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

Purpose:. This study attempts to establish whether biomechanical manipulation through distraction can result in fracture union. Method:. A retrospective clinical audit of 15 patients with delayed or hypertrophic non-unions treated successfully with closed distraction in circular external fixation. Average time to union, complications and complication rates were also reviewed. Inclusion criteria: all patients with delayed or hypertrophic non-union, treated by closed distraction between 2004 and 2011. Results:. Fifteen patients included in the study. The average time to union was 188 days. The most common complication was local pin tract sepsis. The most serious complication was a broken fixation ring that needed replacement. Conclusion:. Biomechanical fracture strain is calculated by dividing the fracture gap distance by the change in the fracture distance i.e. FRACTURE STRAIN = DIFFERENCE IN L/L Hypertrophic non-union occurs when the fracture strain is more than 10 %. This formula shows that by increasing the fracture gap, the fracture strain will decrease This concept is contrary to the current practice of compressing the fracture. This study shows that distraction can be used to manipulate the biomechanical circumstances that dictate the development of fracture non-union. Furthermore ring fixators are ideal devices to use for biomechanical manipulation

Introduction: Metastases in multiple myeloma are typically lytic and when non-union occurs it is usually atrophic. Methods: We report a lady of 67 years who was diagnosed with myeloma 9 years previously. She presented with a sudden onset of pain in her right forearm. Plain radiographs demonstrated a lytic lesion typical of multiple myeloma with an undisplaced pathological fracture in her right ulna. The fracture was treated in a short arm cast for 6 weeks and then by mobilisation. The underlying bone deposit was treated subsequently by external beam irradiation. Results: Nine months later she was re-referred to the orthopaedic oncology service with marked forearm pain particularly on rotation. Radiographs demonstrated a hypertrophic non-union of the pathological fracture with a typical elephant’s hoof appearance. The fracture was stabilised using a Foresight ulnar nail (Smith and Nephew, Warwick, UK). Discussion: Whilst non-unions in metastatic malignancy are typically atrophic, just occasionally hypertrophic non-unions can occur. Management principles remain the same with stabilisation of the entire bone and early mobilisation being appropriate

During fracture repair, a number of growth factors and cytokines are present at elevated levels at the fracture site such as Transforming Growth Factor Beta (TGF-), Fibroblast Growth Factor (FGF) and Platelet Derived Growth Factor (PDGF). The aim of the study was to investigate the presence of these growth factors in healing fractures and fracture non-unions, in order to test the hypothesis that atrophic non-unions express a lower level of growth factors than hypertrophic non-unions and healing fractures. Biopsies were taken from the fracture site of 23 patients (mean age 46) with uninfected non-unions, 12 patients with hypertrophic (mean 13.8 months after fracture) and 11 patients with atrophic (mean 16.5 months after fracture). A comparison group of biopsies from early fracture callus (one to four weeks after fracture) in five patients with healing fractures was also included. Five-micron paraffin sections were immunohistochemically stained for TGF-, FGF-II and PDGF. Growth factors were then assessed in six different cell types. Fibroblasts, endothelial cells and macrophages were found to express TGF-, FGF-II and PDGF in all three-fracture groups. Osteoblasts, osteoclasts and chondrocytes were not present in the healing fracture group. The growth factor expression in osteoblasts, osteoclasts and chondrocytes in the non-union groups were found to be variable, however, the expression of these growth factors appeared to be less in the atrophic non-unions than hypertrophic non-unions. The expression of these growth factors was found to be less in the atrophic non-union group than the hypertrophic non-union group in osteoblasts, osteoclasts and chondrocytes. These results may have relevance for new therapies that can be aimed at delivering growth factors to treat fracture non-unions. By further investigation of the differential expression of these growth factors it may be possible to determine which factors are likely to stimulate fracture healing

Purpose. To assess the efficacy of the treatment of the aseptic hypertrophic nonunion of the tibia and the secondary deformities by distraction-osteogenesis. Material Methods: Between 1998 and 2006, 28 patients with a hyperthrophic tibia nonunion were treated by distraction or compression-distraction depending on the mobility of the nonunion. The mean age of the patients was 37.5 years (range 24 to 68) and the average number of previous surgeries on the affected limb was 2 (from 1 to 4). No active bone infection or history of infection was recorded in this series. Closed distraction was applied in 11 patients, closed distraction – compression in 8 and osteoclasis following by distraction in 9 patients. In all cases an external fixation device (19 circular, 11 monolateral external frames) modified to meet the nonunion requirements was used. Results: Distraction or distraction-compression resulted in solid union in all patients (mean time to union 8.4 months, mean follow up 5 years). The external fixator remained in place for an average of 8.2 months (range 7 to 11.5 months). Mean leg length discrepancy 2.5 cm and mean angular deformity 12° were also corrected on the same procedure. Conclusions: Treatment of the tibia nonunion by callus distraction or distraction – compression leads to successfully results. The procedure and the frame have to be individualized according to the nonunion pathology and the secondary tibia deformities

Introduction and Aims: Distraction osteogenesis can be used to stimulate healing in hypertrophic non-unions (HNU). We evaluated the use of closed (without opening the non-union) Ilizarov distraction for HNU with associated angulation, malrotation, and shortening. Method: Sixty-seven consecutive patients (mean age, 38.3 years) with 71 HNU were treated (1988–2001) using Ilizarov distraction. Patients had undergone an average of five previous operations. HNU classified as stiff (< 5 degrees mobility) were distracted, and those classified as partially mobile (5–20 degrees mobility) were first compressed and then distracted. Results: Non-unions included: 59 tibiae, six femora, two radii, and five ankle arthrodeses. Mean limb length discrepancy, 3.5cm; mean deformity, 16°; history of osteomyelitis, six cases. Closed distraction alone was successful in achieving union in 61 cases (86%) (mean follow-up, six years; mean time to union, eight months). Union rate was 91.6% (55 of 60 cases) for stiff HNU and only 54% (six of 11 cases) for partially mobile HNU. Distraction treatment alone failed to achieve union in 10 cases. In seven, union was achieved after bone grafting. Two required resection of infected non-union with bone transport to achieve union. One had persistent non-union. There were numerous superficial pin infections and three deep infections. Two cases had deformity at proximal tibial lengthening osteotomy site. Conclusion: Closed distraction is safe and reliable for stimulating union in stiff HNU. It is especially effective in a scarred limb that has undergone previous operations. It allows for simultaneous correction of deformity and length. Main disadvantage is lengthy time spent in external fixator

Aims

This study aimed to investigate the effect of solute carrier family 20 member 2 (SLC20A2) gene mutation (identified from a hereditary multiple exostoses family) on chondrocyte proliferation and differentiation.

Tibial nonunion represents a spectrum of conditions which are challenging to treat, and optimal management remains unclear despite its high rate of incidence. We present 44 consecutive patients with 46 stiff tibial nonunions, treated with hexapod external fixators and distraction to achieve union and gradual deformity correction. There were 31 men and 13 women with a mean age of 35 years (18 to 68) and a mean follow-up of 12 months (6 to 40). No tibial osteotomies or bone graft procedures were performed. Bony union was achieved after the initial surgery in 41 (89.1%) tibias. Four persistent nonunions united after repeat treatment with closed hexapod distraction, resulting in bony union in 45 (97.8%) patients. The mean time to union was 23 weeks (11 to 49). Leg-length was restored to within 1 cm of the contralateral side in all tibias. Mechanical alignment was restored to within 5° of normal in 42 (91.3%) tibias. Closed distraction of stiff tibial nonunions can predictably lead to union without further surgery or bone graft. In addition to generating the required distraction to achieve union, hexapod circular external fixators can accurately correct concurrent deformities and limb-length discrepancies.

Cite this article: Bone Joint J 2015;97-B:1417–22.

This review is aimed at clinicians appraising preclinical trauma studies and researchers investigating compromised bone healing or novel treatments for fractures. It categorises the clinical scenarios of poor healing of fractures and attempts to match them with the appropriate animal models in the literature.

We performed an extensive literature search of animal models of long bone fracture repair/nonunion and grouped the resulting studies according to the clinical scenario they were attempting to reflect; we then scrutinised them for their reliability and accuracy in reproducing that clinical scenario.

Models for normal fracture repair (primary and secondary), delayed union, nonunion (atrophic and hypertrophic), segmental defects and fractures at risk of impaired healing were identified. Their accuracy in reflecting the clinical scenario ranged greatly and the reliability of reproducing the scenario ranged from 100% to 40%.

It is vital to know the limitations and success of each model when considering its application.

Osteoarthritis (OA) is the most common joint disease, which is characterized by a progressive loss of proteoglycans and the destruction of extracellular matrix (ECM), leading to a loss of cartilage integrity and joint function. During OA development, chondrocytes alter ECM synthesis and change their gene expression profile including upregulation of hypertrophic markers known from the growth plate. Although physiological mechanical loading can support cartilage formation and maintenance, mechanical overload represents one major risk factor for OA development. To date, little is known on how an OA-like hypertrophic chondrocyte phenotype alters the response of cartilage tissue to mechanical loading. The aim of this study was to investigate whether a hypertrophic phenotype change of chondrocytes affects the response to physiological mechanical loading and to reveal differences compared to normal control cartilage. Cartilage replacement tissue was generated using human articular chondrocytes (normal control cartilage, n=3–5) or human mesenchymal stromal cells which develop a hypertrophic phenotype similar to the one observed in OA (OA cartilage model, n=3–6). Cells were seeded in a collagen type I/III carrier and attached to a beta-TCP bone replacement phase, building an osteochondral unit for simulation of natural conditions. After 21 and 35 days of chondrogenic (re)differentiation, a single physiological mechanical compression episode (1 Hz, 25 %, 3 h) was applied, imitating three hours of normal walking in ten-minute intervals. Proteoglycan and collagen synthesis, gene expression and activation of signaling pathways were assessed. Cartilage replacement tissue of both groups had similar proteoglycan and collagen type II content as well as hardness properties. During (re)differentiation, both cell types showed a comparable upregulation of the chondrogenic marker genes COL2A1 and ACAN. As expected, hypertrophic marker genes (COL10A1, ALPL, MEF2C, IBSP) were only upregulated in the OA cartilage model. Mechanotransduction in both tissues was confirmed by load-induced activation of pERK1/2 signaling. While the 3 h loading episode significantly increased proteoglycan synthesis in normal control cartilage at day 35, the same protocol resulted in a suppression of proteoglycan and collagen synthesis in the OA cartilage model, which was accompanied by a downregulation of COL2A1 gene expression. In addition, hypertrophic marker genes COL10A1, ALPL and IBSP were significantly reduced after loading. Along lower load-induced SOX9 mRNA and protein stimulation in the OA cartilage tissue, a weaker induction of mechanosensitive BMP2, BMP6, FOS and FOSB gene expression was observed. While stable cartilage showed anabolic effects after physiological loading, the hypertrophic chondrocytes reacted with a reduced extracellular matrix synthesis. This could be explained by a lower mechanoinduction of the BMP signaling cascade and insufficient SOX9 stimulation. Progressive OA development could thus be influenced by a reduced mechanocompetence of osteoarthritic chondrocytes

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

During OA the homeostasis of healthy articular chondrocytes is dysregulated, which leads to a phenotypical transition of the cells, further influenced by external stimuli. Chondrocytes sense those stimuli, integrate them at the intracellular level and respond by modifying their secretory and molecular state. This process is controlled by a complex interplay of intracellular factors. Each factor is influenced by a myriad of feedback mechanisms, making the prediction of what will happen in case of external perturbation challenging. Hampering the hypertrophic phenotype has emerged as a potential therapeutic strategy to help OA patients (Ripmeester et al. 2018). Therefore, we developed a computational model of the chondrocyte's underlying regulatory network (RN) to identify key regulators as potential drug targets. A mechanistic mathematical model of articular chondrocyte differentiation was implemented with a semi-quantitative formalism. It is composed of a protein RN and a gene RN(GRN) and developed by combining two strategies. First, we established a mechanistic network based on accumulation of decades of biological knowledge. Second, we combined that mechanistic network with data-driven modelling by inferring an OA-GRN using an ensemble of machine learning methods. This required a large gene expression dataset, provided by distinct public microarrays merged through an in-house pipeline for cross-platform integration. We successfully merged various micro-array experiments into one single dataset where the biological variance was predominant over the batch effect from the different technical platforms. The gain of information provided by this merge enabled us to reconstruct an OA-GRN which subsequently served to complete our mechanistic model. With this model, we studied the system's multi-stability, equating the model's stable states to chondrocyte phenotypes. The network structure explained the occurrence of two biologically relevant phenotypes: a hypertrophic-like and a healthy-like phenotype, recognized based on known cell state markers. Second, we tested several hypotheses that could trigger the onset of OA to validate the model with relevant biological phenomena. For instance, forced inflammation pushed the chondrocyte towards hypertrophy but this was partly rescued by higher levels of TGF-β. However, we could annihilate this rescue by concomitantly mimicking an increase in the ALK1/ALK5 balance. Finally, we performed a screening of in-silico (combinatorial) perturbations (inhibitions and/or over-activations) to identify key molecular factors involved in the stability of the chondrocyte state. More precisely, we looked for the most potent conditions for decreasing hypertrophy. Preliminary validation experiments have confirmed that PKA activation could decrease the hypertrophic phenotype in primary chondrocytes. Importantly the in-silico results highlighted that targeting two factors at the same time would greatly help reducing hypertrophic changes. A priori testing of conditions with in-silico models may cut time and cost of experiments via target prioritization and opens new routes for OA combinatorial therapies

Introduction. Cell-based therapy is needed to overcome the lacking intrinsic ability of cartilage to heal. Generating cartilage tissue from human bone marrow-derived stromal cells (MSC) is limited by up-regulation of COL10, ALP and other hypertrophy markers in vitro and calcifying cartilage at heterotopic sites in vivo. MSC hypertrophic differentiation reflects endochondral ossification, unable to maintain a stable hyaline stage, as observed by redifferentiation of articular chondrocytes (AC). Several transcription factors (TF), are held responsible for hypertrophic development. SOX9, the master regulator of chondrogenesis is also, alongside MEF2C, regulating hypertrophic chondrocyte maturation and COL10 expression. RUNX2/3 are terminal markers driving chondrocyte hypertrophy, and skeletogenesis. However, so far regulation of these key fate determining TFs has not been studied thoroughly on mRNA and protein level through chondrogenesis of human MSC. To fill this gap in knowledge, we aim to uncover regulation of SOX9, RUNX2/3, MEF2C and other TFs related to hypertrophy during MSC chondrogenesis in vitro and in comparison to the gold standard AC redifferentiation. Methods. Expression of SOX9, RUNX2/3 and MEF2C was compared before and during 6-week chondrogenic re-/differentiation of human MSC and AC on mRNA level via qRT-PCR and protein level via Western-Blotting. Chondrogenesis was evaluated by histology at d42 and expression of chondrogenic markers like COL2. Hypertrophic development was characterized by ALP activity and expression of hypertrophic markers like COL10. Results. Hypertrophic development, characterized by upregulation of COL10, high COL10/COL2 ratios and ALP activity, was confirmed in MSC and absent in AC. MSC started into differentiation with less SOX9 before induction, while higher RUNX2/3 was observed compared to AC. During MSC chondrogenesis SOX9 and MEF2C steadily increased on mRNA and protein level. Surprisingly, although RUNX2 mRNA level increased in MSC over 42 days, RUNX2 protein remained undetectable. During AC redifferentiation, SOX9 levels remained high on mRNA and protein level while RUNX2/3 and MEF2C remained low. Conclusion. After expansion and before applying chondrogenic stimuli, a chondrogenic priming with more SOX9 and lower RUNX2/3 was found in AC. In contrast osteochondral priming with higher RUNX2/3 and lower SOX9 levels was observed in MSC which could set the stage for endochondral development, leading to hypertrophy. Dynamic regulation of RUNX2/3 and MEF2C at lower SOX9 background levels separated MSC from AC differentiation over 42 days. Adjusting transcription factor levels in MSC could be essential for creating a protocol leading to diminished hypertrophy of MSC during chondrogenesis

Aims. The aim of our study is to investigate the effect induced by alternated mechanical loading on Notch-1 in mandibular condylar cartilage (MCC) of growing rabbits. Methods. A total of 64 ten-day-old rabbits were randomly divided into two groups according to dietary hardness: normal diet group (pellet) and soft diet group (powder). In each group, the rabbits were further divided into four subgroups by feeding time: two weeks, four weeks, six weeks, and eight weeks. Animals would be injected 5-bromo-2′-deoxyuridine (BrdU) every day for one week before sacrificing. Histomorphometric analysis of MCC thickness was performed through haematoxylin and eosin (HE) staining. Immunochemical analysis was done to test BrdU and Notch-1. The quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were used to measure expression of Notch-1, Jagged-1, and Delta-like 1 (Dll-1). Results. The thickness of MCC in the soft diet group was thinner than the one in normal diet group. Notch-1 was restricted in fibrous layer, proliferative layer, and hypertrophic layer. The expression of Notch-1 increased from two weeks to six weeks and then fell down. Notch-1 in normal diet group was higher than that in soft diet group in anterior part of MCC. The statistical differences of Notch-1 were shown at two, four, and six weeks (p < 0.05). The result of western blot and quantitative real-time PCR (qRT-PCR) showed the expression of Dll-1 and Jagged-1 rose from two to four weeks and started to decrease at four weeks. BrdU distributed in all layers of cartilage and subchondral bone. The number of BrdU-positive cells, which were less in soft diet group, was decreasing along with the experiment period. The significant difference was found at four, six, and eight weeks in anterior and posterior parts (p < 0.05). Conclusion. The structure and proliferation of MCC in rabbits were sensitive to dietary loading changes. The proper mechanical loading was essential for transduction of Notch signalling pathway and development of mandibular condylar cartilage. Cite this article: Bone Joint Res 2021;10(7):437–444

Abstract. Introduction. Recurrent groin pain following periacetabular osteotomy (PAO) is a challenging problem. The purpose of our study was to evaluate the position and dynamics of the psoas tendon as a potential cause for recurrent groin pain following PAO. Methods. Patients with recurrent groin pain following PAO were identified from a single surgeon series. A total of 13 patients with 18 hips (4.7%) out of a 386 PAO, had recurrent groin pain. Muscle path of the psoas tendon was accurately represented using 3D models from CT data were created with Mimics software. A validated discrete element model using rigid body springs was used to predict psoas tendon movement during hip circumduction and walking. Results. Five out of the 18 hips did not show any malformations at the osteotomy site. Thirteen hips (72%) showed malformation secondary to callus at the superior pubic ramus. These were classified into: osteophytes at the osteotomy site, hypertrophic callus or non-union and malunion at the osteotomy. Mean minimal distance of the psoas tendon to osteophytes was found to be 6.24 mm (n=6) and to the osteotomy site was 14.18 mm (n=18). Conclusions. Recurrent groin pain after PAO needs a thorough assessment. One need to have a high suspicion of psoas issues as a cause. 3D CT scan may be necessary to identify causes related to healing of the pubic osteotomy. Dynamic ultrasound of the psoas psoas tendon may help in evaluating for psoas impingement as a cause of recurrent groin pain in these cases

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

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

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

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

INTRODUCTION. The generation of cartilage from progenitor cells for the purpose of cartilage repair is often hampered by unwanted hypertrophic differentiation of the generated tissue due to endochondral ossification. Continuing on our earlier studies, our goal is to further improve the engineering of hyaline cartilage for the treatment of a cartilage defect in our in vivo model for subperiosteal generation of cartilage, by tuning the differentiation status of the generated cartilage and prevent hypertrophic differentiation. As a healthy cartilage matrix contains high amounts of aggrecan we hypothesise that aggrecan supplementation of the bio-gel used in the generation of the subperiosteal cartilage, mimics the composition of the extracellular matrix environment of cartilage with potential beneficial properties for the engineered cartilage. METHODS. A 2% (m/v) low melting agarose was injected between the bone and periosteum at the upper medial side of the tibia of both legs of New Zealand white rabbits (DEC 2012–151). The agarose was left unloaded (n=7) or supplemented (n=7) with 2% (w/v) bovine aggrecan (Sigma-Aldrich). After 14 days, rabbits were euthanised. Generated subperiosteal cartilage tissue was analysed for weight, GAG and DNA content. In addition, RT-qPCR and (immuno)histochemistry was performed for key markers of different phases of endochondral ossification. RESULTS. The nett weight of the generated subperiosteal cartilage tissue was not significantly different between groups, nor was the GAG content different. No significant differences in chondrogenic marker expression (COL2A1, SOX9, ACAN and PTHrP) were detected. Interestingly, gene expression levels of hypertrophic markers COL10A1 and ALPL were significantly decreased. COL1A1 expression was not significantly different between groups. DISCUSSION. In summary, generation of subperiosteal cartilage was successful when an agarose bio-gel was injected beneath the periosteum. The addition of aggrecan to the bio-gel did not result in differences in weight or GAG content in cartilage samples between conditions. However, lower levels of hypertrophic markers were observed, while leaving chondrogenic marker expression unaltered. These data show the potential of aggrecan to favourably influence the subperiosteal microenvironment for the in vivo generation of hyaline cartilage for the optimisation of cartilage regenerative medicine approaches

Introduction. Human Mesenchymal stem cells (hMSCs) are a promising source for articular cartilage repair. Unfortunately, under in vitro conditions, chondrogenically differentiated hMSCs have the tendency to undergo hypertrophy similar to growth plate chondrocytes. Retinoic acid (RA) signalling plays a key role in growth plate hypertrophy. Whilst RA agonists block chondrogenesis and foster hypertrophy during later stages, RAR inverse agonists (IA) enhance chondrogenesis when applied early in culture. Therefore, we hypothesized that treatment with RAR IA will attenuate hypertrophy in chondrogenically differentiated hMSCs. To test this hypothesis, we analysed early (initial chondrogenic differentiation) and late treatment (hypertrophy stage) of hMSCs with an RAR IA. Methods. Pellets of passage 2 hMSCs were formed in V-bottom well plates by centrifugation and pre-differentiated in a chemically defined medium containing 10ng/mL TGFß (CM+) for 14 days. Thereafter, pellets were cultured for an additional 14 days under 6 conditions: CM+, CM- (w/out TGFß), and hypertrophic medium (CM- with 25 ng/ml BMP 4, w/out dexamethasone). Each of these first three conditions was additionally supplemented with the RA receptor (RAR) inverse agonist BMS493 (BMS) at 2μM after 14 days of chondrogenic pre-differentiation. One additional BMP4 group was supplemented with BMS from the beginning of chondrogenic differentiation until day 14. The pellets were assessed for gene expression (Col 2, Col 10, Col 1 and MMP13) and histologically using dimethyl methylene blue (DMMB), alkaline phosphatase staining (ALP) and collagen II and X immunohistochemistry. Results. Hypertrophy was reduced by addition of BMS at day 14 and further reduced by addition from the beginning. BMS treatment resulted in smaller cells under hypertrophic conditions, higher collagen II content in chondrogenic groups and reduction in collagen X production and ALP activity in every condition. Gene expression data for hypertrophic markers, collagen X and MMP13, were upregulated under the influence of BMP4 but a distinct downregulation in MMP13 expression was shown upon addition of BMS during the late stage differentiation and further reduced upon addition during early stage chondrogenesis. Furthermore, Collagen X expression was reduced by early BMS treatment. Discussion. The treatment with the RAR IA, BMS, attenuated hypertrophic changes in chondrogenically differentiated hMSCs as demonstrated by histology, immunohistochemistry and PCR. These findings suggest an additional approach to attenuate hypertrophy in chondrogenically differentiated hMSCs. Current studies are exploring the timing and dose of BMS to most efficaciously prevent hypertrophy

Design criteria for tissue-engineered materials in regenerative medicine include robust biological effectiveness, off-the-shelf availability, and scalable manufacturing under standardized conditions. For bone repair, existing strategies rely on primary autologous cells, associated with unpredictable performance, limited availability and complex logistic. Here, we report the manufacturing of engineered and devitalized human hypertrophic cartilage (HyC) as cell-free material inducing bone formation by recapitulating the developmental process of endochondral ossification. Our strategy relies on a customized human mesenchymal line expressing Bone Morphogenetic Protein-2 (BMP-2), critically required for robust chondrogenesis and concomitant extracellular matrix (ECM) enrichment. Following apoptosis-driven devitalization, lyophilization and storage, the resulting material exhibited unprecedented osteoinductive properties, unmatched by synthetic delivery of BMP-2 or by living engineered grafts. Scalability and pre-clinical efficacy were demonstrated by bioreactor-based production and subsequent orthotopic assessment. Our findings exemplify the broader paradigm of customized ECMs, engineered to activate specific regenerative processes by programming human cell lines as biological factory units

Nonunions occur in situations with interrupted fracture healing process and indicate conditions where the fracture has no potential to heal without further intervention. Per definition, no healing is detected nine months post operation and there is no visible progress of healing over the last three months. The classification of nonunions as hypertrophic, oligotrophic, atrophic and pseudoarthosis, as well as aseptic or septic, identifies mechanical and biological requirements for fracture healing that have not been met. The overall treatment strategy comprises identification and elimination of the problems. However, current clinical methods to determine the state of healing are based on highly subjective radiographic evaluation or clinical examination. A data collection telemetric system for objective continuous measurement of the load carried by a bridging smart implant was developed to assess the mechanical stability and monitor bone healing in complicated fracture situations. The first results from a clinical trial show that the system is capable to offer early warning of nonunions or poor fracture healing. Nonunions are often multifactorial in nature and not just related to a biomechanical problem. Their successful treatment requires consideration of both biological and mechanical aspects. Disturbed vascularity and stability are the most important factors. Infection could be another complicating factor resulting in unpredictable long-time treatment. New technologies for monitoring of fracture healing in addition to radiographic evaluation and clinical examination seem to be promising for early detection of nonunions

INTRODUCTION. The generation of cartilage from progenitor cells for the purpose of cartilage repair is often hampered by unwanted ossification of the generated tissue due to endochondral ossification. Our in vitro data show that celecoxib is able to suppress the hypertrophic differentiation phase of endochondral ossification in differentiating human bone marrow stem cells via inhibition of prostaglandin signalling. Continuing on our earlier studies our goal is to further improve the engineering of hyaline cartilage for the treatment of cartilage defects, by determining if celecoxib released from poly(D,L-lactic acid)microspheres is able to prevent unwanted ossification in an in vivo model for the subperiosteal cartilage generation. METHODS. A 2% (m/v) low melting agarose was injected between the bone and periosteum at the upper medial side of the tibia of both legs of New Zealand white rabbits (DEC 2012–151). The agarose was left unloaded or (n=8) or loaded (n=7) with celecoxib-loaded PGLA microspheres (poly(D,L-lactic acid) microspheres were loaded with 20% (w/w) Celecoxib (Pfizer)). Fourteen days post-injection, rabbits were euthanised. The developed subperiosteal cartilage tissue was analysed for weight, GAG and DNA content. In addition, RT-qPCR and (immuno)histochemistry were performed for key markers of different phases of endochondral ossification. RESULTS. The Functional release of celecoxib from poly(D,L-lactic acid) microspheres was confirmed in vitro by decreased prostaglandin E2 levels in cell culture. The subperiosteal cartilage tissue from the celecoxib group was significantly higher in weight and DNA content as compared to the control condition. GAG content was not significantly different between groups. No significant differences in chondrogenic marker expression (COL2A1, SOX9, ACAN and PTHrP) were detected, but levels of hypertrophic markers COL10A1, RUNX2 and ALPL were significantly decreased. COL1A1 expression was not significantly different between groups. DISCUSSION. In summary, subperiosteal generation of cartilage was successful when an agarose bio-gel was injected subperiosteally. Supplementation of the agarose gel with celecoxib-loaded microspheres favourably changed the weight of the generated cartilage tissue, combined with significantly lower expression levels of indicators of chondrocyte hypertrophy, while leaving chondrogenic differentiation capacity unaltered. These data hold the promise that local supplementation of celecoxib during in vivo cartilage regeneration protects the tissue from adverse hypertrophic differentiation

Osteoarthritis (OA) is the most common degenerative joint disease causing joint immobility and chronic pain. Treatment is mainly based on alleviating pain and reducing disease progression. During OA progression the chondrocyte undergoes a hypertrophic switch in which extracellular matrix (ECM) -degrading enzymes are released, actively degrading the ECM. However, cell biological based therapies to slow down or reverse this katabolic phenotype are still to be developed. Bone morphogenetic protein 7 (BMP-7) has been shown to have OA disease-modifying properties. BMP-7 suppresses the chondrocyte hypertrophic and katabolic phenotype and may be the first biological treatment to target the chondrocyte phenotype in OA. However, intra-articular use of BMP-7 is at risk in the proteolytic and hydrolytic joint-environment. Weekly intra-articular injections are necessary to maintain biological activity, a frequency unacceptable for clinical use. Additionally, production of GMP-grade BMP-7 is challenging and expensive. To enable its clinical use, we sought for BMP-7 mimicking peptides better compatible with the joint-environment while still biologically active and which potentially can be incorporated in a drug-delivery system. We hypothesized that human BMP-7 derived peptides are able to mimic the disease modifying properties of the full-length human BMP-7 protein on the OA chondrocyte phenotype. A BMP-7 peptide library was synthesized consisting of overlapping 20-mer peptides with 18 amino-acids overlap between sequential peptides. OA human articular chondrocytes (HACs) were isolated from OA cartilage from total knee arthroplasty (n=18 donors). HACs were exposed to BMP-7 (1 nM) or BMP-7 library peptides at different concentrations (1, 10, 100 or 1000 nM). Gene-expression levels of important chondrogenic-, hypertrophic-, cartilage degrading- and inflammatory mediators were determined by RT-qPCR. GAG and ALP activity were determined using a colorimetric assay and PGE levels were measured by EIA. During the BMP-7 peptide library screening human BMP-7 derived peptides were screened for their full-length human BMP-7 mimicking properties at different concentrations (1, 10, 100 or 1000nM) on a pool of human chondrocytes. Gene expression as well as GAG, ALP and PGE2 level analysis revealed two distinct peptide regions in the BMP-7 protein based on their pro-chondrogenic and anti-OA phenotype actions on human OA chondrocytes. The two most promising peptides were further analysed for their OA chondrocyte disease modifying properties in the presence of OA synovial fluid, showing similar OA phenotype suppressive activity. Conclusively, we successfully identified two peptide regions in the BMP-7 protein with in vitro OA suppressive actions. Further biochemical fine-tuning of the peptides, and in vivo evaluation, will potentially result in the first peptide-based experimental OA treatment, addressing the hypertrophic and katabolic chondrocyte phenotype in OA

INTRODUCTION. Endochondral ossification in the growth plate is directly responsible for skeletal growth and its de novo bone-generating activity. Growth plates are vulnerable to disturbances that may lead to abnormal skeletal development. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used analgesics but have been reported to impair endochondral ossification-driven fracture healing. Despite the general awareness that NSAIDs affect endochondral ossification, the consequences of NSAIDs on skeletal development are unknown. We hypothesise that the NSAID celecoxib leads to impaired growth plate development and consequently impairs skeletal development. METHODS. Healthy skeletally immature (5 weeks old) C57BL/6 mice were treated for ten weeks with celecoxib (daily oral administration 10 mg/kg) or placebo (water) (institutional approval 2013–094) (n=12 per group). At 15 weeks postnatally, total growth plate thickness, the thickness of specific growth plate zones, (immuno)histological analysis of extracellular matrix composition in the growth plate, cell number and cell size, longitudinal bone growth and bone micro-architecture by micro-CT were analysed. Inhibition of COX-2 activity was confirmed by determining PGE2 levels in plasma using an ELISA. RESULTS. No significant difference in total growth plate thickness or thickness of the resting zone, proliferative or hypertrophic zone was found between groups. Staining of growth plate extracellular matrix components revealed, however, a significantly higher proteoglycan content and less collagen type II staining in the proliferative zone. In the hypertrophic zone of the growth plates of celecoxib treated mice collagen type X was hardly detectable as compared to placebo mice. In addition, a significantly decreased cell number was observed in the hypertrophic zone of the growth plate and cells were significantly smaller in the celecoxib group. Micro-CT analysis of the subchondral bone region directly beneath the growth plate showed significantly higher bone density, bone volume density and trabecular thickness following celecoxib treatment. Despite the detected differences in extracellular matrix composition of the growth plate, no difference was found in the length of the tibia in celecoxib treated mice. DISCUSSION. In summary, there are no measurable differences found in murine skeletal formation as a result of treatment with celecoxib in this study. However, there are notable phenotypic features found in the maturation of the growth plate (hypertrophic zone and subchondral bone) as a result from the celecoxib treatment, of which the potential consequences we do not yet understand. SIGNIFICANCE. When follow-up actions from the use of celecoxib on the growing individual are found this may warrant re-evaluation for the use of celecoxib in these individuals

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

Introduction and Objective. Current cartilage repair strategies lack adequate tissue integration capacity and often present mechanical failure at the graft-to-host tissue junction. The design of multilayered osteochondral tissue engineering (TE) constructs is an attractive approach to overcome these problems. However, calcium ion-release from resorbable bone-replacement materials was suggested to compromise chondrogenic differentiation of adjacent cartilage tissue and it is unclear whether articular chondrocytes (AC) or mesenchymal stroma cells (MSC) are more sensitive to such conditions. Aim of the study was to compare how elevated calcium levels affect cartilage matrix production during re-differentiation of AC versus chondrogenic differentiation of MSC. The results of this study will help to identify the ideal cell source for growth of neocartilage adjacent to a calcified bone replacement material for design of multilayered osteochondral TE approaches. Materials and Methods. Expanded human AC and MSC (6–12 donors per group) were seeded in collagen type I/III scaffolds and cultured under standard chondrogenic conditions at control (1.8mM) or elevated (8.0mM) CaCl2 for 35 days. Proteoglycan and collagen production were assessed via radiolabel-incorporation, ELISA, qPCR and Western blotting. Differences between groups or cell types were calculated using the non-parametric Wilcoxon or Mann-Whitney U test, respectively, with p < 0.05 considered significant. Results. Elevated calcium significantly reduced GAG synthesis (63% of control, p=0.04) and chondrogenic marker expression of AC, lowering the GAG/DNA content (47% of control, p=0.004) and collagen type II deposition (24% of control, p=0.05) of neocartilage compared to control conditions. Opposite, at elevated calcium levels MSC-derived chondrocytes significantly increased GAG synthesis (130% of control, p=0.02) and collagen type II content (160% of control, p=0.03) of cartilage compared to control tissue. Chondrogenic and hypertrophic marker expression was insensitive to calcium levels in MSC-derived chondrocytes. As a result, maturation under elevated calcium allowed for a significantly higher GAG/DNA content in MSC-derived samples compared to AC constructs, although under control conditions both groups developed similarly. Conclusions. AC and MSC showed an opposite reaction to elevation of calcium levels regarding cartilage matrix production and we propose MSC as a preferred cell source to grow chondrocytes in vicinity to calcified bone replacement materials. Since MSC remained prone to hypertrophy under elevated calcium, trizonal cartilage TE constructs, where an AC-layer is separated from the bone replacement phase by an intermediate layer of MSC appear as an ideal design for multilayered osteochondral TE with respect to calcium sensitivity of cells and protection of the upper cartilage layer from hypertrophy

Heterotopic ossi?cation is the abnormal formation of bone in soft tissues and is a frequent complication of hip replacement surgery. Heterotopic ossi?cations are described to develop via endochondral ossification and standard treatment is administration of indomethacin. It is currently unknown how indomethacin influences heterotopic ossi?cation on a molecular level, therefore we aimed to determine whether indomethacin might influence heterotopic ossi?cation via impairing the chondrogenic phase of endochondral ossification. ATDC5, human bone marrow stem cells (hBMSCs) and rabbit periosteal agarose cultures were employed as progenitor cell models; SW1353, human articular chondrocytes and differentiated ATDC5 cells were used as matured chondrocyte cell models. All cells were cultured in the presence of (increasing) concentrations of indomethacin. The action of indomethacin was confirmed by decreased PGE2 levels in all experiments, and was determined by specific PGE2 ELISA. Gene- and protein expression analyses were employed to determine chondrogenic outcome. Progenitor cell models differentiating in the chondrogenic lineage (ATDC5, primary human bone marrow stem cells and ex vivo periosteal agarose cultures) were treated with increasing concentrations of indomethacin and a dose-dependent decrease in gene- and protein expression of chondrogenic and hypertrophic markers as well as decreased glycosaminoglycan content was observed. Even when hypertrophic differentiation was provoked the addition of indomethacin resulted in decreased hypertrophic marker expression. Interestingly, when mature chondrocytes were treated with indomethacin, a clear increase in collagen type 2 expression was observed. Similarly, when ATDC5 cells and bone marrow stem cells were pre-differentiated to obtain a chondrocyte phenotype and indomethacin was added from this time point onwards, low concentrations of indomethacin also resulted in increased chondrogenic differentiation. Indomethacin induces differential effects on in vitro endochondral ossification, depending on the chondrocyte's differentiation stage, with complete inhibition of chondrogenic differentiation as the most pronounced action. This observation may provide a rationale behind the elusive mode of action of indomethacin in the treatment of heterotopic ossifications

Chondromodulin-I (ChM-I) is a bifunctional autocrine regulator of cartilage, initially isolated from fetal bovine epiphyseal cartilage. 1. ChM-I stimulates matrix synthesis of chondrocytes, but inhibits the growth of endothelial cells. 1,. 2. thus ChM-I may be one of the anti-angiogenic molecules present in cartilage. In fetal bovine bone, ChM-I was expressed by all epiphyseal and growth plate chondrocytes except hypertrophic chondrocytes and was present in the matrix throughout the epiphysis and the growth plate, except the hypertrophic zone . 2,. 3. , consistent with its proposed role as anti-angiogenic factor. To examine the possible role of chondromodulin-I in relation to angiogenesis at the vascular front, we studied the immunolocalisation in femoral growth plates from young and mature rats (2–16 weeks) as well as aged rats (62–80 weeks), using a rabbit polyclonal antibody raised against the entire region of mature human recombinant ChM-I. In 2-week old rats, ChM-I was synthesised by all epiphyseal chondrocytes and strong immunostaining was found in the matrix. In the growth plates, ChM-I staining was present in chondrocytes and matrix of the reserve, proliferating and maturing zones with loss of staining in the hypertrophic zone. However, ChM-I was also present where cartilage canals had penetrated into the chondroepiphysis. In 4–16 week old rats, there was a progressive change in the localisation of ChM-I. Hypertrophic chondrocytes also became positive for ChM-I, while cellular staining gradually disappeared from the other zones. By 12–16 weeks, very strong immunostaining was present almost exclusively on the inner perimeter of the lacunae of hypertrophic chondrocytes. As lacunae were opened at the vascular front, ChM-I initially remained on the cartilage-side of the lacunae, and then disappeared completely. In aged rats, very little ChM-I was present in the cells and matrix of the growth plates, except where remodelling had occurred or chondrocytes had become re-activated. The rate of longitudinal growth in rats is high between 1–5 weeks, then declines until skeletal maturity at approximately 12 weeks, after which a very slow rate of growth continues until 26 weeks. In young rats, the loss of ChM-I in the hypertrophic zone was as expected for an anti-angiogenic factor, i.e. loss was required before vascular invasion could take place. However, the same did not apply to cartilage canal formation, since there was no loss of ChM-I around cartilage canals. The change in the localisation of ChM-I in mature rats, in particular the very intense immunolocalisation around hypertrophic chondrocytes, might be related to the reduced rate of growth. It is possible that rapid vascular invasion must be slowed down in these growth plates and that ChM-I prevented vascular invasion until degraded by proteases, such as MMP-9. The relative absence of ChM-I in the stationary growth plates of aged rats suggests that other anti-angiogenic factors prevent vascular invasion in these growth plates

Dysplasia Epiphysealis Hemimelica (DEH) also known as Trevor's Disease is a rare developmental disorder resulting in cartilaginous overgrowth of the epiphysis of long bones. DEH is usually diagnosed in children between two and eight years old and it is three times more often diagnosed in boys. The most reported complaints are pain, limitation in range of motion, and deformity or swelling of the affected joint. Treatment of symptomatic lesions consists of surgical resection of the lesion, resulting in good long-term results. Based on histological evaluation, DEH is often described as an osteochondroma or an osteochondroma-like lesion, although there are clinical, radiological and genetic differences between DEH and osteochondromas. To investigate the hypothesis that DEH and osteochondromas are histologically identical, two cases of DEH and two cases of osteochondromas in patients with Hereditary Multiple Osteochondroma (HMO) are compared at histological level. Tissue samples from patients with a histopathologically confirmed diagnosis of DEH were compared with two age and gender matched patients diagnosed with HMO. After tissue sampling and processing, (immuno)histological stainings were performed for Collagen type II, Collagen type X, Sox-9 and Safranin-O. Histologically, clumping of chondrocytes in a fibrillar matrix, a thick disorganized cartilage cap and ossification centres with small amounts of unresorbed cartilage were observed in DEH. In contrast, chondrocyte organisation in cartilage of osteochondromas displays characteristics of the normal growth plate. In addition, differences in expression of collagen type II, collagen type X and Sox9 were observed. Collagen type II was expressed in the extracellular matrix surrounding proliferative and hypertrophic chondrocytes in osteochondromas, while weak expression was observed in the entire cartilage cap in DEH. Collagen type X was not expressed in DEH, while expressed in the pericellular matrix surrounding hypertrophic chondrocytes in osteochondromas. Staining for Sox9 was positive in the hypertrophic chondrocytes in osteochondromas, while expressed in the nuclei of all chondrocyte clusters in DEH. Both morphological and immunohistological differences were observed in histological sections of DEH and osteochondromas. These findings reject our hypothesis, and supports the earlier observed clinical, radiological and genetic differences and implies a different aetiology between DEH and osteochondroma formation in HMO

Growth plates taken from five- to 20-week-old Japanese white rabbits were immunostained for c-Myc protein. This was localised both in the proliferating zone and upper hypertrophic zone at five weeks, whereas after ten weeks it was found mostly in the lower hypertrophic zone. The proliferating chondrocytes tended to show nuclear staining and the hypertrophic cells cytoplasmic staining, although the terminal hypertrophic chondrocytes sometimes expressed the protein in their nuclei. In the younger rabbits, c-Myc co-localised with proliferating cell nuclear antigen, whereas in the hypertrophic zone of older rabbits, it was present in some chondrocytes the nuclei of which also contained DNA breaks. Our study suggests that, in the rabbit growth plate, c-Myc is associated with different cellular processes, depending on the age and the developmental stage of the chondrocytes

Introduction and Objective. Klinefelter Syndrome (KS, karyotype 47,XXY) is the most frequent chromosomal aneuploidy in males, as well as the most common cause of infertility in men. Patients suffer from a lack of testosterone, i.e. hypergonadotropic hypogonadism provoking infertility, but KS men also show an increased predisposition to osteoporosis and a higher risk of bone fracture. In a mouse model for human KS, bone analysis of adult mice revealed a decrease in bone mass that could not be rescued by testosterone replacement, suggesting a gene dosage effect originating from the supernumerary X-chromosome on bone metabolism. Usually, X chromosome inactivation (XCI) compensates for the dosage imbalance of X-chromosomal genes between sexes. Some studies suggested that expression of genes that escape silencing of the supernumerary X-chromosome (e.g. androgen receptor) has an impact on sex differences, but may also cause pathological changes in males. As a promising new such candidate for a musculoskeletal escape gene, we identified the integral membrane protein (ITM) 2a, which is encoded on the X-chromosome and related to enchondral ossification. The aim of the project was to characterize systemic bone loss in the course of aging in our KS mouse model, and whether the supernumerary X-chromosome causes differences in expression of genes related to bone development. Materials and Methods. Bone structure of 24 month (=aged) old male wild type (WT) and 41, XXY mice (B6Ei.Lt-Y) were analysed by μCT. Afterwards bones were paraffin embedded and cut. In addition, tissue of brain, liver, kidney, lung and heart were also isolated and embedded for IHC staining. Using an anti-ITM2a antibody, expression and cellular localization of ITM2a was evaluated. IHC was also performed on musculoskeletal tissue of WT embryos (E18.5) and neonatal mice to determine possible age-related differences. Results. In 24 month old mice, the analysis of the lumbar vertebrae revealed a significantly lower BV/TV, trabecular bone volume and trabecular number in the XXY- group compared to WT. Trabecular thickness appeared lower but did not reach significance, with the cortical thickness being significantly higher in the XXY- group. High expression of ITM2a was detected in bone slices of both karyotypes in the chondrocytes inside the growth plate, as well as in megakaryocytes and leucocytes as well as endothelial cells of blood vessels inside the bone marrow. Osteocytes, along with erythrocytes and erythropoetic stem cells were negative for ITM2a. Other organs that showed ITM2a positive staining were kidney (blood vessels), heart (muscle) and brain (different structures). Liver and lung tissue were negative for ITM2a. No obvious difference in the intensity of the ITM2a-expression was observed between the WT and the XXY-karyotype. Analyses of embryotic bone tissue (WT) showed high expression of ITM2a in proliferating, hypertrophic and resting chondrocytes in the growth plates of tibia and femur. In comparison, the neonatal animals (WT) did not show any protein-expression in chondrocytes. Furthermore, within the metaphysis of both, embryotic and neonatal bones, endothelial cells and osteoblasts were ITM2a-positive. Further analyses of bones and tissues from young mice (4–6 month) are ongoing. Conclusions. Bone analyses revealed a significant reduction in trabecular bone mass along with fewer and thinner trabeculae in XXY mice compared to the WT, especially in the spine. ITM2a expression was visible in different cell types inside the bone, and in addition, different expression patterns at different stages of development (embryonic/neonatal) were observed. However, we have not found a significant difference in the quantity of ITM2a between tissues of XXY-karyotypes and WT. Further analyses of X-chromosomal encoded and therefore dysregulated modulators in XXY-karyotype mice and patients may reveal new sex chromosomal effector proteins in bone metabolism

Introduction. Current cell-based treatments and marrow stimulating techniques to repair articular cartilage defects are limited in restoring the tissue in its native composition. Despite progress in cartilage tissue engineering and chondrogenesis in vitro, the main limitation of this approach is the progression towards hypertrophy during prolonged culture in pellets or embedded in biomaterials. The objectives of this study were (A) to compare human bone marrow-derived mesenchymal stromal cells (hMSC) chondrogenesis and hypertrophy in pellet culture from single cells or cell spheroids and (B) to investigate the effect of tyramine-modified hyaluronic acid (THA) and collagen I (Col) content in composite hydrogels on the chondrogenesis and hypertrophy of encapsulated hMSC spheroids. Materials and Methods. Pellet cultures were prepared either from hMSC single cells (250’000 cells/pellet) or hMSC spheroids (282 cells/spheroid) at the same final cell concentration (250’000 cells/pellet = 887 spheroids/pellet). The effect of polymer concentration on encapsulated hMSC spheroids (887 spheroids/hydrogel) was investigated in THA-Col hydrogels (50μl) at the following concentrations (THA-Col mg/ml): Group (1) 12.5–2.5, (2) 16.7–1.7, (3) 12.5–1.7, (4) 16.7–2.5 mg/ml. All samples were cultured for 21 days in standard chondrogenic differentiation medium containing 10ng/ml TGF-β1. Chondrogenic differentiation and hypertrophy of both pellet cultures and hMSCs spheroids encapsulated in THA-Col were analysed using gene expression analysis (Aggrecan (ACAN), COL1A1, COL2A1, COL10A1), dimethylmethylene-Blue assay to quantify glycosaminoglycans (GAGs) retained in the samples and (immuno-) histological staining (Safranin-O, collagen II, aggrecan) on day 1 and day 21 (n=3 donors). Results. The culture of hMSCs in pellets based on single cells or spheroids resulted in an increase in chondrogenic-associated markers COL2A1 (2’900–3’400-fold) and ACAN (45–47-fold) compared to respective samples on day 1 in both groups. GAGs increased in spheroid pellets to 21.2±3.4 mg/ml and in single cell pellets to 20.8±6.6 mg/ml on day 21. Comparing the levels of hypertrophic markers, single cell pellets showed 7-fold and 20-fold higher expression of COL1A1 and COL10A1 than spheroid pellets on day 21. The encapsulation of hMSC spheroids in THA-Col resulted in an upregulation of chondrogenic-associated markers and GAG content in all hydrogels with differences in cell differentiation related to the Col and THA polymer ratio, while level of hypertrophy was comparable in all groups with values similar to the spheroid pellet group. Spheroids embedded in hydrogels with lower THA content (group 1 and 3) resulted in more pronounced chondrogenic phenotype marked by upregulation of COL2A1 (3’200–4’500-fold) and ACAN (152–179-fold) relative to the respective samples on day 1. Spheroids embedded in higher THA content hydrogels (group 2 and 4) showed less pronounced chondrogenesis marked by lower upregulation of COL2A1 (980–1800-fold) and ACAN (25–68-fold, relative to day 1 samples). This was confirmed by quantification of GAGs, increasing from 2.5±1.9 and 2.5±1.7 mg/ml (day 1) to 11.4±2.5 and 9.9±3.8 mg/ml on day 21 for groups 1 and 4, respectively. (Immuno-) histological stainings resulted in a more homogenous staining in lower THA content hydrogels compared to a more local matrix deposition in samples with higher THA content. Conclusion. The reduced level of hypertrophy in hMSC pellets prepared from cell spheroids compared to single cell pellets at same cell count might be related to the packing density of the cells with cells being more densely packed in single cell pellets compared to pellets from spheroids. Investigating the effect of polymer ratios on chondrogenesis, it seems that the THA content is the driving factor influencing hMSC chondrogenesis rather than Col content in THA-Col composites at comparable mechanical properties. This study highlights the feasibility to use hMSC spheroids as alternative approach to study in vitro chondrogenic differentiation and the suitability to investigate the effect of biomaterial composition on chondrogenesis and hMSC hypertrophy

Introduction: In the growth plate, chondrocyte swelling (hypertrophy) is a crucial event during endochondral ossification and bone lengthening, accounting for ~80% of the increase in bone length (. 1. ,. 3. ). The swelling is dramatic (~10x) and closely regulated. Failure of chondrocyte hypertrophy may underlie the chondrodysplasias of the vertebrate skeleton (. 1. ). However, the mechanisms which control cell swelling are poorly understood although there must be a key role for chondrocyte osmolyte transporters which are sensitive to an increase in cell volume. We have used confocal scanning laser microscopy (CLSM) to study volume regulation by living in situ growth plate chondrocytes at varying degrees of hypertrophy. Methods: Bovine growth plates were taken from the ends of young (~12d) bovine ribs. In situ growth plate chondrocytes at the proliferative through to hypertrophic stages were fluorescently-labelled (calcein-AM; 5μM), imaged (Zeiss CLSM510) and volumes determined quantitatively as described (. 2. ). An acute osmotic challenge (280-140mOsm) was delivered by perfusion to determine volume-regulatory capacity by cells in the various zones. Results: The resting volumes of proliferative and hypertrophic cells were 550±63μm. 3. and 5227±1974μm. 3. respectively. Reducing osmolarity resulted in a rapid (within ~1min) cell swelling, proliferative and hypertrophic chondrocytes increasing in volume by 126±2% and 146±5% (n=5) respectively. Chondrocytes within the proliferative zone then recovered in volume by ~60% over the following 20mins (p=0.04), whereas no volume recovery was detected in hypertrophic cells (p=0.94). Conclusions: For the increase in growth plate chondrocyte volume to produce hypertrophy it is essential that the membrane transporters which normally prevent cell swelling are suppressed, otherwise the increase in volume will be compromised. These results suggest that chondrocyte hypertrophy is associated with reduced activity of the swelling-stimulated osmolyte transporter whereas the pathway is active in proliferating chondrocytes. Changes in the activity of this pathway are likely to be an important component in the control of chondrocyte hypertrophy. It is clear that the contributions of other membrane transporters in mediating chondrocyte swelling must be identified in order to understand the overall hypertrophic process

Introduction. The hematoma occurring at a fracture site is known to play an important role in fracture healing. Previously, we demonstrated that fracture hematoma contained multilineage mesenchymal progenitor cells. On the other hand, the process of fracture healing is associated by two different mechanisms, intramembranous and endochondral. However, there are no reports proving the details about cellular analysis in the process of endochondoral ossification. Hypothesis. We hypothesized that one of the cell origins for endochondral ossification after fracture was hematoma. Materials & Methods. Fracture hematoma was obtained during osteosynthesis. Hematoma-derived cells were isolated and cultured for 5-weeks of chondrogenic induction followed by 2-weeks hypertrophic induction using pellet culture system. The pellets were analyzed histologically and immunohistochemically. The gene expression levels of chondrogenic, hypertrophic, osteogenic and angiogenic markers were measured by real-time PCR. Results. The histological and immunohistochemical analysis revealed that the Hematoma-derived cells differentiated into hypertrophic chondrocytes through chondrocytes, and finally differentiate into calcifying chondrocytes. The same trend was seen in the gene expression using real-time PCR analysis. Discussion & Conclusions. Our results suggest that fracture hematoma may be an origin of cells which play key roles in the process of endochondoral ossification during fracture healing

Osteoarthritis (OA) is a progressive and degenerative joint disease resulting in changes to articular cartilage. In focal early OA defects, autologous chondrocyte implantation (ACI) has a 2-fold failure rate due to poor graft integration and presence of inflammatory factors (e.g. Interleukin-1β). Bone marrow derived mesenchymal stem cells (MSCs) are an alternative cell source for cell-based treatments due to their chondrogenic capacity, though in vivo implantation leads to bone formation. In vivo, chondrocytes reside under an oxygen tension between 2–7% oxygen or physioxia. Physioxia enhances MSC chondrogenesis with reduced hypertrophic marker (collagen X and MMP13) expression compared to hyperoxic conditions (20% oxygen). This study sought to understand whether implantation of physioxic preconditioned MSCs improves cartilage regeneration in an early OA defect model compared to hyperoxic MSCs. Bone marrow extracted from New Zealand white rabbits (male: 5–6 months old; n = 6) was split equally for expansion under 2% (physioxia) or 20% (hyperoxia) oxygen. Chondrogenic pellets (2 × 105 cells/pellet) formed at passage 1 were cultured in the presence of TGF-β1 under their expansion conditions and measured for their wet weight and GAG content after 21 days. During bone marrow extraction, a dental drill (2.5mm diameter) was applied to medial femoral condyle on both the right and left knee and left untreated for 6 weeks. Following this period, physioxia and hyperoxia preconditioned MSCs were seeded into a hyaluronic acid (TETEC) hydrogel. Fibrous tissue was scraped and then MSC-hydrogel was injected into the right (hyperoxic MSCs) and left (physioxia MSCs) knee. Additional control rabbits with drilled defects had fibrous tissue scrapped and then left untreated without MSC-hydrogel treatment for the duration of the experiment. Rabbits were sacrificed at 6 (n = 3) and 12 (n = 3) weeks post-treatment, condyles harvested, decalcified in 10% EDTA and sectioned using a cryostat. Region of interest was identified; sections stained with Safranin-O/Fast green and evaluated for cartilage regeneration using the Sellers scoring system by three blinded observers. Physioxic culture of rabbit MSCs showed significantly shorter doubling time and greater cell numbers compared to hyperoxic culture (∗p < 0.05). Furthermore, physioxia enhanced MSC chondrogenesis via significant increases in pellet wet weight and GAG content (∗p < 0.05). Implantation of physioxic preconditioned MSCs showed significantly improved cartilage regeneration (Mean Sellers score = 7 ± 3; ∗p < 0.05) compared to hyperoxic MSCs (Sellers score = 12 ± 2) and empty defects (Sellers score = 17 ± 3). Physioxia enhances in vitro rabbit MSC chondrogenesis. Subsequent in vivo implantation of physioxia preconditioned MSCs improved cartilage regeneration in an early OA defect model compared to hyperoxic MSCs. Future studies will investigate the mechanisms for enhanced in vivo regeneration using physioxia preconditioned MSCs

The December 2015 Trauma Roundup. 360 . looks at: Delay to surgery in hip fracture; Hexapod fixators in the management of hypertrophic tibial nonunions; Thromboembolism after nailing pathological fractures; Tibial plateau fracture patterns under the spotlight; The health economic effects of long bone nonunion; Adverse outcomes in trauma; The sacral screw in children; Treating the contralateral SUFE

Summary. Indomethacin has differential effects on chondrogencic outcome depending on differentiation stage. Introduction. Heterotopic ossification (HO) is the abnormal formation of bone in soft tissues and is a frequent complication of hip replacement surgery. The standard treatment to prevent HO is administration of the NSAID indomethacin. HOs are described to develop via endochondral ossification. As it is currently unknown how indomethacin prevents HO, we aimed to define whether indomethacin might influence HO via impairing the chondrogenic phase of endochondral ossification. Materials. ATDC5, human bone marrow stem cells (hBMSCs) and rabbit periosteal agarose cultures were employed as progenitor cell models; SW1353, human articular chondrocytes and differentiated ATDC5 cells were used as matured chondrocyte cell models. All cells were cultured in the presence of (increasing) concentrations of indomethacin. The action of indomethacin was confirmed by decreased PGE. 2. levels in all experiments, and was determined by specific PGE. 2. ELISA. Gene- and protein expression analyses were employed to determine chondrogenic outcome. Results. A dose-dependent decrease in expression of Col2a1, Col10a1 and GAG content was observed when progenitor ATDC5 cells differentiating in the chondrogenic lineage were treated with increasing concentrations of indomethacin. These results were confirmed on primary hBMSCs and ex vivo periosteal agarose cultures. Even when hypertrophic differentiation of ATDC5 cells was provoked by BMP-2 (30ng/ml) the addition of indomethacin resulted in decreased hypertrophic marker expression. Interestingly, when adult chondrocytes (SW1353 and primary human articular chondrocytes) were treated with indomethacin, a clear increase in Col2a1 expression was observed. Similarly, when ATDC5 cells were differentiated for 10 days to obtain a chondrocyte phenotype and indomethacin was added from this time point onwards, low concentrations of indomethacin also resulted in increased Col2a1 expression. Conclusions. Indomethacin (dose-dependently) prevents chondrogenic and hypertrophic differentiation from progenitor cells. In addition we found thatindomethacin (in low concentrations) is able to increase the chondrogenic phenotype of maturated chondrocytes. Together, these data indicate that indomethacin has differentiation stage-dependent effects on chondrogenic differentiation and part of the HO-preventing action of indomethacin might be contributed to inhibition of chondrogenic differentiation

Purpose of the study: Data concerning the teres minor in the context of rotator cuff tears is scarce despite the fact that this muscle plays a crucial role in the event of an infraspinaus tear. The purpose of this study was to analyse the computed tomography and magnetic resonance images of the teres minor muscle in rotator cuff tears. Material and method: The aspect of the teres minor muscle was studied in 1624 shoulders with rotator cuff tears. The axial and sagittal CT or MRI aspect was noted as normal, hypertrophic, atrophic or absent (Walch classification). We searched for correlations with the tendon torn, tear mechanism (trauma, degeneration, mixed), gender, dominance, time to imaging, age at imaging, and fatty infiltration of the supraspinatus, infraspinatus and subscapularis. Results: The teres minor was noted normal in 90.9% of the shoulder, hypertrophic in 5.2%, atrophic in 3.2% and absent in 0.2%. The type of cuff tear had a significant impact (p< 0.0005). Regarding the aspect of the teres minor muscle, the highest percentages were as follows: normal for isolated supraspinatus tears, hypertrophic for supraspinatus and subscapularis tears, atrophic for posterosuperior (supraspinatus, infraspinatus ± teres minor) tears and absent for massive tears involving the supraspinatus, the infraspinatus ± the subscapularis (p< 0.0005). The correlation was statistically significant between the aspect of the teres minor and the grade of fatty infiltration of the infraspinatus (p< 0.0005) and the subscapularis (p< 0.0005). Discussion: The aspect of an atrophic or absent teres minor, which correlates with the trumpet sign in massive tears, was only found in 3.5% of the tears. There would thus be few indications for latissimus dorsi transfer to restore active external rotation

Summary Statement. This study reports that hMSC can be manipulated in order to engineer a bone organ, characterised by mature osseous and vascular components and capable to recruit, host and maintain functional HSCs. Introduction. Bone tissue engineering strategies are typically based on methods involving adult human Mesenchymal Stromal Cells (hMSC) in a process resembling intramembranous ossification. However, most bones develop and repair through endochondral ossification. In addition, endochondral ossification presents several advantages for regenerative purposes such as osteogenic activity, capability to drive formation of the Hematopoietic Stem Cell (HSC) niche, resistance to hypoxia, intrinsic vasculogenic potential and, consequently, efficiency of engraftment. In this study, we aimed at developing an endochondral bone organ model characterised by functional osseous and hematopoietic compartments by using hMSC. Materials & Methods. Expanded hMSC were seeded onto 8 mm diameter, 2 mm thick collagen sponges (UltrafoamTM, Davol Inc.), cultured for vitro under defined chondrogenic (3 weeks) and hypertrophic (2 weeks) conditions and then implanted ectopically in subcutaneous pouches in nude mice. Consistently with the normal process of bone regeneration, which requires an inflammatory environment, we added IL-1β to the hypertrophic medium and assessed its effect on in vitro mineralization, hypertrophy, extracellular matrix processing and in vivo remodeling/bone formation. Samples were analyzed by histology, IHC, Luminex® assays, ISH for human Alu repeats and µCT. Bone marrow cells, extracted after 12 weeks from the implanted samples were analyzed by flow cytometry and transplanted into lethally irradiated congenic animals to asses functionality of the engrafted bone marrow. Results. In vitro, samples showed a mineralised collar, rich in Collagen I and BSP, and a hypertrophic core, rich in proteoglycans and Collagen X. In vivo, extensive remodeling occurred, with mature vessel ingrowth (CD31+, NG2+, α-SMA+) and osteoclast (TRAP+ and MMP9+ multinucleated cells). Bone formation displayed a peculiar topography: at the periphery of the samples, perichondral bone was formed, corresponding to the in vitro pre-mineralised outer ring; in the core of the samples, endochondral bone was formed, corresponding to the in vitro non-mineralised cartilaginous areas. Human cells could be still detected after 12 weeks in vivo, mainly in the bone in the core of the samples. IL-1β resulted in (i) enhanced MMP13 endogenous activity; (ii) enhanced osteoclasts activity by increased M-CSF levels and RANKL/OPG ratio; (iii) faster vascularization; (iv) larger regions of bone marrow, possibly because of an increased synthesis of SDF1, IL-8, M-CSF and MCP-1. Murine bone marrow cells in the newly generated bone included phenotypically and functionally defined HSC at a comparable frequency than normal bones of the same mice. Discussion/Conclusion. We reported the generation of an ectopic “bone organ” with a size, structure and functionality comparable to native bones by appropriately primed hMSC. The use of hMSC and IL-1β makes this model closer to bone regeneration than to bone development. The work, provides a model useful for fundamental and translational studies on bone development and regeneration, as well as for the modeling of normal and malignant hematopoiesis

The superficial zone (SFZ) of articular cartilage has unique structural and biomechanical features, and is important for joint long-term function. Previous studies have shown that TGF-β/Alk5 signaling upregulating PRG4 expression maintains articular cartilage homeostasis. However, the exact role and molecular mechanism of TGF-β signaling in SFZ of articular cartilage homeostasis are still lacking. In this study, a combination of in vitro and in vivo approaches were used to elucidate the role of Alk5 signaling in maintaining the SFZ of articular cartilage and preventing osteoarthritis initiation. Mice with inducible cartilage SFZ-specific deletion of Alk5 were generated to assess the role of Alk5 in OA development. Alterations in cartilage structure were evaluated histologically. The chondrocyte apoptosis and cell cycle were detected by TUNEL and Edu staining, respectively. Isolation, culture and treatment of SFZ cells, the expressions of genes associated with articular cartilage homeostasis and TGF-β signaling were analyzed by qRT-PCR. The effects of TGF-β/Alk5 signaling on proliferation and differentiation of SFZ cells were explored by cells count and alcian blue staining. In addition, SFZ cells isolated from C57 mice were cultured in presence of TGF-β1 or SB505124 for 7 days and transplanted subcutaneously in athymic mice. Postnatal cartilage SFZ-specific deletion of Alk5 induced an OA-like phenotype with degradation of articular cartilage, synovial hyperplasia as well as enhanced chondrocyte apoptosis, overproduction of catabolic factors, and decreased expressions of anabolic factors in chondrocytes. qRT-PCR and IHC results confirmed that Alk5 gene was effectively deleted in articular cartilage SFZ cells. Next, the PRG4-positive cells in articular cartilage SFZ were significantly decreased in Alk5 cKO mice compared with those in Cre-negative control mice. The mRNA expression of Aggrecan and Col2 were decreased, meanwhile, expression of Mmp13 and Adamts5 were significantly increased in articular cartilage SFZ cells of Alk5 cKO mice. In addition, Edu and TUNEL staining results revealed that slow-cell cycle cell number and increase the apoptosis positive cell in articular cartilage SFZ of Alk5 cKO mice compared with Cre-negative mice, respectively. Furthermore, all groups of SFZ cells formed ectopic solid tissue masses 1 week after transplantation. Histological examination revealed that the TGF-β1-pretreated tissues was composed of small and round cells and was positive for alcian blue staining, while the SB505124-pretreated tissue contained more hypertrophic cells though it did stain with alcian blue. TGF-β/alk5 signaling is an essential regulator of the superficial layer of articular cartilage by maintaining chondrocyte number, its differentiation properties, and lubrication function. Furthermore, it plays a critical role in protecting cartilage from OA initiation

Adult articular cartilage mechanical functionality is dependent on the unique zonal organization of its tissue. Current mesenchymal stem cell (MSC)-based treatment has resulted in sub-optimal cartilage repair, with inferior quality of cartilage generated from MSCs in terms of the biochemical content, zonal architecture and mechanical strength when compared to normal cartilage. The phenotype of cartilage derived from MSCs has been reported to be influenced by the microenvironmental biophysical cues, such as the surface topography and substrate stiffness. In this study, the effect of nano-topographic surfaces to direct MSC chondrogenic differentiation to chondrocytes of different phenotypes was investigated, and the application of these pre-differentiated cells for cartilage repair was explored. Specific nano-topographic patterns on the polymeric substrate were generated by nano-thermal imprinting on the PCL, PGA and PLA surfaces respectively. Human bone marrow MSCs seeded on these surfaces were subjected to chondrogenic differentiation and the phenotypic outcome of the differentiated cells was analyzed by real time PCR, matrix quantification and immunohistological staining. The influence of substrate stiffness of the nano-topographic patterns on MSC chondrogenesis was further evaluated. The ability of these pre-differentiated MSCs on different nano-topographic surfaces to form zonal cartilage was verified in in vitro 3D hydrogel culture. These pre-differentiated cells were then implanted as bilayered hydrogel constructs composed of superficial zone-like chondro-progenitors overlaying the middle/deep zone-like chondro-progenitors, was compared to undifferentiated MSCs and non-specifically pre-differentiated MSCs in a osteochondral defect rabbit model. Nano-topographical patterns triggered MSC morphology and cytoskeletal structure changes, and cellular aggregation resulting in specific chondrogenic differentiation outcomes. MSC chondrogenesis on nano-pillar topography facilitated robust hyaline-like cartilage formation, while MSCs on nano-grill topography were induced to form fibro/superficial zone cartilage-like tissue. These phenotypic outcomes were further diversified and controlled by manipulation of the material stiffness. Hyaline cartilage with middle/deep zone cartilage characteristics was derived on softer nano-pillar surfaces, and superficial zone-like cartilage resulted on softer nano-grill surfaces. MSCs on stiffer nano-pillar and stiffer nano-grill resulted in mixed fibro/hyaline/hypertrophic cartilage and non-cartilage tissue, respectively. Further, the nano-topography pre-differentiated cells possessed phenotypic memory, forming phenotypically distinct cartilage in subsequent 3D hydrogel culture. Lastly, implantation of the bilayered hydrogel construct of superficial zone-like chondro-progenitors and middle/deep zone-like chondro-progenitors resulted in regeneration of phenotypically better cartilage tissue with higher mechanical function. Our results demonstrate the potential of nano-topographic cues, coupled with substrate stiffness, in guiding the differentiation of MSCs to chondrocytes of a specific phenotype. Implantation of these chondrocytes in a bilayered hydrogel construct yielded cartilage with more normal architecture and mechanical function. Our approach provides a potential translatable strategy for improved articular cartilage regeneration using MSCs

Purpose of the study: The process of bone lengthening involves three phases: a latence period, distraction, then healing. The healing phase required stability maintained by an external fixator (EF) for 1.16 months/cm lengthening. This time exposes the patient to serious complications. The objective is to accelerate the healing phase in order to shorten the time the patient has to wear the EF. The effect of BMP on osteogenesis in distraction remains a controversial issue. This work was conducted to evaluate the benefit provided by rhBMP-2 for healing the regenerate bone after distraction. Material and methods: Thirty-nine subadult male rabbits were selected at random. On day 0, a tibial osteotomy was performed followed by installation of a M101 EF. After the latency period of seven days, the distraction began at the rate of 0.5mm/12 h for 21 days. At day 28, at the end of distraction, a new operation was performed and three groups of 13 individuals were created at random. The first group received no material, the second a collagen type 1 sponge, and the third group a collagen type 1 sponge soaked in 100 μg/kg rhBMP-2. The animals were monitored with x-rays, absorptiometry and ultrasound for the qualitative and quantitative analysis. Histological and biomechanical analyses were performed at two months. Results: Our complication rate was 41%. Qualitative analysis of the x-rays showed, in group 3, the development of more or less voluminous and dense, sometimes hypertrophic calluses. The progression curves of the bone mineral content showed higher values in group 3. The bone mineral content curves remained nevertheless parallel for the three groups. The calluses were thus denser in group 3 but with an early peak density. Groups 1 and 2 had equivalent radiographic and absorptiometric results. The statistical analysis of the imaging findings is ongoing. The histology and biomechanical exams are being performed. Discussion: The preliminary results show that rhBMP-2 used early in the healing phase enables formation of more dense and hypertrophic calluses. rhBMP-2 does not acceleration the rate of callus formation but stimulates its mineralization. Use of a collagen sponge alone had no effect on healing. Analysis of the histological and mechanic properties observed in the three groups will provide a more precise description of the hypertrophic and strongly mineralized calluses. Conclusion: Our early results show superior bone mineralization in the treated group

Aim: There have been increasingly publications about the complicated disease of patello-femoral joints after total knee arthroplasty (TKA). We have treated soft tissue impingement under the patella after TKA by arthroscopic surgery and investigated the findings and efficacy of the treatment. Materials and Methods: 6 patients and 8 knees which showed soft tissue impingement of patello-femoral joints after TKA. Surgical arthroscopy was performed and impinging soft tissues were classified and the efficacy of arthroscopic treatment were evaluated. Results: We classified the patients with soft tissue impingement under the patella into three groups: (I) patellar clunk syndrome; the isolated fibrous nodule located suprapatellar lesion, without the other fibrous tissues causing the impingement, (II) impinging hypertrophic synovitis; generalized hypertrophic synovitis, no fibrous nodule, and (III) the combined type of (I)+(II), the suprapatellar fibrous nodule with generalized hypertrophic synovitis. Therapeutic efficacy was that in the category of type I two were good-excellent, in type II three were fair and one was poor, and in type III two were fair. Conclusions: Better results were obtained in type I (a patellar clunk syndrome) than type II (impingement synovitis)

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.

While mesenchymal stromal cells (MSCs) are a very attractive cell source for cartilage regeneration, an inherent tendency to undergo hypertrophic maturation and endochondral ossification; as well as insufficient extracellular matrix production still prevent their clinical application in cell –based cartilage repair therapies. We recently demonstrated that intermittent treatment of MSC with parathyroid hormone-related protein (PTHrP) during in vitro chondrogenesis significantly enhanced extracellular matrix deposition and concomitantly reduced hypertrophy (1) opposite to constant PTHrP treatment, which strongly suppressed chondrogenesis via the cAMP/PKA pathway (2). Since signal timing seemed to be decisive for an anabolic versus catabolic outcome of the PTHrP treatment, we here aimed to investigate the role of PTHrP pulse frequency, pulse duration and total weekly exposure time in order to unlock the full potential of PTHrP pulse application to enhance and control MSC chondrogenesis. Human bone marrow-derived MSC were subjected to in vitro chondrogenesis for six weeks. From day 7–42, cells were additionally exposed to 2.5 nM PTHrP(1–34) pulses or left untreated (control). Pulse frequency was increased from three times per week (3×6h/week) to daily, thereby maintaining either pulse duration (6h/d, total 42 h/week) or total weekly exposure time (2.6h/d, total 18 h/week). A high frequency of PTHrP-treatment (daily) was important to significantly increase extracellular matrix deposition and strongly suppress ALP activity by 87 %; independent of the pulse duration. A long pulse duration was, however, critical for the suppression of the hypertrophic marker gene IHH, while MEF2C and IBSP were significantly suppressed by all tested pulse duration and frequency protocols. COL10A1, RUNX2 and MMP13 mRNA levels remained unaffected by intermittent PTHrP. A drop of Sox9 levels and a decreased proliferation rate after 6 hours of PTHrP exposure on day 14 indicated delayed chondroblast formation. Decreased IGFBP-2, -3 and -6 expression as well as decreased IGFBP-2 protein levels in culture supernatants suggested IGF-I-related mechanisms behind anabolic matrix stimulation by intermittent PTHrP. The significant improvement of MSC chondrogenesis by the optimization of intermittent PTHrP application timing revealed the vast potential of PTHrP to suppress hypertrophy and stimulate chondrogenic matrix deposition. A treatment with PTHrP for 6 hours daily emerged as the most effective treatment mode. IGF-I and Sox-9 related mechanisms are suggested behind anabolic effects and delayed chondroblasts formation, respectively. Thus, similar to the established osteoporosis treatment, daily injections of PTHrP may become clinically relevant to support cartilage repair strategies relying on MSCs like subchondral bone microfracturing and autologous MSC implantation

A 30-year-old man presented with pain and limitation of movement of the right hip. The symptoms had failed to respond to conservative treatment. Radiographs and CT scans revealed evidence of impingement between the femoral head-neck junction and an abnormally large anterior inferior iliac spine. Resection of the hypertrophic anterior inferior iliac spine was performed which produced full painless restoration of function of the hip. Hypertrophy of the anterior inferior iliac spine as a cause of femoro-acetabular impingement has not previously been described

Introduction. The results of repair and reconstruction of lesions found in the abductor muscles and tendons during posterior approach to primary total hip arthroplasty (THA) were reported in 2018. During the course of this series it became apparent that the extent of damage in the abductor tendons and muscles usually was obscured by the hypertrophic greater trochanteric bursa, especially the deep layer adherent to the greater trochanter and abductor muscles. The purpose of this study was to evaluate the surgeon's ability to see these lesions during standard posterior approach, and also to describe the dissection necessary to fully expose them. Patients and Methods. A total of 525 patients (525 hips) underwent primary THA through posterior approach between 2009 and 2013. Fifty-four patients had mild chronic damage to the tendon. Forty-one patients had severe damage with major avulsion of the gluteus medius and minimus muscles. Results. None of the hips with normal abductor muscle structure had a dense trochanteric bursa to obscure the abductor attachment to bone, but in 35 hips (85.3%) with significant abductor tendon avulsion, the lesions in the tendons were hidden from view until the greater trochanteric bursa was dissected. Splitting the deep layer of the trochanteric bursa in line with the fibers of the abductor muscles was necessary to reveal avulsion of the tendons from the edge of the greater trochanter. Only six hips (14.6%) had abductor tendon avulsion from the greater trochanter that could be seen without removing the trochanteric bursa tissue. Conclusion. The posterior approach offered excellent exposure of the abductor mechanism during primary THA, but the avulsion defects in the gluteus medius and minimus muscles remained obscured until the outer layer of the greater trochanteric bursa was completely removed and the deep fibrous layer was dissected adequately to expose the tendon attachment to bone. For any tables or figures, please contact the authors directly

The changes occurring in ligamentum flavum in lumbar spine stenosis are a matter of long–standing controversy. More recently, some studies showed that the posterior spinal structures, including hypertrophied ligamentum flavum, play a major role in the pathogenesis of the lumbar stenosis. To investigate the pathogenesis of the degenerative changes of the ligamentum flavum occurring in lumbar spine stenosis, yellow ligament cells from patients with lumbar spine stenosis were cultured for the first time and subjected to biochemical, histochemical and immunohistochemical study. Samples of ligamentum flavum were collected from 4 patients undergoing surgery for lumbar stenosis (mean age 47.2 years). Cell cultures were obtained from each patient and maintained in Dulbecco’s modified essential medium-10% fetal calf serum. Cell characterization was histochemically (Gomori’s and von Kossa staining), immunohistochemically (anti-type I, -type II, -type III and -type X collagen, anti-S100 protein, anti-fibronectin, anti-osteonectin and anti-osteocalcin), biochemically (cAMP activity after human parathyroid hormone stimulation) assessed. Samples collected from 2 age-matched patients who underwent surgery for lumbar fractures were used as controls. Stenotic ligamentum flavum cells expressed high levels of alkaline phosphatase activity and produced a mineralized matrix rich in type I, type III and type X collagen, fibronectin, osteonectin, and osteocalcin. Stimulation with parathyroid hormone increased intracellular cAMP concentration. These findings indicate that there was significant evidence of osteoblast-like activity in these cells. Staining for type II and type X collagen, and S-100 protein reflected the proliferation of hypertrophic chondrocyte-like cells, confirmed with the co-localization of alkaline phosphatase and collagen type II. Cultures from control patients showed nor hypertrophic chondrocytic nor osteoblastic features. Our data demonstrated the presence of hypertrophic chondrocytes with an osteoblast-like activity in human stenotic ligamentum flavum. The osteoblast-like activity could have a role in the pathophysiology of the heterotopic ossification of ligamentum flavum in lumbar spine stenosis