2,4,5 and 6 were differentially and significantly upregulated by the HC-Ob-EVs. Fibronectin (FN), Fibulin-1 and −2, and Laminins (α4 & γ1), which are amongst the early non-collagenous proteins to form the ECM, were differentially and significantly upregulated in the OI-Ob-EVs. We concluded that the persistent expression of Fibronectin (FN), Fibulin-1 and −2, and Laminins in OI-Ob-EVs might indicate the presence of an immature ECM that the OI-Obs are trying to organize. ECM mineralization is largely dependent on the presence of an organized mature ECM, and this being compromised in OI bone environment, may be a contributor to the bone fragility seen in these patients. Annexins, which are calcium-binders that are vital for ECM mineralization, were significantly downregulated in the OI-Ob-EVs and this may be a further contributor to ECM mineralization impairment and bone fragility

A novel injectable hydrogel based on DNA and silicate nanodisks was fabricated and optimized to obtain a suitable drug delivery platform for biomedical applications. Precisely, the hydrogel was designed by combining two different type of networks: a first network (type A) made of interconnections between neighboring DNA strands and a second one (type B) consisting of electrostatic interactions between the silicate nanodisks and the DNA backbone. The silicate nanodisks were introduced to increase the viscosity of the DNA physical hydrogel and improve their shear-thinning properties. Additionally, the silicate nanodisks were selected to modulate the release capability of the designed network. DNA 4% solutions were heated at 90°C for 45 seconds and cooled down at 37°C degree for two hours. In the second step, the silicate nanodisks suspension in water at different concentrations (0.1 up to 0.5%) were then mixed with the pre-gel DNA hydrogels to obtain the nanocomposite hydrogels. Rheological studies were carried out to investigate the shear thinning properties of the hydrogels. Additionally, the hydrogels were characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and X-ray photoelectron microscopy. The hydrogels were loaded with the osteoinductive drug dexamethasone and its release was tested in vitro in phosphate buffer pH 7.4. The drug activity upon release was tested evaluating the osteogenic differentiation of human adipose derived stem cells (hASCs) in vitro through analysis of main osteogenic markers and quantification of alkaline phosphatase activity and calcium deposition. Finally, the hydrogels were tested in vivo and injected into cranial defects in rats to assess their biocompatibility and bone regeneration potential. The inclusion of the silicate nanodisks increased the viscosity of the hydrogels and the best results were obtained with the highest concentration of the nanoclay (0.5%). The hydrogels possessed shear-thinning properties as demonstrated by cyclic strain sweep tests and were able to recover their original storage modulus G' upon removal of strain. Such improvement in the injectable properties of the formulated hydrogels was mainly attributed to the formation of electrostatic interactions between the silicate nanodisks and the phosphate groups of the DNA backbone as confirmed by XPS analysis of the O, N, and P spectra. Additionally, laponite was able to sustain the release of the osteoinductive drug dexamethasone which was instead completely released from the DNA-based hydrogels after a week. The drug after being released was still active and promoted the osteogenic differentiation of hASCs as confirmed by ALP expression and expression of main osteogenic markers including ALP and COLA1. Finally, the gels proved to be biocompatible in vivo when injected into cranial defects and promoted bone formation at the periphery of the defect after a month post-treatment. A novel injectable shear-thinning DNA-based hydrogel was characterized and tested for its drug delivery properties. The hydrogel can promote the sustain release of a small molecule like dexamethasone and be biocompatible in vitro and in vivo. Due to these promising findings, the designed system could find also applicability for the delivery of growth factors or other therapeutic molecules

Osteoarthritis (OA) is the fastest growing global health problem, with a total joint replacement being the only effective treatment for patients with end stage OA. Many groups are examining the use of bone marrow or adipose derived mesenchymal stem cells (MSCs) to repair cartilage, or modulate inflammation to promote healing, however, little efficacy in promoting cartilage repair, or reducing patient symptoms over temporary treatments such as micro-fracture has been observed. There is a growing body of literature demonstrating that MSCs derived from the synovial lining of the joint are superior in terms of chondrogenic differentiation and while improvements in clinical outcome measures have been observed with synovial MSCs, results from clinical studies are still highly variable. Based on our results, we believe this variability in clinical studies with MSCs results in part from the isolation, expansion and re-injection of distinct MSCs subtypes in normal vs. OA tissues, each with differing regenerating potential. However, it remains unknown if this heterogeneity is natural (e.g. multiple MSC subtypes present) or if MSCs are influenced by factors in vivo (disease state/stage). Therefore, in this study, we undertook an ‘omics’ screening approach on MSCs from normal and OA knee synovial tissue. Specifically, we characterized their global proteome and genomic expression patterns to determine if multiple MSC from normal and OA joints are distinct at the protein/gene expression level and/if so, what proteins/genes are differentially expressed between MSCs derived from normal and OA synovial tissue. Synovium tissue was collected from OA patients undergoing joint replacement and normal cadaveric knees. The in vitro adipogenic, chondrogenic and osteogenic differentiation potential of the MSCs was analyzed via qPCR and histology. Fully characterized MSC populations where then analyzed through an unbiased shotgun proteomics, and microarray analysis. Synovial MSCs isolated from both OA and normal knees demonstrated similar multipotent differentiation capacity. Likewise, both OA and normal MSCs display the typical MSCs cell surface marker profile in vitro (CD90+, CD44+, CD73+, CD105+). Using shotgun proteomics, 7720 unique peptides corresponding to 2183 proteins were identified and quantified between normal and OA MSCs. Of these 2183 proteins, 994 were equally expressed in normal and OA, MSCs, 324 were upregulated in OA MSCs (with 50 proteins exclusively expressed in OA MSCs), 630 proteins were upregulated in normal MSCs (with 16 proteins exclusively expressed in normal MSCs). Microarray analysis of normal and OA MSCs demonstrated a similar result in where, 967 genes were differentially expressed between normal and OA MSCs, with 423 genes upregulated in OA, and 544 genes upregulated in normal MSCs. In this project, we have demonstrated that although normal and OA synovial derived MSCs demonstrate similar multipotent differentiation potential and cell surface markers expression, these cells demonstrated significant differences at the molecular level (protein and gene expression). Further research is required to determine if these differences influence functional differences in vitro and/or in vivo and what drives this dramatic change in the regulatory pathways within normal vs. OA synovial MSCs

Aim. The increasing incidence of orthopaedic methicillin-resistant Staphylococcus aureus (MRSA) infections represents a significant therapeutic challenge. Being effective against MRSA, the role of vancomycin may become more important in the orthopaedic setting in the years to come. Nonetheless, vancomycin bone and soft tissue penetration during infection remains unclear. We assessed the effect of a traumatically induced, implant-associated acute osteomyelitis on vancomycin bone penetration in a porcine model. Method. In eight pigs, implant-associated osteomyelitis was induced on day 0, using a Staphylococcus aureus strain. Following administration of 1,000 mg of vancomycin on day 5, vancomycin concentrations were obtained with microdialysis for eight hours in the implant bone cavity, in cancellous bone adjacent to the implant cavity, in subcutaneous adipose tissue (SCT) adjacent to the implant cavity, and in healthy cancellous bone and healthy SCT in the contralateral leg. Venous blood samples were also obtained. The extent of infection and inflammation was evaluated by post-mortem computed tomography scans, C-reactive protein serum levels and cultures of blood and swabs. Results. In relation to all the implant cavities, bone destruction was found. Ranging from 0.20 to 0.74, tissue penetration, expressed as the ratio of tissue to plasma area under the concentration-time curve from 0 to the last measured value, was incomplete for all compartments except for healthy SCT. The lowest penetration was found in the implant cavity. Conclusions. Staphylococcus aureus implant-associated osteomyelitis was found to reduce vancomycin bone penetration, especially in the implant cavity. These findings suggest that it may be unsafe to rely solely on vancomycin therapy when treating acute osteomyelitis. Particularly when metaphyseal cavities are present, surgical debridement seems necessary

Introduction. Trabecular Titanium is a biomaterial characterized by a regular three-dimensional hexagonal cell structure imitating trabecular bone morphology. Components are built via Electron Beam Melting technology in aone- step additive manufacturing process. This biomaterial combines the proven mechanical properties of Titanium with the elastic modulus provided by its cellular solid structure (Regis 2015 MRS Bulletin). Several in vitro studies reported promising outcomes on its osteoinductive and osteoconductive properties: Trabecular Titanium showed to significantly affect osteoblast attachment and proliferation while inhibiting osteoclastogenesis (Gastaldi 2010 J Biomed Mater Res A, Sollazzo 2011 ISRN Mater Sci); human adipose stem cells were able to adhere, proliferate and differentiate into an osteoblast-like phenotype in absence of osteogenic factors (Benazzo 2014 J Biomed Mater Res A). Furthermore, in vivo histological and histomorphometric analysis in a sheep model indicated that it provided bone in-growth in cancellous (+68%) and cortical bone (+87%) (Devine 2012 JBJS). A multicentre prospective study was performed to assess mid-term outcomes of acetabular cups in Trabecular Titanium after Total Hip Arthroplasty (THA). Methods. 89 patients (91 hips) underwent primary cementless THA. There were 46 (52%) men and 43 (48%) women, with a median (IQR) age and BMI of 67 (57–70) years and 26 (24–29) kg/m2, respectively. Diagnosis was mostly primary osteoarthritis in 80 (88%) cases. Radiographic and clinical evaluations (Harris Hip Score [HHS], SF-36) were performed preoperatively and at 7 days, 3, 6, 12, 24 and 60 months. Bone Mineral Density (BMD) was determined by dual-emission X-ray absorptiometry (DEXA) according to DeLee &Charnley 3 Regions of Interest (ROI) postoperatively at the same time-points using as baseline the measureat 1 week. Statistical analysis was carried out using Wilcoxon test. Results. Median (IQR) HHS and SF-36 improved significantly from 48 (39–61) and 49 (37–62) preoperatively to 99 (96–100) and 76 (60–85) at 60 mo. (p≤0.0001). Radiographic analysis showed evident signs of bone remodelling and biological fixation, with presence of superolateral and inferomedial bone buttress, and radial trabeculae in ROI I/II. All cups resulted radiographically stable without any radiolucent lines. The macro-porous structure of this biomaterial generates a high coefficient of friction (Marin 2012 Hip Int), promoting a firm mechanical interlocking at the implant-bone interface which could be already observed in the operating room. BMD initially declined from baseline at 7 days to 6 months. Then, BMD slightly increased or stabilized in all ROIs up to 24 months, while showing evidence of partial decline over time with increasing patient' age at 60 months, although without any clinical significance in terms of patients health status or implant stability. Statistical significant correlations in terms of bone remodeling were observed between groups of patients on the basis of gender and age (p≤0.05). No revision or implant failure was reported. Conclusions. All patients reported significant improvements in quality of life, pain relief and functional recovery. Radiographic evaluation confirmed good implant stability at 60 months. These outcomes corroborate the evidence reported on these cups by orthopaedic registries and literature (Perticarini 2015 BMC Musculoskelet Disord; Bistolfi 2014 Min Ortop)

Aim. Pyogenic spondylodiscitis is associated with prolonged antimicrobial therapy and high relapse rates. Nevertheless, tissue pharmacokinetic studies of relevant antimicrobials in both prophylactic and therapeutic situations are still sparse. Previous approaches based on bone biopsy and discectomy exhibit important methodological limitations. The objective of this study was therefore to assess the concentration of cefuroxime in intervertebral disc (IVD), vertebral body cancellous bone, subcutaneous adipose tissue (SCT) and plasma pharmacokinetics after single dose administration by use of microdialysis (MD) in a large animal model. Method. Ten female pigs were assigned to receive 1,500 mg of cefuroxime intravenously over 15 min. Measurements of cefuroxime were obtained from plasma, SCT, the vertebral cancellous bone and the IVD for 8 hours thereafter. MD was applied for sampling in solid tissues. The cefuroxime concentration in both the MD and plasma samples was determined using ultra-high performance liquid chromatography. Results. For both the IVD and the vertebral cancellous bone, the area under the concentration-curve from zero to the last measured value was significantly lower than that of free plasma. Tissue penetration of cefuroxime was incomplete for the IVD, whereas for vertebral cancellous bone and SCT it was not. Furthermore, the penetration of cefuroxime from plasma to IVD was delayed. Additionally, a noticeable prolonged elimination rate of cefuroxime in the IVD was found. The maximal concentration and the elimination of cefuroxime were reduced in IVD compared to both SCT and vertebral cancellous bone. Due to this delay in elimination of cefuroxime, the time with concentrations above the minimal inhibitory concentration (T>MIC) was significantly higher in IVD than in SCT, vertebral cancellous bone and free plasma for MICs up to 6 μg/ml. Conclusions. MD was successfully applied for serial assessment of the concentration of cefuroxime in the IVD and the vertebral cancellous bone. Penetration of cefuroxime from plasma to IVD was found to be incomplete and delayed, but due to a prolonged elimination, the best results regarding T>MIC was found in IVD

Obtaining primary wound healing in total joint arthroplasty (TJA) is essential to a good result. Wound healing disturbances (WHD) can occur and the consequences can be devastating to the patient and to the surgeon. Determination of the host healing capacity can be useful in predicting complications. Cierney and Mader classified patients as Type A: no healing compromises and Type B: systemic or local healing compromise factors present. Local factors include traumatic arthritis with multiple previous incisions, extensive scarring, lymphodema, poor vascular perfusion, and excessive local adipose deposition. Systemic compromising factors include diabetes, rheumatic diseases, renal or liver disease, immunocompromise, steroids, smoking, and poor nutrition. Low serum albumin, total lymphocyte count, and low transferrin increase WHD. In high risk situations the surgeon should encourage positive patient choices such as smoking cessation and nutritional supplementation to modify healing responses. Use of tourniquet in obese patients also increases WHD. Careful planning of incisions, particularly in patients with scarring or multiple previous operations, is productive. Around the knee the vascular viability is better in the medial flap. Thusly, use the most lateral previous incision, do minimal undermining, and handle tissue meticulously. We do all potentially complicated TKA's without tourniquet to enhance blood flow and tissue viability. The use of perioperative anticoagulation will increase wound problems. If wound drainage or healing problems do occur, immediate action is required. Deep sepsis can be ruled out with a joint aspiration and cell count (less than 2500), differential (less than 60% polys), and negative culture and sensitivity. All hematomas should be evacuated and necrosis or dehiscence should be managed by debridement to obtain a live wound

Obtaining primary wound healing in Total Joint Arthroplasty (TJA) is essential to a good result. Wound healing problems can occur and the consequences can be devastating to the patient and to the surgeon. Determination of the host healing capacity can be useful in predicting complications. Cierney and Mader classified patients as Type A: no healing compromises and Type B: systemic or local healing compromises factors present. Local factors include traumatic arthritis with multiple previous incisions, extensive scarring, lymphedema, poor vascular perfusion, and excessive local adipose deposition. Systemic compromising factors include diabetes, rheumatic diseases, renal or liver disease, immunocompromise, steroids, smoking, and poor nutrition. In high risk situations the surgeon should encourage positive patient choices such as smoking cessation and nutritional supplementation to elevate the total lymphocyte count and total albumin. Careful planning of incisions, particularly in patients with scarring or multiple previous operations, is productive. Around the knee the vascular viability is better in the medial flap. Thusly, use the most lateral previous incision, do minimal undermining, and handle tissue meticulously. We do all potentially complicated TKAs without tourniquet to enhance blood flow and tissue viability. The use of peri-operative anticoagulation will increase wound problems. If wound drainage or healing problems do occur immediate action is required. Deep sepsis can be ruled out with a joint aspiration and cell count [less than 2500], differential [less than 60% polys], and negative culture and sensitivity. All hematomas should be evacuated and necrosis or dehiscence should be managed by debridement to obtain a live wound