Tourniquet is widely used in extremity surgery. In order to prevent surgical site infection, correct timing of antimicrobial prophylaxis and tourniquet inflation is important. We aimed to evaluate the time for which the free drug concentration of cefuroxime is maintained above the minimal inhibitory concentration (T>MIC) in subcutaneous tissue and calcaneal cancellous bone during three clinically relevant tourniquet application scenarios. Twenty-four female pigs were included. Microdialysis catheters were placed for sampling of cefuroxime concentrations bilaterally in calcaneal cancellous bone and subcutaneous tissue, and a tourniquet cuff was applied on a randomly picked leg of each pig. Subsequently, the pigs were randomized into three groups to receive 1.5 g of cefuroxime by intravenous injection 15 min prior to tourniquet inflation (Group A), 45 min prior to tourniquet inflation (Group B), and at the tourniquet release (Group C). The tourniquet duration was 90 min in all groups. Dialysates and venous blood samples were collected eight-hours postcefuroxime administration. Cefuroxime concentrations were maintained above the clinical breakpoint MIC for Staphylococcus aureus (4 μg/mL) in calcaneal cancellous bone and subcutaneous tissue throughout the 90 min tourniquet duration in Group A and B. Cefuroxime administration at tourniquet release (Group C) resulted in concentrations above 4 μg/mL for a minimum of 3.5 hours in the tissues on the tourniquet side. There were no significant differences in the T>MIC (4 μg/mL) in subcutaneous tissue or calcaneal cancellous bone between the three groups. However, Group A tended toward shorter T>MIC in tourniquet calcaneal cancellous bone compared to Group C (p=0.08). We conclude that administration of cefuroxime (1.5 g) in the 15–45 min window prior to tourniquet inflation resulted in sufficient calcaneal cancellous bone and subcutaneous tissue concentrations throughout the 90 min tourniquet application. If the target is to maintain postoperative cefuroxime concentrations above relevant MIC values, our results suggest that a second dose of cefuroxime should be administered at tourniquet release

Introduction. Recently, oxidative stress has been implicated in the development of osteonecrosis. Here we focused on vitamins with marked antioxidant potency to see whether their use might prevent the development of osteonecrosis associated with corticosteroid administration. Methods. Fifteen male Japanese white rabbits weighing about 3.5 kg were injected once into the right gluteal muscle with methylprednisolone (MPSL) 40 mg/kg (S Group). Ten other rabbits, in addition, received consecutive daily intravenous injections of vitamin E 50 mg/kg starting from the day of MPSL administration (E Group), and 10 other animals similarly received consecutive daily intravenous injections of vitamin C 30 mg/kg (C Group). All animals were euthanized 2 weeks after MPSL administration, and femurs were extracted, and stained with hematoxylin-eosin. Blood levels of glutathione (GSH) were also measured. Results. In S Group, the osteonecrosis development rate was 93%, in contrast to 60% in C Group, and none in E Group (P<0.05). Also, GSH levels in both S and C Groups abruptly decreased from the 1st day after MPSL administration, whereas, in E Group, the decline in GSH levels was significantly suppressed on days 1 and 3 after MPSL administration (P<0.05). Conclusion. Vitamin E significantly inhibited the decrease in blood GSH levels noted in the groups not receiving it. Since GSH reflects oxidative stress in vivo, vitamin E administration may be preventative in the setting of this kind of corticosteroid-induced osteonecrosis rabbit model

INTRODUCTION. Adequate osseointegration of knee resurfacing implants for the treatment of focal cartilage defects is an important prerequisite for good clinical outcomes. Inadequate initial fixation and sustained micromotion may lead to osteolysis and ultimately implant failure. PET/CT with the bone seeking tracer 18F-sodium fluoride (18F-NaF) allows for localisation and quantification of abnormalities in bone metabolism. 18F-NaF PET/CT has been shown to correlate with loosening of implants in the hip and spine. Here, we asses osseointegration of the knee resurfacing implants using micro-computed tomography (µCT) and correlate µCT parameters to 18F-NaF uptake on PET/CT scans taken 3 and 12 weeks after surgery. We hypothesize that 18F-NaF uptake at 12 weeks and its relative decrease between 3 and 12 weeks correlates with osseointegration at 12 weeks postoperatively. Polymer implants with Young”s moduli approximately equal to- and below the Young's modulus of bone, with- and without surface modification were used in this study next to a control metal implant. METHODS. Five different osteochondral implants were implanted bilaterally in critically-sized osteochondral defects in 16 goats. At 3 and 12 weeks postoperatively, a 10-minute static PET/CT-scan (Philips, Gemini TF PET/CT) was made 60 minutes after intravenous injection of 18F-NaF. Image processing resulted in an overall bone metabolism parameter, i.e. standardized uptake value (SUV). A cylindrical region of interest was drawn around each implant to obtain the maximum SUV (SUVmax). Bone quality parameters were quantified in a cylinder surrounding the implant using µCT after sacrifice as a measure for osseointegration. The in vivo 18F-NaF PET/CT uptake parameters were correlated to the bone quality parameters. RESULTS. Implant osseointegration strongly varied for the different implants. Some implant groups exhibited very poor osseointegration with clear signs of osteolysis, while titanium implants exhibited good osseointegration. A strong correlation was observed between bone quality parameters as determined using µCT and SUVmax at 12 weeks. The SUVmax of the implants with poor osseointegration remained high, while implants with good osseointegration showed a relative decrease in SUVmax between 3 and 12 weeks. CONCLUSION. This study suggests that the SUVmax of PET/CT 12 weeks after surgery correlates well for the quality of osseointegration assessed on µCT 12 weeks after surgery. De relative decrease of SUVmax between the given time points had a strong correlation with the degree of osseointegration. In this study, large differences in the quality of osseointegration were observed. The role of surface modification, elasticity and micromotion still remain to be determined as well as if 18F-NaF is sensitive enough to discriminate between smaller differences and what the optimum time point would be to predict the ultimate osseointegration

The clinical success of posterior lumbar interbody fusion (PLIF) may be limited by pseudarthrosis, defined as the absence of solid fusion 1 year after surgery. Currently, CT is used to diagnose pseudarthrosis but is not able to be conclusive earlier than 1 year after surgery. No non-invasive technique is available to reliably assess bone graft incorporation in the early phase after PLIF. Positron Emission Tomography (PET) is a nuclear imaging modality that is able to identify changes at the cellular and molecular level in an early stage, well before manifestation of anatomical changes. PET/CT with the bone seeking tracer . 18. F-fluoride allows localization and quantification of bone metabolism. This study investigates whether an . 18. F-fluoride PET/CT scan early after PLIF is able to predict the fusion status at 1 year postoperative on CT. Twenty patients after PLIF were enrolled after written informed consent. At 6 weeks and at 1 year after PLIF, intravenous injection of . 18. F-fluoride was followed by a static scan at 60 minutes (Philips, Gemini TF PET/CT). Processing of images resulted in a bone metabolism parameter i.e. standardized uptake value (SUV). This parameter was determined for 3 regions of interest (ROIs): the intervertebral disc space (IDS) and the upper and lower endplate (UE and LE, respectively) of the operated segment. Interbody fusion was scored on a diagnostic CT scan made 1 year postoperatively and was defined as the amount of complete bony bridges between vertebrae, i.e 0, 1 or 2. Based on these scores, patients were divided in either the pseudarthrosis group (score 0) or the fusion group (scores 1 and 2). Differences between groups were analyzed using the independent samples Mann-Whitney U-test. Ten patients were classified as pseudarthrosis (0 bridges: n=10) and 10 patients as fused (1 bridge: n=5, 2 bridges: n=5). Patients in the pseudarthrosis group showed significantly lower bone metabolism values in the IDS on the 6 weeks PET/CT scan compared to patients in the fusion group (SUV. IDS,6w. 13.3±5.62 for pseudarthrosis and 22.6±6.42 for the fusion group, p=0.003), whereas values at the endplates were similar (SUV. UE,6w. 20.3±5.85 for pseudarthrosis and 21.6±4.24 for the fusion group, p=0.282). Furthermore, only in the pseudarthrosis group, bone metabolism in the IDS was significantly lower than at the endplates (p=0.006). In the fusion group, bone metabolism in the IDS and at the endplates was similar (p=0.470). The PET/CT scan at 1 year postoperative showed that in the pseudarthrosis group, bone metabolism of the IDS remained lower compared to the endplates (SUV. IDS,1y. 13.2±4.37, SUV. UE,1y. 16.4±5.33, p=0.004), while in the fusion group, IDS and endplate bone metabolism was similar (SUV. IDS,1y. 13.6±2.91, SUV. UE,1y. 14.4±3.14, p=0.397). This study shows that low bone metabolism values in the IDS of the operated segment as seen on . 18. F-fluoride PET/CT 6 weeks after PLIF, is related to development of pseudarthrosis 1 year postoperatively. These results suggest that . 18. F-fluoride PET/CT might be an early diagnostic tool to identify patients prone to develop pseudarthrosis after PLIF

The effect of zoledronic acid on bone ingrowth was examined in an animal model in which porous tantalum implants were placed bilaterally within the ulnae of seven dogs. Zoledronic acid in saline was administered via a single post-operative intravenous injection at a dose of 0.1 mg/kg. The ulnae were harvested six weeks after surgery. Undecalcified transverse histological sections of the implant-bone interfaces were imaged with backscattered scanning electron microscopy and the percentage of available pore space that was filled with new bone was calculated. The mean extent of bone ingrowth was 6.6% for the control implants and 12.2% for the zoledronic acid-treated implants, an absolute difference of 5.6% (95% confidence interval, 1.2 to 10.1) and a relative difference of 85% which was statistically significant. Individual islands of new bone formation within the implant pores were similar in number in both groups but were 69% larger in the zoledronic acid-treated group. The bisphosphonate zoledronic acid should be further investigated for use in accelerating or enhancing the biological fixation of implants to bone

Summary. The two-step labeling protocol using Lysostaphin and bio-orthogonal click chemistry for staining bacteria is described. The click protocol is efficient in labeling staphylococci and is non-toxic. This protocol promises the efficient of infections that are difficult to assess by conventional imaging. Introduction. Infection diagnostics in clinics is time consuming, invasive and relays on microbiological cultures. New probes and labeling protocols enabling rapid and specific detection of infection in vivo shall improve the situation. We investigated the potential of a new click labeling protocol to detect staphylococci. Azido (N3) - modified Lysostaphin and DIBO (Di-benzocyclooctyne) - dye were used in the two-step bacteria-labeling protocol. N3 and DIBO were the counterparts of the bioorthogonal “click” reaction. In the first step, Lysostaphin-N3 bound to Staphylococcus aureus. In the second step, N3 clicked to DIBO thus achieving S. aureus selective labeling. Methods. S. aureus NCTC 10788 and E. coli NCTC 12241 (from National Collection of Type Cultures), primary sheep osteoblasts and C57BL/6 mice were used for this study. DIBO-Alexa488 (Invitrogen ®), DyeLight488 (Thermofisher ®), NHS-N3 (Lumiprobe ®), Lysostaphin (Sigma-Aldrich ®) were purchased. In vitro we used standard microbiological protocols to assess antimicrobial and labeling activity of the “click” probe (Lysostaphin-N3 plus DIBO-dye), one-step-labeled Lysostaphin-Dye and non-labeled Lysostaphin. Flow cytometry, Fluorescence microscopy, and Spectrophotometry were employed to measure binding of the probes to bacteria. The cytotoxicity of the probes on osteoblasts was performed using Presto Blue Cell Viability test (Invitrogen ®). In vivo we used Fluorescence Intravital Microscopy and mice with dorsal skin-fold chambers (approved by the local governmental animal care committee). Subsequently to anesthesia each mouse received S. aureus strain Cowan I intravenously. This was followed by intravenous injections of the test probes. Results. Lysostaphin-N3 partially lost its antimicrobial property if compare to Lysostaphin alone, but still bound to S. aureus efficiently and clicked DIBO-dye afterwards. There was no significant cytotoxicity of “click” reagents on sheep osteoblasts. In vitro the two-step labeling with Lysostaphin-Click was more efficient than the one-step labeling with Lysostaphin-Dye. In vivo the two-step and one-step labeling differed rather qualitatively. In the two-step protocol the “click” probe labeled bacteria adherent to the blood vessels and bacteria extravasated into the soft tissue around 30 minutes post-injection. In the one-step protocol bacteria were labeled quickly (within minutes) in the blood flow and on the blood vessels but not in the soft tissue. Discussion/Conclusion. Effective labeling bacteria with the click probe could be useful for a quantitative and selective assessment of infection, in particular for staphylococci infections, which are common in orthopedic implants but often difficult to detect. The one and two-step protocols successfully labeled bacteria in mice but the labeling was different. In the one-step protocol bacteria were labeled in the blood flow quickly; in the two-step protocol bacteria adherent to the blood vessels and extravasated into the soft tissue bacteria were labeled with a delay

Objectives

The aim of this study was to investigate the effect of hyperglycaemia on oxidative stress markers and inflammatory and matrix gene expression within tendons of normal and diabetic rats and to give insights into the processes involved in tendinopathy.

Osteoporosis is a systemic skeletal disorder characterized by reduced bone mass and deterioration of bone microarchitecture, which results in increased bone fragility and fracture risk. Casein kinase 2-interacting protein-1 (CKIP-1) is a protein that plays an important role in regulation of bone formation. The effect of CKIP-1 on bone formation is mainly mediated through negative regulation of the bone morphogenetic protein pathway. In addition, CKIP-1 has an important role in the progression of osteoporosis. This review provides a summary of the recent studies on the role of CKIP-1 in osteoporosis development and treatment.

Cite this article: X. Peng, X. Wu, J. Zhang, G. Zhang, G. Li, X. Pan. The role of CKIP-1 in osteoporosis development and treatment. Bone Joint Res 2018;7:173–178. DOI: 10.1302/2046-3758.72.BJR-2017-0172.R1.

Objectives

Mesenchymal stem cells have the ability to differentiate into various cell types, and thus have emerged as promising alternatives to chondrocytes in cell-based cartilage repair methods. The aim of this experimental study was to investigate the effect of bone marrow derived mesenchymal stem cells combined with platelet rich fibrin on osteochondral defect repair and articular cartilage regeneration in a canine model.

We investigated the effect of locally administered bisphosphonate on distraction osteogenesis in a rabbit model and evaluated its systemic effect. An osteotomy on the right tibia followed by distraction for four weeks was performed on 47 immature rabbits. They were divided into seven equal groups, with each group receiving a different treatment regime. Saline and three types of dosage of alendronate (low, 0.75 μg/kg; mid, 7.5 μg/kg and high 75 μg/kg) were given by systemic injection in four groups, and saline and two dosages (low and mild) were delivered by local injection to the distraction gap in the remaining three groups. The injections were performed five times weekly during the period of distraction.

After nine weeks the animals were killed and image analysis and mechanical testing were performed on the distracted right tibiae and the left tibiae which served as a control group. The local low-dose alendronate group showed a mean increase in bone mineral density of 124.3 mg/cm³ over the local saline group (analysis of variance, p < 0.05) without any adverse effect on the left control tibiae.

The findings indicate that the administration of local low-dose alendronate could be an effective pharmacological means of improving bone formation in distraction osteogenesis.

Objectives

In order to ensure safety of the cell-based therapy for bone regeneration, we examined in vivo biodistribution of locally or systemically transplanted osteoblast-like cells generated from bone marrow (BM) derived mononuclear cells.

Platelet-leucocyte gel (PLG), a new biotechnological blood product, has hitherto been used primarily to treat chronic ulcers and to promote soft-tissue and bone regeneration in a wide range of medical fields. In this study, the antimicrobial efficacy of PLG against Staphylococcus aureus (ATCC 25923) was investigated in a rabbit model of osteomyelitis. Autologous PLG was injected into the tibial canal after inoculation with Staph. aureus. The prophylactic efficacy of PLG was evaluated by microbiological, radiological and histological examination. Animal groups included a treatment group that received systemic cefazolin and a control group that received no treatment.

Treatment with PLG or cefazolin significantly reduced radiological and histological severity scores compared to the control group. This result was confirmed by a significant reduction in the infection rate and the number of viable bacteria. Although not comparable to cefazolin, PLG exhibited antimicrobial efficacy in vivo and therefore represents a novel strategy to prevent bone infection in humans.

An understanding of the remodelling of tendon is crucial for the development of scientific methods of treatment and rehabilitation. This study tested the hypothesis that tendon adapts structurally in response to changes in functional loading. A novel model allowed manipulation of the mechanical environment of the patellar tendon in the presence of normal joint movement via the application of an adjustable external fixator mechanism between the patella and the tibia in sheep, while avoiding exposure of the patellar tendon itself. Stress shielding caused a significant reduction in the structural and material properties of stiffness (79%), ultimate load (69%), energy absorbed (61%), elastic modulus (76%) and ultimate stress (72%) of the tendon compared with controls. Compared with the material properties the structural properties exhibited better recovery after re-stressing with stiffness 97%, ultimate load 92%, energy absorbed 96%, elastic modulus 79% and ultimate stress 80%. The cross-sectional area of the re-stressed tendons was significantly greater than that of stress-shielded tendons.

The remodelling phenomena exhibited in this study are consistent with a putative feedback mechanism under strain control. This study provides a basis from which to explore the interactions of tendon remodelling and mechanical environment.

Little is known about the increase in length of tendons in postnatal life or of their response to limb lengthening procedures. A study was carried out in ten young and nine adult rabbits in which the tibia was lengthened by 20% at two rates 0.8 mm/day and 1.6 mm/day.

The tendon of the flexor digitorum longus (FDL) muscle showed a significant increase in length in response to lengthening of the tibia. The young rabbits exhibited a significantly higher increase in length in the FDL tendon compared with the adults. There was no difference in the amount of lengthening of the FDL tendon at the different rates. Of the increase in length which occurred, 77% was in the proximal half of the tendon.

This investigation demonstrated that tendons have the ability to lengthen during limb distraction. This occurred to a greater extent in the young who showed a higher proliferative response, suggesting that there may be less need for formal tendon lengthening in young children.

The feasibility of bone transport with bone substitute and the factors which are essential for a successful bone transport are unknown. We studied six groups of 12 Japanese white rabbits. Groups A to D received cylindrical autologous bone segments and groups E and F hydroxyapatite prostheses. The periosteum was preserved in group A so that its segments had a blood supply, cells, proteins and scaffold. Group B had no blood supply. Group C had proteins and scaffold and group D had only scaffold. Group E received hydroxyapatite loaded with recombinant human bone morphogenetic protein-2 and group F had hydroxyapatite alone.

Distraction osteogenesis occurred in groups A to C and E which had osteo-conductive transport segments loaded with osteo-inductive proteins. We conclude that scaffold and proteins are essential for successful bone transport, and that bone substitute can be used to regenerate bone.

Sex hormones play important roles in the regulation of the proliferation, maturation and death of chondrocytes in the epiphyseal growth plate. We have investigated the effects of male castration on the cell kinetics of chondrocytes as defined by the numbers of proliferating and dying cells. The growth plates of normal rabbits and animals castrated at eight weeks of age were obtained at 10, 15, 20 and 25 weeks of age.

Our study suggested that castration led to an increase in apoptosis and a decrease in the proliferation of chondrocytes in the growth plate. In addition, the number of chondrocytes in the castrated rabbits was less than that of normal animals of the same age.

We developed a new porous scaffold made from a synthetic polymer, poly(DL-lactide-co-glycolide) (PLG), and evaluated its use in the repair of cartilage. Osteochondral defects made on the femoral trochlear of rabbits were treated by transplantation of the PLG scaffold, examined histologically and compared with an untreated control group.

Fibrous tissue was initially organised in an arcade array with poor cellularity at the articular surface of the scaffold. The tissue regenerated to cartilage at the articular surface. In the subchondral area, new bone formed and the scaffold was absorbed. The histological scores were significantly higher in the defects treated by the scaffold than in the control group (p < 0.05).

Our findings suggest that in an animal model the new porous PLG scaffold is effective for repairing full-thickness osteochondral defects without cultured cells and growth factors.

We report the effects of local administration of osteogenic protein-1 on the biomechanical properties of the overstretched anterior cruciate ligament in an animal model. An injury in the anterior cruciate ligament was created in 45 rabbits. They were divided into three equal groups. In group 1, no treatment was applied, in group II, phosphate-buffered saline was applied around the injured ligament, and in group III, 12.5 μg of osteogenic protein-1 mixed with phosphate-buffered saline was applied around the injured ligament. A control group of 15 rabbits was assembled from randomly-selected injured knees from among the first three groups. Each rabbit was killed at 12 weeks.

The maximum load and stiffness of the anterior cruciate ligament was found to be significantly greater in group III than either group 1 (p = 0.002, p = 0.014) or group II (p = 0.032, p = 0.025). The tensile strength and the tangent modulus of fascicles from the ligament were also significantly greater in group III than either group I (p = 0.002, p = 0.0174) or II (p = 0.005, p = 0.022).

The application of osteogenic protein-1 enhanced the healing in the injured anterior cruciate ligament, but compared with the control group the treated ligament remained lengthened. The administration of osteogenic protein-1 may have a therapeutic role in treating the overstretched anterior cruciate ligament.

We used a canine intercalary bone defect model to determine the effects of recombinant human osteogenic protein 1 (rhOP-1) on allograft incorporation. The allograft was treated with an implant made up of rhOP-1 and type I collagen or with type I collagen alone.

Radiographic analysis showed an increased volume of periosteal callus in both test groups compared with the control group at weeks 4, 6, 8 and 10. Mechanical testing after 12 weeks revealed increased maximal torque and stiffness in the rhOP-1 treated groups compared with the control group.

These results indicate a benefit from the use of an rhOP-1 implant in the healing of bone allografts. The effect was independent of the position of the implant. There may be a beneficial clinical application for this treatment.

We produced large full-thickness articular cartilage defects in 33 rabbits in order to evaluate the effect of joint distraction and autologous culture-expanded bone-marrow-derived mesenchymal cell transplantation (ACBMT) at 12 weeks. After fixing the knee on a hinged external fixator, we resected the entire surface of the tibial plateau. We studied three groups: 1) with and without joint distraction; 2) with joint distraction and collagen gel, and 3) with joint distraction and ACBMT and collagen gel.

The histological scores were significantly higher in the groups with ACBMT collagen gel (p < 0.05). The area of regenerated soft tissue was smaller in the group allowed to bear weight (p < 0.05). These findings suggest that the repair of large defects of cartilage can be enhanced by joint distraction, collagen gel and ACBMT.