ABSTRACT
The conical screen mill (comill) is investigated as a dry-coating process for flowability and bulk density enhancement of pharmaceutical powders. In this study, the effectiveness of the comill is improved by using modified screens with reduced open area. In comparison to the screens provided by the comill manufacturer, the modified screens increase mean residence time of the process and improve the extent of flowability and bulk density enhancement. The effectiveness of the comill as a dry-coating process is demonstrated using Avicel PH 105, a fine grade of microcrystalline cellulose, as a model cohesive powder. The process is evaluated thoroughly using a lab scale comill and scalability is demonstrated using a manufacturing scale model. The use of the modified screens is also compared against the so-called "multi-pass" approach in which material is passed through the comill, collected, and passed through once or several times. While the "multi-pass" approach is offered as a simple method to increase mean residence time and to improve process effectiveness, the use of the modified screens is shown to be the superior approach. Due to the ubiquitous use of the comill and the improvement in effectiveness attained in this study, dry-coating is shown to be a practical and readily implemented process for the pharmaceutical industry.
Subject(s)
Drug Industry , Silicon Dioxide , Silicon Dioxide/chemistry , Particle Size , Powders/chemistry , Drug Compounding/methodsABSTRACT
Pharmaceutical tablets can be susceptible to damage such as edge chipping or erosion of the core during the tablet coating process. The intersection of certain process parameters, equipment design, and tablet properties may induce more significant tablet damage such as complete tablet fracture. In this work, a hybrid predictive approach was developed using discrete element method (DEM) modeling and lab-based tablet impact experiments to identify conditions that may lead to tablet breakage events. The approach was extended to examine potential modifications to the coating equipment and process conditions in silico to mitigate the likelihood of tablet breakage during future batches. The approach is shown to enhance process understanding, identify optimal process conditions within development constraints, and de-risk the manufacture of future tablet coating batches.
Subject(s)
Chemistry, Pharmaceutical/methods , Tablets/chemical synthesis , Computer Simulation , Forecasting , Tablets/standardsABSTRACT
Transplantation of functional islets encapsulated in stable biomaterials has the potential to cure Type I diabetes. However, the success of these materials requires the ability to quantitatively evaluate their stability. Imaging techniques that enable monitoring of biomaterial performance are critical to further development in the field. X-ray phase-contrast (XPC) imaging is an emerging class of X-ray techniques that have shown significant promise for imaging biomaterial and soft tissue structures. In this study, XPC imaging techniques are shown to enable three dimensional (3D) imaging and evaluation of islet volume, alginate hydrogel structure, and local soft tissue features ex vivo. Rat islets were encapsulated in sterile ultrapurified alginate systems produced using a high-throughput microfluidic system. The encapsulated islets were implanted in omentum pouches created in a rodent model of type 1 diabetes. Microbeads were imaged with XPC imaging before implantation and as whole tissue samples after explantation from the animals. XPC microcomputed tomography (µCT) was performed with systems using tube-based and synchrotron X-ray sources. Islets could be identified within alginate beads and the islet volume was quantified in the synchrotron-based µCT volumes. Omental adipose tissue could be distinguished from inflammatory regions resulting from implanted beads in harvested samples with both XPC imaging techniques. Individual beads and the local encapsulation response were observed and quantified using quantitative measurements, which showed good agreement with histology. The 3D structure of the microbeads could be characterized with XPC imaging and failed beads could also be identified. These results point to the substantial potential of XPC imaging as a tool for imaging biomaterials in small animal models and deliver a critical step toward in vivo imaging.
Subject(s)
Foreign Bodies/physiopathology , Hydrogel, Polyethylene Glycol Dimethacrylate/chemistry , Image Processing, Computer-Assisted/methods , Islets of Langerhans/pathology , Microscopy, Phase-Contrast/methods , Microspheres , Alginates/chemistry , Animals , Biocompatible Materials/chemistry , Glucuronic Acid/chemistry , Hexuronic Acids/chemistry , Islets of Langerhans/diagnostic imaging , Male , Rats , Rats, Inbred Lew , Rats, Inbred WF , X-Ray MicrotomographyABSTRACT
Three dimensional imaging techniques are needed for the evaluation and assessment of biomaterials used for tissue engineering and drug delivery applications. Hydrogels are a particularly popular class of materials for medical applications but are difficult to image in tissue using most available imaging modalities. Imaging techniques based on X-ray Phase Contrast (XPC) have shown promise for tissue engineering applications due to their ability to provide image contrast based on multiple X-ray properties. In this manuscript, we investigate the use of XPC for imaging a model hydrogel and soft tissue structure. Porous fibrin loaded poly(ethylene glycol) hydrogels were synthesized and implanted in a rodent subcutaneous model. Samples were explanted and imaged with an analyzer-based XPC technique and processed and stained for histology for comparison. Both hydrogel and soft tissues structures could be identified in XPC images. Structure in skeletal muscle adjacent could be visualized and invading fibrovascular tissue could be quantified. There were no differences between invading tissue measurements from XPC and the gold-standard histology. These results provide evidence of the significant potential of techniques based on XPC for 3D imaging of hydrogel structure and local tissue response.
Subject(s)
Contrast Media/pharmacology , Fibrin , Hydrogels , Implants, Experimental , Muscle, Skeletal , Polyethylene Glycols , Tomography, X-Ray/methods , Animals , Fibrin/chemistry , Fibrin/pharmacokinetics , Hydrogels/chemistry , Hydrogels/pharmacology , Male , Muscle, Skeletal/diagnostic imaging , Polyethylene Glycols/chemistry , Polyethylene Glycols/pharmacology , Porosity , Rats , Rats, Inbred LewABSTRACT
Gradients of soluble factors play an important role in many biological processes, including blood vessel assembly. Gradients can be studied in detail in vitro, but methods that enable the study of spatially distributed soluble factors and multi-cellular processes in vivo are limited. Here, we report on a method for the generation of persistent in vivo gradients of growth factors in a three-dimensional (3D) biomaterial system. Fibrin loaded porous poly (ethylene glycol) (PEG) scaffolds were generated using a particulate leaching method. Platelet derived growth factor BB (PDGF-BB) was encapsulated into poly (lactic-co-glycolic acid) (PLGA) microspheres which were placed distal to the tissue-material interface. PLGA provides sustained release of PDGF-BB and its diffusion through the porous structure results in gradient formation. Gradients within the scaffold were confirmed in vivo using near-infrared fluorescence imaging and gradients were present for more than 3 weeks. The diffusion of PDGF-BB was modeled and verified with in vivo imaging findings. The depth of tissue invasion and density of blood vessels formed in response to the biomaterial increased with magnitude of the gradient. This biomaterial system allows for generation of sustained growth factor gradients for the study of tissue response to gradients in vivo.
Subject(s)
Biocompatible Materials/pharmacology , Blood Vessels/growth & development , Neovascularization, Physiologic/drug effects , Proto-Oncogene Proteins c-sis/pharmacology , Animals , Becaplermin , Blood Vessels/drug effects , Blood Vessels/physiology , Collagen/metabolism , Diffusion , Humans , Hydrogel, Polyethylene Glycol Dimethacrylate/pharmacology , Male , Porosity , Rats, Inbred Lew , Regeneration/drug effects , Solubility , Tissue Scaffolds/chemistryABSTRACT
PURPOSE: To compare perioperative outcomes, specifically hospital length of stay (LOS), after retroperitoneal and conventional transperitoneal robot-assisted partial nephrectomy (RAPN). PATIENTS AND METHODS: We retrospectively compared consecutive patients with a posterior renal mass undergoing retroperitoneal RAPN (n=116) versus transperitoneal RAPN (n=97) at our institution between July 2007 and March 2014. The surgical approach was based on patient and tumor characteristics, history of abdominal surgery, and surgeon preference. The primary outcome was postoperative LOS, and secondary outcomes included complication rate, inpatient narcotic pain medication use, and inpatient antiemetic use. RESULTS: Baseline patient and tumor characteristics were similar between groups. A significantly great proportion of patients undergoing retroperitoneal RAPN had LOS equal to 1 day (57% vs 10%, P<0.01). Complication rates were similar between groups (P=0.37). Median pain medication use was also similar between groups (P=0.85). A significantly greater proportion of retroperitoneal RAPN patients, however, needed no antiemetics postoperatively (59% vs 43%, P=0.02). On multivariate analysis, transperitoneal RAPN was a significant predictor of LOS greater than 1 day (odds ratio=7.4, P<0.01), when controlling for age, sex, body mass index, patient comorbidity, previous abdominal surgery, baseline kidney function, nephrometry score, and tumor size. CONCLUSIONS: For patients with posterior renal masses, retroperitoneal RAPN significantly reduces their hospital LOS when compared with transperitoneal RAPN.
Subject(s)
Kidney Neoplasms/surgery , Nephrectomy/instrumentation , Patient Discharge , Retroperitoneal Space/surgery , Robotic Surgical Procedures/methods , Adult , Aged , Body Mass Index , Female , Humans , Length of Stay , Male , Middle Aged , Multivariate Analysis , Nephrectomy/methods , Retrospective Studies , Treatment OutcomeABSTRACT
Rapid and controlled vascularization within biomaterials is essential for many applications in regenerative medicine. The extent of vascularization is influenced by a number of factors, including scaffold architecture. While properties such as pore size and total porosity have been studied extensively, the importance of controlling the interconnectivity of pores has received less attention. A sintering method was used to generate hydrogel scaffolds with controlled pore interconnectivity. Poly(methyl methacrylate) microspheres were used as a sacrificial agent to generate porous poly(ethylene glycol) diacrylate hydrogels with interconnectivity varying based on microsphere sintering conditions. Interconnectivity levels increased with sintering time and temperature with resultant hydrogel structure showing agreement with template structure. Porous hydrogels with a narrow pore size distribution (130-150 µm) and varying interconnectivity were investigated for their ability to influence vascularization in response to gradients of platelet-derived growth factor-BB (PDGF-BB). A rodent subcutaneous model was used to evaluate vascularized tissue formation in the hydrogels in vivo. Vascularized tissue invasion varied with interconnectivity. At week 3, higher interconnectivity hydrogels had completely vascularized with twice as much invasion. Interconnectivity also influenced PDGF-BB transport within the scaffolds. An agent-based model was used to explore the relative roles of steric and transport effects on the observed results. In conclusion, a technique for the preparation of hydrogels with controlled pore interconnectivity has been developed and evaluated. This method has been used to show that pore interconnectivity can independently influence vascularization of biomaterials.
Subject(s)
Hydrogels/chemistry , Microspheres , Neovascularization, Physiologic/drug effects , Proto-Oncogene Proteins c-sis , Tissue Scaffolds/chemistry , Animals , Becaplermin , Male , Polyethylene Glycols/chemistry , Polymethyl Methacrylate/chemistry , Porosity , Proto-Oncogene Proteins c-sis/chemistry , Proto-Oncogene Proteins c-sis/pharmacology , Rats , Rats, Inbred LewABSTRACT
BACKGROUND: Wound healing and skin tissue engineering are mediated, in part, by interactions between cells and the extracellular matrix (ECM). A subset of the ECM, basement membranes (BM), plays a vital role in regulating proper skin healing and function. METHODS: ECM-rich, tissue-specific hydrogels were extracted and assembled from dermis samples. These hydrogels contain BM proteins vital to skin regeneration, including laminin ß3, collagen IV, and collagen VII. The extracts could be assembled to form hydrogels by either temperature or pH mechanism, with the mechanical properties and structure varying with the mechanism of assembly. A wound healing model was developed to investigate the ability of these hydrogels to enhance healing with a single application in vivo. RESULTS: The pH, but not temperature gels were easily applied to the wounds. There were no signs of increased inflammation due to the application of the hydrogels. The width of granulation tissue at the first week was reduced (p = 0.064) relative to controls with the application of hydrogel. There were no changes in wound closure rates or vessel density. CONCLUSIONS: Dermis-derived hydrogels contain BM proteins important for skin regeneration. They can be easily applied, but their poor mechanical strength and rapid degradation may hinder their biological effects.
Subject(s)
Dermis/physiology , Wound Healing/drug effects , Basement Membrane/metabolism , Collagen/metabolism , Extracellular Matrix/metabolism , Hydrogels , Tissue Engineering , Wound Healing/physiologyABSTRACT
Tissues engineered in bioreactor systems have been used clinically to replace damaged tissues and organs. In addition, these systems are under continued development for many tissue engineering applications. The ability to quantitatively assess material structure and tissue formation is critical for evaluating bioreactor efficacy and for preimplantation assessment of tissue quality. Techniques that allow for the nondestructive and longitudinal monitoring of large engineered tissues within the bioreactor systems will be essential for the translation of these strategies to viable clinical therapies. X-ray Phase Contrast (XPC) imaging techniques have shown tremendous promise for a number of biomedical applications owing to their ability to provide image contrast based on multiple X-ray properties, including absorption, refraction, and scatter. In this research, mesenchymal stem cell-seeded alginate hydrogels were prepared and cultured under osteogenic conditions in a perfusion bioreactor. The constructs were imaged at various time points using XPC microcomputed tomography (µCT). Imaging was performed with systems using both synchrotron- and tube-based X-ray sources. XPC µCT allowed for simultaneous three-dimensional (3D) quantification of hydrogel size and mineralization, as well as spatial information on hydrogel structure and mineralization. Samples were processed for histological evaluation and XPC showed similar features to histology and quantitative analysis consistent with the histomorphometry. These results provide evidence of the significant potential of techniques based on XPC for noninvasive 3D imaging engineered tissues grown in bioreactors.
Subject(s)
Alginates/chemistry , Biocompatible Materials/chemistry , Bioreactors , Calcification, Physiologic , Tissue Engineering/methods , X-Ray Microtomography/methods , Cells, Cultured , Glucuronic Acid/chemistry , Hexuronic Acids/chemistry , Humans , Mesenchymal Stem Cells , Microscopy, Phase-Contrast , SynchrotronsABSTRACT
Three-dimensional (3D) cell culture assays are important tools in the study of vessel assembly. Current techniques for quantitative analysis of vascular network structure have provided important insight into 3D vessel assembly. However, these methods typically require immunohistochemical staining, which requires sample destruction, or fluorescent cell labeling, which may alter cell behavior. The methods also may require sophisticated and expensive microscopy. More robust, easily quantifiable techniques are needed for imaging vascular networks non-invasively. We present an imaging method based on widefield optical sectioning and digital deconvolution (WOSD) that enables imaging of vascular networks in 3D culture without the use of cell labeling, staining, or sample destruction. WOSD can be performed using a standard optical microscope and allows non-invasive 3D monitoring of vascular network formation. This method is illustrated by imaging vascular networks in a 3D hydrogel system. WOSD enabled production of quantifiable 3D images of the network structure. Accuracy of the technique was evaluated by comparing data from WOSD with confocal images of fixed and fluorescently stained samples. Data for vessel length, diameter, and density are consistent between the two methods. The WOSD approach can be applied using standard laboratory equipment and shows great promise for use in analysis of 3D vascular network formation.
Subject(s)
Blood Vessels/anatomy & histology , Blood Vessels/growth & development , Imaging, Three-Dimensional/methods , Neovascularization, Physiologic , Algorithms , Blood Vessels/cytology , Coculture Techniques , Computer Systems , Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells , Humans , Microscopy/methods , Microscopy, Confocal/methods , Models, Cardiovascular , Myocytes, Smooth Muscle/cytologyABSTRACT
Proteolytically degradable poly(ethylene) glycol (PEG) hydrogels have been investigated as tissue engineering scaffolds; however, cell invasion and tissue regeneration are limited by the rate of cell-mediated degradation due to the small mesh size of the resultant crosslinked network. Gelatin leaching is combined with photopolymerization to form porous matrix-metalloproteinase (MMP)-sensitive PEG scaffolds under cytocompatible conditions in the presence of cells. Gelatin leaching allows control over pore size and porosity through selectivity of gelatin bead particle size and porogen loading, respectively. Increases in porogen loading lead to increased porosity, decreased compressive modulus and degradation time, and enhanced proliferation of encapsulated vascular smooth muscle cells.
Subject(s)
Acrylates/chemistry , Hydrogels/chemistry , Hydrogels/chemical synthesis , Polyethylene Glycols/chemistry , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Analysis of Variance , Cell Proliferation , Fluorescence , Gelatin/chemistry , Kinetics , Matrix Metalloproteinases/chemistry , Microscopy, Phase-Contrast , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/physiology , Particle Size , PorosityABSTRACT
Chinese hamster ovary (CHO) cells have been one of the most widely used host cells for the manufacture of therapeutic recombinant proteins. An effective and efficient clinical cell line development process, which could quickly identify those rare, high-producing cell lines among a large population of low and non-productive cells, is of considerable interest to speed up biological drug development. In the glutamine synthetase (GS)-CHO expression system, selection of top-producing cell lines is based on controlling the balance between the expression level of GS and the concentration of its specific inhibitor, l-methionine sulfoximine (MSX). The combined amount of GS expressed from plasmids that have been introduced through transfection and the endogenous CHO GS gene determine the stringency and efficiency of selection. Previous studies have shown significant improvement in selection stringency by using GS-knockout CHO cells, which eliminate background GS expression from the endogenous GS gene in CHOK1SV cells. To further improve selection stringency, a series of weakened SV40E promoters have been generated and used to modulate plasmid-based GS expression with the intent of manipulating GS-CHO selection, finely adjusting the balance between GS expression and GS inhibitor (MSX) levels. The reduction of SV40E promoter activities have been confirmed by TaqMan RT-PCR and GFP expression profiling. Significant productivity improvements in both bulk culture and individual clonal cell line have been achieved with the combined use of GS-knockout CHOK1SV cells and weakened SV40E promoters driving GS expression in the current cell line generation process. The selection stringency was significantly increased, as indicated by the shift towards higher distribution of producing-cell populations, even with no MSX added into cell culture medium. The potential applications of weakened SV40E promoter and GS-knockout cells in development of targeted integration and transient CHO expression systems are also discussed.
Subject(s)
CHO Cells/metabolism , Promoter Regions, Genetic , Recombinant Proteins/biosynthesis , Animals , CHO Cells/cytology , Cricetinae , Cricetulus , Gene Expression/drug effects , Gene Knockout Techniques , Genetic Vectors , Glutamate-Ammonia Ligase/antagonists & inhibitors , Glutamate-Ammonia Ligase/genetics , Glutamate-Ammonia Ligase/metabolism , Humans , Methionine Sulfoximine/pharmacology , Recombinant Proteins/genetics , Simian virus 40/genetics , TransfectionABSTRACT
Biomaterials are employed in the fields of tissue engineering and regenerative medicine (TERM) in order to enhance the regeneration or replacement of tissue function and/or structure. The unique environments resulting from the presence of biomaterials, cells, and tissues result in distinct challenges in regards to monitoring and assessing the results of these interventions. Imaging technologies for three-dimensional (3D) analysis have been identified as a strategic priority in TERM research. Traditionally, histological and immunohistochemical techniques have been used to evaluate engineered tissues. However, these methods do not allow for an accurate volume assessment, are invasive, and do not provide information on functional status. Imaging techniques are needed that enable non-destructive, longitudinal, quantitative, and three-dimensional analysis of TERM strategies. This review focuses on evaluating the application of available imaging modalities for assessment of biomaterials and tissue in TERM applications. Included is a discussion of limitations of these techniques and identification of areas for further development.
Subject(s)
Biocompatible Materials/chemistry , Magnetic Resonance Imaging , Molecular Imaging , Regenerative Medicine/methods , Tissue Engineering/methods , HumansABSTRACT
The spatial presentation of immobilized extracellular matrix (ECM) cues and matrix mechanical properties play an important role in directed and guided cell behavior and neovascularization. The goal of this work was to explore whether gradients of elastic modulus, immobilized matrix metalloproteinase (MMP)-sensitivity, and YRGDS cell adhesion ligands are capable of directing 3D vascular sprout formation in tissue engineered scaffolds. PEGDA hydrogels were engineered with mechanical and biofunctional gradients using perfusion-based frontal photopolymerization (PBFP). Bulk photopolymerized hydrogels with uniform mechanical properties, degradation, and immobilized biofunctionality served as controls. Gradient hydrogels exhibited an 80.4% decrease in elastic modulus and a 56.2% decrease in immobilized YRGDS. PBFP hydrogels also demonstrated gradients in hydrogel degradation with degradation times ranging from 10-12 hours in the more crosslinked regions to 4-6 hours in less crosslinked regions. An in vitro model of neovascularization, composed of co-culture aggregates of endothelial and smooth muscle cells, was used to evaluate the effect of these gradients on vascular sprout formation. Aggregate invasion in gradient hydrogels occurred bi-directionally with sprout alignment observed in the direction parallel to the gradient while control hydrogels with homogeneous properties resulted in uniform invasion. In PBFP gradient hydrogels, aggregate sprout length was found to be twice as long in the direction parallel to the gradient as compared to the perpendicular direction after three weeks in culture. This directionality was found to be more prominent in gradient regions of increased stiffness, crosslinked MMP-sensitive peptide presentation, and immobilized YRGDS concentration.
Subject(s)
Extracellular Matrix/metabolism , Hydrogels/chemistry , Matrix Metalloproteinases/metabolism , Polyethylene Glycols/chemistry , Biomechanical Phenomena , Cell Culture Techniques , Elastic Modulus , Extracellular Matrix/chemistry , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Matrix Metalloproteinases/chemistry , Peptides/chemistry , Tissue Engineering , Tissue ScaffoldsABSTRACT
Porous hydrogels of poly(ethylene glycol) (PEG) have been shown to facilitate vascularized tissue formation. However, PEG hydrogels exhibit limited degradation under physiological conditions which hinders their ultimate applicability for tissue engineering therapies. Introduction of poly(L-lactic acid) (PLLA) chains into the PEG backbone results in copolymers that exhibit degradation via hydrolysis that can be controlled, in part, by the copolymer conditions. In this study, porous, PEG-PLLA hydrogels were generated by solvent casting/particulate leaching and photopolymerization. The influence of polymer conditions on hydrogel architecture, degradation and mechanical properties was investigated. Autofluorescence exhibited by the hydrogels allowed for three-dimensional, non-destructive monitoring of hydrogel structure under fully swelled conditions. The initial pore size depended on particulate size but not polymer concentration, while degradation time was dependent on polymer concentration. Compressive modulus was a function of polymer concentration and decreased as the hydrogels degraded. Interestingly, pore size did not vary during degradation contrary to what has been observed in other polymer systems. These results provide a technique for generating porous, degradable PEG-PLLA hydrogels and insight into how the degradation, structure, and mechanical properties depend on synthesis conditions.
Subject(s)
Hydrogels/chemistry , Polyethylene Glycols/chemistry , Animals , Biocompatible Materials , Biomechanical Phenomena , Cell Line , Fibroblasts/metabolism , Focal Adhesions , Mice , Particle Size , PorosityABSTRACT
Natural hydrogels have been investigated for three-dimensional tissue reconstruction and regeneration given their ability to emulate the structural complexity of multi-component extracellular matrices (ECM). Hydrogels rich in ECM can be extracted and assembled from soft tissues, retain a composition specific to the tissue source, and stimulate vascularized tissue formation. However, poor mechanical properties and rapid degradation hinder their performance in regenerative applications. This study investigates the effect of glutaraldehyde (GA) crosslinking on the mechanical properties, biological activity, and degradation of dermis-isolated ECM-rich hydrogels. Compression tests indicated that hydrogel elastic moduli and yield stress values increased significantly with GA exposure time. Lyophilization was shown to decrease yield stress values with respect to non-lyophilized gels. Crosslinked ECM, unlike non-crosslinked gels, was resistant to pepsin degradation in vitro. In a rodent subcutaneous implant model, crosslinking for 0.5 hours or longer drastically slowed degradation relative to controls. Inflammation was low and mature vascularized granulation tissue was observed in all gels, with an increase in vessel density at 1 week in crosslinked gels relative to controls. These results support the potential use of dermis-derived hydrogels as materials for tissue engineering applications and suggest that crosslinking can enhance mechanical properties and prolong hydrogel lifetime while promoting vascularized tissue formation.
Subject(s)
Cross-Linking Reagents/pharmacology , Dermis/chemistry , Hydrogels/chemistry , Hydrogels/pharmacology , 3T3 Cells , Animals , Blood Vessels/drug effects , Cell Death/drug effects , Cell Survival/drug effects , Compressive Strength/drug effects , Elastic Modulus/drug effects , Glutaral/pharmacology , Implants, Experimental , Mice , Pepsin A/metabolism , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Rats , Rats, Sprague-Dawley , Stress, Mechanical , Subcutaneous Tissue/drug effectsABSTRACT
Cell behavior is guided by the complex interplay of matrix mechanical properties as well as soluble and immobilized biochemical signals. The development of synthetic scaffolds that incorporate key functionalities of the native extracellular matrix (ECM) for support of cell proliferation and tissue regeneration requires that stiffness and immobilized concentrations of ECM signals within these biomaterials be tuned and optimized prior to in vitro and in vivo studies. A detailed experimental sensitivity analysis was conducted to identify the key polymerization conditions that result in significant changes in both elastic modulus and immobilized YRGDS within visible light photopolymerized poly(ethylene glycol) diacrylate hydrogels. Among the polymerization conditions investigated, single as well as simultaneous variations in N-vinylpyrrolidinone and precursor concentrations of acryl-PEG3400-YRGDS resulted in a broad range of the hydrogel elastic modulus (81-1178 kPa) and YRGDS surface concentration (0.04-1.72 pmol cm(-2)). Increasing the YRGDS surface concentration enhanced fibroblast cell adhesion and proliferation for a given stiffness, while increases in the hydrogel elastic modulus caused decreases in cell adhesion and increases in proliferation. The identification of key polymerization conditions is critical for the tuning and optimization of biomaterial properties and the controlled study of cell-substrate interactions.
Subject(s)
Cell Adhesion/physiology , Cell Proliferation , Extracellular Matrix/chemistry , Oligopeptides/pharmacokinetics , Polyethylene Glycols/chemistry , Acrylates/chemistry , Acrylates/radiation effects , Animals , Biomimetic Materials/chemistry , Elastic Modulus , Hardness/physiology , Hydrogels/chemistry , Hydrogels/radiation effects , Ligands , Light , Materials Testing , Mice , NIH 3T3 Cells , Photochemistry/methods , Polyethylene Glycols/radiation effects , Polymers/chemistry , Polymers/radiation effects , Surface Properties , Tensile Strength/physiologyABSTRACT
Vascular network formation within biomaterial scaffolds is essential for the generation of properly functioning engineered tissues. In this study, a method is described for generating composite hydrogels in which porous poly(ethylene glycol) (PEG) hydrogels serve as scaffolds for mechanical and structural support, and fibrin is loaded within the pores to induce vascularized tissue formation. Porous PEG hydrogels were generated by a salt leaching technique with 100-150-µm pore size and thrombin (Tb) preloaded within the scaffold. Fibrinogen (Fg) was loaded into pores with varying concentrations and polymerized into fibrin due to the presence of Tb, with loading efficiencies ranging from 79.9% to 82.4%. Fibrin was distributed throughout the entire porous hydrogels, lasted for greater than 20 days, and increased hydrogel mechanical stiffness. A rodent subcutaneous implant model was used to evaluate the influence of fibrin loading on in vivo response. At weeks 1, 2, and 3, all hydrogels had significant tissue invasion, but no difference in the depth of invasion was found with the Fg concentration. Hydrogels with fibrin loading induced more vascularization, with a significantly higher vascular density at 20 mg/mL (week 1) and 40 mg/mL (weeks 2 and 3) Fg concentration compared to hydrogels without fibrin. In conclusion, we have developed a composite hydrogel that supports rapid vascularized tissue ingrowth, and thus holds great potential for tissue engineering applications.
Subject(s)
Blood Vessels/cytology , Blood Vessels/growth & development , Fibrin/pharmacology , Hydrogels/chemistry , Polyethylene Glycols/chemistry , Tissue Engineering/instrumentation , Tissue Scaffolds , Animals , Biocompatible Materials/chemical synthesis , Blood Vessels/drug effects , Drug Carriers/chemical synthesis , Equipment Design , Male , Materials Testing , Porosity , Rats , Rats, Inbred Lew , Tissue Engineering/methodsABSTRACT
BACKGROUND: Plaque vulnerability depends, in part, on composition. Imaging techniques are needed that can aid the prediction of plaque stability. High-contrast images of soft-tissue structure have been obtained with x-ray phase-contrast (PC) imaging. This research investigates multiple image radiography (MIR), an x-ray PC imaging technique, for evaluation of human carotid artery plaques. METHODS: Carotid plaques were imaged with ultrasound and subsequently excised and formalin fixed. MIR imaging was performed. By using synchrotron radiation, conventional radiographs were acquired for comparison. Image texture measures were computed for soft-tissue regions of the plaques. RESULTS: Ultrasound evaluation identified plaques as homogeneous without calcifications. MIR images revealed complex heterogeneous structure with multiple microcalcifications consistent with histology, and possessed more image texture in specific regions than conventional radiographs (P < .05). MIR refraction images allowed imaging of the geometric structure of tissue interfaces within the plaques, while scatter images contained more texture in soft-tissue regions than absorption or refraction images. CONCLUSIONS: X-ray PC imaging better depicts plaque soft-tissue heterogeneity than ultrasound or conventional radiographs. MIR imaging technique should be investigated further as a viable imaging technique to identify high-risk plaques.
Subject(s)
Carotid Stenosis/diagnostic imaging , Plaque, Atherosclerotic/diagnostic imaging , Tomography, X-Ray Computed , Carotid Stenosis/pathology , Humans , In Vitro Techniques , Plaque, Atherosclerotic/pathology , UltrasonographyABSTRACT
Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.