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Large osteochondral defects are a major challenge in orthopedics, for which osteochondral allograft (OCA) transplantation is nowadays considered as an option, especially in young patients. However, a major issue with OCA is the need for graft storage, which ensures adequate cartilage integrity over time. The aim of this study was to test how long a Ringer-based storage solution can provide good graft quality after explantation and thus meet the requirements for OCA. For this purpose, human osteochondral allografts of the knee and ankle were analyzed. Live/Dead analysis was performed and glycosaminoglycan, as well as hydroxyproline content, were measured as crucial chondrocyte integrity factors. Furthermore, biomechanical tests focusing on stress relaxation and elastic compression modulus were performed. The critical value of 70% living chondrocytes, which corresponds to a number of 300 cells/mm², was reached after an average of 16 weeks of storage. In addition, a constant cell shrinkage was observed over time. The amount of glycosaminoglycan and hydroxyroline showed a slight and constant decrease over time, but no significant differences when compared from Day 0 to the values at Weeks 40-43. Biomechanical testing also revealed no significant differences at the different time points. Therefore, the results show that the Ringer-based storage solution at 4°C is able to provide a chondrocyte survival of 70% until Week 16. This is comparable to previously published storage solutions. Therefore, the study contributes to the establishment of a Ringer-based osteochondral allograft transplantation system for countries where medium-based storage solution cannot be approved., (© 2024 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Biological bioprostheses such as grafts, patches, and heart valves are often derived from biological tissue like the pericardium. These bioprostheses can be of xenogenic, allogeneic, or autologous origin. Irrespective of their origin, all types are pre-treated via crosslinking to render the tissue non-antigenic and mechanically strong or to minimize degradation. The most widely used crosslinking agent is glutaraldehyde. However, glutaraldehyde-treated tissue is prone to calcification, inflammatory degradation, and mechanical injury, and it is incapable of matrix regeneration, leading to structural degeneration over time. In this work, we are investigating an alternative crosslinking method for an intraoperative application. The treated tissue's crosslinking degree was evaluated by differential scanning calorimetry. To confirm the findings, a collagenase assay was conducted. Uniaxial tensile testing was used to assess the tissue's mechanical properties. To support the findings, the treated tissue was visualized using two-photon microscopy. Additionally, fourier transform infrared spectroscopy was performed to study the overall protein secondary structure. Finally, a crosslinking procedure was identified for intraoperative processing. The samples showed a significant increase in thermal and enzymatic stability after treatment compared to the control, with a difference of up to 22.2 °C and 100%, respectively. Also, the tissue showed similar biomechanics to glutaraldehyde-treated tissue, showing greater extensibility, a higher failure strain, and a lower ultimate tensile strength than the control. The significant difference in the structure band ratio after treatment is proof of the introduction of additional crosslinks compared to the untreated control with regard to differences in the amide-I region. The microscopic images support these findings, showing an alteration of the fiber orientation after treatment. For collagen-based biomaterials, such as pericardial tissue, the novel phenolic crosslinking agent proved to be an equivalent alternative to glutaraldehyde regarding tissue characteristics. Although long-term studies must be performed to investigate superiority in terms of longevity and calcification, our novel crosslinking agent can be applied in concentrations of 1.5% or 2.0% for the treatment of biomaterials.

Background: Tissue engineered bioscaffolds based on decellularized composites have gained increasing interest for treatment of various diaphragmatic impairments, including muscular atrophies and diaphragmatic hernias. Detergent-enzymatic treatment (DET) constitutes a standard strategy for diaphragmatic decellularization. However, there is scarce data on comparing DET protocols with different substances in distinct application models in their ability to maximize cellular removal while minimizing extracellular matrix (ECM) damage., Methods: We decellularized diaphragms of male Sprague Dawley rats with 1 % or 0.1 % sodium dodecyl sulfate (SDS) and 4 % sodium deoxycholate (SDC) by orbital shaking (OS) or retrograde perfusion (RP) through the vena cava. We evaluated decellularized diaphragmatic samples by (1) quantitative analysis including DNA quantification and biomechanical testing, (2) qualitative and semiquantitative analysis by proteomics, as well as (3) qualitative assessment with macroscopic and microscopic evaluation by histological staining, immunohistochemistry and scanning electron microscopy., Results: All protocols produced decellularized matrices with micro- and ultramorphologically intact architecture and adequate biomechanical performance with gradual differences. The proteomic profile of decellularized matrices contained a broad range of primal core and ECM-associated proteins similar to native muscle. While no outstanding preference for one singular protocol was determinable, SDS-treated samples showed slightly beneficial properties in comparison to SDC-processed counterparts. Both application modalities proved suitable for DET., Conclusion: DET with SDS or SDC via orbital shaking or retrograde perfusion constitute suitable methods to produce adequately decellularized matrices with characteristically preserved proteomic composition. Exposing compositional and functional specifics of variously treated grafts may enable establishing an ideal processing strategy to sustain valuable tissue characteristics and optimize consecutive recellularization. This aims to design an optimal bioscaffold for future transplantation in quantitative and qualitative diaphragmatic defects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Pulmonary hypertension worsens outcome in left heart disease. Stiffening of the pulmonary artery may drive this pathology by increasing right ventricular dysfunction and lung vascular remodeling. Here we show increased stiffness of pulmonary arteries from patients with left heart disease that correlates with impaired pulmonary hemodynamics. Extracellular matrix remodeling in the pulmonary arterial wall, manifested by dysregulated genes implicated in elastin degradation, precedes the onset of pulmonary hypertension. The resulting degradation of elastic fibers is paralleled by an accumulation of fibrillar collagens. Pentagalloyl glucose preserves arterial elastic fibers from elastolysis, reduces inflammation and collagen accumulation, improves pulmonary artery biomechanics, and normalizes right ventricular and pulmonary hemodynamics in a rat model of pulmonary hypertension due to left heart disease. Thus, targeting extracellular matrix remodeling may present a therapeutic approach for pulmonary hypertension due to left heart disease., (© 2023. The Author(s).)

Bite force measuring devices that are generally suitable for edentulous patients or patients undergoing mandibular reconstruction are missing. This study assesses the validity of a new bite force measuring device (prototype of loadpad ® , novel GmbH) and evaluates its feasibility in patients after segmental mandibular resection. Accuracy and reproducibility were analyzed with two different protocols using a universal testing machine (Z010 AllroundLine, Zwick/Roell, Ulm, Germany). Four groups were tested to evaluate the impact of silicone layers around the sensor: no silicone ("pure"), 2.0 mm soft silicone ("2-soft"), 7.0 mm soft silicone ("7-soft") and 2.0 mm hard silicone ("2-hard"). Thereafter, the device was tested in 10 patients prospectively who underwent mandibular reconstruction using a fibula free flap. Average relative deviations of the measured force in relation to the applied load reached 0.77% ("7-soft") to 5.28% ("2-hard"). Repeated measurements in "2-soft" revealed a mean relative deviation of 2.5% until an applied load of 600 N. Maximum bite force decreased postoperatively by 51.8% to a maximum mean bite force of 131.5 N. The novel device guarantees a high accuracy and degree of reproducibility. Furthermore, it offers new opportunities to quantify perioperative oral function after reconstructive surgery of the mandible also in edentulous patients.

In fractures of the mandible, osteosynthesis with titanium plates is considered the gold standard. Titanium is an established and reliable material, its main disadvantages being metal artefacts and the need for removal in case of osteosynthesis complications. Magnesium, as a resorbable material with an elastic modulus close to cortical bone, offers a resorbable alternative osteosynthesis material, yet mechanical studies in mandible fracture fixation are still missing. The hypothesis of this study was that magnesium miniplates show no significant difference in the mechanical integrity provided for fracture fixation in mandible fractures under load-sharing indications. In a non-inferiority test, a continuous load was applied to a sheep mandible fracture model with osteosynthesis using either titanium miniplates of 1.0 mm thickness (Ti1.0), magnesium plates of 1.75 mm (Mg1.75), or magnesium plates of 1.5 mm thickness (Mg1.5). No significant difference (p > 0.05) was found in the peak force at failure, stiffness, or force at vertical displacement of 1.0 mm between Mg1.75, Mg1.5, and Ti1.0. This study shows the non-inferiority of WE43 magnesium miniplates compared to the clinical gold standard titanium miniplates.

Objective  This study assessed differences between fully- and partially-threaded screws in the initial interfragmentary compression strength. Our hypothesis was that there would be an increased loss in initial compression strength with the partially-threaded screw. Methods  A 45-degree oblique fracture line was created in artificial bone samples. The first group (FULL, n  = 6) was fixed using a 3.5-mm fully-threaded lag screw, while the second group (PARTIAL, n  = 6) used a 3.5-mm partially-threaded lag screw. Torsional stiffness for both rotational directions were evaluated. The groups were compared based on biomechanical parameters: angle-moment-stiffness, time-moment-stiffness, maximal torsional moment (failure load), and calibrated compression force based on pressure sensor measurement. Results  After loss of one PARTIAL sample, no statistically significant differences in calibrated compression force measurement were observed between both groups: [median (interquartile range)] FULL: 112.6 (10.5) N versus PARTIAL: 106.9 (7.1) N, Mann-Whitney U-test: p  = 0.8). In addition, after exclusion of 3 samples for mechanical testing (FULL n  = 5, PARTIAL n  = 4), no statistically significant differences were observed between FULL and PARTIAL constructs in angle-moment-stiffness, time-moment-stiffness, nor maximum torsional moment (failure load). Conclusion  There is no apparent difference in the initial compression strength (compression force or construct stiffness or failure load) achieved using either fully- or partially-threaded screws in this biomechanical model in high-density artificial bone. Fully-threaded screws could, therefore, be more useful in diaphyseal fracture treatment. Further research on the impact in softer osteoporotic, or metaphyseal bone models, and to evaluate the clinical significance is required., Competing Interests: Conflito de Interesses Os autores não têm conflitos de interesse a declarar., (Sociedade Brasileira de Ortopedia e Traumatologia. This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. ( https://creativecommons.org/licenses/by-nc-nd/4.0/ ).)

In recent years, an increase in periprosthetic femur fractures has become apparent due to the increased number of hip replacements. In the case of Vancouver type B1 fractures, locking plate systems offer safe procedures. This study compared the distal lateral femur plate (LOQTEQ ® , aap Implantate AG) with a standard L.I.S.S. LCP ® (DePuy Synthes) regarding their biomechanical properties in fixation of periprosthetic femur fractures after hip arthroplasty. We hypothesized that the new LOQTEQ system has superior stability and durability in comparison. Eighteen artificial left femurs were randomized in two groups (Group A: LOQTEQ ® ; Group B: L.I.S.S. LCP ® ) and tested until failure. Failure was defined as 10° varus deformity and catastrophic implant failure (loosening, breakage, progressive bending). Axial stiffness, loads of failure, cycles of failure, modes of failure were recorded. The axial stiffness in Group A with 73.4 N/mm (SD +/- 3.0) was significantly higher ( p = 0.001) than in Group B (40.7 N/mm (SD +/- 2.8)). Group A resists more cycles than Group B until 10° varus deformity. Catastrophic failure mode was plate breakage in Group A and bending in Group B. In conclusion, LOQTEQ ® provides higher primary stability and tends to have higher durability.

Background: The intra-articular graft force (IAGF) in anterior cruciate ligament reconstruction decreases quickly over the first hours after surgery. Nevertheless, little is known about whether the initial extra-articular tensioning force (EATF) and screw diameter affect the graft force after fixation., Purpose: To investigate the effects of different EATFs on the IAGF of a soft tissue graft fixated via a bioabsorbable interference screw over 100 minutes after fixation and to evaluate the effects of different screw diameters within 1 mm of the tunnel width during this process., Study Design: Controlled laboratory study., Methods: In this biomechanical study, a porcine quadruple-strand soft tissue graft was inserted into the tibial anterior cruciate ligament tunnel. On the extra-articular side, 3 loads were applied during retrograde insertion of the bioabsorbable interference screw (6, 7, and 8 mm): 20 N, 80 N, and maximum manual EATF (N max ). Nine study groups consisting of 10 tibiae each were created to test the effects of different EATFs and screw sizes. The IAGF was measured up to 100 minutes after the EATF was released., Results: An EATF ≥80 N resulted in a larger IAGF for all screw sizes at 100 minutes. There were no significant associations between the IAGF at 100 minutes and different screw diameters. Inserting the tibial screw significantly increased the IAGF in all groups, with the exception of N max applied in groups with 7- or 8-mm screws. When compared with the end of screw insertion, after the release of the EATF, the IAGF dropped by 55% to 77 % at 100 minutes., Conclusion: An initial EATF ≥80 N is associated with a significantly larger IAGF at 100 minutes in this cadaveric simulation. The IAGF in soft tissue grafts decreased substantially after the retrograde placement of an interference screw. A recommendation regarding screw diameter with respect to the IAGF cannot be given., Clinical Relevance: To obtain a higher residual graft force after bioabsorbable interference screw fixation, an initial EATF ≥80 N should be applied according to this model. The significant decrease in graft force after the release of the EATF indicates that the reconstructed knee cannot be mechanically stabilized after the surgery.

Radical resection remains the only curative treatment option in pancreatic cancer. Postoperative pancreatic fistulas (POPF) occur in up to 30% of patients leading to prolonged hospital-stay, increased cost of care and morbidity and mortality. Mechanical properties of the pancreas are associated with POPF. The aim of this study is to analyze the role of extracellular matrix (ECM) and tissue mechanics in the risk of POPF. Biopsies of 41 patients receiving a partial pancreas-resection are analyzed. Clinical data, ECM components and mechanical properties are correlated with POPF. Preoperative cholestasis is correlated with reduced risk of POPF, which comes along with a dilatation of the pancreatic duct and significantly higher content of collagen I. Patients developing POPF exhibited a degenerated tissue integrity, with significantly lower content of fibronectin and a trend for lower collagen I, III, IV and hyaluronic acid. This correlated with a soft tactile sensation of the surgeon during the intervention. However, this was not reflected with tissue mechanics evaluated by ex vivo uniaxial compression testing, where a significantly higher elastic modulus and no effect on the stress relaxation time were found. In conclusion, patients with cholestasis seem to have a lower risk for POPF, and an increase in collagen I. A degenerated matrix with lower content of structural ECM components correlates with increased risk of POPF. However, ex vivo uniaxial compression testing failed to clearly explain the link of ECM properties and POPF., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: To compare the mechanical strength of antegrade versus retrograde lag screw fixation of anterior column acetabular fractures., Methods: Standardised anterior column fractures were created in synthetic pelvis models and stabilised by either antegrade (ANTE, n = 4) or retrograde (RETRO, n = 4) anterior column screw fixation. In a validated setup, a cyclic loading protocol was applied with increasing axial force (750 cycles, 250-750 N) followed by load to failure. Construct survival, energy absorbed, construct stiffness, and load to failure were assessed. Descriptive and opto-metric methods were used to describe the mode of failure., Results: All constructs failed with loads below 1500 N. With regard to energy absorbed until failure, the ANTE group resisted to 3.763 × 10 5  N*cycles (range 3.760 × 10 5 -3.763 × 10 5 ) and the RETRO group to 3.762 × 10 5  N*cycles (range 3.761 × 10 5 -3.765 × 10 5 ; p = 1.0). The load to failure was 1254 N (range 977-1299) in the ANTE group and 1234 N (range 1087-1456) in the RETRO group (p = 1.0). Construct stiffness with 250 N was not different between the two groups (ANTE 192 N/mm vs. RETRO 215 N/mm, p = 0.486). In all samples, the mode of failure was a transiliac fracture with screw breakout due to rotation of the pubic fragment around the axis of the screw with a range of rotational motion [ROM] during cyclic testing of 0.96° in one ANTE sample and 0.82° in one RETRO sample for 750 N, and ROM at failure of 2.53° in one ANTE sample and 2.23° in one RETRO sample. There was some plastic deformation of the screws in all cases but no breakage., Conclusions: In this in vitro mechanical study, antegrade screw fixation of an anterior column acetabular fracture was not different in construct survival, load to failure, stiffness, and mode of failure when compared to retrograde screw fixation., (© 2019. Springer-Verlag GmbH Germany, part of Springer Nature.)

Interfragmentary compression, a major principle of fracture treatment, is clinically not quantified and might be lost quickly even without functional loads. We designed an experimental study hypothesizing that (1) compression can be controlled using either lag screw or compression plate, and expecting similar initial compression, (2) loss of interfragmentary compression through relaxation within one hour is reduced with neutralization locking plate next to lag screw compared to compression plate. Twelve ovine femora (N=6) and humeri (N=6) were assigned into groups: Group 1 received a 45° oblique osteotomy at mid-diaphysis and was fixated using a 3.5 mm interfragmentary lag screw and locking compression plate (3.5 mm LCP, DePuy Synthes) as neutralization plate. Group 2 received a transverse osteotomy and was fixated with dynamic compression using compression plate (LCP). Interfragmentary pressure and relative bone fragment displacements were recorded over one hour. Median loss of compression over one hour time (relaxation) were 0.52% in Group 1, and 0.17% in Group 2 (p>0.05). Median rotational displacements amounted to 0.46° for Group 1, and 0.31° for Group 2, and axial displacement to a median of -20 μm in Group 1 and 25 μm in Group 2. Ovine bone interfragmentary stress relaxation maintains compression over the first hour for lag screw with neutralization plate for an oblique fracture line or compression plate for a transverse fracture line. Measured compression forces around 100 N could be overcome by physiological tension loading in bending or torsion, necessitating for instance tension band plating, additional lag screws or absolutive stability., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2020. Published by Elsevier Ltd.)

Background: Many patients suffering from peripheral arterial disease (PAD) are dependent on bypass surgery. However, in some patients no suitable replacements (i.e. autologous or prosthetic bypass grafts) are available. Advances have been made to develop autologous tissue engineered vascular grafts (TEVG) using endothelial colony forming cells (ECFC) obtained by peripheral blood draw in large animal trials. Clinical translation of this technique, however, still requires additional data for usability of isolated ECFC from high cardiovascular risk patients. Bovine carotid arteries (BCA) were decellularized using a combined SDS (sodium dodecyl sulfate) -free mechanical-osmotic-enzymatic-detergent approach to show the feasibility of xenogenous vessel decellularization. Decellularized BCA chips were seeded with human ECFC, isolated from a high cardiovascular risk patient group, suffering from diabetes, hypertension and/or chronic renal failure. ECFC were cultured alone or in coculture with rat or human mesenchymal stromal cells (rMSC/hMSC). Decellularized BCA chips were evaluated for biochemical, histological and mechanical properties. Successful isolation of ECFC and recellularization capabilities were analyzed by histology., Results: Decellularized BCA showed retained extracellular matrix (ECM) composition and mechanical properties upon cell removal. Isolation of ECFC from the intended target group was successfully performed (80% isolation efficiency). Isolated cells showed a typical ECFC-phenotype. Upon recellularization, co-seeding of patient-isolated ECFC with rMSC/hMSC and further incubation was successful for 14 (n = 9) and 23 (n = 5) days. Reendothelialization (rMSC) and partial reendothelialization (hMSC) was achieved. Seeded cells were CD31 and vWF positive, however, human cells were detectable for up to 14 days in xenogenic cell-culture only. Seeding of ECFC without rMSC was not successful., Conclusion: Using our refined decellularization process we generated easily obtainable TEVG with retained ECM- and mechanical quality, serving as a platform to develop small-diameter (< 6 mm) TEVG. ECFC isolation from the cardiovascular risk target group is possible and sufficient. Survival of diabetic ECFC appears to be highly dependent on perivascular support by rMSC/hMSC under static conditions. ECFC survival was limited to 14 days post seeding.

Background: Tibial-sided graft fixation is thought to be critical for the success of anterior cruciate ligament (ACL) reconstruction. Nevertheless, little is known about the graft force after fixation during the first 24 hours after surgery or the influence of screw diameter and length during this time., Purpose: To investigate the force, over the course of 24 hours, in soft tissue grafts secured with a tibial interference screw and to evaluate the effect of different screw diameters (7, 8, and 9 mm) and lengths (25 and 30 mm) on the force in these grafts., Study Design: Controlled laboratory study., Methods: Quadruple-strand flexor tendon grafts were fixed with bioabsorbable interference screws in 60 porcine tibiae. Grafts were pretensioned at 80 N over 10 minutes, and screws were inserted outside-in while a preload force of 80 N was applied. Different screw lengths (25 and 30 mm) and diameters (7, 8, and 9 mm), resulting in 6 groups with 10 specimens each, were tested. After release of the preload, graft force was recorded over 24 hours., Results: A significant decrease in graft force progressed in all groups over the 24-hour period. In total, a median loss of 75 N (IQR, 68-79 N) compared with the initial loading force was observed. Compared with the loading force of 80 N, this corresponded to a median loss of 91%. No significant differences in the remaining graft force could be found among the 6 different screw length and diameter groups after 10 minutes, 100 minutes, or 24 hours., Conclusion: Graft force in soft tissue grafts secured with a tibial interference screw decreased substantially over the first 24 hours after fixation. Neither the screw diameter nor the screw length affected the decrease in graft force. This raises substantial questions regarding the remaining fixation strength in vivo., Clinical Relevance: It should not be expected that ACL reconstruction can mechanically restabilize an injured knee as would an intact ACL. Reconstructed knees should be protected from mechanical overload in the early postoperative period., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: The screws used in this study were provided by the Richard Wolf GmbH. S.K. has received speaking fees from Smith & Nephew. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto., (© The Author(s) 2020.)

Defects in the extracellular matrix protein fibrillin-1 that perturb transforming growth factor beta (TGFβ) bioavailability lead to Marfan syndrome (MFS). MFS is an autosomal-dominant disorder, which is associated with connective tissue and skeletal defects, among others. To date, it is unclear how biological sex impacts the structural and functional properties of bone in MFS. The aim of this study was to investigate the effects of sex on bone microarchitecture and mechanical properties in mice with deficient fibrillin-1, a model of human MFS. Bones of 11-week-old male and female Fbn1 mgR/mgR mice were investigated. Three-dimensional micro-computed tomography of femora and vertebrae revealed a lower ratio of trabecular bone volume to tissue volume, reduced trabecular number and thickness, and greater trabecular separation in females vs. males. Three-point bending of femora revealed significantly lower post-yield displacement and work-to-fracture in females vs. males. Mechanistically, we found higher Smad2 and ERK1/2 phosphorylation in females vs. males, demonstrating a greater activation of TGFβ signaling in females. In summary, the present findings show pronounced sex differences in the matrix and function of bones deficient in fibrillin-1 microfibrils. Consequently, sex-specific analysis of bone characteristics in patients with MFS may prove useful in improving the clinical management and life quality of these patients, through the development of sex-specific therapeutic approaches.

Bioactive coatings have the potential to improve the bony integration of mechanically loaded orthopedic ceramic implants. Using the concept of mimicking the natural bone surface, four different coatings of varying thickness on a zirconia toughened alumina (ZTA) ceramic implant were investigated regarding their osseointegration in a drill-hole model in sheep. The hypothesis that a bioactive coating of ZTA ceramics would facilitate cancellous bone integration was investigated. The bioactive coatings consisted of either a layer of covalently bound multi phosphonate molecules (chemical modification = CM), a nano hydoxyapatite coating (HA), or two different bioactive glass (BG) coatings in micrometer thickness, forming a hydroxyl-carbonate apatite layer on the implant surface in vivo (dip-coated 45S5 = DipBG; sol-gel 70S30C = SGBG). Coated surfaces were characterized by scanning electron microscopy and X-ray photoelectron spectroscopy. After 12 weeks, osseointegration was evaluated via mechanical push-out testing and histology. HA enhanced the maximum push-out force (HA: mean 3573.85 ± 1119.91 N; SGBG: mean 1691.57 ± 986.76 N; p = 0.046), adhesive shear strength (HA: mean 9.82 ± 2.89 MPA; SGBG: mean 4.57 ± 2.65 MPA; p = 0.025), and energy release rate (HA: mean 3821.95 ± 1474.13 J/mm 2 ; SGBG: mean 1558.47 ± 923.47 J/mm 2 ; p = 0.032) compared to SGBG. The implant-bone interfacial stiffness increased by CM compared to SGBG coating (CM: mean 6258.06 ± 603.80 N/mm; SGBG: mean 3565.57 ± 1705.31 n/mm; p = 0.038). Reduced mechanical osseointegration of SGBG coated implants could be explained histologically by a foreign body reaction surrounding the implants.

Amphotericin B is used for local delivery from polymethylmethacrylate to treat fungal prosthetic joint infections. The optimal amphotericin B formulation and the influence of different poragens in the bone cements are unknown. To investigate the necessary amount of amphotericin B in the bone cement to prevent Candida biofilm several amphotericin B formulations were studied: non-liposomal and liposomal with or without poragen gentamicin. For the non-liposomal formulation, standard bile salt, the sodium deoxycholate, was used and additionally N-methyl-D-glucamine/palmitate was applied. The activity of the released amphotericin B was tested against C. albicans, C. glabrata, C. parapsilosis and C. krusei biofilms with application of the isothermal calorimeter and standard microbiological methods. Compressive strength was measured before and after antifungal elution from the cements. There is less aggregated N-methyl-D-glucamine/palmitate amphotericin B released but its antifungal activity is equivalent with the deoxycholate amphotericin B. The minimum quantity of antifungal preventing the Candida biofilm formation is 12.5 mg in gram of polymer powder for both non-liposomal formulations. The addition of gentamicin reduced the release of sodium deoxycholate amphotericin B. Gentamicin can be added to N-methyl-D-glucamine/palmitate amphotericin B in order to boost the antifungal release. When using liposomal amphotericin B more drug is released. All amphotericin B formulations were active against Candida biofilms. Although compressive strength slightly decreased, the obtained values were above the level of strength recommended for the implant fixation. The finding of this work might be beneficial for the treatment of the prosthetic joint infections caused by Candida spp., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The adapter protein myeloid differentiation primary response gene 88 (MyD88) links the intracellular domains of interleukin receptors 1 and 18, and most Toll-like receptors (TLRs) to interleukin 1 receptor associated kinase (IRAK) signaling and subsequent NF-κB-mediated transcription. Previous work showed that mice with global deficiency of MyD88 (MyD88 -/- ) have osteopenic cancellous bone along with a reduction in osteoblastic but also osteoclastic surfaces. To further elucidate the role of MyD88 in bone, we utilized mice with osteoclast-restricted MyD88 expression in bone (MyD88 OC ). Bones of MyD88 OC and wild type (wt) mice were examined by microCT analysis. Mechanical properties of bones were tested by three-point bending, and gene expression measured using quantitative real-time polymerase chain reaction. In MyD88 OC mice, no osteopenic traits were observed, however, a drastic reduction in geometric parameters was detected. In trabecular bone a loss of connectivity density (-44%, p less than 0.0001) was measured and in cortical bone Imax (-31%, p less than 0.0001), Imin (-20%, p less than 0.001), J (-26%, p less than 0.0001) were reduced. Mechanical testing showed increased load to failure (77%, p less than 0.01) and decreased deflection at failure (-68%, p less than 0.01) of the femur. On the molecular level, relative gene expression analysis showed a (-29%, p less than 0.01) reduction in receptor activator of nuclear factor κ B ligand (RANKL) and no difference in osteoprotegerin (OPG) or RANK. Further, the bone resorption markers cathepsin K (CTSK) and tartrate-resistant acid phosphatase 5 (TRAP) were unchanged. In contrast, the bone formation markers collagen type 1 (COL1A1) and osteocalcin (OC) were decreased by -72% (p less than 0.0001) and -82% (p less than 0.0001), respectively. Together, our data suggests that the function of MyD88 in osteoclasts is sufficient to maintain bone mass, while it fails to preserve bone geometry, likely through dysfunctions in osteoblasts.

Bone formation as well as bone healing capacity is known to be impaired in the elderly. Although bone formation is outpaced by bone resorption in aged individuals, we hereby present a novel path that considerably impacts bone formation and architecture: Bone formation is substantially reduced in aged individual owing to the experience of the adaptive immunity. Thus, immune-aging in addition to chronological aging is a potential risk factor, with an experienced immune system being recognized as more pro-inflammatory. The role of the aging immune system on bone homeostasis and on the bone healing cascade has so far not been considered. Within this study mice at different age and immunological experience were analyzed toward bone properties. Healing was assessed by introducing an osteotomy, immune cells were adoptively transferred to disclose the difference in biological vs. chronological aging. In vitro studies were employed to test the interaction of immune cell products (cytokines) on cells of the musculoskeletal system. In metaphyseal bone, immune-aging affects bone homeostasis by impacting bone formation capacity and thereby influencing mass and microstructure of bone trabeculae leading to an overall reduced mechanical competence as found in bone torsional testing. Furthermore, bone formation is also impacted during bone regeneration in terms of a diminished healing capacity observed in young animals who have an experienced human immune system. We show the impact of an experienced immune system compared to a naïve immune system, demonstrating the substantial differences in the healing capacity and bone homeostasis due to the immune composition. We further showed that in vivo mechanical stimulation changed the immune system phenotype in young mice toward a more naïve composition. While this rescue was found to be significant in young individuals, aged mice only showed a trend toward the reconstitution of a more naïve immune phenotype. Considering the immune system's experience level in an individual, will likely allow one to differentiate (stratify) and treat (immune-modulate) patients more effectively. This work illustrates the relevance of including immune diagnostics when discussing immunomodulatory therapeutic strategies for the progressively aging population of the industrial countries.

During bone healing, tissue formation processes are governed by mechanical strain. Sost/sclerostin, a key Wnt signaling inhibitor and mechano-sensitive pathway, is downregulated in response to mechanical loading. Sclerostin neutralizing antibody (SclAb) increases bone formation. Nevertheless, it remains unclear whether sclerostin inhibition can rescue bone healing in situations of mechanical instability, which otherwise delay healing. We investigated SclAb's influence on tissue formation in a mouse femoral osteotomy, stabilized with rigid or semirigid external fixation. The different fixations allowed different magnitudes of interfragmentary movement during weight bearing, thereby influencing healing outcome. SclAb or vehicle (veh) was administeredand bone healing was assessed at multiple time points up to day 21 postoperatively by in vivo micro-computed tomography, histomorphometry, biomechanical testing, immunohistochemistry, and gene expression. Our results show that SclAb treatment caused a greater bone volume than veh. However, SclAb could not overcome the characteristic delayed healing of semirigid fixation. Indeed, semirigid fixation resulted in delayed healing with a prolonged endochondral ossification phase characterized by increased cartilage, lower bone volume fraction, and less bony bridging across the osteotomy gap than rigid fixation. In a control setting, SclAb negatively affected later stages of healing under rigid fixation, evidenced by the high degree of endosteal bridging at 21 days in the rigid-SclAb group compared with rigid-veh, indicating delayed fracture callus remodeling and bone marrow reconstitution. Under rigid fixation, Sost and sclerostin expression at the gene and protein level, respectively, were increased in SclAb compared with veh-treated bones, suggesting a negative feedback mechanism. Our results suggest that SclAb could be used to enhance overall bone mass but should be carefully considered in bone healing. SclAb may help to increase bone formation early in the healing process but not during advanced stages of fracture callus remodeling and not to overcome delayed healing in semirigid fixation. © 2018 American Society for Bone and Mineral Research., (© 2018 American Society for Bone and Mineral Research.)

Successful fracture healing requires a tight interplay between mechanical and biological cues. In vitro studies illustrated that mechanical loading modulates bone morphogenetic protein (BMP) signaling. However, in the early phases of large bone defect regeneration in vivo, the underlying mechanisms leading to this mechanosensation remained unknown. We investigated the interaction of BMP2 stimulation and mechanical boundary conditions in a rat critical-sized femoral defect model (5 mm) stabilized with three distinctly different external fixator stiffness. Defects were treated with 5 μg rhBMP2 loaded on an absorbable collagen sponge. Early matrix alignment was monitored by second-harmonic generation imaging. Bony bridging of defects and successive healing was monitored by histology at day 7 and day 14 as well as in vivo microCT at days 10, 21, and 42 post-operation. Femora harvested at day 42 were characterized mechanically assessing torsional load to failure ex vivo. At tissue level, differences between groups were visible at day 14 with manifest bone formation in the microCT. Histologically, we observed prolonged chondrogenesis upon flexible fixation, whereas osteogenesis started earlier after rigid and semirigid fixation. At later time points, there was a boost of bone tissue formation upon flexible fixation, whereas other groups already displayed signs of tissue maturation. Based on gene expression profiling, we analyzed the mechanobiological interplay. Already at day 3, these analyses revealed differences in expression pattern, specifically of genes involved in extracellular matrix formation. Gene regulation correlating with fixator stiffness was pronounced at day 7 comprising genes related to immunological processes and cellular contraction. The influence of loading on matrix contraction was further investigated and confirmed in a 3D bioreactor. Taken together, we demonstrate an early onset of mechanical conditions influencing BMP2-induced defect healing and shed light on gene regulatory networks associated with extracellular matrix organization and contraction that seemed to directly impact healing outcomes. © 2018 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Purpose: Clinical reports on meniscal scaffolds seem promising, albeit relatively paucity exists regarding their biomechanical behavior. The aim of the study is to delineate the impact of differing suture materials and the type as well as the temperature of the irrigation fluid on the pull-out strength of a polyurethane meniscal scaffold (Actifit ® )., Materials and Methods: 128 specimens were utilized with horizontal sutures and uniaxial load-to-failure testing was performed. We compared two different suture materials-polydioxanone (PDS) and non-absorbable, braided polyester sutures (NABP)-as well as two common irrigation fluids-lactated Ringer's and electrolyte-free, hypotonic Mannitol-Sorbitol. All specimens were further evaluated according to two different temperatures [room temperature (20 °C) and near-core body temperature (37 °C)]., Results: Mean load-to-failure was 53.3 ± 6.5 N. There was no significant difference between the NABP and the PDS group. Ringer group showed a significantly higher load-to-failure compared to Purisole (P = .0002). This was equivalent for both PDS (P = .0008) and NABP sutures (P = .0008). Significantly higher failure loads could be established for the 37° group (P = .041); yet, this difference was neither confirmed for the PDS or in in the NABP subgroup. Only the subgroup using Purisole at 37° showed significantly higher failure loads compared to 20° (P = .017)., Conclusions: This study underlines the potential to improve pull-out strength during implantation of an Actifit ® scaffold by alteration of the type of irrigation fluid. Lactated Ringer solution provided the highest construct stability in regard to load-to-failure testing and should be considered whenever implantation of a polyurethane meniscal scaffold is conducted.

Gerodermia osteodysplastica (GO) is a segmental progeroid disorder caused by loss-of-function mutations in the GORAB gene, associated with early onset osteoporosis and bone fragility. A conditional mouse model of GO (Gorab Prx1 ) was generated in which the Gorab gene was deleted in long bones. We examined the biomechanical/functional relevance of the Gorab Prx1 mutants as a premature aging model by characterizing bone composition, tissue-level strains, and whole-bone morphology and mechanical properties of the tibia. MicroCT imaging showed that Gorab Prx1 tibiae had an increased anterior convex curvature and decreased cortical cross-sectional area, cortical thickness and moments of inertia, compared to littermate control (LC) tibiae. Fourier transform infrared (FTIR) imaging indicated a 34% decrease in mineral/matrix ratio and a 27% increase in acid phosphate content in the posterior metaphyseal cortex of the Gorab Prx1 tibiae (p < .05), suggesting delayed mineralization. In vivo strain gauge measurement and finite element analysis showed ∼two times higher tissue-level strains within the Gorab Prx1 tibiae relative to LC tibiae when subjected to axial compressive loads of the same magnitude. Three-point bending tests suggested that Gorab Prx1 tibiae were weaker and more brittle, as indicated by decreasing whole-bone strength (46%), stiffness (55%), work-to-fracture (61%) and post-yield displacement (47%). Many of these morphological and biomechanical characteristics of the Gorab Prx1 tibia recapitulated changes in other animal models of skeletal aging. Future studies are necessary to confirm how our observations might guide the way to a better understanding and treatment of GO., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: The role of meniscus scaffolds remains controversial as failure rates remain high. The aim of this study was to evaluate the pullout strength of different suture materials used for fixation of the Collagen Meniscus Implant (CMI) regarding different suture materials, and type or temperature of irrigation fluid., Methods: One-hundred and twelve specimens were utilized with horizontal sutures and mounted to a dedicated test device. Loads were applied perpendicular to the CMI, until failure. Two differing suture materials - polydioxanone (PDS) and non-absorbable, braided polyester sutures (NABP) - were evaluated. Additionally, two common irrigation fluids - lactated Ringer's and electrolyte-free, hypotonic Mannitol-Sorbitol solution - were evaluated. Specimens were further evaluated according to different temperatures of the irrigation fluid. Half of the constructs were tested at room temperature (20°C) and half were evaluated at near-core body temperature (37°C)., Results: PDS sutures showed a significantly higher load-to-failure compared to NABP sutures (P=0.0008). Regarding the type of irrigation fluid, the electrolyte-free Mannitol-Sorbitol solution showed a significantly higher load-to-failure compared to the overall Ringer group (P b 0.0001). This was equivalent for both the PDS (P=0.015) and for the NABP sutures (P b 0.0001). The temperature of the irrigation fluid did not significantly influence load-to-failure., Conclusions: PDS sutures and electrolyte-free Mannitol-Sorbitol irrigation fluid provided the best biomechanical properties regarding load-to-failure testing. This study underlines the potential to improve construct stability for the CMI by alteration of the suture material and the type of irrigation fluid, which should be considered whenever scaffold fixation is conducted., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: New tissue engineering strategies for bone regeneration need to be investigated in a relevant preclinical large animal model before making the translation into human patients. Therefore, our interdisciplinary group established a simplified large animal screening model for intramembranous bone defect regeneration in cancellous and cortical bone., Methods: Related to a well-established model of cancellous drill hole defect regeneration in sheep, both the proximal and distal epimetaphyseal regions of the femur and the humerus were used bilaterally for eight drill hole cancellous defects (Ø 6 mm, 15 mm depth). Several improvements of the surgical procedure and equipment for an easier harvest of samples were invented. For the inclusion of cortical defect regeneration, a total of eight unicortical diaphyseal drill holes (6 mm Ø) were placed in the proximal-lateral and distal-medial parts of the metacarpal (MC) and metatarsal (MT) diaphyseal bone bilaterally. Acting moments within a normal gait cycle in the musculoskeletal lower limb model were compared with the results of the biomechanical in vitro torsion test until failure to ensure a low accidental fracture risk of utilized bones (ANOVA, p < 0.05). The model was tested in vivo, using thirteen adult, female, black-face sheep (Ø 66 kg; ± 5 kg; age ≥ 2.5 years). In a two-step surgical procedure 16 drill holes were performed for the investigation of two different time points within one animal. Defects were left empty, augmented with autologous cancellous bone or soft bone graft substitutes., Results: The in vitro tests confirmed this model a high comparability between drilled MC and MT bones and a high safety margin until fracture. The exclusion of one animal from the in vivo study, due to a spiral fracture of the left MC bone led to a tolerable failure rate of 8 %., Conclusions: As a screening tool, promising biomaterials can be tested in this cancellous and cortical bone defect model prior to the application in a more complex treatment site.

Introduction: Local application of bone morphogenetic proteins (BMPs) at the fracture site is known to stimulate bone regeneration. However, recent studies illustrate that the BMP-initiated mineralization may be enhanced by additional mechanical stimulation. Therefore, bone healing was monitored in vivo in order to investigate the effect of mechanical loading on the initiation and maturation of mineralization after cytokine treatment. We hypothesized that the mechanical stimulation would further enhance the efficacy of BMP2 treatment., Method: Female Sprague-Dawley rats underwent a 5-mm defect, stabilized with an external fixator. Type I collagen scaffolds containing 50 μg of BMP2 diluted in a solvent or solvent only were placed into the defects. The BMP2-treated specimens and control specimens were then each divided into two groups: one that underwent mechanical loading and a nonloaded group. In vivo loading began immediately after surgery and continued once per week for the entire 6-week experimental period. For all groups, the newly formed callus tissue was quantitatively evaluated first by in vivo microcomputed tomography at 2, 4, and 6 weeks and further by histologic or histomorphometric analysis at 6 weeks postoperation., Results: Mechanical stimulation with BMP2 treatment significantly enhanced mineralized tissue volume and mineral content at 2 weeks. Histological analysis demonstrated a significantly greater area of fibrous connective tissue including bone marrow in the stimulated group, suggesting reconstitution of the endosteal canal and more advanced bone remodeling present in the mechanical loaded group. Both groups receiving BMP2 underwent massive bone formation, achieving bony bridging after only 2 weeks, while both control groups, receiving solvent only, revealed a persisting nonunion, filled with fibrous connective tissue, prolapsed muscle tissue, and a sealed medullary canal at week 6., Conclusion: Mechanical loading further enhanced the efficacy of BMP2 application evidenced by increased mineralized tissue volume and mineralization at the stage of bony callus bridging. These data suggest that already a minimal amount of mechanical stimulation through load bearing or exercise may be a promising adjunct stimulus to enhance the efficacy of cytokine treatment in segmental defects. Further studies are required to elucidate the mechanistic interplay between mechanical and biological stimuli.

This study reports that treatment of osseous defects with different growth factors initiates distinct rates of repair. We developed a new method for monitoring the progression of repair, based upon measuring the in vivo mechanical properties of healing bone. Two different members of the bone morphogenetic protein (BMP) family were chosen to initiate defect healing: BMP-2 to induce osteogenesis, and growth-and-differentiation factor (GDF)-5 to induce chondrogenesis. To evaluate bone healing, BMPs were implanted into stabilised 5 mm bone defects in rat femurs and compared to controls. During the first two weeks, in vivo biomechanical measurements showed similar values regardless of the treatment used. However, 2 weeks after surgery, the rhBMP-2 group had a substantial increase in stiffness, which was supported by the imaging modalities. Although the rhGDF-5 group showed comparable mechanical properties at 6 weeks as the rhBMP-2 group, the temporal development of regenerating tissues appeared different with rhGDF-5, resulting in a smaller callus and delayed tissue mineralisation. Moreover, histology showed the presence of cartilage in the rhGDF-5 group whereas the rhBMP-2 group had no cartilaginous tissue. Therefore, this study shows that rhBMP-2 and rhGDF-5 treated defects, under the same conditions, use distinct rates of bone healing as shown by the tissue mechanical properties. Furthermore, results showed that in vivo biomechanical method is capable of detecting differences in healing rate by means of change in callus stiffness due to tissue mineralisation.