Macrophages play a critical role in innate immunity by promoting or inhibiting tissue inflammation and repair. Classically, macrophages can differentiate into either pro-inflammatory (M1) or pro-reparative (M2) phenotypes in response to various stimuli. Therefore, this study aimed to address how extracellular vesicles (EVs) derived from polarized macrophages can affect the inflammatory response of tendon cells.

For that purpose, human THP-1 cells were stimulated with lipopolysaccharide (LPS), and interleukins -4 and -13 (IL- 4, IL-13), to induce macrophages polarization into M1, M2, and hybrid M1/M2 phenotypes. Subsequently, the EVs were isolated from the culture medium by ultracentrifugation. The impact of these nanovesicles on the inflammation and injury scenarios of human tendon-derived cells (hTDCs), which had previously been stimulated with interleukin- 1 beta (IL-1ß) to mimic an inflammatory scenario, was assessed.

We were able to isolate three different nanovesicles populations, showing the typical shape, size and surface markers of EVs. By extensively analyzing the proteomic expression profiles of M1, M2, and M1/M2, distinct proteins that were upregulated in each type of macrophage-derived EVs were identified. Notably, most of the detected pro- inflammatory cytokines and chemokines had higher expression levels in M1-derived EVs and were mostly absent in M2-derived EVs. Hence, by acting as a biological cue, we observed that M2 macrophage-derived EVs increased the expression of the tendon-related marker tenomodulin (TNMD) and tended to reduce the presence of pro-inflammatory markers in hTDCs. Overall, these preliminary results show that EVs derived from polarized macrophages might be a potential tool to modulate the immune system responses becoming a valuable asset in the tendon repair and regeneration fields worthy to be further explored.

Worldwide, tendon disorders are one of the main causes of disability that decrease the quality of life of individuals and represent a substantial economic burden on society. Currently, the main therapies used for tendon injuries are not able to restore tendon functionality, and due to tendons' hypovascular and hypocellular nature, they present a reduced healing capacity, which also limits the success of the available therapies. In order to discover new therapies, extracellular vesicles (EVs), key players in cell-cell communication, have been widely explored for tissue engineering and regenerative medicine applications. Thus, the aim of this study is to assess the role of EVs derived from platelets in stem cell tenogenic commitment using a bioengineered tendon in vitro model for potential use as tendon therapeutic agents. Biomimetic platelet-derived EVs were produced by freeze-thaw cycles of platelets and isolation at different centrifugation speed. To recreate the architecture of tendons, a 3D system consisting of electrospun anisotropic nanofiber scaffolds coated with collagen encapsulating human adipose stem cells (hASCs) and different types of platelet-derived EVs, were produced. Then, the influence of the tendon-mimetic constructs and the distinct EVs populations in the hASCs tenogenic differentiation were assessed over culture time. We observed that the hASCs on the nanofibrous tendon scaffolds, show high cytoskeleton anisotropic organization that is characteristic of tenocytes. Moreover, acting as biological cues, platelet-derived EVs boosted hASCs tenogenic commitment, supported by the increased gene expression of tendon-related markers (SCX and TNMD). Additionally, EVs enhanced the deposition of tendon like extracellular matrix (ECM), as evidenced by the increased gene expression of ECM-related markers such as COL1, COL3, DCN, TNC, and MMP-3, which are fundamental for ECM synthesis and degradation balance. Moreover, EVs induced lower collagen matrix contraction on hASCs, which has been related with lower myofibroblast differentiation. Overall, the results revealed that EVs are capable of modulating stem cells' behavior boosting their tenogenic commitment, through the increased expression of healthy tendon cell markers, potentiating ECM deposition and decreasing cell contractility. Therefore, platelet EVs are a promising biochemical tool, worthy to be further explored, as paracrine signaling that might potentiate tendon repair and regeneration.

Common tendon injuries impair healing, leading to debilitation and an increased re-rupture risk. The impact of oxygen-sensing pathways on repair mechanisms, vital in regulating inflammation and fibrosis, remains unclear despite their relevance in tendon pathologies. Recent studies show that pulsed electromagnetic field (PEMF) reduce inflammation in human tendon cells (hTDCs) and in hypoxia-induced inflammation. We investigated the hypoxia's impact (1% and 2% oxygen tension) using magnetic cell sheet constructs (IL-1β-magCSs) primed with IL-1β. IL-1β-magCSs were exposed to low OT (1h, 4h,6h) in a hypoxic chamber. To confirm the role of PEMF (5Hz, 4mT, 50% duty cycle) on hypoxia modulation, IL-1β-magCSs, previously exposed to OT, were 1h-stimulated with PEMF. Our results show a significant increase in HIF- 1a and HIF-2a expression on IL-1β-magCSs after exposure to 2%-OT at all time points, compared to 1%- OT and normoxia. TNFa, IL-6, and IL-8 expression increased after 6 hours of 1%-OT exposure. PEMF stimulation of hypoxic IL-1β-magCSs led to decreased pro-inflammatory genes and increased anti-inflammatory (IL-4,IL-10) expression compared to unstimulated magCSs. IFN-g, TNF-α, and IL-6 release increased after 6 hours, regardless of %-OT, while IL-10 levels tended to rise after PEMF stimulation at 2%-OT. Also, NFkB expression was increased on IL-1β-magCSs exposed to 4 h and 6 h of 2%-OT, suggesting a link between NFkB and the production of pro-inflammatory factors. Moreover, PEMF stimulation showed a significantly decreased NFkB level in IL-1β-magCSs.

Overall, low OT enhances expression of hypoxia-associated genes and inflammatory markers in IL-1β-magCSs with the involvement of NFkB. PEMF modulates the response of magCSs, previously conditioned to hypoxia and to inflammatory triggers, favouring expression of anti-inflammatory genes and proteins, supporting PEMF impact in pro-regenerative tendon strategies.

Acknowledgements: ERC CoG MagTendon(No.772817), FCT under the Scientific Employment Stimulus-2020.01157.CEECIND. Thanks to Hospital da Prelada for providing tendon tissue samples (Portugal), and TERM

RES Hub (Norte-01-0145-FEDER-022190).

Tendons and tendon-to-bone entheses don't usually regenerate after injury, and the hierarchical organization of such tissues makes them challenging sites of study for tissue engineers. In this study, we have tried a novel approach using miRNA and a bioactive bioink to stimulate the regeneration of the enthesis. microRNAs (miRNAs) are short, non-coding sequences of RNA that act as post-transcriptional regulators of gene and protein expression [1]. Mimics or inhibitors of specific miRNAs can be used to restore lost functions at the cell level or improve healing at the tissue level [2,3]. We characterized the healing of a rat patellar enthesis and found that miRNA-16-5p was upregulated in the fibrotic portion of the injured tissue 10 days after the injury. Based on the reported interactions of miRNA-16-5p with the TGF-β pathway via targeting of SMAD3, we aimed to explore the effects of miRNA-16-5p mimics on the tenogenic differentiation of adipose-derived stem cells (ASCs) encapsulated in a bioactive bioink [4,5]. Bioinks with different properties are used for the 3D printing of biomimetic constructs. By integrating cells, materials, and bioactive molecules it is possible to tailor the regenerative capacity of the ink to meet the particular requirements of the tissue to engineer [5]. Here we have encapsulated ASCs in a gelatin-methacryloyl (GelMa) bioink that incorporates miR-16-5p mimics and magnetically responsive microfibers (MRFs). When the bioink is crosslinked in the presence of a magnetic field, the MRFs align unidirectionally to create an anisotropic construct with the ability to promote the tenogenic differentiation of the encapsulated ASCs. Additionally, the obtained GelMA hydrogels retained the encapsulated miRNA probes, which permitted the effective 3D transfection of the ASC and therefore, the regulation of gene expression, allowing to investigate the effects of the miR-16-5p mimics on the tenogenic differentiation of the ASCs in a biomimetic scenario.

MicroRNA (miR) delivery to regulate chronic inflammation hold extraordinary promise, with new therapeutic possibilities emanating from their ability to fine-tune multiple target gene regulation pathways which is an important factor in controlling aberrant inflammatory reactions in complex multifactorial disease. However, several hurdles have prevented advancements in miR-based therapies. These include off-target effects of miRs, limited trafficking, and inefficient delivery. We propose a magnetically guided nanocarrier to transport therapeutically relevant miRs to assist self- resolving inflammation processes at injury sites and reduce the impact of chronic inflammation- related diseases such as tendinopathies. The high prevalence, significant socio-economic burden and increasing recognition of dysregulated immune mediated pathways in tendon disease provide a compelling rationale for exploring inflammation-targeting strategies as novel treatments in this condition. By combining cationic polymers, miR species (e.g., miR 29a, miR155 antagonist), and magnetic nanoparticles in the form of magnetoplexes with highly efficient magnetofection procedures, we developed inexpensive, easy-to-fabricate, and biocompatible systems with competent miR-binding and fast cellular uptake into different types of human cells, namely macrophages and tendon-derived cells. The system was shown to be cell-compatible and to successfully modulate the expression and production of inflammatory markers in tendon cells, with evidence of functional pro-healing changes in immune cell phenotypes. Hence, magnetoplexes represent a simple, safe, and non-viral nanoplatform that enables contactless miR delivery and high- precision control to reprogram cell profiles toward improved pro-regenerative environments.

Acknowledgements: ERC CoG MagTendon No.772817; FCT Doctoral Grant SFRD/BD/144816/2019, and TERM

RES Hub (Norte-01-0145-FEDER-022190).

Chronic inflammatory events have been associated to almost every chronic disease, including cardiovascular-, neurodegenerative- and autoimmune- diseases, cancer, and host-implant rejection. Given the toll of chronic inflammation in healthcare and socioeconomical costs developing strategies to resolve and control chronic states of inflammation remain a priority for the significant benefit of patients.

Macrophages (Mφ) hold a central role both in the initiation and resolution of inflammatory events, assuming different functional profiles. The outstanding features of Mφ counting with the easy access to tissues, and the extended networking make Mφ excellent candidates for precision therapy. Moreover, sophisticated macrophage-oriented systems could offer innovative immune-regulatory alternatives to effectively regulate chronic environments that traditional pharmacological agents cannot provide.

We propose magnetically assisted systems for balancing Mφ functions at the injury site. This platform combines polymers, inflammatory miRNA antagonists and magnetically responsive nanoparticles to stimulate Mφ functions towards pro-regenerative phenotypes. Strategies with magnetically assisted systems include contactless presentation of immune-modulatory molecules, cell internalization of regulatory agents for functional programming via magnetofection, and multiple payload delivery and release.

Overall, Mφ-oriented systems stimulated pro-regenerative functions of Mφ supporting magnetically assisted theranostic nanoplatforms for precision therapies, envisioning safer and more effective control over the distribution of sensitive nanotherapeutics for the treatments of chronical inflammatory conditions.

Acknowledgements: ERC CoG MagTendon No.772817; FCT Doctoral Grant SFRD/BD/144816/2019, and TERM

RES Hub (Norte-01-0145-FEDER-022190).

A major obstacle in biofabrication is replicating the organization of the extracellular matrix and cellular patterns found in anisotropic tissues within bioengineered constructs. While magnetically-assisted 3D bioprinting techniques have the potential to create scaffolds that mimic natural biological structures, they currently lack the ability to accurately control the dispersion of magnetic substances within the bioinks without compromising the fidelity of the intended composite. To overcome this dichotomy, the concepts of magnetically- and matrix-assisted 3D bioprinting are combined here. This method preserves the resolution of printed structures by keeping low viscosity bioinks uncrosslinked during printing, which allows for the arrangement of magnetically-responsive microfibers without compromising the structural integrity of the design. Solidification is induced after the microfibers are arranged in the desired pattern. Furthermore, the precise design of these magnetic microfillers permits the utilization of low levels of inorganic materials and weak magnetic field strengths, which reduces the potential risks that may be associated with their use. The effectiveness of this approach is evaluated in the context of tendon tissue engineering, and the results demonstrate that combining the tendons like anisotropic fibrous microstructure with remote magneto-mechanical stimulation during in vitro maturation provides both biochemical and biophysical cues that effectively guide human adipose-derived stem cells towards a tenogenic phenotype In summary, the developed strategy allows the fabrication of anisotropic high-resolution magnetic composites with remote stimulation functionalities, opening new horizons for tissue engineering applications.

Acknowledgments: ERC Grant CoG MagTendon nr 772817, BioChips PoC project nr 10106930, (PD/BD/129403/2017), (CEECIND/01375/2017), (2020.03410.CEECIND), (2022.05526.PTDC), (ED481B2019/025).

Tendon diseases are prevalent health concerns for which current therapies present limited success, in part due to the intrinsically low regenerative ability of tendons. Therefore, tissue engineering presents a potential to improve this outcome. Here, we hypothesize that a concurrent control over both biophysical and biochemical stimuli will boost the tenogenic commitment of stem cells, thus promoting regeneration. To achieve this, we combine molecularly imprinted nanoparticles (MINPs), which act as artificial amplifiers for endogenous growth factor (GF) activity, with bioinspired anisotropic hydrogels² to manufacture 3D tenogenic constructs. MINPs were solid phase-imprinted using a TGF-β3 epitope as template and their affinity for the target was assessed by SPR and dot blot. Magnetically-responsive microfibers were produced by cryosectioning electrospun meshes containing iron oxide nanoparticles. The constructs were prepared by encapsulating adipose tissue-derived stem cells (ASCs), microfibers, and MINPs within gelatin hydrogels, while aligning the microfibers with an external magnetostatic field during gelation. This allows an effective modulation of hydrogel fibrillar topography, mimicking the native tissue's anisotropic architecture. Cell responses were analyzed by multiplex immunoassay, quantitative polymerase chain reaction, and immunocytochemistry. MINPs showed an affinity for the template comparable to monoclonal antibodies. Encapsulated ASCs acquired an elongated shape and predominant orientation along the alignment direction. Cellular studies revealed that combining MINPs with aligned microfibers increased TGF-β signaling via non-canonical Akt/ERK pathways and upregulated tendon-associated gene expression, contrasting with randomly oriented gels. Immunostaining of tendon-related proteins presented analogous outcomes, corroborating our hypothesis.

Our results thus demonstrate that microstructural cues and biological signals synergistically direct stem cell fate commitment, suggesting that this strategy holds potential for improving tendon healing and might be adaptable for other biological tissues. The proposed concept highlights the GF-sequestering ability of MINPs which allows a cost-effective alternative to recombinant GF supplementation, potentially decreasing the translational costs of tissue engineering strategies.

Acknowledgements: The authors acknowledge the funding from the European Union's Horizon 2020 under grant No. 772817; from FCT/MCTES for scholarships PD/BD/143039/2018 & COVID/BD/153025/2022 (S.P.B.T.), and PD/BD/129403/2017 (S.M.B.), co-financed by POCH and NORTE 2020, under the Portugal 2020 partnership agreement through the European Social Fund, for contract 2020.03410.CEECIND (R.M.A.D.) and project 2022.05526.PTDC; and from Xunta de Galicia for grant ED481B2019/025 (A.P.).

Relevant in vitro models emulating tendinopathies are highly needed to study these diseases and develop better treatments. We have recently proposed a new strategy that allows the automated 3D writing of microphysiological systems (MPS) embedded into its own biomimetic fibrillar support platform based on the self-assembling of cellulose nanocrystals (CNCs). Here, we explored this CNC platform for writing humanized in vitro tendon models using tendon decellularized extracellular matrix (dECM)-based bioinks to closely recapitulate the biophysical and biochemical cues of tendon cell niche and self-induce the tenogenic differentiation of stem cells. The proposed concept was further explored to study the crosstalk between the tendon core and vascular compartment.

Porcine flexor tendons were decellularized to produce the dECM bioink hydrogel. hASCs were used as cell source and the bioink was directly printed within the CNC fluid gel. Tendon constructs were co-printed with compartmentalized microvascular structures to evaluate the cellular crosstalk with endothelial cells. The tendon-on-chip models showed high cell viability and proliferation during culture up to 21 days, and the synergy between dECM cues and printed patterns induced anisotropic cell organization similar to tendon tissues. Gene and protein analysis showed upregulation of the most important tendon related markers on tendon constructs, demonstrating that the biophysical and biochemical cues of dECM induced hASCs commitment toward tenogenic phenotype. In co-culture system, chemotaxis induced endothelial cells migration toward the tendon compartment, but without significant infiltration. Gene and protein expression results suggest that the cellular crosstalk established in this MPS with endothelial cells boosted hASCs tenogenesis, emulating tendon development stages.

Overall, the proposed system might be promising for the automated fabrication of organotypic tendon-on-chip models that will be a valuable new tool to study tendon physiology, pathology, or the effect of drugs for the treatment of tendinopathy.

Acknowledgments: EU H2020 for ERC-2017-CoG-772817; ERC-PoC-BioCHIPs-101069302; FCT/MCTES for 2022.05526.PTDC, 2020.03410.CEECIND, and PD/BD/129403/2017

Tendons display poor intrinsic healing properties and are difficult to treat[1]. Prior in vitro studies[2] have shown that, by targeting the Activin A receptor with magnetic nanoparticles (MNPs), it is possible to remotely induce the tenogenic differentiation of human adipose stem cells (hASCs). In this study, we investigated the tenogenic regenerative potential of remotely-activated MNPs-labelled hASCs in an in vivo rat model. We consider the potential for magnetic controlled nanoparticle mediated tendon repair strategies.

hASCs were labelled with 250 nm MNPs functionalized with anti-Activin Receptor IIA antibody. Using a rapid curing fibrin gel as delivery method, the MNPs-labelled cells were delivered into a Ø2 mm rat patellar tendon defect. The receptor was then remotely stimulated by exposing the rats to a variable magnetic gradient (1.28T), using a customised magnetic box. The stimulation was performed 1 hour/day, 3 days/week up to 8 weeks. Tenogenesis, iron deposition and collagen alignment were assessed by histological staining and IHC. Inflammation mediators levels were assessed by ELISA and IHC. The presence of human cells in tendons after 4 and 8 weeks was assessed by FISH analysis.

Histological staining showed a more organised collagen arrangement in animals treated with MNPs-labelled cells compared to the controls. IHC showed positive expression of tenomodulin and scleraxis in the experimental groups. Immunostaining for CD45 and CD163 did not detect leukocytes locally, which is consistent with the non-significant levels of the inflammatory cytokines analysis performed on plasma. While no iron deposition was detected in the main organs or in plasma, the FISH analysis showed the presence of human donor cells in rat tendons even after 8 weeks from surgery.

Our approach demonstrates in vivo proof of concept for remote control stem cell tendon repair which could ultimately provide injectable solutions for future treatment.

We are grateful for ERC Advanced Grant support ERC No.789119, ERC CoG MagTendon No.772817 and FCT grant 2020.01157.CEECIND.

Tissue engineering and regenerative medicine (TERM) hold the promise to provide therapies for injured tendons despite the challenging cues of tendon niche and the lack of specific factors to guide regeneration. The emerging potential of magnetic responsiveness and magnetic nanoparticles (MNPs) functionalities offers new perspectives to tackle TERM challenges. Moreover, pulsed electromagnetic field (PEMF) is FDA approved for orthopaedics with potential to control inflammation upon injury. We previously demonstrated that magnetic cell-sheets assisted by PEMF trigger the inflammation resolution by modulating cytokine-enriched environments [1]. To further understand the potential of magnetically assisted living patches, we have recently conducted in vivo studies using a rat patellar defect model.

After labeling of human adipose stem cells with iron oxide MNPs for 16h, magCSs were cultured up to 3 days in α-MEM medium under non-magnetic or PEMF conditions. MagCSs were evaluated by immunocytochemistry, and real time RT-PCR for tendon markers. Cell metabolic activity was also assessed by MTS and ECM proteins quantified by Sirius Red/Fast Green.

The MagCSs effect in ameliorating healing was assessed after implantation in window defects created in the patellar tendon of rats. PEMF was externally applied (3mT, 70Hz) 3d/week for 1h (magnetotherapy). After 4 and 8w, tendons were histologically characterized for immune-detection of tendon and inflammatory markers, and for Perls van Gieson and HE stains. Blood and detoxification organs were screened for inflammatory mediators and biodistribution of MNPs, respectively.

In vitro results suggest that PEMF stimulates cellular metabolic activity, influences protein synthesis and the deposition of collagen and non-collagenous proteins is significantly increased compared to non-magnetic conditions. No adverse reactions, as infection or swelling, were observed after surgery or during follow-up. After 8w, magCSs remained at the implantation site and no MNPs were detected on detoxification organs. Plasma levels of IL1α, β, IL6 and TNFα assessed by multiplex assay were below detectable values (<12.5pg/ml).

Thus, the combination of cell sheets and magnetic technologies hold promise for the development of living tendon substitutes.

Acknowledgement to ERC-COG MagTendon772817, H2020 Achilles 810850, FCT - 2020.01157.CEECIND.

To repair soft tissue, it is vital to ensure that the biomaterial is able to mimic the complex elasticity of the native tissue. It has been demonstrated that substrate stiffness has a huge influence on cellular growth, differentiation, motility and phenotype maintenance. The goal of the present study is to characterize extensively a set of polymeric films with variable mechanical profiles. A range of synthetic biodegradable polymers was selected according to the physico-chemical intrinsic properties of aliphatic polymers. They have similar chemistry (absorbable polyesters made from lactic acid, glycolic acid, trimethylene carbonate, dioxanone & β-caprolactone), however show different mechanical and degradation properties. The films were manufactured by thermal presser and then characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), nuclear magnetic resonance spectroscopy (NMR) and Fourier transform infrared spectroscopy (FTIR). The mechanical properties of the films were assessed by uniaxial tensile tests in wet conditions and also by atomic force microscopy (AFM) to assess the material's stiffness at a micro-level. In vitro assays were performed to assess the cell cytocompatibility, proliferation and differentiation potential of the films. The mechanical properties of the materials are within the range intended for musculoskeletal tissue repair. Biological assays showed good cell adhesion, cell proliferation and cell viability. Stem cells were able to differentiate into adipogenic, osteogenic, chondrogenic and tenogenic lineages. Overall the selection of polymers gave good options for a potential tissue repair scaffold. In the future, the combined effect of stiffness and topography will be assessed on cell phenotype maintenance.

Unresolved inflammatory processes in tendon healing have been related to the progression of tendinopathies. Thus, the management of tendon injuries may rely on cell-based strategies to identify and modulate tendon inflammatory cues. Pulsed electromagnetic field (PEMF) has been approved by FDA for orthopedics therapies and has been related to a reduction in pain and to improve healing. However, the influence of PEMF in tendon healing remains largely unknown. Human tendon resident cells (hTDCs) were cultured in an inflammatory environment induced by exogenous supplementation of IL-1β and their response assessed after exposure to different PEMF treatments. This study demonstrates that IL-1β induced up-regulation of pro-inflammatory factors (IL-6 and TNFα) and extracellular matrix components (MMP−1, −2, −3) whereas reduces the expression of TIMP-1, suggesting IL-1β as a candidate inflammation model to study hTDCs response to inflammation cues. Moreover, in both homeostatic and inflammatory environments, hTDCs respond differently to PEMF treatment suggesting that cells are sensitive to magnetic field parameters such as strength (1.5 – 5mT), frequency (5–17Hz) and duration (10–50% duty cycle, dc). Among the conditions studied, PEMF treatment with 4mT/5Hz/50%dc suppresses the inflammatory response of hTDCs to the IL-1β stimulation, as evidenced by the decreases amount of IL-6, TNFα and downregulation of MMP-1, −2, −3 and COX-2, IL-8, IL-6, TNFα genes. These results demonstrate the potential of PEMF, in particular 4mT/5Hz/50%dc PEMF in treating tendon inflammation suppressing the inflammatory stimulation induced by IL-1β, which may be beneficial for tendon healing strategies.

Tendon and ligament injuries represent highly prevalent and unmet clinical challenge that may significantly benefit from tissue engineering therapeutic strategies, once optimal cell source and biomolecules regulating tendon homeostasis are properly defined. Herein, we aimed to evaluate the expression of tendon/ligament markers in two novel cell populations, namely human dental pulp stem cells (DPSCs) and periodontal ligament cells (PDLCs), in response to supplementation with TGF-β ligands relevant for tendon development and healing, as well as under standard tri-lineage differentiation conditions. DPSCs and PDLCs were isolated from sound human permanent molars removed for orthodontic reasons. Pulp tissue and periodontal ligament were minced and digested with collagenase (3mg/mL) and cells were expanded in α-MEM supplemented with 10% fetal bovine serum (basal medium). To evaluate the susceptibility of DPSCs and PDLCs to tenogenic induction, cells were seeded at density of 1000 cells/cm² and cultured up to 21 days in basal medium or media supplemented with TGF-β3 (10ng/ml), or GDF-5 (50 ng/ml). Cell response was evaluated weakly by analysis of expression of tendon, bone and cartilage markers, employing real time RT-PCR and immunocytochemistry. A significant increase in collagen I and collagen III expression was observed with the culture progression in all conditions, with abundant matrix being deposited by day 14. A significant upregulation of scleraxis expression was demonstrated in response to supplementation with TGF-β3 in both cell populations, when compared to basal medium and medium with GDF-5. It was concluded that TGF-β3 may represent an effective inducer of stem cell tenogenic differentiation.

Introduction

We report 10-year clinical outcomes of a prospective randomised controlled study on uni-compartmental knee arthroplasty using an active constraint robot.

Measuring the clinical impact of CAOS systems has generally been based around surrogate radiological measures with currently few long-term functional follow-up studies reported. We present 10 year clinical follow up results of robotic vs conventional surgery in UKA.

The aim of this study was to assess the incidence of low-grade infections in total hip arthroplasty revisions and the clinical outcomes after two-time revision surgery.

Retrospective study of total hip arthroplasty revision surgery between January of 2012 and December of 2013. Inclusion criteria: two-time revision surgery, PCR (pre and post-op) white blood cells count and microbiological culture. The diagnosis of low-grade infection was based on the Academy of Orthopedic Surgeons’ (AAOS) guidelines. All patients were evaluated with the Harris Hip Score (HHS).

Between this period were revised a total of 79 hips, none of them bilateral, of these fourteen full-filled the inclusion criteria, eight women and six men. The mean time between primary arthroplasty and revision surgery was 4.5 years. The interface was metal-on-polyethilene in thirteen and metal-on-metal in one. All patients had at least three episodes at the urgency department with permanent hip pain in the last six months. Ten have done a course of antibiotics previous to surgery. Intra-articular pus was present in four patients. The agent isolated was S. Aureus in five and S. Epidermidis in two. An elevated PCR and leucocytosis was present in all patients. The mean PCR was 18.1 and leucocytes countage 7600. The HHS was good in four patients, fair in seven patients and poor in three. These three patients with poor had another surgical intervention due to recurrent dislocation.

Periprosthetic joint infection is a common complication after total hip arthroplasty (THA). The incidence is approximately 1% after primary replacement and about 4% revision arthroplasty. As stated by Hanssen successful treatment outcomes require precise assessment of the infecting organism, the immune status of the patient, and the condition of the bone and soft tissues around the joint. With this study we tried to establish a protocol in our service: patient with a THA, interface metal-on-polyethilene, persistent coxalgia and elevated PCR will be diagnosed with low-grade infection, however we need further revision of our experience.

Surgical treatment of distal tibia fractures is usually associated with extensive soft tissue compromise and high complication rates (infection, delayed or non-union, ankle stiffness and osteoarthritis). Wound infection is one of the most common complication (deep infection rates up to 15%) and can develop into an infected non-union.

In 1973, Papineau described a staged technique for treating infected non-union of long bones, consisting of (1) surgical debridement of necrotic tissue, temporary splinting, specific antibiotic treatment, postoperative wet-to-dry wound dressing changes; (2) packing of the bone defect with cortico-cancellous autograft; (3) closure of the soft-tissue wound by a flap or secondary intent.

The authors aim to report a clinical case of a successful treatment of a distal tibia infected non-union with the Papineau technique and negative-pressure wound therapy.

Woman, 56 years-old, referred to Orthopaedic consultation on October 2013 for wound dehiscence and infection with a methicillin-resistant Staphylococcus aureus, one month after open reduction and internal fixation of an open distal tibia fracture.

On November 2013 she underwent surgical debridement, removal of osteosynthesis material, osteotaxis with external fixator, negative-pressure wound therapy and antibiotic treatment with intravenous vancomycin 1g 12/12h (1st stage of Papineau procedure)

On December, she underwent autologous iliac crest cancellous bone grafting and wound care, daily irrigated with saline solution (2nd stage).

On February 2014, she underwent a partial thickness skin graft for wound closure (3rd stage).

On April, the external fixator was removed and there was still no evidence of union. She had pain, disuse osteoporosis, ankle and midfoot stiffness, and was sent to physical therapy.

On April 2015, she can full weight bear with mild pain, the soft tissue envelope is in good condition, the fracture has united and she has an Ankle AOFAS Score of 83.

The Papineau technique has been used for the management of infected non-unions with bony defects, with high success rates. Complete necrotic tissue debridement and targeted antibiotics are fundamental for obtaining a viable and healthy tissue, able to receive the bone graft. Negative-pressure wound therapy is important in reducing the bacterial load, improving the microcirculation and enhancing the granulation tissue.

In the present case, the combination of the two techniques probably acted together in achieving successful eradication of the infection, reconstruction of the bone defect and soft tissue closure.

Surgical Site Infection (SSI) is one of the most frequent nosocomial infections and depends on many factors: patient, microorganism, antiseptic solution use, antibiotic prophylaxis, hand scrubbing, wound care or hospital stay lenght.

With the present paper the authors aim to study the SSI incidence after Total Knee (TKA) or Hip Arthroplasty (THA).

All patients who underwent primary TKA or THA between January 2011 and May 2012 at our institution were considered. Patients who died within 1 year after the procedure of unrelated causes were excluded. Data collected included ASA classification, type of procedure, total and post-operative hospital stay, type and duration of antibiotic prophylaxis.

Data were collected from the consultation at 1 month and 1 year post-operative, clinical registries and telephone interview. SSI was defined according to the Centers for Disease Control and Prevention criteria. Suspected cases of SSI included antibiotic administration longer than 5 days or absence of antibiotic prescription, hospital stay after the procedure longer than 9 days, patient referring infection symptoms, and clinical data reports of infection or re-intervention.

During the studied period and after exclusion of 5 cases, a total of 251 surgeries (104 TKA, 147 THA) were performed, of which 2 were urgent.

For both TKA and THA, the average total hospital stay was 9 days (8 days post-operative). The majority were American Society of Anesthesiologists (ASA) classification 2 and 3.

There were 4 SSI (1,60%), 2 TKA (1,92%) and 2 THA (1,36%), all of them after discharge. Their average total hospital stay was 10,5 days.

Antibiotic prophylaxis was used in 93% of the patients (97,4% a cephalosporin), with an average length of 5,7 days.

Recommended hospital stay after a TKA or THA is about 5 days. On HELICS-CIRURGIA 2006–2010 report it was 10 days, similar to ours. In infected patients, our total hospital stay was lower (10,5 vs 26).

Recommended duration of antibiotic prophylaxis is 24h. On HELICS-CIRURGIA more than 50% had it for more than 24h, which also happened with us; our antibiotic coverage was similar.

Comparing to HELICS-CIRURGIA, the predominance of ASA 2 and 3 classifications was similar, but the overall SSI rate was lower (1,6% vs 2,24%).

We conclude we must reduce hospital stay and antibiotic duration and keep the surveillance of SSI after TKA or THA.

Last year at CAOS UK we reported on the development of the Acrobot® Navigation System for accurate computer-assisted hip resurfacing surgery. This paper describes the findings of using the system in the clinical setting and includes the improvements that have been made to expedite the procedure. The aim of our system is to allow accurate planning of the surgery and precise placement of the prosthesis in accordance with the plan, with a zero intra-operative time penalty in comparison to the standard non-navigated technique.

The system uses a pre-operative CT-based plan to allow the surgeon to have full 3D knowledge of the patient’s anatomy and complete control over the sizes and positions of the components prior to surgery.

At present the navigation system is undergoing final clinical evaluation prior to a clinical study designed to demonstrate the accuracy of outcome compared with the conventional technique. Whilst full results are not yet available, this paper describes the techniques that are being used to evaluate accuracy by comparing pre-operative CT-based plans with post-operative CT scans, and gives initial results.

This approach provides a true measure of procedure outcome by measuring what was achieved against what was planned in 3D. The measure includes all the sources of error present within the procedure protocol, therefore these results represent the first time that the outcome of a navigated orthopaedic procedure has been measured accurately.

We used computer tomography (CT) to measure the outcome of knee-arthroplasty in our prospective double-blind randomised controlled study of our active constraint robotic system ACROBOT.

All patients in our trial had pre-operative CT scan and proprietary software used to plan the size, position and orientation of the implants. Post operatively a further CT scan was performed and measurement studies performed using 3 different methods of manipulating the CT dicom data.

Method 1, a quick and simple method of implant assessment that measures the varus-valgus orientation of the implants relative to the axes of the long bones

Two landmarks each are used to define the individual mechanical axis for both the femur and tibia, for consistency these landmarks are the very ones used in the planning stage on the pre-operative CT.

Landmarks are then placed on the implants in order to measure their tilt relative to the mechanical axes. An appropriate Hounsfield threshold (2800) was used to image the metal components. The angle between the individual mechanical axis and the prosthetic component was calculated.

Method 2, detailed and accurate comparisons between the planned and achieved component positions in 3D are made. Co-registration of the precisely planned CT based models with surface models from the post-op scan gives real measurements of implant position enabling the measurement of the accuracy of component in an all six degrees of freedom giving both translation and rotation errors in all three planes.

The process of alignment was achieved by surface-to-surface registration. An implementation of the iterative closest point algorithm was used to register matching surfaces on the objects to be registered. A polygon mesh of the implant, provided by the manufacturer, defined the surface shape of each size of implant. This was used both to define the planned position and to register to the post-operative scan. Method 3, in this study we quantified post-operative error in knee arthroplasty using one value for each component whilst retaining 3D perspective.

The position of the prosthetic components in the post-op scan is calculated and individual transformation matrix computed which is matched to the transformation matrices for the planned components.

The pre-operative CT based component positions were co-registered to the post-operative CT scan and values for the intersection (volumetric) between the digitised images (both planned and achieved) were calculated. Both the co-registered femoral and tibial component’s intersection was quantified with software packages supporting Boolean volume analysis

Method 1, the sum of the two, independently measured, angles allows an estimate of the post-operative alignment of the load bearing axes in the two bones.

Method 2, 3D CT allows precise measurements of the achieved position for each component in all three planes. Six values, three angular and three translational, define the achieved component position relative to the planned position.

Method 3, the greater the percentage intersection between the planned and achieved images, the greater the accuracy of the surgery. Owing to the shape of the components (large articular surface) large intersections demonstrate more accurate reconstruction of the joint line.

In the recent past the lack of a sufficiently accurate tool to plan and measure the accuracy of component placement has resulted in an inability to detect and study radiological and functional outliers and hence the hypnotised relationship between prosthetic joint placement and outcome has been difficult to prove.

CT offers us the ability to accurately describe the actual position and deviation from plan of component placement in knee arthroplasty. Whilst X-ray has the intrinsic problems of perspective distortion magnification errors and orientation uncertainties CT can be used to define ‘true’ planes for two dimensional (2D) measurements and permits the comparison in three dimensions (3D) between the planned and achieved component positions.