Sodium hyaluronate supplemented culture medium combined with joint-simulating mechanical loading improves chondrogenic differentiation of human mesenchymal stem cells.

In vitro models aim to recapitulate the in vivo situation. To more closely mimic the knee joint environment, current in vitro models need improvements to reflect the complexity of the native tissue. High molecular weight hyaluronan (hMwt HA) is one of the most abundant bioactive macromolecules in healthy synovial fluid, while shear and dynamic compression are two joint-relevant mechanical forces. The present study aimed at investigating the concomitant effect of joint-simulating mechanical loading (JSML) and hMwt HA-supplemented culture medium on the chondrogenic differentiation of primary human bone-marrow-derived mesenchymal stem cells (hBM-MSCs). hBM-MSC chondrogenesis was investigated over 28 d at the gene expression level and total DNA, sulphated glycosaminoglycan, TGF-ß1 production and safranin O staining were evaluated. The concomitant effect of hMwt HA culture medium and JSML significantly increased cartilage-like matrix deposition and sulphated glycosaminoglycan synthesis, especially during early chondrogenesis. A stabilisation of the hBM-MSC-derived chondrocyte phenotype was observed through the reduced upregulation of the hypertrophic marker collagen X and an increase in the chondrogenic collagen type II/X ratio. A combination of JSML and hMwt HA medium better reflects the complexity of the in vivo synovial joint environment. Thus, JSML and hMwt HA medium will be two important features for joint-related culture models to more accurately predict the in vivo outcome, therefore reducing the need for animal studies. Reducing in vitro artefacts would enable a more reliable prescreening of potential cartilage repair therapies.

Long term outcomes of biomaterial-mediated repair of focal cartilage defects in a large animal model.

The repair of focal cartilage defects remains one of the foremost issues in the field of orthopaedics. Chondral defects may arise from a variety of joint pathologies and left untreated, will likely progress to osteoarthritis. Current repair techniques, such as microfracture, result in short-term clinical improvements but have poor long-term outcomes. Emerging scaffold-based repair strategies have reported superior outcomes compared to microfracture and motivate the development of new biomaterials for this purpose. In this study, unique composite implants consisting of a base porous reinforcing component (woven poly(ε-caprolactone)) infiltrated with 1 of 2 hydrogels (self-assembling peptide or thermo-gelling hyaluronan) or bone marrow aspirate were evaluated. The objective was to evaluate cartilage repair with composite scaffold treatment compared to the current standard of care (microfracture) in a translationally relevant large animal model, the Yucatan minipig. While many cartilage-repair studies have shown some success in vivo, most are short term and not clinically relevant. Informed by promising 6-week findings, a 12-month study was carried out and those results are presented here. To aid in comparisons across platforms, several structural and functionally relevant outcome measures were performed. Despite positive early findings, the long-term results indicated less than optimal structural and mechanical results with respect to cartilage repair, with all treatment groups performing worse than the standard of care. This study is important in that it brings much needed attention to the importance of performing translationally relevant long-term studies in an appropriate animal model when developing new clinical cartilage repair approaches.

Transcriptional activation of ENPP1 by osterix in osteoblasts and osteocytes.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the main source of extracellular pyrophosphate. Along with tissue-nonspecific alkaline phosphatase (TNAP), ENPP1 plays an important role in balancing bone mineralisation. Although well established in pre-osteoblasts, the regulating mechanisms of ENPP1 in osteoblasts and osteocytes remain largely unknown. Using bioinformatic methods, osterix (Osx), an essential transcription factor in osteoblast differentiation and osteocyte function, was found to have five predicted binding sites on the ENPP1 promoter. ENPP1 and Osx showed a similar expression profile both in vitro and in vivo. Over-expression of Osx in MC3T3-E1 and MLO-Y4 cells significantly up-regulated the expression of ENPP1 (p < 0.05). The consensus Sp1 sequences, located in the proximal ENPP1 promoter, were identified as Osx-regulating sites using promoter truncation experiments and chromatin immunoprecipitation (ChIP) assays. The p38-mitogen-activated protein kinase (MAPK) signalling pathway was demonstrated to be responsible for ENPP1 promoter activation by Osx. Runt-related transcription factor 2 (Runx2) was confirmed to have synergistic effects with Osx in activating ENPP1 promoter. Taken together, these results provided evidence of the regulating mechanisms of ENPP1 transcription in osteoblasts and osteocytes.

Differences in human mesenchymal stem cell secretomes during chondrogenic induction.

Mesenchymal stem cells (MSCs) can be induced towards chondrogenesis through the application of chondrogenic stimuli such as transforming growth factor-ß (TGF-ß) or by multiaxial mechanical load. Previous work has showed that the chondrogenic effect of multiaxial load on MSCs is mediated by the endogenous production of TGF-ß1 by stimulated cells. This work compared the effects of TGF-ß1 stimulation and multiaxial mechanical load on the secretomes of stimulated cells. MSCs were seeded into fibrin-poly(ester-urethane) scaffolds and chondrogenically stimulated with either TGF-ß1 or mechanical load. The culture media was collected and analysed for 174 proteins using a cytokine antibody array. The results of the secretome analysis were then confirmed at a gene expression level by real-time PCR. As results implicated nitric oxide (NO), the media nitrite content was also determined as an indirect measurement of media NO levels. Results showed that TGF-ß1 stimulation and mechanical load lead to similar changes in factors such as BLC, VEGF and MMP13, whilst differences in detected levels were seen for factors including leptin, MDC, MIP3α and LAP. Gene expression analysis confirmed significant changes in four factors: angiopoietin 2, GROα, MMP13 and osteoprotegerin. After one week in culture the media nitrite content was significantly higher in loaded groups than both control and TGF-ß1 stimulated groups, suggesting this may be a major therapeutic target. These data show that despite clear similarities, TGF-ß1 stimulation and load have distinct effects on MSCs and are not analogous. This study has identified a number of potentially novel targets for tissue engineering, these data may also be useful for improving rehabilitation protocols e.g. after microfracture.

Tissue engineering and regenerative approaches to improving the healing of large bone defects.

Despite the high innate regenerative capacity of bone, large osseous defects fail to heal and remain a clinical challenge. Healing such defects requires the formation of large amounts of bone in an environment often rendered hostile to osteogenesis by damage to the surrounding soft tissues and vasculature. In recent years, there have been intensive research efforts directed towards tissue engineering and regenerative approaches designed to overcome this multifaceted challenge. In this paper, we describe and critically evaluate the state-of-the-art approaches to address the various components of this intricate problem. The discussion includes (i) the properties of synthetic and natural scaffolds, their use in conjunction with cell and growth factor delivery, (ii) their vascularisation, (iii) the potential of gene therapies and (iv) the role of the mechanical environment. In particular, we present a critical analysis of where the field stands, and how it can move forward in a coordinated fashion.

A surprisingly poor correlation between in vitro and in vivo testing of biomaterials for bone regeneration: results of a multicentre analysis.

New regenerative materials and approaches need to be assessed through reliable and comparable methods for rapid translation to the clinic. There is a considerable need for proven in vitro assays that are able to reduce the burden on animal testing, by allowing assessment of biomaterial utility predictive of the results currently obtained through in vivo studies. The purpose of this multicentre review was to investigate the correlation between existing in vitro results with in vivo outcomes observed for a range of biomaterials. Members from the European consortium BioDesign, comprising 8 universities in a European multicentre study, provided data from 36 in vivo studies and 47 in vitro assays testing 93 different biomaterials. The outcomes of the in vitro and in vivo experiments were scored according to commonly recognised measures of success relevant to each experiment. The correlation of in vitro with in vivo scores for each assay alone and in combination was assessed. A surprisingly poor correlation between in vitro and in vivo assessments of biomaterials was revealed indicating a clear need for further development of relevant in vitro assays. There was no significant overall correlation between in vitro and in vivo outcome. The mean in vitro scores revealed a trend of covariance to in vivo score with 58 %. The inadequacies of the current in vitro assessments highlighted here further stress the need for the development of novel approaches to in vitro biomaterial testing and validated pre-clinical pipelines.

A systematic review of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry compared to routine microbiological methods for the time taken to identify microbial organisms from positive blood cultures.

Bloodstream infections are a significant source of mortality and morbidity. Patient outcomes are improved by rapid identification of the causative pathogen and administration of appropriate antimicrobial therapy. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry has recently emerged as an alternative to microbiological identification. It is important to establish whether the costs of MALDI-TOF are justified by more timely identification and appropriate therapy, reduced length of stay and reduced hospital costs. We undertook a systematic review of the literature comparing MALDI-TOF and routine methods for the identification of the aetiological agent in patients with known or suspected bloodstream infection. The primary outcome of the review was the 'time to identify' organisms. Information on related measures such as 'time to appropriate antimicrobial treatment' and downstream hospital cost was also collected where reported. Ten of 775 articles identified met the inclusion criteria. All included studies were observational. MALDI-TOF identification was at least 24 h faster than routine methods in most circumstances. MADLI-TOF was associated with a reduction in downstream hospital costs and length of stay in studies reporting these outcomes. The observational studies reviewed provide evidence of potentially substantial time savings of MALDI-TOF in pathogen identification and instigation of appropriate therapy, which may also reduce hospital stay. Due to the small number of studies, all at relatively high risk of bias, this cannot be considered as definitive evidence of the impact of MALDI-TOF. More and better evidence, including impact on patient health and cost-effectiveness, is required.

Role and regulation of RUNX2 in osteogenesis.

Runt-related transcription factor 2 (RUNX2) is a transcription factor closely associated with the osteoblast phenotype. While frequently referred to, the complexity of its regulation and its interactions within the osteoblast differentiation pathway are often overlooked. This review aims to summarise the knowledge of its regulation at the transcriptional, translational and post-translational level. In addition, the regulation of RUNX2 by factors commonly used during osteogenic studies will be discussed.

Enhancing inflammatory and chemotactic signals to regulate bone regeneration.

Stem cells have become the fundamental element in regenerative medicine due to their inherent potential to differentiate into various cell types, and the ability to produce various bioactive molecules, including growth factors, cytokines and extracellular matrix molecules. In vivo, the secretion of tropic factors is modulated by chemotactic and inflammatory factors. In this study, we analysed the influence of a 2 h stimulation of mesenchymal stem cells (MSCs) with interleukin-1ß (IL1ß), granulocyte-colony stimulating factor (GCSF), stromal cell-derived factor 1 (SDF1) and stem cell factor (SCF). Our results demonstrated that this short stimulation exerts pronounced effects on the expression of multiple cytokine genes and proteins in MSC cells 48 and 72 h later. IL1ß strongly promoted the secretion of a wide range of proteins with chemotactic, proinflammatory and angiogenic properties, whereas SCF regulated the expression of proteins involved in proliferation, chondrogenesis and ECM regulation. This demonstrates that the changes in secretome can be directed towards a desired final functional outcome by selection of the most appropriate cytokine. Moreover, the expression pattern of Wnt signalling pathway components suggested the differential regulation of this pathway by IL1ß and SCF. Altogether, the robust paracrine action of MSCs can be achieved within a just 2 h treatment, which would be feasible within the operating theatre during a single surgical procedure. These results suggest that integrating inflammatory modulation in bone tissue engineering, by modifying the MSC secretome by way of a short stimulus, would provide a more targeted approach than administering unmodified MSCs alone.

A vision on the future of articular cartilage repair.

An AO Foundation (Davos, Switzerland) sponsored workshop "Cell Therapy in Cartilage Repair" from the Symposium "Where Science meets Clinics" (September 5-7, 2013, Davos) gathered leaders from medicine, science, industry, and regulatory organisations to debate the vision of cell therapy in articular cartilage repair and the measures that could be taken to narrow the gap between vision and current practice. Cell-based therapy is already in clinical use to enhance the repair of cartilage lesions, with procedures such as microfracture and articular chondrocyte implantation. However, even though long term follow up is good from a clinical perspective and some of the most rigorous randomised controlled trials in the regenerative medicine/orthopaedics field show beneficial effect, none of these options have proved successful in restoring the original articular cartilage structure and functionality in patients so far. With the remarkable recent advances in experimental research in cell biology (new sources for chondrocytes, stem cells), molecular biology (growth factors, genes), biomaterials, biomechanics, and translational science, a combined effort between scientists and clinicians with broad expertise may allow development of an improved cell therapy for cartilage repair. This position paper describes the current state of the art in the field to help define a procedure adapted to the clinical situation for upcoming translation in the patient.

Barriers and strategies for the clinical translation of advanced orthopaedic tissue engineering protocols.

Research in orthopaedic tissue engineering has intensified over the last decade and new protocols continue to emerge. The clinical translation of these new applications, however, remains associated with a number of obstacles. This report highlights the major issues that impede the clinical translation of advanced tissue engineering concepts, discusses strategies to overcome these barriers, and examines the need to increase incentives for translational strategies. The statements are based on presentations and discussions held at the AO Foundation-sponsored symposium "Where Science meets Clinics 2013" held at the Congress Center in Davos, Switzerland, in September, 2013. The event organisers convened a diverse group of over one hundred stakeholders involved in clinical translation of orthopaedic tissue engineering, including scientists, clinicians, healthcare industry professionals and regulatory agency representatives. A major point that emerged from the discussions was that there continues to be a critical need for early trans-disciplinary communication and collaboration in the development and execution of research approaches. Equally importantly was the need to address the shortage of sustained funding programs for multidisciplinary teams conducting translational research. Such detailed discussions between experts contribute towards the development of a roadmap to more successfully advance the clinical translation of novel tissue engineering concepts and ultimately improve patient care in orthopaedic and trauma surgery.

In search of an osteoblast cell model for in vitro research.

The process of bone formation, remodelling and healing involves a coordinated action of various cell types. Advances in understanding the biology of osteoblast cells during these processes have been enabled through the use of various in vitro culture models from different origins. In an era of intensive bone tissue engineering research, these cell models are more and more often applied due to limited availability of primary human osteoblast cells. While they are a helpful tool in developing novel therapies or biomaterials; concerns arise regarding their phenotypic state and differences in relation to primary human osteoblast cells. In this review we discuss the osteoblastic development of some of the available cell models; such as primary human, rat, mouse, bovine, ovine and rabbit osteoblast cells; as well as MC3T3-E1, MG-63 and SaOs-2 cell lines, together with their advantages and disadvantages. Through this, we provide suggestions on the selection of the appropriate and most relevant osteoblast model for in vitro studies, with specific emphasis on cell-material based studies.

A combination of shear and dynamic compression leads to mechanically induced chondrogenesis of human mesenchymal stem cells.

There is great interest in how bone marrow derived stem cells make fate decisions. Numerous studies have investigated the role of individual growth factors on mesenchymal stem cell differentiation, leading to protocols for cartilage, bone and adipose tissue. However, these protocols overlook the role of biomechanics on stem cell differentiation. There have been various studies that have applied mechanical stimulation to constructs containing mesenchymal stem cells, with varying degrees of success. One critical fate decision is that between cartilage and bone. Articular motion is a combination of compressive, tensile and shear deformations; therefore, one can presume that compression alone is unlikely to be a sufficient mechanical signal to generate a cartilage-like tissue in vitro. Within this study, we aimed to determine the role of shear on the fate of stem cell differentiation. Specifically, we investigated the potential enhancing effect of surface shear, superimposed on cyclic axial compression, on chondrogenic differentiation of human bone marrow-derived stem cells. Using a custom built loading device we applied compression, shear or a combination of both stimuli onto fibrin/polyurethane composites in which human mesenchymal stem cells were embedded, while no exogenous growth-factors were added to the culture medium. Both compression or shear alone was insufficient for the chondrogenic induction of human mesenchymal stem cells. However, the application of shear superimposed upon dynamic compression led to significant increases in chondrogenic gene expression. Histological analysis detected sulphated glycosaminoglycan and collagen II only in the compression and shear group. The results obtained may provide insight into post-operative care after cell therapy involving mesenchymal stromal cells.

Evaluation of the influence of platelet-rich plasma (PRP), platelet lysate (PL) and mechanical loading on chondrogenesis in vitro.

The aim of this work is to investigate the capability of PRP as an adjuvant therapy to autologous chondrocyte implantation (ACI) in combination with multi-axial load with respect to cartilage regeneration. Articular cartilage shows poor repair capacity and therapies for cartilage defects are still lacking. Well-established operative treatments include ACI, and growing evidence shows the beneficial effects of PRP. Platelets contain numerous growth factors, among them transforming growth factor beta (TGF-ß). Dynamic mechanical loading is known to be essential for tissue formation, improving extracellular matrix (ECM) production. For our ACI model monolayer expanded human chondrocytes were seeded into polyurethane scaffolds and embedded in fibrin (hChondro), in PRP-Gel (PRP), or in fibrin with platelet lysate (PL), which was added to the media once a week with a concentration of 50 vol%. The groups were either exposed to static conditions or multi-axial forces in a ball-joint bioreactor for 1 h per day over 2 weeks, mimicking ACI under physiological load. The culture medium was collected and analyzed for glycosaminoglycan (GAG), nitrite and transforming growth factor beta 1 (TGF-ß1) content. The cell-scaffold constructs were collected for DNA and GAG quantification; the expression of chondrogenic genes, TGF-ß and related receptors, as well as inflammatory genes, were analyzed using qPCR. Loading conditions showed superior chondrogenic differentiation (upregulation of COL2A1, ACAN, COMP and PRG4 expression) than static conditions. PRP and PL groups combined with mechanical loading showed upregulation of COL2A1, ACAN and COMP. The highest amount of total TGF-ß1 was quantified in the PL group. Latent TGF-ß1 was activated in all loaded groups, while the highest amount was found in the PL group. Load increased TGFBR1/TGFBR2 mRNA ratio, with further increases in response to supplements. In general, loading increased nitrite release into the media. However, over time, the media nitrite content was lower in the PL group compared to the control group. Based on these experiments, we conclude that chondrogenic differentiation is strongest when simulated ACI is performed in combination with dynamic mechanical loading and PRP-gel or PL supplementation. An inflammatory reaction was reduced by PRP and PL, which could be one of the major therapeutic effects. Loading presumably can enhance the action of TGF-ß1, which was predominantly activated in loaded PL groups. The combination of load and PRP represents an effective and promising synergy concerning chondrocyte-based cartilage repair.

Pellet culture model for human primary osteoblasts.

In vitro monolayer culture of human primary osteoblasts (hOBs) often shows unsatisfactory results for extracellular matrix deposition, maturation and calcification. Nevertheless, monolayer culture is still the method of choice for in vitro differentiation of primary osteoblasts. We believe that the delay in mature ECM production by the monolayer cultured osteoblasts is determined by their state of cell maturation. A functional relationship between the inhibition of osteoblast proliferation and the induction of genes associated with matrix maturation was suggested within a monolayer culture model for rat calvarial osteoblasts. We hypothesize, that a pellet culture model could be utilized to decrease initial proliferation and increase the transformation of osteoblasts into a more mature phenotype. We performed pellet cultures using hOBs and compared their differentiation potential to 2D monolayer cultures. Using the pellet culture model, we were able to generate a population of cuboidal shaped central osteoblastic cells. Increased proliferation, as seen during low-density monolayer culture, was absent in pellet cultures and monolayers seeded at 40,000 cells/cm2. Moreover, the expression pattern of phenotypic markers Runx2, osterix, osteocalcin, col I and E11 mRNA was significantly different depending on whether the cells were cultured in low density monolayer, high density monolayer or pellet culture. We conclude that the transformation of the osteoblast phenotype in vitro to a more mature stage can be achieved more rapidly in 3D culture. Moreover, that dense monolayer leads to the formation of more mature osteoblasts than low-density seeded monolayer, while hOB cells in pellets seem to have transformed even further along the osteoblast phenotype.

Impact of rapid microbial identification on clinical outcomes in bloodstream infection: the RAPIDO randomized trial.

OBJECTIVES: Bloodstream infection has a high mortality rate. It is not clear whether laboratory-based rapid identification of the organisms involved would improve outcome. METHODS: The RAPIDO trial was an open parallel-group multicentre randomized controlled trial. We tested all positive blood cultures from hospitalized adults by conventional methods of microbial identification and those from patients randomized (1:1) to rapid diagnosis in addition to matrix-assisted desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) performed directly on positive blood cultures. The only primary outcome was 28-day mortality. Clinical advice on patient management was provided to members of both groups by infection specialists. RESULTS: First positive blood culture samples from 8628 patients were randomized, 4312 into rapid diagnosis and 4136 into conventional diagnosis. After prespecified postrandomization exclusions, 2740 in the rapid diagnosis arm and 2810 in the conventional arm were included in the mortality analysis. There was no significant difference in 28-day survival (81.5% 2233/2740 rapid vs. 82.3% 2313/2810 conventional; hazard ratio 1.05, 95% confidence interval 0.93-1.19, p 0.42). Microbial identification was quicker in the rapid diagnosis group (median (interquartile range) 38.5 (26.7-50.3) hours after blood sampling vs. 50.3 (47.1-72.9) hours after blood sampling, p < 0.01), but times to effective antimicrobial therapy were no shorter (respectively median (interquartile range) 24 (2-78) hours vs. 13 (2-69) hours). There were no significant differences in 7-day mortality or total antibiotic consumption; times to resolution of fever, discharge from hospital or de-escalation of broad-spectrum therapy or 28-day Clostridioides difficile incidence. CONCLUSIONS: Rapid identification of bloodstream pathogens by MALDI-TOF MS in this trial did not reduce patient mortality despite delivering laboratory data to clinicians sooner.

Syndesmotic fixation in unstable ankle fractures: Does early post-operative weight bearing affect radiographic outcomes?

OBJECTIVE: To analyse whether early post-operative full weight bearing following syndesmotic ankle fixation affected radiographic outcomes suggestive of diastasis. DESIGN: Retrospective comparative cohort study over a two year period. SETTING: Level 1 trauma centre PATIENTS/PARTICIPANTS: 152 consecutive patients sustaining an unstable ankle fracture requiring syndesmotic stabilisation were included. Exclusions were 49 patients who had trimalleolar fixation without syndesmosis screws, one patient who had concomitant ankle and talar fracture. Five patients were lost to follow up and eleven patients were followed up in other centres. A total of 86 patients were analysed INTERVENTION: Protected or full weight bearing. MAIN OUTCOME MEASUREMENT: The primary outcome measure was early diastasis. The secondary outcomes were late diastasis, wound complications and re-operation. Analysis of variance was used for the predictor variable of weight bearing status. We assumed a priori that p values of less than 0.05 were significant. RESULTS: Median age was 36 (IQR 30), with 54 males and 32 females. Median follow up was 12 weeks (IQR 6). There was no significant difference when comparing weight bearing status and change in radiographic measurements intra-operatively compared to 6 and 12 week follow up radiographs (tibiofibular clear space p = 0.799, tibiofibular overlap p = 0.733 and medial clear space p = 0.261). CONCLUSION: After surgical stabilization of an unstable syndesmotic injury, full weight bearing did not lead to syndesmotic diastasis in the early post-operative period. Full weight bearing is recommended following ankle fixation which includes syndesmotic fixation.

Orthoplastic reconstruction of type IIIB open tibial fractures retaining debrided devitalized cortical segments: the Bristol experience 2014 to 2018.

AIMS: Type IIIB open tibial fractures are devastating high-energy injuries. At initial debridement, the surgeon will often be faced with large bone fragments with tenuous, if any, soft-tissue attachments. Conventionally these are discarded to avoid infection. We aimed to determine if orthoplastic reconstruction using mechanically relevant devitalized bone (ORDB) was associated with an increased infection rate in type IIIB open tibial shaft fractures. PATIENT AND METHODS: This was a consecutive cohort study of 113 patients, who had sustained type IIIB fractures of the tibia following blunt trauma, over a four-year period in a level 1 trauma centre. The median age was 44.3 years (interquartile range (IQR) 28.1 to 65.9) with a median follow-up of 1.7 years (IQR 1.2 to 2.1). There were 73 male patients and 40 female patients. The primary outcome measures were deep infection rate and number of operations. The secondary outcomes were nonunion and flap failure. RESULTS: In all, 44 patients had ORDB as part of their reconstruction, with the remaining 69 not requiring it. Eight out of 113 patients (7.1%) developed a deep infection (ORDB 1/44, non-ORDB 7/69). The median number of operations was two. A total of 16/242 complication-related reoperations were undertaken (6.6%), with 2/16 (12.5%) occurring in the ORDB group. CONCLUSION: In the setting of an effective orthoplastic approach to type IIIB open diaphyseal tibial fractures, using mechanically relevant debrided devitalized bone fragments in the definitive reconstruction appears to be safe. Cite this article: Bone Joint J 2019;101-B:1002-1008.

Medication-related osteonecrosis of the jaw in a minipig model: Parameters for developing a macroscopic, radiological, and microscopic grading scheme.

OBJECTIVES: To devise a macroscopic, radiological, and histological scale for assessing pathological changes associated with medication-related osteonecrosis of the jaw in a minipig model. MATERIALS AND METHODS: Medication-related osteonecrosis of the jaw was induced in Göttingen minipigs by weekly intravenous administration of bisphosphonate (zoledronic acid) combined with a tooth extraction procedure. Controls either did not receive zoledronic acid or did not undergo tooth extraction. After 20 weeks, minipigs were euthanized and underwent computed tomography and micro-computed tomography scanning. The mandible underwent additional histological examination. RESULTS: The most consistent macroscopic findings in animals that had developed bisphosphonate-related osteonecrosis of the jaw (BRONJ) were necrotic, denuded bone, and formation of fistula and pus. Under radiological examination, impaired extraction socket healing, decrease in attenuation of bone beneath the extraction site, and periosteal reaction were observed. Under histological examination, demineralization of the extracellular bone matrix, denuding of bone, and osteonecrosis were recorded. CONCLUSION: These parameters were used to develop a scoring system for grading BRONJ.

A simple, lanthanide-based method to enhance the transduction efficiency of adenovirus vectors.

Based upon the powerful bridging and charge-masking properties of lanthanide cations (Ln3+), we have investigated their use to improve the transduction efficiency of adenovirus vectors. Using a luciferase marker gene, it was possible to increase transgene expression by the murine mesenchymal stem cell line C3H10T(1/2) by up to four log orders when using very low multiplicities of infection in conjunction with Ln3+; La3+ was superior to Gd3+, Y3+ and Lu3+ in this regard. All Ln3+ were more effective than Ca2+. Flow cytometry, using a green fluorescent protein marker gene, confirmed that La3+ increased both the percentage of transduced cells and the level of transgene expression per cell. Transduction of primary cultures of a variety of different mesenchymal cells from human, rabbit, bovine and rat sources, as well as gene transfer to synovium and muscle in vivo, was also greatly enhanced. Our findings suggest that this lanthanide-based method holds much promise for expediting both experimental and clinical applications of gene transfer with adenoviral vectors.