Incorporation of Cell-Adhesive Proteins in 3D-Printed Lipoic Acid-Maleic Acid-Poly(Propylene Glycol)-Based Tough Gel Ink for Cell-Supportive Microenvironment.

In extrusion-based 3D printing, the use of synthetic polymeric hydrogels can facilitate fabrication of cellularized and implanted scaffolds with sufficient mechanical properties to maintain the structural integrity and physical stress within the in vivo conditions. However, synthetic hydrogels face challenges due to their poor properties of cellular adhesion, bioactivity, and biofunctionality. New compositions of hydrogel inks have been designed to address this limitation. A viscous poly(maleate-propylene oxide)-lipoate-poly(ethylene oxide) (MPLE) hydrogel is recently developed that shows high-resolution printability, drug-controlled release, excellent mechanical properties with adhesiveness, and biocompatibility. In this study, the authors demonstrate that the incorporation of cell-adhesive proteins like gelatin and albumin within the MPLE gel allows printing of biologically functional 3D scaffolds with rapid cell spreading (within 7 days) and high cell proliferation (twofold increase) as compared with MPLE gel only. Addition of proteins (10% w/v) supports the formation of interconnected cell clusters (≈1.6-fold increase in cell areas after 7-day) and spreading of cells in the printed scaffolds without additional growth factors. In in vivo studies, the protein-loaded scaffolds showed excellent biocompatibility and increased angiogenesis without inflammatory response after 4-week implantation in mice, thus demonstrating the promise to contribute to the printable tough hydrogel inks for tissue engineering.

Erectile Dysfunction Treatment Using Stem Cell Delivery Patch in a Cavernous Nerve Injury Rat Model.

Erectile dysfunction (ED) is a common and feared complication of radical prostatectomy (RP) for prostate cancer. Recently, tissue engineering for post-prostatectomy ED has been attempted in which controlled interactions between cells, growth factors, and the extracellular matrix (ECM) are important for the structural integrity if nerve regeneration. In this study, we evaluated the effects of a biomechanical ECM patch on the morphology and behavior of human bone marrow-derived mesenchymal stem cells (hBMSCs) in a bilateral cavernous nerve injury (BCNI) rat model. The ECM patch, made of decellularized human fibroblast-derived ECM (hFDM) and a biocompatible polyvinyl alcohol (PVA) hydrogel, was tested with human bone marrow-derived mesenchymal stem cells (hBMSCs) on a bilateral cavernous nerve injury (BCNI) rat model. In vitro analysis showed that the hFDM/PVA + hBMSCs patches significantly increased neural development markers. In vivo experiments demonstrated that the rats treated with the hFDM/PVA patch had higher ICP/MAP ratios, higher ratios of smooth muscle to collagen, increased nNOS content, higher levels of eNOS protein expression, and higher cGMP levels compared to the BCNI group. These results indicate that the hFDM/PVA patch is effective in promoting angiogenesis, smooth muscle regeneration, and nitrergic nerve regeneration, which could contribute to improved erectile function in post-prostatectomy ED.

Assessment of Esophageal Reconstruction via Bioreactor Cultivation of a Synthetic Scaffold in a Canine Model.

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.

Control of maleic acid-propylene diepoxide hydrogel for 3D printing application for flexible tissue engineering scaffold with high resolution by end capping and graft polymerization.

BACKGROUND: Control of 3D printing of highly tough hydrogel inks with adequate printability, scaffold fidelity and mechanical properties are highly desirable for biomedical and tissue engineering applications. However, developing a biocompatible tough ink with high-resolution printability, biodegradability, self-healing, adhesion, and integration with surrounding tissues is a big challenge in 3D printing. The aim of this study was to develop extrusion-based 3D printing of viscous hydrogel composing of maleic acid and propylene diepoxide by controlling continuous mechanisms of condensation and radical polymerization. METHODS: The molecular weight of highly adhesive propagating poly(malate-co-propylene oxide) copolymer was controlled by capping its growing chain with mono-functional lipoic acid with different compositions during condensation reaction to form lipoic acid capped gel (LP-capped gel). Poly(ethylene oxide)-diacrylate, PEGDA, is graft-polymerized to the LP-capped backbone polymer (MPLE gel) by UV irradiation during 3D printing process to control the properties of gel printability, mechanical properties, and cell adhesiveness and post-printing fidelity of the printed scaffolds with high resolution and mechanical properties (MPLE scaffold). The scaffolds in complex geometries have been printed out in diverse forms with addition of model drugs with different molecular weights and chemical structures. Both the highly adhesive LP-capped gel and printing-controlled MPLE gel/scaffolds are diversely characterized and compared with for their applications to the extrusion-based printability, including biocompatibility, self-healing, drug releasing, adhesiveness, multi-layered high-resolution printing. Further in vitro/in vivo tests were done to observe cytotoxicity, immune response and tissue formation by using different cells in mice model. RESULTS: LP-capped hydrogel from maleic acid and propylene diepoxide gel showed control of gel properties with lipoic acid with one function group of thiol during condensation reaction, and the ratio at 1:0.3 (w/v) between LP-capped gel and PEGDA was chosen for the optimal results during radical polymerization process for 3D printing at high resolution (90-140 µm in strut thickness) with various complex geometries (lattice, rhombus, and honeycomb). The hydrogel showed excellent properties of self-healing, mechanical strength, biocompatibility, etc. In addition, the long-term release profiles of bioactive molecules were well-controlled by incorporating drugs of high molecular bovine serum albumin (BSA, 21 days, 98.4 ± 0.69%), or small molecule ornidazole (ORN, 14 days, 97.1 ± 1.98%) into the MPLE gel scaffolds for the tests of potential therapeutic applications. More importantly, the MPLE gels represents excellent in vitro cyto-compatibility against osteoblast-like cells (MC3T3) with viability value at 96.43% ± 7.48% over 7 culturing days. For in-vivo studies, the flexible MPLE scaffolds showed significant improvement on angiogenesis with minor inflammatory response after 4-week implantation in mice. CONCLUSION: The MPLE gel inks was well-controlled for the fabrication of flexible complex tissue engineering scaffold with high resolutions, shear-thinning, 3D printability and post-printing fidelity, by modulating the composition of the highly adhesive LP-capped gel and inert PEGDA as well as end capping of lipoic acid to the propagating poly(malate-co-propylene oxide) copolymer. The gel ink demonstrated its excellent printability, in vitro/in vivo biocompatibility and mechanical properties as well as sustained drug release from the gel.

Alternative non-oral nutrition in a rat model: a novel modified gastrostomy technique.

The gastrostomy technique is essential for esophageal reconstruction using a scaffold. To date, there are no established methods to supply nutrients through a gastrostomy tube in rats. The purpose of this study was to analyze the feasibility of a newly modified gastrostomy technique for non-oral nutrition in an adult rat model. We modified the gastrostomy technique for adult rats in a few different ways. (1) The external opening for food injection was made at the midpoint between the ears to prevent damage due to self-harm behaviour. (2) An imbedded subcutaneous tunnel was created between the internal and external openings of the gastrostomy. We compared the efficacy and safety between groups with a T-tube for biliary drainage (TT group, n=14) and a conventional silicone Foley catheter (FC group, n=7) as optimal gastrostomy tubes for in a rat model. We also evaluated the feasibility of the heparin cap connector at the end of gastrostomy tube to control food supply in the TT group (with a cap, n=7; without a cap, n=7). No mortality was observed in the TT group with a cap, whereas most rats in the FC group died within 2 weeks after the procedure. Weight loss decreased significantly in the TT group with a cap compared with all the other groups. The appearance and attitude scores were significantly better in the TT group with a cap. In addition, histologic analysis showed that the TT group a cap showed a marked decrease over time in tissue fibrosis and macrophages compared with the other experimental groups. Therefore, gastrostomy using a silicone T-tube plugged with a cap proved to be a stable and effective option for non-oral feeding in an adult rat model.

Three-Dimensional Printed Design of Antibiotic-Releasing Esophageal Patches for Antimicrobial Activity Prevention.

Pharyngoesophageal defects can cause exposure to various bacterial flora and severe inflammation. We fabricated a biodegradable polycaprolactone (PCL) patch composed of both thin film and three-dimensional (3D) printed lattice, and then investigated the efficacy of pharyngoesophageal reconstruction by using 3D printed antibiotic-releasing PCL patches that inhibited early inflammation by sustained tetracycline (TCN) release from both thin PCL films and printed rods implanted in esophageal partial defects. PCL was 3D printed in lattice form on a presolution casted PCL thin film at ∼100 µm resolution. TCN was loaded onto the PCL-printed patches by 3D printing a mixture of TCN and PCL particles melted at 100°C. TCN exhibited sustained release in vitro for over 1 month. After loading TCN, the patches showed decreased tensile strength and Young's modulus, and less than 20% TCN was slowly released from the 2.5% TCN-loaded PCL patches over 150 days. Cytotoxicity tests of extract solutions from patch samples demonstrated excellent in vitro cell compatibility. Antibiotic-releasing PCL patches were then transplanted into partial esophageal defects in rats. Microcomputed tomography analysis revealed no leak of orally injected contrast agent in the entire esophagus. Tissue remodeling was examined through histological responses of M1 and M2 macrophages. In particular, the 1% and 3% TCN patch groups exhibited significant muscle layer regeneration by desmin immunostaining. Further histological and immunofluorescence analyses revealed that the 1% and 3% TCN patch groups exhibited the best esophageal regeneration according to reepithelialization, neovascularization, and elastin texture around the implanted sites. Our antibiotic-releasing patch successfully consolidates the regenerative potential of esophageal muscle and mucosa and the antibacterial activity of TCN for 3D esophageal reconstruction. Impact statement Anastomosis site leakage and necrosis after pharyngoesophageal transplantation inevitably causes mortality because the mediastinum and neck compartments become contaminated. Herein, we present antibiotic-releasing pharyngoesophageal patch that prevents saliva leakage and has an antimicrobial effect. We have demonstrated antibiotic release profile and mechanical properties for esophageal transplantation. Upon esophageal transplantation of antibiotic-releasing polycaprolactone patches, antimicrobial effects and muscle regeneration around the graft sites were clearly identified in the group containing 1% and 3% of tetracycline. The esophageal graft led to the remarkable recovery throughout reepithelialization, neovascularization, and elastin texture of around the implanted sites. We believe that current system is capable of various applications that require antibacterial in vivo.

Regeneration of irradiation-damaged esophagus by local delivery of mesenchymal stem-cell spheroids encapsulated in a hyaluronic-acid-based hydrogel.

Radiation therapy (RT) is a typical treatment for head and neck cancers. Generally, prolonged irradiation of the esophagus causes esophageal fibrosis due to increased reactive oxygen species and proinflammatory cytokines. This study was designed to determine whether catechol-functionalized hyaluronic acid (HA-CA) hydrogel-encapsulated human mesenchymal stem-cell spheroids (MSC-SPs) could ameliorate damage to the esophagus in a mouse model of radiation-induced esophageal fibrosis. MSC-SPs were cultured in concave microwells 600 µm in diameter at a cell density of 1 × 106 cells per mL. Most cells formed spheroids with a 100-300 µm size distribution in concave microwells. MSC-SPs were well maintained in the HA gel, and live-dead staining confirmed that most cells survived. The HA gel containing the MSC-SPs was then injected into the damaged esophageal layer. Inflammatory signs or adverse tissue reactions were not observed after esophageal injection of HA-gel-encapsulated MSC-SPs. Based on Masson's trichrome staining at 4 and 12 weeks postinjection, the inner esophageal layer (IEL) was significantly thinner in the MSC-SP + HA gel group compared to those in the other experimental groups. While the saline and HA gel treatments made the esophageal muscles loose and thick, the MSC-SP + HA gel group showed bundles of tightly packed esophageal muscles, as assayed by desmin immunostaining. qPCR analysis showed that epithelial genes tended to increase over time in the MSC-SP + HA gel group, and the expression of most fibrosis-related genes decreased. This study proposes the potential of using HA-CA-hydrogel-encapsulated MSC-SPs as a promising therapy against radiation-induced esophageal fibrosis.

Experimental investigation of esophageal reconstruction with electrospun polyurethane nanofiber and 3D printing polycaprolactone scaffolds using a rat model.

BACKGROUND: We evaluated the outcome of esophageal reconstructions using tissue-engineered scaffolds. METHOD: Partial esophageal defects were reconstructed with the following scaffolds; animals were grouped (n = 7 per group) as follows: (a) normal rats; (b) rats implanted with three-dimensional printing (3DP) polycaprolactone (PCL) scaffolds; (c) with human adipose-derived mesenchymal stem cell (ADSC)-seeded 3DP PCL scaffolds; (d) with polyurethane (PU)-nanofiber(Nf) scaffolds; and (e) with ADSC-seeded PU-Nf scaffolds. RESULTS: The esophageal defects were successfully repaired; however, muscle regeneration was greater in the 3DP PCL + ADSC groups than in the PU-Nf + ADSC groups (P < .001). Regeneration of the epithelium was greater in PU-Nf and PU-Nf + ADSC groups than in the 3DP PCL and 3DP PCL + ADSC groups (P < .001). CONCLUSION: A tendency for more re-epithelization was observed with the PU-Nf scaffolds, while more muscle regeneration was achieved with the 3DP PCL scaffolds.

Self-crosslinking hyaluronic acid-carboxymethylcellulose hydrogel enhances multilayered 3D-printed construct shape integrity and mechanical stability for soft tissue engineering.

One of the primary challenges in extrusion-based 3D bioprinting is the ability to print self-supported multilayered constructs with biocompatible hydrogels. The bioinks should have sufficient post-printing mechanical stability for soft tissue and organ regeneration. Here, we report on the synthesis, characterization and 3D printability of hyaluronic acid (HA)-carboxymethylcellulose (CMC) hydrogels cross-linked through N-acyl-hydrazone bonding. The hydrogel's hydrolytic stability was acquired by the effects of both the prevention of the oxidation of the six-membered rings of HA, and the stabilization of acyl-hydrazone bonds. The shear-thinning and self-healing properties of the hydrogel allowed us to print different 3D constructs (lattice, cubic and tube) of up to 50 layers with superior precision and high post-printing stability without support materials or post-processing depending on their compositions (H7:C3, H5:C5 and H3:C7). Morphological analyses of different zones of the 3D-printed constructs were undertaken for verification of the interconnection of pores. Texture profile analysis (TPA) (hardness (strength), elastic recovery, etc) and cyclic compression studies of the 3D-printed constructs demonstrated exceptional elastic properties and fast recovery after 50% strain, respectively, which have been attributed to the addition of CMC into HA. A model drug quercetin was released in a sustained manner from hydrogels and 3D constructs. In vitro cytotoxicity studies confirmed the excellent cyto-compatibility of these gels. In vivo mice studies prove that these biocompatible hydrogels enhance angiogenesis. The results indicate that controlling the key properties (e.g. self-crosslinking capacity, composition) can lead to the generation of multilayered constructs from 3D-bioprintable HA-CMC hydrogels capable of being leveraged for soft tissue engineering applications.

Transplantation of a 3D-printed tracheal graft combined with iPS cell-derived MSCs and chondrocytes.

For successful tracheal reconstruction, tissue-engineered artificial trachea should meet several requirements, such as biocompatible constructs comparable to natural trachea, coverage with ciliated respiratory mucosa, and adequate cartilage remodeling to support a cylindrical structure. Here, we designed an artificial trachea with mechanical properties similar to the native trachea that can enhance the regeneration of tracheal mucosa and cartilage through the optimal combination of a two-layered tubular scaffold and human induced pluripotent stem cell (iPSC)-derived cells. The framework of the artificial trachea was fabricated with electrospun polycaprolactone (PCL) nanofibers (inner) and 3D-printed PCL microfibers (outer). Also, human bronchial epithelial cells (hBECs), iPSC-derived mesenchymal stem cells (iPSC-MSCs), and iPSC-derived chondrocytes (iPSC-Chds) were used to maximize the regeneration of tracheal mucosa and cartilage in vivo. After 2 days of cultivation using a bioreactor system, tissue-engineered artificial tracheas were transplanted into a segmental trachea defect (1.5-cm length) rabbit model. Endoscopy did not reveal granulation ingrowth into tracheal lumen. Alcian blue staining clearly showed the formation of ciliated columnar epithelium in iPSC-MSC groups. In addition, micro-CT analysis showed that iPSC-Chd groups were effective in forming neocartilage at defect sites. Therefore, this study describes a promising approach for long-term functional reconstruction of a segmental tracheal defect.

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats.

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. 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. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Stretchable ECM Patch Enhances Stem Cell Delivery for Post-MI Cardiovascular Repair.

Current cell-based therapies administered after myocardial infarction (MI) show limited efficacy due to subpar cell retention in a dynamically beating heart. In particular, cardiac patches generally provide a cursory level of cell attachment due to the lack of an adequate microenvironment. From this perspective, decellularized cell-derived ECM (CDM) is attractive in its recapitulation of a natural biophysical environment for cells. Unfortunately, its weak physical property renders it difficult to retain in its original form, limiting its full potential. Here, a novel strategy to peel CDM off from its underlying substrate is proposed. By physically stamping it onto a polyvinyl alcohol hydrogel, the resulting stretchable extracellular matrix (ECM) membrane preserves the natural microenvironment of CDM, thereby conferring a biological interface to a viscoelastic membrane. Its various mechanical and biological properties are characterized and its capacity to improve cardiomyocyte functionality is demonstrated. Finally, evidence of enhanced stem cell delivery using the stretchable ECM membrane is presented, which leads to improved cardiac remodeling in a rat MI model. A new class of material based on natural CDM is envisioned for the enhanced delivery of cells and growth factors that have a known affinity with ECM.

Mechanical stimuli enhance simultaneous differentiation into oesophageal cell lineages in a double-layered tubular scaffold.

The tissue-engineered oesophagus serves as an alternative and promising therapeutic approach for long-gap oesophageal replacement. This study proposes an advanced in vitro culture platform focused on construction of the oesophagus by combining an electrospun double-layered tubular scaffold, stem cells, biochemical reagents, and biomechanical factors. Human mesenchymal stem cells were seeded onto the inner and outer surfaces of the scaffold. Mechanical stimuli were applied with a hollow organ bioreactor along with different biochemical reagents inside and outside of the scaffold. Electrospun fibres in a tubular scaffold were found to be randomly and circumferentially oriented for the inner and outer surfaces, respectively. Amongst the two types of mechanical stimuli, the intermittent shear flow that can simultaneously cause circumferential stretching due to hydrostatic pressure, and shear stress caused by flow on the inner surface, was found to be more effective for simultaneous differentiation into epithelial and muscle lineage than steady shear flow. Under these conditions, the expression of epithelial markers on the inner surface was significantly observed, although it was minimal on the outer surface. Muscle differentiation showed the opposite expression pattern. Meanwhile, the mechanical tests showed that the strength of the scaffold was improved after incubation for 14 days. We have developed a potential platform for tissue-engineered oesophagus construction. Specifically, simultaneous differentiation into epithelial and muscle lineages can be achieved by utilizing the double-layered scaffold and appropriate mechanical stimulation.

Stem Cells Seeded on Multilayered Scaffolds Implanted into an Injured Bladder Rat Model Improves Bladder Function.

Background: To investigate whether human adipose-derived stem cells (hADSCs) seeded on multilayered poly (l-lactide-co-ɛ-caprolactone) (PLCL) sheets improve bladder function in a rat model of detrusor smooth muscle-removed bladder. Methods: Male rats were randomly divided into 4 groups: Normal, injury (detrusor smooth muscle-removed bladder), PLCL (detrusor smooth muscle-removed bladder implanted with PLCL sheets), and PLCL + ADSC (detrusor smooth muscle-removed bladder implanted with PLCL sheets seeded with hADSCs). Four weeks after the treatment, physiological, histological, immunohistochemical, and immunoblot analyses were performed. Results: hADSCs were compatible with PLCL sheets. Further, the physiological study of PLCL + ADSC group showed significant improvement in compliance and contractility suggesting the functional improvement of the bladder. Histological, immunohistochemical and immunoblot analyses revealed the uniform distribution of hADSCs in between PLCL sheets as well as differentiation of hADSCs into smooth muscle cells (SMC) which is illustrated by the expression of SMC markers. Conclusion: hADSCs seeded on the multilayered PLCL sheets has the potential to differentiate into SMC, thus facilitating the recovery of compliance and contractility of the injured bladder.

Injectable basic fibroblast growth factor-loaded alginate/hyaluronic acid hydrogel for rejuvenation of geriatric larynx.

Increase in the geriatric population has led to an increase in the number of elderly patients with laryngeal atrophy and dysfunction. Symptoms of voice change, dysphagia, and aspiration pneumonia negatively influence patient's health status, quality of life, and life span. Injection laryngoplasty used to treat laryngeal dysfunctions does not recover intrinsic functions of the larynx. Thus, we fabricated an injectable basic fibroblast growth factor (bFGF)-loaded alginate (ALG)/hyaluronic acid (HA) hydrogel for inducing rejuvenation of geriatric laryngeal muscles. Optimal in situ-forming bFGF-loaded ALG/HA hydrogel for injection laryngoplasty was prepared and the release profile of bFGF was analyzed. For in vivo analysis, the bFGF-loaded ALG/HA hydrogel was injected into the laryngeal muscles of 18-month-old Sprague-Dawley rats. The rejuvenation efficacy of bFGF-loaded ALG/HA hydrogel in geriatric laryngeal muscle tissues 4- and 12-weeks post-injection was evaluated by quantitative polymerase chain reaction (qPCR), histology, immune-fluorescence staining and functionality analysis. The bFGF-loaded ALG/HA hydrogel induced an increase in the expression of myogenic regulatory factor-related genes, hypertrophy of muscle fiber, proliferation of muscle satellite cells, and angiogenesis and decreased interstitial fibrosis. Administration of the bFGF-loaded ALG/HA hydrogel caused successful glottal gap closure. Thus, the bFGF-loaded ALG/HA hydrogel could be a promising candidate for laryngoplasty aimed at rejuvenating geriatric larynx. STATEMENT OF SIGNIFICANCE: In this manuscript, optimal in situ-forming bFGF-loaded ALG/HA hydrogel for injection laryngoplasty was prepared and the release profile of bFGF was analyzed. Herein, we introduced the materials and methods of injection laryngoplasty for geriatric rat experiment. In addition, we studied effects of bFGF-loaded ALG/HA hydrogel on the therapeutic rejuvenation of geriatric rat larynx. The bFGF-loaded ALG/HA hydrogel induced an increase in the expression of myogenic regulatory factor-related genes, hypertrophy of muscle fiber, proliferation of muscle satellite cells, and angiogenesis and decreased interstitial fibrosis. Furthermore, our functional analysis through the high-speed camera setup demonstrated that the administration of the bFGF-loaded ALG/HA hydrogel induced successful glottal gap closure. Thus, the bFGF-loaded ALG/HA hydrogel could be a promising candidate for injection laryngoplasty with therapeutic effects.

Comparative studies on thin polycaprolactone-tricalcium phosphate composite scaffolds and its interaction with mesenchymal stem cells.

BACKGROUND: Hybrid scaffolds combining biodegradable polymers and ceramic particles for control of cell adhesion and proliferation are interesting materials for tissue engineering applications. Combinations of biodegradable polymers and ceramics are to provide higher beneficial functionalities to tissue engineering scaffolds with addition of different cell specific bio-factors. Many such hybrid combinations have been reported by several researchers around the world by using various methods and solvents as well as bioactive matrix polymers to fabricate such biomaterials. However, thin hybrid scaffolds with high porosity, cell adhesion factors and biodegradability, as well as the ability to support stem cells often require tedious processes like electrospinning, freeze drying, etc. A simple method to develop porous biodegradable hybrid scaffolds with proper cell adhesion factors is still the need of the hour in tissue engineering and regenerative medicine. METHOD: Thin biodegradable poly(ε-caprolactone) (PCL) based hybrid scaffolds were developed in combination with α-tricalcium phosphate (TCP) particles, gelatin and fibronectin separately and the fabricated scaffolds were evaluated systematically using human mesenchymal stem cells (hMSCs) for tissue engineering applications. A simple modified solvent casting method combined with gas foaming process was used to develop porous thin hybrid structures and compared their properties with those of corresponding non-porous hybrid scaffolds. The TCP particles distribution, morphology, biodegradability and functional groups of the different hybrid scaffolds were analyzed using energy-dispersive X-ray spectroscopy (EDX), light microscopy/scanning electron microscopy (SEM), buffer solutions and Fourier-transform infrared spectroscopy (FTIR), respectively The cellular and tissue regeneration behaviors such as in vitro cell attachment (live/dead assay), cell proliferation (CCK-8 assay) and histological studies were performed using hMSCs. RESULTS: Thin PCL-based hybrid scaffolds were fabricated using modified solvent casting method. Homogeneous distribution of TCP particles in the scaffolds were confirmed by EDX. Cellular interactions of the hybrid scaffolds demonstrated overall higher cell adhesion, proliferation and tissue regeneration on the non-porous thin films of PCL-TCP, PCL-TCP-gelatin and PCL-TCP-fibronectin. Coating of fibronectin was remarkable in induction of cell adhesion and proliferation. CONCLUSIONS: The experimental results revealed that diversely designed PCL-TCP thin hybrid films showed high cell interaction and proliferation with hMSCs. From the results of the cell viability, attachment, proliferation and histological analyses as well as their biodegradation and coating effects, we conclude that these thin PCL-TCP hybrid films are suitable for tissue engineering applications.

Tissue-Engineered Esophagus via Bioreactor Cultivation for Circumferential Esophageal Reconstruction.

The use of biomaterials for circumferential esophageal repair is technically challenging in a rat model, and an optimal scaffold implantation technique with nutritional support is essential. The purpose of this study was to investigate the effects of three-dimensional printed esophageal grafts and bioreactor cultivation on muscle regeneration and reepithelialization from circumferential esophageal defects in a rat model. Here, we designed an artificial esophagus that can enhance the regeneration of esophageal mucosa and muscle through the optimal combination of a two-layered tubular scaffold and mesenchymal stem cell-based bioreactor system. The graft was verified by the performance comparison with an omentum-cultured esophageal scaffold. We also applied a new surgical anastomosis technique and a thyroid gland flap over the implanted scaffold to improve graft survival. Although no regenerated mucosal layer was observed around the implants of the control group, histological examination of the regenerative esophagi along the scaffold revealed that the bioreactor system and omentum-cultured groups showed more than 80% of the mucosal regeneration without a fistula. The regenerated tissues showed that the integration of the esophageal scaffold and its native esophageal tissue was intact and were covered with layers of stratified squamous epithelium with several newly developed blood vessels. Therefore, this study describes a novel approach for circumferential esophageal reconstruction. Impact Statement In vivo functional esophageal reconstruction remains challenging due to anastomosis site leakage and necrosis of the implanted scaffold in a circumferential esophageal defect. Therefore, it is necessary to develop a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle without leakage of the esophageal anastomosis. In this study, we proposed an intriguing strategy that combines a mesenchymal stem cell-seeded tubular scaffold with a bioreactor system for esophageal reconstruction and introduced a new surgical anastomosis technique with the thyroid gland flap over the implanted scaffold to improve graft survival. We believe that this system should be a powerful platform for circumferential replacement of the esophagus in a rat model.

A terpolymeric hydrogel of hyaluronate-hydroxyethyl acrylate-gelatin methacryloyl with tunable properties as biomaterial.

Here, we report synthesis of a terpolymeric covalently crosslinked hydrogel of hyaluronate (HA) as biomaterial with elasticity, mechanical properties and cell interactions via conventional free radical polymerization technique. To provide elasticity and mechanical properties, 2-hydroxyethyl acrylate (HEA) was grafted in HA, while to tune cellular interactions, gelatin methacryloyl (GM) was used as crosslinker. The composition and probable structure of the terpolymer (HA-g-pHEA-x-GM) were analysed by FTIR, 1H HR-MAS-NMR, and TGA analyses. The SEM and texture analyses of hydrogel showed interconnected micro-porous network and high mechanical properties, respectively. In vitro biocompatibility was studied against human chondrocytes, whereas, in vivo biocompatibility and tissue regeneration were confirmed using mouse model. The hydrogel releases model protein-bovine serum albumin, and corticosteroid drug-dexamethasone in a sustain way at pH 7.4 and 37 °C. Overall, the tunable mechanical properties, micro-porous network, and cytocompatibility of the HA-g-pHEA-x-GM hydrogel highlights its potential applicability in cartilage tissue engineering and drug delivery.

Combinational effects of mechanical forces and substrate surface characteristics on esophageal epithelial differentiation.

Even the efficacy of substrate and mechanical stimuli in addition to biochemical cues have been recognized in many studies of stem cell differentiation, few studies have been reported on the differentiation into esophageal epithelial cells. Therefore, the aim of this study was set to propose a method of differentiating stem cells into esophageal epithelial cells according to biochemical reagent concentration, substrate properties, and mechanical forces. After the concentration of all-trans retinoic acid was determined as 5 µM by a baseline experiment, the degree of differentiation was compared in three different kinds of substrates: cover glass, polyurethane (PU) membrane, and electrospun PU sheet (ePU). Then, on the substrate showing the more positive results, that is, ePU, two types of mechanical forces, intermittent hydrostatic pressure (IHP), and shear stress (SS), were applied individually at different magnitudes for the latter 7 days of an overall incubation period of 14 days. Following various biological assays, the lower IHP (50 mmHg) resulted in greater positive effects than the others. Even with cessation of the mechanical force, the relevant markers were remarkably increased. Although the range of factors regulating differentiation was limited, this study nonetheless demonstrated the combinational effects of mechanical force along with substrate type for the first time in related studies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 552-560, 2019.

Regeneration of Paralyzed Vocal Fold by the Injection of Plasmid DNA Complex-Loaded Hydrogel Bulking Agent.

Various growth factor delivery systems were used in the treatment of glottal insufficiency; however, relatively little attention has been paid to a gene delivery system for aspects of active vocal fold (VF) regeneration. Herein, we present a plasmid DNA (pDNA; bFGF gene encoding) complex-loaded alginate (ALG)/hyaluronic acid (HA) mixture hydrogel dispersed with polycaprolactone (PCL) microspheres that can enhance simultaneous regeneration of VF muscle and lamina propria, as well as have a bulking effect on atrophied VF. We have demonstrated long-term efficacy of bFGF synthesized from pDNA complex-transfected cells in vitro. PCL microspheres-dispersed ALG/HA hydrogel (with or without pDNA complex loading) are injected into rabbit VFs with recurrent laryngeal nerve denervation. The PCL microspheres dispersed in the hydrogel bulking agents remain stable at the applied site, leading to constant medialization of the paralyzed VF without significant initial volume loss even after 24 weeks. A regenerative effect for collagen deposition and HA synthesis around the injected site, which are major components of VF tissue, is well confirmed in the pDNA-complex-loaded hydrogel group. Moreover, the compensation of atrophied VFs also leads to the contact of bilateral VF and the remarkable recovery of voice function in the pDNA-complex-loaded group. Based on the results, pDNA (bFGF encoding) complex-loaded hydrogel dispersed with PCL microspheres may be employed as a bioactive bulking agent for the treatment of glottal insufficiency.