The June 2024 Foot & Ankle Roundup³⁶⁰ looks at: First MTPJ fusion in young versus old patients; Minimally invasive calcaneum Zadek osteotomy and the effect of sequential burr passes; Comparison between Achilles tendon reinsertion and dorsal closing wedge calcaneal osteotomy for the treatment of insertional Achilles tendinopathy; Revision ankle arthroplasty – is it worthwhile?; Tibiotalocalcaneal arthrodesis or below-knee amputation – salvage or sacrifice?; Fusion or replacement for hallux rigidus?.

The reliable production of _in vitro_ chondrocytes that faithfully recapitulate _in vivo_ development would be of great benefit for orthopaedic disease modelling and regenerative therapy(1,2). Current efforts are limited by off-target differentiation, resulting in a heterogeneous product, and by the lack of comparison to human tissue, which precludes detailed evaluation of _in vitro_ cells(3,4). We performed single-cell RNA-sequencing of long bones dissected from first-trimester fetal limbs to form a detailed ‘atlas’ of endochondral ossification. Through 100-gene in-situ sequencing, we placed each sequenced cell type into its anatomical context to spatially resolve the process of endochondral ossification. We then used this atlas to perform deconvolution on a series of previously published bulk transcriptomes generated from _in vitro_ chondrogenesis protocols to evaluate their ability to accurately produce chondrocytes. We then applied single-nuclear RNA-sequencing to cells from the best performing protocol collected at multiple time points to allow direct comparison between the differentiation of _in vitro_ and _in vivo_ cells. We captured 275,000 single fetal cells, profiling the development of chondrocytes from multipotent mesenchymal progenitors to hypertrophic cells at full transcriptomic breadth. Using this atlas as the ground truth for evaluating _in vitro_ cells, we found substantial variability in cell states produced by each protocol, with many showing little similarity to _in vivo_ cells, and all exhibiting off-target differentiation. Trajectory alignment between _in vivo_ and _in vitro_ single-cell data revealed key differences in gene expression dynamics between _in vitro_ and _in vivo cells,_ with several osteoblastic transcription factors erroneously unregulated _in vitro,_ including _FOXO1._. Using this information, we inhibited _FOXO1_ in culture to successfully increase chondrocyte yield _in vitro._. This study presents a new framework for evaluating tissue engineering protocols, using single-cell data to drive improvement and bring the prospect of true engineered cartilage closer to reality

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Pigment epithelium-derived factor (PEDF) is known to induce several types of tissue regeneration by activating tissue-specific stem cells. Here, we investigated the therapeutic potential of PEDF 29-mer peptide in the damaged articular cartilage (AC) in rat osteoarthritis (OA).

In osteoarthritis, chondrocytes acquire a hypertrophic phenotype that contributes to matrix degradation. Inflammation is proposed as trigger for the shift to a hypertrophic phenotype. Using in vitro culture of human chondrocytes and cartilage explants we could not find evidence for a role of inflammatory signalling activation. We found, however, that tissue repair macrophages may contribute to the onset of hypertrophy (doi: 10.1177/19476035211021907) Intra-articularly injected triamcinolone acetonide to inhibit inflammation in a murine model of collagenase-induced osteoarthritis, increased synovial macrophage numbers and osteophytosis, confirming the role of macrophages in chondrocyte hypertrophy occurring in osteophyte formation (doi: 10.1111/bph.15780). In search of targets to inhibit chondrocyte hypertrophy, we combined existing microarray data of different cartilage layers of murine growth plate and murine articular cartilage after induction of collagenase-induced osteoarthritis. We identified common differentially expressed genes and selected those known to be associated to inflammation. This revealed EPHA2, a tyrosine kinase receptor, as a new target. Using in silico, in vitro and in vivo models we demonstrated that inhibition of EPHA2 might be a promising treatment for osteoarthritis. Recently, single cell RNA-seq. has revealed detailed information about different populations of chondrocytes in articular cartilage during osteoarthritis. We re-analysed a published scRNA-seq data set of healthy and osteoarthritic cartilage to obtain the differentially expressed genes in the population of hypertrophic chondrocytes compared to the other chondrocytes, applied pathway analyses and then used drug databases to search for upstream inhibitors of these pathways. This drug repurposing approach led to the selection of 6 drugs that were screened and tested using several in vitro models with human chondrocytes and cartilage explants. In this lecture I will present this sequence of studies to highlight different approaches and models that can be used in the quest for a disease modifying drug for osteoarthritis

Bone healing outcome is highly dependent on the initial mechanical fracture environment [1]. In vivo, direct bone healing requires absolute stability and an interfragmentary strain (IFS) below 2% [2]. In the majority of cases, however, endochondral ossification is engaged where frequency and amplitude of IFS are key factors. Still, at the cellular level, the influence of those parameters remains unknown. Understanding the regulation of naïve hMSC differentiation is essential for developing effective bone healing strategies. Human bone-marrow-derived MSC (KEK-ZH-NR: 2010–0444/0) were embedded in 8% gelatin methacryol. Samples (5mm Ø x 4mm) were subjected to 0, 10 and 30% compressive strain (5sec compression, 2hrs pause sequence for 14 days) using a multi-well uniaxial bioreactor (RISystem) and in presence of chondro-permissive medium (CP, DMEM HG, 1% NEAA, 10 µM ITS, 50 µg/mL ascorbic acid, and 100 mM Dex). Cell differentiation was assessed by qRT-PCR and histo-/immunohistology staining. Experiments were repeated 5 times with cells from 5 donors in duplicate. ANOVA with Tukey post-hoc correction or Kurskal-Wallis test with Dunn's correction was used. Data showed a strong upregulation of hypertrophic related genes COMP, MMP13 and Type 10 collagen upon stimulation when compared to chondrogenic SOX9, ACAN, Type 2 collagen or to osteoblastic related genes Type 1 Collagen, Runx2. When compared to chondrogenic control medium, cells in CP with or without stimulation showed low proteoglycan synthesis as shown by Safranine-O-green staining. In addition, the cells were significantly larger in 10% and 30% strain compared to control medium with 0% strain. Type 1 and 10 collagens immunostaining showed stronger Coll 10 expression in the samples subjected to strain compared to control. Uniaxial deformation seems to mainly promote hypertrophic-like chondrocyte differentiation of MSC. Osteogenic or potentially late hypertrophic related genes are also induced by strain. Acknowledgments: Funded by the AO Foundation, StrainBot sponsored by RISystemAG & PERRENS 101 GmbH

The signaling molecule prostaglandin E2 (PGE2), synthesized by cyclooxygenase-2 (COX-2), is immunoregulatory and reported to be essential for skeletal stem cell function. Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in osteoarthritis (OA) analgesia, but cohort studies suggested that long-term use may accelerate pathology. Interestingly, OA chondrocytes secrete high amounts of PGE2. Mesenchymal stromal cell (MSC) chondrogenesis is an in vitro OA model that phenocopies PGE2 secretion along with a hypertrophic OA-like cell morphology. Our aim was to investigate cause and effects of PGE2 secretion in MSC-based cartilage neogenesis and hypertrophy and identify molecular mechanisms responsible for adverse effects in OA analgesia. Human bone marrow-derived MSCs were cultured in chondrogenic medium with TGFβ (10ng/mL) and treated with PGE2 (1µM), celecoxib (COX-2 inhibitor; 0.5µM), AH23848/AH6809 (PGE2 receptor antagonists; 10µM), or DMSO as a control (n=3–4). Assessment criteria were proteoglycan deposition (histology), chondrocyte/hypertrophy marker expression (qPCR), and ALP activity. PGE2 secretion was measured (ELISA) after TGFβ withdrawal (from day 21, n=2) or WNT inhibition (2µM IWP-2 from day 14; n=3). Strong decrease in PGE2 secretion upon TGFβ deprivation or WNT inhibition identified both pathways as PGE2 drivers. Homogeneous proteoglycan deposition and COL2A1 expression analysis showed that MSC chondrogenesis was not compromised by any treatment. Importantly, hypertrophy markers (COL10A1, ALPL, SPP1, IBSP) were significantly reduced by PGE2 treatment, but increased by all inhibitors. Additionally, PGE2 significantly decreased ALP activity (2.9-fold), whereas the inhibitors caused a significant increase (1.3-fold, 1.7-fold, 1.8-fold). This identified PGE2 as an important inhibitor of chondrocyte hypertrophy. Although TGFβ and WNT are known pro-arthritic signaling pathways, they appear to induce a PGE2-mediated antihypertrophic effect that can counteract pathological cell changes in chondrocytes. Hampering this rescue mechanism via COX inhibition using NSAIDs thus risks acceleration of OA progression, indicating the need of OA analgesia adjustment

Displaced fractures of the distal radius in children are usually reduced under sedation or general anaesthesia to restore anatomical alignment before the limb is immobilized. However, there is growing evidence of the ability of the distal radius to remodel rapidly, raising doubts over the benefit to these children of restoring alignment. There is now clinical equipoise concerning whether or not young children with displaced distal radial fractures benefit from reduction, as they have the greatest ability to remodel. The Children’s Radius Acute Fracture Fixation Trial (CRAFFT), funded by the National Institute for Health and Care Research, aims to definitively answer this question and determine how best to manage severely displaced distal radial fractures in children aged up to ten years.

Cite this article: Bone Joint J 2024;106-B(1):16–18.

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The aim of this study was to reassess the rate of neurological, psoas-related, and abdominal complications associated with L4-L5 lateral lumbar interbody fusion (LLIF) undertaken using a standardized preoperative assessment and surgical technique.

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Therapeutic agents that prevent chondrocyte loss, extracellular matrix (ECM) degradation, and osteoarthritis (OA) progression are required. The expression level of epidermal growth factor (EGF)-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) in damaged human cartilage is significantly higher than in undamaged cartilage. However, the effect of EDIL3 on cartilage is still unknown.

Osteoarthritis (OA) is mainly caused by ageing, strain, trauma, and congenital joint abnormalities, resulting in articular cartilage degeneration. During the pathogenesis of OA, the changes in subchondral bone (SB) are not only secondary manifestations of OA, but also an active part of the disease, and are closely associated with the severity of OA. In different stages of OA, there were microstructural changes in SB. Osteocytes, osteoblasts, and osteoclasts in SB are important in the pathogenesis of OA. The signal transduction mechanism in SB is necessary to maintain the balance of a stable phenotype, extracellular matrix (ECM) synthesis, and bone remodelling between articular cartilage and SB. An imbalance in signal transduction can lead to reduced cartilage quality and SB thickening, which leads to the progression of OA. By understanding changes in SB in OA, researchers are exploring drugs that can regulate these changes, which will help to provide new ideas for the treatment of OA.

Cite this article: Bone Joint Res 2023;12(9):536–545.

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Implantation of ultra-purified alginate (UPAL) gel is safe and effective in animal osteochondral defect models. This study aimed to examine the applicability of UPAL gel implantation to acellular therapy in humans with cartilage injury.

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To explore the novel molecular mechanisms of histone deacetylase 4 (HDAC4) in chondrocytes via RNA sequencing (RNA-seq) analysis.

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by progressive cartilage degradation, synovial membrane inflammation, osteophyte formation, and subchondral bone sclerosis. Pathological changes in cartilage and subchondral bone are the main processes in OA. In recent decades, many studies have demonstrated that activin-like kinase 3 (ALK3), a bone morphogenetic protein receptor, is essential for cartilage formation, osteogenesis, and postnatal skeletal development. Although the role of bone morphogenetic protein (BMP) signalling in articular cartilage and bone has been extensively studied, many new discoveries have been made in recent years around ALK3 targets in articular cartilage, subchondral bone, and the interaction between the two, broadening the original knowledge of the relationship between ALK3 and OA. In this review, we focus on the roles of ALK3 in OA, including cartilage and subchondral bone and related cells. It may be helpful to seek more efficient drugs or treatments for OA based on ALK3 signalling in future.

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This study reports mid-term outcomes after periacetabular osteotomy (PAO) exclusively in a borderline hip dysplasia (BHD) population to provide a contrast to published outcomes for arthroscopic surgery of the hip in BHD.

Introduction. A significant burden of disease exists with respect to critical sized bone defects; outcomes are unpredictable and often poor. There is no absolute agreement on what constitutes a “critically-sized” bone defect however it is widely considered as one that would not heal spontaneously despite surgical stabilisation, thus requiring re-operation. The aetiology of such defects is varied. High-energy trauma with soft tissue loss and periosteal stripping, bone infection and tumour resection all require extensive debridement and the critical-sized defects generated require careful consideration and strategic management. Current management practice of these defects lacks consensus. Existing literature tells us that tibial defects 25mm or great have a poor natural history; however, there is no universally agreed management strategy and there remains a significant evidence gap. Drawing its origins from musculoskeletal oncology, the Capanna technique describes a hybrid mode of reconstruction. Mass allograft is combined with a vascularised fibula autograft, allowing the patient to benefit from the favourable characteristics of two popular reconstruction techniques. Allograft confers initial mechanical stability with autograft contributing osteogenic, inductive and conductive capacity to encourage union. Secondarily its inherent vascularity affords the construct the ability to withstand deleterious effects of stressors such as infection that may threaten union. The strengths of this hybrid construct we believe can be used within the context of critical-sized bone defects within tibial trauma to the same success as seen within tumour reconstruction. Methodology. Utilising the Capanna technique in trauma requires modification to the original procedure. In tumour surgery pre-operative cross-sectional imaging is a pre-requisite. This allows surgeons to assess margins, plan resections and order allograft to match the defect. In trauma this is not possible. We therefore propose a two-stage approach to address critical-sized tibial defects in open fractures. After initial debridement, external fixation and soft tissue management via a combined orthoplastics approach, CT imaging is performed to assess the defect geometry, with a polymethylmethacrylate (PMMA) spacer placed at index procedure to maintain soft tissue tension, alignment and deliver local antibiotics. Once comfortable that no further debridement is required and the risk of infection is appropriate then 3D printing technology can be used to mill custom jigs. Appropriate tibial allograft is ordered based on CT measurements. A pedicled fibula graft is raised through a lateral approach. The peroneal vessels are mobilised to the tibioperoneal trunk and passed medially into the bone void. The cadaveric bone is prepared using the custom jig on the back table and posterolateral troughs made to allow insertion of the fibula, permitting some hypertrophic expansion. A separate medial incision allows attachment of the custom jig to host tibia allowing for reciprocal cuts to match the allograft. The fibula is implanted into the allograft, ensuring nil tension on the pedicle and, after docking the graft, the hybrid construct is secured with multi-planar locking plates to provide rotational stability. The medial window allows plate placement safely away from the vascular pedicle. Results. We present a 50-year-old healthy male with a Gustilo & Anderson 3B proximal tibial fracture, open posteromedially with associated shear fragment, treated using the Capanna technique. Presenting following a fall climbing additional injuries included a closed ipsilateral calcaneal and medial malleolar fracture, both treated operatively. Our patient underwent reconstruction of his tibia with the above staged technique. Two debridements were carried out due to a 48-hour delay in presentation due to remote geographical location of recovery. Debridements were carried out in accordance with BOAST guidelines; a spanning knee external fixator applied and a small area of skin loss on the proximal medial calf reconstructed with a split thickness skin graft. A revision cement spacer was inserted into the metaphyseal defect measuring 84mm. At definitive surgery the external fixator was removed and graft fixation was extended to include the intra-articular fragments. No intra-operative complications were encountered during surgeries. The patient returned to theatre on day 13 with a medial sided haematoma. 20ml of haemoserous fluid was evacuated, a DAIR procedure performed and antibiotic-loaded bioceramics applied locally. Samples grew Staphylococcus aureus and antibiotic treatment was rationalised to Co-Trimoxazole 960mg BD and Rifampicin 450mg BD. The patient has completed a six-week course of Rifampicin and continues on suppressive Co-Trimoxazole monotherapy until planned metalwork removal. There is no evidence of ongoing active infection and radiological evidence of early union. The patient is independently walking four miles to the gym daily and we believe, thus far, despite accepted complications, we have demonstrated a relative early success. Conclusions. A variety of techniques exist for the management of critical-sized bone defects within the tibia. All of these come with a variety of drawbacks and limitations. Whilst acceptance of a limb length discrepancy is one option, intercalary defects of greater than 5 to 7cm typically require reconstruction. In patients in whom fine wire fixators and distraction osteogenesis are deemed inappropriate, or are unwilling to tolerate the frequent re-operations and potential donor site morbidity of the Masqualet technique, the Capanna technique offers a novel solution. Through using tibial allograft to address the size mismatch between vascularised fibula and tibia, the possible complication of fatigue fracture of an isolated fibula autograft is potentially avoidable in patients who have high functional demands. The Capanna technique has demonstrated satisfactory results within tumour reconstruction. Papers report that by combining the structural strength of allograft with the osteoconductive and osteoinductive properties of a vascularised autograft that limb salvage rates of greater than 80% and union rates of greater than 90% are achievable. If these results can indeed be replicated in the management of critical-sized bone defects in tibial trauma we potentially have a treatment strategy that can excel over the more widely practiced current techniques

Virtual mechanical testing is a method for measuring bone healing using finite element models built from computed tomography (CT) scans. Previously, we validated a dual-zone material model for ovine fracture callus that differentiates between mineralized woven bone and soft tissue based on radiodensity. 1. The objective of this study was to translate the dual-zone material model from sheep to two important clinical scenarios: human tibial fractures in early-stage healing and late-stage nonunions. CT scans for N = 19 tibial shaft fractures were obtained prospectively at 12 weeks post-op. A second group of N = 33 tibial nonunions with CT scans were retrospectively identified. The modeling techniques were based on our published method. 2. The dual-zone material model was implemented for humans by performing a cutoff sweep for both the 12-week and nonunion groups. Virtual torsional rigidity (VTR) was calculated as VTR = ML/φ [N-m. 2. /°], where M is the moment reaction, L is the diaphyseal segment length, and φ is the angle of twist. As the soft tissue cutoff was increased, the rigidity of the clinical fractures decreased and soft tissue located within the fracture gaps produced higher strains that are not predicted without the dual zone approach. The structural integrity of the nonunions varied, ranging from very low rigidities in atrophic cases to very high rigidities in highly calcified hypertrophic cases, even with dual-zone material modeling. Human fracture calluses are heterogeneous, comprising of woven bone and interstitial soft tissue. Use of a dual-zone callus material model may be instrumental in identifying delayed unions during early healing when callus formation is minimal and/or predominantly fibrous with little mineralization. ACKNOWLEDGEMENTS:. This work was supported by the National Science Foundation (NSF) grant CMMI-1943287

Integrin α2β1 is one of the major transmembrane receptors for fibrillary collagen. In native bone we could show that the absence of this protein led to a protective effect against age-related osteoporosis. The objective of this study was to elucidate the effects of integrin α2β1 deficiency on fracture repair and its underlying mechanisms. Standardised femoral fractures were stabilised by an intramedullary nail in 12 week old female C57Bl/6J mice (wild type and integrin α2. -/-. ). After 7, 14 and 28 days mice were sacrificed. Dissected femura were subjected to µCT and histological analyses. To evaluate the biomechanical properties, 28-day-healed femura were tested in a torsional testing device. Masson goldner staining, Alizarin blue, IHC and IF staining were performed on paraffin slices. Blood serum of the animals were measured by ELISA for BMP-2. Primary osteoblasts were analysed by in/on-cell western technology and qRT-PCR. Integrin α2β1 deficient animals showed earlier transition from cartilaginous callus to mineralized callus during fracture repair. The shift from chondrocytes over hypertrophic chondrocytes to bone-forming osteoblasts was accelerated. Collagen production was increased in mutant fracture callus. Serum levels of BMP-2 were increased in healing KO mice. Isolated integrin deficient osteoblast presented an earlier expression and production of active BMP-2 during the differentiation, which led to earlier mineralisation. Biomechanical testing showed no differences between wild-type and mutant bones. Knockout of integrin α2β1 leads to a beneficial outcome for fracture repair. Callus maturation is accelerated, leading to faster recovery, accompanied by an increased generation of extra-cellular matrix material. Biomechanical properties are not diminished by this accelerated healing. The underlying mechanism is driven by an earlier availability of BMP-2, one main effectors for bone development. Local inhibition of integrin α2β1 is therefore a promising target to accelerate fracture repair, especially in patients with retarded healing

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The aim of this study was to assess the safety and clinical outcome of patients with a femoral shaft fracture and a previous complex post-traumatic femoral malunion who were treated with a clamshell osteotomy and fixation with an intramedullary nail (IMN).

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This systematic review aimed to summarize the full range of complications reported following ankle arthroscopy and the frequency at which they occur.

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Eccentric reductions may become concentric through femoral head ‘docking’ (FHD) following closed reduction (CR) for developmental dysplasia of the hip (DDH). However, changes regarding position and morphology through FHD are not well understood. We aimed to assess these changes using serial MRI.