We have developed a novel technique to analyse bone, using imaging mass cytometry (IMC) without the constraints of using immunofluorescent histochemistry. IMC can measure the expression of over 40 proteins simultaneously, without autofluorescence. We analysed mitochondrial respiratory chain (RC) protein deficiencies in human bone which are thought to contribute to osteoporosis with increasing age. Osteoporosis is characterised by reduced bone mineral density (BMD) and fragility fractures. Humans accumulate mitochondrial mutations and RC deficiency with age and this has been linked to the changing phenotype in advancing age and age-related disease. Mitochondrial mutations are detectable from the age of 30 onwards, coincidently the age BMD begins to decline. Mitochondria contain their own genome which accumulates somatic variants at around 10 times the rate of nuclear DNA. Once these mutations exceed a threshold, RC deficiency and cellular dysfunction occur. The PolgD257A/D257A mouse model expresses a proof-reading deficient version of PolgA, a mtDNA polymerase. These mice accumulate mutations 3-5 times higher than wild-type mice showing enhanced levels of age-related osteoporosis and RC deficiency in osteoblasts. Bone samples were analysed from young and old patients, developing a protocol and analysis framework for IMC in bone tissue sections to analyse osteoblasts in-situ for RC deficiency. Samples from the femoral neck of 10 older healthy volunteers aged 40 – 85 were compared with samples from young patients aged 1-19. We have identified RC complex I defect in osteoblasts from 6 of the older volunteers, complex II defects in 2 of the older volunteers, complex IV defect in just 1 older volunteer, and complex V defect in 4 of the older volunteers. These observations are consistent with the PolgD257A/D257A mouse-model and suggest that RC deficiency, due to age-related pathogenic mitochondrial DNA mutations, may play a significant role in the pathogenesis of human age-related osteoporosis

The expression of the mechanosensor, integrin αvβ3, is reduced in osteoporotic bone cells compared to controls. MLO-Y4 osteocytes experience altered mechanotransduction under estrogen deficiency and it is unknown whether this is associated with defective αvβ3 expression or signalling. The objectives of this study are to (1) investigate αvβ3 expression and spatial organisation in osteocytes during estrogen deficiency, and (2) establish whether altered responses of osteocytes under estrogen deficiency correlate to defective αvβ3 expression and functionality. MLO-Y4 cells were cultured as follows: Ctrl (no added estradiol), E+ (10nM 17β-estradiol for 5 days), and Ew (10nM 17β-estradiol for 3 days and withdrawal for 2 days). Cells were cultured with/without 0.5µM IntegriSense750 (αvβ3 antagonist). Laminar oscillatory fluid flow of 1Pa at 0.5Hz was applied for 1hr. αvβ3 content was quantified using an ELISA. The location and quantity of αvβ3 and focal-adhesions was determined by immunocytochemistry. Estrogen withdrawal under static conditions led to lower cell and focal-adhesion area (p<0.05), compared to E+ cells. Fluid flow led to higher αvβ3 content (p<0.05) in all groups, compared to static counterparts, with αvβ3 blocking altering this response. Fluid flow on Ew cells had the highest αvβ3 levels (p<0.05), but αvβ3 did not localise at focal-adhesions sites. Cell morphologies were similar after treatment with the αvβ3 antagonist to the Ew group. These results suggest there are fewer functional focal-adhesion sites at which αvβ3 integrins localise to facilitate mechanotransduction. To further understand these results, we are analysing osteocyte mechanotransduction by quantifying PGE2 and gene expression (COX-2, RANKL, OPG, SOST)

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

Nuclear factor erythroid 2–related factor 2 (Nrf2) is a crucial transcription factor to maintain cellular redox homeostasis, but is also affecting bone metabolism. As the association between Nrf2 and osteoporosis in elderly females is not fully elucidated, our aim was to shed light on the potential contribution of Nrf2 to the development of age-dependent osteoporosis using a mouse model.

Female wild-type (WT, n=18) and Nrf2-knockout (KO, n=12) mice were sacrificed at different ages (12 weeks=young mature adult, and 90 weeks=old), morphological cortical and trabecular properties of femoral bone analyzed by micro-computed tomography (µCT), and compared to histochemistry. Mechanical properties were derived from quasi-static compression tests and digital image correlation (DIC) used to analyze full-field strain distribution. Bone resorbing cells and aromatase expression by osteocytes were evaluated immunohistochemically and empty osteocyte lacunae counted in cortical bone. Wilcoxon rank sum test was used for data comparison and differences considered statistically significant at p<0.05.

When compared to old WT mice, old Nrf2-KO mice revealed a significantly reduced trabecular bone mineral density (BMD), cortical thickness (Ct.Th), cortical area (Ct.Ar), and cortical bone fraction (Ct.Ar/Tt.Ar). Surprisingly, these parameters were not different in skeletally mature young adult mice. Metaphyseal trabeculae were thin but present in all old WT mice, while no trabecular bone was detectable in 60% of old KO mice. Occurrence of empty osteocyte lacunae did not differ between both groups, but a significantly higher number of osteoclast-like cells and fewer aromatase-positive osteocytes were found in old KO mice. Furthermore, female Nrf2-KO mice showed an age-dependently reduced fracture resilience when compared to age-matched WT mice.

Our results confirmed lower bone quantity and quality as well as an increased number of bone resorbing cells in old female Nrf2-KO mice. Additionally, aromatase expression in osteocytes of old Nrf2-KO mice was compromised, which may indicate a chronic lack of estrogen in bones of old Nrf2-deficient mice. Thus, chronic Nrf2 loss seems to contribute to age-dependent progression of female osteoporosis.

Introduction. Osteonecrosis (ON) is a bone disease characterized by death of osteocytes and loss of associated hematopoietic elements usually occurring as focal lesions in weight bearing joints such as the hip. The pathophysiology of the disease is still unclear and osteonecrosis can be viewed as both a vascular and a bone disease. The number of mesenchymal stem cells (precursors of osteoblastic cells) has been shown to be depressed in patients with osteonecrosis. Also, the proliferation rate of the osteoblastic cells in the proximal femur may be depressed. These findings raised the possibility that osteonecrosis might be a disease of bone cells or bone metabolism. On this basis, we started this study to evaluate bone metabolism status among patients with osteonecrosis. Methods. In a prospective study, we evaluated 110 patients with osteonecrosis at the time of the diagnosis for vitamin D, parathormone, osteocalcin, and c-telopeptide measurements. DEXA was performed in all patients as well. We excluded from this study patients with sickle cell anemia (n=5), Gaucher disease (n=1), on hemodialysis (n=14), and who were already treated for osteoporosis (n=8). Results. There were 20 women and 90 men (mean age 47 ± 11 years). Twenty percent of the patients had unilateral osteonecrosis of the femoral head, 61 % of the patients had bilateral osteonecrosis, and 20 % had multifocal disease. Risk factors were corticosteroid and alcohol abuse. Vitamin D deficiency (<15 ng/ml) was found in 60 % of the patients and vitamin D insufficiency (15 to 30 ng/ml) was found in 15% of the patients. Secondary hyperparathyroidism (>55pg/ml) was present in only 7 patients. Patients with alcohol abuse had significantly lower vitamin D concentration than the other patients: 11.9 ± 8.7 vs. 20.8 ± 9.2 ng/ml (p=0.005). Among 90 samples, 45 showed an osteocalcin level below the normal range (<14 ng/ml). Most of the patients had a normal level of C-telopeptide. Patients with corticosteroid-associated osteonecrosis had significantly lower osteocalcin levels than others osteonecrotic patients: 14.1 ± 5.3 vs 22.7 ± 13.0 ng/ml (p=0.015). Bone mineral density measurements were obtained for 60 patients and showed a T-score < -1.5 at the lumbar site and < 1.8 at the hip. Conclusion. Patients with osteonecrosis have a high prevalence of vitamin D deficiency without secondary hyperparathyroidism. They also display a low bone turnover confirmed by low osteocalcin levels and normal levels of C-telopeptide. Osteonecrosis is also associated with severe osteopenia

Abstract

Introduction. The proteoglycan aggrecan is a major component of the cartilaginous matrices which provides resistance against compressive forces. Spontaneously occurring functional null mutations in the aggrecan gene (Acan) in various species lead to perinatal chondrodysplasia. The aim of the present study was to investigate the cellular and biomechanical properties of the cartilaginous growth plate, and the development of intervertebral disc in a novel, experimentally induced aggrecan mutant mouse strain carrying an insertion in exon 5 of the Acan gene. Methods. The novel aggrecan mutant mice were generated by inserting a loxP site into exon 5 (E5i) by homologous recombination in ES cells. Wild type and homozygous mutant (Acan-E5i/E5i) mice were analyzed by skeletal staining, histology and immunohistochemistry. Proliferation and survival were assessed by phosphorylated histone H3 immunostaining and TUNEL assay, respectively. Shape index (SI) in the proliferative zone (PZ) of the growth plate (GP) was calculated as a ratio of the long and short axes of the cells. Orientation of the PZ chondrocytes was characterized by the angle between the cell long axis and longitudinal direction of the bone growth. Imaging and stiffness measurements were performed by atomic force microscopy (AFM). Results. Acan-E5i/E5i mice are characterized by severe dwarfism, short snout, protruding tongue, cleft palate, and die at birth due to respiratory failure. On sections the cartilage of mutant mice appeared as tightly packed chondrocytes surrounded by a compressed matrix. At E18.5 and E14.5, the mutant PZ consisted of rounded (SI=1.71 at E18.5; SI=1.72 at E14.5) non-oriented chondrocytes, compared to the wild type PZ with flattened (SI=3.92 at E18.5; SI=3.90 at E14.5), columnar cells oriented with right angle to the longitudinal axis of the growth. At E13, the shape and orientation of mutant chondrocytes were similar to control. AFM at E14.5 and E18.5 demonstrated a stiffer matrix with denser collagen network in the mutant compared to wild type. The mutant cartilage had increased apoptosis and reduced proliferation rate at E18.5. The IVDs development appeared normal at E13.5-E14.5, however, the IVD was severely malformed at E18.5. Discussion. We have shown that aggrecan deficiency impairs cartilage biomechanics and results in a stiffer matrix. The altered mechanical properties might be responsible for the disorganization of mutant GP and compression of the IVD at around birth. Interestingly, the altered matrix mechanics is dispensable for early flattening and orientation of GP proliferative chondrocytes. In summary, aggrecan is essential for proper cartilage cytoarchitecture and morphogenesis by ensuring the suitable mechanical environment

To report the case of an asymptomatic simultaneous bilateral neck of femur fracture following vitamin D deficiency which was missed, misdiagnosed and treated for coexisting severe bilateral osteoarthritis knee. A male aged 62 years presented with severe osteoarthritis of both knee joints confining him to bed about eight weeks prior to presentation. The patient did not have any complaints pertaining to his hip joints/axial skeleton. Examination of the hip joints revealed only crepitus with absence of straight leg rising. Radiological survey showed bilateral displaced fracture neck of femur. He had elevated serum alkaline phosphatase; 119IU/L(N:39–117IU/L), decreased Serum 25 (OH) Vit D level;6.03ng/ml(N:7.6–75ng/ml), decreased spot urinary calcium;78mg/day(N:100–300mg/day) with normal serum calcium, phosphorus and highly raised parathormone levels;142.51pg/ml(N:12–72pg/ml). Tc-99 Bone scan showed increased radiotracer uptake in both the hip joints and knee joints. Bone Mineral Density was in favour of osteoporosis. Biopsy fromthe heads of both femurs also revealed osteoporosis. Bilateral staged total hip arthroplasty was done and he was put on Vitamin D replacement therapy. Patient was on regular monthly follow-up for intial one year and three monthly follow-up thereafter. At present with three year follow-up patient is community ambulant with a walking frame. Despite medical advice patient had denied total knee arthroplasty for osteoarthritis of his knee joints. Asymptomatic simultaneous bilateral neck of femur fracture is a rare injury and poses a diagnostic challenge to the treating orthopaedic surgeon with its bizarre clinical picture. Similar presentation of metabolic bone disease can be easily missed without a proper screening, keeping in mind a high index of suspicion for the above disorders. Besides proper clinical examination of both hip and knee joint should be performed in patients presenting with bilateral knee pain. A good functional outcome may be achieved with prompt surgical intervention and medical treatment

Histone modifications critically contribute to the epigenetic orchestration of bone development - in part by modifying accessibility of genes to transcription factors. Based on the previous finding that histone H2A deubiquitinase 2A-DUB/Mysm1 interacts with the p53-axis in hematopoiesis and tissue development, we here analyzed the molecular and cellular mechanisms of Mysm1-p53 interplay in bone development.

The bone phenotype of 4–5 week-old Mysm1-/- (MKO), Mysm1-/-p53-/- (DKO) and corresponding wildtype (WT) mice was determined using µCT and histology. Primary osteoblasts, mesenchymal stem cells (MSCs) and osteoclasts were isolated from long bones to assess cell proliferation, differentiation, apoptosis and activity. Statistics: one-way ANOVA, p<0.05.

MKO mice displayed an osteopenic bone phenotype compared to WT (BV/TV: 5.7±2.9 vs. 12.5±4.2, TbN: 1.3±0.6 vs. 2.7±0.7 1/mm, respectively), and these effects were abolished in DKO mice (BV/TV: 17.8±2.6, TbN: 3.7±0.4 1/mm). MKO mice compared to WT also showed both in vitro and in vivo disturbed osteoclast formation (in vitro: 1.5±1.2 vs. 9.9±1.8 OcN/mm2, in vivo OcN/BPm: 1.4±1.0 vs. 3.0±0.7 cells/mm, respectively) accompanied by increased apoptosis and DNA damage; additional p53 knockout attenuated these effects (7.8±1.8 OcN/mm2 and OcN/BPm: 2.2±1.0 cells/mm). Primary osteoblasts from both MKO and DKO mice showed decreased expression of the transcription factor Runx2 and of the osteogenic markers. ChIP-Seq analysis revealed direct binding of Mysm1 to Runx2 promoter regions in osteoblasts, implying that Mysm1 here regulates osteogenic differentiation. In contrast, MKO-MSCs differentiation did not differ from WT, but DKO-MSCs displayed a significantly increased expression of Alpl, Bglap and Runx2. The different effects of Mysm1-/- in MSCs and osteoblasts presumably resulted from the lower expression level of Mysm1 in MSCs in comparison to mature osteoblasts.

Thus, our data demonstrate that H2A deubiquitinase Mysm1 is essential for the epigenetic control of bone development via distinct mechanisms: 1) In osteoclasts, Mysm1 is involved in maturation of osteoclast progenitors and osteoclast survival. 2) In osteoblasts, Mysm1 directly controls Runx2 expression, thereby explaining osteopenic phenotype of MKO mice. 3) In MSCs, Mysm1 may play an inferior role due to low expression level. However, loss of p53 increases Runx2 expression during MSC differentiation, leading to normal bone formation in DKO mice.

Normal function of the patellofemoral joint is maintained by a complex interaction between soft tissues and articular surfaces. No quantitative data have been found on the relative contributions of these structures to patellar stability. Eight knees were studied using a materials testing machine to displace the patella 10 mm laterally and medially and measure the force required. Patellar stability was tested from 0° to 90° knee flexion with the quadriceps tensed to 175 N. Four conditions were examined: intact, vastus medialis obliquus relaxed, flat lateral condyle, and ruptured medial retinaculae. Abnormal trochlear geometry reduced the lateral stability by 70% at 30° flexion, while relaxation of vastus medialis obliquus caused a 30% reduction. Ruptured medial retinaculae had the largest effect at 0° flexion with 49% reduction. There was no effect on medial stability. There is a complex interaction between these structures, with their contributions to loss of lateral patellar stability varying with knee flexion.

TRIM32 is a candidate gene at the 9q33.1 genetic susceptibility locus for hip osteoarthritis (OA). Increased cartilage degradation typical of OA has previously been demonstrated in Trim32 knockout mice.

Our aim is to investigate the role of TRIM32 in human and murine articular tissue.

TRIM32 expression in human articular cartilage was examined by immunostaining. TRIM32 expression was compared in femoral head chondrocytes from patients with and without primary hip OA (n=6/group) and examined by Western blotting. Aggrecanolysis by femoral head explants from Trim32 knockout (T32KO) and wild-type (WT) mice was compared following stimulation with IL1α or retinoic acid (RA) and was assessed by DMMB assay (n=4/group). Expression of chondrocyte phenotype markers was measured by qPCR and compared between articular chondrocytes from WT and T32KO mice following catabolic (IL1α/TNFα) or anabolic (Oncostatin-M (OSM)/IGF1) stimulation.

TRIM32 expression was demonstrated in human articular cartilage; TRIM32 expression by chondrocytes was reduced in patients with hip OA (p=0.03). Greater aggrecanolysis occurred in cartilage explants from T32KO mice after treatment with no stimulation (p=0.03), IL1α (p=0.02), and RA (p=0.001). Unstimulated T32KO chondrocytes expressed reduced Col2a1 (p=8.53×10⁻⁵), and Sox9 (p=2.35×10⁻⁶). Upon IL1α treatment, T32KO chondrocytes expressed increased Col10a1 (p=0.0003). Upon anabolic stimulation, T32KO chondrocytes expressed increased Col2a1 (OSM: p=0.001; IGF: p=0.001), and reduced Sox9 (OSM: p=0.0002; IGF: p=0.0006).

These results indicate that altered TRIM32 expression in human articular tissue is associated with OA, and that Trim32 knockout results in increased cartilage degradation in murine femoral head explants. Predisposition to cartilage degeneration with reduced Trim32 expression may involve increased chondrocyte hypertrophy upon catabolic cytokine stimulation and dysregulation of Col2a1 and Sox9 expression upon anabolic stimulation.

Neoangiogenesis drives the replacement of mineralised cartilage by trabecular bone during bone growth regulated by molecules like e.g. VEGF, OPG and RANKL. The Heparan sulfate proteoglycan Syndecan-1 (Sdc1) plays a role in the interaction of osteoclasts and osteoblasts and the development of blood vessels. We expected Sdc1 to have an influence on bone structure and vessel development. Therefore, bone structure and angiogenesis at the growth plate in mice was compared and the influence of Syndecan-1 deficiency was characterised. Animals: Femura of male and female C57BL/6 WT (5♀, 6♂) and Sdc1-/- (9♀, 5♂) mice were used for native bone analysis at 4 month age. Histology: Bone structure was analysed using microCT scans with a resolution of 9µm. Vascularisation was visualised using an anti-Endomucin antibody in 80µm thick cryosections. In vitro angiogenesis: Bone marrow isolates were used to generate endothelial progenitor cells by sequential cultivation on fibronectin. Microvessel development was analysed 4h after plating on matrigel. Bone structure in male Sdc1 deficient mice was significantly reduced compare to male WT, whereas female mice of both genotypes did not differ. Sdc1 deficient mice at the age of 4 month showed a high decrease in the number of vessel bulbs at the chondro-osseous border (growth plate) compared to WT mice. However, no sex related differences were shown. Quantification of microvessel outgrowth of endothelial cells revealed a decreased amount of sprouting, but increased length of microvessels of Sdc1-/- cells compared to WT. Syndecan-1 has a significant impact on neoangiogenesis at the chondro-osseous border of the native bone, but the impact of Syndecan-1 deficiency on the loss of bone structure was significantly higher in male mice. This emphasises the importance to further characterise the function of Syndecan-1 regulated processes during enchondral ossification in a sex dependent manner

Duchenne muscular dystrophy (DMD) is a prevalent childhood neuromuscular disease characterized by progressive skeletal and cardiac muscle degeneration due to dystrophin protein deficiency. Despite ongoing drug development efforts, no cure exists, with limited success in preclinical studies. To expedite DMD drug development, we introduce an innovative organ-on-a-chip (OOC) platform. This microfluidic device sustains up to six 3D patient-derived skeletal muscle tissues, enabling real-time evaluation of anti-DMD treatments. Our in vitro model recreates myotube integrity loss, a hallmark of DMD, by encapsulating myogenic precursors in a fibrin-composite matrix using a PDMS casting mold. Continuous contractile regimes mimic sarcolemmal instability, monitored through tissue contractibility and Creatine Kinase (CK) levels—an established marker of muscle damage. We further enhance our platform with a nanoplasmonic CK biosensor, enabling rapid, label-free, and real-time sarcolemmal damage assessment. Combining these elements, our work demonstrates the potential of OOCs in accelerating drug development for DMD and similar neuromuscular disorders

Introduction and Objective. Global prevalence of obesity has risen almost three-fold between 1975 and 2016. Alongside the more well-known health implications of obesity such as cardiovascular disease, cancer and type II diabetes, is the effect of male obesity on testosterone depletion and hypogonadism. Hypogonadism is a well-known contributor to the acceleration of bone loss during aging, and obesity is the single biggest risk factor for testosterone deficiency in men. Understanding the micro and macro structural changes to bone in response to testosterone depletion in combination with a high fat ‘Western’ diet, will advance our understanding of the relationship between obesity and bone metabolism. This study investigated the impact of surgically induced testosterone depletion and subsequent testosterone treatment upon bone remodelling in mice fed a high fat diet. Materials and Methods. Male ApoE. −/−. mice were split into 3 groups at 7 weeks of age and fed a high fat diet: Sham surgery with placebo treatment, orchiectomy surgery with placebo treatment, and orchiectomy surgery with testosterone treatment. Surgeries were performed at 8 weeks of age, followed by fortnightly testosterone treatment via injection. Mice were sacrificed at 25 weeks of age. Tibiae were collected and scanned ex-vivo at 4.3μm on a SkyScan 1272 Micro-CT scanner (Bruker). Left tibiae were used for assessment of trabecular and cortical Volumes of Interest (VOIs) 0.2mm and 1.0mm respectively from the growth-plate bridge break. Tibiae were subsequently paraffin embedded and sectioned at 4μm prior to immunohistochemical evaluation of alkaline phosphatase. Results. Trabecular bone volume and mineral density were significantly reduced in orchiectomised mice compared to sham-operated controls; and these parameters were normalised to control levels in orchiectomised mice treated with testosterone. In contrast, Trabecular thickness was significantly higher in testosterone depleted animals. Cortical bone parameters and body weights did not significantly differ between groups. Levels of alkaline phosphatase did not differ significantly in cortical or trabecular osteoblasts between groups. Conclusions. Findings suggest that testosterone deficiency significantly reduces trabecular bone parameters, and testosterone therapy may be a useful intervention for the loss of bone mass in testosterone deficient males. These results indicate that testosterone therapy may be useful for the treatment of trabecular bone frailty in testosterone deficient males. Observed changes in trabecular bone do not appear to be due to decreased mineralisation caused by osteoblast alkaline phosphatase. Ongoing work includes histology analysis to elucidate the mechanisms underpinning the changes seen in the bones of testosterone deficient animals

Introduction and Objective. The surgical strategy for acetabular component revision is determined by available host bone stock. Acetabular bone deficiencies vary from cavitary or segmental defects to complete discontinuity. For segmental acetabular defects with more than 50% of the graft supporting the cup it is recommended the application of reinforcement ring or ilioischial antiprotrusio devices. Acetabular reconstruction with the use of the antiprotrusion cage (APC) and allografts represents a reliable procedure to manage severe periprosthetic deficiencies with highly successful long-term outcomes in revision arthroplasty. Objective. We present our experience, results, critical issues and technical innovations aimed at improving survival rates of antiprotrusio cages. Materials and Methods. From 2004 to 2019 we performed 69 revisions of the acetabulum using defrosted morcellized bone graft and the Burch Schneider anti-protrusion cage. The approach was direct lateral in 25 cases, direct anterior in 44. Patients were re-evaluated with standard radiography and clinical examination. Results. Eight patients died from causes not related to surgery, and two patients were not available for follow up. Five patients were reviewed for, respectively, non-osseointegration of the ring, post-traumatic loosening with rupture of the screws preceded by the appearance of supero-medial radiolucency, post-traumatic rupture of the distal flange, post-traumatic rupture of the cemented polyethylene-ceramic insert, and dislocation treated with new dual-mobility insert. Among these cases, the first three did not show macroscopic signs of osseointegration of the ring, and the only areas of stability were represented by the bone-cement contact at the holes in the ring. Although radiographic studies have shown fast remodeling of the bone graft and the implant survival range from 70% to 100% in the 10-year follow up, the actual osseointegration of the ring has yet to be clarified. To improve osseointegration of the currently available APC whose metal surface in contact with the bone is sandblasted, we combined the main features of the APC design long validated by surgical experience with the 3D-Metal Technology for high porosity of the external surface already applied to and validated with the press fit cups. The new APC design is produced with the 3D-Metal technology using Titanium alloy (Ti6Al4V ELI) that Improves fatigue resistance, primary stability and favorable environment for bone graft ingrowth. We preview the results of the first cases with short-term follow up. Conclusions. Acetabular reconstruction with impacted morcellized bone graft and APC is a current and reliable surgical technique that allows the restoration of bone loss with a high survival rate of the implant in the medium to long term. The new 3D Metal Cage is designed to offer high friction for the initial stability. The high porosity of the 3D Metal structure creates a favorable environment for bone growth, thus providing valid secondary fixation reproducing the results achieved with the 3D metal press fit cup

Renal Osteodystrophy is a type of metabolic bone disease characterized by bone mineralization deficiency due to electrolyte and endocrine abnormalities. Patients with chronic kidney disease (CKD) are more likely to experience falls and fractures due to renal osteodystrophy and the high prevalence of risk factors for falls. Treatment involves medical management to resolve the etiology of the underlying renal condition, as well as management (and prevention) of pathological fractures. A 66-year-old female patient, with severe osteoporosis and chronic kidney disease undergoing haemodialysis, has presented with multiple fractures along the years. She was submitted to bilateral proximal femoral nailing as fracture treatment on the left and prophylactically due to pathological bone injury on the right, followed by revision of the left nail with a longer one after varus angulation and fracture distal to the nail extremity. Meanwhile, the patient suffered a pathological fracture of the radial and cubital diaphysis and was submitted to conservative treatment with cast, with consolidation of the fracture. Posteriorly, she re-fractured these bones after a fall and repeated the conservative treatment. Clinical management: There is a multidisciplinary approach to manage the chronic illness of the patient, including medical management to resolve the etiology and consequences of her chronic kidney disease, pain control, conservative or surgical fracture management and prevention of falls. The incidence of chronic renal disease is increasing and the patients with this condition live longer than previously and are more physically active. Thus, patients may experience trauma as a direct result of increased physical activity in a setting of weakened pathologic bone. Their quality of life is primarily limited by musculoskeletal problems, such as bone pain, muscle weakness, growth retardation, and skeletal deformity. A multidisciplinary approach is required to treat these patients, controlling their chronic diseases, managing fractures and preventing falls

The aim of this study was to perform a systematic review of the literature on Gustilo-Anderson (GA) type IIIB open tibial shaft (AO-42) injuries to determine the consistency of reporting in the literature. A search of PubMed, EMBASE and Cochrane Central Register of Controlled Trials was performed to identify relevant studies published from January 2000 to January 2021 using the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) statement. The study was registered using the PROSPERO International prospective register of systematic reviews. Patient/injury demographics, management and outcome reporting were recorded. There were 32 studies that met the inclusion criteria with a total of 1,947 patients (70.3% male, 29.7% female). There were 6 studies (18.8%) studies that reported on comorbidities and smoking, with mechanism of injury reported in 22 (68.8%). No studies reported on all operative criteria included, with only three studies (9.4%) reporting for time to antibiotics, 14 studies (43.8%) for time from injury to debridement and nine studies (28.1%) for time to definitive fixation. All studies reported on the rate of deep infection, with a high proportion documenting union rate (26/32, 81.3%). However, only two studies reported on mortality or on other post-operative complications (2/32, 6.3%). Only 12 studies (37.5%) provided any patient reported outcomes. This study has demonstrated a deficiency and a lack of standardized variable and outcome reporting in the orthopaedic literature for Gustilo-Anderson type IIIB open tibial shaft fractures. We propose a future international collaborative Delphi process is needed to standardize

Traumatic acute or chronic tendon injuries are a wide clinical problem in modern society, resulting in important economic burden to the health system and poor quality of life in patients. Due to the low cellularity and vascularity of tendon tissue the repair process is slow and inefficient, resulting in mechanically, structurally, and functionally inferior tissue. Tissue engineering and regenerative medicine are promising alternatives to the natural healing process for tendon repair, especially in the reconstruction of large damaged tissues. The aim of TRITONE project is to develop a smart, bioactive implantable 3D printed scaffold, able to reproduce the structural and functional properties of human tendon, using FDA approved materials and starting from MSC and their precursor, MPC cell mixtures from human donors. Total cohort selected in the last 12 months was divided in group 1 (N=20) of subjects with tendon injury and group 2 (N=20) of healthy subject. Groups were profiled and age and gender matched. Inclusion criteria were age>18 years and presence of informed consent. Ongoing pregnancy, antihypertensive treatment, cardiovascular diseases, ongoing treatment with anti-aggregants, acetylsalicylic-acid or lithium and age<18 years were exclusion criteria. Firstly, we defined clinical, biological, nutritional life style and genetic profile of the cohort. The deficiency of certain nutrients and sex hormonal differences were correlated with tendon-injured patients. It was established the optimal amount of MPC/MSC human cell (collected from different patients during femoral neck osteotomy). Finally, most suitable biomaterials for tendon regeneration and polymer tendon-like structure were identified. Hyaluronic acid, chemical surface and soft-molecular imprinting (SOFT-MI) was used to functionalize the scaffold. These preliminary results are promising. It will be necessary to enroll many more patients to identify genetic status connected with the onset of tendinopathy. The functional and structural characterization of smart bioactive tendon in dynamic environment will represent the next project step

The management of comminuted metaphyseal fractures is a technical challenge and satisfactory outcomes of such fixations often remain elusive. The small articular fragments and bone loss often make it difficult for standard fixation implants for proper fixation. We developed a novel technique to achieve anatomical reduction in multiple cases of comminuted metaphyseal fractures at different sites by employing the cantilever mechanism with the help of multiple thin Kirschner wires augmented by standard fixation implants. We performed a retrospective study of 10 patients with different metaphyseal fractures complicated by comminution and loss of bone stock. All patients were treated with the help of cantilever mechanism using multiple Kirschner wires augmented by compression plates. All the patients were operated by the same surgeon between November 2020 to March 2021 and followed up till March 2023. Surgical outcomes were evaluated according to the clinical and radiological criteria. A total of 10 patients were included in the study. Since we only included patients with highly unstable and comminuted fractures which were difficult to fix with traditional methods, the number of patients in the study were less. All 10 patients showed satisfactory clinical and radiological union at the end of the study with good range of motion. One of the patient in the study had post-operative wound complication which was managed conservatively with regular dressings and oral antibiotics. Comminuted metaphyseal fractures might differ in pattern and presentation with every patient and there can be no standard treatment for all. The cantilever technique of fracture fixation is based on the principle of cantilever mechanism used in bridges and helps achieve good anatomical reduction and fixation. It provides a decent alternative when standard modes of fixation don't give desired result owing to comminuted nature of fractures and deficiency of bone stock

Introduction and Objective. Neoangiogenesis drives the replacement of mineralized cartilage by trabecular bone during bone growth regulated by molecules like e.g. VEGF, OPG and RANKL and the close interaction of progenitors of osteoblasts, chondrocytes, endothelial cells and osteoclasts/chondroclasts. The Heparan sulfate proteoglycan Syndecan-1 (Sdc-1) plays a role in the interaction between osteoclasts and osteoblasts and the development of blood vessels. As the processes of osteogenesis and angiogenesis are closely related to each other in bone, we expected Sdc-1 to have an influence on vessel structure during aging. Therefore, angiogenesis at the growth plate in mice of different ages was compared and the influence of Syndecan-1 deficiency was characterized. Materials and Methods. Animals: C57BL/6 (WT) and Sdc1−/− mice were used for native bone analysis at 4, 12 and 18 month age. Femura were dissected, cryoprotected and embedded. Histology: Embedded bones were sectioned into 80um thick slices so that the 3D network of the vascularization of the bone could be visualized using an anti-Endomucin antibody and DAPI as counter staining. For semi-automatical quantification of the vessel bulbs we used a custom made software. In vitro angiogenesis: For aortic ring assay, aortic tissue was isolated from 4 month old mice, cut into 0.5mm rings and embedded in collagen type I matrix. Microvessel outgrowth was quantified after 6 days of culture. Results. We verified our custom-made software using slices of WT mice and showed that there is no variation of the number of bulbs with regard to the width of the growth plate in periphery versus center zones in all age groups which indicates a homogeneous distribution of angiogenesis throughout the interface of cartilage to newly forming bone. Furthermore, in both, WT and Sdc-1 deficient mice the number of bulbs decreased significantly with age. However, Sdc-1 knockout mice at the age of 4 and 12 month showed a highly significant decrease in angiogenesis close to the growth plate compared to WT mice, whereas in older mice these differences were gone. Quantification of microvessel outgrowth of aortic tissue revealed a significant decrease in number of vessels from rings taken from Syndecan-1 deficient mice compared to WT mice. Conclusions. Syndecan-1 has a significant impact on neoangiogenesis in vitro and in vivo during aging as demonstrated at the chondro-osseous border of the native bone, emphasizing the importance to further characterize the function of Syndecan-1 regulated processes during enchondral ossification