Fracture healing is a spatially controlled process involving crosstalk of multiple tissues. To precisely capture and understand molecular mechanism underlying impaired healing, there is a need to integrate spatially-resolved molecular analyses into preclinical fracture healing models. I will present our recent data obtained by spatial transcriptomics of musculoskeletal samples from fracture healing studies in mice. Subsequently, I will show how spatial transcriptomics can be integrated into multimodal approaches in preclinical fracture healing models. In combination with established in vivo imaging and emerging omics techniques, spatially-resolved analyses have the potential to elucidate the molecular mechanisms underlying impaired healing with optimization of treatments

Gram-negative prosthetic joint infections (GN-PJI) present unique challenges in management due to their distinct pathogenesis of biofilm formation on implant surfaces. To date, there are no animal models that can fully recapitulate how a biofilm is challenged in vivo in the setting of GN-PJI. The purpose of this study is to establish a clinically representative GN-PJI in vivo model that can reliably depict biofilm formation on titanium implant surface. We hypothesized that the biofilm formation on the implant surface would affect the ability of the implant to be osseointegrated. The model was developed using a 3D-printed, medical-grade titanium (Ti-6Al-4V), monoblock, cementless hemiarthroplasty hip implant. This implant was used to replace the femoral head of a Sprague-Dawley rat using a posterior surgical approach. To induce PJI, two bioluminescent Pseudomonas aeruginosa (PA) strains were utilized: a reference strain (PA14-lux) and a mutant strain that is defective in biofilm formation (DflgK-lux). PJI development and biofilm formation was quantitatively assessed in vivo using the in vivo imaging system (IVIS), and in vitro using the viable colony count of the bacterial load on implant surface. Magnetic Resonance Imaging (MRI) was acquired to assess the involvement of periprosthetic tissue in vivo, and the field emission scanning electron microscopy (FE-SEM) of the explanted implants was used to visualize the biofilm formation at the bone-implant interface. The implant stability, as an outcome, was directly assessed by quantifying the osseointegration using microCT scans of the extracted femurs with retained implants in vitro, and indirectly assessed by identifying the gait pattern changes using DigiGaitTM system in vivo. A localized prosthetic infection was reliably established within the hip joint and was followed by IVIS in real-time. There was a quantitative and qualitative difference in the bacterial load and biofilm formation between PA14 and DflgK. This difference in the ability to persist in the model between the two strains was reflected on the gait pattern and implant osseointegration. We developed a novel uncemented hip hemiarthroplasty GN-PJI rat model. This model is clinically representative since animals can bear weight on the implant. PJI was detected by various modalities. In addition, biofilm formation correlated with implant function and stability. In conclusion, the proposed in vivo GN-PJI model will allow for more reliable testing of novel biofilm-targeting therapetics

Introduction. Gram-negative prosthetic joint infections (GN-PJI) present unique challenges in management due to their distinct pathogenesis of biofilm formation on implant surfaces. The purpose of this study is to establish a clinically representative GN-PJI model that can reliably recapitulate biofilm formation on titanium implant surface in vivo. We hypothesized that biofilm formation on an implant surface will affect its ability to osseointegrate. Methods. The model was developed using 3D-printed titanium hip implants, to replace the femoral head of male Sprague-Dawley rats. GN-PJI was induced using two bioluminescent Pseudomonas aeruginosa strains: a reference strain (PA14-lux) and a mutant biofilm-defective strain (ΔflgK-lux). Infection was monitored in real-time using the in vivo imaging system (IVIS) and Magnetic Resonance Imaging (MRI). Bacterial loads on implant surface and in periprosthetic tissues were quantified utilizing viable-colony-count. Field-emission scanning-electron-microscopy of the explanted implants was used to visualize the biofilm formation at the bone-implant-interface. The implant stability, as an outcome, was directly assessed by quantifying the osseointegration in vitro using microCT scan, and indirectly assessed by identifying the gait pattern changes using DigiGait. TM. system in vivo. Results. Localized infection was established within the hip joint and was followed by IVIS in real-time. There was a quantitative and qualitative difference in the bacterial load and biofilm formation between PA14-lux and ΔflgK-lux. This difference in the ability to persist in the model between the two strains was reflected in the gait pattern and implant osseointegration. Conclusions. We developed a novel uncemented hip hemiarthroplasty, GN-PJI rat model. To date, the proposed in vivo biofilm-based model is the most clinically representative for GN-PJI since animals can bear weight on the implant and poor osseointegration correlates with biofilm formation. In addition, localized PJI was detected by various modalities. Clinical Relevance. The proposed in vivo GN-PJI model will allow for more reliable testing of novel biofilm-targeting therapeutics

To investigate temporal changes in synovial lymphatic system (SLS) drainage function after Anterior cruciate ligament (ACL) injury, a non-invasive ACL rupture model was used to induce the PTOA phenotype without altering the SLS structure. We have created a non-invasive ACL rupture model in the right knee (single overload impact) of 12- week-old C57bl/6 male mice to mimic the ACL rupture-induced PTOA development. 70 kDa-TxRedDextran were injected into the right knee of the mice at 0, 1, 2, and 4 wks post modeling (n=5/group), and the fluorescence signal distribution and intensity were measured by the IVIS system at 1 and 6 hrs post-injection. After 24 hrs, the drainage lymph nodes and whole knee joint were harvested and subjected to ex vivo IVIS imaging and immunofluorescence detection respectively. Manual ACL rupture was induced by 12N overloaded force and validated by a front drawer test. Intraarticular clearance of TxRed-Dextran detected by the IVIS was significantly reduced at 1, and 2 wks at a level of 43% and 55% respectively but was not significantly different from baseline levels at 4 wks (89%). TxRed-Dextran signal in draining lymph nodes was significantly reduced at 1 week at the level of but not for 2 and 4 wks compared to baseline levels (week 1–29%, week 2–50%, week 4–94%). TxRed-Dextran particle was significantly enriched in the synovium at 1, 2 wks but was not significantly different from baseline levels at 4 wks rupture-post ACL rupture (Particle numbers: Sham Ctrl-34 ±14, week 1, 113 ± 17; week 2, 89 ± 13; week 4, 46 ± 18; mean ± SD). We observed the drainage function of SLS significantly decreased at 1 and 2 wks after the ACL rupture, and was slowly restored at 4 wks post-injury in a non-invasive ACL rupture model. Early impairment of SLS drainage function may lead to accumulation of inflammatory factors and promote PTOA progression

Cartilage diseases have a significant impact on the patient's quality of life and are a heavy burden for the healthcare system. Better understanding, early detection and proper follow-up could improve quality of life and reduce healthcare related costs. Therefore, the aim of this study was to evaluate if difference between osteoarthritic (OA) and non-osteoarthritic (non-OA) knees can be detected quantitatively on cartilage and subchondral bone levels with advanced but clinical available imaging techniques. Two OA (mean age = 88.3 years) and three non-OA (mean age = 51.0 years) human cadaveric knees were scanned two times. A high-resolution peripheral quantitative computed tomography (HR-pQCT) scan (XtremeCT, Scanco Medical AG, Switzerland) was performed to quantify the bone microstructure. A contrast-enhanced clinical CT scan (GE Revolution Evo, GE Medical Systems AG, Switzerland) was acquired with the contrast agent Visipaque 320 (60 ml) to measure cartilage. Subregions dividing the condyle in four parts were identified semi-automatically and the images were segmented using adaptive thresholding. Microstructural parameters of subchondral bone and cartilage thickness were quantified. The overall cartilage thickness was reduced by 0.27 mm between the OA and non-OA knees and the subchondral bone quality decreased accordingly (reduction of 33.52 % in BV/TV in the layer from 3 to 8 mm below the cartilage) for the femoral medial condyle. The largest differences were observed at the medial part of the femoral medial condyle both for cartilage and for bone parameters, corresponding to clinical observations. Subchondral bone microstructural parameters and cartilage thickness were quantified using in vivo available imaging and apparent differences between the OA and non-OA knees were detected. Those results may improve OA follow-up and diagnosis and could lead to a better understanding of OA. However, further in vivo studies are needed to validate these methods in clinical practice

Aims

In this investigation, we administered oxidative stress to nucleus pulposus cells (NPCs), recognized DNA-damage-inducible transcript 4 (DDIT4) as a component in intervertebral disc degeneration (IVDD), and devised a hydrogel capable of conveying small interfering RNA (siRNA) to IVDD.

Aims

Transcription factor nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of chronic inflammatory diseases in all organ systems. Despite its importance, NF-κB targeted drug therapy to mitigate chronic inflammation has had limited success in preclinical studies. We hypothesized that sex differences affect the response to NF-κB treatment during chronic inflammation in bone. This study investigated the therapeutic effects of NF-κB decoy oligodeoxynucleotides (ODN) during chronic inflammation in male and female mice.

Aims

This study aimed to define the histopathology of degenerated humeral head cartilage and synovial inflammation of the glenohumeral joint in patients with omarthrosis (OmA) and cuff tear arthropathy (CTA). Additionally, the potential of immunohistochemical tissue biomarkers in reflecting the degeneration status of humeral head cartilage was evaluated.

Aims

Biofilm-related infection is a major complication that occurs in orthopaedic surgery. Various treatments are available but efficacy to eradicate infections varies significantly. A systematic review was performed to evaluate therapeutic interventions combating biofilm-related infections on in vivo animal models.

Background; We investigated, as a neoadjuvant to surgical therapy, the effect of a gene therapy of the primary tumour on the progression of minimal residual disease to overt liver metastases. The gene construct coding for the immunostimulatory molecules GM-CSF and B7-1 was delivered to the growing tumour by electroporation in Balb/C mice. Methods; JBS fibrosarcomas were induced subcutaneously and were randomised at 80mm3 to control and treatment groups. One day prior to treatment, the portal circulation was seeded with tumour cells. Gene delivery was assessed by in vivo imaging, cytokine measurement and anti–tumour cytotoxicity (in vitro and in vivo). Responses were determined by liver examination. Results; Gene expression and cytokine production was evident in treated tumours. Development of liver metastases was inhibited by neoadjuvant therapy in all 8 animals, in comparison to none of the control animals (n = 6) (average liver weight=0.99 g vs. 1.748 g. p< 0.03.) Metastases were confirmed histologically. Cytotoxicity studies and rechallenge confirmed development of specific T cell antitumour responses after gene therapy. Conclusions; Immunogene therapy of the primary tumour induces effective anti–tumoural responses and inhibits the development of liver metastases. This strategy could be developed for neoadjuvant therapy of some human cancers

Summary Statement. Increased Dkk-1 signaling is associated with OA occurrence and joint microenvironment damage. Interruption of Dkk1 action is beneficial to improve OA knees. Introduction. Osteoarthritis (OA) is a leading cause of disability and healthcare financial burden for total knee arthroplasty, rehabilitation, and disability. Inappropriate mechanical stress, immunological, or biochemical regulation reportedly disturbs homeostasis among cartilage, synovium and subchondral bone microstructure that contributes to OA pathogenesis. Control of joint-deleterious factor action is an emerging strategy to ameliorate OA-induced joint deterioration. Dickkopf-1 (Dkk-1) is a potent inhibitor for Wnt/β-catenin signaling regulation of tissue development and remodeling in physiological or pathological contexts. Dkk-1 also acts as a master deleterious factor that represses osteoblast differentiation capacity and bone repair. Associations among Dkk-1 expression, chondrocyte fate, synovial fibroblast behavior or OA incidence are merit of characterization. Patients & Methods. Cartilage, synovial tissue and fluid were harvested from informed consent OA patients underwent arthroplasty and patient with knee injuries without OA changes as controls. Primary chondrocyte cultures and synovial fibroblasts were treated with inflammatory cytokines or Dkk-1 antisense oligonucleotide or monoclonal antibodies. Knees in experimental animals were subjected to anterior cruciate ligament transection- or intra-articular collagenase injection to induce OA. Joint inflammation, integrity and subchondral bone microstructure in knees as well gait profiles were quantified using 2-deoxyglucose-probed near-infrared in vivo image, µCT, catwalk and histomorphometric analyses. Results. In clinical vignettes, patients with end-stage OA knee had higher abundances of Dkk-1 in cartilage, synovial tissue, and synovial fluid compared to control patients. Disruption of DKk-1 signaling ameliorated the promoting effects of inflammatory cytokines on the survival and cartilage matrix synthesis in primary cartilage chondrocyte cultures. Of interest, Dkk-1 neutralization attenuated the excessive angiogenic activities and matrix metalloproteinase secretion in primary synovial fibroblasts of OA knees. Dkk-1 modulation of survival or metabolic activities in chondrocytes and synovial fibroblasts were through β-catenin-dependent and -independent signaling pathways. Moreover, increased Dkk-1 expression in lesion sites and sera was associated with the incidence of femoral head osteonecrosis. Loss of Dkk-1 action alleviated bone cell apoptosis in osteonecrotic bone microenvironments. In experimental OA knee models, knockdown of Dkk-1 alleviated articular cartilage damage as evidenced by improved Mankin score in OA knees. Dkk-1 disruption also alleviated the adverse effects of OA on subchondral bone exposure and loss of trabecular bone volume and mineral acquisition in injured joints. Loss of Dkk-1 function reduced joint inflammation, vessel number, leukocyte infiltration in synovium compartment of OA joint and improved gait profiles of affected limbs. Conclusion. Dkk-1 signaling is associated with the OA knee occurrence and accelerates apoptosis, matrix degradation and angiogenic activities in chondrocytes and synovial fibroblasts of OA joint. Dkk-1 interference alleviates the promoting effects of OA on cartilage, synovial and subchondral bone remodeling. Blocking the deleterious actions of Dkk-1 in joint microenvironment will be a prospective molecular regime beneficial for retarding excessive joint deterioration in OA knees

Purpose. To achieve 3D kinematic analysis of total knee arthroplasty (TKA), 2D/3D registration techniques, which use X-ray fluoroscopic images and computer-aided design model of the knee implants, have been applied to clinical cases. In previous feature-based registration methods, only edge contours originated from knee implants are assumed to be extracted from X-ray images before 2D/3D registration. Due to the influence of bone and bone-cement close to knee implants, however, edge detection methods extract unwanted spurious edges and noises in clinical images. Thus, time-consuming and labor-intensive manual operations are often necessary to remove the unwanted edges. It has been a serious problem for clinical applications, and there is a strong demand for development of improved method. The purpose of this study was to develop a pose estimation method to perform accurate 2D/3D registration even if spurious edges and noises exist in knee images. Methods. Our 2D/3D registration technique is based on a feature-based algorithm, and contour points from X-ray images are extracted by Gaussian Laplacian filter and zero crossing methods. The basic principle of the algorithm is that the 3D pose of a model can be determined by projecting rays from contour points in an image back to the X-ray focus and noting that all of these rays are tangential to the model surface. Therefore, 3D poses are estimated by minimizing the sum of Euclidean distances between all projected rays and the model surface. Additionally, we introduce robust statistics into the 3D pose estimation method to perform accurate 2D/3D registration even if spurious edges and noises exist in knee images. The robust estimation method employs weight functions to reduce the influence of spurious edges and noises. The weight functions are defined for each contour point, and optimization is performed after the weight functions are multiplied to a cost function. Experimental results. The accuracy and stability validation were performed using in vivo images. The effects of robust estimation were evaluated by comparison with non-robust estimation. One image contained spurious edges and noises, and the other image didn't (they were erased manually). We applied robust and non-robust methods to each image (300 frames). As correct poses, we used the poses which were got by applying previous method to the contour images which spurious edges and noises didn't exist. The root mean square errors (RMSE) and success rate were calculated, and the success rate was defined as the rate of satisfying clinical required accuracy (error is less than 1mm, 1 degree). As results of the experiments, when non-robust method was applied to contour images in which spurious edges and noises exist, RMSE was too large and success rate was 0 %. However, when robust method was applied to the same images, RMSE was less than 1 mm, 1 degree, and the success rate was about 60 percent. Fig. 1 shows typical result of the experiment. Conclusions. We have developed a robust 3D kinematic estimation method of TKA from X-ray images, and the method was found to be helpful for analyzing TKA kinematics without labor-intensive operations

Background. Large bone defects still challenge the orthopaedic surgeon. Local vascularity at the site of the fracture has an important influence on the healing procedure. Vascular endothelial growth factor (VEGF) and it's receptor (VEGFR2) are potent inducer of angiogenesis during the fracture healing. Aim of the present study was the investigation of critical size fracture (CSF) healing in VEGFR2-luc mice using tailored scaffolds. Methods. CSFs were performed and stabilised in mouse femur using an external fixator. The fracture was bridged using a synthetic 3D printed scaffold with a defined porosity to promote regeneration. The ß-tricalciumphosphate (ßTCP) and strontium doped ß-tricalciumphosphate (ßTCP+Sr) scaffolds were investigated for their regenerative potential. The expression levels of VEGFR2 could be monitored non-invasively via in vivo bioluminescence imaging for 2 months. After the longitudinal measurements the animals were euthanised for an in depth histological endpoint analysis. The different scaffold induced tissue regeneration was quantified for both, the ßTCP and the ßTCP+Sr group. Results. Expression levels of VEGFR2 were significantly higher in the ßTCP+Sr group when compared to the ßTCP, control and sham group. Both types of scaffolds significantly enhanced new bone formation when compared to the sham group. The ßTCP+Sr scaffolds showed a significantly greater regenerative potential. Conclusions. This standardised defect model mimics a clinically relevant situation to study the regenerative effects of biomaterials on bone. Moreover, the rate of regeneration correlates with the VEGFR2 expression levels, what affirms the usability of our method for longitudinal fracture healing studies. As in line with relevant literature, it could be shown that strontium does have an enhancing effect on bone regeneration. Consequently, strontium doped scaffolds might be a useful addition in the surgeon's spectrum of methods. Level of Evidence. Experimental

Aims

High-energy injuries can result in multiple complications, the most prevalent being infection. Vancomycin powder has been used with increasing frequency in orthopaedic trauma given its success in reducing infection following spine surgery. Additionally, large, traumatic injuries require wound coverage and management by dressings such as negative pressure wound therapy (NPWT). NPWT has been shown to decrease the ability of antibiotic cement beads to reduce infection, but its effect on antibiotic powder is not known. The goal of this study was to determine if NPWT reduces the efficacy of topically applied antibiotic powder.

INTRODUCTION. Trabecular Titanium. ™. (TT) is a novel material with a structure similar to trabecular bone, already used for prosthetic clinical applications. Being the bone-implant interface the weakest point during the initial healing period, the association of TT with a hydrogel enriched with progenitor cells and osteoinductive factors may represent a promising strategy to improve prosthesis osteointegration. In a previous in vitro study we evaluated the ability of an ammidated carboxymethylcellulose hydrogel (CMCA) and of TT enriched with CMCA to support bone marrow mesenchymal stem cells (BMSCs) viability and osteogenic differentiation [1]. The aim of this study was to evaluate in vivo if the association of TT with CMCA enriched with strontium chloride (SrCl. 2. ) and BMSCs could ameliorate TT osteointegration. METHODS. This study combines TT with CMCA, SrCl. 2. and BMSCs. To mimic prosthesis-bone implants, TT discs were seeded with human BMSCs predifferentiated in osteogenic medium, then press-fit into engineered bone. A total of 36 athymic mice were implanted subcutaneously, each animal received 2 constructs as un-seeded TT and TT+CMCA or cell seeded TT+BMSCs and TT+CMCA+BMSCs. After 4, 8 and 12 weeks, osteodeposition, bone mineral density (BMD) and osteointegration were evaluated by fluorescence imaging, micro-CT, SEM, histology and pull-out tests. RESULTS. Micro-CT analysis demonstrated the homogeneity of the engineered bone in all experimental groups, supporting the reproducibility of our novel engineered model. Macroscopic evaluation of explanted constructs after 4 weeks revealed their integration with mice subcutaneous structures. In pull-out biomechanical tests, increases in extraction energy and peak force from 4 to 12 weeks were observed in all the experimental groups, except TT+CMCA. TT+CMCA+BMSCs showed the highest value of peak force and the greatest increase in comparison to samples explanted at 4 weeks. In vivo fluorescence imaging showed osteodeposition activity inside the constructs, observation confirmed by the ex-vivo analyses revealing a higher activity in TT+BMSCs and in TT+CMCA+BMSCs in comparison to acellularized TT samples. SEM evaluation of ECM deposition at the interface between bone scaffolds and TT disks revealed a significant difference between TT+CMCA+BMSCs and the other experimental groups with the former showing an almost complete filling of the space between the integration surfaces already after 4 weeks. In histomorphometric analyses of tissue ingrowth at 8 weeks, TT+BMSCs and TT+CMCA+BMSCs showed a greater tissue ingrowth compared to TT and TT+CMCA samples. DISCUSSION. Several efforts have been made to improve osteointegration with particular attention to critical cases such as implant revision surgeries. The association of porous structures with osteoinductive factors enriched hydrogels and stem cells represents a novel and promising strategy for more effective osteointegration to reduce prosthesis mobilization risks. Our results demonstrate that the association of Trabecular Titanium. ™. with a SrCl. 2. enriched hydrogel and BMSCs increases the production of ECM and may thus represent a valid approach to accelerate prosthesis osteointegration. Further validation of these data will include construct implantation in large animal orthotopic models to better mimic surgical procedures

Introduction. Ten explanted pyrolytic carbon components of a number of finger prostheses were obtained at revision surgery for wear analysis. Implants were removed for either dislocation or failure of fixation. Hypothesis Failure of the components was due to wear from the articulating surfaces, as occurs in many hip and knee prostheses. Methods. The articulating surfaces were examined using a ZYGO NewView 5000 non-contact profilometer with a resolution of 1nm, to determine the roughness average (RA) of the surface. A total of 86 RA measurements were taken. Detailed images of the surface displayed as a 3D map of were acquired. The RA values for each component were averaged and compared against the British standard for orthopaedic implants, which states that the articulating surfaces of devices made of metal or ceramic should have RA values lower than 0.050 µm. Results. The low surface roughness demonstrated that the vast majority of the articulating surfaces of the components were relatively unworn with RA values lower than British standard, even following use in vivo. ZYGO images showed light unidirectional scratching on four of the explanted components, but despite the scratching, the RA values of these components were still low (<0.050 µm) showing that this was superficial damage. No other significant damage was observed. Discussion. Due to the lack of damage on the articulating surfaces and the low RA values recorded the failure of these prostheses is not considered to be wear related. Significance This is the first report of ex vivo analysis of pyrolytic carbon finger prostheses

Objectives

Electromagnetic fields (EMF) are widely used in musculoskeletal disorders. There are indications that EMF might also be effective in the treatment of osteoporosis. To justify clinical follow-up experiments, we examined the effects of EMF on bone micro-architectural changes in osteoporotic and healthy rats. Moreover, we tested the effects of EMF on fracture healing.

Objectives

Numerous complications following total knee replacement (TKR) relate to the patellofemoral (PF) joint, including pain and patellar maltracking, yet the options for in vivo imaging of the PF joint are limited, especially after TKR. We propose a novel sequential biplane radiological method that permits accurate tracking of the PF and tibiofemoral (TF) joints throughout the range of movement under weightbearing, and test it in knees pre- and post-arthroplasty.

The August 2012 Spine Roundup³⁶⁰ looks at: neural tissue and polymerising bone cement; a new prognostic score for spinal metastases from prostatic tumours; recovery after spinal decompression; spinal tuberculosis; unintended durotomy at spinal surgery; how carrying a load on your head can damage the cervical spine; and how age changes your lumbar spine.