Spatially Resolved Molecular Analysis of Host Response to Medical Device Implantation Using the 3D OrbiSIMS Highlights a Critical Role for Lipids.

A key goal for implanted medical devices is that they do not elicit a detrimental immune response. Macrophages play critical roles in the modulation of the host immune response and are the cells responsible for persistent inflammatory reactions to implanted biomaterials. Two novel immune-instructive polymers that stimulate pro- or anti-inflammatory responses from macrophages in vitro are investigated. These also modulate in vivo foreign body responses (FBR) when implanted subcutaneously in mice. Immunofluorescent staining of tissue abutting the polymer reveals responses consistent with pro- or anti-inflammatory responses previously described for these polymers. Three Dimensional OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) analysis to spatially characterize the metabolites in the tissue surrounding the implant, providing molecular histology insight into the metabolite response in the host is applied. For the pro-inflammatory polymer, monoacylglycerols (MG) and diacylglycerols (DG) are observed at increased intensity, while for the anti-inflammatory coating, the number of phospholipid species detected decreased, and pyridine and pyrimidine levels are elevated. Small molecule signatures from single-cell studies of M2 macrophages in vitro correlate with the in vivo observations, suggesting potential for prediction. Metabolite characterization by the 3D OrbiSIMS is shown to provide insight into the mechanism of bio-instructive materials as medical devices and to inform on the FBR to biomaterials.

Label-free Chemical Characterization of Polarized Immune Cells in vitro and Host Response to Implanted Bio-instructive Polymers in vivo Using 3D OrbiSIMS.

The Three-dimensional OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) is a secondary ion mass spectrometry instrument, a combination of a Time of Flight (ToF) instrument with an Orbitrap analyzer. The 3D OrbiSIMS technique is a powerful tool for metabolic profiling in biological samples. This can be achieved at subcellular spatial resolution, high sensitivity, and high mass-resolving power coupled with MS/MS analysis. Characterizing the metabolic signature of macrophage subsets within tissue sections offers great potential to understand the response of the human immune system to implanted biomaterials. Here, we describe a protocol for direct analysis of individual cells after in vitro differentiation of naïve monocytes into M1 and M2 phenotypes using cytokines. As a first step in vivo, we investigate explanted silicon catheter sections as a medical device in a rodent model of foreign body response. Protocols are presented to allow the host response to different immune instructive materials to be compared. The first demonstration of this capability illustrates the great potential of direct cell and tissue section analysis for in situ metabolite profiling to probe functional phenotypes using molecular signatures. Details of the in vitro cell approach, materials, sample preparation, and explant handling are presented, in addition to the data acquisition approaches and the data analysis pipelines required to achieve useful interpretation of these complex spectra. This method is useful for in situ characterization of both in vitro single cells and ex vivo tissue sections. This will aid the understanding of the immune response to medical implants by informing the design of immune-instructive biomaterials with positive interactions. It can also be used to investigate a broad range of other clinically relevant therapeutics and immune dysregulations. Graphical overview.

Cefazolin Loaded Oxidized Regenerated Cellulose/Polycaprolactone Bilayered Composite for Use as Potential Antibacterial Dural Substitute.

Oxidized regenerated cellulose/polycaprolactone bilayered composite (ORC/PCL bilayered composite) was investigated for use as an antibacterial dural substitute. Cefazolin at the concentrations of 25, 50, 75 and 100 mg/mL was loaded in the ORC/PCL bilayered composite. Microstructure, density, thickness, tensile properties, cefazolin loading content, cefazolin releasing profile and antibacterial activity against S. aureus were measured. It was seen that the change in concentration of cefazolin loading affected the microstructure of the composite on the rough side, but not on the dense or smooth side. Cefazolin loaded ORC/PCL bilayered composite showed greater densities, but lower thickness, compared to those of drug unloaded composite. Tensile modulus was found to be greater and increased with increasing cefazolin loading, but tensile strength and strain at break were lower compared to the drug unloaded composite. In vitro cefazolin release in artificial cerebrospinal fluid (aCSF) consisted of initial burst release on day 1, followed by a constant small release of cefazolin. The antibacterial activity was observed to last for up to 4 days depending on the cefazolin loading. All these results suggested that ORC/PCL bilayered composite could be modified to serve as an antibiotic carrier for potential use as an antibacterial synthetic dura mater.

Single-Cell Metabolic Profiling of Macrophages Using 3D OrbiSIMS: Correlations with Phenotype.

Macrophages are important immune cells that respond to environmental cues acquiring a range of activation statuses represented by pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes at each end of their spectrum. Characterizing the metabolic signature (metabolic profiling) of different macrophage subsets is a powerful tool to understand the response of the human immune system to different stimuli. Here, the recently developed 3D OrbiSIMS instrument is applied to yield useful insight into the metabolome from individual cells after in vitro differentiation of macrophages into naïve, M1, and M2 phenotypes using different cytokines. This analysis strategy not only requires more than 6 orders of magnitude less sample than traditional mass spectrometry approaches but also allows the study of cell-to-cell variance. Characteristic metabolites in macrophage subsets are identified using a targeted lipid and data-driven multivariate approach highlighting amino acids and other small molecules. The diamino acids alanylasparagine and lipid sphingomyelin SM(d18/16:0) are uniquely found in M1 macrophages, while pyridine and pyrimidine are observed at increased intensity in M2 macrophages, findings which link to known biological pathways. The first demonstration of this capability illustrates the great potential of direct cell analysis for in situ metabolite profiling with the 3D OrbiSIMS to probe functional phenotype at the single-cell level using molecular signatures and to understand the response of the human body to implanted devices and immune diseases.

Evaluation of tissue ingrowth and reaction of a porous polyethylene block as an onlay bone graft in rabbit posterior mandible.

PURPOSE: A new form of porous polyethylene, characterized by higher porosity and pore interconnectivity, was developed for use as a tissue-integrated implant. This study evaluated the effectiveness of porous polyethylene blocks used as an onlay bone graft in rabbit mandible in terms of tissue reaction, bone ingrowth, fibrovascularization, and graft-bone interfacial integrity. METHODS: Twelve New Zealand white rabbits were randomized into 3 treatment groups according to the study period (4, 12, or 24 weeks). Cylindrical specimens measuring 5 mm in diameter and 4.5 mm in thickness were placed directly on the body of the mandible without bone bed decortication, fixed in place with a titanium screw, and covered with a collagen membrane. Histologic and histomorphometric analyses were done using hematoxylin and eosin-stained bone slices. Interfacial shear strength was tested to quantify graft-bone interfacial integrity. RESULTS: The porous polyethylene graft was observed to integrate with the mandibular bone and exhibited tissue-bridge connections. At all postoperative time points, it was noted that the host tissues had grown deep into the pores of the porous polyethylene in the direction from the interface to the center of the graft. Both fibrovascular tissue and bone were found within the pores, but most bone ingrowth was observed at the graft-mandibular bone interface. Bone ingrowth depth and interfacial shear strength were in the range of 2.76-3.89 mm and 1.11-1.43 MPa, respectively. No significant differences among post-implantation time points were found for tissue ingrowth percentage and interfacial shear strength (P>0.05). CONCLUSIONS: Within the limits of the study, the present study revealed that the new porous polyethylene did not provoke any adverse systemic reactions. The material promoted fibrovascularization and displayed osteoconductive and osteogenic properties within and outside the contact interface. Stable interfacial integration between the graft and bone also took place.

Performance evaluation of bilayer oxidized regenerated cellulose/poly ε-caprolactone knitted fabric-reinforced composites for dural substitution.

Ideally, alloplastic dural substitute should have functional properties resembling human dura mater and retain a watertight closure to prevent cerebrospinal leakage. Therefore, functional properties for successful dural closure application of newly developed bilayer oxidized regenerated cellulose knitted fabric/poly ε-caprolactone knitted fabric-reinforced composites were studied and compared with human cadaveric dura mater and three commercial dural substitutes including two collagen matrices and one synthetic poly-L-lactide patch. It was found that oxidized regenerated cellulose knitted fabric/poly ε-caprolactone knitted fabric-reinforced composites uniquely contained a bilayer structure consisting of micropores distributed within the relatively dense microstructure. Density, tensile properties and stitch tear strength of oxidized regenerated cellulose knitted fabric/poly ε-caprolactone knitted fabric-reinforced composites were found to be closed to human cadaveric dura mater than those of dense-type and porous-type dural substitutes. Water tightness performance in both sutured and non-sutured forms of oxidized regenerated cellulose knitted fabric/poly ε-caprolactone knitted fabric-reinforced composites was slightly inferior to human cadaveric dura mater, but still better than those of commercial dural substitutes. This study revealed that oxidized regenerated cellulose knitted fabric/poly ε-caprolactone knitted fabric-reinforced composite showed better functional properties than typical dural substitutes and was found to be a good candidate for being employed as a dural substitute. The role and relationship of both microstructure and the type of materials on the functional properties and water tightness of the dural substitutes were also elucidated.

Bilayer oxidized regenerated cellulose/poly ε-caprolactone knitted fabric-reinforced composite for use as an artificial dural substitute.

A novel bilayer knitted fabric-reinforced composite for potentially being used as a dural substitute was developed by solution infiltration of oxidized regenerated cellulose knitted fabric (ORC) with poly ε-caprolactone (PCL) solution at various concentrations ranging 10-40 g/100 mL. It was found that the density of all formulations did not differ significantly and was lower than that of the human dura. Microstructure of the samples typically comprised a bilayer structure having a nonporous PCL layer on one side and the ORC/PCL composite layer on another side. Tensile modulus and strength of the samples initially decreased with increasing PCL solution concentration for up to 20 g/100 mL and re-increased again with further increasing PCL solution concentration. Strain at break of all formulations were not significantly different. Watertight test revealed that all composites could prevent leakage at the pressure within the normal range of intracranial pressure. In vitro degradation study revealed that the weight loss percentage and change in tensile properties of all samples displayed biphasic profile comprising an initially rapid decrease and followed by a gradual decrease with incubation times afterward. Micro and macro porous channels were observed to be in situ generated in the composite layer by ORC dissolution and PCL resorption during degradation while nonporous layer remained relatively unchanged. The degradation rate was found to decrease with increasing PCL solution concentration. In vitro biocompatibility using alamar blue assay on selected samples showed that fibroblasts could attach and proliferate well at all incubation periods.

Influence of process parameters on the content of biomimetic calcium phosphate coating on titanium: a Taguchi analysis.

In this study, a statistical design of experimental methodology based on Taguchi orthogonal design has been used to study the effect of various processing parameters on the amount of calcium phosphate coating produced by such technique. Seven control factors with three levels each including sodium hydroxide concentration, pretreatment temperature, pretreatment time, cleaning method, coating time, coating temperature and surface area to solution volume ratio were studied. X-ray diffraction revealed that all the coatings consisted of the mixture of octacalcium phosphate (OCP) and hydroxyapatite (HA) and the presence of each phase depended on the process conditions used. Various content and size (-1-100 µm) of isolated spheroid particles with nanosized plate-like morphology deposited on the titanium surface or a continuous layer of plate-like nanocrystals having the plate thickness in the range of -100-300 nm and the plate width in the range of 3-8 µm were formed depending on the process conditions employed. The optimum condition of using sodium hydroxide concentration of 1 M, pretreatment temperature of 70 degrees C, pretreatment time of 24 h, cleaning by ultrasonic, coating time of 6 h, coating temperature of 50 degrees C and surface area to solution volume ratio of 32.74 for producing the greatest amount of the coating formed on the titanium surface was predicted and validated. In addition, coating temperature was found to be the dominant factor with the greatest contribution to the coating formation while coating time and cleaning method were significant factors. Other factors had negligible effects on the coating performance.