Ex vivo organotypic bone slice culture reveals preferential chondrogenesis after sustained growth plate injury.

Postnatal bone growth primarily relies on chondrocyte proliferation and osteogenic differentiation within the growth plate (GP) via endochondral ossification. Despite its importance, the GP is vulnerable to injuries, affecting 15-30 % of bone fractures. These injuries may lead to growth discrepancies, influence bone length and shape, and negatively affecting the patient's quality of life. This study aimed to investigate the molecular and cellular physiological and pathophysiological regeneration following sustained growth plate injury (GPI) in an ex vivo rat femur organotypic culture (OTC) model. Specifically, focusing on postnatal endochondral ossification process. 300 µm thick ex vivo bone cultures with a 2 mm long horizontal GPI was utilized. After 15 days of cultivation, gene expression analysis, histological and immunohistochemistry staining's were conducted to analyze key markers of endochondral ossification. In our OTCs we observed a significant increase in Sox9 expression due to GPI at day 15. The Ihh-PTHrP feedback loop was affected, favoring chondrocyte proliferation and maturation. Ihh levels increased significantly on day 7 and day 15, while PTHrP was downregulated on day 7. GPI had no impact on osteoclast number and activity, but gene expression analysis indicated OTCs' efforts to inhibit osteoclast differentiation and activation, thereby reducing bone resorption. In conclusion, our study provides novel insights into the molecular and cellular mechanisms underlying postnatal bone growth and regeneration following growth plate injury (GPI). We demonstrate that chondrocyte proliferation and differentiation play pivotal roles in the regeneration process, with the Ihh-PTHrP feedback loop modulating these processes. Importantly, our ex vivo rat femur organotypic culture model allows for the detailed investigation of these processes, providing a valuable tool for future research in the field of skeletal biology and regenerative medicine.

Newly Developed Resorbable Magnesium Biomaterials for Orbital Floor Reconstruction in Caprine and Ovine Animal Models-A Prototype Design and Proof-of-Principle Study.

BACKGROUND: orbital floor fractures have not been reconstructed using magnesium biomaterials. METHODS: To test technical feasibility, ex vivo caprine and ovine heads (n = 5) were used. Head tissues were harvested from pubescent animals (n = 5; mean age: 3.2 years; mean mass: 26.3 kg) and stored below 11 degrees for 7-10 days. All procedures were performed in a university animal resource facility. Two experienced maxillofacial surgeons performed orbital floor procedures in both orbits of all animals in a step-by-step preplanned dissection. A transconjunctival approach was chosen to repair the orbital floor with three different implants (i.e., magnesium implants; titanium mesh; and polydioxanone or PDO sheets). The position of each implant was evaluated by Cone-beam computed tomography (CBCT). RESULTS: Axial, coronal, and sagittal plane images showed good positioning of the magnesium plates. The magnesium plates had a radiographic visibility similar to that of the PDO sheets but lower than that of the titanium mesh. CONCLUSIONS: The prototype design study showed a novel indication for magnesium biomaterials. Further testing of this new biomaterial may lead to the first resorbable biomaterial with good mechanical properties for extensive orbital wall defects.

The combined effect of zinc and calcium on the biodegradation of ultrahigh-purity magnesium implants.

Magnesium (Mg)-based implants are promising candidates for orthopedic interventions, because of their biocompatibility, good mechanical features, and ability to degrade completely in the body, eliminating the need for an additional removal surgery. In the present study, we synthesized and investigated two Mg-based materials, ultrahigh-purity ZX00 (Mg-Zn-Ca; <0.5 wt% Zn and <0.5 wt% Ca, in wt%; Fe-content <1 ppm) and ultrahigh-purity Mg (XHP-Mg, >99.999 wt% Mg; Fe-content <1 ppm), in vitro and in vivo in juvenile healthy rats to clarify the effect of the alloying elements Zn and Ca on mechanical properties, microstructure, cytocompatibility and degradation rate. Potential differences in bone formation and bone in-growth were also assessed and compared with state-of-the-art non-degradable titanium (Ti)-implanted, sham-operated, and control (non-intervention) groups, using micro-computed tomography, histology and scanning electron microscopy. At 6 and 24 weeks after implantation, serum alkaline phosphatase (ALP), calcium (Ca), and Mg level were measured and bone marrow stromal cells (BMSCs) were isolated for real-time PCR analysis. Results show that ZX00 implants have smaller grain size and superior mechanical properties than XHP-Mg, and that both reveal good biocompatibility in cytocompatibilty tests. ZX00 homogenously degraded with an increased gas accumulation 12 and 24 weeks after implantation, whereas XHP-Mg exhibited higher gas accumulation already at 2 weeks. Serum ALP, Ca, and Mg levels were comparable among all groups and both Mg-based implants led to similar relative expression levels of Alp, Runx2, and Bmp-2 genes at weeks 6 and 24. Histologically, Mg-based implants are superior for new bone tissue formation and bone in-growth compared to Ti implants. Furthermore, by tracking the sequence of multicolor fluorochrome labels, we observed higher mineral apposition rate at week 2 in both Mg-based implants compared to the control groups. Our findings suggest that (i) ZX00 and XHP-Mg support bone formation and remodeling, (ii) both Mg-based implants are superior to Ti implants in terms of new bone tissue formation and osseointegration, and (iii) ZX00 is more favorable due to its lower degradation rate and moderate gas accumulation.

Immunological reaction to magnesium-based implants for orthopedic applications. What do we know so far? A systematic review on in vivo studies.

Magnesium-based implants (Mg) became an attractive candidate in orthopedic surgery due to their valuable properties, such as osteoconductivity, biodegradability, elasticity and mechanical strength. However, previous studies on biodegradable and non-biodegradable metal implants showed that these materials are not inert when placed in vivo as they interact with host defensive mechanisms. The aim of this study was to systematically review available in vivo studies with Mg-based implants that investigated immunological reactions to these implants. The following questions were raised: Do different types of Mg-based implants in terms of shape, size and alloying system cause different extent of immune response? and; Are there missing links to properly understand immunological reactions upon implantation and degradation of Mg-based implants? The database used for the literature research was PubMed (U.S. National Library of Medicine) and it was undertaken in the end of 2021. The inclusion criteria comprised (i) in vivo studies with bony implantation of Mg-based implants and (ii) analysis of the presence of local immune cells or systemic inflammatory parameters. We further excluded any studies involving coated Mg-implants, in vitro studies, and studies in which the implants had no bone contact. The systematic search process was conducted according to PRISMA guidelines. Initially, the search yielded 225 original articles. After reading each article, and based on the inclusion and exclusion criteria, 16 articles were included in the systematic review. In the available studies, Mg-based implants were not found to cause any severe inflammatory reaction, and only a mild to moderate inflammatory potential was attributed to the material. The timeline of foreign body giant cell formation showed to be different between the reviewed studies. The variety of degradation kinetics of different tested implants and discrepancies in studies regarding the time points of immunological investigations impair the conclusion of immunological reactions. This may be induced by different physical properties of an implant such as size, shape and alloying system. Further research is essential to elucidate the underlying mechanisms by which implant degradation affects the immune system. Also, better understanding will facilitate the decision of patients whether to undergo surgery with new device implantation.

Pilot Study and Preliminary Results of Biodegradable Intramedullary Nailing of Forearm Fractures in Children.

(1) Background: Diaphyseal forearm fractures are a common injury in children and adolescents. When operative treatment is needed, elastic stable intramedullary nailing (ESIN) is the most common surgical procedure. Although there is no clear evidence, hardware removal after fracture healing is performed in many patients. Often, the primary minimal invasive incision needs to be widened during implant removal. In order to decrease the burden of care of pediatric fractures, significant efforts were made to develop biodegradable implants, which make hardware removal unnecessary. Our study will conduct an observational trial on the clinical use of the Activa IM-Nail™ in forearm fractures in children between 3 and 13 years of age. The objective of this trial is to evaluate the risks and benefits of the Activa IM-Nail™. Among other objectives, the rate of refracture will be determined. (2) Methods: An international Europe-based, multicenter, prospective, single-arm, open-label study will be performed to ascertain the rate of refracture and to determine the subjective benefits of Activa IM-Nail™ for patients, parents and other caregivers. The study will include clinical follow-up including early post-operative complication, radiographs until bony healing and an additional follow-up after 1 year. At this stage, preliminary results and early complications on 76 patients are analyzed in this study and presented. (3) Results: As of April 2022, 76 patients were enrolled as per study protocol. There were 31 girls (40.8%) and 45 boys (59.2%). The mean age at the time of inclusion was 8.9 years (±2.4 years). The mean operation time was 58.9 ± 22.9 min (range, 15-119 min). The mean follow-up time was 8.9 ± 5.1 months (range, 0.2-18.6). Up to now, one refracture has occurred in one child falling from a height of about one meter 7 months after index surgery (1/76; 1.3%). (4) Conclusion: The research project assesses the safety and effectiveness of Activa IM-Nails™ as part of the surgical treatment of dislocated forearm fractures in children in the context of a PMCF study. The use of Activa IM-Nails™ with regard to various objectives, including postoperative complications and refracture rate, seems to be equal to the standard titan ESIN procedure compared to the literature. Preliminary results are encouraging and are made available.

[Supracondylar humeral fractures in childhood]. / Suprakondyläre Humerusfraktur im Kindesalter.

The classification of supracondylar humeral fractures in German-speaking areas is carried out according to von Laer, which has been appropriated from the AO system and has the advantage that it can be used to derive the treatment. When indicated immediate surgery is given preference over a delayed treatment. The result is controlled by functional tests directly during the operation. Instability of the fracture and correct placement of the Kirschner (K) wires are challenging. Alternatives are an external fixator and elastically stable intramedullary nailing (ESIN). Concomitant injuries initially affect the median nerve and the brachial artery and secondarily the radial nerve. Lesions of the ulnar nerve are mostly a postoperative complication. The bony consolidation is achieved after 3-4 weeks and afterwards implant removal can be safely carried out. Embedded K­wires and ESIN are removed after 3-6 months, depending on the surgical capacity and complaints of the patient.

The influence of biodegradable magnesium implants on the growth plate.

Mg-based biodegradable materials are considered promising candidates in the paediatric field due to their favourable mechanical and biological properties and their biodegrading potential that makes a second surgery for implant removal unnecessary. In many cases the surgical fixation technique requires a crossing of the growth plate by the implant in order to achieve an adequate fragment replacement or fracture stabilisation. This study investigates the kinetics of slowly and rapidly degrading Mg alloys in a transphyseal rat model, and also reports on their dynamics in the context of the physis and consecutive bone growth. Twenty-six male Sprague-Dawley rats received either a rapidly degrading (ZX50; n = 13) or a slowly degrading (WZ21; n = 13) Mg alloy, implanted transphyseal into the distal femur. The contralateral leg was drilled in the same manner and served as a direct sham specimen. Degradation behaviour, gas formation, and leg length were measured by continuous in vivo micro CT for up to 52 weeks, and additional high-resolution µCT (HRS) scans and histomorphological analyses of the growth plate were performed. The growth plate was locally destroyed and bone growth was significantly diminished by the fast degradation of ZX50 implants and the accompanying release of large amounts of hydrogen gas. In contrast, WZ21 implants showed homogenous and moderate degradation performance, and the effect on bone growth did not differ significantly from a single drill-hole defect. STATEMENT OF SIGNIFICANCE: This study is the first that reports on the effects of degrading magnesium implants on the growth plate in a living animal model. The results show that high evolution of hydrogen gas due to rapid Mg degradation can damage the growth plate substantially. Slow degradation, however, such as seen for WZ21 alloys, does not affect the growth plate more than drilling alone, thus meeting one important prerequisite for deployment in paediatric osteosynthesis.

Resorbable implants in pediatric fracture treatment.

Pediatric osteosynthesis has developed over the last 20 years, thereby reducing medical and economic burden, including long and expensive hospitalization. Currently, conventional and rigid alloying systems such as titanium are used for stabilization of bone fractures in children. In many cases, implants must be removed, as otherwise growth would be impeded. Biodegradable implant materials exhibit beneficial properties and would make a second removal surgery unnecessary. In the following article, we will give an overview of implant materials that are currently used in pediatric traumatology with a focus on Mg-based implants. Furthermore, we will discuss current scientific knowledge on resorbable implants, including results from pre-clinics and clinics.

Runx2 mediated Induction of Novel Targets ST2 and Runx3 Leads to Cooperative Regulation of Hypertrophic Differentiation in ATDC5 Chondrocytes.

Knowledge concerning expression and function of Suppression of Tumorigenicity 2 (ST2) in chondrocytes is at present, limited. Analysis of murine growth plates and ATDC5 chondrocytes indicated peak expression of the ST2 transmembrane receptor (ST2L) and soluble (sST2) isoforms during the hypertrophic differentiation concomitant with the expression of the hypertrophic markers Collagen X (Col X), Runx2 and MMP-13. Gain- and loss-of-function experiments in ATDC5 and primary human growth plate chondrocytes (PHCs), confirmed regulation of ST2 by the key transcription factor Runx2, indicating ST2 to be a novel Runx2 target. ST2 knock-out mice (ST2-/-) exhibited noticeable hypertrophic zone (HZ) reduction in murine growth plates, accompanied by lower expression of Col X and Osteocalcin (OSC) compared to wild-type (WT) mice. Likewise, ST2 knockdown resulted in decreased Col X expression and downregulation of OSC and Vascular Endothelial Growth Factor (VEGF) in ATDC5 cells. The ST2 suppression was also associated with upregulation of the proliferative stage markers Sox9 and Collagen II (Col II), indicating ST2 to be a new regulator of ATDC5 chondrocyte differentiation. Runx3 was, furthermore, identified as a novel Runx2 target in chondrocytes. This study suggests that Runx2 mediates ST2 and Runx3 induction to cooperatively regulate hypertrophic differentiation of ATDC5 chondrocytes.

The potential of isotopically enriched magnesium to study bone implant degradation in vivo.

This pilot study highlights the substantial potential of using isotopically enriched (non-radioactive) metals to study the fate of biodegradable metal implants. It was possible to show that magnesium (Mg) release can be observed by combining isotopic mass spectrometry and isotopic pattern deconvolution for data reduction, even at low amounts of Mg released a from slowly degrading 26Mg enriched (>99%) Mg metal. Following implantation into rats, structural in vivo changes were monitored by µCT. Results showed that the applied Mg had an average degradation rate of 16±5µmyear-1, which corresponds with the degradation rate of pure Mg. Bone and tissue extraction was performed 4, 24, and 52weeks after implantation. Bone cross sections were analyzed by laser ablation inductively coupled plasma mass spectrometry (ICP-MS) to determine the lateral 26Mg distribution. The 26Mg/24Mg ratios in digested tissue and excretion samples were analyzed by multi collector ICP-MS. Isotope pattern deconvolution in combination with ICP-MS enabled detection of Mg pin material in amounts as low as 200ppm in bone tissues and 20ppm in tissues up to two fold increased Mg levels with a contribution of pin-derived Mg of up to 75% (4weeks) and 30% (24weeks) were found adjacent to the implant. After complete degradation, no visual bone disturbance or residual pin-Mg could be detected in cortical bone. In organs, increased Δ26Mg/24Mg values up to 16‰ were determined compared to control samples. Increased Δ26Mg/24Mg values were detected in serum samples at a constant total Mg level. In contrast to urine, feces did not show a shift in the 26Mg/24Mg ratios. This investigation showed that the organism is capable of handling excess Mg well and that bones fully recover after degradation. STATEMENT OF SIGNIFICANCE: Magnesium alloys as bone implants have faced increasing attention over the past years. In vivo degradation and metabolism studies of these implant materials have shown the promising application in orthopaedic trauma surgery. With advance in Mg research it has become increasingly important to monitor the fate of the implant material in the organism. For the first time, the indispensible potential of isotopically enriched materials is documented by applying 26Mg enriched Mg implants in an animal model. Therefore, the spatial distribution of pin-Mg in bone and the pin-Mg migration and excretion in the organism could be monitored to better understand metal degradation as well as Mg turn over and excretion.

Chondrogenic differentiation of ATDC5-cells under the influence of Mg and Mg alloy degradation.

Biodegradable magnesium (Mg)-based materials are a potential alternative to permanent implants for application in children. Nevertheless effects of those materials on growth plate cartilage and chondrogenesis have not been previously evaluated. In vitro differentiation of ATDC5 cells was evaluated under the influence of pure Mg (PMg), Mg with 10wt% of gadolinium (Mg-10Gd) and Mg with 2wt% of silver (Mg-2Ag) degradation products (extracts) and direct cell culture on the materials. Gene expression showed an inhibitory effect on ATDC5 mineralization with the three extracts and a chondrogenic potential of Mg-10Gd. Cells cultured in Mg-10Gd and Mg-2Ag extracts showed the same proliferation and morphology than cells cultured in growth conditions. Mg-10Gd induced an increase in production of ECM and a bigger cell size, similar to the effects found with differentiation conditions. An increased metabolic activity was observed in cells cultured under the influence of Mg-10Gd extracts, indicated by an acidic pH during most of the culture period. After 7days of culture on the materials, ATDC5 growth, distribution and ECM synthesis were higher on Mg-10Gd samples, followed by Mg-2Ag and PMg, which was influenced by the homogeneity and composition of the degradation layer. This study confirmed the tolerance of ATDC5 cells to Mg-based materials and a chondrogenic effect of Mg-10Gd. Further studies in vitro and in vivo are necessary to evaluate cell reactions to those materials, as well as the effects on bone growth and the biocompatibility of the alloying system in the body.

Time-Dependent Changes in T1 during Fracture Healing in Juvenile Rats: A Quantitative MR Approach.

Quantitative magnetic resonance imaging (qMRI) offers several advantages in imaging and determination of soft tissue alterations when compared to qualitative imaging techniques. Although applications in brain and muscle tissues are well studied, its suitability to quantify relaxation times of intact and injured bone tissue, especially in children, is widely unknown. The objective observation of a fracture including its age determination can become of legal interest in cases of child abuse or maltreatment. Therefore, the aim of this study is the determination of time dependent changes in intact and corresponding injured bones in immature rats via qMRI, to provide the basis for an objective and radiation-free approach for fracture dating. Thirty-five MR scans of 7 Sprague-Dawley rats (male, 4 weeks old, 100 ± 5 g) were acquired on a 3T MRI scanner (TimTrio, Siemens AG, Erlangen, Germany) after the surgical infliction of an epiphyseal fracture in the tibia. The images were taken at days 1, 3, 7, 14, 28, 42 and 82 post-surgery. A proton density-weighted and a T1-weighted 3D FLASH sequence were acquired to calculate the longitudinal relaxation time T1 of the fractured region and the surrounding tissues. The calculation of T1 in intact and injured bone resulted in a quantitative observation of bone development in intact juvenile tibiae as well as the bone healing process in the injured tibiae. In both areas, T1 decreased over time. To evaluate the differences in T1 behaviour between the intact and injured bone, the relative T1 values (bone-fracture) were calculated, showing clear detectable alterations of T1 after fracture occurrence. These results indicate that qMRI has a high potential not only for clinically relevant applications to detect growth defects or developmental alterations in juvenile bones, but also for forensically relevant applications such as the dating of fractures in cases of child abuse or maltreatment.

Effects of Corroded and Non-Corroded Biodegradable Mg and Mg Alloys on Viability, Morphology and Differentiation of MC3T3-E1 Cells Elicited by Direct Cell/Material Interaction.

This study investigated the effect of biodegradable Mg and Mg alloys on selected properties of MC3T3-E1 cells elicited by direct cell/material interaction. The chemical composition and morphology of the surface of Mg and Mg based alloys (Mg2Ag and Mg10Gd) were analysed by scanning electron microscopy (SEM) and EDX, following corrosion in cell culture medium for 1, 2, 3 and 8 days. The most pronounced difference in surface morphology, namely crystal formation, was observed when Pure Mg and Mg2Ag were immersed in cell medium for 8 days, and was associated with an increase in atomic % of oxygen and a decrease of surface calcium and phosphorous. Crystal formation on the surface of Mg10Gd was, in contrast, negligible at all time points. Time-dependent changes in oxygen, calcium and phosphorous surface content were furthermore not observed for Mg10Gd. MC3T3-E1 cell viability was reduced by culture on the surfaces of corroded Mg, Mg2Ag and Mg10Gd in a corrosion time-independent manner. Cells did not survive when cultured on 3 day pre-corroded Pure Mg and Mg2Ag, indicating crystal formation to be particular detrimental in this regard. Cell viability was not affected when cells were cultured on non-corroded Mg and Mg alloys for up to 12 days. These results suggest that corrosion associated changes in surface morphology and chemical composition significantly hamper cell viability and, thus, that non-corroded surfaces are more conducive to cell survival. An analysis of the differentiation potential of MC3T3-E1 cells cultured on non-corroded samples based on measurement of Collagen I and Runx2 expression, revealed a down-regulation of these markers within the first 6 days following cell seeding on all samples, despite persistent survival and proliferation. Cells cultured on Mg10Gd, however, exhibited a pronounced upregulation of collagen I and Runx2 between days 8 and 12, indicating an enhancement of osteointegration by this alloy that could be valuable for in vivo orthopedic applications.

Postoperative paediatric pain prevalence: A retrospective analysis in a university teaching hospital.

BACKGROUND: Overall pain prevalence in paediatric patients is well documented, but relatively little attention has been paid to pain prevalence and intensity on specific postoperative days within the first week following an operation. OBJECTIVES: To evaluate reported pain prevalence on the day of surgery and each day during the following week and to analyse pain trajectories. DESIGN: Retrospective study. SETTING: Single centre university hospital. PARTICIPANTS: 815 postoperative children and adolescents (age≤18 years) were included (female: 36%, age 9.8±5.8). Children with ear, nose, throat (e.g. tonsillectomy), eye (e.g. strabismus repair) or dental surgery (e.g. dental extraction) were treated at other departments and therefore were not included in this study. METHODS: Retrospective analysis of the overall and clinically relevant (pain score ≥4/10) postoperative pain prevalence in children and adolescents during the first week after surgery. Possible influencing factors (age, sex, body mass index, type of anaesthesia, type of surgery and duration of surgery) on pain trajectories are analysed using mixed model techniques. RESULTS: Overall, 36% of 815 analysed children and adolescents suffered from pain ≥4 during their entire hospital stay. Compared to the day of surgery, the number of patients with pain ≥4 was slightly higher on day 1 after surgery (21% vs. 25%, respectively). In self-reported pain intensity rating (done for patients age≥4 years) the type of surgery (p<.001) was the only significant variable influencing pain intensity. In observational pain assessment (age<4 years) pain scores increased with patient's age (p=.004). In this patient group, pain intensity ratings did not differ between types of surgery (p=.278). CONCLUSION: Type of surgery is an important predictor for self-reported pain intensity ratings in children but not for observational pain assessment in younger children. In younger children observational pain assessment ratings increase with age.

Clinical impact of interleukin 6 as a predictive biomarker in the early diagnosis of postoperative systemic inflammatory response syndrome after major thoracic surgery: A prospective clinical trial.

BACKGROUND: Postoperative systemic inflammatory response syndrome and sepsis are associated with high morbidity and mortality rates. Early detection of postoperative systemic inflammatory response syndrome improves the outcome. The aim of this study was to evaluate the feasibility of interleukin 6 as a predictive biomarker in the early diagnosis of postoperative systemic inflammatory response syndrome after a major thoracic operation. METHODS: A total of 94 patients were enrolled in this prospective, clinical, single-center study. The enrolled subjects underwent either lung resection or esophageal operation. Interleukin 6, procalcitonin, C-reactive protein, and leucocytes were measured sequentially before, during, and after the operation. These levels were compared between patients who developed postoperative systemic inflammatory response syndrome and those who did not. RESULTS: The enrollees who completed the study included of 55 males (79.7%) and 14 females (20.3%) with a mean age of 60.9 years. Twenty patients (29.0%) developed systemic inflammatory response syndrome at a median time of 33.0 hours postoperatively. In cases of postoperative systemic inflammatory response syndrome, interleukin 6 was the most predictive biomarker, showing a striking increase on the day of operation and preceding the median onset of postoperative systemic inflammatory response syndrome, which occurred the next day (P ≤ .001). Peak procalcitonin and C-reactive protein occurrence were significantly delayed at 24 hours (P = .012) and 48 hours (P = .012). There was no mortality 30 days postoperatively. CONCLUSION: Interleukin 6 is a reliable predictor of postoperative systemic inflammatory response syndrome, and it is able to detect postoperative system inflammatory response syndrome before the onset of related clinical symptoms. When identifying patients at high risk, it would be beneficial to include interleukin 6 in conventional postoperative monitoring, particularly after extended surgical resection.

The impact of item order and sex on self-report of pain intensity in children.

OBJECTIVE: When questionnaires are used for pain assessment, the order effect must be taken into consideration; that is, whether the answer to a pain item is influenced by answers given to previous items. This study aimed to evaluate possible order effects in children and adolescents when answering pain items. Furthermore, the possible influence of sex on order effects was analyzed. METHOD: Three pain items (worst, at movement, and at rest) were given in 6 different orders. Two hundred and 46 postoperative hospitalized children and adolescents (age range of 11-18 years, mean of 14.4 years; female: 41%, duration of surgery range of 2-274 min) participated in this study. Each item order was answered by 40-43 children and adolescents. RESULTS: While there were no general order effects, we observed a sex-specific order effect. The position of pain at rest (p = .034) and pain on movement (p = .036) items had different influences on worst pain values in female patients compared to male patients. A sex-specific influence on pain at rest values was only caused by the position of pain on movement items (p = .036). CONCLUSION: The fact that male and female pediatric patients are differently influenced by the order of pain items has to be considered in planning a questionnaire. As more specific items (pain on movement) are less influenced by the item order effect, they should be preferred.

The origin points of the knee collateral ligaments: an MRI study on paediatric patients during growth.

PURPOSE: Different femoral origins for both the medial collateral ligament (MCL) and the lateral collateral ligament (LCL) have been reported in the growing skeleton (epiphyseal and metaphyseal). Knowledge about the exact attachment sites is mandatory for anatomically correct reconstruction. This study assesses the femoral origins of the knee collateral ligaments in skeletally immature individuals using magnetic resonance imaging (MRI). METHODS: MRIs of 336 knee joints (median age 15 years (range 2-18 years), m = 209 and f = 127) were retrospectively analysed to assess the distances between the femoral origins of the MCL and LCL to the distal femoral growth plate. In 175 patients, the body sizes were additionally retrieved from medical records. RESULTS: Both MCL and LCL ligament origins were invariably located on the epiphysis. Mean MCL origin-growth plate distance was 9.6 mm (SD 2.1 mm; range 2.2-13.6 mm) in boys and 8.6 mm (SD 1.5 mm; range 3.4-12.0 mm) in girls. Mean LCL origin-growth plate distance was 9.3 mm (SD 1.8 mm; range 4.3-13.0 mm) in boys and 8.2 mm (SD 1.5 mm; range 3.4-11.8 mm) in girls. The distance between the growth plate and both collateral ligaments as well as the length of the LCL correlated positively with patients' age and body size (MCL R(2) = 0.673 and 0.556, LCL R (2) = 0.734 and 0.645, LCL length R(2) = 0.589 and 0.741; all p < 0.001). CONCLUSIONS: During growth, the femoral origins of the MCL and the LCL are constantly located on the distal femoral epiphysis. There is a linear increase in the distances from the ligaments' origins to the growth plate according to age and body size. This new information may be of clinical importance for reconstructive surgery of the knee's collateral ligaments.

Extra-virgin olive oil diet and mild physical activity prevent cartilage degeneration in an osteoarthritis model: an in vivo and in vitro study on lubricin expression.

Mediterranean diet includes a relatively high fat consumption mostly from monounsaturated fatty acids mainly provided by olive oil, the principal source of culinary and dressing fat. The beneficial effects of olive oil have been widely studied and could be due to its phytochemicals, which have been shown to possess anti-inflammatory properties. Lubricin is a chondroprotective glycoprotein and it serves as a critical boundary lubricant between opposing cartilage surfaces. A joint injury causes an initial flare of cytokines, which decreases lubricin expression and predisposes to cartilage degeneration such as osteoarthritis. The aim of this study was to evaluate the role of extra-virgin olive oil diet and physical activity on inflammation and expression of lubricin in articular cartilage of rats after injury. In this study we used histomorphometric, histological, immunocytochemical, immunohistochemical, western blot and biochemical analysis for lubricin and interleukin-1 evaluations in the cartilage and in the synovial fluid. We report the beneficial effect of physical activity (treadmill training) and extra-virgin olive oil supplementation, on the articular cartilage. The effects of anterior cruciate ligament transection decrease drastically the expression of lubricin and increase the expression of interleukin-1 in rats, while after physical activity and extra-virgin olive oil supplemented diet, the values return to a normal level compared to the control group. With our results we can confirm the importance of the physical activity in conjunction with extra-virgin olive oil diet in medical therapy to prevent osteoarthritis disease in order to preserve the articular cartilage and then the entire joint.

Towards a better understanding of bone bridge formation in the growth plate - an immunohistochemical approach.

The growth plate at the end of long bones is the cartilaginous organ responsible for longitudinal bone growth in children. Trauma to the growth plate, i.e. fractures, can severely impair longitudinal bone growth, leading to growth disorders due to destruction of the epiphyseal circulation and formation of a bone bridge. From the clinical experience it is known that in some patients this bone bridge eventually disappears during the growth process. However, the molecular mechanisms involved in bone bridge formation and dissolution have not been clarified yet. The aim of this study was to investigate the spatial and temporal protein level of molecules potentially involved in these processes, i.e. RANKL, OPG, DKK-1, Coll 10, BMP-2 and IL-6, in an experimental rat model using an immunohistochemical approach. The results from our study suggest that bone bridge formation might be an early event starting immediately after growth plate injury and involving several pro-osteoblastic molecules, i.e. IL-6, BMP-2 as well as OPG and Coll X. In the late studied time points 3- and 9-month post-injury expression of anti-osteoblastic proteins, i.e. DKK1 and RANKL, was increased. This indicates that bone bridge dissolution might be a late event and potentially linked to Wnt signaling inhibition and RANK/RANKL signaling activation.