Effect of different decellularization protocols on reendothelialization with human cells for a perfused renal bioscaffold of the rat.

BACKGROUND: Scaffolds for tissue engineering can be received by whole organ decellularization while maintaining the site-specific extracellular matrix and the vascular tree. One among other decellularization techniques is the perfusion-based method using specific agents e.g. SDS for the elimination of cellular components. While SDS can disrupt the composition of the extracellular matrix and impair the adherence and growth of site-specific cells there are indications that xenogeneic cell types may benefit from protein denaturation by using higher detergent concentrations. The aim of this work is to investigate the effect of two different SDS-concentrations (i.e. 0.66% and 3%) on the ability of human endothelial cells to adhere and proliferate in an acellular rat kidney scaffold. MATERIAL AND METHODS: Acellular rat kidney scaffold was obtained by perfusion-based decellularization through the renal artery using a standardized protocol including SDS at concentrations of 0.66% or 3%. Subsequently cell seeding was performed with human immortalized endothelial cells EA.hy 926 via the renal artery. Recellularized kidneys were harvested after five days of pressure-controlled dynamic culture followed sectioning, histochemical and immunohistochemical staining as well as semiquantitative analysis. RESULTS: Efficacy of decellularization was verified by absence of cellular components as well as preservation of ultrastructure and adhesive proteins of the extracellular matrix. In semiquantitative analysis of recellularization, cell count after five days of dynamic culture more than doubled when using the gentle decellularization protocol with a concentration of SDS at 0.66% compared to 3%. Detectable cells maintained their endothelial phenotype and presented proliferative behavior while only a negligible fraction underwent apoptosis. CONCLUSION: Recellularization of acellular kidney scaffold with endothelial cells EA.hy 926 seeded through the renal artery benefits from gentle decellularization procedure. Because of that, decellularization with a SDS concentration at 0.66% should be preferred in further studies and coculture experiments.

Improving mandibular reconstruction by using topology optimization, patient specific design and additive manufacturing?-A biomechanical comparison against miniplates on human specimen.

In this study, topology optimized, patient specific osteosynthesis plates (TOPOS-implants) are evaluated for the mandibular reconstruction using fibula segments. These shape optimized implants are compared to a standard treatment with miniplates (thickness: 1.0 mm, titanium grade 4) in biomechanical testing using human cadaveric specimen. Mandible and fibula of 21 body donors were used. Geometrical models were created based on automated segmentation of CT-scans of all specimens. All reconstructions, including cutting guides for osteotomy as well as TOPOS-implants, were planned using a custom-made software tool. The TOPOS-implants were produced by electron beam melting (thickness: 1.0 mm, titanium grade 5). The fibula-reconstructed mandibles were tested in static and dynamic testing in a multi-axial test system, which can adapt to the donor anatomy and apply side-specific loads. Static testing was used to confirm mechanical similarity between the reconstruction groups. Force-controlled dynamic testing was performed with a sinusoidal loading between 60 and 240 N (reconstructed side: 30% reduction to consider resected muscles) at 5 Hz for up to 5 · 105 cycles. There was a significant difference between the groups for dynamic testing: All TOPOS-implants stayed intact during all cycles, while miniplate failure occurred after 26.4% of the planned loading (1.32 · 105 ± 1.46 · 105 cycles). Bone fracture occurred in both groups (miniplates: n = 3, TOPOS-implants: n = 2). A correlation between bone failure and cortical bone thickness in mandible angle as well as the number of bicortical screws used was demonstrated. For both groups no screw failure was detected. In conclusion, the topology optimized, patient specific implants showed superior fatigue properties compared to miniplates in mandibular reconstruction. Additionally, the patient specific shape comes with intrinsic guiding properties to support the reconstruction process during surgery. This demonstrates that the combination of additive manufacturing and topology optimization can be beneficial for future maxillofacial surgery.

Effects of focal metallic implants on opposing cartilage - an in-vitro study with an abrasion test machine.

BACKGROUND: For focal cartilage defects, biological repair might be ineffective in patients over 45 years. A focal metallic implant (FMI) (Hemi-CAP Arthrosurface Inc., Franklin, MA, USA) was designed to reduce symptoms. The aim of this study was to evaluate the effects of a FMI on the opposing tibial cartilage in a biomechanical set-up. It is hypothesized that a FMI would not damage the opposing cartilage under physiological loading conditions. METHODS: An abrasion machine was used to test the effects of cyclic loading on osteochondral plugs. The machine applied a compressive load of 33 N and sheared the samples 10 mm in the anteroposterior direction by 1 Hz. Tibial osteochondral plugs from porcine knees were placed in opposition to a FMI and cycled for 1 or 6 h. After testing each plug was fixed, stained and evaluated for cartilage damage. RESULTS: After 1 h of loading (n = 6), none of the osteochondral plugs showed histologic signs of degradation. After 6 h of loading (n = 6) three samples had histologic signs of injury in the tangential zone (grade 1) and one had signs of injury in the transitional and deep zones (grade 2). Exploration for 6 h resulted in significant more cartilage damage compared to the shorter exploration time (p = 0.06). However, no significant difference between saline and hyaluronic acid was evident (p = 0.55). CONCLUSION: Under physiologic loading conditions, contact with a FMI leads to cartilage damage in the opposing articular cartilage in six hours. In clinical practice, a thorough analysis of pre-existing defects on the opposing cartilage is recommended when FMI is considered.

Polydioxanone Threads for Facial Rejuvenation: Analysis of Quality Variation in the Market.

BACKGROUND: Beside botulinum-toxin injections and hyaluronic acid fillers, thread lifts have established themselves as the third column of minimally invasive facial rejuvenation. Most commonly, barbed threads for this approach are made out of polydioxanone, a material known for decades from application in resorbable sutures. The clinical efficacy and the putative material safety of polydioxanone have fueled the popularity of thread lifts. METHODS: The present study highlights significant variation among six commercially available threads in microstructure, tensile strength, elasticity, anchoring capacity in human tissue, and biocompatibility. RESULTS: Despite their license to be marketed and sold in the European Union, some products performed significantly worse than others on material testing, and even displayed cytotoxic characteristics. CONCLUSION: The results of this study are highly relevant for clinicians and may be linked to various typical side effects of polydioxanone threads for facial rejuvenation.

Integrated additive design and manufacturing approach for the bioengineering of bone scaffolds for favorable mechanical and biological properties.

Additive manufacturing (AM) presents the possibility of personalized bone scaffolds with unprecedented structural and functional designs. In contrast to earlier conventional design concepts, e.g. raster-angle, a workflow was established to produce scaffolds with triply periodic minimal surface (TPMS) architecture. A core challenge is the realization of such structures using melt-extrusion based 3D printing. This study presents methods for generation of scaffold design files, finite element (FE) analysis of scaffold Young's moduli, AM of scaffolds with polycaprolactone (PCL), and a customized in vitro assay to evaluate cell migration. The reliability of FE analysis when using computer-aided designed models as input may be impeded by anomalies introduced during 3D printing. Using micro-computed tomography reconstructions of printed scaffolds as an input for numerical simulation in comparison to experimentally obtained scaffold Young's moduli showed a moderate trend (R 2 = 0.62). Interestingly, in a preliminary cell migration assay, adipose-derived mesenchymal stromal cells (AdMSC) migrated furthest on PCL scaffolds with Diamond, followed by Gyroid and Schwarz P architectures. A similar trend, but with an accelerated AdMSC migration rate, was observed for PCL scaffolds surface coated with calcium-phosphate-based apatite. We elaborate on the importance of start-to-finish integration of all steps of AM, i.e. design, engineering and manufacturing. Using such a workflow, specific biological and mechanical functionality, e.g. improved regeneration via enhanced cell migration and higher structural integrity, may be realized for scaffolds intended as temporary guiding structures for endogenous tissue regeneration.

Full biomechanical mapping of the ovine knee joint to determine creep-recovery, stiffness and thickness variation.

BACKGROUND: Clinical cartilage repair strategies can be tested using the sheep model as suggest by the European Medicines Agency. To characterize variation within the joint a full biomechanical mapping is necessary. The aim of this study is to establish a loading model, to map regional differences within the knee and determine reference areas for area specific replacement techniques. METHODS: A porous indenter was selected to evaluate 22 defined test locations (femoral condyles, tibia plateau, patella, femoral groove) on ovine knees (n = 7). A high-dynamic force-controlled micro creep and creep-recovery indentation test system applied five loading (0.11 MPa) and unloading (5.6 kPa) cycles for 60 s each and recorded creep-recovery. Needle indentation was used to measure cartilage thickness and calculate total strain. FINDINGS: Steady state behaviour was observed from the third cycle and further evaluated. Little variation of stiffness in N/mm was found within the patella (4.3SD0.5) and femoral groove (8.1SD0.7) compared to larger variations in the femur (7.9SD2.0) and tibia (7.5SD3.2). Creep indentation showed values of 14.5%(SD2.7%) for the patella and 17.4%(SD3%) for the femoral grove opposed to 13.4%(SD4.3%) for the femoral condyles and 21.8%(SD6.6%) for the tibia plateau. Similar trends were observed analysing creep-recovery. Values were normalized to cartilage thickness which ranged between 0.36 mm and 1.14 mm. INTERPRETATION: Our setup allows a reliable evaluation of zonal differences. Homogenous biomechanical behaviour is found within the patella and femoral groove whereas significant biomechanical variation within the femoral condyles and tibia plateau indicates the need for site-specific cartilage repair products.

Effect of Lower Limb Alignment in Medial Meniscus-Deficient Knees on Tibiofemoral Contact Pressure.

BACKGROUND: Degenerative medial meniscal tears and subsequent partial meniscal resection compromise meniscal function and lead to an overload of the medial compartment. In addition, lower limb alignment plays a key role in load distribution between the medial and lateral knee compartments, and varus alignment is a potential risk factor for medial osteoarthritis. PURPOSE/HYPOTHESIS: The purpose of this biomechanical study was to investigate the effect of valgus and varus alignment on peak pressure and contact area in knees with concomitant horizontal medial meniscal tears and subsequent leaflet resection. It was hypothesized that varus alignment in combination with meniscal loss leads to the highest peak pressure within the medial compartment. STUDY DESIGN: Controlled laboratory study. METHODS: Six fresh-frozen human cadaveric knees were axially loaded using a 1000-N compressive load in full extension with the mechanical axis rotated to intersect the tibial plateau at 40%, 45%, 50%, 55%, and 60% of its width (TPW) to simulate varus and valgus alignment. Tibiofemoral peak contact pressure and contact area of the medial and lateral compartments were determined using pressure-sensitive foils in each of 4 different meniscal conditions: intact, 15-mm horizontal tear of the posterior horn, inferior leaflet resection, and resection of both leaflets. RESULTS: The effect of alignment on peak pressure (normalized to the neutral axis) within the medial compartment in cases of an intact meniscus was measured as follows: varus shift resulted in a mean increase in peak pressure of 18.5% at 45% of the TPW and 37.4% at 40% of the TPW, whereas valgus shift led to a mean decrease in peak pressure of 8.7% at 55% of the TPW and 23.1% at 60% of the TPW. Peak pressure changes between the intact meniscus and resection within the medial compartment was less in valgus-aligned knees (0.21 MPa at 60% TPW, 0.59 MPa at 50% TPW, and 0.76 MPa at 40% TPW). Contact area was significantly reduced after partial meniscal resection in the neutral axis (intact, 553.5 ± 87.6 mm2; resection of both leaflets, 323.3 ± 84.2 mm2; P < .001). This finding was consistent in any alignment. CONCLUSION: Both partial medial meniscal resection and varus alignment led to an increase in medial compartment peak pressure. Valgus alignment prevented medial overloading by decreasing contact pressure even after partial meniscal resection. A horizontal meniscal tear did not influence peak pressure and contact area even in varus alignment. CLINICAL RELEVANCE: As a clinical consequence, partial meniscal resection should be avoided to maintain the original biomechanical behavior, and the mechanical axis should be taken into account if partial meniscectomy is necessary.

3D grating-based X-ray phase-contrast computed tomography for high-resolution quantitative assessment of cartilage: An experimental feasibility study with 3T MRI, 7T MRI and biomechanical correlation.

OBJECTIVE: Aim of this study was, to demonstrate the feasibility of high-resolution grating-based X-ray phase-contrast computed tomography (PCCT) for quantitative assessment of cartilage. MATERIALS AND METHODS: In an experimental setup, 12 osteochondral samples were harvested from n = 6 bovine knees (n = 2 each). From each knee, one cartilage sample was degraded using 2.5% Trypsin. In addition to PCCT and biomechanical cartilage stiffness measurements, 3T and 7T MRI was performed including MSME SE T2 and ME GE T2* mapping sequences for relaxationtime measurements. Paired t-tests and receiver operating characteristics (ROC) curves were used for statistical analyses. RESULTS: PCCT provided high-resolution images for improved morphological cartilage evaluation as compared to 3T and 7T MRI. Quantitative analyses revealed significant differences between the superficial and the deep cartilage layer for T2 mapping as well as for PCCT (P<0.05). No significant difference was detected for PCCT between healthy and degraded samples (P>0.05). MRI and stiffness measurements showed significant differences between healthy and degraded osteochondral samples. Accuracy in the prediction of cartilage degradation was excellent for MRI and biomechanical analyses. CONCLUSION: In conclusion, high-resolution grating-based X-ray PCCT cartilage imaging is feasible. In addition to MRI and biomechanical analyses it provides complementary, water content independent, information for improved morphological and quantitative characterization of articular cartilage ultrastructure.

Chemically Modified Messenger RNA: Modified RNA Application for Treatment of Achilles Tendon Defects.

Different regenerative medicine approaches for tendon healing exist. Recently, especially gene therapy gained popularity. However, potential mutagenic and immunologic effects might prevent its translation to clinical research. Chemically modified mRNA (cmRNA) might bypass these limitations of gene therapy. Therefore, the purpose of this study was to evaluate the early healing properties of Achilles tendon defects in rats treated with basic fibroblast growth factor (bFGF) cmRNA. Forty male Lewis rats were used for the study and randomly assigned to two study groups: (1) treatment with cmRNA coding for bFGF and (2) noncoding cmRNA control. Protein expression was measured using in vivo bioluminescence imaging at 24, 48, and 72 h, as well as 14 days. Animals were euthanized 2 weeks following surgery. Biomechanical, histological, and immunohistological analyses were performed with the significance level set at p < 0.05. Protein expression was evident for 3 days. At 14 days, bioluminescence imaging revealed only little protein expression. Biomechanically, tendons treated with bFGF cmRNA showed a construct stiffness closer to the healthy contralateral side when compared with the control group (p = 0.034), without any significant differences in terms of load to failure. Hematoxylin and eosin staining detected no side effects of the treatment, as signs of inflammation, or necrosis. Furthermore, it revealed the shape of the nuclei to be more oval in the bFGF group in the tendon midsubstance (p = 0.043) with a reduced cell count (p = 0.035). Immunohistological staining for type I, II, III, and IV collagen did not differ significantly between the two groups. In conclusion, this pilot study demonstrates the feasibility of a novel messenger RNA (mRNA)-based therapy for Achilles tendon defects using chemically modified mRNA coding for bFGF.

In Vitro Analysis of Cartilage Regeneration Using a Collagen Type I Hydrogel (CaReS) in the Bovine Cartilage Punch Model.

OBJECTIVE: Limitations of matrix-assisted autologous chondrocyte implantation to regenerate functional hyaline cartilage demand a better understanding of the underlying cellular/molecular processes. Thus, the regenerative capacity of a clinically approved hydrogel collagen type I implant was tested in a standardized bovine cartilage punch model. METHODS: Cartilage rings (outer diameter 6 mm; inner defect diameter 2 mm) were prepared from the bovine trochlear groove. Collagen implants (± bovine chondrocytes) were placed inside the cartilage rings and cultured up to 12 weeks. Cartilage-implant constructs were analyzed by histology (hematoxylin/eosin; safranin O), immunohistology (aggrecan, collagens 1 and 2), and for protein content, RNA expression, and implant push-out force. RESULTS: Cartilage-implant constructs revealed vital morphology, preserved matrix integrity throughout culture, progressive, but slight proteoglycan loss from the "host" cartilage or its surface and decreasing proteoglycan release into the culture supernatant. In contrast, collagen 2 and 1 content of cartilage and cartilage-implant interface was approximately constant over time. Cell-free and cell-loaded implants showed (1) cell migration onto/into the implant, (2) progressive deposition of aggrecan and constant levels of collagens 1 and 2, (3) progressively increased mRNA levels for aggrecan and collagen 2, and (4) significantly augmented push-out forces over time. Cell-loaded implants displayed a significantly earlier and more long-lasting deposition of aggrecan, as well as tendentially higher push-out forces. CONCLUSION: Preserved tissue integrity and progressively increasing cartilage differentiation and push-out forces for up to 12 weeks of cultivation suggest initial cartilage regeneration and lateral bonding of the implant in this in vitro model for cartilage replacement materials.

Effect of Statistically Iterative Image Reconstruction on Vertebral Bone Strength Prediction Using Bone Mineral Density and Finite Element Modeling: A Preliminary Study.

Statistical iterative reconstruction (SIR) using multidetector computed tomography (MDCT) is a promising alternative to standard filtered back projection (FBP), because of lower noise generation while maintaining image quality. Hence, we investigated the feasibility of SIR in predicting MDCT-based bone mineral density (BMD) and vertebral bone strength from finite element (FE) analysis. The BMD and FE-predicted bone strength derived from MDCT images reconstructed using standard FBP (FFBP) and SIR with (FSIR) and without regularization (FSIRB0) were validated against experimental failure loads (Fexp). Statistical iterative reconstruction produced the best quality images with regard to noise, signal-to-noise ratio, and contrast-to-noise ratio. Fexp significantly correlated with FFBP, FSIR, and FSIRB0. FFBP had a significant correlation with FSIRB0 and FSIR. The BMD derived from FBP, SIRB0, and SIR were significantly correlated. Effects of regularization should be further investigated with FE and BMD analysis to allow for an optimal iterative reconstruction algorithm to be implemented in an in vivo scenario.

Improving results in rat fracture models: enhancing the efficacy of biomechanical testing by a modification of the experimental setup.

BACKGROUND: Animal fracture models, primarily performed in rats, are crucial to investigate normal and pathological bone healing. However, results of biomechanical testing representing a major outcome measure show high standard deviations often precluding statistical significance. Therefore, the aim of our study was a systematical examination of biomechanical characteristics of rat femurs during three-point bending. Furthermore, we tried to reduce variation of results by individually adapting the span of bearing and loading areas to the bone's length. METHODS: We examined 40 paired femurs of male Wistar-rats by DXA (BMD and BMC of the whole femur) and pQCT-scans at the levels of bearing and loading areas of the subsequent biomechanical three-point bending test. Individual adjustment of bearing and loading bars was done respecting the length of each specimen. Subgroups of light (< 400 g, n = 22) and heavy (> 400 g, n = 18) animals were formed and analysed separately. We furthermore compared the results of the individualised bending-setting to 20 femurs tested with a fix span of 15 mm. RESULTS: Femurs showed a length range of 34 to 46 mm. The failure loads ranged from 116 to 251 N (mean 175.4 ± 45.2 N; heavy animals mean 221 ± 18.9 N; light animals mean 138.1 ± 16.4 N) and stiffness ranged from 185 N/mm to 426 N/mm (mean 315.6 ± 63 N/mm; heavy animals mean 358.1 ± 34.64 N/mm; light animals mean 280.8 ± 59.85 N/mm). The correlation of densitometric techniques and failure loads was high (DXA R2 = 0.89 and pQCT R2 = 0.88). In comparison to femurs tested with a fix span, individual adaptation of biomechanical testing homogenized our data significantly. Most notably, the standard deviation of failure loads (221 ± 18.95 N individualized setting vs. 205.5 ± 30.36 N fixed) and stiffness (358.1 ± 34.64 N/mm individualized setting vs. 498.5 ± 104.8 N/mm fixed) was reduced by at least one third. CONCLUSIONS: Total variation observed in any trait reflects biological and methodological variation. Precision of the method hence affects the statistical power of the study. By simply adapting the setting of the biomechanical testing, interindividual variation could be reduced, which improves the precision of the method significantly.

Non-linear optical microscopy and histological analysis of collagen, elastin and lysyl oxidase expression in breast capsular contracture.

BACKGROUND: Capsular contracture is one of the most common complications in surgical interventions for aesthetic breast augmentation or post-mastectomy breast reconstruction involving the use of silicone prostheses. Although the precise cause of capsular contracture is yet unknown, the leading hypothesis is that it is caused by long-term unresolved foreign body reaction towards the silicone breast implant. To authors' best knowledge, this is the first study that elucidates the presence of lysyl oxidase (LOX)-an enzyme that is involved in collagen and elastin crosslinking within fibrous capsules harvested from patients with severe capsular contracture. It was hypothesized that over-expression of LOX plays a role in the irreversible crosslinking of collagen and elastin which, in turn, stabilizes the fibrous proteins and contributes to the progression of capsular contracture. METHODS: Eight fibrous capsules were collected from patients undergoing capsulectomy procedure, biomechanical testing was performed for compressive Young's moduli and evaluated for Type I and II collagen, elastin and LOX by means of non-linear optical microscopy and immunohistology techniques. RESULTS: Observations revealed the heterogeneity of tissue structure within and among the collected fibrous capsules. Regardless of the tissue structure, it has been shown that LOX expression was intensified at the implant-to-tissue interface. CONCLUSION: Our results indicate the involvement of LOX in the initiation of fibrous capsule formation which ultimately contributes towards the progression of capsular contracture.

Repair of the lateral posterior meniscal root improves stability in an ACL-deficient knee.

PURPOSE: To investigate the stabilizing effect of a lateral meniscus posterior root repair in an ACL and root deficient knee. METHODS: The hypothesis of the current study was that a sequential transection of the posterior root and the meniscofemoral ligaments in an ACL-deficient knee increases rotational instability, and conversely, a repair of the meniscus root reduces the internal tibial rotation. Therefore, eight human knee joints were tested in a robotic setup (5 N m internal torque, 50 N m anterior translation load). Five conditions were tested: intact, ACL cut, ACL cut + lateral meniscus posterior root tear (LMRT), ACL cut + LMRT + transection of the MFL and ACL cut + lateral meniscus root repair. The angles of internal tibial rotation as well as anterior tibial translation were recorded. RESULTS: Transection of the lateral meniscus posterior root increased the internal tibial instability as compared to the ACL-insufficient state. A significant increase was detected in 60° and 90° of flextion. Sectioning of the meniscofemoral ligament further destabilized the knees significantly at all flexion angles as compared to the ACL-deficient state. Even in 30°, 60° and 90° a significant difference was detected as compared to the isolated root tear. A tibial fixation of the lateral meniscus root reduced the internal tibial rotation in all flexion angles and led to a significant decrease of internal tibial rotation in 30° and 90° as compared to the transection of the root and the MFL. The anterior tibial translation was increased in all conditions as compared to the native state. CONCLUSION: A lateral meniscus root repair can reduce internal tibial rotation in the ACL-deficient knee. To check the condition of the lateral posterior meniscus root attachment is clinical relevant as a lateral meniscus root repair might improve rotational stability.

Multidetector Computed Tomography Imaging: Effect of Sparse Sampling and Iterative Reconstruction on Trabecular Bone Microstructure.

Multidetector computed tomography-based trabecular bone microstructure analysis ensures promising results in fracture risk prediction caused by osteoporosis. Because multidetector computed tomography is associated with high radiation exposure, its clinical routine use is limited. Hence, in this study, we investigated in 11 thoracic midvertebral specimens whether trabecular texture parameters are comparable derived from (1) images reconstructed using statistical iterative reconstruction (SIR) and filtered back projection as criterion standard at different exposures (80, 150, 220, and 500 mAs) and (2) from SIR-based sparse sampling projections (12.5%, 25%, 50%, and 100%) and equivalent exposures as criterion standard. Twenty-four texture features were computed, and those that showed similar values between (1) filtered back projection and SIR at the different exposure levels and (2) sparse sampling and equivalent exposures and reconstructed with SIR were identified. These parameters can be of equal value in determining trabecular bone microstructure with lower radiation exposure using sparse sampling and SIR.

Whole bone testing in small animals: systematic characterization of the mechanical properties of different rodent bones available for rat fracture models.

OBJECTIVES: Rat fracture models are extensively used to characterize normal and pathological bone healing. Despite, systematic research on inter- and intra-individual differences of common rat bones examined is surprisingly not available. Thus, we studied the biomechanical behaviour and radiological characteristics of the humerus, the tibia and the femur of the male Wistar rat-all of which are potentially available in the experimental situation-to identify useful or detrimental biomechanical properties of each bone and to facilitate sample size calculations. METHODS: 40 paired femura, tibiae and humeri of male Wistar rats (10-38 weeks, weight between 240 and 720 g) were analysed by DXA, pQCT scan and three-point-bending. Bearing and loading bars of the biomechanical setup were adapted percentually to the bone's length. Subgroups of light (skeletal immature) rats under 400 g (N = 11, 22 specimens of each bone) and heavy (mature) rats over 400 g (N = 9, 18 specimens of each bone) were formed and evaluated separately. RESULTS: Radiologically, neither significant differences between left and right bones, nor a specific side preference was evident. Mean side differences of the BMC were relatively small (1-3% measured by DXA and 2.5-5% by pQCT). Over all, bone mineral content (BMC) assessed by DXA and pQCT (TOT CNT, CORT CNT) showed high correlations between each other (BMC vs. TOT and CORT CNT: R2 = 0.94-0.99). The load-displacement diagram showed a typical, reproducible curve for each type of bone. Tibiae were the longest bones (mean 41.8 ± 4.12 mm) followed by femurs (mean 38.9 ± 4.12 mm) and humeri (mean 29.88 ± 3.33 mm). Failure loads and stiffness ranged from 175.4 ± 45.23 N / 315.6 ± 63.00 N/mm for the femurs, 124.6 ± 41.13 N / 260.5 ± 59.97 N/mm for the humeri to 117.1 ± 33.94 N / 143.8 ± 36.99 N/mm for the tibiae. Smallest interindividual differences were observed in failure loads of the femurs (CV% 8.6) and tibiae (CV% 10.7) of heavy animals, light animals showed good consistency in failure loads of the humeri (CV% 7.7). Most consistent results of both sides (left vs. right) in failure loads were provided by the femurs of light animals (mean difference 4.0 ± 2.8%); concerning stiffness, humeri of heavy animals were most consistent (mean difference of 6.2 ± 5%). In general, the failure loads showed strong correlations to the BMC (R2 = 0.85-0.88) whereas stiffness correlated only moderate, except for the humerus (BMC vs. stiffness: R2 = 0.79). DISCUSSION: Altogether, the rat's femur of mature specimens showed the most accurate and consistent radiological and biomechanical results. In synopsis with the common experimental use enabling comparison among different studies, this bone offers ideal biomechanical conditions for three point bending experiments. This can be explained by the combination of a superior aspect ratio and a round and long, straight morphology, which satisfies the beam criteria more than other bones tested.

Correction: Viable adhered Staphylococcus aureus highly reduced on novel antimicrobial sutures using chlorhexidine and octenidine to avoid surgical site infection (SSI).

[This corrects the article DOI: 10.1371/journal.pone.0190912.].

Viable adhered Staphylococcus aureus highly reduced on novel antimicrobial sutures using chlorhexidine and octenidine to avoid surgical site infection (SSI).

BACKGROUND: Surgical sutures can promote migration of bacteria and thus start infections. Antiseptic coating of sutures may inhibit proliferation of adhered bacteria and avoid such complications. OBJECTIVES: This study investigated the inhibition of viable adhering bacteria on novel antimicrobially coated surgical sutures using chlorhexidine or octenidine, a critical factor for proliferation at the onset of local infections. The medical need, a rapid eradication of bacteria in wounds, can be fulfilled by a high antimicrobial efficacy during the first days after wound closure. METHODS: As a pretesting on antibacterial efficacy against relevant bacterial pathogens a zone of inhibition assay was conducted with middle ranged concentrated suture coatings (22 µg/cm). For further investigation of adhering bacteria in detail the most clinically relevant Staphylococcus aureus (ATCC®49230™) was used. Absorbable braided sutures were coated with chlorhexidine-laurate, chlorhexidine-palmitate, octenidine-laurate, and octenidine-palmitate. Each coating type resulted in 11, 22, or 33 µg/cm drug content on sutures. Scanning electron microscopy (SEM) was performed once to inspect the coating quality and twice to investigate if bacteria have colonized on sutures. Adhesion experiments were assessed by exposing coated sutures to S. aureus suspensions for 3 h at 37°C. Subsequently, sutures were sonicated and the number of viable bacteria released from the suture surface was determined. Furthermore, the number of viable planktonic bacteria was measured in suspensions containing antimicrobial sutures. Commercially available sutures without drugs (Vicryl®, PGA Resorba®, and Gunze PGA), as well as triclosan-containing Vicryl® Plus were used as control groups. RESULTS: Zone of inhibition assay documented a multispecies efficacy of novel coated sutures against tested bacterial strains, comparable to most relevant S. aureus over 48 hours. SEM pictures demonstrated uniform layers on coated sutures with higher roughness for palmitate coatings and sustaining integrity of coated sutures. Adherent S. aureus were found via SEM on all types of investigated sutures. The novel antimicrobial sutures showed significantly less viable adhered S. aureus bacteria (up to 6.1 log) compared to Vicryl® Plus (0.5 log). Within 11 µg/cm drug-containing sutures, octenidine-palmitate (OL11) showed the highest number of viable adhered S. aureus (0.5 log), similar to Vicryl® Plus. Chlorhexidine-laurate (CL11) showed the lowest number of S. aureus on sutures (1.7 log), a 1.2 log greater reduction. In addition, planktonic S. aureus in suspensions were highly inhibited by CL11 (0.9 log) represents a 0.6 log greater reduction compared to Vicryl® Plus (0.3 log). CONCLUSIONS: Novel antimicrobial sutures can potentially limit surgical site infections caused by multiple pathogenic bacterial species. Therefore, a potential inhibition of multispecies biofilm formation is assumed. In detail tested with S. aureus, the chlorhexidine-laurate coating (CL11) best meets the medical requirements for a fast bacterial eradication. This suture coating shows the lowest survival rate of adhering as well as planktonic bacteria, a high drug release during the first-clinically most relevant- 48 hours, as well as biocompatibility. Thus, CL11 coatings should be recommended for prophylactic antimicrobial sutures as an optimal surgical supplement to reduce wound infections. However, animal and clinical investigations are important to prove safety and efficacy for future applications.

Effect of radiation dose reduction on texture measures of trabecular bone microstructure: an in vitro study.

Osteoporosis is characterized by bone loss and degradation of bone microstructure leading to fracture particularly in elderly people. Osteoporotic bone degeneration and fracture risk can be assessed by bone mineral density and trabecular bone score from 2D projection dual-energy X-ray absorptiometry images. However, multidetector computed tomography image based quantification of trabecular bone microstructure showed significant improvement in prediction of fracture risk beyond that from bone mineral density and trabecular bone score; however, high radiation exposure limits its use in routine clinical in vivo examinations. Hence, this study investigated reduction of radiation dose and its effects on image quality of thoracic midvertebral specimens. Twenty-four texture features were extracted to quantify the image quality from multidetector computed tomography images of 11 thoracic midvertebral specimens, by means of statistical moments, the gray-level co-occurrence matrix, and the gray-level run-length matrix, and were analyzed by an independent sample t-test to observe differences in image texture with respect to radiation doses of 80, 150, 220, and 500 mAs. The results showed that three features-namely, global variance, energy, and run percentage, were not statistically significant ([Formula: see text]) for low doses with respect to 500 mAs. Hence, it is evident that these three dose-independent features can be used for disease monitoring with a low-dose imaging protocol.

BMP-2 gene activated muscle tissue fragments for osteochondral defect regeneration in the rabbit knee.

BACKGROUND: Previously published data indicate that BMP-2 gene activated muscle tissue grafts can repair large bone defects in rats. This innovative abbreviated ex vivo gene therapy is appealing because it does not require elaborative and time-consuming extraction and expansion of cells. Hence, in the present study, we evaluated the potential of this expedited tissue engineering approach for regenerating osteochondral defects in rabbits. METHODS: Autologous muscle tissue grafts from female White New Zealand rabbits were directly transduced with an adenoviral BMP-2 vector or remained unmodified. Osteochondral defects in the medial condyle of rabbit knees were treated with either BMP-2 activated muscle tissue implants or unmodified muscle tissue or remained empty. After 13 weeks, repair of osteochondral defects was examined by biomechanical indentation testing and by histology/imunohistochemistry applying an extended O'Driscoll scoring system and histomorphometry. RESULTS: Biomechanical investigations revealed a trend towards slightly improved mechanical properties of the group receiving BMP-2 activated muscle tissue compared to unmodified muscle treatment and empty defect controls. However, a statistically significant difference was noted only between BMP-2 muscle and unmodified muscle treatment. Also, histological evaluation resulted in slightly higher histological scores and improved collagen I/II ratio without statistical significance in the BMP-2 treatment group. Histomorphometry indicated enhanced repair of subchondral bone after treatment with BMP-2 muscle, with a significantly larger bone area compared to untreated defects. CONCLUSIONS: Gene activated muscle tissue grafts showed potential for osteochondral defect repair. There is room for improvement via the use of appropriate growth factor combinations.