Aims. Given the possible radiation damage and inaccuracy of radiological investigations, particularly in children, ultrasound and superb microvascular imaging (SMI) may offer alternative methods of evaluating new bone formation when limb lengthening is undertaken in paediatric patients. The aim of this study was to assess the use of ultrasound combined with SMI in monitoring new bone formation during limb lengthening in children. Methods. In this retrospective cohort study, ultrasound and radiograph examinations were performed every two weeks in 30 paediatric patients undergoing limb lengthening. Ultrasound was used to monitor new bone formation. The number of vertical vessels and the blood flow resistance index were compared with those from plain radiographs. Results. We categorized the new bone formation into three stages: stage I (early lengthening), in which there was no obvious callus formation on radiographs and ultrasound; stage II (lengthening), in which radiographs showed low-density callus formation with uneven distribution and three sub-stages could be identified on ultrasound: in Ia punctate callus was visible; in IIb there was linear callus formation which was not yet connected and in IIc there was continuous linear callus. In stage III (healing), the bone ends had united, the periosteum was intact, and the callus had disappeared, as confirmed on radiographs, indicating healed bone. A progressive increase in the number of vertical vessels was noted in the early stages, peaking during stages IIb and IIc, followed by a gradual decline (p < 0.001). Delayed healing involved patients with a prolonged stage IIa or those who regressed to stage IIa from stages IIb or IIc during lengthening. Conclusion. We found that the formation of new bone in paediatric patients undergoing limb lengthening could be reliably evaluated using ultrasound when combined with the radiological findings. This combination enabled an improved assessment of the prognosis, and adjustments to the lengthening protocol. While SMI offered additional insights into angiogenesis within the new bone, its role primarily contributed to the understanding of the microvascular environment rather than directly informing adjustments of treatment. Cite this article: Bone Joint J 2024;106-B(7):751–758

Aims

Alcoholism is a well-known detrimental factor in fracture healing. However, the underlying mechanism of alcohol-inhibited fracture healing remains poorly understood.

Introduction: The mechanobiology and response of bone formation to strain under physiological loading is well established, however investigation into exceedingly soft scaffolds relative to cancellous bone is limited. In this study we designed and 3D printed mechanically-optimised low-stiffness implants, targeting specific strain ranges inducing bone formation and assessed their biological performance in a pre-clinical in vivo load-bearing tibial tuberosity advancement (TTA) model. The TTA model provides an attractive pre-clinical framework to investigate implant osseointegration within an uneven loading environment due to the dominating patellar tendon force. A knee finite element model from ovine CT data was developed to determine physiological target strains from simulated TTA surgery. We 3D printed low-stiffness Ti wedge osteotomy implants with homogeneous stiffness of 0.8 GPa (Ti1), 0.6 GPa (Ti2) and a locally-optimised design with a 0.3 GPa cortex and soft 0.1 GPa core (Ti3), for implantation in a 12-week ovine tibial advancement osteotomy (9mm). We quantitatively assessed bone fusion, bone area, mineral apposition rate and bone formation rate. Optimised Ti3 implants exhibited evenly high strains throughout, despite uneven wedge osteotomy loading. We demonstrated that higher strains above 3.75%, led to greater bone formation. Histomorphometry showed uniform bone ingrowthin optimised Ti3 compared to homogeneous designs (Ti1 and Ti2), and greater bone-implant contact. The greatest bone formation scores were seen in Ti3, followed by Ti2 and Ti1. Results from our study indicate lower stiffness and higher strain ranges than normally achieved in Ti scaffolds stimulate early bone formation. By accounting for loading environments through rational design, implants can be optimised to improve uniform osseointegration. Design and 3D printing of exceedingly soft titanium orthopaedic implants enhance strain induced bone formation and have significant importance in future implant design for knee, hip arthroplasty and treatment of large load-bearing bone defects

Heterotopic ossification (HO) is defined as aberrant bone formation in extraskeletal locations. In this process, local stromal cells of mesenchymal origin abnormally differentiate, resulting in pathologic cartilage and bone matrix deposition. However, the specific cell type and mechanisms beyond this process are not well understood, in part due to the heterogeneity of progenitor cells involved. Here, a combination of single cell RNA sequencing (scRNA-Seq) and lineage tracing, defined the extent to which synovial / tendon sheath progenitor cells contribute to HO. For this purpose, a Tppp3 (tubulin polymerization-promoting protein family member 3) inducible reporter model was used, in combination with either Scx (Scleraxis) or Pdgfra (Platelet derived growth factor receptor alpha) reporter animals. Both arthroplasty-induced and tendon injury-mouse experimental HO models were utilized. ScRNA-Seq of tendon-induced traumatic HO suggested that Tppp3 is a progenitor cell marker for either osteochondral or tendon or cells. After HO induction, Tppp3 reporter+ cell population expanded in number and contributed to cartilage and bone formation in tendon and joint-associated HO. Using double reporter animals, we found that both Pdgfra+Tppp3+ and Pdgfra+Tppp3- progenitor cells produced HO-associated cartilage. Finally, the examination of human samples showed a significant population of TPPP3+ cells overlapping with osteogenic markers in areas of HO. Overall, these results provide novel observations that peritenon and synovial progenitor cells undergo abnormal osteochondral differentiation and contribute to heterotopic bone formation after trauma

Polyetheretherketone (PEEK) interbody fusion cages combined with autologous bone graft is the current clinical gold standard treatment for spinal fusion, however, bone graft harvest increases surgical time, risk of infection and chronic pain. We describe novel low-stiffness 3D Printed titanium interbody cages without autologous bone graft and assessed their biological performance in a pre-clinical in vivo interbody fusion model in comparison to the gold standard, PEEK with graft. Titanium interbody spacers were 3D Printed with a microporous (Ti1: <1000μm) and macroporous (Ti2: >1000μm) design. Both Ti1 and Ti2 had an identical elastic modulus (stiffness), and were similar to the elastic modulus of PEEK. Interbody fusion was performed on L2-L3 and L4-L5 vertebral levels in 24 skeletally mature sheep using Ti1 or Ti2 spacers, or a PEEK spacer filled with iliac crest autograft, and assessed at 8 and 16 weeks. We quantitatively assessed bone fusion, bone area, mineral apposition rate and bone formation rate. Functional spinal units were biomechanically tested to analyse range of motion, neutral zone, and stiffness. Results: Bone formation in macroporous Ti2 was significantly greater than microporous Ti1 treatments (p=.006). Fusion scores for Ti2 and PEEK demonstrated greater rates of bone formation from 8 to 16 weeks, with bridging rates of 100% for Ti2 at 16 weeks compared to just 88% for PEEK and 50% for Ti1. Biomechanical outcomes significantly improved at 16 versus 8 weeks, with no significant differences between Ti and PEEK with graft. This study demonstrated that macroporous 3D Printed Ti spacers are able to achieve fixation and arthrodesis with complete bone fusion by 16 weeks without the need for bone graft. These significant data indicate that low-modulus 3D Printed titanium interbody cages have similar performance to autograft-filled PEEK, and could be reliably used in spinal fusion avoiding the complications of bone graft harvesting

Aims. Several artificial bone grafts have been developed but fail to achieve anticipated osteogenesis due to their insufficient neovascularization capacity and periosteum support. This study aimed to develop a vascularized bone-periosteum construct (VBPC) to provide better angiogenesis and osteogenesis for bone regeneration. Methods. A total of 24 male New Zealand white rabbits were divided into four groups according to the experimental materials. Allogenic adipose-derived mesenchymal stem cells (AMSCs) were cultured and seeded evenly in the collagen/chitosan sheet to form cell sheet as periosteum. Simultaneously, allogenic AMSCs were seeded onto alginate beads and were cultured to differentiate to endothelial-like cells to form vascularized bone construct (VBC). The cell sheet was wrapped onto VBC to create a vascularized bone-periosteum construct (VBPC). Four different experimental materials – acellular construct, VBC, non-vascularized bone-periosteum construct, and VBPC – were then implanted in bilateral L4-L5 intertransverse space. At 12 weeks post-surgery, the bone-forming capacities were determined by CT, biomechanical testing, histology, and immunohistochemistry staining analyses. Results. At 12 weeks, the VBPC group significantly increased new bone formation volume compared with the other groups. Biomechanical testing demonstrated higher torque strength in the VBPC group. Notably, the haematoxylin and eosin, Masson’s trichrome, and immunohistochemistry-stained histological results revealed that VBPC promoted neovascularization and new bone formation in the spine fusion areas. Conclusion. The tissue-engineered VBPC showed great capability in promoting angiogenesis and osteogenesis in vivo. It may provide a novel approach to create a superior blood supply and nutritional environment to overcome the deficits of current artificial bone graft substitutes. Cite this article: Bone Joint Res 2023;12(12):722–733

Aims. Acquired heterotopic ossification (HO) is a debilitating disease characterized by abnormal extraskeletal bone formation within soft-tissues after injury. The exact pathogenesis of HO remains unknown. It was reported that BRD4 may contribute to osteoblastic differentiation. The current study aims to determine the role of BRD4 in the pathogenesis of HO and whether it could be a potential target for HO therapy. Methods. Achilles tendon puncture (ATP) mouse model was performed on ten-week-old male C57BL/6J mice. One week after ATP procedure, the mice were given different treatments (e.g. JQ1, shMancr). Achilles tendon samples were collected five weeks after treatment for RNA-seq and real-time quantitative polymerase chain reaction (RT-qPCR) analysis; the legs were removed for micro-CT imaging and subsequent histology. Human bone marrow mesenchymal stem cells (hBMSCs) were isolated and purified bone marrow collected during surgeries by using density gradient centrifugation. After a series of interventions such as knockdown or overexpressing BRD4, Alizarin red staining, RT-qPCR, and Western Blot (Runx2, alkaline phosphatase (ALP), Osx) were performed on hBMSCs. Results. Overexpression of BRD4 enhanced while inhibition of Brd4 suppressed the osteogenic differentiation of hBMSCs in vitro. Overexpression of Brd4 increased the expression of mitotically associated long non-coding RNA (Mancr). Downregulation of Mancr suppressed the osteoinductive effect of BRD4. In vivo, inhibition of BRD4 by JQ1 significantly attenuated pathological bone formation in the ATP model (p = 0.001). Conclusion. BRD4 was found to be upregulated in HO and Brd4-Mancr-Runx2 signalling was involved in the modulation of new bone formation in HO. Cite this article: Bone Joint Res 2021;10(10):668–676

Aims. To develop an early implant instability murine model and explore the use of intermittent parathyroid hormone (iPTH) treatment for initially unstable implants. Methods. 3D-printed titanium implants were inserted into an oversized drill-hole in the tibiae of C57Bl/6 mice (n = 54). After implantation, the mice were randomly divided into three treatment groups (phosphate buffered saline (PBS)-control, iPTH, and delayed iPTH). Radiological analysis, micro-CT (µCT), and biomechanical pull-out testing were performed to assess implant loosening, bone formation, and osseointegration. Peri-implant tissue formation and cellular composition were evaluated by histology. Results. iPTH reduced radiological signs of loosening and led to an increase in peri-implant bone formation over the course of four weeks (timepoints: one week, two weeks, and four weeks). Observational histological analysis shows that iPTH prohibits the progression of fibrosis. Delaying iPTH treatment until after onset of peri-implant fibrosis still resulted in enhanced osseointegration and implant stability. Despite initial instability, iPTH increased the mean pull-out strength of the implant from 8.41 N (SD 8.15) in the PBS-control group to 21.49 N (SD 10.45) and 23.68 N (SD 8.99) in the immediate and delayed iPTH groups, respectively. Immediate and delayed iPTH increased mean peri-implant bone volume fraction (BV/TV) to 0.46 (SD 0.07) and 0.34 (SD 0.10), respectively, compared to PBS-control mean BV/TV of 0.23 (SD 0.03) (PBS-control vs immediate iPTH, p < 0.001; PBS-control vs delayed iPTH, p = 0.048; immediate iPTH vs delayed iPTH, p = 0.111). Conclusion. iPTH treatment mediated successful osseointegration and increased bone mechanical strength, despite initial implant instability. Clinically, this suggests that initially unstable implants may be osseointegrated with iPTH treatment. Cite this article: Bone Joint Res 2022;11(5):260–269

Aim. Bone regeneration following the treatment of Staphylococcal bone infection or osteomyelitis is challenging due to the ability of Staphylococcus aureus to invade and persist within bone cells, which could possibly lead to antimicrobial tolerance and incessant bone destruction. Here, we investigated the influence of Staphylococcal bone infection on osteoblasts metabolism and function, with the underlying goal of determining whether Staphylococcus aureus-infected osteoblasts retain their ability to produce extracellular mineralized organic matrix after antibiotic treatment. Method. Using our in vitro infection model, human osteoblasts-like Saos-2 cells were infected with high-grade Staphylococcus aureus EDCC 5055 strain, and then treated with 8 µg/ml rifampicin and osteogenic stimulators up to 21-days. Results. Immunofluorescence and transmission electron microscopic (TEM) imaging demonstrated the presence of intracellular bacteria within the infected osteoblasts as early as 2 hours post-infection. TEM micrographs revealed intact intracellular bacteria with dividing septa indicative of active replication. The infected osteoblasts showed significant amounts of intracellular bacteria colonies and alteration in metabolic activity compared to the uninfected osteoblasts (p≤0.001). Treatment of S. aureus-infected osteoblasts with a single dose of 8 µg/ml rifampicin sufficiently restored the metabolic activity comparative to the uninfected groups. Alizarin red staining and quantification of the rifampicin-treated infected osteoblasts revealed significantly lower amount of mineralized extracellular matrix after 7-days osteogenesis (p<0.05). Interestingly, prolonged osteogenic stimulation and rifampicin-treatment up to 21 days improved the extracellular matrix mineralization level comparable to the rifampicin-treated uninfected group. However, the untreated (native) osteoblasts showed significantly more quantity of mineral deposits (p≤0.001). Ultrastructural analysis of the rifampicin-treated infected osteoblasts at 21-days osteogenesis revealed active osteoblasts and newly differentiated osteocytes, with densely distributed calcium crystal deposits within the extracellular organic matrix. Moreover, residual colony of dead bacteria bodies and empty vacuoles of the fully degraded bacteria embedded within the mineralized extracellular matrix. Gene expression level of prominent bone formation markers, namely RUNX2, COL1A1, ALPL, BMP-2, SPARC, BGLAP, OPG/RANKL showed no significant difference between the infected and uninfected osteoblast at 21-days of osteogenesis. Conclusions. Staphylococcus aureus bone infection can drastically impair osteoblasts metabolism and function. However, treatment with potent intracellular penetrating antibiotics, namely rifampicin restored the metabolic and bone formation activity of surviving osteoblasts. Delay in early osteogenesis caused by the bacterial infection was significantly improved over time after successful intracellular bacteria eradication

Aim. Antibiotic-loaded biomaterials are often used in dead space management after excision of infected bone. This study assessed the chronological progression of new bone formation in infected defects, filled only with an absorbable, osteoconductive bone void filler with Gentamicin (1). Method. 163 patients were treated for osteomyelitis or infected fractures with a single-stage excision, implantation of antibiotic carrier, stabilisation and wound closure. All had Cierny & Mader Type III (n=128) or Type IV (n=35) infection. No bone grafting was performed in any patient. Patients were followed up for a minimum of 12 months (mean 21.4 months; 12–56). Bone void filling was assessed on serial digitised, standardized radiographs taken immediately after surgery, at 6 weeks, 3, 6 and 12 months and then yearly. Data on defect size, location, degree of void filling, quality of the bone-biomaterial interface and material leakage were collected. Bone formation was calculated at final follow-up, as a percentage of initial defect volume, by determining the bone area on AP and lateral radiographs to the nearest 5%. Results. 138 patients had adequate radiographs for assessment. Infection was eradicated in 95.7%. 2.5% of patients suffered a fracture during follow-up. Overall, bone formation was good (mean 73.8% defect filling), with one quarter of patients having complete defect filling and 87% having more than 50% of the defect healed. Bone formed better in metaphyseal defects compared to diaphyseal defects (mean 79% filling vs 66%; p<0.02). Good interdigitation of the biomaterial with the host bone, seen on the initial radiograph, was associated with more bone formation (77% vs 69%; p=0.021). Leakage of the biomaterial out of the defect reduced mean bone formation from 77% to 62% (p=0.006). 38 cases had radiographs more than 2 years after implantation. In 24 (63.2%), bone formation continued to increase after the first year radiograph. Conclusion. This biomaterial was effective in allowing significant amounts of bone to form in the defect. This removed the need for bone grafting in this series, with a low risk of fracture or recurrent infection. However, bone formation is affected by the site of the lesion and the adequacy of filling at surgery. It is important to achieve good contact between the bone surface and the biomaterial at operation. Bone formation is slow and progresses for at least 2 years after implantation, in two thirds of patients

Growing evidence has suggested that paracrine mechanisms of Mesenchymal stem cell (MSC) may be involved in the underlying mechanism of MSC after transplantation, and extracellular vesicles (EVs) are an important component of this paracrine role. The aim of this study was to investigate the in vitro osteogenic effects of EVs derived from undifferentiated mesenchymal stem cells and from chemically induced to differentiate into osteogenic cells for 7 days. Further, the osteoinductive potential of EVs for bone regeneration in rat calvarial defects was assessed. We could isolate and characterize EVs from naïve and osteogenic-induced MSCs. Proteomic analysis revealed that EVs contained distinct protein profiles, with Osteo-EVs having more differentially expressed proteins with osteogenic properties. EVs were found to enhance the proliferation and migration of cultured MSC. In addition, the study found that Osteo-EVs/MEM combination scaffolds could enhance greater bone formation after 4 weeks as compared to native MEM loaded with serum-free media. The study suggests that EVs derived from chemically osteogenic-induced MSCs for 7 days can significantly enhance both the osteogenic differentiation activity of cultured hMSCs and the osteoinductivity of MEM scaffolds. The results indicate that Osteo-MSC-secreted nanocarriers-EVs combined with MEM scaffolds can be used for repairing bone defects

1. In two dogs, approximately one to two years and three to four months of age, an experimental comparison was made between the calcium accretion rate as defined by the Bauer-Carlsson-Lindquist equation, and the bone formation rate determined by double tetracycline labelling. 2. The overall calcium accretion rate was determined from the specific activity of the blood plasma, and the urinary and faecal excretion of isotope, following an intravenous tracer dose of Ca. 45. A time of five days after injection was used for the calculation of accretion rates, but data for shorter times of calculation are included. 3. Local accretion rates were obtained for different parts of the skeleton by determining the specific activities of bone samples at the end of the experiment. 4. The amount of isotope the uptake of which was not related to new bone formation (the diffuse component) was determined autoradiographically. 5. Local values for appositional growth rate and bone formation rate were obtained, using sections of undecalcified bone specimens, by measuring the linear separation between two tetracycline bone markers and the area of new bone enclosed by them. 6. In the older dog, the measurements for cortical bone showed that the accretion rate was two to three times as great as the bone formation rate: the observed diffuse component was sufficient to account for the greater part of this difference. Measurement of the bone formation rate for cancellous bone presented difficulties, but the approximate values obtained suggested that the accretion rate and the bone formation rate were of about the same order for this tissue. 7. In the younger dog, the bone formation rate could be determined only in cortical bone: at the sites studied, the values for the accretion rate and the bone formation rate did not differ by more than 20 per cent. It is suggested that this is due partly to the low specific activity of the diffuse component in this young animal, and partly to the relatively large amounts of new bone formed during the period of the experiment. 8. Despite the important differences between the rates of calcium accretion and bone formation that were found to exist in regions where there was only a small amount of new bone formation, there was a strong correlation between the two rates. The value of the accretion rate as a parameter of bone metabolism is clear

Aims. To verify whether secretory leucocyte protease inhibitor (SLPI) can promote early tendon-to-bone healing after anterior cruciate ligament (ACL) reconstruction. Methods. In vitro: the mobility of the rat bone mesenchymal stem cells (BMSCs) treated with SLPI was evaluated by scratch assay. Then the expression levels of osteogenic differentiation-related genes were analyzed by real-time quantitative PCR (qPCR) to determine the osteogenic effect of SLPI on BMSCs. In vivo: a rat model of ACL reconstruction was used to verify the effect of SLPI on tendon-to-bone healing. All the animals of the SLPI group and the negative control (NC) group were euthanized for histological evaluation, micro-CT scanning, and biomechanical testing. Results. SLPI improved the migration ability of BMSCs and upregulated the expression of genes related to osteogenic differentiation of BMSCs in vitro. In vivo, the SLPI group had higher histological scores at the tendon-bone interface by histological evaluation. Micro-CT showed more new bone formation and bone ingrowth around the grafted tendon in the SLPI group. Evaluation of the healing strength of the tendon-bone connection showed that the SLPI group had a higher maximum failure force and stiffness. Conclusion. SLPI can effectively promote early tendon-to-bone healing after ACL reconstruction via enhancing the migration and osteogenic differentiation of BMSCs. Cite this article: Bone Joint Res 2022;11(7):503–512

Aims. Bone regeneration during treatment of staphylococcal bone infection is challenging due to the ability of Staphylococcus aureus to invade and persist within osteoblasts. Here, we sought to determine whether the metabolic and extracellular organic matrix formation and mineralization ability of S. aureus-infected human osteoblasts can be restored after rifampicin (RMP) therapy. Methods. The human osteoblast-like Saos-2 cells infected with S. aureus EDCC 5055 strain and treated with 8 µg/ml RMP underwent osteogenic stimulation for up to 21 days. Test groups were Saos-2 cells + S. aureus and Saos-2 cells + S. aureus + 8 µg/ml RMP, and control groups were uninfected untreated Saos-2 cells and uninfected Saos-2 cells + 8 µg/ml RMP. Results. The S. aureus-infected osteoblasts showed a significant number of intracellular bacteria colonies and an unusual higher metabolic activity (p < 0.005) compared to uninfected osteoblasts. Treatment with 8 µg/ml RMP significantly eradicated intracellular bacteria and the metabolic activity was comparable to uninfected groups. The RMP-treated infected osteoblasts revealed a significantly reduced amount of mineralized extracellular matrix (ECM) at seven days osteogenesis relative to uninfected untreated osteoblasts (p = 0.007). Prolonged osteogenesis and RMP treatment at 21 days significantly improved the ECM mineralization level. Ultrastructural images of the mineralized RMP-treated infected osteoblasts revealed viable osteoblasts and densely distributed calcium crystal deposits within the extracellular organic matrix. The expression levels of prominent bone formation genes were comparable to the RMP-treated uninfected osteoblasts. Conclusion. Intracellular S. aureus infection impaired osteoblast metabolism and function. However, treatment with low dosage of RMP eradicated the intracellular S. aureus, enabling extracellular organic matrix formation and mineralization of osteoblasts at later stage. Cite this article: Bone Joint Res 2022;11(5):327–341

Osteoporosis affects millions globally and current anti-catabolic treatments are limited by significant side-effects. Osteoporosis arises when skeletal stem cells (SSC) no longer sufficiently replenish osteoblasts, leading to net bone loss. A key regulator of SSC behaviour is physical loading, yet the mechanisms by which SSCs sense and respond to changes in their mechanical environment are virtually unknown. Primary cilia are nearly ubiquitous ‘antennae-like’ cellular organelles that have very recently emerged as extracellular chemo/mechano-sensors and thus, are strong candidates to play an important role in regulating SSC responses in bone. This paper will demonstrate that the SSC primary cilium plays an important role in loading-induced bone formation via initial chemosensation and transduction of the potent chemokine TGFβ1 regulating SSC recruitment to the bone surface and secondly it will be shown that the primary cilium is a cAMP responsive mechanosensor directly regulating SSC mechanotransduction via localisation of adenylyl cyclase 6 to the ciliary microdomain. Finally, it will be shown that targeting the cilium therapeutically can be an effective approach to enhance both biochemical and biophysically induced SSC osteogenesis contributing to bone formation, demonstrating a novel anabolic therapy for bone loss diseases such as osteoporosis

Introduction and Objective. Calcium phosphates are among the most commonly used bone graft substitute materials. Compositions containing predominantly monetite (∼84.7%) with smaller additions of beta-tricalcium phosphate (β-TCP; ∼8.3%) and calcium pyrophosphate (Ca-PP; ∼6.8%) have previously been demonstrated to exhibit osteoinductive properties. Such a multi-component calcium phosphate bioceramic was fashioned in the form of hollowed-out, dome-shaped devices (15 mm diameter, 4 mm height), each reinforced with a 3D printed Ti6Al4V ELI frame. With the aim to induce bone formation beyond the skeletal envelope, these devices were investigated in vivo using a sheep (Ovis aries) occipital bone model. Materials and Methods. The bioceramic composition was prepared from a mixture of β-TCP/dicalcium pyrophosphate and monocalcium phosphate monohydrate powders mixed with glycerol. The Ti6Al4V ELI frame was positioned into a dome-shaped mould and bioceramic paste was poured over the frame and allowed to set, in sterile water, prior to removal from the mould. In adult female sheep (n=7), the devices were positioned directly over the bone and stabilised using self-drilling screws. After 52 weeks, the devices were retrieved, resin embedded, and used for X-ray micro-computed tomography (micro-CT), histology, backscattered electron scanning electron microscopy (BSE-SEM), energy dispersive X-ray spectroscopy (EDX), micro-Raman spectroscopy, and Fourier transform infrared spectroscopy (FTIR). Results. The bioceramic composition (Ca/P: ∼0.85 at. %) transforms to carbonated apatite (Ca/P: ∼1.2 at. %, Mg/Ca: ∼0.03 at. %), in vivo, largely at the expense of monetite and Ca-PP whereas β-TCP remains detectable. Discrete particles of Ca-PP are identified by correlative BSE-SEM and micro-Raman spectroscopy. Together with chemical transformation, physical degradation is evident within the bulk of the bioceramic. Beyond the confines of the skeletal envelope, de novo bone occupies ∼53–84% (∼73 ± 11%; mean ± standard deviation) of the hollowed-out space. Low porosity and the arrangement of remodelled bone into a concentric lamellar pattern is indicative of cortical-like structure. Such areas are typically surrounded by yet unremodelled, and microstructurally disordered, woven bone that stains intensely with blue cationic dyes, owing to relatively higher acid phosphate content. This pattern indicates a recurring sequence of woven bone formation followed by remodelling. Bone formation is also visible within the bioceramic. Recently remodelled and areas of ongoing remodelling are identified by relatively lower mineral density than the surrounding woven bone. Dendritic extensions of osteocytes appear to extend into the bioceramic surface. Both micro-Raman spectroscopy and FTIR reveal little, if any, detectable difference between the mineral and organic phases of the extracellular matrix, between de novo and native bone. Conclusions. The bioceramic composition undergoes physical degradation, but remains largely intact by 52 weeks in vivo, and only partially transforms to carbonated apatite. In addition to very high bone volume within the hollowed-out bioceramic device, the overall composition and microstructure of de novo bone are similar to native bone. Notably, the mineral phase of bone in response to, and in direct contact with the β-TCP, monetite, and Ca-PP, remains exclusively carbonated apatite

Purpose. Vitamin D is a key regulator of bone homeostasis. The enzyme CYP24A1 is responsible for transforming vitamin D into 24,25(OH)2vitD. The putative biological activity of 24,25(OH)2vitD remains unclear. Previous studies showed an increase in the circulating levels of this metabolite following a fracture in chicks. Our laboratory has engineered a mouse model deficient for the Cyp24a1 gene for studying the role of 24,25(OH)2vitD. We set out to study the role of 24,25(OH)2vitD in endochondral and intramembranous bone formation in fracture repair in this mouse model based on the results of the chick fracture repair study. Method. Wild-type and mutant Cyp24a1 gene deficient mice were subjected to two different surgical procedures to simulate bone development and fracture repair. To mimic endochondral ossification, we devised a modified technique to perform intramedullary nailing of a mouse tibia followed by an induced fracture. To evaluate intramembranous ossification, we applied distraction osteogenesis to a mouse tibia using a mini Ilizarov external fixator apparatus. Histomorphometric parameters and gene expression differences in fracture repair between the mutant mice and the wild-type controls were measured using micro computed tomography, histology and reverse-transcription quantitative PCR (RT-qPCR) respectively. Results. Quantitative histomorphometric results showed a delay in endochondral fracture repair in the mutant mice calluses as compared to the wild-type mice calluses. In the same model, gene expression of type X collagen in the callus was higher in the wild-type mice. These significant differences were fully rescued by injecting the mutant mice with exogenous 24,25(OH)2vitD. In the intramembranous bone formation model, we found a trend towards reduced bone formation in the gap created by the distraction process in the mutant mice as compared to the wild-type mice. However, the differences did not reach statistical significance. Conclusion. Our results support a role for 24,25(OH)2vitD in fracture repair which is more dominant in a chondrocyte-mediated bone formation pathway like endochondral ossification. Although our results did not reach statistical significance in the intramembranous ossification model, the observed trend suggests a potential role as well. Further study of the role of 24,25(OH)2vitD in bone healing has the potential to support novel approaches in accelerating bone formation and fracture repair

Objectives. We have observed clinical cases where bone is formed in the overlaying muscle covering surgically created bone defects treated with a hydroxyapatite/calcium sulphate biomaterial. Our objective was to investigate the osteoinductive potential of the biomaterial and to determine if growth factors secreted from local bone cells induce osteoblastic differentiation of muscle cells. Materials and Methods. We seeded mouse skeletal muscle cells C2C12 on the hydroxyapatite/calcium sulphate biomaterial and the phenotype of the cells was analysed. To mimic surgical conditions with leakage of extra cellular matrix (ECM) proteins and growth factors, we cultured rat bone cells ROS 17/2.8 in a bioreactor and harvested the secreted proteins. The secretome was added to rat muscle cells L6. The phenotype of the muscle cells after treatment with the media was assessed using immunostaining and light microscopy. Results. C2C12 cells differentiated into osteoblast-like cells expressing prominent bone markers after seeding on the biomaterial. The conditioned media of the ROS 17/2.8 contained bone morphogenetic protein-2 (BMP-2 8.4 ng/mg, standard deviation (. sd. ) 0.8) and BMP-7 (50.6 ng/mg, . sd. 2.2). In vitro, this secretome induced differentiation of skeletal muscle cells L6 towards an osteogenic lineage. Conclusion. Extra cellular matrix proteins and growth factors leaking from a bone cavity, along with a ceramic biomaterial, can synergistically enhance the process of ectopic ossification. The overlaying muscle acts as an osteoinductive niche, and provides the required cells for bone formation. Cite this article: D. B. Raina, A. Gupta, M. M. Petersen, W. Hettwer, M. McNally, M. Tägil, M-H. Zheng, A. Kumar, L. Lidgren. Muscle as an osteoinductive niche for local bone formation with the use of a biphasic calcium sulphate/hydroxyapatite biomaterial. Bone Joint Res 2016;5:500–511. DOI: 10.1302/2046-3758.510.BJR-2016-0133.R1

Bone morphogenetic proteins (BMPs) are able to induce osteogenic differentiation in many cells, including muscle cells. However, the actual contribution of muscle cells to bone formation and repair is unclear. Our objective was to examine the capacity of myogenic cells to contribute to BMP-induced ectopic bone formation and fracture repair. Osteogenic gene expression was measured by quantitative PCR in osteoprogenitors, myoblasts, and fibroblasts following BMP-2 treatment. The MyoD-Cre x ROSA26R and MyoD-Cre x Z/AP mouse strains were used to track the fate of MyoD+ cells in vivo. In these double-transgenic mice, MyoD+ progenitors undergo a permanent recombination event to induce reporter gene expression. Ectopic bone was produced by the intramuscular implantation of BMP-7. Closed tibial fractures and open tibial fractures with periosteal stripping were also performed. Cellular contribution was tracked at one, two and three week time points by histological staining. Osteoprogenitors and myoblasts exhibited comparable expression of early and late bone markers; in contrast bone marker expression was considerably less in fibroblasts. The sensitivity of cells to BMP-2 correlated with the expression of BMP receptor-1a (Bmpr1a). Pilot experiments using the MyoD-Cre x Rosa26R mice identified a contribution by MyoD expressing cells in BMP-induced ectopic bone formation. However, false positive LacZ staining in osteoclasts led us to seek alternative systems such as the MyoD-cre x Z/AP mice that have negligible background staining. Initially, a minor contribution from MyoD expressing cells was noted in the ectopic bones in the MyoD-cre x Z/AP mice, but without false positive osteoclast staining. Soft tissue trauma usually precedes the formation of ectopic bone. Hence, to mimic the clinical condition more precisely, physical injury to the muscle was performed. Traumatising the muscle two days prior to BMP-7 implantation: (1) induced MyoD expression in quiescent satellite cells; (2) increased ectopic bone formation; and (3) greatly enhanced the number of MyoD positive cells in the ectopic bone. In open tibial fractures the majority of the initial callus was MyoD+ indicating a significant contribution by myogenic cells. In contrast, closed fractures with the periosteum intact had a negligible myogenic contribution. Myoblasts but not fibroblasts were highly responsive to BMP stimulation and this was associated with BMP receptor expression. Our transgenic mouse models demonstrate for the first time that muscle progenitors can significantly contribute to ectopic bone formation and fracture repair. This may have translational applications for clinical orthopaedic therapies

Summary. A promising approach to stimulate in vivo bone formation by using our newly developed magnesium-based bone substitutes, which can be an alternative to treat the patients with bone loss in addition to the anticatabolic drugs and growth factors. Introduction. Bone impairment arising from osteoporosis as well as other pathological diseases is a major health problem. Anti-catabolic drugs such as bisphosphonates and other biological agents such as bone morphogenetic proteins and insulin-like growth factor can theoretically apply to stimulate bone formation. However, the formation of more brittle bone and uncontrolled release rate are still a challenge nowadays. Hence, we propose to stimulate bone formation by using a newly developed magnesium-based bone substitute. Indeed, the presence of magnesium ions can stimulate bone growth and healing by enhancing osteoblastic activity. This study aims to investigate the mechanical, in vitro and in vivo properties of this novel bone substitute. Methods. The bone substitutes were prepared by incorporating 9% TMSPM-treated Mg granules (i.e. 45μm & 150μm) into biodegradable polymer, polycaprolactone (PCL). The TMSPM silane-coupling agent treatment was used to protect the Mg particles from rapid degradation. Compression test was performed to study the mechanical properties of the bone substitute by using the MTS machine. A 7-day stimulated body fluid (SBF) immersion test was conducted to test their bioactivity. The surface composition was checked by energy dispersive x-ray spectroscopy (EDX) after immersion. The cytocompatibility and osteogenic differentiation properties of the bone substitutes were studied by MTT, ALP assays and qRT-PCR with the use of MC3T3-E1 pre-osteoblasts. Finally, the in vivo response of the bone substitutes was evaluated by using rat model of 2 months. Micro-CT was used to monitor the volume change of bone formation. Pure PCL was used as the control. Results. At least 36% higher compressive modulus was found on the new bone substitutes as compared to pure PCL. Calcium and phosphate deposition were detected on the Mg bone substitutes but not on pure PCL after 7-day SBF immersion. Significantly higher cell viabilities and specific ALP activities were found on the new bone substitutes as compared to pure PCL. Additionally, significantly higher ALP, Type I collagen, osteopontin and Runx2 expressions were found on the Mg-based substitutes at different time points. Finally, more than 15% new bone was found on the Mg bone substitutes after 1 week of post-operation and 40% higher after 3 weeks. Discussion/Conclusion. The increased compressive moduli of the Mg-based bone substitutes suggested that the mechanical property of PCL could be enhanced by incorporating Mg granules and the values fall within the range of cancellous bone (50 – 800 MPa). Moreover, the detection of the calcium and phosphate on the bone substitutes showed that they might possess osteoinductivity. The in vitro study showed the enhanced cytocompatibility and osteogenic differentiation properties of the new bone substitutes, which was possibly due to the effect of Mg ions release. Our previous study showed that only a low level of Mg ions (i.e. 50ppm) is able to stimulate the growth and differentiation of osteoblasts. Hence, this suggested the importance of controlling the release of Mg ions. This also explained why more new bone formation was found on the new bone substitutes than pure PCL during animal implantation. Hence, all the data presented here suggested our new bone substitutes maybe a potential candidate to stimulate new bone formation

Introduction. This study investigated the binding agent Calcium/Sodium Alginate fibre gel and the addition of autogenic bone marrow aspirate (BMA) on bone growth into a porous HA scaffold implanted in an ovine femoral condyle critical-sized defect. Our hypothesis was that Alginate fibre gel would have no negative effect on bone formation and osteoconduction within the scaffold and that BMA would augment the incorporation of the graft with the surrounding bone at 6 and 12 weeks post implantation. Methods. 24, 8mm x 15mm defects were filled with either porous HA granules, porous HA granules + Alginate fibre gel (HA putty) or porous HA granules + Alginate fibre gel + BMA (HA putty +BMA) and remained in vivo for 6 and 12 weeks (n=4). 1ml of bone marrow aspirate per cm3 of graft was used. Image analysis quantified bone apposition rates, bone ingrowth, bone-implant contact and quantity of graft. Mann Whitney U tests were used for statistical analysis where p<0.05 was considered significant. Results. Highest bone formation were measured in the 12 week HA putty+BMA group (1.57±0.24(micromillimetres/day). HA granules at 12 weeks encouraged the greatest increase in bone formation (33.56±3.53%). Smaller amounts of bone was measured in the 6 week HA putty+BMA group (8.57±2.86%). Bone formation in the HA group at 12 weeks was significantly higher when compared with the HA putty (p= 0.043) and the HA putty+BMA group (p= 0.043). At both the 6 and 12 week time point, highest bone-implant contact was seen in the HA granules group (59.34±10.89% and 72.65±3.38% respectively) when compared with both the HA putty (p=0.018) and HA putty+BMA (p=0.047). Results showed no significant difference in the amount of implant remaining when each group was compared. Conclusions. Results from this study showed that the inclusion of BMA did not augment bone growth to the scaffold or increase its osteoconductive capacity when combined with Calcium/Sodium Alginate fibre gel. Further research is necessary to optimise Calcium/Sodium Alginate fibre gel when used to bind HA granules and to investigate the effect of BMA with this type of HA alone

Summary Statement. The present study demonstrates the beneficial effects of strontium (Sr) modified calcium phosphate cement to improve new bone formation in a metaphyseal osteoporotic fracture defects in rats compared to calcium phosphate cement and empty defects. Keywords: strontium, fracture, calcium phosphate, bone formation. Introduction. Impaired fracture healing with subsequent implant failure is a dramatic problem in osteoporotic fractures. Biomaterials are of interest to stimulate fracture healing in osteoporotic defects and the objective of the current study is to investigate the effects of Strontium modified calcium phosphate cement (SrCPC) in a critical-size metaphyseal fracture defect of osteoporotic rats compared to calcium phosphate (CPC) and empty defect control group. Methods. 45 female Sprague-Dawley rats were randomized into 3 groups: SrCPC, CPC and empty defect (n=15 for each). A combinatorial approach of multi-deficiency diet for 3 months after bilateral ovariectomy was used for induction of osteoporosis. Left femur of all animals underwent a 4mm wedge-shaped metaphyseal osteotomy that was internally fixed with a T-shaped plate. The defect was then either filled with CPC or SrCPC and internally stabilised with a T shaped mini-plate. Empty defect served as a control. After 6 weeks femora were harvested followed by histological, histomorphometrical, immunohistochemical (bone-morphogenic protein 2, osteocalcin and osteoprotegerin), and molecular biology analysis (alkaline phosphatase, collagen10a1 and osteocalcin) to demonstrate the effects of the biomaterials on new bone formation. Time of flight secondary ion mass spectrometry (TOF-SIMS) technology was used to assess the distribution of released strontium ions and calcium appearance of newly formed bone. Results. Histomorphometric analysis showed a statistically significant increase in the bone formation at the tissue-implant interface in the SrCPC group (p<0.001). A statistically significantly more cartilage and unmineralised bone formation was also seen in the SrCPC group in comparision to the CPC group alone (p<0.05) and also to the empty defect (p<0.05) in the former fracture defect zone. These data were confirmed by the immunohistochemistry results which revealed an increase in bone-morphogenic protein 2, osteocalcin and osteoprotegerin and an increase in expression of genes responsible for bone formation viz. alkaline phosphatase, collagen10a1 and osteocalcin. TOF-SIMs analysis showed a higher release of Sr from the SrCPC into the interface region and related to a higher calcium content in this area compared to CPC. Discussion/Conclusion. SrCPC treatment showed enhanced new bone formation in a metaphyseal osteoporotic fracture defect of rats after 6 weeks compared to CPC-filled and empty defects in histomorphometry, immunochemistry and gene expression analysis. Strontium ranelate is a well-known anti-osteoporotic drug increasing bone formation and reducing bone resorption. As revealed by TOF-SIMS release of Sr out of the the SrCPC cement is most likely attributable for new bone formation. Therefore, Sr seems to be a good candidate not only for systemic treatment in osteoporosis but also in Sr-modification of biomaterials for local stimulation of new bone formation in osteoporotic fracture defects

Objectives. An experimental rabbit model was used to test the null hypothesis, that there is no difference in new bone formation around uncoated titanium discs compared with coated titanium discs when implanted into the muscles of rabbits. Methods. A total of three titanium discs with different surface and coating (1, porous coating; 2, porous coating + Bonemaster (Biomet); and 3, porous coating + plasma-sprayed hydroxyapatite) were implanted in 12 female rabbits. Six animals were killed after six weeks and the remaining six were killed after 12 weeks. The implants with surrounding tissues were embedded in methyl methacrylate and grinded sections were stained with Masson-Goldners trichrome and examined by light microscopy of coded sections. Results. Small amounts of bone were observed scattered along the surface of five of the 12 implants coated with porous titanium, and around one out of 12 porous coated surfaces with Bonemaster. No bone formation could be detected around porous coated implants with plasma-sprayed hydroxyapatite. Conclusion. Porous titanium coating is to some degree osteoinductive in muscles

1. The effect of immobilisation on bone formation has been investigated in the rat. As chlortetracycline has the property of being deposited preferentially in areas of newly deposited bone, its uptake can be used as a reflection of osseous formation. 2. One hind limb of albino rats was immobilised either by section of the second, third and fourth lumbar nerve trunks or by section of tendo calcaneus and ligamentum patellae. The incorporation of chlortetracycline was determined quantitatively in the femur and tibia of both hind limbs at intervals after immobilisation. 3. Tetracycline uptake is expressed in terms of bone weight, this being most important in order to obtain correct values. 4. A comparison between the tetracycline uptake and the weight of the bones gives information about the rate of bone destruction. 5. In animals immobilised by nerve section three phases can be distinguished: a first phase with diminished bone formation, a second with increased formation and increased destruction and a third phase with diminished bone formation. Both mechanisms, decreased formation and increased destruction, are therefore important in the production of immobilisation osteoporosis; their relative importance depending upon the duration of the immobilisation. 6. In animals immobilised by tendon section the mechanical function becomes restored in the second week as a result of healing: this explains the rapid compensation for the initial loss of weight of the bones accompanied by a marked increase in tetracycline uptake. 7. These results are discussed and compared with information in the literature. Variations in bone formation and destruction rates with time could explain the varied results reported by other authors

Bone formation proceeds through two distinct processes. One involves the deposition of bone by osteoblasts (intramembranous ossification) and another through the remodeling of an intermediate cartilaginous matrix formed by chondrogenic differentiation of mesenchymal stem/stromal cells (MSCs) aggregates – a process called endochondral ossification (EO). EO is responsible for formation of long bones during development and most prevalent during facture repair upon callus formation. In adult bone injuries MSCs from periosteum form bone via EO whereas MSCs from bone marrow are primarily differentiate to osteoblast in vivo. We hypothesized that the unique biophysical and biochemical properties of bone mineral phase has a role in programming MSCs. Using a biomimetic bone like apatite (BBHAp) as surrogate for bone mineral phase, we studied the chondrogenic differentiation of human marrow derived MSCs and observed that the BBHAp dictates MSCs fate and strictly dictates the pathway of bone formation in vivo. Through exhaustive dissection of the signaling pathways at play, a prominent role of PTH1R in modulating the effects imposed by the BBHAp has been unraveled. These fundamental insights gained in how bone microenvironment might alter fate of MSCs has important implications for bone repair and regeneration therapies

The use of designer scaffolds to deliver biologically active osteogenic growth factors such as recombinant human bone morphogenetic protein-2 (rhBMP-2) to the sites of tissue regeneration in for example orthopaedics, has tremendous therapeutic implications. The aims of this study were to generate biomimetic biodegradable porous osteogenic scaffolds using a supercritical fluid process to encapsulate rhBMP-2, and to examine the ability of the scaffolds to promote human osteoprogenitor differentiation and bone formation in vitro and in vivo. The rhBMP-2 encapsulated in Poly(-lactic acid) (PLA) scaffolds (100ng/mg PLA) were generated using an innovative supercritical fluid mixing method. The bioactivity of rhBMP-2 encapsulated PLA scaffolds were confirmed by induction of the C2C12 promyoblast cell line into the osteogenic lineage as detected by alkaline phosphatase expression. No induction of alkaline phosphatase-positive cells was observed using blank scaffolds. BMP-2 released from encapsulated constructs promoted adhesion, migration, expansion and differentiation of human osteoprogenitor cells on 3-D scaffolds. Enhanced matrix synthesis and cell differentiation on growth factor encapsulated scaffolds was observed following culture of human osteoprogenitors on explants of chick femoral bone wedge defects in an ex vivo model of bone formation developed using the chick chorioallantoic membrane model. In vivo studies using diffusion chamber implantation and subcutaneous implantation of human osteoprogenitors on rhBMP-2 encapsulated scaffolds showed morphologic evidence of new bone matrix and cartilage formation in athymic mice as assessed by x-ray analysis, immunocytochemistry and birefringence. These studies provide evidence of controlled release of BMP-2 from biodegradable polymer scaffolds initiating new bone formation in vivo. The generation of 3-D biomimetic structures incorporating osteoinductive factors such as BMP-2 indicates their potential for de novo bone formation that exploits cell-matrix interactions and, significantly, realistic delivery protocols for growth factors in musculo-skeletal tissue engineering

We used interconnected porous calcium hydroxyapatite ceramic to bridge a rabbit ulnar defect. Two weeks after inducing the defect we percutaneously injected rabbit bone marrow-derived mesenchymal stromal cells labelled with ferumoxide. The contribution of an external magnetic targeting system to attract these cells into the ceramic and their effect on subsequent bone formation were evaluated. This technique significantly facilitated the infiltration of ferumoxide-labelled cells into ceramic and significantly contributed to the enhancement of bone formation even in the chronic phase. As such, it is potentially of clinical use to treat fractures, bone defects, delayed union and nonunion

We measured the extent and rate of new bone formation over an 18-month period before, during and after the lengthening of ten leg segments in six patients aged between 8 and 18 years, using dual-energy X-ray absorptiometry (DEXA). New bone formation could be identified within one week of the start of distraction. As lengthening proceeded, the bone density of the gap fell, reaching minimum values at the time of maximal distraction. Consolidation of the regenerating bone was started 1 to 2 weeks later in the tibia, and 2.5 to 3.0 weeks later in the femur. The rate of mineral accretion in new bone was significantly greater in the tibia than in the femur (16 +/- 1.86%/month, and 11 +/- 1.1%/month respectively; mean +/- SEM). There was significant osteoporosis distal to the osteotomy, more in the tibia than in the femur, particularly on the side of the fixator. The bone mineral density of the distal segment remained low at the time of fixator removal (44.2 +/- 5.58% and 61.0 +/- 4.2% of the control values at the tibia and femur respectively) and was only partially reversed by subsequent weight-bearing. We conclude that dual-energy X-ray absorptiometry provides an objective and quantitative assessment of new bone formation during leg lengthening. The technique also allows the measurement of the distraction gap and the assessment of leg alignment from the high-resolution images. Its use may decrease the requirements for conventional radiography

Introduction: The current practice of impaction allograft to fill large defects in revision total hip replacements is sometimes useful but clinical results are inconsistent. Other studies have shown that addition of mesenchymal stem cells (MSC) in blocks of hydroxyapatite (HA) scaffold can enhance new bone formation in a critical sized defect. However, no study has been conducted on combined MSCs with morselised allograft and HA granules. It is hypothesized that impaction of allograft or HA granules seeded with MSCs or osteoprogenitors will enhance new bone formation compared with the groups without MSCs. Materials and Methods: Six sheep were used for the study. Each sheep received 8 scaffolds which were embedded in both paraspinal muscles. Groups were: 1) 3.5g allograft, 2) 3.5g allograft with MSCs, 3) 3.5g allograft with osteoblasts; 4) 3.5g of 50:50 allograft/ HA, 5) 3.5g of 50:50 allograft/HA with MSCs, 6) 3.5g of 50:50 allograft/HA with osteoblasts; 7) a block of HA, 8) a block of HA with MSCs. The experimental scaffolds were seeded with either 10x106 MSCs/ml or 10x106 MSC-derived osteoprogenitors/ml, in 3ml autologous plasma. Grafts were impacted twenty times at 3KN. At eight weeks, samples were sectioned for histology analysis. Areas of new bone formation were measured as percentage to total available spaces. ANOVA was used for statistical analysis. Results: Addition of MSCs increased new bone formation in allograft (4.98%), allograft/HA (5.15%) and HA block (7.09%) compared with their controls at 2.24%, 1.96% and 1.96% respectively. Statistical study showed significant increase in 50:50 allograft/HA with MSCs compared with 50:50 allograft/HA only (p=0.046) and 50:50 allograft/HA with osteoprogenitors (p=0.028). No difference was found in allograft groups. For the HA block groups, addition of MSCs showed a significant new bone increase compared to the control (p=0.028). Conclusion: Addition of MSCs to the allograft and HA granules will enhance new bone formation after impaction which can be used for revision total hip replacements, especially when allograft and HA is mixed. However, addition of osteoprogenitors has not achieved the similar results. This study encourages a further clinical investigation of impaction tissue-engineered graft to repair bone defects in revision total joint replacements

Introduction: Current treatments for osteoporosis do not completely eliminate the risk of fracture and bone loss may continue even at a low level. Enhanced bone formation and mineralization could minimize the risk of fracture in osteoporosis and prevent the pain and associated morbidity in these patients. Bone morphogenetic protein-type 2 has been successfully used to promote bone formation and to augment fracture repair in general and in the spine in particular [1]. The aim of this study was to increase local bone formation and mineralization in osteopenic vertebrae by administration of recombinant human morphogenetic proteins (rhBMP-2) in an ovine model. Methods: Osteoporosis was induced in ten skeletally mature sheep with ovariectomy, low calcium diet and weekly steroid injection. Bone mineral density (BMD) of the lumbar spine was assessed monthly by DXA. When the BMD of the lumbar spine was reduced by at least 25% the induction treatment was stopped and pellets containing inert carrier alone (control) or rhBMP-2 in either slow or fast release formulation were implanted directly into three adjacent lumbar vertebrae of each animal in a random order. BMD was assessed at regular intervals and two and three months later five animals were euthanized and the lumbar spines were collected for histomorphometric analysis using the SkyScan 1076 Micro CT (SkyScan, Belgium). Significant differences between BMD and bone morphometric data (including trabecular bone volume, separation and number) were examined using ANOVA and Tukey’s test with significance set at P< 0.05. Results: After five months of induction treatment BMD in the lumbar spines of all animals was reduced by at least 25% (p< 0.05). BMD increased insignificantly after cessation of the induction treatment but remained lower than the initial values. As there were no significant differences the histomorphometric data after two and three months were pooled. The trabecular bone volume in the vicinity of both the slow and fast release BMP implants increased by over 15% compared with the control (p< 0.05). Trabecular separation was reduced over 13% and trabecular number around both types of pellets increased by over 12% compared to the control (NS). Discussion: This animal model provides an opportunity to evaluate systemic and local treatments for osteoporosis. The significant increase in bone formation adjacent to the implants as early as two months suggests that rhBMP-2 in either formulation improves bone quality at sites with high risk of fracture. The impact of the fast and slow release BMPs implants were not significantly different

The relationship between heterotopic bone formation and the morphological type of osteoarthritis was examined after 43 hip replacements. Of the 43 hips studied, nine were atrophic, 19 were normotrophic, and 15 were hypertrophic. The incidence of heterotopic bone formation in the atrophic type was 11%, in the normotrophic type 32%, and in the hypertrophic type 87%. The difference between each type was statistically significant (p less than 0.001)

The purpose of this study was to determine the benefit and risk of NSAID-based prophylaxis for ectopic bone formation amongst patients undergoing total hip replacement (or revision) surgery. A double-blind randomised placebo-controlled clinical trial, stratified by treatment site and surgery (primary or revision), was conducted in 20 orthopaedic surgery centres in Australia and New Zealand. 902 patients, undergoing elective primary or revision total hip replacement surgery, were randomly allocated to 14 days treatment with ibuprofen (1200mg daily) or matching placebo commenced within 24 hours of surgery. Patients were only excluded if there was, in the opinion of the responsible physician, a definite indication or contra-indication for treatment with an NSAID during the 14 day study treatment period. Outcomes were assessed six to 12 months after surgery and included changes in self-reported hip pain and physical function (WOMAC), physical performance measures and radiographic evidence of ectopic bone formation. There was only a 6% loss to follow-up for self-report measures and a 12% loss to follow-up for radiographs. Six to twelve months after surgery, there were no significant differences between the ibuprofen and placebo groups for improvements in hip pain (mean difference, 95% confidence interval: −0.1, −0.4 to 0.2, p=0.6) or physical function (−0.1, −0.4 to 0.2, p=0.5), despite a much reduced risk of ectopic bone formation (relative risk 0.69, 95% confidence interval 0.56 to 0.83) associated with ibuprofen. There was a significantly increased risk of major bleeding complications during the admission period (2.09, 1.00 to 4.39). These data, from the largest-ever trial of prophylaxis against ectopic bone formation, do not support the use of routine NSAIDs-based prophylaxis for patients undergoing total hip replacement surgery

Objective. Superior bone ingrowth and resistance to bacterial infection are ideal for orthopaedic implants. We compared new bone formation, strength of bone bonding, and infection rates between silicon nitride ceramic (Si3N4; abbreviated SiN), medical-grade PEEK (PEEK), and titanium (Ti) in rat calvariae. PEEK and Ti are used in spinal and arthroplasty implants respectively, while SiN is a non-oxide ceramic used in spinal implants for the past 4 years. Methods. Specimens of 10 mm × 10 mm by 1.75 mm size were implanted into experimental calvarial defects in 2-year old Wistar rats using standard surgical techniques (n's: SiN=48; PEEK=24; Ti=24). One group of animals was immediately inoculated with 1 × 104 Staphylococcus epidermidis; control animals received saline only. After sacrifice at 3 days, 7 days, 14 days, and 3 months post-inoculation (n=4 rats per time period), one calvarial sample each for PEEK and Ti, and two samples for SiN (per bacterial condition and time point) were retrieved for histology; remaining samples were used for sample push-out testing with a Micro Tester 5848 (Instron) with a 1-kN load cell, using published techniques. New bone formation was measured with tetracycline double-labeling at 11 and 4 days before the 14-day and 3-month time periods. Results. Of the materials tested, 3-month bone ingrowth and periprosthetic infection rates were most favorable for SiN (Fig. 1). At 90 days post-implantation without bacteria, new bone growth in calvariae with SiN was ∼69% compared with 24% and 36% for PEEK and Ti, respectively. With bacterial inoculation, new bone growth was 21%, 26% and 41% for PEEK, Ti, and SiN, respectively. At 3-months, live bacteria were observed for PEEK (88%) and Ti (21%), while no bacteria were present around SiN (Fig. 2). Push-out strength was greater for SiN when compared to Ti and PEEK (Fig. 3). Conclusions. Superior bone formation, bone ingrowth, and bacterial resistance were associated with SiN when compared to Ti and PEEK. SiN proved effective in inhibiting bacteria and promoting osteogenesis in experimental osseous defects, when compared to Ti and PEEK. These observations are most likely related to the hydrophilic properties of SiN that contributed to the adsorption of proteins known to decrease bacteria attachment and growth (vitronectin and fibronectin). We conclude that SiN may be a superior biomaterial for the development of orthopaedic implants

Bone Morphogenetic Protein 7 (BMP7) is a powerful osteoinductive substance that could stimulate bone formation in difficult conditions including distraction osteogenesis. However, to be effective, large unphysiological doses are required. Blocking the expression of BMP antagonists could amplify the effects of BMP7, allowing smaller doses of BMP7 to be used without altering its osteogenic potential. In this study, BMP7 antagonist Noggin was shown to be upregulated following BMP7 injection in a rabbit distraction osteogenesis model suggesting a role for Noggin in controlling BMP7 activity. Blocking Noggin expression may thus permit smaller doses of BMP7 to be used effectively. Distraction osteogenesis (DO) is an excellent method to form new bone. However, the long duration the external fixator has to be kept on until the new bone consolidates, could lead to numerous problems. BMP7 may accelerate bone formation in DO. However, large doses of BMP7 may be necessary. In this study, we investigated the expression of BMP7 antagonist Noggin in DO. Noggin may control BMP7 activity through a negative feedback mechanism. Blocking Noggin may amplify the effects of BMP7, thus permitting the use of smaller doses of BMP7 effectively in DO. Using smaller doses of BMP7 – while maintaining its powerful effects – may decrease side effects and render this drug more affordable economically. Noggin is normally expressed in DO. Its expression is upregulated by local application of BMP7. Its expression is co-localized to the same cells that express BMP7 and its receptors. The right tibia of sixteen rabbits was lengthened using a uniplanar fixator. The rabbits were divided into two groups: one received seventy-five micrograms recombinant BMP7 and the other placebo. All injections were performed one week after start of distraction. Rabbits were sacrificed ten minutes, one day, two days and two weeks following the injections. The expression of Noggin was studied in the distracted tissue by immunohistochemistry. Noggin may play a role in DO. Blocking its action may have huge clinical implications, by permitting the use of smaller – but equally effective – amounts of BMP7. Funding: CIHR, FRSQ and Shriners of North America

1. Bones consist essentially of bundles of collagenous fibres united by a cementing substance in which the inorganic material lies in the form of minute plate-like crystals. 2. During weight bearing and muscle action bones as a whole are deformed to a variable extent. Periods of deformation are followed by periods of relaxed pressure during which the bones tend to return to their normal form. 3. These variations in deformation and elastic recoil set up alternating pressures and tensions within the bones along the bone cyrstal encrusted fibres which make up the trabeculae, lamellae and Haversian systems, and these alternating phases of compression and tension stimulate the activity of osteoblasts so that bone formation predominates over bone resorption. 4. These alterations of pressure and tension are intermittent and reciprocal in nature and do not, as postulated by the trajectorial theory, involve different trabeculae, nor is it necessary to consider whether tension or pressure is the more important phase in determining bone deposition. 5. The pressure exerted by cysts, tumours, erupting teeth, etc., is of a quite different nature, as is the response to trauma or callus formation in the healing of fractures. These processes are essentially vascular phenomena involving localised areas of bony tissue and not bones as mechanical units

1. A case of polyarteritis nodosa is reported, the presenting manifestation of which was subperiosteal new bone formation in both legs. 2. The effect of cortisone on the symptoms is recorded. 3. The histology is reported after biopsy of bone and adjacent tissue. 4. Skeletal involvement in polyarteritis nodosa is believed to be hitherto unrecorded

1. Three patients with localised periosteal new bone formation associated with periosteal arteritis and other evidence of systemic lupus erythematosus are described. 2. Systemic steroid therapy was valuable in the management of this condition

1 . Normal and diseased bone was obtained by biopsy from five patients suffering from Paget's disease. The tissue was studied by histology, microradiography and quantitative fluorescence microscopy using tetracycline markers. Study of the morphological changes showed that two of the biopsies could be regarded as normal, while one was osteoporotic; two biopsy specimens were in the porotic phase of Paget's disease and the remaining five were in the sclerotic phase. 2. The tetracycline markers were used to measure the linear rate at which bone was deposited on individual surfaces (appositional growth rate) in µ per day and the percentage volume of new bone added to the total volume of bone per day (bone formation rate). The values obtained for appositional growth rate in all the biopsies were of the order of 1 µ per day, but slightly higher values were obtained in the diseased tissue of each individual. The bone formation rate in normal bone from the proximal femur was about 0·04 per cent per day, about 0·13 per cent per day in the porotic phase, and about 0·4 per cent per day in the sclerotic phase of Paget's disease. 3. Although these values must be accepted with some reservation, there seems to be no doubt that there is an upper limit of about 1 µ per day to the rate of deposition of bone on an individual bone surface; this suggests that in Paget's disease the osteoblast behaves as a normal cell

1. A patient with ectopic bone in the upper arm associated with multiple congenital anomalies is reported. 2. The previous cases of congenital abnormalities in patients with ectopic bone formation have been indicated and the problem of etiology has been discussed

1. In five series of experiments in eighty-two rabbits we succeeded in causing rarefaction of the calcaneum of all the animals soon after it was relieved from muscular compressing forces; new bone was generated when the calcaneum was subjected again to the stresses and strains of muscle contraction. 2. We found evidence that during muscle action pressure forces are transmitted through the bone, and that the presence or absence of these pressure forces conditions the balance between bone formation and bone removal. 3. In the calcaneum of the rabbit lack of muscular action seems to be the most important factor inducing osteoporosis. It is possible that the origin of post-traumatic osteoporosis has the same basis. 4. In our experiments bone rarefaction was characterised by a great increase in the vascularity of the bone; this increase ceased when the bone reached its final precarious bone density. Thus, vascular over-activity accompanied the removal of bone; but bone reconstruction was also seen to be accompanied by a more localised increase in vascularity. 5. From our experiments we cannot suggest that the inhibition of muscle contraction accompanying Sudeck's syndrome is responsible for this disorder, because we were unable in our animals to cause any of the other signs characteristic of Sudeck's bone atrophy. But the constancy with which we caused bone atrophy by the removal of muscle action may possibly help to explain the mechanism of bone absorption accompanying Sudeck's disease

Introduction. Enhanced angiogenesis and osteogenesis may provide new strategies for the treatment of osteonecrosis. Methods. Synergistic effects of vascular endothelial growth factor (VEGF) and bone morphogenetic protein - 6 (BMP-6) on in vitro osteogenic differentiation and in vivo ectopic bone formation mediated by a cloned mouse bone marrow stromal cell line, D1, previously isolated from Balb/c mice in our laboratory, were determined. Results. When human VEGF and BMP-6 genes both were expressed in D1 cells, significant increase in alkaline phosphatase activity was observed as compared to D1 cells in control groups. In the in vivo study, D1 cells transfected with hVEGF and hBMP-6 were loaded onto a 3-D PLAGA (polylactic-co-glycolic acid) scaffold and implanted subcutaneously in Balb/c mice. Micro-CT analysis of the retrieved implants clearly indicated the synergistic interaction of VEGF with BMP-6 as greater ectopic bone formation was observed in the VEGF plus BMP-6 group as compared to VEGF or BMP-6 alone. In addition, histology of the implants showed enhanced blood vessel formation with VEGF treatments. Conclusion. This study demonstrated the synergistic interaction of VEGF with BMP-6 during osteogenesis in vitro and in vivo. The results indicated that this novel combination of therapeutic growth factors should be investigated further as a potential treatment of osteonecrosis

Aim: To examine the effect of the bisphosphonate zoledronic acid in doses of 0.1mg/kg on new bone formation and stress shielding in a distraction-osteogenesis model in New Zealand white rabbits. Method: Thirty male rabbits underwent a right tibial osteotomy at eight weeks of age. Distraction of the osteotomy by 0.75mm/day was performed for two weeks followed by four weeks for consolidation. Group I was given saline infusions, Group II zoledronic acid at surgery, and Group III received zoledronic acid at surgery and again at two weeks. DXA scans evaluated BMC and BMD. Quantitative computerised tomography measured the cross-sectional areas. Four-point bend testing of both distracted and non-operated tibiae was performed in a standardised fashion. Results: Bone mineral accretion between two and four weeks was significantly higher in treated versus saline groups, and was better maintained at six weeks (P< 0.01 ANOVA). Stress shielding osteopaenia that was seen in surrounding bone segments in Group I (controls) was abolished in the treated groups. By six weeks there was a 49% and 59% increase in cross sectional area of new bone in Groups II and III respectively (P< 0.01 ANOVA). Group II tibiae were 29% stronger in four-point bending, while Group III were 89% stronger than Group I (P< 0.01 ANOVA). There was little detectable effect on the non-operated tibiae. Conclusions: Zoledronic acid administration significantly increased the rate and amount of new bone formation and its mineralisation. The increases in bone formation and retention translated to a significant, dose-dependent increase in strength. Further research into the role of zoledronic acid in orthopaedic surgery is indicated

Purpose: Previous studies have shown the utility of the bisphosphonate zoledronic acid (ZA) by systemic administration and local delivery for enhancing local bone formation with porous implants. The purpose of this study was to quantify the long term effect of local delivery of ZA on bone growth within and around porous tantalum implants one year after surgery. Method: Hydroxyapatite coated porous tantalum (Trabecular Metal. ™. , Zimmer Inc) implants measuring 9 mm in diameter and 90 mm in length were used in a canine bilateral femoral intramedullary model. Commercially pure ZA (Novartis Pharma) of either 0.05 mg or 0.20 mg ZA was applied to implants. Bilateral surgery was performed on 10 dogs – all 10 with a control implant on one side and 5 each with either a 0.05 mg or 0.20 mg ZA-dosed implant on the contralateral side. After one year, the femora were harvested and processed for undecalcified thin section histology and backscattered scanning electron microscopy. Statistical analysis was done using the student’s t tests and multiple two-level hierarchical models. Results: The 160 histologic sections revealed that compared with controls, there was more intramedullary bone around implants dosed with both 0.05 mg ZA (+91%, p< 0.001) and 0.20 mg ZA (+115%, p< 0.001). Bone ingrowth was present in all sections and was more abundant within 1.5mm of the implant periphery. The 0.20 mg ZA dose resulted in more net intramedullary bone formation than the 0.05 mg dose (+41%, p< 0.006). The mean extent of bone ingrowth for implants dosed with 0.20 mg ZA was significantly greater than controls (+32%, p< 0.003) and also greater than for implants dosed with 0.05 mg ZA (+47% for the area within 1.5 mm of the periphery, p< 0.002). Conclusion: This study demonstrated that the enhanced net bone formation that occurs due to local elution of ZA from porous implants was sustained out to 1 year after surgery. A notable dose response was also demonstrated. The peri-implant response was confined to within a few millimeters of the implant suggesting that ZA elution remains localized. This study supports the concept of using ZA-dosed implants for enhancing net bone formation within and around noncemented implants

Aims: We tested the hypothesis whether vascular endothelial growth factor (VEGF-A) gene transfer is an appropriate way to enhance recruitment and activity of osteoblasts in vivo. Methods: We tested plasmid/ liposome and adenoviral gene transfer vectors in vitro and selected adenoviruses for in vivo experiments. Adenovirus vectors containing VEGF-A or lacZ genes (1.4x10. 10. pfu) were injected locally into right distal femurs of New Zealand White rabbits. Saline was injected into all contralateral distal femurs. One and three weeks after the gene transfers femurs were collected for analyzes. Trabecular bone hard tissue histo-morphometry was performed to analyze the effect of gene transfer on bone turnover. Results: X-Gal staining showed that up to twenty percent of the bone marrow cells were transfected. When compared to unilateral lacZ transfected trabecular bone at one week time point, VEGF-A bone had 8% less bone volume, 90% higher osteoblast number, 100% higher osteoblast surface, 125% higher osteoid volume and 70% less resorption surface. Corresponding parameters were 70% higher bone volume, 7% higher osteo-blast number, 30% higher osteoblast surface, 22% higher osteoid volume and 49% less resorption surface at week three. Conclusions: Our results suggest that adenovirus-mediated VEGF-A gene transfer induces bone formation via increasing osteoblast activity and maybe useful for the treatment of osteoporosis and other diseases that required efþcient osteogenic therapy

The thrombin-related peptide, TP508, is a synthetic 23 amino acid peptide, which represents the receptor binding domain of thrombin. TP508 mimics thrombin by interacting with receptors on cells involved in tissue repair. TP508 has been shown to enhance revascularization of injured tissue, and promote soft tissue wound healing, cartilage repair, and fracture repair. The aim of this study is to (1) test the effect of TP508 on bone regeneration during distraction osteogenesis; (2) study the chemotactic effect of TP508 on human osteoblasts. Unilateral tibial osteoectomies were performed and stabilized with MX100 Orthofix lengthener in 5 male adult NZW rabbits. After 7 days, distraction was initiated at rates of 1.4 mm / day for 6 days. TP508 (100 μg/ml, n=2; 10 μg/ml, n=1) or saline (300 μl, n=2) was injected into the osteotomy / lengthening gap at days 1, 7 and 14 post surgery. Animals were sacrificed at 2 weeks after leg lengthening. Bone formation in the regenerate was assessed by radiography, quantitative computed tomography (pQCT) and histology. For chemotaxis studies, MG63 cells were cultured on glass cover slips for three days, and then inverted onto a Dunn chamber slide and sealed with dental wax. Gradients of TP508 (1, 10, 100 μg/ml) were added to the outer well and plain medium to the inner well. A sequence of images of the cells between the wells was taken via a CCD camera for 9 hours at interval of 10 minutes. Movements of individual cells were tracked and statistically analysed by a specially written Macro program. The Rayleigh test for unimodal clustering was used to determine the directional chemotactic movements. The radiographic evaluation indicated a significant increase in new bone in the distraction regenerate in the TP508 treated groups at 1 and 2 weeks. pQCT images at 2 weeks demonstrated more advanced bone formation in the TP508 treated animals compared to the control. The mean total bone mineral density (BMD) of the regenerate, obtained from 3 slices was significantly greater (p = 0.019, t-test) in the TP508 treated group (BMD = 479.20 +/− 35.57 mg/ccm) than that in the saline control group (BMD = 355 +/− 2.83 mg/ccm). The histological evaluation supported the radiographic and the pQCT results. For chemotaxis study, no directional movements of the cells were found in the controls, whereas the MG63 cells were strongly chemotactic to TP508 at 1, 10 and 100 μg/ml concentrations. This preliminary study shows that administration of TP508 enhances bone formation during distraction osteogenesis in the rabbit. The findings also show that TP508 has a chemotactic effect on osteoblasts, consistent with the effect of TP508 on fracture repair. A large animal study is in the process to confirm these findings and explore the underlying mechanisms

Aims: The present study examined the effect of ade-novirus-mediated recombinant human BMP-2 (RAd-BMP-2) gene therapy combined with bioactive glass (BG) microspheres in promotion of new bone formation. Methods: Harlan Dawley female rats (n=72) underwent unilateral surgery of right or left tibia in a random order. A round cortical window (. −. 2.8 mm) was drilled into the anteromedial cortex of the proximal tibia. A smaller unicortical hole (. −. 1.0 mm) was drilled 5 mm distally. Bone marrow was removed and the medullary space between the cortical holes was þlled with BG microspheres. Adenoviral vectors RAdBMP-2 carrying the BMP-2 gene or RAdLacZ harbouring the E. coli LacZ reporter gene were injected locally into the medullary spaces. The control defects were þlled with BG microspheres only. Empty control defects were left to heal without any þlling. The rats were killed 4 days, 2 and 8 weeks after surgery and the tibias were harvested for analyses. At each time point, six animals were used for pQCT, radiography, BEI-SEM and histomorphometric analyses. Results: All BG-þlled defects showed a time-related increase of intramedullary new bone. At 8 weeks, there was signiþcantly more new bone in defects treated with BG and RAdBMP-2 gene than in defects left to heal without þlling (p=0.003) (BG + RAdBMP-2: 25.0 ± 6.0% and empty control defects: 12.3 ± 3.8%). Also defects þlled with BG only showed higher new bone formation than empty control defects, but this was not statistically signiþcant (p=0.10) (BG: 19.9 ± 7.3%). Conclusions: The current study showed that local BMP-2 gene therapy enhances new bone formation on bioactive glass microspheres

Aim

In this study we investigated the effects of non-steroidal anti-inflammatory drugs (NSAIDs) with different cyclooxygenase (COX) selectivity on orthopaedic device-related infections (ODRIs) in a rat model. Specifically, we aimed to measure the impact of NSAID therapy on bone changes, bacterial load, and cytokine levels after treatment with antibiotics. In addition, we compared the effects of long vs short-term celecoxib (a COX-2 inhibitor) treatment on the same outcomes.

We investigated the effects of non-steroidal anti-inflammatory drugs (NSAIDs) with different cyclooxygenase (COX) selectivity on orthopaedic device-related infections (ODRIs) in a rat model. We aimed to measure the impact of NSAID therapy on bone changes, bacterial load, and cytokine levels after treatment with antibiotics. We also compared the effects of long vs short-term celecoxib (a COX-2 inhibitor) treatment on the same outcomes.

Skeletally mature female Wistar rats were implanted with Staphylococcus epidermidis- contaminated polyetheretherketone (PEEK) screws in the proximal right tibia and monitored for 7 days. All animals received subcutaneous antibiotics (rifampicin plus cefazolin) for two weeks from day 7 to 21. In phase I of the study, rats were randomly assigned to receive 28 days of oral treatment with acetylsalicylic acid, ibuprofen, celecoxib, or vehicle control. In phase II, an additional group received seven days of celecoxib treatment from day 0 to 7. Bone changes were monitored using in vivo micro-CT and histology. Quantitative bacteriology was performed at euthanasia. Plasma samples were collected to measure cytokine levels on days 0, 6, 20, and 28.

Combination antibiotic therapy resulted in treatment success in 85.71% of cases, while the addition of long-term celecoxib treatment reduced it to 45.45%. Long-term celecoxib treatment significantly reduced bone loss (33.85% mean difference [95% CI 14.12–53.58], p=0.0004 on day 6 bone fraction) and periosteal reaction (0.2760 μm mean difference [95% CI 0.2073–0.3448], p<0.0001 on day 14 periosteal thickness) during early infection compared to the control group. Short- term celecoxib treatment showed similar radiological results without a reduction in treatment success (88.9%). No differences in the inflammatory markers were observed.

Our findings highlight the potential benefits of short-term use of celecoxib in improving bone fraction during the early post-infection period without impairing the efficacy of antibiotic therapy

Several bisphosphonates are now available for the treatment of osteoporosis. Porous hydroxyapatite/collagen (HA/Col) composite is an osteoconductive bone substitute which is resorbed by osteoclasts. The effects of the bisphosphonate alendronate on the formation of bone in porous HA/Col and its resorption by osteoclasts were evaluated using a rabbit model. Porous HA/Col cylinders measuring 6 mm in diameter and 8 mm in length, with a pore size of 100 μm to 500 μm and 95% porosity, were inserted into a defect produced in the lateral femoral condyles of 72 rabbits. The rabbits were divided into four groups based on the protocol of alendronate administration: the control group did not receive any alendronate, the pre group had alendronate treatment for three weeks prior to the implantation of the HA/Col, the post group had alendronate treatment following implantation until euthanasia, and the pre+post group had continuous alendronate treatment from three weeks prior to surgery until euthanasia. All rabbits were injected intravenously with either saline or alendronate (7.5 μg/kg) once a week. Each group had 18 rabbits, six in each group being killed at three, six and 12 weeks post-operatively. Alendronate administration suppressed the resorption of the implants. Additionally, the mineral densities of newly formed bone in the alendronate-treated groups were lower than those in the control group at 12 weeks post-operatively. Interestingly, the number of osteoclasts attached to the implant correlated with the extent of bone formation at three weeks. In conclusion, the systemic administration of alendronate in our rabbit model at a dose-for-weight equivalent to the clinical dose used in the treatment of osteoporosis in Japan affected the mineral density and remodelling of bone tissue in implanted porous HA/Col composites

Type 1 diabetes mellitus (DM) is associated with a decreased bone formation. Osteoblastic expression of parathyroid hormone-related protein(PTHrP) -an important modulator of osteoblast differentiation- decreases in age-related osteopenia. We here examined the putative role of PTHrP on the decreased osteoblastic function in DM. We performed marrow ablation in the tibiae of diabetic mice after streptozotocin injection (glycemia > 300mg/dl). Some mice were treated with PTHrP(1–36) (100 ng/g/every other day, s.c.) or vehicle for 2 weeks. Both tibiae were then removed for histological evaluation or total RNA isolation. In vitro, MC3T3-E1 cells were grown in differentiation medium (a-MEM), with or without high glucose(HG) (25 mM) (or mannitol, as osmotic control), supplemented (or not) with PTHrP(1–36) (100 nM). In some experiments, anti-PTHrP N-terminal antibody C13 (1:100) or PTHrP(7–34) (1 μM) were added to normal-glucose medium. RANKL secretion was measured in the cell-conditioned medium by ELISA. Gene expression was analyzed by real-time PCR. DM induced a 10–15% weight loss and a decrease (20–40%;p< 0.05) in the gene expression of the following osteoblastic factors in the regenerating tibia for 6 days: PTHrP, the PTH/PTHrP type1 receptor (PTH1R), osteocalcin, VEGF and its receptors 1 and 2; and in the OPG/RANKL ratio, related to an increased PPAR-γ mRNA expression. Compared to control mice, the regenerating tibia of DM mice showed a 5-fold increase in adipocyte number, and a decreased osteoblast number and osteoid surface. In MC3T3-E1 cells, HG decreased (20–40%) the OPG/RANKL ratio and the gene expression of both PTHrP/PTH1R and VEGF systems. PTHrP(1–36) reversed these HG-related effects in vivo and in vitro. Similar inhibitory effects were induced by a neutralizing PTHrP antibody or the antagonist PTHrP(7–34) in these cells in normal glucose. In conclusion, a deficit in PTHrP production by osteoblasts seems to be at least in part responsible for the DM-related decreased bone formation in mice

We have evaluated in vivo a novel, polymer-based, matrix for tissue engineering of bone. A segmental defect of 15 mm was created in the ulna of New Zealand white rabbits to determine the regenerative properties of a porous polylactide-co-glycolide matrix alone and in combination with autogenous marrow and/or the osteoinductive protein, BMP-7. In this study four implant groups were used: 1) matrix alone; 2) matrix with autogenous marrow; 3) matrix with 20 μg of BMP-7; and 4) matrix with 20 μg of BMP-7 and autogenous marrow. The results showed that the degree of bone formation was dependent on the properties of the graft material. The osteoconductive sintered matrix structure showed significant formation of bone at the implant-bone interface. The addition of autogenous marrow increased the penetration of new bone further into the central area of the matrix and also increased the degree of revascularisation. The osteoinductive growth factor BMP-7 induced penetration of new bone throughout the entire structure of the implant. The most effective treatment was with the combination of marrow cells and osteoinductive BMP-7

It has long been recognised that the periosteal membrane has osteogenic capability and experimental studies have concluded that periosteum transplanted to a distant site could also be osteogenic. This ability of periosteum to generate new bone at distant sites may have clinical application. In the laboratory setting however periosteal flaps in animals have demonstrated variable results. Little clinical work using the technique of periosteal transfer has been reported, with only individual case reports in the literature. A clinical review of a series of three fracture patients in whom vascularised periosteal transfer has been used is presented. Cases involved a primary bony defect at the fracture site (first metatarsal), established non-union (tibia) or post-traumatic AVN (talar dome). The technique is described and clinical follow-up of the patients is presented. In each instance evidence of lasting new bone formation was demonstrated clinically and radiologically. The efficacy of this technique in forming new bone is demonstrated. The technique may have utility alongside other techniques in cases where new bone is required

Summary Statement. An autologous thrombin activated 3-fold PRP, mixed with a biphasic calcium phosphate at a 1mL:1cc ratio, is beneficial for early bone healing in older age sheep. Introduction. The management of bone defects continues to present challenges. Upon activation, platelets secrete an array of growth factors that contribute to bone regeneration. Therefore, combining platelet rich plasma (PRP) with bone graft substitutes has the potential to reduce or replace the reliance on autograft. The simple, autologous nature of PRP has encouraged its use. However, this enthusiasm has failed to consistently translate to clinical expediency. Lack of standardisation and improper use may contribute to the conflicting outcomes reported within both pre-clinical and clinical investigations. This study investigates the potential of PRP for bone augmentation in an older age sheep model. Specifically, PRP dose is controlled to provide clearer indications for its clinical use. Methods. Eighty 11mm diameter defects of 20mm in depth were created in the cancellous bone within the epiphyseal region of the medial proximal tibia and distal femur of twenty five-year-old sheep. The defects were treated with three doses of an autologous thrombin activated PRP combined with a biphasic calcium phosphate (BCP). Activated platelet poor plasma (PPP) and the BCP alone provided reference groups, while the autograft and empty defects served as controls. All animals were sacrificed at four weeks post-operatively for radiographic assessment, micro-computed tomography quantification, histological assessment, histomorphometric quantification of new bone area and bone ingrowth, and weekly fluorochrome bone label quantification. TGF-β1 concentrations were quantified using enzyme-linked immunosorbent assays. Results. The PRP had a 2.9-fold (0.4) increase in platelet concentration, a 0.57-fold (0.09) decrease in leukocytes, and a 0.65-fold (0.11) decrease in fibrinogen. After activation, the PRP had an 8.9-fold (1.5) increase in TGF-β1 serum concentration above baseline. Eleven (11) mm diameter cancellous bone defects in the hind legs of five-year-old sheep do not spontaneously heal within four weeks. PRP dose had a significant effect on the radiographic grade. The highest dose of PRP treatment had a significantly greater micro-CT BV/TV over the BCP alone (PRP: 30.6±1.8%; BCP: 24.5±0.1%). All doses of PRP treatment were significantly greater than the BCP alone for both the histomorphometric new bone area (PRP: 14.5±1.3%; BCP: 9.7±1.5%) and bone ingrowth depth (PRP: 2288±210µm; BCP:1151±268µm). From week two onwards, PRP had a significant effect on the weekly bone ingrowth over BCP, however, autograft had the greatest amount of fluorescently labelled bone within the first three weeks. PRP has a significant effect on the shape and density of osteoblasts within the central region of the defect compared to the BCP alone, however, was not significantly different to autograft. TGF-β1 appeared a better predictor of healing outcomes than platelet concentration, however both had relatively weak correlations (r<.324). Conclusion. PRP induces new bone formation with a dose dependant response at four weeks when used with a biphasic calcium phosphate in older age sheep

We have studied prospectively the effect of indomethacin on the development of heterotopic ossification (HO) after the internal fixation of acetabular fractures. After operation 107 patients randomly received either a six-week course of indomethacin or no treatment against HO. Plain radiographs of 101 patients at a mean of 7.9 months after surgery showed HO in 47.4% of the 57 patients who received indomethacin and in 56.8% of the 44 who did not. This difference was not statistically significant. Heterotopic ossification of Brooker class II or more was seen in four patients (7%) with prophylaxis and in one without (p = 0.51). Measurements of the volume of HO on 3-D CT reconstructions showed a median value of 1.5 cm. 3. in patients with indomethacin and 4.0 cm. 3. in those without (p = 0.28). When only the 57 patients in whom the operation was carried out through either a Kocher-Langenbeck or an extended iliofemoral approach were included the indomethacin group showed a median volume of 1.7 cm. 3. compared with 3.6 cm. 3. On plain radiographs Brooker class II or above was seen in 9.4% of the patients receiving indomethacin and in 4.8% of those who did not. Indomethacin was therefore not effective in preventing ectopic bone formation after surgery for acetabular fractures. There was a significant association of male gender with volume of HO using a non-parametric analysis of variance

Ex vivo gene transfer of osteogenic factors into multipotential stem cells offers potentially important therapeutic implications in a variety of musculoskeletal diseases. One possible approach is the development of a cellular vehicle, namely bone morphogenetic protein (BMP)-producing bone marrow cells, created using adenoviral gene transfer. These transduced cells provide local delivery of BMP for bone formation. The aims of this study were to study the feasibility of gene transfer to human bone osteoprogenitor cells, using adenoviral vectors. Specifically, the aims were to study the efficacy of transduction with an adenoviral vector expressing BMP-2 and then to determine the ability of the transduced cells to produce active BMP-2 and to generate bone ex vivo. Primary human bone marrow osteoprogenitor cells were expanded in culture and infected with AxCALacZ, a replication-deficient adenoviral vector carrying the E. coli lacZ gene, with a range of multiplicity of infection (MOI) of 6.25 to100. Transduced cells showed positive staining for β-galactosidase using X-Gal with an efficiency close to 100%. Uninfected cells showed no β-galactosidase activity. Efficiency was independent from MOI, however cells infected at the lower MOIs expressed lower levels of β-galactosidase. Following confirmation that primary bone marrow cells could be infected by adenoviral constructs, additional osteoprogenitors were infected with AxCAOBMP-2, a vector carrying the human BMP-2 gene, at a multiplicity of infection of 10–20. In order to determine BMP-2 activity, conditioned media from bone marrow cells expressing BMP-2 was added to promyoblast C. 2. C. 12. cells. The promyoblast C. 2. C. 12. cells are exquisitely sensitive to BMP-2 with induction of alkaline phosphatase activity (ED. 50. 20 nM) in a dose-dependant manner. Alkaline phosphatase activity was induced following culture with conditioned media from BMP-2 expressing cells, in a dose dependant manner, confirming successful secretion of active BMP-2. Immunohistochemical staining for alka- line phosphatase in C. 2. C. 12. cells also confirmed the bio-chemical observations. Media from uninfected control human bone marrow cells failed to produce a similar effect. The concentration of BMP-2 in the media was estimated to be 5–10 nM/10. 7. cells. To examine whether adenoviral transfection affected the osteoblast phenotype and their ability to mineralise in vitro, adenovirally-transduced bone marrow cells expressing BMP-2 were seeded onto poly(-lactic acid co÷glycolic acid) (75:25) porous scaffolds (provided by K. Shakesheff and S. Howdle; Nottingham University) and cultured for up to 6 weeks. Expression of alkaline phosphatase activity, type I collagen formation, as well as the synthesis of osteoblast stimulating factor-1 confirmed bone cell differentiation and maintenance of the osteoblast phenotype in extended culture for up to 6 weeks. These results indicate the ability to deliver active BMP-2 using human bone marrow osteoprogenitor cells following adenoviral infection. The maintenance of osteoblast phenotype in extended culture and generation of mineralised 3-D scaffolds containing such constructs offers a realistic approach to tissue engineer bone for orthopaedic applications

Carbonate-substituted hydroxyapatite (CHA) is more osteoconductive and more resorbable than hydroxyapatite (HA), but the underlying mode of its action is unclear. We hypothesised that increased resorption of the ceramic by osteoclasts might subsequently upregulate osteoblasts by a coupling mechanism, and sought to test this in a large animal model.

Defects were created in both the lateral femoral condyles of 12 adult sheep. Six were implanted with CHA granules bilaterally, and six with HA. Six of the animals in each group received the bisphosphonate zoledronate (0.05 mg/kg), which inhibits the function of osteoclasts, intra-operatively.

After six weeks bony ingrowth was greater in the CHA implants than in HA, but not in the animals given zoledronate. Functional osteoclasts are necessary for the enhanced osteoconduction seen in CHA compared with HA.

Objective: To evaluate the effects of a new potent bisphosphonate on the formation, mineralisation, density, and mechanical properties of bone in distraction osteogenesis. Methods: Thirty immature New Zealand White rabbits had a 10.5 millimetre lengthening of their tibia performed over 2 weeks using an Orthofix M-100 fixator. Ten control rabbits received saline only; 10 received the new bisphosphonate at the time of surgery, and 10 received a second dose at the end of distraction. Bone mineral content (BMC) and density (BMD) measurements were made at two, four and six weeks. Quantitative CT analysis of regenerate, proximal and distal bone, and corresponding segments in the non-operated limb was performed after culling. Mechanical testing was by 4-point bending. Results: Bone mineral accrual was significantly faster in both treatment groups (ANOVA p< 0.01). BMD increased in all treated animals (ANOVA p< 0.01). Cross sectional area of regenerate at six weeks was increased by 49% in the single dosed group versus controls and by 59% in the re-dosed group. (ANOVA p< 0.01). BMC of the regenerate was increased by 92% in the single dose group and by 111% in the re-dosed group (ANOVA p< 0.01). Moment of inertia of the regenerate was significantly increased in both treated groups (ANOVA p< 0.05). The difference between single dose and controls was significant (p< 0.05), the difference between re-dosed and single dosed was not (p=0.5). Conclusion: Bisphosphonate therapy significantly increased new bone formation, bone mineralisation and mechanical properties. Osteoporotic effects were reversed. This effect could have wide ranging implications for many orthopaedic practices

Introduction: The aim of the current investigation to study the inherent ability of biomaterial scaffolds to regenerate bone defects without osteoinductive growth factors. We have developed a biosynthetic hybrid scaffold that mimics the biofunctionality of the provisional fibrin matrix which regulated the initial stages of in vivo bone regeneration. The material is comprised of a fibrinogen backbone and polyethylene glycol (PEG) cross-links that regulate the strength, durability, and degradation of the matrix during the healing process. Precise control over the degradability of the hydrogel scaffold provides the ability to systematically regulate the cellular infiltration associated with fracture healing. Furthermore, improved physical strength (over purified native fibrin clots) enables superior handling properties and stable in situ fixation. Materials & Methods: In the current study, a 7-mm critical size defect is created in the right tibia of female Sprague-Dawley rats (age 3–4 months); an external fixator is placed proximal and distal to the mid-section of the tibia. Pre-cast fibrinogen-PEG cylindrical hydro-gels (3-mm dia, 7-mm long) are placed into the site of the defect. Three different hydrogel compositions are tested: 1:1, 1:2, and 1:3 fibrinogen to PEG. Independent experiments demonstrate that higher concentrations of PEG give the hydrogels slower degradation kinetics. Radiographs, post operative and during follow-up, and histological evaluation were done. Results & Discussion: Both radiography and histological evaluation reveals extensive and widespread periosteal new bone formation. Post-operative radiographs show the formation of a periosteal callus in the gap region of treated animals after five weeks compared to immediately following excision (Figure 1, right). Five weeks post-operatively, histological sections stained with H& E reveal a thick covering of newly formed and moderately differentiated lamellar-fibred bone alongside lengthy stretches of the original cortex. There are large amounts of closely packed trabeculae of recently deposited, woven-fibered bone wherever there are empty spaces of the hydrogel scaffold. These trabeculae join at their perimeters with the preexisting bone. We also demonstrate a clear relationship between the composition of the hydrogel and the synthesis of new bone in the defect site. In conclusion, we demonstrate the formation of newly synthesized bone in critical size defects in the rat tibia using a biomimetic hydrogel scaffold without the use of exogenous growth factors

Current bone grafts include allograft and autografts, both of which have limitations. Tissue engineering biotechnology has shown considerable promise in improving grafts. A competent graft material should ideally have osteoconductive and osteoinductive properties and comprise of bone forming cells and osteoinductive growth factors. In this study, we have evaluated the in vitro formation of bone and have used human demineralised bone matrix [DBM] and human insoluble collagenous matric [ICM] as scaffolds for mesenchymal stem cells [MSCs] and osteogenic protein [OP-1]. The objective was to determine whether combined addition of OP-1 and MSCs resulted in a superior bone graft substitute by improving the inherent osteoinductive property. DBM and ICM were prepared and combined with rhOP [1.4 mg/0.25 mg of bone] and MSCs [1 x 105/ ml]. Statistically significant differences in MSC proliferation were seen between materials with and without OP-1 [P< 0.05}, n=8] in DBM on day 1, and both DBM and ICM on day 7 and 14. Enhanced osteogenic differentiation was observed in the presence of OP-1 when compared to DBM alone and on DBM and ICM with OP-1. In conclusion MSCs and OP-1 can be seeded together on DBM and ICM and Von Kossa staining and X-ray analysis confirmed in vitro de novo bone formation, with DBM + MSCs + OP-1 being more successful in this regard. Conclusion: To date, no other study, to the author’s knowledge, has used MSCs and OP-1 together on a graft material; this funding, therefore, has very important clinical implications

Purpose: Autologous bone grafting is considered the gold standard for multiple orthopaedic indications, including non-union of fractures and other bone defects. Previously autograft was most commonly harvested from the iliac crest, with an estimated complication rate of greater than 10%. New technology, the RIA system, allows harvest of graft material from the medullary canal of the femur. The purpose of this study is to examine the osteo-inductive properties of this human femoral bone graft obtained using the RIA system and the RIA filtrate combined with chronOS (Tricalcium Phosphate). This study will examine whether these materials will induce bone growth when implanted in a rat sub-muscular pouch model. Method: Three samples were collected from each human subject. These included:. femoral bone graft obtained using the RIA {n=10}. chronOS washed with RIA filtrate {n=10}and. a mixture of these two materials {n=10}. chronOS (alone) was used as control {n=10}. These materials were implanted into a sub-muscular pouch in athymic rats (to eliminate rejection of the xenograft). Rat serum levels of BMP-2, VEG-F, TGF-β and IL-10 were obtained at days 7, 14, 21 and 28. Rats were sacrificed at day 28 and radiographic and histologic examinations and histomorphometric analyses were performed. Results: Overall, there were no significant differences in BMP-2, VEG-F, TGF-β and IL-10 levels either between groups or between time points. Average serum values for BMP-2 decreased over time for all groups. Histologically and radiographically, all four materials induced new bone production. chronOS alone produced the greatest volume of new bone while RIA reamings alone produced the least. Histological analysis demonstrated formation of normal bone. Conclusion: The RIA system allowed for harvest of femoral bone graft. This graft induced bone formation and increased osteogenic protein levels when implanted in the rat model. The RIA filtrate, when combined with Tricalcium Phosphate, is equally osteoinductive. Combining reamings with filtrate material may allow large volumes of graft to be produced using this system. This new technology may allow the collection of large volume, osteoinductive grafts without the complications previously described for iliac crest bone graft harvesting

The pathogenesis of Calcifying Tendinitis (CT) is still not well established. Prognostic factors for outcome could not yet be identified. The purpose of this study was to evaluate the histologic features of calcific deposits (CD) and their correlation with radiologic and clinical findings.

122 patients with a radiologically confirmed CD were prospectively scheduled for arthroscopic shoulder surgery. According to their radiologic appearance (RA) the CD were graded as fluffy or sharply demarcated. Arthroscopic removal of the deposit was performed and biopsies were taken and embedded in methylmethacry-late. Sections were stained and also immunohistology was performed. Shoulder function was assessed with the Constant score (CS) and the SST.

Three distinct histologic stages (HS) of the CDs could be divided: calcification (I), fibrotic organisation (II) and ossification (III). Biopsies revealed 42x (34%) HS I, 18x (15%) HS II and 62x (51%) HS III deposits. 90% of the CD were located in the SSP tendon. 12 months after the operation the CS and the SST showed a significant improvement (p< 0.01). Forty percent of the patients with ossification (III) of the CD underwent unsuccessful shock wave therapy before. The preoperative RA as well as the HS of the CD did not predispose to postoperative outcome.

In this study three definite histologic stages of Calcifying Tendinitis were identified that have not been described previously. We underline the hypothesis that CT is an active cell mediated tissue process which can lead to production of primitive bone.

Chronic non-specific tenosynovitis of the posterior tibial tendon is a well-known clinical entity, characterised by pain, swelling and tenderness behind the medial malleolus. There are no reports in the literature of any case associated with any radiological abnormality. Three such cases are discussed, each presenting with the clinical findings typical of the syndrome but with associated radiological changes. These changes may pose diagnostic problems and several primary conditions need to be excluded. Surgical decompression of the tendon may be necessary to relieve symptoms, to prevent erosion and rupture of the tendon, and to establish the diagnosis.

Introduction: Distraction osteogenesis is a widely utilised orthopaedic procedure; however prolonged treatment time and considerable disuse osteoporosis remain problematic, with decreases of 44% to 61% in bone mineral density reported in adjacent bone. Refracture rates of 10–20% are reported after frame removal.

We set out to examine the role of bisphosphonates in protecting the bone against stress-shielding related osteopaenia during distraction osteogenesis. We used a NZW rabbit model with 2 weeks distraction to 10.5 mm then 4 weeks consolidation. We achieved positive results in the initial trial using the bisphosphonate pamidronate (Novartis). Not only were we able to abolish the decrease in BMD in the surrounding bone, we noted an increase in the mineral properties and strength of the new bone.

Moving on to zoledronic acid (Novartis), a third generation bisphosphonate designed for use in malignant hypercalcaemia and bone metastases, we achieved even more promising results. In a study of thirty rabbits, we gave saline to 10 controls, 0.1 mg/kg zoledronic acid to 10 rabbits at surgery and 10 further rabbits received 0.1 mg/kg zoledronic acid at surgery and at two weeks. The animals were scanned by DXA at 2, 4 and 6 weeks, and by QCT after culling. Mechanical testing was performed by destructive 4-point bend tests.

Second-weekly DXA scans documented faster mineral accrual after distraction between 2 and 4 weeks in both treatment groups (ANOVA p< 0.01). In the control group, the BMD in the segments around the lengthening fell by 0.16 g/cm2 between the 2nd and 6th week. The BMD showed a net increase over the same time period in all treated animals (ANOVA p< 0.01).

The cross sectional area of the regenerate at six weeks as measured by QCT was increased by 49% in the zoledronate group versus controls and by 59% in the re-dosed zoledronate group. (ANOVA p< 0.01). The final (6 week) BMC of the regenerate was increased by 92% in the zoledronate group versus controls and by 111% in the re-dosed zoledronate group (ANOVA p< 0.01). Bone mineral density was increased by a lesser but significant degree to normal values (28% and 34% respectively, ANOVA p< 0.01).

Four point bend testing revealed increases in peak load of 29% in the single dose and 89% in the re-dosed group (ANOVA p< 0.01).

Two patients are presented, one with congenital pseudarthrosis of the tibia, and one who had not united a distraction gap of 5 cm at six months, who were treated with pamidronate. Both showed successful responses in line with our research findings.

A clinical trial is being set up to establish a scientific case for bisphosphonate use in patients undergoing distraction osteogenesis with the aim of possible earlier frame removal and less refractures. Further research in other areas of bone healing is also planned.

To test the hypothesis that: CERAMENT[™]|G (C-G) would improve new bone growth and decrease infection rate after debridement as compared with 1) CERAMENT|BONE VOID FILLER (CBVF) and 2) no void filler in a rat osteomyelitis model.

72 Sprague Dawley rats were injected with 1.5 × 10∧6 CFU of S. aureus into a drill hole in the right tibia. After 3 weeks, the osteomyelitic defect was debrided, and filled with either: 1) C-G (n=32), 2) CBVF (n=20), or 3) nothing (n=20). 6 weeks after the second surgery, 20 rats from each group were sacrificed and the right tibias were harvested. A long-term group (n=12) of C-G treated rats were also sacrificed at 6 months after the second surgery. The tissues were sonicated and the colony forming units in the sonicate were quantified by serial dilutions and culture. MicroCT was used to quantify the new bone growth (BV/TV) in the debrided osteomyelitic void. Histological samples were analyzed for the presence of a neutrophil response by a blinded pathologist.

(*: p<0.05)

Positive cultures in:

○ 30% of animals treated with CBVF

Neutrophil reaction in:

○ 35% of animals treated with CBVF

The BV/TV in:

○ C-G treated rats was 24% greater than CBVF treated rats (*)

Animals sacrificed at 6 months which were treated with C-G did not have any evidence of infection by culture or histology. The bone mass of the implanted limb was higher than the contralateral (non-operated) side.

CERAMENT|G decreased the rate of infection and increased new bone growth as compared with both CBVF and no void filler in a debrided osteomyelitic environment. Animals treated with C-G at 6 months showed no evidence of infection and retained a higher bone mass relative to the contralateral (non-operated) side.

This study supports the use of CERAMENT|G as a readily available void filler which could be used in osteomyelitic environments after debridement.

We studied the quantity and rate of formation of new bone during lengthening of 17 limb segments in 10 patients using dual-energy X-ray absorptiometry (DEXA), ultrasonography and radiography. Whereas new bone was detected by both DEXA and ultrasonography within 1 to 2 weeks of distraction, it was not visible on the radiographs until 4 to 8 weeks. Limb alignment and gap measurement were accurately assessed by DEXA without the need for standard radiographs or scanograms. With ultrasound the distraction gap appeared as an echolucent window which narrowed progressively producing a hyper-reflecting line after which further consolidation could not be assessed. As measured by DEXA the density of the new bone at this stage was approximately 45% of control values and did not represent normal cortication. Whereas ultrasound could be used to identify defects in mineralisation and to determine when to dynamise the fixator system, DEXA could measure the quantity and rate of formation of bone throughout lengthening.

Aims: In a recent study, chemical microroughening of bioactive glass surface was shown to promote attachment of osteoblastic cells and osseointegration of porous bioactive glass implant. The current in vivostudy employed molecular biologic techniques to clarify the osteogenic effects of smooth and microrough glass surfaces. Methods:Using a rat model, a portion of the medullary canal in the proximal tibia was evacuated and filled with microroughened or smooth bioactive glass microspheres. The primary bone healing response and subsequent remodelling were analysed at 1, 2, and 8 weeks, respectively. The expression of various genes for the bone matrix components (type I collagen, osteocalcin, osteopontin, osteonectin) and proteolytic enzymes (cathepsin K, MMP-9) were determined by Northern analysis. Results: The microroughened bioactive glass microspheres were found to induce higher mRNA levels for osteopontin and lower levels for osteonectin at 2 weeks after operation when compared to smooth control micropheres. At 8 weeks, the MMP-9 expression levels were significantly higher with microroughened bioactive glass microspheres. Conclusion: Microroughening of the bioactive glass surface triggered temporal changes in the expression of specific genes.

Aim:. The objective of this study was to improve the osseoinductive capacity of human demineralised bone matrix (DBM) and human insoluble collagenous matrix (ICM), following incorporation of recombinant human osteogenic protein 1 (rhOP-1) and human mesenchymal stem cells (MSCs). Ethical Committee approval has been obtained by our Institution. Methods: Recombinant human osteogenic protein Ð 1 (400ng/0.25g of bone) was seeded onto DBM and ICM together with human MSCs (1 x 105). Cellular proliferation was quantitatively evaluated in vitrousing Alamar Blue and 3H-TdR assays. Quantitative cellular differentiation was assessed using the alkaline phosphatase assay. Von Kossa staining, X-ray analysis, and PCR were used for qualitative evaluation of cellular differentiation. Qualitative analysis of proliferation and differentiation was assessed using scanning electron microscopy (SEM). Results: MSC proliferation and differentiation down the osteogenic lineage was observed on DBM and ICM in the presence of OP-1, and also on DBM alone. Alamar blue and 3H-TdR assays conþrmed that MSC proliferation occurred on both DBM and ICM, with the values being signiþcantly greater with addition of OP-1. The ALP activity showed that MSCs differentiated into osteo-blasts on DBM alone, and on DBM and ICM with OP-1. In all cases, OP-1 had a signiþcant effect on MSCs. Discussion: DBM and ICM when seeded with MSCs and OP-1 provide an improved osteoconductive and osteoinductive graft material resulting in de novo-bone formation.

Aims. Distraction osteogenesis (DO) is a useful orthopaedic procedure employed to lengthen and reshape bones by stimulating bone formation through controlled slow stretching force. Despite its promising applications, difficulties are still encountered. Our previous study demonstrated that pulsed electromagnetic field (PEMF) treatment significantly enhances bone mineralization and neovascularization, suggesting its potential application. The current study compared a new, high slew rate (HSR) PEMF signal, with different treatment durations, with the standard Food and Drug Administration (FDA)-approved signal, to determine if HSR PEMF is a better alternative for bone formation augmentation. Methods. The effects of a HSR PEMF signal with three daily treatment durations (0.5, one, and three hours/day) were investigated in an established rat DO model with comparison of an FDA-approved classic signal (three hrs/day). PEMF treatments were applied to the rats daily for 35 days, starting from the distraction phase until termination. Radiography, micro-CT (μCT), biomechanical tests, and histological examinations were employed to evaluate the quality of bone formation. Results. All rats tolerated the treatment well and no obvious adverse effects were found. By comparison, the HSR signal (three hrs/day) treatment group achieved the best healing outcome, in that endochondral ossification and bone consolidation were enhanced. In addition, HSR signal treatment (one one hr/day) had similar effects to treatment using the classic signal (three three hrs/day), indicating that treatment duration could be significantly shortened with the HSR signal. Conclusion. HSR signal may significantly enhance bone formation and shorten daily treatment duration in DO, making it a potential candidate for a new clinical protocol for patients undergoing DO treatments. Cite this article: Bone Joint Res 2021;10(12):767–779

Successful application of patient derived cells to engineer vascularized bone grafts is often hampered by low cell numbers and lengthy in vitro expansion. With sound induced morphogenesis (SIM), local cell density enhancement was shown to improve microvasculature formation at lower cell concentration than conventional methods [1]. SIM takes advantage of hydrodynamic forces that act on cells to arrange them within a hydrogel. Following, we are evaluating the potential of cell-hydrogel biografts with high local cell density to improve the therapeutic efficacy in clinical scenarios such as anastomosis or bone formation within non-union fractures. To assess anastomosis, human umbilical vein endothelial cells (HUVEC) and human mesenchymal stromal cells (MSC) were mixed at a 1:1 ratio in PEG-based or Dextran-based hydrogels at a final concentration of 2×10. 6. cells×mL. -1. For ectopic bone formation, MSC were resuspended in PEG-based hydrogels at 2×10. 6. or 5×10. 6. cells×mL. -1. , with or without BMP-2. Cells were assembled into distinct patterns at a frequency of 60 Hz. Four biografts of 4 × 9 mm. 2. were implanted at the back of nude mice (total of 7 animals) and harvested after 2 or 8 weeks. Explants were fixed and imaged as whole constructs or embedded in paraffin for histological analysis. Upon explantation, microscopic evaluation indicated that HUVEC were retained within the PEG-hydrogel after 2 weeks and formed a pre-vascular network. In the second study, ectopic bone formation was more pronounced in areas of higher local cell density based on visual inspection. Ongoing experiments are further characterizing bone formation by micro-CT and histological evaluation. Our results indicate that local cell density enhancement by sound requires a lower initial cell concentration than conventional, static seeding methods to achieve comparable microvasculature structures or local osteogenesis

Introduction and Objective. Bone is a tissue which continually regenerates and also having the ability to heal after injuries however, healing of large defects requires intensive surgical treatment. Bioactive glasses are unique materials that can be utilized in both bone and skin regeneration and repair. They are degradable in physiological fluids and have osteoconductive, osteoinductive and osteostimulative properties. Osteoinductive growth factors such as Bone Morphogenetic Proteins (BMP), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), Transforming Growth Factor (TGF) are well known to stimulate new bone formation and regeneration. Unfortunately, the synthesis of these factors is not cost- effective and, the broad application of growth factors is limited by their poor stability in the scaffolds. Instead, it is wise to incorporate osteoinductive nanomaterials such as graphene nanoplatelets into the structures of synthetic scaffolds. In this study, borate-based 13-93B3 bioactive glass scaffolds were prepared by polymer foam replication method and they were coated with graphene-containing poly (ε-caprolactone) layer to support the bone repair and regeneration. Materials and Methods. Effects of graphene concentration (1, 3, 5, 10 wt%) on the healing of rat segmental femur defects were investigated in vivo using male Sprague–Dawley rats. Fabricated porous bioactive glass scaffolds were coated by graphene- containing polycaprolactone solution using dip coating method. The prepared 0, 1, 3, 5 and 10 wt% graphene nanoparticle-containing PCL-coated composite scaffolds were designated as BG, 1G-P-BG, 3G-P-BG, 5G-P-BG and 10G-P-BG, for each group (n: 4) respectively. Histopathological and immunohistochemical (bone morphogenetic protein, BMP-2; smooth muscle actin, SMA and alkaline phosphatase, ALP) examinations were made after 4 and 8 weeks of implantation. Results. Results showed that after 8-weeks of implantation both cartilage and bone formation were observed in all animal groups. After 4 and 8 weeks of implantation the both osteoblast and osteoclast numbers were significantly higher in the group 4 compared to the control group. Bone formation was significant starting from 1 wt% graphene-coated bioactive glass implanted group and highest amount of bone formation was obtained in group containing 10 wt% graphene (p<0.001). Newly formed vessels expressed this marker and increased vascularization was observed in 8- weeks period compared to the 4-weeks period. In addition, an increase in new vessel formation were observed in graphene-coated scaffold implanted groups compared to the control group. While cartilage tissue was observed in control group, bone formation percentages were significant in graphene-coated scaffold implanted groups. Highest amount of bone formation occurred in group 4 (10 % wt G-C). Conclusions. Additionally, the presence of graphene nanoplatelets enhanced the BMP-2, SMA and ALP levels compared to the bare bioactive glass scaffolds. It was concluded that pristine graphene-coated bioactive glass scaffolds improve osteointegration and bone formation in rat femur defect when compared to bare bioglass scaffolds

Abstract. Objectives. The term heterotopic ossification (HO) describes lamellar bone formation within soft tissues following injury. A genome-wide scan of patients after hip arthroplasty has identified that variation within the lncRNA CASC20 is associated with HO susceptibility. Previous findings in our lab have demonstrated upregulation of CASC20 during BMP2-induced osteodifferentiation of adipose-derived stem cells (hMAD) alongside osteodifferentiation markers, RUNX2 and OSX. We hypothesize that CASC20 is a novel regulator of bone formation and aim to investigate CASC20 function in bone formation. Methods. 1) We used miRanda prediction algorithm and the ENCORI database to respectively predict which miRNAs CASC20 interacts with and to select for experimentally validated miRNAs. 2) We characterized the expression and functional role of CASC20-interacting miRNAs by respectively analyzing publicly available datasets (GSE107279 and pubmed.ncbi.nlm.nih.gov/26175215/) and by using Gene Ontology (GO) analysis. 3) We overexpressed CASC20 in hMAD using a lentiviral system and tested the effect of CASC20 overexpression in osteodifferentiation and expression of putative CASC20-interacting miRNAs. Results. 1) We identified 64 experimentally validated miRNAs that are predicted to interact with CASC20. 2) GO analysis revealed that the most frequently targeted molecular functions included SMADs, MAPKK and other kinase activities known to play a central role in osteo and chondrogenesis. We found 10 miRNAs including hsa-miR-485-3p that demonstrated down-regulation in both osteo- and chondrogenesis. 3) We found that CASC20-overexpression augmented the osteodifferentiation of hMAD measured in mineralization using Alizarin Red S. CASC20 overexpression increased the expression of osteogenic marker ALP and decreased the expression of hsa-miR-485-3p. Conclusion. Here we show how CASC20 may regulate bone formation by acting as a competitive endogenous RNA (ceRNA). We are currently using CASC20 overexpression model in osteo- and chondrogenesis, and testing CASC20-miRNA interaction to establish the underlying mechanism for the observed associations

Critical size bone defects are frequently caused by accidental trauma, oncologic surgery, and infection. Distraction osteogenesis (DO) is a useful technique to promote the repair of critical size bone defects. However, DO is usually a lengthy treatment, therefore accompanied with increased risks of complications such as infections and delayed union. Herein, we developed an innovative intramedullary biodegradable magnesium (Mg) nail to accelerate bone regeneration in critical size bone defect repair during DO. We observed that Mg nail induced almost 4-fold increase of new bone formation and over 5-fold of new vessel formation at 2 weeks after distraction. Mg nail upregulated the expression of calcitonin gene-related peptide (CGRP) in the new bone as compared with the DO alone group. We further revealed that blockade of the sensory nerve by overdose capsaicin blunted Mg nail enhanced critical size bone defect repair during the DO process. Moreover, inhibitors/antagonist of CGRP receptor, FAK, and VEGF receptor blocked the Mg nail stimulated vessel and bone formation. In summary, we revealed, for the first time, a CGRP-FAK-VEGF signaling axis linking sensory nerve and endothelial cells, which may be the main mechanism underlying Mg-enhanced critical size bone defect repair when combined with DO, suggesting a great potential of Mg implants in reducing DO treatment time for clinical applications

Bone defects require implantable graft substitutes, especially porous and biodegradable biomaterial for tissue regeneration. The aim of this study was to fabricate and assess a 3D-printed biodegradable hydroxyapatite/calcium carbonate scaffold for bone regeneration. Materials and methods:. A 3D-printed biodegradable biomaterial containing calcium phosphate and aragonite (calcium carbonate) was fabricated using a Bioplotter. The physicochemical properties of the material were characterised. The materials were assessed in vitro for cytotoxicity and ostegenic potential and in vivo in rat intercondylar Φ3mm bone defect model for 3 months and Φ5mm of mini pig femoral bone defects for 6 months. The results showed that the materials contained hydroxyapatite and calcium carbonate, with the compression strength of 2.49± 0.2 MPa, pore size of 300.00 ± 41mm, and porosity of 40.±3%. The hydroxyapatite/aragonite was not cytotoxic and it promoted osteogenic differentiation of human umbilical cord matrix mesenchymal stem cells in vitro. After implantation, the bone defects were healed in the treatment group whereas the defect of controlled group with gelatin sponge implantation remained non-union. hydroxyapatite/aragonite fully integrated with host bone tissue and bridged the defects in 2 months, and significant biodegradation was followed by host new bone formation. After implantation into Φ5mm femoral defects in mini pigs hydroxyapatite/aragonite were completed degraded in 6 months and fully replaced by host bone formation, which matched the healing and degradation of porcine allogenic bone graft. In conclusion, hydroxyapatite/aragonite is a suitable new scaffold for bone regeneration. The calcium carbonate in the materials may have played an important role in osteogenesis and material biodegradation

Background. Current clinical treatment for spinal instability requires invasive spinal fusion with cages and screw instrumentation. We previously reported a novel injectable hydrogel (Bgel), which supports the delivery and differentiation of mesenchymal stem cells (MSCs) to bone forming cells and supports bone formation in vivo. Here, we investigated whether this system could be utilised to induce bone formation within intervertebral disc tissue as a potential injectable spinal fusion approach. Methodology. Bovine and Human Nucleus pulpous tissue explants were injected with Bgel with and without MSCs. Tissue samples were cultured under hypoxia (5%) in standard culture media for 4 weeks. Cell viability, histological assessment of matrix deposition, calcium formation, and cell phenotype analysis using immunohistochemistry for NP matrix and bone markers. Results. Following injection of B-gel into tissue explants following culture for 4 weeks, cells were visualized within the regions of the B-gel. Demonstrating that native cells were able to migrate into regions of B-gel. Increased collagen deposition was seen in tissue explants injected with Bgel, with increased collagen type I and X but decreased collagen type II staining in explants injected with Bgel. Tissue explants, in the absence of Bgel, showed limited calcium deposition, which was increased in B-gel injected explants. Furthermore, disc cells increased expression of bone markers (alkaline phosphatase & osteocalcin), but decreased NP matrix (Aggrecan and Collagen type II) following Bgel injection. Conclusion. This system could have potential to support spinal fusion via direct injection into the disc. Conflict of interest: C Le Maitre & C Sammon are inventors on the hydrogel discussed. Funding: This work was funded by GrowMed Tech Proof of Concept funding

A novel EP4 selective agonist (KMN-159) was developed [1] and has been proven that it can act as an osteopromotive factor to repair critical-size femoral bone defects in rats at a dose-dependent manner [2]. Based on its osteopromotive properties, we hypothesized that KMN-159 could also aid in bone formation for spinal fusion. Therefore, the aim of this study was to investigate its spinal fusion effect in a dorsolateral spinal fusion model in rats. This study was performed on 192, 10-week-old male Wistar rats. The rats were randomized into 8 groups (n = 12 per group): 1) SHAM (negative control), 2) MCM (scaffold only), 3) MCM + 20 µg BMP-2 (positive control), 4-8) MCM + 0.2, 2, 20, 200 or 2000 µg KMN-159. A posterolateral intertransverse process spinal fusion at L4 to L5 was performed bilaterally by implanting group dependent scaffolds (see above) or left empty in the SHAM group (protocol no. 25-5131/474/38). Animals were euthanized after 3 weeks and 6 weeks for µCT and biomechanical testing analysis. The results showed that KMN-159 promoted new bone formation in a dose-dependent manner at 3 weeks and 6 weeks as verified by µCT. The biomechanical testing showed that the dose of 20, 200 and 2000 µg KMN-159 groups obtained comparable strength with BMP-2 group, which higher than SHAM, MCM and lower doses of 0.2 and 2 µg KMN-159 groups. In conclusion, KMN-159 could be a potential replacement of BMP-2 as a novel osteopromotive factor for spinal fusion. Acknowledgements: We are grateful to Ulrike Heide, Anna-Maria Placht (assistance with surgeries) as well as Suzanne Manthey & Annett Wenke (histology)

Bone homeostasis is a highly regulated process involving pathways in bone as WNT, FGF or BMP, but also requiring support from surrounding tissues as vessels and nerves. In bone diseases, the bone-vessel-nerve triad is impacted. Recently, new players appeared as regulators of bone homeostasis: microRNAs (miRNA). Five miRNAs associated with osteoporotic fractures are already known, among which miR-125b is decreasing bone formation by downregulating human mesenchymal stem cells (hMSCs) differentiation. Other miRNAs, as miR-214 (in cluster with miR-199a), are secreted by osteoclasts to regulate osteoblasts and inhibit bone formation. This forms a very complex regulatory network. hMSCs and osteoblasts (n=3) were transfected with mimic/antagomiR of miR-125b, miR-199a-5p or miR-214, or with a scrambled miRNA (negative control) in osteogenic differentiation calcium-enriched medium (Ca++). Mineralization was assessed by Alizarin Red/CPC staining, miRNA expression by qPCR and protein by western blotting. Exposure of hMSCs or osteoblasts to Ca++ increased mineralization compared to basal medium. hMSCs transfected with miR-125b mimic in Ca++ presented less mineralization compared to scramble. This correlated with decreased levels of BMPR2 and RUNX2. hMSCs transfected with miR-125b inhibitor presented higher mineralization. Interestingly, hMSCs transfected with miR-214 mimic in Ca++ presented no mineralization while miR-214 inhibitor increased mineralization. No differences were observed in hMSCs transfected with miR-199a-5p modulators. On the contrary, osteoblasts transfected with miR-199a-5p mimic present less mineralization than scrambled-transfected and same was observed for miR-214 and miR-125b mimics. We highlight that miR-125b and miR-214 decrease mineralization of hMSCs in calcium-enriched medium. We noticed that miR-199a-5p is able to regulate mineralization in osteoblasts but not in hMSCs suggesting that this effect is cell-specific. Interestingly, the cluster miR-199a/214 is known as modulator of vascular function and could thus contribute to bone remodeling via different ways. With this work we slightly open the door to possible therapeutic approaches for bone diseases

Metabolic bone diseases, such as osteoporosis and osteopetrosis, result from an imbalanced bone remodeling process. In vitro bone models are often used to investigate either bone formation or resorption independently, while in vivo, these processes are coupled. Combining these processes in a co-culture is challenging as it requires finding the right medium components to stimulate each cell type involved without interfering with the other cell type's differentiation. Furthermore, differentiation stimulating factors often comprise growth factors in supraphysiological concentrations, which can overshadow the cell-mediated crosstalk and coupling. To address these challenges, we aimed to recreate the physiological bone remodeling process, which follows a specific sequence of events starting with cell activation and bone resorption by osteoclasts, reversal, followed by bone formation by osteoblasts. We used a mineralized silk fibroin scaffold as a bone-mimetic template, inspired by bone's extracellular matrix composition and organization. Our model supported osteoclastic resorption and osteoblastic mineralization in the specific sequence that represents physiological bone remodeling. We also demonstrated how culture variables, such as different cell ratios, base media, and the use of osteogenic/osteoclast supplements, and the application of mechanical load, can be adjusted to represent either a high bone turnover system or a self-regulating system. The latter system did not require the addition of osteoclastic and osteogenic differentiation factors for remodeling, therefore avoiding growth factor use. Our in vitro model for bone remodeling has the potential to reduce animal experiments and advance in vitro drug development for bone remodeling pathologies like osteoporosis. By recreating the physiological bone remodeling cycle, we can investigate cell-cell and cell-matrix interactions, which are essential for understanding bone physiology and pathology. Furthermore, by tuning the culture variables, we can investigate bone remodeling under various conditions, potentially providing insights into the mechanisms underlying different bone disorders

Bone defects and fractures, caused by injury, trauma or tumour resection require hospital treatment and temporary loss of mobility, representing an important burden for societies and health systems worldwide. Autografts are the gold standard for promoting new bone formation, but these may provide insufficient material and lead to donor site morbidity and pain. We previously showed that Fibrinogen (Fg) scaffolds promote bone regeneration in vivo (1), and that modifying them with 10mM of Magnesium (Mg) ions modulates macrophage response in vitro and in vivo (2). Also, we showed that Extracellular Vesicles (EV) secreted by Dendritic Cells (DC) recruit Mesenchymal Stem/Stromal Cells (MSC)(3). Herein, we aim to functionalize FgMg scaffolds with DC-EV, to promote recruitment and osteogenic differentiation of MSC. Scaffolds were produced by freeze-drying (2). Ethical permission was sought for all studies. Primary human peripheral blood monocyte-derived DC were cultured, their secreted EV were isolated by differential (ultra)-centrifugation and characterised by transmission electron microscopy and nanoparticle tracking analysis (3). Bone marrow MSC were used to determine the impact of EV-functionalized scaffolds through migration assays and their osteogenic differentiation was assessed by Alizarin Red staining. Fg and FgMg scaffolds functionalized with EV were characterized. Fg and FgMg scaffolds functionalized with DC-secreted EV were more efficient at recruiting MSC than scaffolds alone. MSC cultured on FgMg scaffolds showed significantly increased calcium deposits, in comparison with those cultured on Fg scaffolds. Fg scaffold modification by Mg promotes MSC osteogenic differentiation, while their functionalization with DC-secreted EV acts to promote MSC recruitment. This renders the FgMg-EV functionalized scaffolds an attractive material to promote new bone formation. Acknowledgments: Work funded by Orthoregeneration Network (ON Pilot Grant Spine 2021, EVS4Fusion). MCT supported by ERASMUS+ program

Patients with bone and muscle weakness from disuse have higher risk of fracture and worse post-injury mortality rates. The goal of this current study was to better inform post-fracture rehabilitation strategies by investigating if physical remobilization following disuse by hindlimb unloading improves osteochondral callus formation compared to continued disuse by hindlimb suspension (HLS). We hypothesized that continued HLS would impair callus bone and cartilage formation and that physical rehabilitation after HLS would increase callus properties. All animal procedures were approved by the VCU IACUC. Skeletally mature, male and female C57BL/6J mice (18 weeks) underwent HLS for 3 weeks. Mice then had their right femur fractured by open surgical dissection (stabilized with 24-gauge pin). Mice were then either randomly assigned to continued HLS or allow normal physical weight-bearing remobilization (HLS + R). Mice allowed normal cage activity throughout the experiment served as controls (GC). All mice were sacrificed 14-days following fracture with 4-8 mice (male and female) per treatment. Data analyzed by respective ANOVA with Tukey post-hoc (*p< 0.05; # p < 0.10). Male and female mice showed conserved and significant decreases in hindlimb callus bone formation from continued HLS versus HLS + R. Combining treatment groups regardless of mouse sex, histological analyses using staining on these same calluses demonstrated that HLS resulted in trends toward decreased cartilage cross-sectional area and increased osteoclast density in woven bone versus physically rehabilitated mice. In support of our hypothesis, physical remobilization increases callus bone formation following fracture compared to continued disuse potentially due to increased endochondral ossification and decreased bone resorption. In all, partial weight-bearing exercise immediately following fracture may improve callus healing compared to delayed rehabilitation regimens that are frequently used

Various approaches have been implemented to enhance bone regeneration, including the utilization of autologous platelet-rich plasma and bone morphogenetic protein-2. The objective of this study was to evaluate the impact of Marburg Bone Bank-derived bone grafts in conjunction with platelet-rich plasma (PRP), recombinant human bone morphogenetic protein-2 (rhBMP-2), and zoledronic acid (ZA) on osteogenesis within rabbit bone defects. Methodology. Bone defects (5mm in diameter) were created in the femurs of 96 male rabbits. The animals were allocated into five groups: (1) bone graft + PRP (BG + PRP), (2) bone graft + 5μg rhBMP-2 (BG + rhBMP-2), (3) bone graft + 5μg ZA (BG + ZA), (4) bone graft + 10μg rhBMP-2 + 5μg ZA (BG + rhBMP-2 + ZA), and (5) bone graft (BG). Marburg Bone Bank-processed human femoral head allografts were utilized for bone grafting. The rabbits were euthanized at 14-, 30-, and 60-days post-surgery, and their femurs underwent histopathological and histomorphometric assessments. Results. Histomorphometric analysis revealed significantly enhanced de novo osteogenesis within the bone allografts in the BG + PRP and BG + rhBMP-2 groups compared to the BG, BG + ZA, and BG + rhBMP-2 + ZA groups at 14 and 30 days (p < 0.05). However, on day 60, the BG + rhBMP-2 group exhibited elevated osteoclastic activity (early resorption). The local co-administration of ZA with thermally treated grafts impeded both bone graft resorption and new bone formation within the bone defect across all time points. The addition of ZA to BG + rhBMP-2 resulted in diminished osteogenic activity compared to the BG + rhBMP-2 group (p < 0.000). Conclusion. The study findings indicated that the combination of PRP and rhBMP-2 with Marburg bone grafts facilitates early-stage osteogenesis in bone defect healing. Incorporating ZA into the thermally treated bone graft hinders both graft resorption and de novo bone formation

Bone regeneration is an area of acute medical need, but its clinical success is hampered by the need to ensure rapid vascularization of osteogenic grafts. Vascular Endothelial Growth Factor (VEGF) is the master regulator of vascular growth and during bone development angiogenesis and osteogenesis are physiologically coupled through so-called angiocrine factors produced by blood vessels. However, how to exploit this process for therapeutic bone regeneration remains a challenge (1). Here we will describe recent work aiming at understanding the cross-talk between vascular growth and osteogenesis under conditions relevant for therapeutic bone regeneration. To this end we take advantage of a unique platform to generate controlled signalling microenvironments, by the covalent decoration of fibrin matrices with tunable doses and combinations of engineered growth factors. The combination of human osteoprogenitors and hydroxyapatite in these engineered fibrin matrices provides a controlled model to investigate how specific molecular signals regulate vascular invasion and bone formation in vivo. In particular, we found that:. 1). Controlling the distribution of VEGF protein in the microenvironment is key to recapitulate its physiologic function to couple angiogenesis and osteogenesis (2);. 2). Such coupling is exquisitely dependent on VEGF dose and on a delicate equilibrium between opposing effects. A narrow range of VEGF doses specifically activates Notch1 signaling in invading blood vessels, inducing a pro-osteogenic functional state called Type H endothelium, that promotes differentiation of surrounding mesenchymal progenitors. However, lower doses are ineffective and higher ones paradoxically inhibit both vascular invasion and bone formation (Figure 1) (3);. 3). Semaphorin3a (Sema3a) acts as a novel pro-osteogenic angiocrine factor downstream of VEGF and it mediates VEGF dose-dependent effects on both vascular invasion and osteogenic progenitor stimulation. In conclusion, vascularization of osteogenic grafts is not simply necessary in order to enable progenitor survival. Rather, blood vessels can actively stimulate bone regeneration in engineered grafts through specific molecular signals that can be harnessed for therapeutic purposes. Acknowledgements: This work was supported in part by the European Union Horizon 2020 Program (Grant agreement 874790 – cmRNAbone). For any figures and tables, please contact the authors directly

Failure of osseointegration and periprosthetic joint infection (PJI) are the two main reasons of implant failure after total joint replacement (TJR). Nanofiber (NF) implant surface coating represents an alternative local drug eluting device that improves osseointegration and decreases the risk of PJI. The purpose of this study was to investigate the therapeutic efficacies of erythromycin (EM)-loaded coaxial PLGA/PCL-PVA NF coating in a rat S. aureus-infected tibia model. NF coatings with 100mg and 1000mg EM were prepared. NF without EM was included as positive control. 56 Sprague Dawley rats were divided into 4 groups. A titanium pin (1.0-mm x 8 mm) was placed into the tibia through the intercondylar notch. S. aureus (SA) was introduced by both direct injection of 10 μl broth (1 × 10. 4. CFU) into the medullary cavity and single dip of Ti pins into a similar solution prior to insertion. Rats were sacrificed at 8 and 16 weeks after surgery. The outcome measurements include μCT based quantitative osteolysis evaluation and hard tissue histology. Results: EM-NF coating (EM100 and EM1000) reduced osteolysis at 8 and 16 weeks, compared to EM0 and negative control. The effective infection control by EM-NFs was further confirmed by hard tissue section analysis. The Bone implant contact (BIC) and bone area fraction Occupancy (BAFO) within 200 µm of the surface of the pins were used to evaluate the osseointegration and new bone formation around the implants. At 16 weeks, the bone implant contact (BIC) of EM 100 (35.08%) was higher than that of negative control (3.43%) and EM0 (0%). The bone area fraction occupancy within 200 µm (BAFO) of EM100 (0.63 mm2) was higher than that of negative control (0.390 mm2) and EM0 (0.0 mm. 2. ). The BAFO of EM100 was also higher than that of EM1000 (0.3mm. 2. ). There was much less osteolysis observed with EM100 and EM1000 NF coatings at 16 weeks, as compared to EM0 positive control, p=0.08 and p=0.1, respectively. Osseointegration and periprosthetic bone formation was enhanced by EM-NFs, especially EM100. Data from this pilot study is promising for improving implant surface fabrication strategies

Abstract. Purpose. It is becoming apparent that mesenchymal stem cells (MSCs) do not directly contribute to mesenchymal tissue regeneration. Pre-clinical attempts to repair large bone defects in big animal models have been hampered by poor MSCs survival after implantation which impedes their direct or indirect effects. Based on previous work, we hypothesized that a venous axial vascularization of the scaffold supporting MSCs or their combination with fresh bone marrow (BM) aspirate would improve their in vivo survival. Methods. Cross-shape profile tubular microporous monetite implants (12mm long, 5mm large) as two longitudinal halves were produced by 3D powder printing. They were implanted around the femoral veins of Wistar rats and loaded with 1mL of BM aspirate either alone or supplemented by 10. 7. MSCs. This was compared with BM-free scaffolds loaded only with 10. 7. MSCs. After 8 weeks bone formation were investigated by micro-CT, scanning electron microscopy, histology and immunohistochemistry. Results. Little bone formation was observed within the scaffold when it was only loaded with MSCs surprisingly. Coupling MSCs, autologous BM and venous perfusion of the scaffold led to a higher volume of new bone than BM alone suggesting that MSCs augmented the bone formation capacity of BM aspirate or enhanced its survival post implantation. Conclusion. Subcutaneous bone formation within 3D-printed implant that mixed of BM with or without MSCs was successfully achieved for the first time by venous perfusion. The inability of MSCs to form differentiated tissues by their own was confirmed in this study; however, contact between MSCs and BM cells and MSCs paracrine secretome (e.g., cytokines, chemokines, extracellular vesicles) may have induced immunomodulatory effects (e.g., macrophages polarization, Treg cells) that triggered bone formation. This approach, if translatable to large animal models, offers immediate clinical value as well as an insight into the role of immune system in tissue regeneration. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported: I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Novel biomaterials are being developed and studied, intended to be applied as bone graft substitute materials. Typically, these materials are being tested in in vitro setups, where among others their cytotoxicity and alkaline phosphatase activity (as a marker for osteoblastic differentiation) are being evaluated. However, it has been reported that in vitro tests correlate poorly with in vivo results and therefore many promising biomaterials may not reach the clinic as a bone graft substitute product. One of the reasons for the poor correlation, may be the minimal complexity of the in vitro tests, as compared to the in vivo environment. Ex vivo models, mimicking the natural tissue environment whilst maintaining control of culture parameters, may be a promising alternative to assess biomaterials for bone formation. Assess the possibility of an ex vivo culture platform to test biomaterials on their potential to stimulate new bone formation. Osteochondral plugs (cylinders n=10, Ø 10 mm, height 15 mm) were drilled from fresh porcine knees, from the slaughterhouse. A bone defect (Ø 6 mm) was created and which was filled with a biomaterial graft (S53P4 bioactive glass (n=3); collagen sponges loaded with BMP-2 (n=3, as positive control)) or kept empty (n=4). The explants were cultured in custom-made two-chamber bioreactors for six weeks (LifeTec Group BV). Cartilage and bone were physically separated, similar to the in vivo situation, by a sealing ring. The two tissues were cultured in separate compartments, allowing for specific culture medium for each tissue. Medium was changed every 2–3 days and weekly micro computed tomography (µCT) images were obtained to longitudinally monitor the formation of new bone. An MTT assay was performed on half of the samples after six weeks of culture. The other samples were fixed for histology, to determine which cells were present after six weeks. The MTT metabolic assay showed that a number of cells in the bone were viable after six weeks. The further away from the border, the fewer living cells were observed. The cells in the cartilage also survived. No significant bone formation was observed with µCT in either of groups, even though abundant bone formation was expected in the BMP-2 group. Explanations of the negative results of the positive group might be that too few viable cells remain after six weeks, or that the cells that are still present are not able to form bone. No significant bone formation was observed in the bone defects in osteochondral explants that were cultured with, or without, biomaterials for six weeks. However, the platform showed that it is capable to successfully culture osteochondral explants for six weeks. Histology needs to be performed to evaluate which cells were present at the end of the culture and this will be compared to the cells present directly after drilling the explants

Introduction. Patients with metastatic spinal cord compression (MSCC) or unstable spinal lesions warrant early surgical consultation. In multiple myeloma, chemotherapy and radiotherapy have the potential to decompress the spinal canal effectively in the presence of epidural lesions. Mechanical stability conferred by bracing may potentiate intraosseous and extraosseous bone formation, thus increasing spinal stability. This study aims to review the role of non-operative management in myeloma patients with a high degree of spinal instability, in a specialist tertiary centre. Methods. Retrospective analysis of a prospectively collected database of 83 patients with unstable myelomatous lesions of the spine, defined by a Spinal Instability Neoplastic Score (SINS) of 13–18. Data collected include patient demographics, systemic treatment, neurological status, radiological presence of cord compression, most unstable vertebral level and presence of intraosseous and extraosseous bone formation. Post-treatment scores were calculated based on follow-up imaging which was carried out at 2 weeks for cord compression and 12 weeks for spinal instability. A paired t-test was used to identify any significant difference between pre- and post-treatment SINS and linear regression was used to assess the association between variables and the change in SINS. Results. A significant reduction in SINS was observed from a pre-treatment average score of 14 to a score of 9, following treatment for myeloma (p<0.001). A higher initial score and a younger age were associated with a larger overall reduction in SINS (p<0.001 and p=0.02 respectively). No single variable (bisphosphates, chemotherapy, radiotherapy and steroids) had a significant association with SINS reduction. 25 (30%) patients had spinal cord compression, all of which showed radiological resolution of cord compression at 2 weeks. No patients developed neurological deterioration during treatment and all patients had an improvement in their pain scores. 64 (77%) patients had evidence of intraosseous and/or extraosseous bone formation on their follow-up scan. Conclusion. Non-operative management in the form of bracing and systemic therapy is a safe and effective treatment for spinal instability and spinal cord compression in myeloma. Treatment of unstable myelomatous lesions of the spine with or without cord compression should not follow traditional guidelines for MSCC. The decision to adopt a non-operative approach in this cohort of patients should ideally be made in a tertiary centre with expertise in multiple myeloma and in a multidisciplinary setting

Aims. Surgeons and most engineers believe that bone compaction improves implant primary stability without causing undue damage to the bone itself. In this study, we developed a murine distal femoral implant model and tested this dogma. Methods. Each mouse received two femoral implants, one placed into a site prepared by drilling and the other into the contralateral site prepared by drilling followed by stepwise condensation. Results. Condensation significantly increased peri-implant bone density but it also produced higher strains at the interface between the bone and implant, which led to significantly more bone microdamage. Despite increased peri-implant bone density, condensation did not improve implant primary stability as measured by an in vivo lateral stability test. Ultimately, the condensed bone underwent resorption, which delayed the onset of new bone formation around the implant. Conclusion. Collectively, these multiscale analyses demonstrate that condensation does not positively contribute to implant stability or to new peri-implant bone formation. Cite this article:Bone Joint Res. 2020;9(2):60–70

Intermittently administered parathyroid hormone (PTH 1-34) has been shown to promote bone formation in both human and animal studies. The hormone and its analogues stimulate both bone formation and resorption, and as such at low doses are now in clinical use for the treatment of severe osteoporosis. By varying the duration of exposure, parathyroid hormone can modulate genes leading to increased bone formation within a so-called ‘anabolic window’. The osteogenic mechanisms involved are multiple, affecting the stimulation of osteoprogenitor cells, osteoblasts, osteocytes and the stem cell niche, and ultimately leading to increased osteoblast activation, reduced osteoblast apoptosis, upregulation of Wnt/β-catenin signalling, increased stem cell mobilisation, and mediation of the RANKL/OPG pathway. Ongoing investigation into their effect on bone formation through ‘coupled’ and ‘uncoupled’ mechanisms further underlines the impact of intermittent PTH on both cortical and cancellous bone. Given the principally catabolic actions of continuous PTH, this article reviews the skeletal actions of intermittent PTH 1-34 and the mechanisms underlying its effect. Cite this article: L. Osagie-Clouard, A. Sanghani, M. Coathup, T. Briggs, M. Bostrom, G. Blunn. Parathyroid hormone 1-34 and skeletal anabolic action: The use of parathyroid hormone in bone formation. Bone Joint Res 2017;6:14–21. DOI: 10.1302/2046-3758.61.BJR-2016-0085.R1

Aims. The use of 3D-printed titanium implant (DT) can effectively guide bone regeneration. DT triggers a continuous host immune reaction, including macrophage type 1 polarization, that resists osseointegration. Interleukin 4 (IL4) is a specific cytokine modulating osteogenic capability that switches macrophage polarization type 1 to type 2, and this switch favours bone regeneration. Methods. IL4 at concentrations of 0, 30, and 100 ng/ml was used at day 3 to create a biomimetic environment for bone marrow mesenchymal stromal cell (BMMSC) osteogenesis and macrophage polarization on the DT. The osteogenic and immune responses of BMMSCs and macrophages were evaluated respectively. Results. DT plus 30 ng/ml of IL4 (DT + 30 IL4) from day 3 to day 7 significantly (p < 0.01) enhanced macrophage type 2 polarization and BMMSC osteogenesis compared with the other groups. Local injection of IL4 enhanced new bone formation surrounding the DT. Conclusion. DT + 30 IL4 may switch macrophage polarization at the appropriate timepoints to promote bone regeneration. Cite this article: Bone Joint Res 2021;10(7):411–424

The aim of the ongoing projects was to demonstrate the efficacy of autologous bone marrow derived stem cells (MSC) combined with biomaterial to induced new bone formation in a randomized multicenter controlled clinical trial. Patients with a need for bone reconstruction of residual edentulous ridges in both the mandible and maxilla due to bone defects with a vertical loss of alveolar bone volume and/or knife edge ridges (≤ than 4,5 mm) unable to provide adequate primary stabilization for dental implants were included in the clinical study. Autologous bone marrow MSC were expanded, loaded on BCP and used to augment the alveolar ridges. After five months bone biopsies were harvested at the implant position site and implants were installed in the regenerated bone. The implants were loaded after 8–12 weeks. Safety, efficacy, quality of life and success/survival were assessed. Five clinical centers, 4 different countries participated. Bone grafts harvested from the ramus of the mandibles were used as control in the projects

Aims. Bone turnover markers (BTMs) follow distinct trends after fractures and limited evidence suggests differential levels in BTMs in patients with delayed healing. The effect of vitamin D, and other factors that influence BTMs and fracture healing, is important to elucidate the use of BTMs as surrogates of fracture healing. We sought to determine whether BTMs can be used as early markers of delayed fracture healing, and the effect of vitamin D on BTM response after fracture. Methods. A total of 102 participants aged 18 to 50 years (median 28 years (interquartile range 23 to 35)), receiving an intramedullary nail for a tibial or femoral shaft fracture, were enrolled in a randomized controlled trial comparing vitamin D. 3. supplementation to placebo. Serum C-terminal telopeptide of type I collagen (CTX; bone resorption marker) and N-terminal propeptide of type I procollagen (P1NP; bone formation marker) were measured at baseline, six weeks, and 12 weeks post-injury. Clinical and radiological fracture healing was assessed at three months. Results. CTX and P1NP concentrations peaked at six weeks in all groups. Elevated six-week CTX and P1NP were associated with radiological healing at 12 weeks post-injury (odds ratio (OR) 10.5; 95% confidence interval 2.71 to 53.5, p = 0.002). We found no association between CTX or P1NP and functional healing. Baseline serum 25(OH)D showed a weak inverse relationship with P1NP (p = 0.036) and CTX (p = 0.221) at 12 weeks, but we observed no association between vitamin D supplementation and either BTM. Conclusion. Given the association between six-week BTM concentrations and three-month radiological fracture healing, CTX and P1NP appear to be potential surrogate markers of fracture healing. Cite this article: Bone Joint Res 2022;11(4):239–250

Fragility fractures are skeletal complications associated with type 2 diabetes (T2D) causing disability, hospitalization, impaired quality of life, and increased mortality. Increased circulating sclerostin and accumulation of advanced glycation end-products (AGEs) are two potential mechanisms underlying low bone turnover and increased fracture risk. We have recently shown that T2D affects the expression of genes controlling bone formation (SOST and RUNX2) and that accumulation of AGEs is associated with impaired bone formation in T2D. We hypothesized that Wnt/B- catenin target genes are down-regulated in bone of T2D subjects as a consequence of decreased SOST and AGEs accumulation. To this end, we studied gene expression in extracts of bone samples obtained from femoral heads of 14 subjects with relatively well-controlled T2D (HbA1c 6.5±1.7%) and 21 control, non-diabetic postmenopausal women (age >65 years) undergoing hip replacement. There were no differences in age (73.2± .8 vs. 75.2±8.5 years) or BMI (27.7±5.6 vs. 29.9±5.4 kg/m2) between control and T2D groups, respectively. Expression of LEF1 mRNA was significantly lower in T2D compared to non-diabetic subjects (p=0.002), while DKK1 was not different between groups (p=0.108). Correlation analysis showed that DKK1 (r2=0.038; p=0.043) and HbA1c (r2=0.503; p=0.048) increased with age in T2D. COL1A1 mRNA trended lower in T2D compared to controls (p=0.056). Bone volume (9,333 ± 1,443 vs. 15,53 ± 2,442 mm2; p=0.048), mineralized volume (9,278 ± 1,418 vs. 15,45 ± 2,444 mm. 2. ; p=0.048) and BV/TV (0,2125 ± 0,03114 vs. 0,3719 ± 0,03196 %; p=0.002) measured by bone histomorphometry were lower in T2D compared to controls. Our data show that even in patients with relatively good glycemic control, T2D decreases expression of Wnt/B-catenin target genes andCOL1A1, associated with decreased bone density. These results may help understand the mechanisms underlying bone fragility in T2D

Growth factors produced by inflammatory cells and mesenchymal progenitors are required for proper bone regeneration. Signaling pathways activated downstream of these proteins work in concert and synergistically to drive osteoblast and/or chondrocyte differentiation. While dysregulation can result in abnormal healing, activating these pathways in the correct spatiotemporal context can enhance healing. Bone morphogenetic protein (BMP) signaling is well-recognized as being required for bone regeneration, and BMP is used clinically to enhance bone healing. However, it is imperative to develop new therapeutics that can be used alone or in conjunction with BMP to drive even more robust healing. Notch signaling is another highly conserved signaling pathway involved in tissue development and regeneration. Our work has explored Notch signaling during osteoblastogenesis and bone healing using both in vitro studies with human primary mesenchymal progenitor cells and in vivo studies with genetically modified mouse models. Notch signaling is required and sufficient for osteoblast differentiation, and is required for proper bone regeneration. Indeed, intact Notch signaling through the Jagged-1 ligand is required for BMP induced bone formation. On-going work continues to explore the intersection between BMP and Notch signaling, and determining cell types that express Notch receptors and Notch ligands during bone healing. Our long-term objective is to develop Notch signaling as a clinical therapy to repair bone

Custom 3D printed implants can be anatomically designed to assist in complex surgery of the bony pelvis in both orthopaedic oncology and orthopaedic reconstruction surgery. This series includes patients who had major pelvic bone loss after initially presenting with tumours, fractures or infection after previous total hip arthroplasty. The extent of the bone loss in the pelvis was severe and therefore impossible to be reconstructed by conventional ‘off –the-shelve’ implants. The implant was designed considering the remaining bony structures of the contra-lateral hemi- pelvis, to provide an anatomical, secured support for the reconstructed hip joint. The latter was realised by strategically orientated screws and by porous structures (an integral part of the implant), which stimulates osseointegration. A custom pelvic implant was designed, manufactured and 3D printed. Reconstruction of the pelvis was performed together with a cemented (bipolar bearing) acetabular cup. In some cases, a proximal femoral replacement was also necessary to compensate for bony defects. All patients had sufficient range of motion (ROM) at the hip with post-operative stability. It has been verified, at six and twelve months postoperatively, that there is a strong hold of the implant due to osseointegration. Additionally, in patients whose posterior acetabular wall was missing, it was discovered that the implant assisted in bone formation and covered the entire posterior surface of the implant. All patients in this study managed with this novel treatment option, proved to have a stable pelvic reconstruction with restoration of leg lengths, improvement of strength and independent ambulation at short and medium term follow-up