ABSTRACT
Objectives@#. Using tissue-engineered materials for esophageal reconstruction is a technically challenging task in animals that requires bioreactor training to enhance cellular reactivity. There have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in initial epithelialization in the special environment of peristalsis. The purpose of this study was to evaluate the potential of an artificial esophagus that can enhance the regeneration of esophageal mucosa and muscle through the optimal combination of a double-layered polymeric scaffold and a custom-designed mesenchymal stem cell-based bioreactor system in a canine model. @*Methods@#. We fabricated a novel double-layered scaffold as a tissue-engineered esophagus using an electrospinning technique. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. After 3 days of cultivation using the bioreactor system, tissue-engineered artificial esophagus was transplanted into a partial esophageal defect (5×3 cm-long resection) in a canine model. @*Results@#. Scanning electron microscopy (SEM) showed that the electrospun fibers in a tubular scaffold were randomly and circumferentially located toward the inner and outer surfaces. Complete recovery of the esophageal mucosa was confirmed by endoscopic analysis and SEM. Esophagogastroduodenoscopy and computed tomography also showed that there were no signs of leakage or stricture and that there was a normal lumen with complete epithelialization. Significant regeneration of the mucosal layer was observed by keratin-5 immunostaining. Alpha-smooth muscle actin immunostaining showed significantly greater esophageal muscle regeneration at 12 months than at 6 months. @*Conclusion@#. Custom-designed bioreactor cultured electrospun polyurethane scaffolds can be a promising approach for esophageal tissue engineering.
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BACKGROUND@#Long segmental tracheal repair is challenging in regenerative medicine due to low adhesion of stem cells to tracheal scaffolds. Optimal transplantation of stem cells for tracheal defects has not been established. We evaluated the role of hyaluronic acid (HA) coating of tracheal scaffolds in mesenchymal stem cell (MSC) adhesion and tracheal regeneration in a rabbit model. @*METHODS@#A three-dimensionally printed tubular tracheal prosthesis was incubated with dopa-HA-fluorescein isothiocyanate in phosphate-buffered saline for 2 days. MSCs were incubated with an HA-coated scaffold, and their adhesion was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. HA coated scaffolds with or without MSC seeding were transplanted at the circumferential tracheal defect in rabbits, and survival, rigid bronchoscopy, radiologic findings, and histologic findings were compared between the two groups. @*RESULTS@#HA-coated scaffolds showed better MSC adhesion than non-coated scaffolds. The HA-coated scaffolds with MSC group showed a wider airway and greater mucosal regeneration compared to the HA-coated scaffolds without MSC group. @*CONCLUSION@#HA coating of scaffolds can promote MSC adhesion and tracheal regeneration.
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Objectives@#. A polydioxanone (PDO) stent was developed to treat tracheomalacia in pediatric patients. However, its safety and efficacy need to be verified in animal studies before clinical trials in patients can be conducted. This study evaluated the safety and efficacy of a PDO stent in normal and tracheomalacia-model rabbits. @*Methods@#. In total, 29 New Zealand white rabbits were used: 13 for evaluating the biocompatibility of the PDO stent in normal rabbits and 16 for the creation of a tracheomalacia model. The tracheomalacia model was successfully established in 12 rabbits, and PDO stents were placed in eight of those rabbits. @*Results@#. The PDO stent was successfully positioned in the trachea of the normal rabbits using an endoscopic approach, and its degradation was observed 10 weeks later. The stent fragments did not induce distal airway obstruction or damage, and the mucosal changes that occurred after stent placement were reversed after degradation. The same procedure was performed on the tracheomalacia-model rabbits. The survival duration of the tracheomalacia rabbits with and without stents was 49.0±6.8 and 1.0±0.8 days, respectively. Thus, the PDO stent yielded a significant survival gain (P=0.001). In the tracheomalacia rabbits, stent degradation and granulation tissue were observed 7 weeks after placement, leading to airway collapse and death. @*Conclusion@#. We successfully developed a PDO stent and an endoscopic guide placement system. The degradation time of the stent was around 10 weeks in normal rabbits, and its degradation was accelerated in the tracheomalacia model. The mucosal changes associated with PDO stent placement were reversible. Placement of the PDO stent prolonged survival in tracheomalacia-model rabbits.
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Objectives@#. A polydioxanone (PDO) stent was developed to treat tracheomalacia in pediatric patients. However, its safety and efficacy need to be verified in animal studies before clinical trials in patients can be conducted. This study evaluated the safety and efficacy of a PDO stent in normal and tracheomalacia-model rabbits. @*Methods@#. In total, 29 New Zealand white rabbits were used: 13 for evaluating the biocompatibility of the PDO stent in normal rabbits and 16 for the creation of a tracheomalacia model. The tracheomalacia model was successfully established in 12 rabbits, and PDO stents were placed in eight of those rabbits. @*Results@#. The PDO stent was successfully positioned in the trachea of the normal rabbits using an endoscopic approach, and its degradation was observed 10 weeks later. The stent fragments did not induce distal airway obstruction or damage, and the mucosal changes that occurred after stent placement were reversed after degradation. The same procedure was performed on the tracheomalacia-model rabbits. The survival duration of the tracheomalacia rabbits with and without stents was 49.0±6.8 and 1.0±0.8 days, respectively. Thus, the PDO stent yielded a significant survival gain (P=0.001). In the tracheomalacia rabbits, stent degradation and granulation tissue were observed 7 weeks after placement, leading to airway collapse and death. @*Conclusion@#. We successfully developed a PDO stent and an endoscopic guide placement system. The degradation time of the stent was around 10 weeks in normal rabbits, and its degradation was accelerated in the tracheomalacia model. The mucosal changes associated with PDO stent placement were reversible. Placement of the PDO stent prolonged survival in tracheomalacia-model rabbits.
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BACKGROUND@#Long segmental tracheal repair is challenging in regenerative medicine due to low adhesion of stem cells to tracheal scaffolds. Optimal transplantation of stem cells for tracheal defects has not been established. We evaluated the role of hyaluronic acid (HA) coating of tracheal scaffolds in mesenchymal stem cell (MSC) adhesion and tracheal regeneration in a rabbit model. @*METHODS@#A three-dimensionally printed tubular tracheal prosthesis was incubated with dopa-HA-fluorescein isothiocyanate in phosphate-buffered saline for 2 days. MSCs were incubated with an HA-coated scaffold, and their adhesion was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. HA coated scaffolds with or without MSC seeding were transplanted at the circumferential tracheal defect in rabbits, and survival, rigid bronchoscopy, radiologic findings, and histologic findings were compared between the two groups. @*RESULTS@#HA-coated scaffolds showed better MSC adhesion than non-coated scaffolds. The HA-coated scaffolds with MSC group showed a wider airway and greater mucosal regeneration compared to the HA-coated scaffolds without MSC group. @*CONCLUSION@#HA coating of scaffolds can promote MSC adhesion and tracheal regeneration.
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PURPOSE: To investigate the feasibility of a polycaprolactone (PCL) scaffold fabricated by three-dimensional (3D) printing for tissue engineering applications for tunica albuginea. MATERIALS AND METHODS: PCL scaffolds were fabricated by use of a 3D printing system. Two scaffolds were fabricated that differed in the architecture of the lay-down pattern: a 90°PCL scaffold and a 45°PCL scaffold. Mechanical properties were measured to compare tensile strength between the two scaffold types. The scaffolds were characterized by scanning electron microscope (SEM) images. The scaffolds were seeded with fibroblast cells, and the ability of these scaffolds to support the cells was evaluated by immunofluorescence staining. RESULTS: The PCL scaffolds had well-structured shapes, regular arrays, and good interconnection in SEM images. The horizontal and vertical Young's modulus coefficients were 13 and 12 MPa for the 90°PCL scaffold and 19 and 21 MPa for the 45°PCL scaffold, respectively. Microscopy images revealed that human fibroblast cells covered the entire scaffold surface. Immunofluorescence staining of ER-TR7 confirmed that the fibroblast cells remained viable and proliferated throughout the time course of the culture. CONCLUSIONS: This preliminary study provides experimental evidence for the feasibility of 3D printing of PCL scaffolds for tissue engineering applications of tunica albuginea.
Subject(s)
Humans , Male , Elastic Modulus , Fibroblasts , Fluorescent Antibody Technique , Microscopy , Penis , Printing, Three-Dimensional , Tensile Strength , Tissue EngineeringABSTRACT
Tracheal restenosis is a major obstacle to successful tracheal replacement, and remains the greatest challenge in tracheal regeneration. However, there have been no detailed investigations of restenosis. The present study was performed to analyze the serial changes in recruited inflammatory cells and associated histological changes after tracheal scaffold implantation. Asymmetrically porous scaffolds, which successfully prevented tracheal stenosis in a partial trachea defect model, designed with a tubular shape by electrospinning and reinforced by 3D-printing to reconstruct 2-cm circumferential tracheal defect. Serial rigid bronchoscopy, micro-computed tomography, and histology [H&E, Masson's Trichrome, IHC against a-smooth muscle actin (α-SMA)] were performed 1, 4, and 8 weeks after transplantation. Progressive stenosis developed especially at the site of anastomosis. Neutrophils were the main inflammatory cells recruited in the early stage, while macrophage infiltration increased with time. Recruitment of fibroblasts peaked at 4 weeks and deposition of a-SMA increased from 4 weeks and was maintained through 8 weeks. During the first 8 weeks post-transplantation, neutrophils and macrophages played significant roles in restenosis of the trachea. Antagonists to these would be ideal targets to reduce restenosis and thus play a pivotal role in successful tracheal regeneration.
Subject(s)
Actins , Bronchoscopy , Constriction, Pathologic , Fibroblasts , Inflammation , Macrophages , Neutrophils , Regeneration , Trachea , Tracheal StenosisABSTRACT
We investigated the use of Polycaprolactone (PCL)/β-tricalcium phosphate (β-TCP) composites with applied mechanical stimulation as scaffold for bone tissue engineering. PCL-based three-dimensional (3D) structures were fabricated in a solvent-free process using a 3D-printing technique. The mass fraction of β-TCP was varied in the range 0–30%, and the structure and compressive modulus of the specimens was characterized. The shape and interconnectivity of the pores was found to be satisfactory, and the compressive modulus of the specimens was comparable with that of human trabecular bone. Human mesenchymal stem cells were seeded on the composites, and various biological evaluations were performed over 9 days. With a mass fraction of β-TCP of 30%, differentiation began earlier; however, the cell proliferation rate was lower. Through the use of mechanical stimulation, however, the proliferation rate recovered, and was comparable with that of the other groups. This stimulation effect was also observed in ECM generation and other biological assays. With mechanical stimulation, expression of osteogenic markers was lower on samples with a β-TCP content of 10 wt% than without β-TCP; however, with mechanical stimulation, the sample with a β-TCP content of 30 wt% exhibited significantly greater expression of those markers than the other samples. We found that mechanical stimulation and the addition of β-TCP interacted closely, and that a mass fraction of β-TCP of 30% was particularly useful as a bone tissue scaffold when accompanied by mechanical stimulation.
Subject(s)
Humans , Biological Assay , Bone and Bones , Cell Proliferation , Mesenchymal Stem CellsABSTRACT
BACKGROUND/AIMS: Chronic liver disease is a major widespread cause of death, and whole liver transplantation is the only definitive treatment for patients with end-stage liver diseases. However, many problems, including donor shortage, surgical complications and cost, hinder their usage. Recently, tissue-engineering technology provided a potential breakthrough for solving these problems. Three-dimensional (3D) printing technology has been used to mimic tissues and organs suitable for transplantation, but applications for the liver have been rare. METHODS: A 3D bioprinting system was used to construct 3D printed hepatic structures using alginate. HepG2 cells were cultured on these 3D structures for 3 weeks and examined by fluorescence microscopy, histology and immunohistochemistry. The expression of liver-specific markers was quantified on days 1, 7, 14, and 21. RESULTS: The cells grew well on the alginate scaffold, and liver-specific gene expression increased. The cells grew more extensively in 3D culture than two-dimensional culture and exhibited better structural aspects of the liver, indicating that the 3D bioprinting method recapitulates the liver architecture. CONCLUSIONS: The 3D bioprinting of hepatic structures appears feasible. This technology may become a major tool and provide a bridge between basic science and the clinical challenges for regenerative medicine of the liver.
Subject(s)
Humans , Bioprinting , Cause of Death , Gene Expression , Hep G2 Cells , Immunohistochemistry , Liver , Liver Diseases , Liver Transplantation , Methods , Microscopy, Fluorescence , Printing, Three-Dimensional , Regenerative Medicine , Tissue DonorsABSTRACT
PURPOSE: The major problem in producing artificial livers is that primary hepatocytes cannot be cultured for many days. Recently, 3-dimensional (3D) printing technology draws attention and this technology regarded as a useful tool for current cell biology. By using the 3D bio-printing, these problems can be resolved. METHODS: To generate 3D bio-printed structures (25 mm × 25 mm), cells-alginate constructs were fabricated by 3D bio-printing system. Mouse primary hepatocytes were isolated from the livers of 6–8 weeks old mice by a 2-step collagenase method. Samples of 4 × 10⁷ hepatocytes with 80%–90% viability were printed with 3% alginate solution, and cultured with well-defined culture medium for primary hepatocytes. To confirm functional ability of hepatocytes cultured on 3D alginate scaffold, we conducted quantitative real-time polymerase chain reaction and immunofluorescence with hepatic marker genes. RESULTS: Isolated primary hepatocytes were printed with alginate. The 3D printed hepatocytes remained alive for 14 days. Gene expression levels of Albumin, HNF-4α and Foxa3 were gradually increased in the 3D structures. Immunofluorescence analysis showed that the primary hepatocytes produced hepatic-specific proteins over the same period of time. CONCLUSION: Our research indicates that 3D bio-printing technique can be used for long-term culture of primary hepatocytes. It can therefore be used for drug screening and as a potential method of producing artificial livers.
Subject(s)
Animals , Mice , Collagenases , Drug Evaluation, Preclinical , Fluorescent Antibody Technique , Gene Expression , Hepatocytes , Liver , Liver, Artificial , Methods , Printing, Three-Dimensional , Real-Time Polymerase Chain ReactionABSTRACT
Three-Dimensional (3D) printing technologies have been widely used in the medical sector for the production of medical assistance equipment and surgical guides, particularly 3D bio-printing that combines 3D printing technology with biocompatible materials and cells in field of tissue engineering and regenerative medicine. These additive manufacturing technologies can make patient-made production from medical image data. Thus, the application of 3D bio-printers with biocompatible materials has been increasing. Currently, 3D bio-printing technology is in the early stages of research and development but it has great potential in the fields of tissue and organ regeneration. The present paper discusses the history and types of 3D printers, the classification of 3D bio-printers, and the technology used to manufacture artificial tissues and organs.
Subject(s)
Biocompatible Materials , Classification , Medical Assistance , Printing, Three-Dimensional , Regeneration , Regenerative Medicine , Tissue EngineeringABSTRACT
Study on the Potential of Hydroxapatite Based Bioactive Bone Cement PURPOSE: The purpose of this study is to propose a new bioactive bone cement (BBC) composed of bone powder (hydroxyapatite; HA), chitosan powder, and currently available polymethylmethacrylate (PMMA) bone cement for use in orthopaedic surgeries such as vertebroplasty or bone filler. MATERIALS AND METHODS: Three types of proposed BBCs and a currently available commercial PMMA were tested. In vitro studies the surface morphology, chemical composition, changes in pH value along the time, exothermic temperatures, intrusion and cellular responses were investigated. SEM, radiological and histological examinations were performed in animal studies. RESULTS: The major components of BBCs were C, O, Ca, P, Cl, Si, S, Ba and Mg. The pH values in BBCs decreased after 1 day, however they eventually reached 7.2-7.4. The water absorbency, weight loss, and porosity in BBCs increased more than PMMA more than during degradation (p<0.05). However, the compressive Young's moduli and ultimate compressive strength (UCS) of BBCs were lower than those of PMMA (<0.05). The exothermic temperatures of the BBCs were considerably lower than that of PMMA (p<0.05). In view of setting time, it takes relatively longer for BBCII and III to be solidified than PMMA (p<0.05). The intrusion tests showed that the BBCs were more intrusive than PMMA (p<0.05). The cell proliferation test on BBCII showed that the BBCII was more preferable than the PMMA. No cytotoxic characteristics were found in all BBCs. In the animal test, BBC II was more biocompatible as well as osteoconductible than the PMMA. CONCLUSION: The results of in vitro and animal studies indicated that the proposed BBCs have a potential of clinical application as replacement of the current PMMA bone cements.
Subject(s)
Animals , Bone Cements , Cell Proliferation , Chitosan , Compressive Strength , Durapatite , Hydrogen-Ion Concentration , Polymethyl Methacrylate , Porosity , Vertebroplasty , Water , Weight LossABSTRACT
PURPOSE: The biomechanical responses of degenerative porcine intervertebral disc were compared with those MATERIALS AND METHODS: Two groups were set; Group A (44.0+/-2.8 months old, female) and Group B (6.2 +/-1.3 months old, female). Histological (H&E stain) observations were carried out to see the degeneration for both groups. Then biomechanical responses were investigated by measuring height changes in disc, intradiscal pressure values and relaxation time for each specimen under axial compressive loads. RESULTS: Degenerative changes were confirmed through H&E staining in Group A. The ratios of the nucleus pulposus area to total area were 14.7+/-4.5% and 29.2+/-6.0% in Group A and B, respectively (p=0.000). The decrease rates in disc height were 12.1+/-3.3% and 21.6+/-7.6%, in Group A and B, respectively under the axial compression of 740 N (p=0.000). No significant difference in intradiscal pressure measured in anterior zone between-groups except at axial load of 740N (p> 0.05). However, significant difference in pressure was found in posterolateral zone when the load was 542 N and higher (542 N: p=0.015, 740 N: p=0.010). The average relaxation time for Group A was significantly longer than that for Group B at 740N, i.e., at maximum load (anterior: p=0.010, posterolateral: p=0.014). CONCLUSION: Different biomechanical responses in degenerative disc were confirmed. They are 1) less flexible, 2) slower in energy relaxation under axial loading, and 3) larger portion of the external load were taken up at posterior part of annulus fibrous, especially in degenerative disc.