Experimental validation of a mean field model of mineralized collagen fiber arrays at two levels of hierarchy

In the course of this study, stiffness of a fibril array of mineralized collagen fibrils modeled with a mean field method was validated experimentally at site-matched two levels of tissue hierarchy using mineralized turkey leg tendons (MTLT). The applied modeling approaches allowed to model the properties of this unidirectional tissue from nanoscale (mineralized collagen fibrils) to macroscale (mineralized tendon). At the microlevel, the indentation moduli obtained with a mean field homogenization scheme were compared to the experimental ones obtained with microindentation. At the macrolevel, the macroscopic stiffness predicted with micro finite element (μFE) models was compared to the experimental stiffness measured with uniaxial tensile tests. Elastic properties of the elements in μFE models were injected from the mean field model or two-directional microindentations. Quantitatively, the indentation moduli can be properly predicted with the mean-field models. Local stiffness trends within specific tissue morphologies are very weak, suggesting additional factors responsible for the stiffness variations. At macrolevel, the μFE models underestimate the macroscopic stiffness, as compared to tensile tests, but the correlations are strong.

Collagen XII: Protecting bone and muscle integrity by organizing collagen fibrils.

Collagen XII, largest member of the fibril-associated collagens with interrupted triple helix (FACIT) family, assembles from three identical α-chains encoded by the COL12A1 gene. The molecule consists of three threadlike N-terminal noncollagenous NC3 domains, joined by disulfide bonds and a short interrupted collagen triple helix toward the C-terminus. Splice variants differ considerably in size and properties: "small" collagen XIIB (220 kDa subunit) is similar to collagen XIV, whereas collagen XIIA (350 kDa) has a much larger NC3 domain carrying glycosaminoglycan chains. Collagen XII binds to collagen I-containing fibrils via its collagenous domain, whereas its large noncollagenous arms interact with other matrix proteins such as tenascin-X. In dense connective tissues and bone, collagen XII is thought to regulate organization and mechanical properties of collagen fibril bundles. Accordingly, recent findings show that collagen XII mutations cause Ehlers-Danlos/myopathy overlap syndrome associated with skeletal abnormalities and muscle weakness in mice and humans.

Suitability of Common Collagen Scaffolds for Anterior Cruciate Ligament Repair

Introduction: Anterior cruciate ligament (ACL) injuries are very common; in Germany incidence of ACL ruptures is estimated at 32 per 100 000 in the general population and in the sports community this rate more than doubles. Current gold standard for anterior cruciate lig- ament repair is reconstruction using an autograft [1]. However, this approach has shown some limitations. A new method has been her- alded by the Knee Team at the Bern University Hospital (Inselspital) and the Sonnenhof clinic called Dynamic Intraligamentary Stabilization (DIS), which keeps ACL remnants in place in order to promote biologi- cal healing and makes use of a dynamic screw system [2]. The aim of this study was to investigate the cytocompatibility of collagen patches in combination with DIS to support regeneration of the ACL. The spe- cific hypothesis we tested was whether MSCs would differentiate towards TCs in co-culture. Materials and methods: Primary Tenocytes (TCs) and human bone marrow derived mesenchymal stem cells (MSCs) were harvested from ACL removed during knee prothesis or from bone marrow aspirations (Ethical Permit 187/10). Cells were seeded on two types of three dimensional carriers currently approved for cartilage repair, Novocart (NC, B. Brown) and Chondro-Gide (CG, Geistlich). These scaffolds comprise collagen structures with interconnecting pores originally developed for seeding of chondrocytes in the case of CG. ~40k cells were seeded on punched zylindrical cores of 8 mm in Ø and cultured on CG or NC patches for up to 7 days. The cells were either cultured as TC only, MSC only or co-cultured in a 1:1 mix on the scaffolds and on both sides of culture inserts (PET, high density pore Ø 0.4 mm, BD, Fal- con) with cell-cell contact. We monitored DNA content, GAG and HOP-content, tracked the cells using DIL and DIO fluorescent dyes (Molecular Probes, Life technologies) and confocal laser scanning and SEM microscopy as well as RT-PCR of tenocyte specific markers (i.e. col 1 and 3, TNC, TNMD, SCXA&B, and markers of dedifferentiation ACAN, col2, MMP3, MMP13). Finally, H&E stain was interpreted on cryosections and SEM images of cells on the scaffold were taken. Results: ThecLSMimagesshowedcellproliferationoverthe7dayson both matrices, however, on CG there were much fewer MSCs attached than on NC. SEM images showed a roundish chondrocyte-like pheno- type of cells on CG whereas on NC the phenotype was more teno- cyte-like (Fig. 1). Gene expression of both, MSC and TC seem to confirm a more favorable environment in 3D for both patches rather than monolayer control.

Mesenchymal stem cells and collagen patches for anterior cruciate ligament repair

AIM: To investigate collagen patches seeded with mesenchymal stem cells (MSCs) and/or tenocytes (TCs) with regards to their suitability for anterior cruciate ligament (ACL) repair. METHODS: Dynamic Intraligamentary Stabilization (DIS) utilizes a dynamic screw system to keep ACL remnants in place and promote biological healing, supplemented by collagen patches. How these scaffolds interact with cells and what type of benefit they provide has not yet been investigated in detail. Primary ACL-derived TCs and human bone marrow derived MSCs were seeded onto two different types of 3D collagen scaffolds, Chondro-Gide® (CG) and Novocart® (NC). Cells were seeded onto the scaffolds and cultured for 7 days either as a pure populations or as “premix” containing a 1 : 1 ratio of TCs to MSCs. Additionally, as controls, cells were seeded in monolayers and in co-cultures on both sides of porous high-density membrane inserts (0.4µm). We analyzed the patches by real time polymerase chain reaction (RT-PCR), glycosaminoglycan (GAG), DNA and hydroxy-proline (HYP) content, was determined. To determine cell spreading and adherence in the scaffolds microscopic imaging techniques, i.e. confocal laser scanning microscopy (cLSM) and scanning electron microscopy (SEM), were applied. RESULTS: CLSM and SEM imaging analysis confirmed cell adherence onto scaffolds. The metabolic cell activity revealed that patches promote adherence and proliferation of cells. The most dramatic increase in absolute metabolic cell activity was measured for CG samples seeded with tenocytes or a 1:1 cell premix. Analysis of DNA content and cLSM imaging also indicated MSCs were not proliferating as nicely as tenocytes on CG. The HYP to GAG ratio significantly changed for the premix group, resulting from a slightly lower GAG content, demonstrating that the cells are modifying the underlying matrix. Real-time quantitative polymerase chain reaction data indicated that MSCs showed a trend of differentiation towards a more tenogenic-like phenotype after 7 days. CONCLUSION: CG and NC are both cyto-compatible with primary MSCs and TCs; TCs seemed to perform better on these collagen patches than MSCs.

SMG6 mediated degradation of nonsense mRNA requires phosphorylation-independent interaction with the helicase domain of UPF1

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD targeted mRNAs can be degraded by different routes that all involve phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of three known NMD factors thought to be recruited to nonsense mRNAs by interaction with P-UPF1, leading to eventual mRNA degradation. By MS2-mediated tethering of SMG6 and mutants thereof to a reporter RNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 for inducing RNA decay. Our experiments revealed a phosphorylation-independent interaction between SMG6 and UPF1 that is important for SMG6-mediated mRNA decay and using yeast two hybrid assays, we mapped this interaction to the unique stalk region of the UPF1 helicase domain. This region of UPF1 is essential for SMG6-mediated reporter RNA decay and also for NMD. Our results postulate that besides recruiting SMG6 to its RNA substrates, UPF1 is also required to activate its endonuclease activity.

SMG6 mediated degradation of nonsense mRNA requires phosphorylation-independent interaction with the helicase domain of UPF1

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD targeted mRNAs can be degraded by different routes that all involve phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of three known NMD factors thought to be recruited to nonsense mRNAs by interaction with P-UPF1, leading to eventual mRNA degradation. By MS2-mediated tethering of SMG6 and mutants thereof to a reporter RNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 for inducing RNA decay. Our experiments revealed a phosphorylation-independent interaction between SMG6 and UPF1 that is important for SMG6-mediated mRNA decay and using yeast two hybrid assays, we mapped this interaction to the unique stalk region of the UPF1 helicase domain. This region of UPF1 is essential for SMG6-mediated reporter RNA decay and also for NMD. Our results postulate that besides recruiting SMG6 to its RNA substrates, UPF1 is also required to activate its endonuclease activity.

S-nitroso albumin enhances bone formation in a rabbit calvaria model.

Nitric oxide (NO) is a mediator involved in bone regeneration. We therefore examined the effect of the novel NO donor, S-nitroso human serum albumin (S-NO-HSA) on bone formation in a rabbit calvaria augmentation model. Circular grooves (8 mm diameter, two per animal) were created by a trephine drill in the cortical bone of 40 rabbits and titanium caps were placed on the rabbit calvaria bone filled with a collagen sponge soaked with either 100 μL S-NO-HSA (5%, 20%) or human albumin (5%, 20%). After 4 weeks the titanium hemispheres were subjected to histological and histomorphometric analysis. Bone formation and the volume of the residual collagen sponge were evaluated. S-NO-HSA treatment groups had a significantly higher volume of newly formed bone underneath the titanium hemispheres compared to the albumin control groups (5%: 15.5 ± 4.0% versus 10.6 ± 2.9%; P < 0.05; 20%: 14.0 ± 4.6% versus 6.0 ± 3.8%; P < 0.01). The volume of residual collagen sponge was also significantly lower in the S-NO-HSA groups compared to the control groups (5%: 0.4 ± 0.5% versus 2.6 ± 2.4%; P < 0.05 and 20%: 1.5 ± 2.7% versus 13.0 ± 18.7%; P < 0.01). This study demonstrates for the first time that S-NO-HSA promotes bone formation by slow NO release. Additionally, S-NO-HSA increases collagen sponge degradation.

Clinical outcomes following regenerative therapy of non-contained intrabony defects using a deproteinized bovine bone mineral combined with either enamel matrix derivative or collagen membrane

BACKGROUND The purpose of this study is to compare clinical outcomes in the treatment of deep non-contained intrabony defects (i.e., with ≥70% 1-wall component and a residual 2- to 3-wall component in the most apical part) using deproteinized bovine bone mineral (DBBM) combined with either enamel matrix protein derivative (EMD) or collagen membrane (CM). METHODS Forty patients with multiple intrabony defects were enrolled. Only one non-contained defect per patient with an intrabony depth ≥3 mm located in the interproximal area of single- and multirooted teeth was randomly assigned to the treatment with either EMD + DBBM (test: n = 20) or CM + DBBM (control: n = 20). At baseline and after 12 months, clinical parameters including probing depth (PD) and clinical attachment level (CAL) were recorded. The primary outcome variable was the change in CAL between baseline and 12 months. RESULTS At baseline, the intrabony component of the defects amounted to 6.1 ± 1.9 mm for EMD + DBBM and 6.0 ± 1.9 mm for CM + DBBM sites (P = 0.81). The mean CAL gain at sites treated with EMD + DBBM was not statistically significantly different (P = 0.82) compared with CM + DBBM (3.8 ± 1.5 versus 3.7 ± 1.2 mm). No statistically significant difference (P = 0.62) was observed comparing the frequency of CAL gain ≥4 mm between EMD + DBBM (60%) and CM + DBBM (50%) or comparing the frequency of residual PD ≥6 mm between EMD + DBBM (5%) and CM + DBBM (15%) (P = 0.21). CONCLUSION Within the limitations of the present study, regenerative therapy using either EMD + DBBM or CM + DBBM yielded comparable clinical outcomes in deep non-contained intrabony defects after 12 months.

Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation.

INTRODUCTION Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC) networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. METHODS Bone marrow-derived DCs (BMDCs) were exposed in vitro to 20 or 1,000 nm polystyrene (PS) particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4(+) T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. RESULTS The frequency of PS particle-positive CD11c(+)/CD11b(+) BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4(+) T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. CONCLUSION These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4(+) T-cell stimulating capacity, 20 nm (but not 1,000 nm) PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles.

Impaired DNase1-mediated degradation of neutrophil extracellular traps is associated with acute thrombotic microangiopathies.

BACKGROUND Acute thrombotic microangiopathies (TMAs) are characterized by excessive microvascular thrombosis and are associated with markers of neutrophil extracellular traps (NETs) in plasma. NETs are composed of DNA fibers and promote thrombus formation through the activation of platelets and clotting factors. OBJECTIVE The efficient removal of NETs may be required to prevent excessive thrombosis such as in TMAs. To test this hypothesis, we investigated whether TMAs are associated with a defect in the degradation of NETs. APPROACH AND RESULTS We show that NETs generated in vitro were efficiently degraded by plasma from healthy donors. However, NETs remained stable after exposure to plasma from TMA patients. The inability to degrade NETs was linked to a reduced DNase activity in TMA plasma. Plasma DNase1 was required for efficient NET-degradation and TMA plasma showed decreased levels of this enzyme. Supplementation of TMA plasma with recombinant human DNase1 restored NET-degradation activity. CONCLUSIONS Our data indicates that DNase1-mediated degradation of NETs is impaired in patients with TMAs. The role of plasma DNases in thrombosis is, as of yet, poorly understood. Reduced plasma DNase1 activity may cause the persistence of pro-thrombotic NETs and thus promote microvascular thrombosis in TMA patients. This article is protected by copyright. All rights reserved.

Chondrocytes expressing intracellular collagen type II enter the cell cycle and co-express collagen type I in monolayer culture.

For autologous chondrocyte transplantation, articular chondrocytes are harvested from cartilage tissue and expanded in vitro in monolayer culture. We aimed to characterize with a cellular resolution the synthesis of collagen type II (COL2) and collagen type I (COL1) during expansion in order to further understand why these cells lose the potential to form cartilage tissue when re-introduced into a microenvironment that supports chondrogenesis. During expansion for six passages, levels of transcripts encoding COL2 decreased to <0.1%, whereas transcript levels encoding COL1 increased 370-fold as compared to primary chondrocytes. Flow cytometry for intracellular proteins revealed that chondrocytes acquired a COL2/COL1-double positive phenotype during expansion, and the COL2 positive cells were able to enter the cell cycle. While the fraction of COL2 positive cells decreased from 70% to <2% in primary chondrocytes to passage six cells, the fraction of COL1 positive cells increased from <1% to >95%. In parallel to the decrease of the fraction of COL2 positive cells, the cells' potential to form cartilage-like tissue in pellet cultures steadily decreased. Intracellular staining for COL2 enables for characterization of chondrocyte lineage cells in more detail with a cellular resolution, and it may allow predicting the effectiveness of expanded chondrocytes to form cartilage-like tissue.

A computer-assisted microscopic analysis of bone tissue developed inside a polyactive plymer implanted into an equine articular surface

One of the most promising applications for the restoration of small or moderately sized focal articular lesions is mosaicplasty (MP). Although recurrent hemarthrosis is a rare complication after MP, recently, various strategies have been designed to find an effective filling material to prevent postoperative bleeding from the donor site. The porous biodegradable polymer Polyactive (PA; a polyethylene glycol terephthalate - polybutylene terephthalate copolymer) represents a promising solution in this respect. A histological evaluation of the longterm PA-filled donor sites obtained from 10 experimental horses was performed. In this study, attention was primarily focused on the bone tissue developed in the plug. A computer-assisted image analysis and quantitative polarized light microscopic measurements of decalcified, longitudinally sectioned, dimethylmethylene blue (DMMB)- and picrosirius red (PS) stained sections revealed that the coverage area of the bone trabecules in the PA-filled donor tunnels was substantially (25%) enlarged compared to the neighboring cancellous bone. For this quantification, identical ROIs (regions of interest) were used and compared. The birefringence retardation values were also measured with a polarized light microscope using monochromatic light. Identical retardation values could be recorded from the bone trabeculae developed in the PA and in the neighboring bone, which indicates that the collagen orientation pattern does not differ significantly among these bone trabecules. Based on our new data, we speculate that PA promotes bone formation, and some of the currently identified degradation products of PA may enhance osteo-conduction and osteoinduction inside the donor canal.