Fabrication of biogenic coatings with suitable mechanical properties is a key goal in orthopedics, to overcome the limitations of currently available coatings and improve the clinical results of coated implants compared to uncoated ones. In this paper, biological-like apatite coatings were deposited from a natural bone-apatite source by a pulsed electron deposition technique (PED).

Bone apatite-like (BAL) films were deposited directly from bone targets, obtained by standard deproteinization of bovine tibial cortical shafts and compared to films deposited by sintered stoichiometric-hydroxyapatite targets (HA). Deposition was performed at room temperature by PED in the Ionized Jet Deposition (IJD) version. Half of the samples was annealed at 400°C for 1h (BAL_400 and HA_400). As-deposited and annealed coatings were characterized in terms of composition and crystallinity (XRD, FT-IR), microstructure and morphology (SEM-EDS, AFM) and mechanical properties (nanoindentation and micro-scratch). For the biological tests, human dental pulp stem cells (hDPSCs) were isolated from dental pulp from patients undergoing a routine tooth extraction, plated on the samples (2500 cells/cm2) and cultured for 3 weeks, when the expression of typical osteogenic markers Runx-2, osteopontin, Osx and Osteocalcin in hDPSCs was evaluated.

Results showed that deposition by PED allows for a close transfer of the targets” composition. As-deposited coatings exhibited low cristallinity, that was significantly increased by post-deposition annealing, up to resembling that of biogenic apatite target. As a result of annealing, mechanical properties increased up to values comparable to those of commercial plasma-sprayed HA-coatings.

In vitro biological tests indicated that BAL_400 promotes hDPSCs proliferation to a higher extent compared to non-annealed bone coating and HA-references. Furher, immunofluorescence and western blot analyses revealed that the typical osteogenic markers were expressed, indicating that BAL_400 alone can efficiently promote the osteogenic commitment of the cells, even in absence of an osteogenic medium.

In conclusion, bone-like apatite coatings were deposited by PED, which closely resembled composition and structure of natural-apatite. Upon annealing at 400°C, the coatings exhibited satisfactory mechanical properties and were capable of providing a suitable microenvironment for hDPSCs adherence and proliferation and for them to reach osteogenic commitment.

These results suggest that bone apatite-like thin films obtained by biogenic source may represent an innovative platform to boost bone regeneration in the orthopedic, maxillofacial and odontoiatric field.

Orthopedic implants containing biodegradable magnesium have been used for fracture repair with considerable efficacy; however, the underlying mechanisms by which these implants improve fracture healing remain elusive. Here we show the formation of abundant new bone at peripheral cortical sites after intramedullary implantation of a pin containing ultrapure magnesium into the intact distal femur in rats. This response was accompanied by substantial increases of neuronal calcitonin gene-related polypeptide-a (CGRP) in both the peripheral cortex of the femur and the ipsilateral dorsal root ganglia (DRG). Surgical removal of the periosteum, capsaicin denervation of sensory nerves or knockdown in vivo of the CGRP-receptor-encoding genes Calcrl or Ramp1 substantially reversed the magnesium-induced osteogenesis that we observed in this model. Overexpression of these genes, however, enhanced magnesium-induced osteogenesis. We further found that an elevation of extracellular magnesium induces magnesium transporter 1 (MAGT1)-dependent and transient receptor potential cation channel, subfamily M, member 7 (TRPM7)-dependent magnesium entry, as well as an increase in intracellular adenosine triphosphate (ATP) and the accumulation of terminal synaptic vesicles in isolated rat DRG neurons. In isolated rat periosteum-derived stem cells, CGRP induces CALCRL- and RAMP1-dependent activation of cAMP-responsive element binding protein 1 (CREB1) and SP7 (also known as osterix), and thus enhances osteogenic differentiation of these stem cells. Furthermore, we have developed an innovative, magnesium-containing intramedullary nail that facilitates femur fracture repair in rats with ovariectomy-induced osteoporosis. Taken together, these findings reveal a previously undefined role of magnesium in promoting CGRP-mediated osteogenic differentiation, which suggests the therapeutic potential of this ion in orthopedics.

Histone modifications critically contribute to the epigenetic orchestration of bone development - in part by modifying accessibility of genes to transcription factors. Based on the previous finding that histone H2A deubiquitinase 2A-DUB/Mysm1 interacts with the p53-axis in hematopoiesis and tissue development, we here analyzed the molecular and cellular mechanisms of Mysm1-p53 interplay in bone development.

The bone phenotype of 4–5 week-old Mysm1-/- (MKO), Mysm1-/-p53-/- (DKO) and corresponding wildtype (WT) mice was determined using µCT and histology. Primary osteoblasts, mesenchymal stem cells (MSCs) and osteoclasts were isolated from long bones to assess cell proliferation, differentiation, apoptosis and activity. Statistics: one-way ANOVA, p<0.05.

MKO mice displayed an osteopenic bone phenotype compared to WT (BV/TV: 5.7±2.9 vs. 12.5±4.2, TbN: 1.3±0.6 vs. 2.7±0.7 1/mm, respectively), and these effects were abolished in DKO mice (BV/TV: 17.8±2.6, TbN: 3.7±0.4 1/mm). MKO mice compared to WT also showed both in vitro and in vivo disturbed osteoclast formation (in vitro: 1.5±1.2 vs. 9.9±1.8 OcN/mm2, in vivo OcN/BPm: 1.4±1.0 vs. 3.0±0.7 cells/mm, respectively) accompanied by increased apoptosis and DNA damage; additional p53 knockout attenuated these effects (7.8±1.8 OcN/mm2 and OcN/BPm: 2.2±1.0 cells/mm). Primary osteoblasts from both MKO and DKO mice showed decreased expression of the transcription factor Runx2 and of the osteogenic markers. ChIP-Seq analysis revealed direct binding of Mysm1 to Runx2 promoter regions in osteoblasts, implying that Mysm1 here regulates osteogenic differentiation. In contrast, MKO-MSCs differentiation did not differ from WT, but DKO-MSCs displayed a significantly increased expression of Alpl, Bglap and Runx2. The different effects of Mysm1-/- in MSCs and osteoblasts presumably resulted from the lower expression level of Mysm1 in MSCs in comparison to mature osteoblasts.

Thus, our data demonstrate that H2A deubiquitinase Mysm1 is essential for the epigenetic control of bone development via distinct mechanisms: 1) In osteoclasts, Mysm1 is involved in maturation of osteoclast progenitors and osteoclast survival. 2) In osteoblasts, Mysm1 directly controls Runx2 expression, thereby explaining osteopenic phenotype of MKO mice. 3) In MSCs, Mysm1 may play an inferior role due to low expression level. However, loss of p53 increases Runx2 expression during MSC differentiation, leading to normal bone formation in DKO mice.

MicroRNA´s are regulatory sequences which influence the posttranscriptional synthesis of about 70% of protein encoding genes. In different studies, MicroRNA-146a (miR-146a) was associated with inflammatory and autoimmunological processes. In vitro it was shown, that miR-146a influences the bone metabolism by regulating differentiation of mesenchymal stem cells. The miR-146a deficient mouse starts to develop lymphoproliferative and myeloproliferative disease by 6–8 months of age. In this study, we investigate the influence of miR-146a deficiency on bone structure and stability dependent on age and gender.

Due to the increasing life expectancy the incidence of gonarthrosis, the degeneration of articular cartilage and bone in the knee joint, is increasing worldwide. Although the success rate of knee arthroplasties is high, complications like the loosening of the implant necessitate subsequent treatments. Moreover, the morphology and microstructure of the knee joint varies considerably between patients, therefore the anatomical expertise of orthopedic surgeons is essential. In this analysis we therefore investigate the variation and micro-architectural alterations in subchondral bone in osteoarthritis (OA) patients undergoing a knee replacement surgery.

We investigate OA bone degenerations using clinical X-rays and micro-computed tomography (micro-CT). Tibial bone samples are collected from 100 patients undergoing a total knee arthroplasty at the Klinikum Wels-Grieskirchen. Images are obtained using an industrial micro-CT scanner RayScan 250E. Microstructural parameters include bone volume fraction and cortical thickness of the subcondral bone and are obtained from micro-CT images with isometric voxel sizes of 50 µm.

Using micro-CT, we show a high morphological variation in relation to cortical thickness, both within the respective condyle as well as between the medial and lateral condyle. Cortical thickness seems to correlate with cartilage thickness and knee joint alignment. The results are incorporated into a gonarthrosis database that integrates microstructural parameters via a combined analysis of X-ray and micro-CT data. This database aims to facilitate the assessment of osteoarthritis, i.e. in relation to cartilage degeneration, in future patients on the basis of the investigated patient collective.

Introduction Leg length inequalities (LLIs) are a common finding in every orthopaedic practice. They can be classified into anatomical and functional LLIs. LLIs can e.g. cause gait and balance disabilities, low back pain and functional scoliosis of the spine. In patients with a total hip replacement a higher rate of aseptic loosening of the prosthesis was found when LLIs were present (Gurney 2002). Until today LLIs are treated statically by wooden blocs, which are placed under the shorter extremity, until the pelvis is levelled. However, the correction of LLIs should also be evaluated dynamically to examine the influence of correction onto the spine and pelvis during gait. Therefore, we seek to evaluate in this pilot study the influence of simulated LLIs on spine and pelvis during gait. Methods A total of 30 healthy subjects (17 females & 13 males) with an average age of 24.4 years were measured in this study. First, LLIs (1 to 4 cm) were simulated with the subjects standing on a simulation platform, which height could be controlled, as previously described (Betsch 2012). In addition, a specially designed sandal with different insole heights (1 to 4 cm) was used to simulate LLIs under dynamic condition while subjects were walking on a treadmill. Changes in pelvic position and spinal posture caused by the LLIS were measured using a rasterstereographic system (Formetric 4D motion, Diers International GmbH, Germany). All data were checked for Gaussian distribution by the Chi square test. Student t-tests were used to check for differences between the LLIs. The level of significance was set at p

Mobility plays an important role, in particular for patients with osteoporosis and after trauma surgery, both as an outcome and as treatment. Mobility is closely linked to the patient”s quality of life and exercise is a powerful additional treatment option. In order to be able to generate an evidence base to evaluate various surgical and non-surgical treatment options, objective measurements of patient mobility and exercise over a certain time period are needed. Wearables are a promising candidate, with obvious advantages compared to questionnaires and/or PROs. However, when extracting parameters with wearables, one often faces the problem of algorithms not performing well enough for special cases like slow gait speeds or impaired gait, as they typically appear in this patient group. We plan to further extend the applicability of the actibelt system (3D accelerometer, 100Hz), in particular to improve the measurement precision of real-world walking speed in slow and impaired walking. We are using a special measurement wheel including a rotating 3D accelerometer that allows to capture high quality real-world walking speed and distance measurements, and a mobile high resolution camera system. In a first block 20 patients with osteoporosis were included in the study at the Ludwigs-Maximilians-University”s Department of General, Trauma and Reconstructive Surgery in Munich, Germany and equipped with an actibelt. Patients were asked to walk as “normal” as possible, while wearing their usual apparel, in the building and outside the building. They climbed stairs and had to deal with all unexpected “stop and go” events that appear in real-world walking. Various gait parameters will be extracted from the recorded data and compared to the gold standard. We will then tune the existing algorithms as well as new algorithms (e.g. step detection based on continuous wavelet transformation) to explore potential improvements of both step detection and speed estimation algorithms. Further refinement and validation using real world data is warranted.

The use of hip resurfacing arthroplasty (HRA) has largely regressed due to the fear of metal-on-metal bearings. However committed HRA users continue to assert the functional advantages that a geometry retaining implant would have on a patient”s hip. Currently worldwide, HRA is only recommended to men who demand an active lifestyle. Despite this precarious indication, it is not clear to what extent HRA has on higher activity function. The aim of this study was to determine the functional extent to which could be achieved with HRA. The primary objective is to assess the loading pattern change for patients implanted with HRA at high walking speeds and inclinations. The second objective is to compare their loading features to a healthy group to determine if a normal gait pattern could be achieved.

Between 2012 and 2016, a total of 28 prospective unilateral HRA patients were analysed on an instrumented treadmill from a single centre. All 28 patient patients had a uniform implant type and had no other lower limb operations or disease. Perioperative plain orthogonal radiographs were used to measure hip length and global hip offset change. A healthy control group (n=35) were analysed to compare. All HRA patients gait characteristics were assessed at incrementally higher speeds and inclinations to determine the extent of improvement HRA has on a challenging activity. A Student t-test along with a multivariate analysis was done with significance set at α=0.05. Weight and height variance was accounted with Hof normalisation.

The HRA and control group were reasonably matched for age (57 vs 55yrs), BMI (27 vs 25) and height (175 vs 170cm) respectively. Hip measurements revealed less than 5mm change for all cases. The mean time from initial preoperative gait assessment to postoperative assessment was 30 months (24–48months). The mean top walking speed for controls was 1.97m/s and postoperatively 2.1 m/sec for the HRA group. The significant (p<0.001) loading change during flat walking can be seen with restoration of symmetry. Walking at an inclination demonstrated a marked change during weight acceptance (p<0.001) and a loading pattern returning to near normal.

This prospective study found HRA patients walking faster than age matched controls. They demonstrated a significant change in their loading pattern, by significantly shifting load from the unaffected side to the implanted side. Uphill walking, an activity which requires more hip flexion, demonstrated a change in stance phase which was near normal. This small comparative study confirms near physiological function can be achieved with HRA at higher activity levels.

Use of scaffolds for articular cartilage repair (ACR) has increased over the last years with many biomaterials options suggested for this purpose. It is known that scaffolds for ACR have to be optimally biodegradable with simultaneous promotion of chondrogenesis, favouring hyaline cartilage formation under rather complex biomechanical and physiological conditions. Whereas improvement of the scaffolds by their conditioning with stem cells or adult chondrocytes can be employed in bioreactors, “ideal” scaffolds should be capable of performing such functions directly after implantation. It was previously considered that scaffold structure and composition would be the best if it mimics the structure of native cartilage. However, in this case no clear reparative stimuli are being imposed on the scaffold area, which would drive chondrocytes activity in a desired way.

In this work, we studied new xeno-free, recombinant human type III collagen-laden polylactide (PLA) mesh scaffolds, which have been designed, produced, and biomechanically optimized in vitro and in vivo validated in a porcine and equine model. The scaffolds were additionally assessed for relative performance simulated synovial fluids for both human conditions and veterinary cases.

It was experimentally shown that success of the scaffolds in ACR eventually require lower stiffness than surrounding cartilage yet matching the strain compliance, different in static and dynamic conditions. This ensures an optimal combination of load transfer and oscillatory nutrients supply to the cells, which otherwise is difficult to rely on just with a passive diffusion in avascular cartilage conditions. The results encourage further development of such scaffold structures targeted on their best clinical performance rather than trying to imitate the respective original tissue.

The authors would like to thank Finnish Agency for Innovation (Tekes) for providing financial support to this project. A.Z. also acknowledges Teknos Foundation (Finland) for the scholarship.

The combination of natural polymers with calcium phosphate cements (CPCs) by mimicking the highly mineralized collagen-based matrix of native bone is crucial in order to obtain mechanically compatible injectable bone substitute (IBS) formulations. This combination overcomes the drawbacks of CPCs like high resorbability, poor mechanical properties, and degradability. In this study, methylcellulose (MC) was combined with CPCs because of MC's thermoresponsive behavior which makes MC suitable for IBS application. In addition, gelatin (GEL) was also incorporated to adjust the gelation temperature and to enhance cell adhesion. These polymers combination makes the liquid (L) phase. The powder (P) phase comprised of tetra calcium phosphate (TTCP), dicalcium phosphate dehydrates (DCPD), and calcium sulfate dehydrates (CSD). TTCP and DCPD are commonly studied for the development of bone cements and they lead to high-density products. CSD was added to the powder phase to increase the porosity as well as to enhance mechanical properties of the IBS. TTCP was synthesized using a solid state method. Test tube inversion method was used to adjust the gelation temperature. GEL concentration was kept constant at 5 wt% and MC concentration varied between 1.5 and 12 wt%. The weight fraction of P/L phase was used as 1.8:1 (wt/wt). Synthesized IBS was characterized by using X-Ray Diffraction (XRD), Fourier Transform Infrared Analysis (FTIR), Zeta Particle Size Analysis, rheometry, and thermogravimetric analysis. XRD and FTIR analysis proved that TTCP was successfully synthesized with a particle size of 430.1 nm. The particle size of P phase mixture was measured as 581.1 nm. Based on the test tube inversion tests, weight fraction of MC was chosen as 10 and 12 while the weight fraction of GEL was fixed as 5. FTIR spectra of the liquid phase was showed that there was a hydrophilic interaction between MC and GEL since both Amide I at 1633 cm⁻¹ and β-gylcosides bonds among saccharide units at 900–1230 cm⁻¹ were clearly seen. MC10GEL5/P and MC12GEL5/P were analyzed by the XRD. According to this analysis, only the peaks of TTCP, DCPD, and CSD were observed. From the rheological data obtained from the rheometer, it is evident that all the prepared formulations exhibited Newtonian flow. The measured viscosity of all the investigated formula remained constant with the applied force over time. The MC12GEL5/P had the highest viscosity value due to its high concentration of MC (12% w/v). Results of TG of the synthesized IBS showed two main decomposition steps for the L phase because of the hydrophilic interaction between MC and GEL. The synthesized self-crosslinkable IBS represent promising platforms for future studies in bone tissue engineering. Overall, the presented study identified a novel IBS with suitable viscoelastic properties for non-invasive treatment of bone defects which may ultimately be a substitute for surgery for a wide variety of therapeutic applications.

The intervertebral disc faces high compressive forces during daily activities. Axial compression induces creeping fluid loss and reduction in disc height. With degeneration, disc fluids and height are progressively lost, altering biomechanics. It is assumed that this loss of fluids is caused by a drop in osmolality in the disc due to proteoglycan depletion. Here we investigate the isolated effect of a reduction in osmosis on the biomechanical properties of the intervertebral disc. Continuous diurnal loading was applied to healthy caprine intervertebral discs in a loaded disc culture system for a total of 6 days. We increased testing bath osmolality with two doses of polyethylene-glycol (PEG), thereby reducing the osmotic gradient between the disc and the surrounding fluid. This way we could study the isolated effect of reduced osmosis on axial creep, without damaging the disc. We evaluated: daily creep and recovery, recovery time-constants and compressive stiffness. Additionally, we investigated water content. There was a strong dose-dependent effect of PEG concentration on water content and axial creep behaviour: disc height, amplitude and rate of creep and recovery were all significantly reduced. Axial compressive stiffness of the disc was not affected. Reduction of water content and amplitude of creep and recovery showed similarity to degenerative disc biomechanics. However, the time-constants increased, indicating that the hydraulic permeability was reduced, in contrast to what happens with degeneration. This suggests that besides the osmotic gradient, the permeability of the tissues determines healthy intervertebral disc biomechanics.

Cervical spine fractures are frequent in impact sports, such as rugby union. The consequences of these fractures can be devastating as they can lead to paraplegia, tetraplegia and death. Many studies have been conducted to understand the injury mechanisms but the relationship between player cervical spine posture and fracture pattern is still unclear. The aim of this study was to evaluate the influence of player cervical spine posture on fracture pattern due to an impact load. Nineteen porcine cervical spines (C2 to C6) were dissected, potted in PMMA bone cement and mounted in a custom made rig. They were impacted with a mean load of 6 kN. Eight specimens were tested in an axial position, five in flexion and six in lateral bending. All specimens were micro-CT imaged (Nikon XT225 ST Scanner, Nikon Metrology, UK) before and after the tests, and the images were used to assess the fracture patterns. The injuries were classified according to Allen-Ferguson classification system by three independent observers. The preliminary results showed that the main fracture modalities were consistent with those seen clinically. The main fractures for the axial orientation were observed in C5-C6 level with fractures on the articular process and endplates. These findings support the concept that the fracture patterns are related to the spine position and give an insight for improvements on sports rules in order to reduce the risk of injury.

With around 20–40% of our bodyweight, skeletal muscles are the biggest organ complex of the human body. Being a metabolic active tissue, muscle mass, function and fibertype composition is highly regulated in a tight spatial-temporal manner. In geriatric patients, it is essentially important to understand the underlying mechanisms of the age related losses of fiber size and total number of fibers, as well as fibertype shifting.

To date, there have been few studies dealing with gene expression profiling of skeletal muscles, mostly focusing on age related differences in whole-muscle specimen. Being carried out on mouse or rat limb muscles, most other studies do not represent the conditions of human muscle, due to the differences in fibertype composition. Our study provides a fibertype-specific approach for whole-genome expression analysis in human skeletal muscle.

22 fresh frozen biceps brachii and quadriceps femoris muscle samples were acquired from the muscle bank of the Friedrich-Baur-Institut, Department of Neurology, Ludwig-Maximilians-University, Munich, Germany. Consecutive cross-sections were used for immunohistochemical myosine-heavy-chain-staining and individual fibers were acquired by laser-capture-microdissection. Around 100 cells of each fibertype of each biopsy were dissected, reversely transcribed, pre-amplified and labeled for microarray analysis. Fiber type-specific gene expression was analyzed with ANOVA. Correction for multiple testing was performed using the Benjamini-Hochberg procedure with a conservative threshold and the pathway analysis was carried out using the Ingenuity Pathway Analysis program (QIAGEN).

By comparing type I vs. type IIa, type I vs. type IIx and type IIa vs. type IIx, we could identify 2855, 2865 and 510 differentially expressed genes. As expected, many differentially regulated genes belong to functional groups like cytoskeleton, muscle contraction and energy metabolism, proving the feasibility of our study. However, many genes that are involved in the response to oxidative stress were also differently regulated, showing distinct mechanisms of the different fiber types, of coping with oxidative stress. In consensus with available literature, the relative proportion of type I fibers seemed to increase with age. Despite higher levels of oxidative stress, type I fibers seem to have more efficient antioxidative mechanisms in comparison to type IIa and IIx fibers, which might explain the higher vulnerability of members of the type II family to oxidative stress. Furthermore, genes that are involved in fibertype specification were also regulated differently. However, we could not verify an age-specific activation of pathways involved in fibertype shifting. Whether fibertype shifting is solely due to disproportionate loss of type II fibers, or also in vivo - transdifferentiation of fibers, has to be investigated further.

Worldwide, osteoporosis, causes more than 8.9 million fractures annually, resulting in an osteoporotic fracture every 3 seconds, where 1 in every 3 women and 1 in every 5 men aged over 50 will experience osteoporotic fractures at least once in their lifetime. Vertebral fractures, estimated at 1.4 million/year are among the most common fractures, posing enormous health and socioeconomic challenges to the individual and society at large. Considering that the great majority of individuals at high risk (up to 80%), who have already had at least one osteoporotic fracture, are neither identified nor treated, prediction of the risk factors for vertebral fractures can be of great value for prevention/early diagnosis. Recent studies show that finite element analysis of computed tomography (CT) scans provides noninvasive means to assess fracture risk and has the potential to be clinically implemented upon proper validation. The objective of this study was to develop a voxel-based finite element model using quantitative computed tomography (QCT) images in conjunction with in-vitro experiments to evaluate the strength of the vertebral bodies and predict the fracture risk criteria. A total of 10 vertebrae were dissected from juvenile sheep lumbar spines. The attached soft tissues and posterior elements and facet joints were completely removed, and the upper and lower vertebral bodies were polished using glass paper to provide smooth surfaces. The specimens were wrapped in phosphate buffer saline (PBS) soaked gauze, sealed in plastic bags, and stored in a refrigerator at −22°C. QCT scans of the specimens were captured using a bone density calibration phantom (QRM Co., Moehrendorf, Germany) with three 18 mm cylindrical inserts, providing 0, 100 and 200 mg HA/ccm, respectively. All the specimens, preserved hydrated in PBS solution, were mechanically tested at room temperature using a mechanical testing apparatus (Zwick/Roell, Ulm-Germany). The QCT images were then used to reconstruct the voxel-based FE model employing a custom-developed heterogeneous material mapping code. Five different equations for the correlation of the density and the elastic modulus were used to validate the efficiency of the FE model as compared to the in-vitro experiments. The results of the voxel-based FE models matched well with the in-vitro experiments, with an average error of 11.38 (±4.09)% based on the power law equation. A failure criterion was embedded in the FE models and the initiation of fracture was successfully predicted for all specimens. Further, typical kyphoplasty treatment was simulated in the 5 models to evaluate the application of the validated algorithm in the estimation of the failure patterns. Our novel voxel-based FE model can be used in future studies to predict the outcome of different types of therapeutic modalities/surgeries and estimate fracture risk including postoperative fractures.

Osteoarthritis is a degenerative disease mainly caused by aging, although in younger patients (aged 25 – 50) it can be a consequence of sports-related injuries or trauma. This results in early osteoarthritis with subsequent changes in cartilage extracellular matrix. Cell-based tissue engineering approaches using mesenchymal stem cells (MSCs) are an ideal cell type for the treatment of early osteoarthritc defects. Our group has demonstrated in a clinical study, that interleukin-1β (IL-1β) was expressed in cartilage plugs from patients with early osteoarthritis. In vitro studies have shown that IL-1β inhibits cartilage formation in chondrocytes or MSCs undergoing chondrogenesis. However, these studies show complete inhibition of tissue formation, whereas in the context of early osteoarthritis, cartilage extracellular matrix remains around the defect site. Thus, the present study sought to develop a model mimicking early osteoarthritis using MSCs.

Cartilage injury is generally associated with cytokine release and accumulation of reactive oxygen species. These mediators trigger pathologic behaviour of the surviving chondrocytes, which respond by excessive expression of catabolic enzymes, such as matrix metalloproteinase 13 (MMP-13), reduced synthesis of type II collagen (COL2A1) and apoptosis. In the long run, these pathologic conditions can cause a posttraumatic osteoarthritis. With the objective to attenuate the progressive degradation of the extracellular matrix and, what is more, promote chondroanabolic processes, a multidirectional treatment of trauma-induced pathogenesis was tested for the first time. Therefore, we evaluated the combinations of one anabolic growth factor (IGF-1, FGF18 or BMP7) with the antioxidant N-acetyl cysteine (NAC) in a human ex vivo cartilage trauma model and compared the findings with the corresponding monotherapy. Human cartilage tissue was obtained with informed consent from donors undergoing knee joint replacement (n=24). Only macroscopically intact tissue was used to prepare explants. Cartilage explants were subjected to a blunt impact (0.59 J) by a drop-tower and treated by IGF-1 [100 ng/mL], FGF18 [200 ng/mL] or BMP7 [100 ng/mL] and/or NAC [2 mM] for 7 days. Following parameters were analysed: cell viability (live/dead staining), gene expression (qRT-PCR) as well as biosynthesis (ELISA) of type II collagen and MMP-13. For statistical analysisKruskal-Wallis or One-way ANOVA was used. All data were collected in the orthopedic research laboratory of the University of Ulm, Germany.

Trauma-induced cell death was completely prevented by NAC treatment and FGF18 or BMP7 to a large extent, respectively (p<0.0001). IGF-1 exhibited only poor cell protection. Combination of NAC and FGF18 or BMP7 did not result in enhanced effectiveness; however, IGF-1 significantly reduced NAC-mediated cell protection. While IGF-1 or BMP7 induced collagen type II gene expression (p=0.0069 and p<0.0001, respectively) and its biosynthesis (p<0.0001 and p=0.0131, respectively), NAC or FGF18 caused significant suppression of this matrix component (each p<0.001). Although COL2A1 mRNA was significantly increased by NAC plus IGF-1 (p<0.0001), biosynthesis of collagen type II was generally abolished after multidirectional treatment. Except for IGF-1, all tested therapeutics exhibited chondroprotective qualities, as demonstrated by attenuated MMP-13 expression and breakdown of type II collagen. In combination with IGF-1, NAC-mediated chondroprotection was reduced.

Overall, both chondroanabolic and antioxidative therapy had individual advantages. Since adverse interactions were found by simultaneous application of the therapeutics, a sequential approach might improve the efficacy. In support of this strategy current experiments showed that though cell and chondroprotective effects of NAC were maintained after withdrawal of the antioxidant, type II collagen expression recovered by time.

To overcome the severely limited regenerative capacity of cartilage, bone marrow mesenchymal stromal cells (MSCs) are an attractive cell source that is accessible less invasively and in higher quantity than articular chondrocytes (ACs). However, current in vitro chondrogenic protocols induce MSCs to form transient cartilage reminiscent of growth plate cartilage that becomes hypertrophic and is remodeled into bone. In contrast, under the same conditions, ACs form stable articular-like cartilage. Developmental studies in mice have revealed that TGF-beta/BMP, Wnt, and Hedghog/PTHrP signaling are the major regulators of both, articular cartilage and endochondral bone formation. While the differential regulation of TGF-beta/BMP and Hedgehog/PTHrP in endochondral MSC versus AC chondral differentiation is established knowledge, little is known about Wnt in these cells. Aim of this study was therefore to compare in vitro levels of Wnt network components in MSC-derived endochondral versus AC-derived articular cartilage.

Whole genome expression data comparing human MSCs and ACs at days 0 and 28 of in vitro chondrogenesis were screened for differential expression of Wnt ligands, receptors, co-receptors, activators/inhibitors and signaling molecules. Expression of the most strongly differentially regulated Wnt network genes was studied in detail during in vitro chondrogenesis of MSCs vs ACs via qPCR at days 0, 7, 14, 21, 35, and 42.

During early chondrogenesis, most Wnt components were expressed at low levels in both MSCs and ACs, with two exceptions. MSCs started into chondrogenesis with significantly higher levels of the non-canonical ligand WNT5A. ACs on the other hand expressed significantly higher levels of the canonical antagonist FRZB on day 0. During advancing and late chondrogenesis, MSCs downregulated WNT5A but still expressed it at significantly higher levels at day 42 than ACs. Strong regulation was also evident for WNT11 and the receptor PTK7 which were both strongly upregulated in MSCs. Unlike MSCs, ACs barely regulated these non-canonical Wnt genes. With regard to canonical signaling, only the transcription factor LEF1 showed strong upregulation in MSCs, while FZD9 and FRZB were only slightly upregulated in late MSC chondrogenesis. Again, these genes remained unregulated in ACs.

Our data suggest that a dynamic Wnt network regulation may be a unique characteristic of endochondral MSC differentiation while during AC chondral differentiation Wnt expression remained rather low and stable. Overall, mRNA of the non-canonical Wnt network components were stronger regulated than canonical factors which may indicate that primarily non-canonical signaling is dynamic in endochondral differentiation. Next step is to assess levels of active and total beta-catenin, the canonical Wnt mediator, and to use Wnt antagonists to establish a causal relationship between Wnt signaling and endochondral differentiation.

Dura mater is a thick membrane that is the outermost of the three layers of the meninges that surround the brain and spinal cord. Appropriate dural healing is crucial to prevent cerebrospinal fluid leaks but the entire process has been barely understood so far. Understanding of dural healing and tissue neoformation over the dural grafts, which are usually used for duraplasty, is still partial. Therefore, implantation of decellular dura mater (DM) to recipient from different donor and vitalization with recipient”s mesenchymal stem cells for the treatment of tissue on transplantation process is significant approach. This approach prevents immunological reactions and provides long-term stabilization. According to this study, it is believed that this approach will provide DM healing and become crucial in DM transplantation.

The aim of this study was to develop a new construct by tissue engineering of the human DM based on a decellular allograft. Thus human DM collected from forensic medicine and decellularized using the detergent sodium dodecyl sulfate (SDS) in the multiple process of physical, enzimatic and chemical steps. Decellularization were exposing the tissue to freeze-thaw cycles, incubation in hypotonic tris-HCl buffer, 0.1% (w/v) SDS in hypotonic buffer and hypertonic buffer followed by disinfection using 0.1% (v/v) peracetic acid and final washing in phosphate-buffered saline. As a result of all these processes, cellular components of DM were removed by preserving the extracellular matrix without any significant loss in mechanical properties. Based on the histological analysis of the decellularized DM revealed the absence of visible whole cells. Collagen and glycosaminoglycan (GAG) contents of decellular DM evaluated histological staining by Masson Trichrome and Alcian blue respectively. Also biochemical tests were carried out by spectrophotometry (Quickzym Biosciences, The Netherlands) and total GAG content were analyzed by 1.9 dimethylmethylene blue assay. The histoarchitecture was unchanged, and there were no significant changes of total collagen and GAG content. Biomechanical properties were determined by tensile tests, which has confirmed the retention of biomechanical properties following decellularization. The mean tensile strengths were 7,424±4,20 MPa for control group, 5,254±2,068 MPa for decellularization group. There was no statistically significant difference between tensile strength (p=0,277) and tissue thickness (p=0, 520) for both group.

In conclusion, this study has developed biomechanically functional decellularized DM scaffold for use in DM repair. In addition, this study is a part of the progressing study and additional studies investigating the biocompatibility performance of the decellularized DM scaffold and there is need for in vivo studies.

The biomechanical evaluation of tendon repair with collagen-based scaffolds in rat model is a common method to determine the functional outcome of the tested material. We introduced a magnetic resonance imaging (MRI) approach to verify the biomechanical test data. In present study different collagen scaffolds for tendon repair were examined.

Two collagen test materials: based on bovine stabilized collagen, chemically cross-linked with oriented collagenous fibres (material 1) and based on porcine dermal extracellular matrix, with no cross-linking (material 2) were compared. The animal study was approved by the local review board. Surgery was performed on male Sprague-Dawley rats with a body weight of 400 ± 19 g. Each rat underwent a 5 mm transection of the right Achilles tendon. The M. plantaris tendon was removed. The remaining tendon ends were re-joined with a 5 mm scaffold of either the material 1 or 2. Each scaffold material was sutured into place with two single stiches (Vicryl 4–0, Ethicon) each end. A total of 16 rats (n= 8 each group) were observed for 28 days follow up. The animals were sacrificed and hind limbs were transected proximal to the knee joint. MRI was performed using a 7 Tesla scanner (BioSpec 70/30, Bruker). T2-weighted TurboRARE sequences with an in-plane resolution of 0.12 mm and a slice thickness of 0.7 mm were analysed. All soft and hard tissues were removed from the Achilles tendon-calcaneus-foot complex before biomechanical testing. Subsequently, the specimens were fixed in a materials testing machine (Z1.0, Zwick, Ulm, Germany) for tensile testing. All tendons were preloaded with 1 N and subsequently stretched at a rate of 1 mm/s until complete failure was observed. Non-operated tendons were used as a control (n=4).

After 28 postoperative days, MRI demonstrated that four scaffolds (material 1: n=2, material 2: n=2) were slightly dislocated in the proximal part of hind limb. In total five failures of reconstruction could be detected in the tendon repairs (material 1: n=3, material 2: n=2). Tendons augmented with the bovine material 1 showed a maximum tensile load of 57.9 ± 17.9 N and tendons with porcine scaffold material 2 of 63.1 ± 19.5 N. The native tendons demonstrated only slightly higher loads of 76.6 ± 11.6 N. Maximum failure load of the tendon-scaffold construct in both groups did not differ significantly (p < 0.05). Stiffness of the tendons treated with the bovine scaffold (9.9 ± 3.6 N/mm) and with the porcine scaffold (10.7 ± 2.7 N/mm) showed no differences. Stiffness of the native healthy tendon of the contralateral site was significantly higher (20.2 ± 6.6 N/mm, p < 0.05). No differences in the mechanical properties between samples of both scaffold groups could be detected, regardless of whether the repaired tendon defect has failed or the scaffold has been dislocated.

The results show that MRI is important as an auxiliary tool to verify the biomechanical outcome of tendon repair in animal models.

Evaluation of different biomaterials is being performed with various methods trying to simulate the closest hostile-like in vitro environments. However the complexity of the conditions usually limits practically feasible combination of most relevant chemical, biological, biomechanical parameters in one single test. Many biomaterials and tissue engineering developments rely on high-throughput screening to multiply number of specimens and thus to gather sufficient data. The price to be paid for these methods is limited number of physical readouts, increased inter-specimens scatter, and unavoidable spatial constrains driving the conditions away of the clinical scenarios. For orthopaedic biomaterials this is of a particular concern, as implantation site conditions cannot be squeezed too much without lost of natural-mimicking stimuli.

Here we are presenting another approach based on high-output screening of biomaterials, which is based on the strategy of raising the number of readouts obtainable from every specimen at more clinically-relevant conditions. On the contrary to common methods like ISO 10993 or simplified biomechanical tests, the biomaterials enhanced simulation testing (BEST) evaluates specimens without pre-selected biomaterial model, assessing the whole specimen as would happen in the implantation site. Besides reducing the risk of improper conclusions caused by wrong material model choice, the data processing with non-local method intrinsically includes the test history bypassing common challenges usually seen with hereditary integration. For properly designed experiment, readouts might include invariant moduli, viscous stiffness, fluidity, fluid permittivity and diffusivity (without need for pressure-driven separate tests), fluid source, effective channel size, and swelling pressure (if swelling is present) in addition to conventional biomechanical parameters.

New solutions in advanced and consistent evaluations for biomaterials allow better risks control, shorten lead development time and costs, and compliant with 3R-strategy (2010/63/EC) and new regulatory requirements (2012/0266/COD in EU and FY2017 regulatory priorities by FDA). The approach shown is able to combine scientifically based tests with multi-purpose protocols to secure patient safety by screening of biomaterials under proper conditions.

The authors thank Finnish Agency for Innovations (Tekes) for providing partial financial support.

To date there has been no material for endoprosthetics providing excellent resistance to abrasion and corrosion combined with great tensile strength, fracture toughness, and bending strength, as well as adequate biocompatibility. Carbon-fiber-reinforced silicon carbide (C/SiC, C/C-SiC or C/SiSiC) is as a ceramic compound a potentially novel biomaterial offering higher ductility and durability than comparable oxide ceramics.

Aim of this investigation was to test the suitability of C/SiC ceramics as a new material for bearing couples in endoprosthetics. One essential quality that any new material must possess is biocompatibility. For this project the in-vitro biocompatibility was investigated by using cuboid like scaffolds made of CMC. To determine whether the material is suited as a lubricant partner in endoprosthetics, we measured its abrasion coefficient and wear tolerance against various antibodies. The C/SiC samples tested were produced via the Liquid Silicon Infiltration (LSI) of pyrolized porous fiber preforms made by warm-flow pressing free-flowing granulates on a hydraulic downstroking press with a heated die of the type HPS-S, 1000 kN. After preparation of the composites, the tribological characteristics are determined. Flexural strength was determined at room temperature according to DIN685-3 with an universal testing machine Z100 and the Young”s -modulus was carried out via resonant frequency-damping analysis RFDA. The samples”surface as well as cell adhesion and cell morphology were assessed via ESEM. The human osteoblast-like cell line MG-63 and human ostoeblast were used for cel culture ecperiments (WST, Live/dead, Cytotoxicity, cell morphology). Based on the raw data the mean value and the standard deviation were calculated. The Mann-Whitney-U-Test was used to evaluate the differences between experiment and control samples. The flexural strength at room temperature is approx. 180 MPa, while the elongation at break is about 0.13%. The Young”s modulus is detected between 120 and 150 GPa. The density lies between 2.5 and 3.0 g/cm³. We noted a friction coefficient µ between 0.31. The cell lines exhibited no morphological alterations, and adhered well to the C/SiC samples. Vitality was not impaired by contact with the ceramic composite. Cell growth was observed evenly distributed over a 21-day period. In the future, investigators aiming to apply this composite in endoprosthetics will have to focus on its efficacy in conjunction with sudden, strong demands, and long-term performance in bodily fluids within joint simulators, etc. In conclusion: C/SiC can definitely be considered a new material with genuine potential for use in endoprosthetics.

Nowadays, biomaterials can be used to maintain or replace several functions of the human body being constricted or lost due to tumors, fractures, injuries as well as chronic diseases, infections or simply aging. Titanium and its alloys, i.e. Ti6Al4V are the most common materials (70 to 80%) used for structural orthopedic implants due to their unique combination of good mechanical properties, corrosion resistance and biocompatibility. Addition of β-stabilizers, e. g. niobium (Nb), can improve the mechanical properties of such titanium alloys further, simultaneously offering excellent biocompatibility. Previous studies concerning biocompatibility analyses with niobium and especially Ti-42Nb specimens are rarely described; none for niobium and Ti-42Nb powders examining human cell viability, collagen and interleukin synthesis. In this in vitro study, human osteoblasts were cultured on different roughened niobium specimens (Nb Amperit, Nb Ampertec), Nb sheets and spherical Ti-42Nb (sintered and 3D-printed by selective laser melting, SLM) and compared with forged Ti6Al4V specimens. Furthermore, human osteoblasts were incubated with particulates of the Nb and Ti-42Nb specimens in three particle concentrations over four and seven days to imitate influence of wear debris against the background of osteolysis and aseptic implant loosening. Thereby, the specimens with the roughest surfaces, i.e. Ti-42Nb and Nb Ampertec, revealed excellent and similar results concerning cell viability (WST-1 test, live-dead staining) and collagen-I synthesis superior to forged Ti6Al4V. Examinations with particulate debris disclosed a significant dose-dependent influence of all powders with Nb Ampertec showing the highest decrease of cell viability and collagen-I synthesis. Furthermore, interleukin expression was only slightly increased for all powders. In summary, from a cell-biological point of view Nb Ampertec (sintered Nb) and Ti-42Nb materials seem to be superior alternatives for medical applications compared to common materials like forged Ti6Al4V.

Total Hip Arthroplasty (THA) is a well-established, cost-effective treatment for improving function and alleviating pain in patients who have disabling hip disease with excellent long-term results. Based on the excellent results, there is an ongoing trend for THA to be performed in younger and more active patients, having higher physical demands on their new total joints.

Polyethylene (PE) wear and its biological consequences are one of the main causes of implant failure in THA. Macrophages phagocytise PE wear particles and this will result in osteolysis and loss of periprosthetic bone. The risk of these complications can be estimated in relation to the amount of volumetric wear based on two assumptions: that the number of PE particles dispersed in the peri-prosthetic tissues is controlled by the amount of PE wear; and that the development of osteolysis and the resulting aseptic loosening is triggered by these PE particles. Based on these assumptions, a model was developed to estimate the osteolysis-free life of a THA, depending on the Linear Wear Rate (LWR) and femoral head size of the PE bearing.

A review of the literature was conducted to provide an estimate of the radiologic osteolysis threshold based on the volumetric wear of the PE bearing. This review demonstrates that this radiologic osteolysis threshold is approximated 670 mm3 for conventional PE. The osteolysis-free life of the THA was estimated by simply dividing this threshold volume by the annual Volumetric Wear Rate (VWR) of the bearing. The annual VWR is basically controlled by two parameters: (1) annual LWR and (2) head size, and was calculated by using published formulae.

For 28 mm heads, following osteolysis-free life was determined in function of the annual LWR. LWR: 10 µm/y => 116.6 years / LWR: 25 µm/y => 46.6 years / LWR: 50 µm/y => 23.3 years / LWR: 100 µm/y => 11.6 years. For 40 mm heads, following osteolysis-free life was determined in function of the annual LWR. LWR: 10 µm/y => 57.1 years / LWR: 25 µm/y => 22.9 years / LWR: 50 µm/y => 11.4 years / LWR: 100 µm/y => 5.7 years.

The osteolysis-free life determined by this model is in good agreement with the clinical results of PE bearings having a 28 mm head size and demonstrates that extreme low LWRs are mandatory to assure a descent osteolysis-free life for THA (PE bearings) using large heads, such as 40 mm. For such head sizes, small variations of the LWR may have large impacts on the osteolysis-free life of the THA.

Successful reconstruction of bone defects requires an adequate filling material that supports regeneration and formation of new bone within the treated defect in an optimal fashion. Currently available synthetic bone graft substitutes cannot fulfill all requirements of the highly complex biological processes involved in physiological bone healing. Due their unphysiologically asynchronous biodegradation properties, their specific foreign material-mediated side effects and complications and their relatively modest overall osteogenic potential, their overall clinical performance typically lags behind conventional bone grafts of human origin. However, defect- and pathology specific combination of synthetic bone graft substitutes exhibiting appropriate carrier properties with therapeutic agents and/or conventional bone graft materials allows creation of biologically enhanced composite constructs that can surpass the biological and therapeutic limits even of autologous bone grafts. This presentation introduces a bone defect reconstruction concept based on biological enhancement of optimal therapeutic agent-carrier composites and provides a rationale for an individual, requirement-specific adaptation of a truly patient-specific reconstruction of bone defects. It represents the pinnacle of the bone defect reconstruction pyramid, founded on the basic principles and prerequisites of complete elimination of the underlying pathology, preservation, augmentation or restoration of mechanical stability of the treated bone segment and creation of a biodegradable scaffold with adequate mechanical integrity. It summarises the current body of relevant experimental and clinical research, presents clinical case examples illustrating the various aspects of the proposed concept as well as early clinical results. The author hopes that the theoretical and conceptual framework provided, will help guide future research as well as clinical decision making with respect to this particular field.

Achilles tendon (AT) rupture may lead to complaints of heel pain. In forefoot ulcer patients AT lengthening is used to transfer pressure from forefoot to the heel. The primary aim was to investigate if AT was longer or associated with changes in pedobaric measurements, in particular heel pressure, on the injured leg 4–5 years after the injury.

Confirming clinical evidence, we recently demonstrated in a rodent model that a severe trauma which induces an acute systemic inflammation considerably impairs fracture healing. Interleukin-6 (IL-6) is a key cytokine in posttraumatic inflammation as its serum level correlates with injury severity and mortality. IL-6 signals are transmitted by the transmembrane glycoprotein 130 (gp130) via two distinct mechanisms: firstly, through classic signalling via the membrane-anchored IL-6 receptor and secondly, through trans-signalling using a soluble IL-6 receptor. Whereas IL-6 trans-signalling is considered a danger signal driving inflammation, classic signalling may mediate anti-inflammatory, pro-regenerative processes. The role of the two distinct pathways in bone healing has not yet been elucidated. Here, we studied the function of IL-6 in the pathophysiology of compromised bone healing induced by severe trauma.

Male C57BL/6J mice received an osteotomy of the right femur stabilized with an external fixator. Systemic inflammation was induced by additional blunt chest trauma (TxT) applied immediately after the osteotomy. Mice were injected with either fusion protein sgp130Fc, which selectively inhibits IL-6 trans-signalling, or a neutralizing anti-IL-6 antibody (IL-6 Ab), blocking both signalling pathways. Control mice received vehicle solution. Animals were euthanised 21 days after surgery. Fracture healing was analysed by biomechanical testing, μCT, and histomorphometry (n= 6–9; p=0.05; ANOVA/Fisher LSD post hoc).

Thoracic trauma significantly impaired fracture healing [bending stiffness (EI) −57%, p<0.00]. Treatment with sgp130Fc significantly attenuated bone regeneration as demonstrated by an increased EI (+110%, p<0.00) and a trend of augmented apparent Young”s modulus (+69%, p=0.13) compared to TxT control. Histomorphometric analysis could not detect differences in the amount of bone, confirming µCT results, but revealed a significantly decreased cartilage area after treatment with sgp130Fc (−76%, p=0.01). Inhibition of both signalling pathways with IL-6 Ab, however, did not have any effects.

In conclusion, severe trauma significantly impaired fracture healing, confirming previous studies. Treatment with sgp130Fc ameliorated the negative effects providing evidence that IL-6 trans-signalling triggers the excessive immune response after trauma impairing bone regeneration. Injection of IL-6 Ab did not improve fracture healing thereby implying that classic signalling may rather have beneficial effects.

Osteoarthritis (OA) affects millions of people and is the fastest growing cause of disability worldwide. In order to address this burden, early intervention strategies have been proposed. Therapies that utilise bone marrow stromal cells (BM-MSCs) to induce cartilage repair, either as a cell therapy or by endogenous release by drilling or microfracture, have proved promising. However, limitations include fibrotic features of the regenerated cartilage that may affect mechanical properties and therefore the longevity of such a repair. In order to improve this regenerative technique, further research is required to understand the key players in the repair mechanism. An interaction, which may be important, is that between BM-MSCs and the resident chondrocytes. The aim of this study is to understand the interplay between BM-MSC and resident chondrocytesisolated from different zonal locations within the human knee.

We compared chondrocytes from three different cartilage areas: chondrocytes from 1) the superficial zone (SZ) and 2) the middle-deep (MDZ) zone of non-weight bearing femoral condyles, and from 3) the osteoarthritic zone (OAZ) of patients undergoing knee replacement. First, we evaluated the influence of different chondrocytes on BM-MSCs monolayer in a transwell co-culture, assessing transcript levels of early chondrogenic markers including Sox9 and Col1. Secondly, in a 3D co-culture system, we evaluated how cartilage chips from the three different zones affect the chondrogenic differentiation of BM-MSC pellets. Results indicated that cells from the SZ induce chondrogenic differentiation of BM-MSCs when co-cultured. In contrast, MDZ and OAZ have a negative effect, compared to control conditions. Our findings suggest that chondrocytes from the SZ, a zone which has been reported to reduce with age and may be lost in advanced OA, is important to direct BM-MSCs differentiation towards the chondrogenic fate. This may be relevant to cartilage repair strategies.

The aim of this study is to compare the release of titanium (Ti) and zirconium (Zr) into the tissue surrounding Ti- and ZrO₂-implants.

Methyl methacrylate embedded mini pig maxillae with 6 Ti-implants and 4 ZrO2-implants were analysed after 12-weeks of implantation. The spatial distribution of elemental Ti and Zr in maxillae near implants was assessed with laser ablation (LA)-inductively coupled plasma (ICP)-mass spectrometry (MS). From each maxilla two bone slices adjacent to the implants were measured. The contents of Ti and Zr in these bone slices were determined by ICP-MS and ICP-optical emission spectrometry.

Increased intensity of Ti and Zr could be detected in bone tissues at a distance of 891±398 µm (mean ± SD) from Ti-implants and 927±404 µm from ZrO2-implants. The increased intensity was mainly detected near implant screw threads. The average Ti content detected in 11 bone slices from samples with Ti-implants was 1.67 mg/kg, which is significantly higher than the Ti content detected in 8 slices from samples with ZrO₂-implants. The highest Ti content detected was 2.17 mg/kg. The average Zr content in 4 bone slices from samples with ZrO₂-implants is 0.59 mg/Kg, the other 4 bone slices showed Zr contents below the detection limit (

After 12-weeks of implantation, increased intensity of Ti and Zr can be detected in bone tissues near Ti- and ZrO₂-implants. The results show that Ti content released from Ti-implants is higher than the Zr content released from ZrO₂-implants.

As we grow older, the risk of tendon degeneration and injuries increases, which can result in pain, disability, healthcare cost, and lost productivity. Even after surgical repair the results are often unsatisfactory. The cellular reasons for the differences in the healing potential, however, are not well studied. To get a deeper insight into the biological characteristics of tenocyte-like cells from different patient groups we established a biobank with material from over 150 human donors. The patients/donors suffered from rotator cuff tears and were operated to restore the function. A proportion of the isolated cells showed stem cell-like characteristics and was able to differentiate into the osteoblastic, chondrogenic and adipogenic linage. Investigating the differentiation potential of the cells with regard to donor characteristics, we were able to demonstrate that age, sex but also the “degeneration” has an impact of the cellular potential. A possibility to stimulate the cellular activity is the application of growth factors, as already clinically used for stimulation of bone healing. Therefore, the responsiveness of the cells to the growth factors Bone Morphogenetic protein-2/7 (BMP-2/7) was analysed in vitro. Independent of the donor characteristics, the cells responded to the BMP-stimulation by increased proliferation and collagen-1 synthesis. However, cells isolated from donors with high fatty infiltration of the muscle or older females were less responsive. Looking into the intracellular signalling pathway, the data showed that the BMP-signal is mainly mediated by the canonical-pathway with samd8 playing a major role. This basic research gives first information regarding the differences in tenocytes biology with respect to the donor and is important for the understanding of tendon regeneration and the future development of new treatment strategies.

Tendon injuries are a worldwide problem affecting several age groups and stem cell based therapies hold potential for tendon strategies guiding tendon regeneration.

Tendons rely on mechano-sensing mechanisms that regulate homeostasis and influence regeneration. The mechanosensitive receptors available in cell membranes sense the external stimuli and initiate mechanotransduction processes. Activins are members of the TGF-β superfamily which participate in several tendon biological processes. It is envisioned that the activation of the activin receptor, trigger downstream Smad2/3 pathway thus regulating the transcription of tenogenic genes driving stem cell differentiation.

In this work, we propose to target the Activin receptor type IIA (ActRIIA) in human adipose stem cells (hASCs), inducing hASCs commitment towards the tenogenic lineage. Since mechanotransduction can be remotely triggered through magnetic actuation combined with magnetic nanoparticles (MNPs), we stimulated hASCs tagged complexes using a vertical oscillating magnetic bioreactor (MICA Biosystems Ltd). Carboxyl functionalised MNPs (Micromod) were coated with anti-ActRIIA antibody (Abcam) by carbodiimide activation. hASCs were then cultured with MNPs-anti-ActRIIA for 14days with or without magnetic exposure (1Hz, 1h/every other day). hASCs cultured alone in αMEM (negative control) or in αMEM supplemented with ActivinA (R&D systems) (positive control of ActRIIA activation) were used as experimental controls. The tenogenic commitment of hASCs was assessed by real time RT-PCR, immunocytochemistry and quantification of collagen and non-collagenous proteins. Moreover, the phosphorylation of Smad2/3 was also evaluated on hASCs incubated for 2, 10, or 30min under magnetic stimulated (1Hz) and non-stimulated conditions.

The increased gene expression of tendon related markers and higher ECM proteins deposition suggests that remote magnetic activation of ActRIIA promotes effectively hASCs tenogenic commitment. Furthermore, the detection of phospho-Smad2/3 proteins by ELISA (Cell Signaling Technology) was significantly more intense after 10min in hASCs under magnetic stimulation and in comparison to the control groups. These outcomes suggest that ActRIIA is a mechanosensitive receptor that can be remotely activated upon magnetic stimulation.

In conclusion, remotely activation of MNPs tagged hASCs has potential for modulating tenogenic differentiation of stem cells envisioning successful cell therapies for tendon regeneration.

Cellular therapies play an important role in tendon tissue engineering and regenerative medicine with tenocytes being described as the most prominent cell population for these applications if available in large numbers. However, this is difficult to achieve, because in vitro expansion of tenocytes leads to phenotypic drift and loss of function. Recent work suggests that maintenance of tenogenic phenotype in vitro can be achieved by recapitulating different aspects of the native tendon microenvironment. One approach used to modulate in vitro microenvironment and enhance extracellular matrix (ECM) deposition is macromolecular crowding (MMC). MMC is based on the addition of inert macromolecules to the culture media to mimic the dense extracellular matrix and accelerate the production of ECM-rich substitutes. In addition, as tendon has been described to be a relatively avascular and hypoxic tissue and low oxygen tension can stimulate collagen synthesis and cross-linking through the activation of hypoxia-inducible factor 1-alpha (HIF1-α), we venture to assess the synergistic effect of MMC and low oxygen tension on human tenocyte phenotype maintenance by enhancing deposition of tissue-specific extracellular matrix.

SDS-PAGE and immunocytochemistry analysis, demonstrated that human tenocytes treated with the optimal MMC concentration at 2% oxygen tension showed increased collagen type I synthesis and deposition after 7 days. Moreover, immunocytochemistry for collagen type III, type V, VI, elastin and fibronectin illustrated enhanced deposition when cells were treated with MMC at 2% oxygen tension. In addition, it was shown that low oxygen tension and MMC did not affect the spindle-shape morphology, metabolic activity, proliferation and viability of human tenocytes Collectively, these results suggest that the synergistic effect of optimal macromolecular crowding concentration and low oxygen tension (2%) can accelerate the formation of ECM-rich substitutes, which may stimulate tenogenic phenotype maintenance. Further gene and protein analysis for tendon specific markers should be performed to validate our promising results.

The formation of postoperative adhesions poses a major complication in surgery, especially in the treatment of tendon, where adhesions can result in an alteration of the biomechanical and gliding properties, impeding a proper functioning of the tendon. Current treatments to prevent adhesions in the tendon are mainly based on the use of mechanical barriers which isolate the tendon and prevent fibrin deposition. Despite the positive results in preclinical models, these results have not been translated to clinics. Thus, in this study we propose a porcine peritoneum xenograft as an alternative antiadhesion barrier which integrates a basal membrane, since the presence of a basal membrane together with an epithelium or mesothelium layer prevents the formation of adhesions in vivo. First results have shown the suitability of the porcine peritoneum xenograft as an antiadhesion barrier due to its lower crosslinking ratio (p<0.05) and faster degradation by MMPs in vitro than a commercially available tendon product, which suggest a faster remodelling in vivo. On the other hand, the porcine peritoneum showed higher mechanical properties (p<0.01) and a lower coefficient of friction (p<0.01), characteristics that make the porcine peritoneum an appropriate material for tendon regeneration. Furthermore, the presence in the xenograft of a collagen type IV and laminin network after decellularisation was confirmed with immunohistochemistry, which poses the potential of the porcine peritoneum as antiadhesion device due to the presence of a basal membrane. Preliminary cell assessment experiments showed different morphology of adult dermal fibroblast (ADFs) on the different sides of the material (basal membrane and connective tissue) due to the differences in composition of both layers. Furthermore, the culture of ADFs during 7 days in media conditioned with the porcine peritoneum resulted in higher proliferation and metabolic activity (p<0.05) than those observed in the control and the media conditioned with the commercial product, suggesting the presence of growth factors in the porcine peritoneum which promote the growth of cells. Although positive results have been observed regarding the potential of porcine peritoneum as antiadhesion barrier for tendon regeneration, further studies which assess the influence of the basal membrane on cell behaviour and confirm the potential of the xenograft as antiadhesion barrier are being carried out.

Osteoarthritis (OA) is the most common degenerative joint disease causing joint immobility and chronic pain. Treatment is mainly based on alleviating pain and reducing disease progression. During OA progression the chondrocyte undergoes a hypertrophic switch in which extracellular matrix (ECM) -degrading enzymes are released, actively degrading the ECM. However, cell biological based therapies to slow down or reverse this katabolic phenotype are still to be developed. Bone morphogenetic protein 7 (BMP-7) has been shown to have OA disease-modifying properties. BMP-7 suppresses the chondrocyte hypertrophic and katabolic phenotype and may be the first biological treatment to target the chondrocyte phenotype in OA. However, intra-articular use of BMP-7 is at risk in the proteolytic and hydrolytic joint-environment. Weekly intra-articular injections are necessary to maintain biological activity, a frequency unacceptable for clinical use. Additionally, production of GMP-grade BMP-7 is challenging and expensive. To enable its clinical use, we sought for BMP-7 mimicking peptides better compatible with the joint-environment while still biologically active and which potentially can be incorporated in a drug-delivery system. We hypothesized that human BMP-7 derived peptides are able to mimic the disease modifying properties of the full-length human BMP-7 protein on the OA chondrocyte phenotype.

A BMP-7 peptide library was synthesized consisting of overlapping 20-mer peptides with 18 amino-acids overlap between sequential peptides. OA human articular chondrocytes (HACs) were isolated from OA cartilage from total knee arthroplasty (n=18 donors). HACs were exposed to BMP-7 (1 nM) or BMP-7 library peptides at different concentrations (1, 10, 100 or 1000 nM). Gene-expression levels of important chondrogenic-, hypertrophic-, cartilage degrading- and inflammatory mediators were determined by RT-qPCR. GAG and ALP activity were determined using a colorimetric assay and PGE levels were measured by EIA.

During the BMP-7 peptide library screening human BMP-7 derived peptides were screened for their full-length human BMP-7 mimicking properties at different concentrations (1, 10, 100 or 1000nM) on a pool of human chondrocytes. Gene expression as well as GAG, ALP and PGE2 level analysis revealed two distinct peptide regions in the BMP-7 protein based on their pro-chondrogenic and anti-OA phenotype actions on human OA chondrocytes. The two most promising peptides were further analysed for their OA chondrocyte disease modifying properties in the presence of OA synovial fluid, showing similar OA phenotype suppressive activity.

Conclusively, we successfully identified two peptide regions in the BMP-7 protein with in vitro OA suppressive actions. Further biochemical fine-tuning of the peptides, and in vivo evaluation, will potentially result in the first peptide-based experimental OA treatment, addressing the hypertrophic and katabolic chondrocyte phenotype in OA.

Designs of medical devices are tested for their mechanical behaviour, ability to transfer the load that is normally bore by the healthy tissue, and prove of the resistance to fatigue. The virtual testing in silico is widely accepted technique based on three sets of input data – geometry is normally obtained from CT or MRI scan as well as the tissue density that is translated into mechanical properties of the tissue. The virtual behaviour of the system is controlled by set of constrains accordingly while the third set of data consist of the load that system normally transfers through the load-bearing tissue. The magnitude and character of the load is highly dependent on the physical activity, external loads, physical condition of the subject and its specific factors such as gender, health condition, etc. Most of the published simulations use estimated simplified loads, which barely simulate the real behaviour of the system. The evaluation of the spinal load published some years back estimated a normal (N) and shear force (S) accompanied by the flexing moment (M). Due to lack of experimental possibility we used these data to test the biomechanical response of the lumbar segment with isotropic material models of all tissues.

Then we investigated the possibility to evaluate muscular forces and their recruitment. It is a complex task and even today it is not possible to measure directly in vivo all muscular forces contributing to the movement. The musculo-skeletal system is a statically indeterminate system. The forces can be solved by means of computational modelling based on the measured trajectories of the body motion and additional optimization functions combined with static equations. The trajectories were recorded by the fast camera system in our motion laboratory and consequently exported into an open simulation software that uses a generic skeleton with around two hundreds muscle fascicles. The skeleton was scaled to correspond to our subject's anthropometric data and further scaling to mock-up the generic vertebrae was performed to eliminate discrepancies between the generic and subject's bones. Once these adjustments were done a kinematics and inverse dynamics modules were engage with selected objective function controlling the muscular recruitment that the max. relative muscular force is as small as possible. The 84 muscular forces acting on the segment were exported to a text file in APDL language and uploaded in the Finite Element (FE) database.

The results of FE analysis were compared to the results obtained earlier using N,S,M load [1]. The comparison between the two models shows that the results of segment's total displacement was reduced by 36 percent compared to initial results. The stress and stress intensity increased six times. The identical model with orthotropic material showed reduced displacement by 80 percent and the stress and stress intensity was reduced by 60 percent compared to initial results.

Clinical investigations show that the cervical spine presents wide inter-individual variability, where its motion patterns and load sharing strongly depend on the anatomy. The magnitude and scope of cervical diseases, including disc degeneration, stenosis, and spondylolisthesis, constitute serious health and socioeconomic challenges that continue to increase along with the world”s growing aging population. Although complex exact finite element (FE) modeling is feasible and reliable for biomechanical studies, its clinical application has been limited as it is time-consuming and constrained to the input geometry, typically based on one or few subjects. The objective of this study was twofold: first to develop a validated parametric subject-specific FE model that automatically updates the geometry of the lower cervical spine based on different individuals; and second to investigate the motion patterns and biomechanics associated with typical cervical spine diseases. Six healthy volunteers participated in this study upon informed consent. 26 parameters were identified and measured for each vertebra in the lower cervical spine from Lateral and AP radiographs in neutral, flexion and extension viewpoints in the standing position. The lower cervical FE model was developed including the typical vertebrae (C3-C7), intervertebral discs, facet joints, and ligaments using ANSYS (PA, USA). In order to validate the FE model, the bottom surface of C7 was fixed, and a 73.6N preload together with a 1.8 N.m pure moment were input into the model in both flexion and extension. The results were compared to experimental studies from literature. Disc degeneration disease (DDD) was used as an example, where the geometry of C5-C6 disc was changed in the model to simulate 3 different grades of disc degeneration (mimicking grades 1 to 3), and the resulting biomechanical responses were evaluated. The average ranges of motion (ROM) were found to be 4.84 (±0.73) degrees and 5.36 (±0.68) degrees for flexion and extension for C5-C6 functional unit, respectively, in alignment with literature. The total ROM of the model with disc generation grades 2 and 3 was found to have decreased significantly as compared to the intact model. In contrast, the axial stresses on the degenerated discs were significantly higher than the intact discs for all 3 degeneration grades. Our preliminary results show that this novel validated subject-specific FE model provides a potential valuable tool for noninvasive time and cost effective analyses of cervical spine biomechanical (kinematic and kinetic) changes associated with various diseases. The model also provides an opportunity for clinicians to use quantitative data towards subject-specific informed therapy and surgical planning. Ongoing and future work includes expanding the studied population to investigate individuals with different cervical spine afflictions.

Rib fractures (RF) represent the most common bone fracture after blunt trauma, occurring in 10–20% of all trauma patients and leading to concomitant injuries of the inner organs in severe cases. However, a standardized classification system for serial rib fractures (SRF) does still not exist. Basic knowledge about the facture pattern of SRF would help to predict organ damage, support forensic medical examinations, and provide data for in vitro and in silico studies regarding the thoracic stability. The purpose of our study was therefore to identify specific SRF patterns after blunt chest trauma.

All SRF cases (≥3 subsequent RF) between mid-2008 and end of 2015 were extracted from the CT database of our University Hospital (n=383). Fractures were assigned to anterior, antero-lateral, lateral, postero-lateral, and posterior location within the transverse plane (36° each) using an angular measuring technique (reliability ±2°). Rib level, fracture type (transverse, oblique, multifragment, infracted), as well as degree of dislocation (none, </≥ rib width) were recorded and each related to the cause of accident.

In total, 3747 RF were identified (9.7 per patient, ranging from 3 (n=25) to 33 (n=1)). On average, most RF occurred in crush/burying injuries (15.9, n=13) and pedestrian accidents (12.2, n=14), least in car/truck accidents (8.8, n=76). Altogether, RF gradually increased from rib 1 (n=140) towards rib 5 (n=517) and then decreased towards rib 12 (n=49), showing a bell-shaped distribution. More RF were detected on the left thorax (n=2027) than on the right (n=1720). Overall, most RF were found in the lateral (33%) and postero-lateral (29%) segment. Posterior RF mostly occurred in the lower thorax (63%), whereas anterior (100%), antero-lateral (87%), and lateral (63%) RF mostly appeared in the upper thorax. RF were distributed symmetrically to the sagittal plane, showing a hotspot (up to 98 RF) at rib levels 4 to 7 in the lateral segment and rib level 5 in the antero-lateral segment. In the car/truck accident group, 47% of all RF were in the lateral segment, in case of frontal collision (n=24) even 60%. Fall injuries (n=141) entailed mostly postero-lateral RF (35%). In case of falls >3 m (n=45), 48% more RF were detected on the left thorax compared to the right. CPR related SRF (n=33) showed a distinct fracture pattern, since 70% of all RF were located antero-laterally. Infractions were the most observed fracture type (44%), followed by oblique (25%) and transverse (18%) fractures, while 46% of all RF were dislocated (15% ≥ rib width).

SRF show distinct fracture patterns depending on the cause of accident. Additional data should be collected to confirm our results and to establish a SRF classification system.

The screw fastening torque applied during bone fracture fixation has a decisive influence on subsequent bone healing. Insufficient screw tightness can result in device/construct instability; conversely, excessive torques risk damaging the bone causing premature fixation failure. This effect is even more prominent in osteoporotic bone, a condition associated annually with almost 9 million fractures worldwide. During fracture fixation, screw tightening torque is applied using subjective feel. This approach may not be optimal for patient”s recovery, increasing risk of fixation failure, particularly in osteoporotic bone, and potentially require revision surgical interventions.

Besides bone density, various factors influence the performance of screw fixation. These factors include bone geometry, cortical thickness and time-dependant relaxation behaviour of the bone. If the influence of screw fastening torque on the bone and relationships between these factors was better understood, the surgical technique could be optimised to reduce the risk of complications.

Within this study, we developed an axisymmetric finite element (FE) model of bone screw tightening incorporating viscoelastic behaviour of the cortical bone such as creep and stress relaxation. The model anticipated time-dependent behaviour of the bone for different bone thickness and density after a typical bone fixation screw had been inserted. The idealised model has been developed based on CT scans of bones with varying densities and inserted screws. The model was validated through a series of experiments involving bovine tibiae (4–5 months) to evaluate the evolution of surface strains with time (Ncorr v1.2). Stress distribution was assessed in photoelastic experiments using acrylic analogues. Relaxation tests have been performed in aqueous environment for up to 48 hours to ensure the relaxation would be complete. The creep behaviour (maximum principal strain) was compared against computational predictions. Our early simulations predicted relaxation strains on the surface of the bone to be 1.1% within 24 hours comparing favourably to 1.3% measured experimentally. Stress distribution patterns were in agreement with photoelastic results.

Using experimentally derived viscoelastic properties, the model has the potential to predict creep and stress relaxation patterns after screw insertion with different fastening torques for bones with varying density and geometry. We aim to develop this into a planning tool providing guidance to surgeons for optimal tightening when using screw fixation, particularly in reduced quality bone.