Aim. Autologous-labeled leukocytes combined with sulfur colloid bone marrow scan is the current imaging modality of choice for diagnosing prosthetic joint infection (PJI). Although this technique is reliable, in-vitro leukocyte labeling raises technical difficulties that limit its widespread use and sulfur colloid is increasingly difficult to obtain. Therefore, valid alternatives are needed. The purpose of our study was to determine the clinical value of 99mTc-sulesomab combined with 99mTc-colloidal rhenium sulphide (nanocolloid) bone marrow imaging in the diagnosis of infection in painful total joint arthroplasties. Materials and methods. A retrospective study was conducted on a cohort of 53 patients with painful hip or knee prostheses that underwent 99mTc-sulesomab and 99mTc-nanocolloids sequentially, between January 2008 and December 2016. The combined images were interpreted as positive for infection when there was activity on the sulesomab scan without corresponding activity on the bone marrow scan. The final diagnosis was made with microbiological findings or by clinical follow up of at least 12 months. Results. There were 49 total knee and 4 total hip replacements. Forty of them were women, with an average age of 65 years. Infections were diagnosed in 5 of the 53 patients. An isolated 99mTc-sulesomab scan shows 100% sensitivity but only 29.4% specificity. Combining it with a 99mTc-nanocolloid bone marrow scan, the overall sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 100%, 95.8%, 81.4%, 100% and 96.2% respectively. Conclusion. 99mTc-sulesomab combined with 99mTc-nanocolloid showed to be a useful method for diagnosing prosthetic joint infections. These technically simpler and ready-to-use products may be an alternative to autologous-labeled leukocytes/sulfur colloid marrow scan, although it needs validation at a larger scale

Introduction. Bone marrow stimulation has been a successful treatment option in cartilage repair and microfracture was the procedure of choice since the late 1980s. Despite its success in young and active patients, microfracture has inherent shortcomings such as shallow channels, wall compression, and non-standardized depth and diameter. This in vitro study assessed bone marrow access comparing microfracture, 1 and 2mm K-Wires, 1mm drill, and a recently introduced standardized subchondral bone needling procedure (Nanofracture) that creates 9mm deep and 1mm wide channels. Methods. An adult ovine model was used to assess access to bone the marrow spaces as well as effects on bone following microfracture, nanofracture, K-wire, and drilling following ethical clearance. All bone marrow stimulation techniques were conducted on a full thickness articular cartilage defect on the medial femoral condyles by the same surgeon. The same groups were repeated in vitro in 4 paired ovine distal femurs. MicroCT (Inveon Scanner, Siemens, Germany) was performed using 3D reconstruction and 25 micron slice analysis (MIMICS, Materialise, Belgium). Results. Microfracture elicited shallow depth with bone compression surrounding the channels. Trabecular channel access was limited; the channel depth and diameter were non-standardized and highly user and instrument dependent. Nanofracture demonstrated deep cancellous bone perforation with a high number of open trabecular channels. K-Wire drilling with both diameters resulted in well-defined channel walls, outlined by fine osseous deposits. Trabecular channel access was limited. The diameter of bone perforation is standardized, but depth is defined by visual controls. 1mm drill bit reaming demonstrated better osseous evacuation, but still limited trabecular marrow access. Discussion and Conclusion. Nanofracture resulted in thin, fragmented cancellous bone channels without rotational heat generation. Compared to microfracture, drilling and K-Wire stimulation, nanofracture showed superior bone marrow access with multiple trabecular access channels extending 9mm into subchondral bone

Background. Periprostetic joint infections (PJI) are often difficult to diagnose, to treat and often leave the patient with severe impaired function. The presence of low virulent bacteria is frequently discovered in apparent aseptic revisions of shoulder arthroplasties and pose a challenge to diagnose preoperatively. Dual Isotope In111 Leucocyte/ Tc99 Bone Marrow SPECT CT scan (L/BMS) is considered the radionuclide gold standard in preoperative diagnosing PJI with reported high specificity and sensitivity in hip and knee arthroplasties. Unfortunately, it is labour-intensive and expensive to perform and documentation using L/BMS on shoulder arthroplasties lack. Aim. To investigate if L/BMS succeeds in detecting shoulder PJI compared to tissue cultures obtained perioperatively. Method. All patients referred to a highly-specialised shoulder department with a painful or stiff shoulder-arthroplasty were included in the cohort. To diagnose infection as a possible cause of arthroplasty failure a L/BMS was planned for all patients. If the arthroplasty was revised, 5 tissue biopsies were obtained from the most infection-suspicious site during revision. Biopsies were cultured in broth and on plates for 14 days due to the high frequency of low virulent infection in shoulder revisions. Infection was defined as growth of the same bacteria in 3 or more of 5 the biopsies. Results. During the observation period 71 patients were referred. Revision surgery was performed in 62% of the patients (44/71) of which 29 also had been examined by L/BMS. A microbiological diagnose was available for all. The most predominant organism isolated was P. Acnes. Two patients both had a positive L/BMS and positive cultures. Negative L/BMS and negative cultures were found in 20 patients. The remaining 7 patients had negative L/BMS, but positive cultures. The two patients with a positive L/BMS both showed overt clinical signs of infection. L/BMS show a sensitivity 0.22 95%CI(0–0.49) and specificity 1.00 95%CI(1.00–1.00) in detecting shoulder PJI. The Positive Predictive Value is 1.00 95%CI(1.00–1.00) and Negative Predictive Value 0.74 95%CI(0.57–0.90). No patients infected with P. Acnes resulted in a positive scintigraphy nor had they preoperative or perioperative signs of infection. Conclusion. Only patients with severe infectious symptoms of shoulder PJI resulted in positive L/BMS. Hence, the scan added nothing to the preoperative clinical diagnose. In111 Leucocyte/ Tc99 Bone Marrow SPECT CT scan cannot be recommended as a standard screening procedure when evaluating failed shoulder arthroplasties for possible infection

INTRODUCTION. There is no effective therapy available today that alters the pathobiologic course of osteoarthritis. Recent advances have shown Mesenchymal stem cells to be a potential disease modifying treatment. Considering the tissue differentiation property and vast paracrine effects of MSCs we proposed the present study to find out the safety and efficacy of Mesenchymal stem cells in osteoarthritis of knee joint. METHODS. 12 patients with grade 1and2 bilateral osteoarthritis knee (Ahlbacks radiological grading) were selected. 8–10 ml of bone marrow was aspirated under strict aseptic precautions from the iliac spine. After the stem cell culture and expansion for 4–6 weeks the MSC suspension in 10xPBS was injected directly into the 24 knees by lateral approach. The outcome was evaluated by modified VAS score, WOMAC score, KOOS and MRI measurement of knee articular cartilage integrity by the modified WORMS score. RESULTS. Statistically significant improvement in VAS score, total WOMAC score and total KOOS score was observed from pre injection to 1st follow up at 6 weeks, 2nd follow up at 6 months and final follow up of mean 26.7 months. There was also a significant improvement from 1st follow up to 2nd and final follow up. The modified WORMS score showed a statistically significant decrease of 1.49 %. CONCLUSION. Intra-articular injection of autologous bone marrow derived culture-expanded MSCs can be considered a potential treatment of early osteoarthritis knee which relieves pain, stiffness, improves physical functions, and improves the articular cartilage integrity

Introduction. Problematic bone defects are encountered regularly in orthopaedic practice particularly in fracture non-union, revision hip and knee arthroplasty, following bone tumour excision and in spinal fusion surgery. At present the optimal source of graft to ‘fill’ these defects is autologous bone but this has significant drawbacks including harvest site morbidity and limited quantities. Bone marrow has been proposed as the main source of osteogenic stem cells for the tissue-engineered cell therapy approach to bone defect management. Such cells constitute a minute proportion of the total marrow cell population and their isolation and expansion is a time consuming and expensive strategy. In this study we investigated human bone marrow stem cells as a potential treatment of bone defect by looking at variability in patient osteogenic cell populations as a function of patient differences. We produced a model to predict which patients would be more suited to cell based therapies and propose possible methods for improving the quality of grafts. Methods. Bone marrow was harvested from 30 patients undergoing elective total hip replacement surgery in Musgrave Park Hospital, Belfast (12 males, 18 females, age range 52-82 years). The osteogenic stem cell fraction was cultured and subsequently analysed using colony forming efficiency assays, flow cytometry, fluorescence activated cell sorting and proteomics. Results. The number and proliferative capacity of osteogenic stem cells varied markedly between patients. Statistical analysis revealed significantly better osteogenic capacity in:. male patients. samples in which the growth hormone Fibroblastic Growth Factor-2 was added to culture medium. patients who used the cholesterol lowering agent simvastatin. Patient use of inhaled steroids and NSAIDs were found to have detrimental effects. A statistical model to predict marrow profiles based on these variables was produced. Conclusions. Stem cell based tissue engineering represents the future of the treatment of bone defect. This study provides evidence that inter-patient variability in marrow cell colony forming and proliferation ability can in some way be explained by patient associated factors. Using this knowledge, we can identify which patients would be best suited to this method of treatment and propose techniques for enhancement of their graft profiles

Aim. To evaluate the efficacy of bone marrow derived stromal cells (BMSC) for the treatment of non-unions in fractures. Methods. An ethically approved single centre randomised control trial recruited 35 patients for treatment of non-unions with BMSC during 2006–2010. Autologous BMSC were culture expanded at the Good Manufacturing Practice (GMP) standard Oscell® laboratory in the hospital. Following in vitro expansion- cells in autologous serum and serum alone were randomised for insertion at one of the two fracture sides by StratOs® computer software. Patients and the operating surgeon were blinded to the side of cell insertion. Such method of randomisation created internal controls at the fracture sites- one side receiving the cell (‘test side’) and other, not (‘control’). Serial radiographs extending up to an average of twelve months were evaluated by six independent assessors blinded to side of cell insertion. Callus formation and bridging of fracture was compared for ‘test’ and ‘control’ side. Radiological and clinical outcome at final follow-up was also noted. Results. The study had 21 males and 14 females with a mean age of 51.2±13.2 years (range 18–76). The average duration of non-union was 3±2 years (range 1–10 years) with mean 3.5 (range 1–12) surgical interventions prior to BMSC insertion. Independent assessment of ‘test’ and ‘control’ side revealed that the callus formation and fracture bridging was slow although a trend to improvement on the side of the BMSC insertion was observed at 9–12 months. At final follow-up 22 patients progressed to bony union; 13 patients had persisting non union. Conclusion. BMSC can achieve progression to union in substantial number of cases of resistant non-unions where the alternative is extensive reconstructive procedures or amputations. Larger trials are required to study the pattern of early healing following cell therapy in such cases

Background. A cell-based tissue-engineered construct can be employed for treating meniscal lesions occurring in the non-vascularized inner two-thirds. The objective of this study was to test the hypothesis that both pre-differentiation of human bone marrow derived stromal cells (hBMSCs) into chondrogenic lineage before cell seeding and platelet-rich plasma (PRP) pretreatment on a PLGA mesh scaffold enhances the healing capacity of the meniscus with hBMSCs-seeded scaffolds in vivo. Methods. PRP of 5 donors was mixed and used for the experiments. The woven PLGA mesh scaffold (VicrylTM, Ethicon) measuring 20×8 mm (thickness, 0.2 mm) was prepared. The scaffolds were immersed into 1,000 μl of PRP and were centrifuged at 150g for 10 min. Then, the scaffold was flipped 180° and the same procedure was done for the other side. After washing, the scaffolds were soaked into 1,000 μl of DMEM media. hBMSCs from an iliac crest of 10 patients after informed consent and approval of our IRB were induced into chondrogenic differentiation with chondrogenic media containing 10 ng/ml rhTGF-ß3 in 1.2% alginate bead culture system for 7 days. Then, 2×10. 5. hBMSCs were recovered, seeded onto the scaffold, and cultured under dynamic condition. Based on the presence of pre-differentiation into chondrogenic lineage and the PRP pretreatment, 4 study groups were prepared. (no differentiation without PRP, no differentiation with PRP, chondrogenic differentiation without PRP, chondrogenic differentiation with PRP) Cell number for each cell-seeded scaffold was determined at 24 hours after seeding. Then, scaffolds were placed between human meniscal discs and were implanted subcutaneously in nude mice for 6 weeks (n=10 per group). Results. Cell attachment analysis revealed no significant difference among groups (p>0.05). The average cell number attached on the scaffold was ranged 1.1×10. 5. to 1.2×10. 5. among groups after 24 hours, so the initial cell seeding efficiency was ranged 55 to 60%. Histologic results from the 10 constructs containing hBMSCs undifferentiated and seeded onto non-PRP treated scaffolds revealed none had healed at all. Of the constructs containing hBMSCs undifferentiated and seeded onto PRP-pretreated scaffolds, three menisci healed and seven did not heal. Of the constructs containing hBMSCs pre-differentiated into chondrogenic lineage and seeded onto non-PRP treated scaffolds, six menisci healed and four did not heal. Of the constructs containing hBMSCs pre-differentiated into chondrogenic lineage and seeded onto PRP-pretreated scaffolds, seven menisci healed and three did not heal. Histological evaluation demonstrated a continuous hypercellular new fibrous tissue integrating into the native devitalized meniscus disc tissue in healed samples. The histological outcome between the groups was significant (p<0.05) (Table 1) (Figure 1). Conclusion. hBMSCs, which were differentiated into chondrogenic lineage before cell seeding and attached PRP-pretreated PLGA mesh scaffolds, demonstrated enhanced healing capacity of human meniscus in a meniscal repair mouse model. These findings demonstrate that both pre-differentiation of hBMSCs into chondogenesis and the PLGA scaffold modified by PRP pretreatment provides more biomimetic and biocompatible strategy for cell-mediated meniscal repair. Acknowledgements. This study was supported by Basic Science Research Program through the National Research Foundation of Korea (#2015-01004099)

Background. Despite promising results have shown by osteogenic cell-based demineralized bone matrix composites, they need to be optimized for grafts that act as structural frameworks in load-bearing defects. The purpose of this experiment is to determine the effect of bone marrow mesenchymal stem cells seeding on partially demineralized laser-perforated structural allografts that have been implanted in critical femoral defects. Materials and Methods. Thirty-two wistar rats were divided into four groups according to the type of structural bone allograft; the first: partially demineralized only (Donly), the second: partially demineralized stem cell seeded (DST), the third: partially demineralized laser-perforated (DLP), and the fourth: partially demineralized laser-perforated and stem cell seeded (DLPST). Trans-cortical holes were achieved in four rows of three holes approximated cylindrical holes 0.5 mm in diameter, with centres 2.5 mm apart. P3 MSCs were used for graft seeding. Histologic and histomorphometric analysis were performed at 12 weeks. Results. DLP grafts had the highest woven bone formation, where most parts of laser pores were completely healed by woven bone. DST and DLPST grafts surfaces had extra vessel-ingrowth-like porosities. Furthermore, in the DLPST grafts, a distinct bone formation at the interfaces was noted. Conclusion. This study indicated that surface changes induced by laser perforation, accelerated angiogenesis induction by MSCs, which resulted in endochondral bone formation at the interface. Despite non-optimal results, stem cells showed a tendency to improve osteochondrogenesis, and the process might have improved, if they could have been supplemented with the proper stipulations

Purpose. Bone marrow multi-potent stromal cells represent a heterogenous source of cells with great promise in joint cartilage regenerative medicine. However, due to their low numbers upon harvesting, MSCs need to be expanded without compromising their capacity to form chondrocytes (cartilage cells). To date there is no consensus on how to expand MSCs in order to maximize their potential for cartilage repair and nor are there any specific cell signatures of MSCs with chondrogenic propensity. Emerging evidence suggest that marrow stem cells exist in a hypoxic microenvironment. On this basis and in addition to cartilages natural existence in hypoxic environment (1–7% O2), we hypothesized that MSC expansion under hypoxia will result in the enrichment of MSCs with predilection to chondrocytes compared to expansion under the conventional culture conditions of 21% O2. Method. Bone marrow was harvested from the iliac crest of 4 donors (mean age 43.5 years) post informed consent and local ethical approval. Fifteen million mono-nucleated (MNCs) cells were seeded into T150cm2 culture flasks in the presence of alpha MEM plus 10% FBS and 5 ng/ml FGF2. Similarly, 0.25 million MNCs were seeded in 10cm petri dishes for colony forming unit-fibroblastic (CFU-f) assay. The seeded flasks and petri dishes were cultured under normoxia (21% O2) and hypoxia (3% O2). Petri dished cells were cultured for 14 days and those in flasks were cultured until passage 2 (P2). Developed cell colonies per dish were revealed after crystal violet staining. Colony counts and diameters were recorded. P2 cells were treated with a panel of antibodies for cell surface marker analysis by fluorescent activated cell sorting (FACS) flow cytometry. P2 cell pellets were formed and induced towards cartilage in a defined serum free medium containing TGFβ1. Pellets were cultured for 3 weeks under normoxia and were then processed for histological, biochemical and gene expression analyses. Results. The mean number of cell colonies was 1.25-fold higher after hypoxia culture relative to normoxia. There were no differences in colony diameters. A panel of common protein signatures (CD29, CD90, CD105 and CD151) for stem cells declined in expression after expansion in hypoxia. However, other signatures (CD13, CD34 and CD44) expression level increased under hypoxia, whilst CD73 expression was unchanged. Pellets from hypoxia-expanded MSCs showed on average a 1.4-fold increase in chondrogenic capacity as judged by glycosaminoglycan (GAG) matrix per DNA content relative to normoxia pellets. The gene expression of collagen II, SOX9, aggrecan and matrillin-3 increased by 1.2-, 2-, 1.3- and 1.5-fold, respectively, in pellets formed from hypoxia-expanded stem cells relative to their normoxia counterparts. Conclusion. Expansion of stem cells under hypoxia potentiates their capacity to form cartilage with improved cartilage properties. However, there is a need for signatures to identify stem cells with propensity to form cartilage

Articular cartilage repair remains a challenge in orthopedic surgery, as none of the current clinical therapies can regenerate the functional hyaline cartilage tissue. In this study, we proposed a one-step surgery strategy that uses autologous bone marrow mesenchymal stem cells (MSCs) embedded in type II collagen (Col-II) gels to repair the full thickness chondral defects in minipig models. Briefly, 8 mm full thickness chondral defects were created in both knees separately, one knee received Col-II + MSCs transplantation, while the untreated knee served as control. At 1, 3 and 6 months postoperatively, the animals were sacrificed, regenerated tissue was evaluated by magnetic resonance imaging, macro- and microscopic observation, and histological analysis. Results showed that regenerated tissue in Col-II + MSCs transplantation group exhibited significantly better structure compared with that in control group, in terms of cell distribution, smoothness of surface, adjacent tissue integration, Col-II content, structure of calcified layer and subchondral bone. With the regeneration of hyaline-like cartilage tissue, this one step strategy has the potential to be translated into clinical application

Introduction. According to proposal of Noble, the femoral bone marrow cavity form of patients who underwent Total Hip Arthroplasty (THA) can be classified under 3 categories; those are Stovepipe, Normal and Champagne-fluted. We developed typical sodium chloride femoral model was created by 3D prototyping technique. The purpose was to identify the relationship of pressure zone of the surrounding areas between femoral bone marrow cavity form and hip stem. Materials and Method. As opponent clarified stem design concept Zweymüller type model was used. According to CT data with the patients who underwent THA, the sodium chloride femoral model was custom-made and selected as the representative model based on Noble's 3 categories. Eight models of each category were used to performed mechanical test. Result. In mechanics test, the result of comparison between the contact pressure zones of zone 1–7, significant differences of contact pressure zones were identified between the Stovepipe group and Normal group in zone 3, 4 and 5. In zone 3 and 5, such significant differences were also identified between Champagne-fluted group and Normal group. In Stovepipe group, a significant difference of the contact pressure zone was observed at the proximal and distal. In Champagne-fluted group and the Normal group, a significant difference was observed in the contact pressure in distal femur (3, 4, 5 Zone) and (Zone1, 2, 6, 7) proximal femur. Discussion. Although in most studies Sawbone® is used for femoral models, the focus of this research is of those who possess a characteristic femur with marrow cavity form. Therefore, sodium chloride bone model was used instead. In comparison in terms of applicability between sodium chloride bone model and regular model, the failure of all 24 joints of sodium chloride bone model were unconfirmed in mechanics test. Moreover, the possibility that its performance in mechanics test is equivalent to Sawbone®is considered. The design concept for Zweymüller type achieves the ability to load distribute within a wide range of cortical bone across the middle position to distal femur. It's determined by the concept that a wide range of contact pressure was admitted at middle position and distal femur in the Champagne-fluted group and the Normal group. On the other hand, the contact pressure zone of Stovepipe was not able to meet the expected level at distal femur. The method of this research is control its stress condition within the stem design. By this point, it is considered possible that the stability of various stem design was able to be forecasted and the assessment of stableness was positive. Conclusion. On the basis of Noble's categories, 3 types of bone models were created by 3D prototyping technique, and pressure distribution measurement were performed. The result from the pressure distribution indicated that even in Zweymüller stem had anxiety of securing force in Champagne-fluted type and Stovepipe type canal. We believe the method of in vivo study can develop to assess the stability of implant preoperatively

Objective

The aim of this study was to investigate PDGF release in the peripheral circulation following trauma and to correlate it with the numbers of MSCs in iliac crest bone marrow (BM) aspirate.

Purpose

The biomechanical role of the meniscus in the knee joint is a function of its extracellular matrix which consists of type I collagen throughout, type II collagen in the inner meniscus region and glycosaminoglynated (GAG) proteins of which aggrecan is the most prevaleet. Meniscus reparative capacity is limited, particularly when a defect is located in the inner avascular portion, and menisectomy predisposes the joint to osteoarthritis. Using meniscus cells in tissue engineering strategies has been advocated to generate functional meniscus substitutes. However, meniscus cells, like chondrocytes of cartilage, lose their matrix-forming phenotype during culture expansion. Co-culture of chondrocytes with stem cells has been shown to result in enhanced matrix formation. We hypothesized that meniscus cells in co-culture with stem cells will result in increased matrix formation.

Background

Replacing bone lost as a consequence of trauma or disease is a major challenge in the treatment of musculoskeletal disorders. Tissue engineering strategies seek to harness the potential of stem cells to regenerate lost or damaged tissue. Bone marrow aspirate (BMA) provides a promising autologous source of skeletal stem cells (SSCs) however, previous studies have demonstrated that the concentration of SSCs required for robust tissue regeneration is below levels present in iliac crest BMA, emphasising the need for cell enrichment strategies prior to clinical application.

Purpose

Mesenchymal stromal cells (MSCs) are an attractive choice for regenerative medicine. We previously showed that MSCs enhance wound healing in animals after radiotherapy. The effect of MSCs on tumor growth is not well understood. The potential use of MSCs to enhance wound healing after radiotherapy (RT) and resection of soft tissue sarcoma (STS) is dependent on a satisfactory safety profile to ensure that tumor proliferation does not occur and recurrence is not increased.

The treatment of critical-sized bone defects still remains today a challenge, especially when the surrounding soft, vascularized and innervated tissues have been damaged - a lack of revascularization within the injured site leading to physiological disorders, from delayed healing to osteonecrosis. The axial insertion of a vascular bundle (e.g. arterio-venous loop, AVL) within a synthetic bone filler to initiate and promote its revascularization has been foreseen as a promising alternative to the current strategies (e.g., vascularized free flaps) for the regeneration of large bone defects. In a previous work, we showed that the insertion of a vein in a 3D-printed monetite scaffold induced its higher revascularization than AVL, thus a possible simplification of the surgical procedures (no microsurgery required). Going further, we investigate in this study whether or not the presence of a vein could stimulate the formation of mineralized tissue insides a synthetic scaffold filled with bone marrow and implanted in ectopic site. Monetite scaffolds were produced by additive manufacturing according to a reactive 3D-printing technique co-developed by the authors then thoroughly characterized. Animal study was performed on 14 male Wistar rats. After anesthesia and analgesia, a skin medial incision in rat thigh allowed the site on implantation to be exposed. Bone marrow was collected on the opposite femur through a minimally invasive procedure and the implant was soaked with it. For the control group (N=7), the implant was inserted in the incision and the wound was closed whereas the femoral bundle was dissected and the vein inserted in the implant for the experimental group (N=7). After 8 weeks animals were sacrificed, the implant collected and fixed in a 4% paraformaldehyde solution. Explants were characterized by µCT then embedded in poly-methyl methacrylate prior SEM, histology and immunohistochemistry. Images were analyzed with CT-Analyzer (Bruker) and ImageJ (NIH) and statistical analyses were carried out using SPSS (IBM). Implants were successfully 3D-printed with a +150 µm deviation from the initial CAD. As expected, implants were composed of 63%wt monetite and 37%wt unreacted TCP, with a total porosity of 44%. Data suggested that scaffold biodegradation was significantly higher when perfused by a vein. Moreover, the latter allowed for the development of a dense vascular network within the implant, which is far more advanced than for the control group. Finally, although mineralized tissues were observed both inside and outside the implant for both groups, bone formation appeared to be much more important in the experimental one. The ectopic formation of a new mineralized tissue within a monetite implant soaked with bone marrow seems to be highly stimulated by the simple presence of a vein alone. Although AVL have been studied extensively, little is known about the couple angiogenesis/osteogenesis which appears to be a key factor for the regeneration of critical-sized bone defects. Even less is known about the mechanisms that lead to the formation of a new bone tissue, induced by the presence of a vein only. With this in mind, this study could be considered as a proof of concept for further investigations

Mesenchymal stem cells (MSCs) are capable of forming bone, cartilage and other mesenchymal tissues but are also important modulators of innate and adaptive immune responses. We have capitalized on these important functions to mitigate adverse responses when bone is exposed to pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), or prolonged pro-inflammatory cytokines. Our goal was to optimize osteogenesis and mitigate persistent undesired inflammation by: 1. preconditioning MSCs by short term exposure to lipopolysaccharide (LPS) and Tumor Necrosis Factor alpha (TNF-α), 2. genetic modification of MSCs to overexpress Interleukin 4 (IL-4) either constitutively, or as NFκB-responsive IL-4 over-expression cells, and 3. training the MSCs (innate immune memory) by repeated stimulation with LPS. In the first experiment, bone marrow MSCs and macrophages were isolated from femurs and tibias of C57BL/6 mice. MSCs (1×104 cells) were seeded in 24-well transwell plates in the bottom chamber with MSC growth medium. MSCs were treated with 20 ng/ml TNF-α and 1–20 μg/ml LPS for three days. Primary macrophages (2 × 103 cells) were seeded to the insert of a separate transwell plate and polarized into the M1 phenotype. At day four, MSCs and macrophages were washed and the inserts with M1 macrophages were moved to the plates containing preconditioned MSCs at the bottom of the well. Co-culture was carried out in MSC growth medium for 24h. In the second experiment, bone marrow derived macrophages and MSCs were isolated from femora and tibiae of Balb/c male mice. 5×104 macrophages and 1×104 MSCs were seeded in the bottom well of the 24-well transwell plate. The upper chambers were seeded with unmodified MSCs, MSCs preconditioned with 20 ng/ml TNF-α and 20 mg/ml LPS for 3 days, NFκB-IL4 secreting MSCs (all 5×104 cells), or controls without MSCs. Co-culture was carried out in mixed osteogenic-macrophage media with clinically relevant polyethylene or titanium alloy particles. In the third experiment, bone marrow MSCs and macrophages were collected from femurs and tibias of C57BL/6 male mice. The MSCs were stimulated by LPS, washed out for five days, and re-stimulated by LPS in co-culture with macrophages. First, preconditioned MSCs enhanced anti-inflammatory M2 macrophage (Arginase 1 and CD206) expression, decreased pro-inflammatory M1 macrophage (TNF-α/IL-1Ra ratio) expression, and increased osteogenic markers (alkaline phosphatase expression and matrix mineralization) in co-culture. Second, NFκB-IL4 secreting MSCs decreased pro-inflammatory M1 (TNF-α), increased anti-inflammatory M2 (Arg1, IL-1ra) expression, and enhanced the expression of osteogenic factors Runx2 and alkaline phosphatase, in the presence of particles, compared to other groups. Third, LPS-trained MSCs increased anti-inflammatory (Arginase1 and CD206), and decreased the proinflammatory (TNF-α, IL1b, iNOS, and IL6) marker expression in MSC/macrophage co-culture. Transforming MSCs via the techniques of preconditioning, genetic modification, or training (innate immune memory) can modulate/convert a potentially injurious microenvironment to an anti-inflammatory pro-reconstructive milieu. These effects are highly relevant for bone healing in the presence of adverse stimuli. These concepts using transformed MSCs could also be extended to other organ systems subjected to potentially damaging agents

Title. Longitudinal Intravital Imaging to Quantify the “Race for the Surface” Between Host Immune Cell and Bacteria for Orthopaedic Implants with S. aureus Colonization in a Murine Model. Aim. To assess S. aureus vs. host cell colonization of contaminated implants vis intravital multiphoton laser scanning microscopy (IV-MLSM) in a murine model. Method. All animal experiments were approved by IACUC. A flat stainless steel or titanium L-shaped pin was contaminated with 10. 5. CFU of a red fluorescent protein (RFP) expressing strain of USA300LAC, and surgically implanted through the femur of global GFP-transgenic mice. IV-MLSM was performed at 2, 4, and 6 hours post-op. Parallel cross-sectional CFU studies were performed to quantify the bacteria load on the implant at 2,4,6,12,18 and 24 hours. Results. 1) We developed a high-fidelity reproducible IV-MLSM system to quantify S. aureus and host cell colonization of a bone implant in the mouse femur. Proper placement of all implants were confirmed with in vivo X-rays, and ex vivo photos. We empirically derive the ROI during each imaging session by aggregating the imaged volume which ranges from (636.4um × 636.4um × 151um) = 0.625 +/- 0.014 mm. 3. of bone marrow in a global GFP-transgenic mouse. 2) IV-MLSM imaging acquisition of the “race for the surface”.In vitro MPLSM images of implants partially coated with USA300LAC (RFP-MRSA) were verified by SEM image. Results from IV-MLSM of RFP-MRSA and GFP. +. host cell colonization of the contaminated implants illustrated the mutually exclusive surface coating at 3hrs, which to our knowledge is the first demonstration of “the race for the surface” between bacteria and host cells via intravital microscopy. 3) Quantifying the “race for the surface” with CFU verification of S. aureus on the implant. 3D volumetric rendering of the GFP. +. voxels and RFP+ voxels within the ROI were generated in Imaris. The voxel numbers suggeste that the fight for the surface concludes ∼3hrs post-infection, and then transitions to an aggressive MRSA proliferation phase. The results of WT control demonstrate a significant increase in CFU by 12hrs post-op for both stainless steel (P<0.01) and titanium (P<0.01). Conclusions. We developed IV-MLSM to quantify the “Race for the Surface” between host cells and contaminating S. aureus in a murine femur implant model. This race is remarkably fast, as the implant surface is completely covered with 3hrs, peak bacterial growth on the implant occurs between 2 and 12 hours and is complete by 12hrs

Aim. Debridement, Antibiotics, Irrigation, and implant Retention (DAIR) is a surgical treatment protocol suitable for some patients with fracture related infection (FRI). Clinically relevant pre-clinical models of DAIR are scarce and none have been developed in large animals. Therefore, this project aimed to develop a large animal model for FRI including a DAIR approach and compare outcomes after 2 or 5 weeks of infection. Method. Swiss Alpine sheep (n=8), (2–6 years, 50–80 kg) were included in this study. This study was approved by cantonal Ethical authorities in Chur, Switzerland. A 2 mm osteotomy was created in the tibia and fixed with a 10-hole 5.5 mm steel plate. Subsequently, 2.5 mL of saline solution containing 10. 6. CFU/mL of Staphylococcus aureus MSSA (ATCC 25923) was added over the plate. Sheep were observed for 2 (n=3) or 5 weeks (n=5) until revision surgery, during which visibly infected or necrotic tissues were removed, and the wound flushed with saline. All samples were collected for bacterial quantification. After revision surgery, the sheep were treated systemically for 2 weeks with flucloxacillin and for 4 weeks with rifampicin and cotrimoxazole. After 2 further weeks off antibiotics, the animals were euthanized. Bacteriological culture was performed at the end of the study. Bone cores were isolated from the osteotomy site and processed for Giemsa & Eosin and Brown and Brenn staining. A radiographical examination was performed every second week. Results. Bacteriological evaluation of the retrieved samples during revision surgery showed no significant difference between the 2 vs 5 weeks infection periods in term of total CFU counts. At the end of the study, radiographical examination showed callus formation over the osteotomy site in both groups, although the osteotomy was not completely healed in either group. At euthanasia, the 2 weeks infection group showed a higher soft tissue burden compared to the 5 weeks group, whereby the infection in the 5 weeks group was primarily located in the bone and bone marrow. Conclusions. The large animal model of FRI and DAIR was successfully established. Bacteriological outcomes highlight that the increasing duration of the infection does not change the outcome but the location of the infection from a predominantly soft tissue infection to a deeper bone and intramedullary (IM) channel infection. The debridement of the IM channel could potentially reduce the infection burden by eliminating those bacteria not easily reached by systemic antibiotics, though is not practical using conventional techniques

Previous studies have described an age-dependent distortion of bone microarchitecture for α-CGRP-deficient mice (3). In addition, we observed changes in cell survival and activity of osteoblasts and osteoclasts isolated from young wildtype (WT) mice when stimulated with α-CGRP whereas loss of α-CGRP showed only little effects on bone cell metabolism of cells isolated from young α-CGRP-deficient mice. We assume that aging processes differently affect bone cell metabolism in the absence and presence of α-CGRP. To further explore this hypothesis, we investigated and compared cell metabolism of osteoblasts and bone marrow derived macrophages (BMM)/osteoclast cultures isolated from young (8–12 weeks) and old (9 month) α-CGRP-deficient mice and age matched WT controls. Isolation/differentiation of bone marrow macrophages (BMM, for 5 days) to osteoclasts and osteoblast-like cells (for 7/14/21 days) from young (8–12 weeks) and old (9 month) female α-CGRP−/− and WT control (both C57Bl/6J) mice according to established protocols. We analyzed cell migration of osteoblast-like cells out of femoral bone chips (crystal violet staining), proliferation (BrdU incorporation) and caspase 3/7-activity (apoptosis rate). Alkaline phosphatase (ALP) activity reflects osteoblast bone formation activity and counting of multinucleated (≥ 3 nuclei), TRAP (tartrate resistant acid phosphatase) stained osteoclasts reflects osteoclast differentiation capacity. We counted reduced numbers of BMM from young α-CGRP−/− mice after initial seeding compared to young WT controls but we found no differences between old α-CGRP−/− mice and age-matched controls. Total BMM number was higher in old compared to young animals. Migration of osteoblast-like cells out of bone chips was comparable in both, young and old α-CGRP−/− and WT mice, but number of osteoblast-like cells was lower in old compared to young animals. Proliferation of old α-CGRP−/− BMM was higher when compared to age-matched WT whereas proliferation of old α-CGRP−/− osteoblasts after 21 days of osteogenic differentiation was lower. No differences in bone cell proliferation was detected between young α-CGRP−/− and age-machted WT mice. Caspase 3/7 activity of bone cells from young as well as old α-CGRP−/− mice was comparable to age-matched controls. Number of TRAP-positive multinucleated osteoclasts from young α-CGRP−/− mice was by trend higher compared to age-matched WT whereas no difference was observed in osteoclast cultures from old α-CGRP−/− mice and old WT. ALP activity, as a marker for bone formation activity, was comparable in young WT and α-CGRP−/− osteoblasts throughout all time points whereas ALP activity was strongly reduced in old α-CGRP−/− osteoblasts after 21 days of osteogenic differentiation compared to age-matched WT. Our data indicate that loss of α-CGRP results in a reduction of bone formation rate in older individuals caused by lower proliferation and reduced activity of osteogenic cells but has no profound effects on bone resorption rate. We suggest that the osteopenic bone phenotype described in aged α-CGRP-deficient mice could be due to an increase of dysfunctional matured osteoblasts during aging resulting in impaired bone formation