Aims. The effect of the gut microbiota (GM) and its metabolite on bone health is termed the gut-bone axis. Multiple studies have elucidated the mechanisms but findings vary greatly. A systematic review was performed to analyze current animal models and explore the effect of GM on bone. Methods. Literature search was performed on PubMed and Embase databases. Information on the types and strains of animals, induction of osteoporosis, intervention strategies, determination of GM, assessment on bone mineral density (BMD) and bone quality, and key findings were extracted. Results. A total of 30 studies were included, of which six studies used rats and 24 studies used mice. Osteoporosis or bone loss was induced in 14 studies. Interventions included ten with probiotics, three with prebiotics, nine with antibiotics, two with short-chain fatty acid (SCFA), six with vitamins and proteins, two with traditional Chinese medicine (TCM), and one with neuropeptide Y1R antagonist. In general, probiotics, prebiotics, nutritional interventions, and TCM were found to reverse the GM dysbiosis and rescue bone loss. Conclusion. Despite the positive therapeutic effect of probiotics, prebiotics, and nutritional or pharmaceutical interventions on osteoporosis, there is still a critical knowledge gap regarding the role of GM in rescuing bone loss and its related pathways. Cite this article: Bone Joint Res 2021;10(1):51–59

Antibiotic resistance represents a threat to human health. It has been suggested that by 2050, antibiotic-resistant infections could cause ten million deaths each year. In orthopaedics, many patients undergoing surgery suffer from complications resulting from implant-associated infection. In these circumstances secondary surgery is usually required and chronic and/or relapsing disease may ensue. The development of effective treatments for antibiotic-resistant infections is needed. Recent evidence shows that bacteriophage (phages; viruses that infect bacteria) therapy may represent a viable and successful solution. In this review, a brief description of bone and joint infection and the nature of bacteriophages is presented, as well as a summary of our current knowledge on the use of bacteriophages in the treatment of bacterial infections. We present contemporary published in vitro and in vivo data as well as data from clinical trials, as they relate to bone and joint infections. We discuss the potential use of bacteriophage therapy in orthopaedic infections. This area of research is beginning to reveal successful results, but mostly in nonorthopaedic fields. We believe that bacteriophage therapy has potential therapeutic value for implant-associated infections in orthopaedics. Cite this article: Bone Joint J 2021;103-B(2):234–244

Bone remodelling is mediated through the synchronism of bone resorption (catabolism) by osteoclasts and bone formation (anabolism) by osteoblasts. Imbalances in the bone remodelling cycle represent an underling cause of metabolic bone diseases such as osteoporosis, where bone resorption exceeds formation (1). Current therapeutic strategies to repair osteoporotic bone fractures focus solely in targeting anabolism or supressing catabolism (2). However, these therapeutics do not reverse the structural damage present at the defect site, ultimately leading to impaired fracture healing, making the repair of osteoporotic fractures particularly challenging in orthopaedics. Herein, we focus on investigating a combined versatile pro-anabolic and anti-catabolic effect of Magnesium (Mg. 2+. ) to modulate bone cell behaviour (3), to develop an engineered biomimetic bio-instructive biomaterial scaffold structurally designed to enhance bone formation while impeding pathological osteoclast resorption activities to facilitate better bone healing and promote repair. Pre-osteoblasts MC3T3-E1 (OBs) and osteoclasts progenitors RAW 264.7 (OCs) cell lines were cultured in growth media exposed to increasing concentrations of MgCl. 2. (0, 0.5, 1, 10, 25 and 50mM) and the optimal concentration to concurrently promote the differentiation of OBs and inhibit the differentiation or funtion of RANKL-induced OCs was assessed. We next used Fluorescence Lifetime Imaging Microscopy to investigate changes in the metabolic pathways during OBs and OCs differentiation when exposed to increasing MgCl. 2. concentrations. We developed a range of magnesium-incorporated collagen scaffolds to permit the spatiotemporal release of Mg. 2+. within the established therapeutic window, and to investigate the behaviour of bone cells in a 3D environment. In our results, we reported an increase in the expression of the bone formation markers osteocalcin and osteopontin for OBs exposed to 10mM MgCl. 2. , and a significant downregulation of the osteoclast-specific markers TRAP and cathepsin K in RANKL-induced OCs differentiation when exposed to 25mM MgCl. 2. Moreover, 25mM MgCl. 2. induced changes in the energy metabolism of OCs from a predominantly oxidative phosphorylation towards a more glycolytic pathway suggesting a regulatory effect of Mg. 2+. in the underlying mechanisms of osteoclasts formation and function. The developed porous collagen-magnesium scaffolds significantly reduced the expression of early osteoclastogenic markers RANK and NFkB, and an elevated expression of the osteogenic markers Runx2 and Col1A1 was reported after 7 days. Our research to date has provided evidences to demonstrate the potential of Mg. 2+. to concurrently enhance osteogenesis while inhibiting osteoclastogenesis in vitro, potentially introducing new targets for developing therapies to repair osteoporotic bone fractures

Aims. Flucloxacillin is commonly administered intravenously for perioperative antimicrobial prophylaxis, while oral administration is typical for prophylaxis following smaller traumatic wounds. We assessed the time, for which the free flucloxacillin concentration was maintained above the minimum inhibitory concentration (fT > MIC) for methicillin-susceptible Staphylococcus aureus in soft and bone tissue, after intravenous and oral administration, using microdialysis in a porcine model. Methods. A total of 16 pigs were randomly allocated to either intravenous (Group IV) or oral (Group PO) flucloxacillin 1 g every six hours during a 24-hour period. Microdialysis was used for sampling in cancellous and cortical bone, subcutaneous tissue, and the knee joint. In addition, plasma was sampled. The flucloxacillin fT > MIC was evaluated using a low MIC target (0.5 μg/ml) and a high MIC target (2.0 μg/ml). Results. Intravenous administration resulted in longer fT > MIC (0.5 μg/ml) compared to oral administration, except for cortical bone. In Group IV, all pigs reached a concentration of 0.5 μg/ml in all compartments. The mean fT > MIC (0.5 μg/ml) was 149 minutes (95% confidence interval (CI) 119 to 179; range 68 to 323) in subcutaneous tissue and 61 minutes (95% CI 29 to 94; range 0 to 121) to 106 minutes (95% CI 76 to 136; range 71 to 154) in bone tissue. In Group PO, 0/8 pigs reached a concentration of 0.5 μg/ml in all compartments. For the high MIC target (2.0 μg/ml), fT > MIC was close to zero minutes in both groups across compartments. Conclusion. Although intravenous administration of flucloxacillin 1 g provided higher fT > MIC for the low MIC target compared to oral administration, concentrations were surprisingly low, particularly for bone tissue. Achievement of sufficient bone and soft tissue flucloxacillin concentrations may require a dose increase or continuous administration. Cite this article: Bone Joint Res 2021;10(1):60–67

Objectives. Meropenem may be an important drug in the treatment of open tibial fractures and chronic osteomyelitis. Therefore, the objective of this study was to describe meropenem pharmacokinetics in plasma, subcutaneous adipose tissue (SCT), and cancellous bone using microdialysis in a porcine model. Methods. Six female pigs were assigned to receive 1000 mg of meropenem intravenously over five minutes. Measurements of meropenem were obtained from plasma, SCT, and cancellous bone for eight hours thereafter. Microdialysis was applied for sampling in solid tissues. The meropenem concentrations were determined using ultra-high-performance liquid chromatography. Results. The penetration of meropenem into cancellous bone, expressed as the ratio of plasma to cancellous bone area under the concentration-curve from zero to the last measured value, was incomplete and delayed. The time with concentration above the minimal inhibitory concentration (T. >MIC. ), for an MIC of 0.5 μg/ml, was shorter for cancellous bone in comparison with both plasma and SCT. For MICs above 0.5 μg/ml, T. >MIC. in cancellous bone was only shorter than SCT. Considering an MIC of 4 μg/ml, no animals achieved the target of 40% T. >MIC. in plasma and cancellous bone, while less than 20% achieved it in SCT. Conclusion. The main finding of this study was short T. >MIC. in cancellous bone after intravenous administration of 1000 mg meropenem. Consequently, in order to achieve sufficient tissue concentration in the cases of open tibial fractures and chronic osteomyelitis, supplemental application of meropenem may be necessary. Cite this article: P. Hanberg, A. Lund, K. Søballe, M. Bue. Single-dose pharmacokinetics of meropenem in porcine cancellous bone determined by microdialysis: An animal study. Bone Joint Res 2019;8:342–348. DOI: 10.1302/2046-3758.87.BJR-2018-0308.R1

Objectives. The ability to determine human bone stiffness is of clinical relevance in many fields, including bone quality assessment and orthopaedic prosthesis design. Stiffness can be measured using compression testing, an experimental technique commonly used to test bone specimens in vitro. This systematic review aims to determine how best to perform compression testing of human bone. Methods. A keyword search of all English language articles up until December 2017 of compression testing of bone was undertaken in Medline, Embase, PubMed, and Scopus databases. Studies using bulk tissue, animal tissue, whole bone, or testing techniques other than compression testing were excluded. Results. A total of 4712 abstracts were retrieved, with 177 papers included in the analysis; 20 studies directly analyzed the compression testing technique to improve the accuracy of testing. Several influencing factors should be considered when testing bone samples in compression. These include the method of data analysis, specimen storage, specimen preparation, testing configuration, and loading protocol. Conclusion. Compression testing is a widely used technique for measuring the stiffness of bone but there is a great deal of inter-study variation in experimental techniques across the literature. Based on best evidence from the literature, suggestions for bone compression testing are made in this review, although further studies are needed to establish standardized bone testing techniques in order to increase the comparability and reliability of bone stiffness studies. Cite this article: S. Zhao, M. Arnold, S. Ma, R. L. Abel, J. P. Cobb, U. Hansen, O. Boughton. Standardizing compression testing for measuring the stiffness of human bone. Bone Joint Res 2018;7:524–538. DOI: 10.1302/2046-3758.78.BJR-2018-0025.R1

Aims. Tuberculosis (TB) infection of bones and joints accounts for 6.7% of TB cases in England, and is associated with significant morbidity and disability. Public Health England reports that patients with TB experience delays in diagnosis and treatment. Our aims were to determine the demographics, presentation and investigation of patients with a TB infection of bones and joints, to help doctors assessing potential cases and to identify avoidable delays. Patients and Methods. This was a retrospective observational study of all adults with positive TB cultures on specimens taken at a tertiary orthopaedic centre between June 2012 and May 2014. A laboratory information system search identified the patients. The demographics, clinical presentation, radiology, histopathology and key clinical dates were obtained from medical records. Results. A total of 31 adult patients were identified. Their median age was 37 years (interquartile range (IQR): 29 to 53); 21 (68%) were male; 89% were migrants. The main sites affected were joints (10, 32%), the spine (8, 26%) and long bones (6, 19%); 8 (26%) had multifocal disease. The most common presenting symptoms were pain (29/31, 94%) and swelling (26/28, 93%). ‘Typical’ symptoms of TB, such as fever, sweats and weight loss, were uncommon. Patients waited a median of seven months (IQR 3 to 13.5) between the onset of symptoms and referral to the tertiary centre and 2.3 months (IQR 1.6 to 3.4.)) between referral and starting treatment. Radiology suggested TB in 26 (84%), but in seven patients (23%) the initial biopsy specimens were not sent for mycobacterial culture, necessitating a second biopsy. Rapid Polymerase Chain Reaction-based testing for TB using Xpert MTB/RIF was performed in five patients; 4 (80%) tested positive for TB. These patients had a reduced time between the diagnostic biopsy and starting treatment than those whose samples were not tested (median eight days versus 36 days, p = 0.016). Conclusion. Patients with bone and joint TB experience delays in diagnosis and treatment, some of which are avoidable. Maintaining a high index of clinical suspicion and sending specimens for mycobacterial culture are crucial to avoid missing cases. Rapid diagnostic tests reduce delays and should be performed on patients with radiological features of TB. Cite this article: Bone Joint J 2018;100-B:119–24

Despite its intrinsic ability to regenerate form and function after injury, bone tissue can be challenged by a multitude of pathological conditions. While innovative approaches have helped to unravel the cascades of bone healing, this knowledge has so far not improved the clinical outcomes of bone defect treatment. Recent findings have allowed us to gain in-depth knowledge about the physiological conditions and biological principles of bone regeneration. Now it is time to transfer the lessons learned from bone healing to the challenging scenarios in defects and employ innovative technologies to enable biomaterial-based strategies for bone defect healing. This review aims to provide an overview on endogenous cascades of bone material formation and how these are transferred to new perspectives in biomaterial-driven approaches in bone regeneration. Cite this article: T. Winkler, F. A. Sass, G. N. Duda, K. Schmidt-Bleek. A review of biomaterials in bone defect healing, remaining shortcomings and future opportunities for bone tissue engineering: The unsolved challenge. Bone Joint Res 2018;7:232–243. DOI: 10.1302/2046-3758.73.BJR-2017-0270.R1

Objectives. This systematic review aimed to assess the in vivo and clinical effect of strontium (Sr)-enriched biomaterials in bone formation and/or remodelling. Methods. A systematic search was performed in Pubmed, followed by a two-step selection process. We included in vivo original studies on Sr-containing biomaterials used for bone support or regeneration, comparing at least two groups that only differ in Sr addition in the experimental group. Results. A total of 572 references were retrieved and 27 were included. Animal models were used in 26 articles, and one article described a human study. Osteoporotic models were included in 11 papers. All articles showed similar or increased effect of Sr in bone formation and/or regeneration, in both healthy and osteoporotic models. No study found a decreased effect. Adverse effects were assessed in 17 articles, 13 on local and four on systemic adverse effects. From these, only one reported a systemic impact from Sr addition. Data on gene and/or protein expression were available from seven studies. Conclusions. This review showed the safety and effectiveness of Sr-enriched biomaterials for stimulating bone formation and remodelling in animal models. The effect seems to increase over time and is impacted by the concentration used. However, included studies present a wide range of study methods. Future work should focus on consistent models and guidelines when developing a future clinical application of this element. Cite this article: N. Neves, D. Linhares, G. Costa, C. C. Ribeiro, M. A. Barbosa. In vivo and clinical application of strontium-enriched biomaterials for bone regeneration: A systematic review. Bone Joint Res 2017;6:366–375. DOI: 10.1302/2046-3758.66.BJR-2016-0311.R1

Objectives. The most concerning infection of allografts and operative procedures is methicillin resistant Staphylococcus aureus (MRSA) and no current iontophoresed antibiotics effectively combat this microbe. It was initially hypothesised that iontophoresis of vancomycin through bone would not be effective due to its large molecular size and lack of charge. The aim of this study was to determine whether this was a viable procedure and to find the optimum conditions for its use. . Methods. An iontophoresis cell was set up with varying concentrations of Vancomycin within the medulla of a section of sheep tibia, sealed from an external saline solution. The cell was run for varying times, Vancomycin concentrations and voltages, to gain information on optimisation of conditions for impregnating the graft. Each graft was then sectioned and dust ground from the exposed surface. The dust was serially washed to extract the Vancomycin and concentrations measured and plotted for all variables tested. Results. Vancomycin was successfully delivered and impregnated to the graft using the iontophoresis technique. The first order fit to the whole data set gave a significant result (p = 0.0233), with a significant concentration (p = 0.02774) component. The time component was the next most significant (p = 0.0597), but did not exceed the 95% confidence level. Conclusions. Iontophoresis is an effective method for delivering Vancomycin to allograft bone. The concentrations of the vancomycin solution affected the bone concentration, but results were highly variable. Further study should be done on the effectiveness of delivering different antibiotics using this method. Cite this article: Bone Joint Res 2014;3:101–7

The treatment of chronic osteomyelitis often includes surgical debridement and filling the resultant void with antibiotic-loaded polymethylmethacrylate cement, bone grafts or bone substitutes. Recently, the use of bioactive glass to treat bone defects in infections has been reported in a limited series of patients. However, no direct comparison between this biomaterial and antibiotic-loaded bone substitute has been performed. . In this retrospective study, we compared the safety and efficacy of surgical debridement and local application of the bioactive glass S53P4 in a series of 27 patients affected by chronic osteomyelitis of the long bones (Group A) with two other series, treated respectively with an antibiotic-loaded hydroxyapatite and calcium sulphate compound (Group B; n = 27) or a mixture of tricalcium phosphate and an antibiotic-loaded demineralised bone matrix (Group C; n = 22). Systemic antibiotics were also used in all groups. After comparable periods of follow-up, the control of infection was similar in the three groups. In particular, 25 out of 27 (92.6%) patients of Group A, 24 out of 27 (88.9%) in Group B and 19 out of 22 (86.3%) in Group C showed no infection recurrence at means of 21.8 (12 to 36), 22.1 (12 to 36) and 21.5 (12 to 36) months follow-up, respectively, while Group A showed a reduced wound complication rate. Our results show that patients treated with a bioactive glass without local antibiotics achieved similar eradication of infection and less drainage than those treated with two different antibiotic-loaded calcium-based bone substitutes. Cite this article: Bone Joint J 2014; 96-B:845–50

Bone sarcomas are rare cancers and orthopaedic surgeons come across them infrequently, sometimes unexpectedly during surgical procedures. We investigated the outcomes of patients who underwent a surgical procedure where sarcomas were found unexpectedly and were subsequently referred to our unit for treatment. We identified 95 patients (44 intra-lesional excisions, 35 fracture fixations, 16 joint replacements) with mean age of 48 years (11 to 83); 60% were males (n = 57). Local recurrence arose in 40% who underwent limb salvage surgery versus 12% who had an amputation. Despite achieving local control, overall survival was worse for patients treated with amputation rather than limb salvage (54% vs 75% five-year survival). Factors that negatively influenced survival were invasive primary surgery (fracture fixation, joint replacement), a delay of greater than two months until referral to our oncology service, and high-grade tumours. Survival in these circumstances depends mostly on factors that are determined prior to definitive treatment by a tertiary orthopaedic oncology unit. Limb salvage in this group of patients is associated with a higher rate of inadequate marginal surgery and, consequently, higher local recurrence rates than amputation, but should still be attempted whenever possible, as local control is not the primary determinant of survival. . Cite this article: Bone Joint J 2014;96-B:665–72

Objectives. This study aims to assess the correlation of CT-based structural rigidity analysis with mechanically determined axial rigidity in normal and metabolically diseased rat bone. Methods. A total of 30 rats were divided equally into normal, ovariectomized, and partially nephrectomized groups. Cortical and trabecular bone segments from each animal underwent micro-CT to assess their average and minimum axial rigidities using structural rigidity analysis. Following imaging, all specimens were subjected to uniaxial compression and assessment of mechanically-derived axial rigidity. Results. The average structural rigidity-based axial rigidity was well correlated with the average mechanically-derived axial rigidity results (R. 2. = 0.74). This correlation improved significantly (p < 0.0001) when the CT-based Structural Rigidity Analysis (CTRA) minimum axial rigidity was correlated to the mechanically-derived minimum axial rigidity results (R. 2. = 0.84). Tests of slopes in the mixed model regression analysis indicated a significantly steeper slope for the average axial rigidity compared with the minimum axial rigidity (p = 0.028) and a significant difference in the intercepts (p = 0.022). The CTRA average and minimum axial rigidities were correlated with the mechanically-derived average and minimum axial rigidities using paired t-test analysis (p = 0.37 and p = 0.18, respectively). Conclusions. In summary, the results of this study suggest that structural rigidity analysis of micro-CT data can be used to accurately and quantitatively measure the axial rigidity of bones with metabolic pathologies in an experimental rat model. It appears that minimum axial rigidity is a better model for measuring bone rigidity than average axial rigidity

Aims. Osteoarthritis (OA) is the most prevalent systemic musculoskeletal disorder, characterized by articular cartilage degeneration and subchondral bone (SCB) sclerosis. Here, we sought to examine the contribution of accelerated growth to OA development using a murine model of excessive longitudinal growth. Suppressor of cytokine signalling 2 (SOCS2) is a negative regulator of growth hormone (GH) signalling, thus mice deficient in SOCS2 (Socs2. -/-. ) display accelerated bone growth. Methods. We examined vulnerability of Socs2. -/-. mice to OA following surgical induction of disease (destabilization of the medial meniscus (DMM)), and with ageing, by histology and micro-CT. Results. We observed a significant increase in mean number (wild-type (WT) DMM: 532 (SD 56); WT sham: 495 (SD 45); knockout (KO) DMM: 169 (SD 49); KO sham: 187 (SD 56); p < 0.001) and density (WT DMM: 2.2 (SD 0.9); WT sham: 1.2 (SD 0.5); KO DMM: 13.0 (SD 0.5); KO sham: 14.4 (SD 0.7)) of growth plate bridges in Socs2. -/-. in comparison with WT. Histological examination of WT and Socs2. -/-. knees revealed articular cartilage damage with DMM in comparison to sham. Articular cartilage lesion severity scores (mean and maximum) were similar in WT and Socs2. -/-. mice with either DMM, or with ageing. Micro-CT analysis revealed significant decreases in SCB thickness, epiphyseal trabecular number, and thickness in the medial compartment of Socs2. -/-. , in comparison with WT (p < 0.001). DMM had no effect on the SCB thickness in comparison with sham in either genotype. Conclusion. Together, these data suggest that enhanced GH signalling through SOCS2 deletion accelerates growth plate fusion, however this has no effect on OA vulnerability in this model. Cite this article: Bone Joint Res 2022;11(3):162–170

Aims. The aim of this study is to develop a core set of outcome domains that should be considered and reported in all future trials of childhood limb fractures. Methods. A four-phase study was conducted to agree a set of core outcome domains. Identification of candidate outcome domains were identified through systematic review of trials, and outcome domains relevant to families were identified through semi-structured interviews with 20 families (parent-child pairing or group). Outcome domains were prioritized using an international three-round Delphi survey with 205 panellists and then condensed into a core outcome set through a consensus workshop with 30 stakeholders. Results. The systematic review and interviews identified 85 outcome domains as relevant to professionals or families. The Delphi survey prioritized 30 upper and 29 lower limb outcome domains at first round, an additional 17 upper and 18 lower limb outcomes at second round, and four additional outcomes for upper and lower limb at the third round as important domains. At the consensus workshop, the core outcome domains were agreed as: 1) pain and discomfort; 2) return to physical and recreational activities; 3) emotional and psychosocial wellbeing; 4) complications from the injury and treatment; 5) rturn to baseline activities daily living; 6) participation in learning; 7) appearance and deformity; and 8) time to union. In addition, 9a) recovery of mobility and 9b) recovery of manual dexterity was recommended as a core outcome for lower and upper limb fractures, respectively. Conclusion. This set of core outcome domains is recommended as a minimum set of outcomes to be reported in all trials. It is not an exhaustive set and further work is required to identify what outcome tools should be used to measure each of these outcomes. Adoption of this outcome set will improve the consistency of research for these children that can be combined for more meaningful meta-analyses and policy development. Cite this article: Bone Joint J 2021;103-B(12):1821–1830

Introduction. The non-union of long bones poses a substantial challenge to clinicians and patients alike. The Ilizarov fixation system and Limb Reconstruction System (LRS), renowned for their versatility in managing complex non-unions. The purpose of this retrospective study was to assess the outcomes of acute docking with the bone peg-in-bone technique for the management of non-unions of long bones. The study seeks to evaluate its effectiveness in achieving complete bony union, preserving limb length and alignment, correcting existing deformities, and preventing the onset of new ones. Method. A retrospective analysis of 42 patients was done with infected and non-infected non-unions of long bones who received treatment at a tertiary care hospital between April 2016 to April 2022. We utilized the Association for the Study and Application of Methods of the Ilizarov (ASAMI) scoring system to assess both bone and functional outcomes and measured mechanical lateral distal femoral angle (mLDFA) for the femur and the medial proximal tibial angle (MPTA) for the tibia. Result. In our retrospective study involving 42 patients, a total of 30 patients had post debridement gap of >2 cm and average gap of 4.54 cm (range 1 – 13 cm) and therefore underwent corticotomy and lengthening. The average external fixation time was 6.52 (range 4 – 11 months) and average external fixation index of 2.08 (range 0.4 – 4.5 months/cm). The ASAMI scoring system showed bone result of 38 excellent, 3 good and 1 fair. Functional result of 40 excellent and 2 good outcomes. The post op mLDFA and MPTA were in normal range except in 3 patients which not statistically significant. Conclusion. In conclusion, the use of acute docking provides several advantages such as promoting early fracture healing, increasing stability, shortening treatment time, reducing the number of surgical procedures and reduced number of complications

This study aimed to characterise the microarchitecture of bone in different species of animal leading to the development of a physiologically relevant 3D printed cellular model of trabecular (Tb) and cortical bone (CB). Using high resolution micro-computed tomography (μ-CT) bone samples from multiple species were scanned and analysed before creating in silico models for 3D printing. Biologically relevant printing materials with physical characteristics similar to that of in vivo bone will be selected and tested for printability. Porcine and murine bone samples were scanned using μ-CT, with a resolution of 4.60 μM for murine and 11 μM for porcine and reconstructed to determine the architectural properties of both Tb and CB independently. A region of interest, 1 mm in height, will be used to generate an in-silico 3D model with dimensions (10 mm. 3. ) and suitable resolution before being translated into printable G code using CAD assisted software. A 1 mm section of each bone was analysed, to determine the differences in the microarchitecture with the intent of setting a benchmark for the developmental 3D in vitro model to be comparable against. In contrast, porcine caudal vertebrae (PCV) have an increased volume due to the size of the bone sample. Interestingly, BV/TR for Tb is similar between species in all samples except murine femur. Murine tibia and PCV have a similar Tb. number and thickness, however different SMI shape and separation. μ-CT scanning and analysis permits tessellation of the 3D output which will lead to the generation of an in silico printable model. Biomaterials are currently under optimisation to allow printability and shape integrity to reflect the morphological and physiological properties of bone

Millions of patients each year suffer from challenging non-healing bone defects secondary to trauma or disease (e.g. cancer, osteoporosis or osteomyelitis). Tissue engineering approach to non-healing bone defects has been investigated over the past few decades in a search for a novel solution for critical size bone defects. The success of the tissue engineering approach relies on three main pillars, the right type of cells; and appropriate scaffold; and a biologically relevant biochemical/ biophysical stimuli. When it comes to cells the mesodermal origin of mesenchymal stem cells and its well demonstrated multipotentiality makes it an ideal option to be used in musculoskeletal regeneration. For the presented set of experimental assays, fully characterised (passage 3 to 5)ovine adipose-derived mesenchymal stems cells (Ad-MSC) were cultured either in growth medium (GM) consisting of Dulbecco's Modification of Eagle's Medium (DMEM) supplemented with 10% (v/v) foetal bovine serum and 1% penicillin-streptomycin as a control or in osteogenic differentiation medium (DM), consisting of GM further supplemented with L- ascorbic acid (50 μg/ml), β-glycerophosphate (10 mM) and dexamethasone (100nM). Osteogenic differentiation was assessed biochemically by quantifying alkaline phosphatase (ALP) enzyme activity and alizarin red staining after 3, 7, 14 and 21 days in culture (where 1×105 cells/well were seeded in 24 well-plate, n=6/media type/ time point). Temporal patterns in osteogenic gene expression were quantified using real-time PCR for Runx-2, osteocalcin (OC), osteonectin (ON) and type 1 collagen (Col 1) at days 7, 15 and 21 (where 1×105 cells were seeded in T25 cell culture flasks for RNA extraction, n= 4 / gene/ media type/time point). The morphology of osteogenic cells was additionally evaluated by scanning electron microscopy (SEM) of cells seeded at low-density (1×102 cells) on glass coverslips for 2 weeks in GM or DM. The level of ALP activity of cells grown in osteogenic DM was significantly higher than the control growing in the standard growth medium (p ≤ 0.05) at days 3, 7 and 14. At 21 days there was a sharp drop in ALP values in the differentiating cells. Mineralisation, as evidenced by alizarin red staining, increased significantly by day 14 and then peaked at day 21. Quantitative real-time PCR confirmed early increases in Runx-2, Col 1 and osteonectin, peaking in the second week of culture, while osteocalcin peaked at 21 days of culture. Taken as a whole, these data indicate that ovine-MSCs exhibit a tightly defined pathway of initial proliferation and matrix maturation (up to 14 days), followed by terminal differentiation and mineralisation (days 14 to 21). SEM analysis confirmed the flattened, roughened appearance of these cells and abandoned extracellular matrix which resembled mature osteoblasts. Given the ready availability of adipose tissues, the use of Ad-MSCs as progenitors for bone tissue engineering applications is both feasible and reasonable. The data from this study indicate that Ad-MSCs follow a predictable pathway of differentiation that can be tracked using validated molecular and biochemical assays. Additional work is needed to confirm that these cells are osteogenic in vivo, and to identifying the best combination of scaffold materials and cell culture techniques (e.g. static versus dynamic) to accelerate or stimulate osteogenic differentiation for bone tissue engineering applications

Fracture nonunion is a severe clinical problem for the patient, as well as for the clinician. About 5-20% of fractures does not heal properly after more than six months, with a 19% nonunion rate for tibia, 12% for femur and 13% for humerus, leading to patient morbidity, prolonged hospitalization, and high costs. The standard treatment with iliac crest-derived autologous bone filling the nonunion site may cause pain or hematoma to the patient, as well as major complications such as infection. The application of mesenchymal autologous cells (MSC) to improve bone formation calls for randomized, open, two-arm clinical studies to verify safety and efficacy. The ORTHOUNION * project (ORTHOpedic randomized clinical trial with expanded bone marrow MSC and bioceramics versus autograft in long bone nonUNIONs) is a multicentric, open, randomized, comparative phase II clinical trial, approved in the framework of the H2020 funding programme, under the coordination of Enrique Gòmez Barrena of the Hospital La Paz (Madrid, Spain). Starting from January 2017, patients with nonunion of femur, tibia or humerus have been actively enrolled in Spain, France, Germany, and Italy. The study protocol encompasses two experimental arms, i.e., autologous bone marrow-derived mesenchymal cells after expansion (‘high dose’ or ‘low dose’ MSC) combined to ceramic granules (MBCP™, Biomatlante), and iliac crest-derived autologous trabecular bone (ICAG) as active comparator arm, with a 2-year follow-up after surgery. Despite the COVID 19 pandemic with several lockdown periods in the four countries, the trial was continued, leading to 42 patients treated out of 51 included, with 11 receiving the bone graft (G1 arm), 15 the ‘high dose’ MSC (200x10. 6. , G2a arm) and 16 the ‘low dose’ MSC (100x10. 6. , G2b arm). The Rizzoli Orthopaedic Institute has functioned as coordinator of the Italian clinical centres (Bologna, Milano, Brescia) and the Biomedical Science and Technologies and Nanobiotechnology Lab of the RIT Dept. has enrolled six patients with the collaboration of the Rizzoli’ 3rd Orthopaedic and Traumatological Clinic prevalently Oncologic. Moreover, the IOR Lab has collected and analysed the blood samples from all the patients treated to monitor the changes of the bone turnover markers following the surgical treatment with G1, G2a or G2b protocols. The clinical and biochemical results of the study, still under evaluation, are presented. * ORTHOUNION Horizon 2020 GA 733288

Regeneration of bone defects in elderly patients is limited due to the decreased function of bone forming cells and compromised tissue physiology. Previous studies suggested that the regenerative activity of stem cells from aged tissues can be enhanced by exposure to young systemic and tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells (hiPSCs) can enhance the bone regeneration potential of aged human bone marrow stromal cells (hBMSCs). ECM was engineered from hiPSC-derived mesenchymal-like progenitors (hiPSC-MPs), as well as young (<30 years) and aged (>70 years) hBMSCs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. Three hBMSCs of different ages were cultured on engineered ECMs. Growth and differentiation responses were compared to tissue culture plastic, as well as to collagen and fibronectin coated plates. Decellularized ECMs contained collagens type I and IV, fibronectin, laminin and < 5% residual DNA, suggesting efficient cell elimination. Cultivation of young and aged hBMSCs on the hiPSC-ECM in osteogenic medium significantly increased hBMSC growth and markers of osteogenesis, including collagen deposition, alkaline phosphatase activity, bone sialoprotein expression and matrix mineralization compared to plastic controls and single protein substrates. In aged BMSCs, matrix mineralization was only detected in ECM cultures in osteogenic medium. Comparison of ECMs engineered from hiPSC-MPs and hBMSCs of different ages suggested similar structure, composition and potential to enhance osteogenic responses in aged BMSCs. Engineered ECM induced a higher osteogenic response compared to specific matrix components. Our studies suggest that aged BMSCs osteogenic activity can be enhanced by culture on engineered ECM. hiPSCs represent a scalable cell source, and tissue engineering strategies employing engineered ECM materials could potentially enhance bone regeneration in elderly patients