Antimicrobial Silver Multilayer Coating for Prevention of Bacterial Colonization of Orthopedic Implants.

Due to increasing rates of periprosthetic joint infections (PJI), new approaches are needed to minimize the infection risk. The first goal of this study was to modify a well-established infection model to test surface-active antimicrobial systems. The second goal was to evaluate the antimicrobial activity of a silver multilayer (SML) coating. In vitro tests with SML items showed a >4 Log reduction in a proliferation assay and a 2.2 Log reduction in an agar immersion test (7 d). In the in vivo model blank and SML coated K-wires were seeded with ~2 × 104 CFU of a methicillin-sensitive Staphylococcus epidermidis (MSSE) and inserted into the intramedullary tibial canal of rabbits. After 7 days, the animals were sacrificed and a clinical, microbiological and histological analysis was performed. Microbiology showed a 1.6 Log pathogen reduction on the surface of SML items (p = 0.022) and in loosely attached tissue (p = 0.012). In the SML group 7 of 12 SML items were completely free of pathogens (cure rate = 58%, p = 0.002), while only 1 of 12 blank items were free of pathogens (cure rate = 8%, p = 0.110). No silver was detected in the blood or urine of the SML treated animals and only scarcely in the liver or adjacent lymph nodes. In summary, an in vivo infection model to test implants with bacterial pre-incubation was established and the antimicrobial activity of the SML coating was successfully proven.

Safety of intradiscal injection and biocompatibility of polyester amide microspheres in a canine model predisposed to intervertebral disc degeneration.

Repair of degenerated intervertebral discs (IVD) might be established via intradiscal delivery of biologic therapies. Polyester amide polymers (PEA) were evaluated for in vitro cytotoxicity and in vivo biocompatibility, and thereafter intradiscal application of PEA microspheres (PEAMs) in a canine model predisposed to IVD degeneration at long-term (6 months) follow-up. PEA extracts did not induce cytotoxicity in mouse fibroblast cells (microscopy and XTT assay), while a slight foreign body reaction was demonstrated by histopathology after intramuscular implantation in rabbits. Intradiscal injection of a volume of 40 µL through 26 and 27G needles induced no degenerative changes in acanine model susceptible to IVD disease. Although sham-injected IVDs showed increased CAV1 expression compared with noninjected IVDs, which may indicate increased cell senescence, these findings were not supported by immunohistochemistry, biomolecular analysis of genes related to apoptosis, biochemical and histopathological results. PEAM-injected IVDs showed significantly higher BAX/BCL2 ratio vs sham-injected IVDs suggestive of an anti-apoptotic effect of the PEAMs. These findings were not supported by other analyses (clinical signs, disc height index, T2 values, biomolecular and biochemical analyses, and IVD histopathology). PEAs showed a good cytocompatibility and biocompatibility. PEAMs are considered safe sustained release systems for intradiscal delivery of biological treatments. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 707-714, 2017.

Applicability of a newly developed bioassay for determining bioactivity of anti-inflammatory compounds in release studies--celecoxib and triamcinolone acetonide released from novel PLGA-based microspheres.

PURPOSE: To develop a bio-assay for measuring long-term bioactivity of released anti-inflammatory compounds and to test the bioactivity of celecoxib (CXB) and triamcinolone acetonide (TA) released from a new PLGA-based microsphere platform. METHODS: Human osteoarthritic chondrocytes were plated according to standardized procedures after batch-wise harvest and cultured for 3 days to prevent cell confluency and changes in cell behaviour. Prostaglandin E2 (PGE2) production stimulated by TNFα was used as a parameter of inflammation. A novel microsphere platform based on PTE-functionalised PLGA was used to incorporate CXB and TA. Loaded microspheres were added to transwells overlying the cells, with transfer of the wells to new cell cultures every 3 days. Inhibition of PGE2 production was determined over a period of 21 days. RESULTS: PLGA(75:25)-PTE microspheres were prepared and loaded with CXB and TA at 86 and 97% loading efficiency, respectively. In the bioactivity assay, PGE2 levels induced by TNFα were reduced to an average of 30% using microspheres loaded with 0.1 nmol CXB per transwell; with microspheres loaded with 0.1 nmol TA, PGE2 production was initially reduced to 3% and gradually recovered to 30% reduction. At 1 nmol loading, PGE2 was inhibited to 0-7% for CXB-loaded microspheres, and 0-28% for TA-loaded microspheres. CONCLUSIONS: We present a novel sustained release bioactivity assay which provides an essential link between in vitro buffer-based release kinetics and in vivo application. Novel PLGA-based microspheres loaded with TA and CXB showed efficient anti-inflammatory effects over time.

Hindered amine light stabilizers: An alternative for radiation cross-linked UHMwPE implants.

To reduce wear UHMwPE implants are commonly cross-linked by use of gamma or e-beam irradiation. After irradiation however, radicals are still present that may cause oxidative degradation of the polymer. A way to reduce oxidative degradation could be to add a stabilizer to the polymer. Currently Vitamin E is the state of the art in stabilizing irradiation cross-linked UHMwPE implants. However, this stabilizer has some drawbacks. It interferes with the cross-link chemistry, leading to a lower cross-link density, it will be consumed and it results in the discolouration of the UHMwPE compound due to Vitamin E conversion products. It has been shown that all these drawbacks can be overcome by using HALS stabilizers. Due to their different mechanism of action, HALS stabilizers do not interfere with the cross-link chemistry. As part of the stabilization mechanism the radical scavenging molecules are actually regenerated, which allows for using a lower total concentration. The HALS conversion products do not result in discoloration of the polymer compound. Although their biocompatibilities still have to be evaluated, HALS stabilizers are potential alternative stabilizers for UHMwPE implants.

Characterization of esterified hyaluronan-gelatin polymer composites suitable for chondrogenic differentiation of mesenchymal stem cells.

Composite scaffolds of homogeneously mixed esterified hyaluronan (HY) and gelatin (G) were manufactured with variable component compositions (HY100%; HY95%/G5%; HY70%/G30%). The goals of this study were to analyze the produced composite scaffolds using physical and chemical methods, for example, scanning electron microscopy, IR-spectroscopy, water contact angle, protein assay, and tensile testing as well as to assess the effects of adding gelatin to the composite scaffolds on attachment, proliferation, and chondrogenic differentiation of human mesenchymal stem cells. Numbers of attached cells were significantly higher on the composite material compared to pure hyaluronan at different time points of two-dimensional or three-dimensional cell culture (p< 0.02). In composite scaffolds, a significantly greater amount of cartilage-specific extracellular matrix components was deposited after 28 days in culture (glycosaminoglycan: p < 0.001; collagen: p < 0.001) as compared with 100% hyaluronan scaffolds. Additionally, gelatin-containing composite scaffolds displayed stronger promotion of collagen type II expression than pure hyaluronan scaffolds. The mechanism, based on which gelatin influences cell adhesion, was examined. The effect was inhibited by collagenase treatment of the composites or by addition of alpha5beta1-integrin blocking antibodies to the cell suspension. In summary, the results describe the establishment of a class of composite polymer scaffolds, consisting of esterified hyaluronan and gelatin, which are potentially useful for cell-based tissue engineering approaches using mesenchymal stem cells for chondrogenic differentiation.

Biomaterials/scaffolds. Design of bioactive, multiphasic PCL/collagen type I and type II-PCL-TCP/collagen composite scaffolds for functional tissue engineering of osteochondral repair tissue by using electrospinning and FDM techniques.

Current clinical therapies for traumatic or chronic injuries involving osteochondral tissue result in temporary pain reduction and filling of the defect but with biomechanically inferior repair tissue. Tissue engineering of osteochondral repair tissue using autologous cells and bioactive biomaterials has the potential to overcome the current limitations and results in native-like repair tissue with good integration capabilities. For this reason, we applied two modem biomaterial design techniques, namely, electrospinning and fused deposition modeling (FDM), to produce bioactive poly(epsilon-caprolactone)/collagen (PCL/Col) type I and type II-PCL-tri-calcium phosphate (TCP)/Col composites for precursor cell-based osteochondral repair. The application of these two design techniques (electrospinning and FDM) allowed us to specifically produce the a suitable three-dimensional (3D) environment for the cells to grow into a particular tissue (cartilage and bone) in vitro prior to in vivo implantation. We hypothesize that our new designed biomaterials, seeded with autologous bone marrow-derived precursor cells, in combination with bioreactor-stimulated cell-culture techniques can be used to produce clinically relevant osteochondral repair tissue.

Cyclic, mechanical compression enhances chondrogenesis of mesenchymal progenitor cells in tissue engineering scaffolds.

The effects of cyclic, mechanical compression on human bone marrow-derived mesenchymal progenitor cells undergoing chondrogenic differentiation were examined in this study. Mesenchymal progenitor cells were injected into cylindrical biodegradable scaffolds (hyaluronan-gelatin composites), cultured in a defined, serum-free chondrogenic medium and subjected to cyclic, mechanical compression. Scaffolds were loaded for 4 hours daily in the first 7 days of culture. At 1, 7, 14 and 21 days of culture, scaffolds were harvested for reverse transcriptase Polymerase Chain Reaction (RT-PCR), histology, quantitative DNA, proteoglycan and collagen analysis. Scaffolds loaded for 7 days showed a significant upregulation especially of chondrogenic markers (type II collagen, aggrecan; p<0.0001). No significant difference could be found for DNA content between loaded samples and unloaded controls. At day 1 in culture no significant differences in proteoglycan- and collagen contents could be detected between unloaded and loaded samples. After 21 days the proteoglycan (p<0.001) and collagen contents (p<0.0001) were significantly higher in the loaded samples compared to unloaded controls. By histological analysis (toluidine blue) a higher amount of proteoglycan-rich, extracellular matrix production throughout the matrix could be detected for loaded samples compared to unloaded controls. This study indicates that cyclic, mechanical compression enhances the expression of chondrogenic markers in mesenchymal progenitor cells differentiated in vitro resulting in an increased cartilaginous matrix formation, and suggests that mechanical forces may play an important role in cartilage repair.

Characterization of recombinant soluble carcinoembryonic antigen cell adhesion molecule 1.

Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is a type 1 transmembrane, homotypic cell adhesion protein expressed on epithelial and hematopoietic cells. CEACAM1 has four major isoforms with three or four immunoglobulin (Ig)-like ectodomains and either long or short cytoplasmic domains. In a 3D model of breast epithelial cell morphogenesis, CEACAM1 plays an essential role in lumen formation [J. Cell Sci. 112 (1999) 4193]. Two soluble ectodomain isoforms of CEACAM1 expressed in myeloma cells were immunologically active and highly glycosylated. The molecular weights of the 3-ecto- and 4-ectodomain isoforms were 90 and 110kDa, respectively, and monomers by sedimentation equilibrium centrifugation. Both isoforms were prolate ellipsoids with axial ratios of 6 for the 3-ecto- and 8 for 4-ectodomain isoforms, respectively, by size exclusion chromatography and analytical ultracentrifugation. Both isoforms caused a significant reduction in lumen formation when tested in the 3D model culture system.

Influence of different collagen species on physico-chemical properties of crosslinked collagen matrices.

Collagen-based scaffolds are appealing products for the repair of cartilage defects using tissue engineering strategies. The present study investigated the species-related differences of collagen scaffolds with and without 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS)-crosslinking. Resistance against collagenase digestion, swelling ratio, amino acid sequence, shrinkage temperature, ultrastructural matrix morphology, crosslinking density and stress-strain characteristics were determined to evaluate the physico-chemical properties of equine- and bovine-collagen-based scaffolds. Three-factor ANOVA analysis revealed a highly significant effect of collagen type (p=0.0001), crosslinking (p=0.0001) and time (p=0.0001) on degradation of the collagen samples by collagenase treatment. Crosslinked equine collagen samples showed a significantly reduced swelling ratio compared to bovine collagen samples (p< 0.0001). The amino acid composition of equine collagen revealed a higher amount of hydroxylysine and lysine. Shrinkage temperatures of non-crosslinked samples showed a significant difference between equine (60 degrees C) and bovine collagen (57 degrees C). Three-factor ANOVA analysis revealed a highly significant effect of collagen type (p=0.0001), crosslinking (p=0.0001) and matrix condition (p=0.0001) on rupture strength measured by stress-strain analysis. The ultrastructure, the crosslinking density and the strain at rupture between collagen matrices of both species showed no significant differences. For tissue engineering purposes, the higher enzymatic stability, the higher form stability, as well as the lower risk of transmissible disease make the case for considering equine-based collagen. This study also indicates that results obtained for scaffolds based on a certain collagen species may not be transferable to scaffolds based on another, because of the differing physico-chemical properties.

CEACAM1, a cell-cell adhesion molecule, directly associates with annexin II in a three-dimensional model of mammary morphogenesis.

The epithelial cell adhesion molecule CEACAM1 (carcinoembryonic antigen cell adhesion molecule-1) is down-regulated in colon, prostate, breast, and liver cancer. Here we show that CEACAM1-4S, a splice form with four Ig-like ectodomains and a short cytoplasmic domain (14 amino acids), directly associates with annexin II, a lipid raft-associated molecule, which is also down-regulated in many cancers. Annexin II was identified using a glutathione S-transferase pull-down assay in which the cytoplasmic domain of CEACAM-4S was fused to glutathione S-transferase, the fusion protein was incubated with cell lysates, and isolated proteins were sequenced by mass spectrometry. The interaction was confirmed first by reciprocal immunoprecipitations using anti-CEACAM1 and anti-annexin II antibodies and second by confocal laser microscopy showing co-localization of CEACAM1 with annexin II in mammary epithelial cells grown in Matrigel. In addition, CEACAM1 co-localized with p11, a component of the tetrameric AIIt complex at the plasma membrane, and with annexin II in secretory vesicles. Immobilized, oriented peptides from the cytoplasmic domain of CEACAM1-4S were shown to directly associate with bovine AIIt, which is 98% homologous to human AIIt, with average KD values of about 30 nM using surface plasmon resonance, demonstrating direct binding of functionally relevant AIIt to the cytoplasmic domain of CEACAM1-4S.

A carcinoembryonic antigen-specific diabody produced in tobacco.

The feasibility of using tobacco for production of a recombinant antibody (T84.66/GS8 diabody) directed against the carcinoembryonic antigen (CEA) and used for tumor imaging was investigated. Two constructs were generated for targeting the protein either to the apoplast or to the endoplasmic reticulum. Expression of the diabody in tobacco leaves after vacuum-assisted infiltration of engineered Agrobacteria (agro-infiltration) and in regenerated transgenic tobacco plants was analyzed and compared. Results in terms of protein expression and accumulation between both systems showed a good correlation. His6-tagged T84.66 diabody was readily purified from agro-infiltrated tobacco leaves and from transgenic plants by immobilized metal ion affinity chromatography. The purified protein was analyzed by polyacrylamide gel electrophoresis, Western blot, gel filtration, electrospray mass spectrometry, direct and competition ELISA, electrophoretic mobility shift assay, and staining of CEA-positive colon adenocarcinoma cell line LS174T. Our results demonstrate that tobacco is a competent production system for this clinically relevant diabody.