Assessment of implant surface and instrument insert changes due to instrumentation with different tips for ultrasonic-driven debridement.

BACKGROUND: To assess the changes of implant surfaces of different roughness after instrumentation with ultrasonic-driven scaler tips of different materials. METHODS: Experiments were performed on two moderately rough surfaces (I-Inicell® and II-SLA®), one surface without pre-treatment (III) and one smooth machined surface (IV). Scaler tips made of steel (A), PEEK (B), titanium (C), carbon (D) and resin (E) were used for instrumentation with a standardized pressure of 100 g for ten seconds and under continuous automatic motion. Each combination of scaler tip and implant surface was performed three times on 8 titanium discs. After instrumentation roughness was assessed by profilometry, morphological changes were assessed by scanning electron microscopy, and element distribution on the utmost surface by energy dispersive X-ray spectroscopy. RESULTS: The surface roughness of discs I and II were significantly reduced by instrumentation with all tips except E. For disc III and IV roughness was enhanced by tip A and C and, only for IV, by tip D. Instrumentation with tips B, D and E left extensive residuals on surface I, II and III. The element analysis of these deposits proved consistent with the elemental composition of the respective tip materials. CONCLUSION: All ultrasonic instruments led to microscopic alterations of all types of implants surfaces assessed in the present study. The least change of implant surfaces might result from resin or carbon tips on machined surfaces.

Bone tissue engineering using adipose-derived stem cells and endothelial cells: Effects of the cell ratio.

The microvascular endothelial network is essential for bone formation and regeneration. In this context, endothelial cells not only support vascularization but also influence bone physiology via cell contact-dependent mechanisms. In order to improve vascularization and osteogenesis in tissue engineering applications, several strategies have been developed. One promising approach is the coapplication of endothelial and adipose derived stem cells (ADSCs). In this study, we aimed at investigating the best ratio of human umbilical vein endothelial cells (HUVECs) and osteogenic differentiated ADSCs with regard to proliferation, apoptosis, osteogenesis and angiogenesis. For this purpose, cocultures of ADSCs and HUVECs with ratios of 25%:75%, 50%:50% and 75%:25% were performed. We were able to prove that cocultivation supports proliferation whereas apoptosis was unidirectional decreased in cocultured HUVECs mediated by a p-BAD-dependent mechanism. Moreover, coculturing ADSCs and HUVECs stimulated matrix mineralization and the activity of alkaline phosphatase (ALP). Increased gene expression of the proangiogenic markers eNOS, Flt, Ang2 and MMP3 as well as sprouting phenomena in matrigel assays proved the angiogenic potential of the coculture. In summary, coculturing ADSCs and HUVECs stimulates proliferation, cell survival, osteogenesis and angiogenesis particularly in the 50%:50% coculture.

The Adipose-Derived Stem Cell and Endothelial Cell Coculture System-Role of Growth Factors?

Adequate vascularization is a fundamental prerequisite for bone regeneration, formation and tissue engineering applications. Endothelialization of scaffold materials is a promising strategy to support neovascularization and bone tissue formation. Besides oxygen and nutrition supply, the endothelial network plays an important role concerning osteogenic differentiation of osteoprogenitor cells and consecutive bone formation. In this study we aimed to enhance the growth stimulating, proangiogenic and osteogenic features of the ADSC and HUVEC coculture system by means of VEGFA165 and BMP2 application. We were able to show that sprouting phenomena and osteogenic differentiation were enhanced in the ADSC/HUVEC coculture. Furthermore, apoptosis was unidirectionally decreased in HUVECs, but these effects were not further enhanced upon VEGFA165 or BMP2 application. In summary, the ADSC/HUVEC coculture system per se is a powerful tool for bone tissue engineering applications.

Enhanced vascularization andde novotissue formation in hydrogels made of engineered RGD-tagged spider silk proteins in the arteriovenous loop model.

Due to its low immunogenic potential and the possibility to fine-tune their properties, materials made of recombinant engineered spider silks are promising candidates for tissue engineering applications. However, vascularization of silk-based scaffolds is one critical step for the generation of bioartificial tissues and consequently for clinical application. To circumvent insufficient vascularization, the surgically induced angiogenesis by means of arteriovenous loops (AVL) represents a highly effective methodology. Here, previously established hydrogels consisting of nano-fibrillary recombinant eADF4(C16) were transferred into Teflon isolation chambers and vascularized in the rat AVL model over 4 weeks. To improve vascularization, also RGD-tagged eADF4(C16) hydrogels were implanted in the AVL model over 2 and 4 weeks. Thereafter, the specimen were explanted and analyzed using histology and microcomputed tomography. We were able to confirm biocompatibility and tissue formation over time. Functionalizing eADF4(C16) with RGD-motifs improved hydrogel stability and enhanced vascularization even outperforming other hydrogels, such as fibrin. This study demonstrates that the scaffold ultrastructure as well as biofunctionalization with RGD-motifs are powerful tools to optimize silk-based biomaterials for tissue engineering applications.

Human Umbilical Vein Endothelial Cell Support Bone Formation of Adipose-Derived Stem Cell-Loaded and 3D-Printed Osteogenic Matrices in the Arteriovenous Loop Model.

Introduction: For the regeneration of large volume tissue defects, the interaction between angiogenesis and osteogenesis is a crucial prerequisite. The surgically induced angiogenesis by means of an arteriovenous loop (AVL), is a powerful methodology to enhance vascularization of osteogenic matrices. Moreover, the AVL increases oxygen and nutrition supply, thereby supporting cell survival as well as tissue formation. Adipose-derived stem cells (ADSCs) are interesting cell sources because of their simple isolation, expansion, and their osteogenic potential. This study targets to investigate the coimplantation of human ADSCs after osteogenic differentiation and human umbilical vein endothelial cells (HUVECs), embedded in a vascularized osteogenic matrix of hydroxyapatite (HAp) ceramic for bone tissue engineering. Materials and Methods: An osteogenic matrix consisting of HAp granules and fibrin has been vascularized by means of an AVL. Trials in experimental groups of four settings were performed. Control experiments without any cells (A) and three cell-loaded groups using HUVECs (B), ADSCs (C), as well as the combination of ADSCs and HUVECs (D) were performed. The scaffolds were implanted in a porous titanium chamber, fixed subcutaneously in the hind leg of immunodeficient Rowett Nude rats and explanted after 6 weeks. Results: In all groups, the osteogenic matrix was strongly vascularized. Moreover, remodeling processes and bone formation in the cell-containing groups with more bone in the coimplantation group were proved successful. Conclusion: Vascularization and bone formation of osteogenic matrices consisting of ADSCs and HUVECs in the rat AVL model could be demonstrated successfully for the first time. Hence, the coimplantation of differentiated ADSCs with HUVECs may therefore be considered as a promising approach for bone tissue engineering.

Risks and Benefits of Glioblastoma Resection in Older Adults: A Retrospective Austrian Multicenter Study.

OBJECTIVE: To assess the prognostic profile, clinical outcome, treatment-associated morbidity, and treatment burden of elderly patients with glioblastoma (GBM) undergoing microsurgical tumor resection as part of contemporary treatment algorithms. METHODS: We retrospectively identified patients with GBM ≥65 years of age who were treated by resection at 2 neuro-oncology centers. Survival was assessed by Kaplan-Meier analyses; log-rank tests identified prognostic factors. RESULTS: The study population included 160 patients (mean age, 73.1 ± 5.1 years), and the median contrast-enhancing tumor volume was 31.0 cm3. Biomarker analyses revealed O(6)-methylguanine-DNA methyltransferase-promoter methylation in 62.7% and wild-type isocitrate dehydrogenase in 97.5% of tumors. The median extent of resection (EOR) was 92.3%, surgical complications were noted in 10.0% of patients, and the median postoperative hospitalization period was 8 days. Most patients (60.0%) received adjuvant radio-/chemotherapy. The overall treatment-associated morbidity was 30.6%. The median progression-free and overall survival were 5.4 months (95% confidence interval [CI], 4.6-6.4 months) and 10.0 months (95% CI, 7.9-11.7 months). The strongest predictors for favorable outcome were patient age ≤73.0 years (P = 0.0083), preoperative Karnofsky Performance Status Scale score ≥80% (P = 0.0179), postoperative modified Rankin Scale score ≤1 (P < 0.0001), adjuvant treatment (P < 0.0001), and no treatment-associated morbidity (P = 0.0478). Increased EOR did not correlate with survival (P = 0.5046), but correlated significantly with treatment-associated morbidity (P = 0.0031). CONCLUSIONS: Clinical outcome for elderly patients with GBM remains limited. Nonetheless, the observed treatment-associated morbidity and treatment burden were moderate in the patients, and patient age and performance status remained the strongest predictors for survival. The risks and benefits of tumor resection in the age of biomarker-adjusted treatment concepts require further prospective evaluation.

Designing an improved T-cell mobilising CXCL10 mutant through enhanced GAG binding affinity.

The chemokine CXCL10 is released by a plethora of cells, including immune and metastatic cancer cells, following stimulation with interferon-gamma. It acts via its GPC receptor on T-cells attracting them to various target tissues. Glycosaminoglycans (GAGs) are regarded as co-receptors of chemokines, which enable the establishment of a chemotactic gradient for target cell migration. We have engineered human CXCL10 towards improved T-cell mobilisation by implementing a single site-directed mutation N20K into the protein, which leads to a higher GAG binding affinity compared to the wild type. Interestingly, this mutation not only increased T-cell migration in a transendothelial migration assay, the mutant intensified T-cell chemotaxis also in a Boyden chamber set-up thereby indicating a strong role of T-cell-localised GAGs on leukocyte migration. A CXCL10 mutant with increased GAG-binding affinity could therefore potentially serve as a T-cell mobiliser in pathological conditions where the immune surveillance of the target tissue is impaired, as is the case for most solid tumors.

Molecular dynamics simulations of the chemokine CCL2 in complex with pull down-derived heparan sulfate hexasaccharides.

BACKGROUND: Binding of chemokines to glycosaminoglycans (GAGs) is a crucial step in leukocyte recruitment to inflamed tissues. METHODS: A disaccharide compositional analysis of the HS dp6 fraction in combination with MS analysis of the CCL2-depleted dp6 fraction was the basis for target GAG ligand structure suggestions. Four experimentally-derived heparan sulfate hexasaccharides, two potentially chemokine-specific and two unspecific, have been docked to CCL2. Subsequent 300 ns molecular dynamics simulations were used to improve the docked complexes. RESULTS: Hexasaccharides with four sulfations and no acetylations are suggested for selective and high affinity chemokine binding. Using the Antithromin-III/heparin complex as positive control for docking, we were able to recover the correct complex structure only if the previously liganded ATIII structure was used as input. Since the liganded structure is not known for a CCL2-GAG complex, we investigated if molecular dynamics simulations could improve initial docking results. We found that all four GAG oligosaccharides ended up in close contact with the known binding residues after about 100 ns simulation time. CONCLUSIONS: A discrimination of specific vs. unspecific CCL2 GAG ligands is not possible by this approach. Long-time molecular dynamics simulations are, however, well suited to capture the delicate enthalpy/entropy balance of GAG binding and improve results obtained from docking. GENERAL SIGNIFICANCE: With the comparison of two methods, MS-based ligand identification and molecular modelling, we have shown the current limitations of our molecular understanding of complex ligand binding which is could be due to the numerical inaccessibility of ligand-induced protein conformational changes.

Intrinsic Vascularization of Recombinant eADF4(C16) Spider Silk Matrices in the Arteriovenous Loop Model.

The surgically induced angiogenesis by means of arteriovenous (AV) loops represents a powerful method to significantly enhance vascularization of biomaterials. Regarding tissue engineering applications, spider silk is a promising biomaterial with a good biocompatibility and slow biodegradation. This study aims at investigating vascularization as well as de novo tissue formation of fibrous matrices made of electro-spun (ES) or wet-spun (WS) engineered ADF4(C16) spider silks in the rat AV loop model. Either ES or WS spider silk fibrous matrices were filled into Teflon chambers. Intrinsic vascularization was induced by means of an AV loop. After 4 weeks of vascularization, de novo tissue formation and biocompatibility were analyzed. Regardless of their significantly differing fiber diameters, both ES and WS eADF4(C16) fiber matrices displayed a good biocompatibility and initiated de novo tissue formation as well as vessel formation. Both matrices demonstrated partial vascularization originating from the AV loop, with more vessels in spider silk matrices with lower fiber diameters. We were able to demonstrate intrinsic vascularization of spider silk fibrous matrices by means of the AV loop. Moreover, our study indicates that the adjustment of the fiber diameter of engineered spider silks enables new possibilities to optimize vascularization. Impact Statement Spider silk is a promising biomaterial demonstrating excellent biocompatibility and biodegradation. Biotechnology allows the high-volume production of recombinant spider silk proteins, such as eADF4(C16), with the required purity for biomedical applications. In this study, eADF4(C16) fibrous matrices were produced by either electro- or wet-spinning, resulting in different fiber diameters. Forming an arteriovenous fistula, surgical vascularization of the scaffolds was induced. After 4 weeks, both silks demonstrated a good biocompatibility and tissue formation. The thinner electro-spun fibers displayed a faster biodegradation and vascularization, indicating that the adjustment of the fiber diameter is a valuable tool to fine-tune vascularization and biodegradation.

Optimizing MSLT Specificity in Narcolepsy With Cataplexy.

Study Objectives: Multiple sleep onset rapid eye movement (R) periods (SOREMPs) and a mean sleep latency of ≤8 minutes on the multiple sleep latency test (MSLT) are diagnostic criteria of narcolepsy (NC), but also occur in other conditions with increased sleep pressure, including insufficient sleep syndrome (ISS), sleep-disordered breathing (SDB), or Parkinson's disease (PD). These false positives are common, may create diagnostic uncertainty, and highlight the need for complementary MSLT measures with high specificity for NC. Methods: Detailed analysis of MSLT findings in 56 NC, 83 PD, 89 SDB, and 23 ISS patients, using receiver operating characteristic curves. Results: A positive MSLT (mean sleep latency ≤ 8.0 minutes and ≥2 SOREMPs) was found in 53 NC (95%), 1 PD (1%), 8 SDB (9%), and 12 ISS patients (52%). MSLT-based differentiation between NC and non-NC patients was best when applying a mean R latency of ≤5 minutes (sensitivity/specificity/positive predictive value [PPV]: 49%/95%/96%) or a mean percentage of sleep stage R ≥ 40% (sensitivity/specificity/PPV: 60%/100%/100%) as cutoffs. When analyzing all 252 naps with SOREMPs in isolation, the combination of both R latency of ≤5 minutes and R percentage of ≥50% yielded a sensitivity/specificity/PPV of 50%/99%/99%. In addition, a sleep stage sequence with R occurring prior to N2 was more common in NC than in non-NC (71% vs. 32%, p < .001), and in combination with R percentage of ≥50% yielded a sensitivity/specificity/PPV of 53%/96%/97%. Conclusions: A better characterization of R sleep by latency, duration, and sleep stage sequence facilitates detection of false positives and, hence, contributes to a higher MSLT specificity in NC.

Designing a mutant CCL2-HSA chimera with high glycosaminoglycan-binding affinity and selectivity.

Chemokines like CCL2 mediate leukocyte migration to inflammatory sites by binding to G-protein coupled receptors on the target cell as well as to glycosaminoglycans (GAGs) on the endothelium of the inflamed tissue. We have recently shown that the dominant-negative Met-CCL2 mutant Y13A/S21K/Q23R with improved GAG binding affinity is highly bio-active in several animal models of inflammatory diseases. For chronic indications, we have performed here a fusion to human serum albumin (HSA) in order to extend the serum half-life of the chemokine mutant. To compensate a potential drop in GAG-binding affinity due to steric hindrance by HSA, a series of novel CCL2 mutants was generated with additional basic amino acids which were genetically introduced at sites oriented towards the GAG ligand. From this set of mutants, the Met-CCL2 variant Y13A/N17K/S21K/Q23K/S34K exhibited high GAG-binding affinity and a similar selectivity as wild type (wt) CCL2. From a set of different HSA-chemokine chimeric constructs, the linked HSA(C34A)(Gly)4Ser-Met-CCL2(Y13A/N17K/S21K/Q23K/S34K) fusion protein was found to show the best overall GAG-binding characteristics. Molecular modeling demonstrated an energetically beneficial fold of this novel protein chimera. This was experimentally supported by GdmCl-induced unfolding studies, in which the fusion construct exhibited a well-defined secondary structure and a transition point significantly higher than both the wt and the unfused CCL2 mutant protein. Unlike the wt chemokine, the quaternary structure of the HSA-fusion protein is monomeric according to size-exclusion chromatography experiments. In competition experiments, the HSA-fusion construct displaced only two of seven unrelated chemokines from heparan sulfate, whereas the unfused CCL2 mutant protein displaced five other chemokines. The most effective concentration of the HSA-fusion protein in inhibiting CCL2-mediated monocyte attachment to endothelial cells, as detected in the flow chamber, was 8.6 µg/ml. This novel HSA-fusion protein exhibits not only high affinity but also selective displacement of chemokines from GAGs binding. HSA is therefore proposed to be a highly promising scaffold candidate for therapeutic, GAG-targeting chemokine mutants.

Bacterial expression and functional reconstitution of human heparanase.

Human heparanase is a heparan sulfate degrading enzyme located in the extracellular matrix playing a decisive role in angiogenesis and tumor metastasis. Translated as a 65 kDa inactive prae-form, the protein is processed into an 8 kDa and a 50 kDa subunit which form a non-covalently associated active heterodimer. We have expressed the two subunits separately in Escherichia coli which yielded active human heparanase upon reconstitution. The two purified subunits folded independently and secondary structure analysis by far-UV CD spectroscopy gave 33.1/11.1% α/ß content for the 50 kDa subunit and 6.9/49% α/ß content for the 8 kDa subunit. This heparanase expression system is easy and can be used for efficient screening for enzyme inhibitors.