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Current approaches for the engineering of osteochondral grafts are associated with poor tissue formation and compromised integration at the interface between the cartilage and bone layers. Many researchers have attempted to provide osteochondral grafts of combined cartilage and bone for osteochondral repair to help overcome the limitations of standard procedures. Solid freeform fabrication is recognized as a promising tool for creating tissue engineering scaffolds due to advantages such as superior interconnectivity and a highly porous structure. This study aimed to develop a three-dimensional plotting system to enable the manufacturing of a biphasic graft consisting cartilage and subchondral bone for application to osteochondral defects. The material advantages of both synthetic (poly L lactide-co-polyglycolide) and natural (alginate) polymers were combined for a supporting frame and cell printing. Specifically, in order to promote the maturity of the osteochondral graft in our study, cartilage-derived ECM (cECM) or hydroxyapatate (HA) substances blended with alginate was plotted together with human fetal cartilage-derived progenitor cells in the cartilage or subchondral bone layer under a multi-nozzle deposition system. Notably, a plotted biphasic graft shows good integration between cartilage and subchondral bone layers without structural separation. Furthermore, the non-toxicity of the cECM and HA substances were proved from a live/dead assay of plotted cell-laden alginate. A fabricated osteochondral graft with cECM and HA substances showed dominant cartilage and bone tissue formation in a differentiation assay. Future studies should be done to modify the alginate physical properties for long-lasting structural stability.

Naturally derived biomaterials are rarely used in advanced rapid prototyping technology despite their superior biocompatibility. The main problem of natural material plotting is the high sensitivity of materials concentration and viscosity on the plotting nozzle. The aim of the current study was to develop a three dimensional (3D) plotting system capable of dispensing extracellular matrix (ECM)-c (ECM powder blended collagen) and manufacture various shapes of ECM-c scaffolds to apply for irregular defects. We had adapted a powder-based plotting approach to print the stable 3D construct using only cartilage derived ECM materials. This study successfully developed the plotting method for high viscous ECM-c material and showed the 3D plotted scaffolds with high interconnected pores as well as complex shape. Furthermore, cell culture results proved that plotted ECM-c scaffolds were able to provide a suitable environment for cell attachment, proliferation and chondrogenesis. This study shows the 3D printing feasibility of ECM natural material has demonstrated as a first time. We believe our results will offer a meaningful step toward the 3D scaffold printing based on natural ECM materials for future organ printing.

Objectives Tracheal reconstruction with tissue-engineering technique has come into the limelight in the realm of head and neck surgery. We intended to evaluate the plausibility of allogenic chondrocytes cultured with porcine cartilage-derived substance (PCS) scaffold for partial tracheal defect reconstruction. Methods Powder made from crushed and decellularized porcine articular cartilage was formed as 5 mm × 12 mm (height × diameter) scaffold. Chondrocytes from rabbit articular cartilage were expanded and cultured with PCS scaffold. After 7 weeks culture, the scaffolds were implanted on a 5 mm × 10 mm artificial tracheal defect in six rabbits. Two, four and eight weeks postoperatively, the sites were evaluated endoscopically, radiologically, histologically and functionally. Results None of the six rabbits showed any sign of respiratory distress. Endoscopic examination did not show any collapse or blockage of the reconstructed trachea and the defects were completely covered with regenerated respiratory epithelium. Computed tomography showed good luminal contour of trachea. Postoperative histologic data showed that the implanted chondrocyte successfully formed neo-cartilage with minimal inflammatory response and granulation tissue. Ciliary beat frequency of regenerated epithelium was similar to those of normal adjacent mucosa. Conclusions The shape and function of reconstructed trachea using allogenic chondrocytes cultured with PCS scaffold was restored successfully without any graft rejection.

In this study, we examined whether porcine articular cartilage (PAC) is a suitable and effective anti-adhesive material. PAC, which contained no non-collagenous tissue components, was collected by mechanical manipulation and decellularization of porcine knee cartilage. The PAC film for use as an anti-adhesive barrier was easily shaped into various sizes using homemade silicone molds. The PAC film was cross-linked to study the usefulness of the anti-adhesive barrier shape. The cross-linked PAC (Cx-PAC) film showed more stable physical properties over extended periods compared to uncross-linked PAC (UnCx-PAC) film. To control the mechanical properties, Cx-PAC film was thermally treated at 45 degrees C or 65 degrees C followed by incubation at room temperature. The Cx-PAC films exhibited varying enthalpies, ultimate tensile strength values, and contact angles before and after thermal treatment and after incubation at room temperature. Next, to examine the anti-adhesive properties, human umbilical vein endothelial cells (HUVECs) were cultured on Cx-PAC and thermal-treated Cx-PAC films. Scanning electron microscopy, fluorescence, and MTT assays showed that HUVECs were well adhered to the surface of the plate and proliferated, indicating no inhibition of the attachment and proliferation of HUVECs. In contrast, Cx-PAC and thermal-treated Cx-PAC exhibited little and/or no cell attachment and proliferation because of the inhibition effect on HUVECs. In conclusion, we successfully developed a Cx-PAC film with controllable mechanical properties that can be used as an anti-adhesive barrier.

Recombinant human transforming growth factor beta-3 (rhTGF-β3) is a key regulator of chondrogenesis in stem cells and cartilage formation. We have developed a novel drug delivery system that continuously releases rhTGF-β3 using a multilayered extracellular matrix (ECM) membrane. We hypothesize that the sustained release of rhTGF-β3 could activate stem cells and result in enhanced repair of cartilage defects. The properties and efficacy of the ECM multilayer-based delivery system (EMLDS) are investigated using rhTGF-β3 as a candidate drug. The bioactivity of the released rhTGF-s3 was evaluated through chondrogenic differentiation of mesenchymal stem cells (MSCs) using western blot and circular dichroism (CD) analyses in vitro. The cartilage reparability was evaluated through implanting EMLDS with endogenous and exogenous MSC in both in vivo and ex vivo models, respectively. In the results, the sustained release of rhTGF-s3 was clearly observed over a prolonged period of time in vitro and the released rhTGF-β3 maintained its structural stability and biological activity. Successful cartilage repair was also demonstrated when rabbit MSCs were treated with rhTGF-β3-loaded EMLDS ((+) rhTGF-β3 EMLDS) in an in vivo model and when rabbit chondrocytes and MSCs were treated in ex vivo models. Therefore, the multilayer ECM membrane could be a useful drug delivery system for cartilage repair.

Tissue engineering using a biocompatible scaffold with various cells might be a solution for tracheal reconstruction. We investigated the plausibility of using mesenchymal stem cells (MSCs) seeded on a porcine cartilage powder (PCP) scaffold for tracheal defect repair. PCP made with minced and decellularized porcine articular cartilage was molded into a 5 × 12 mm (height × diameter) scaffold. MSCs from young rabbit bone marrow were expanded and cultured with the PCP scaffold. After 7 weeks culture, the tracheal implants were transplanted on a 5 × 10 mm tracheal defect in six rabbits. 6 and 10 weeks postoperatively, the implanted area was evaluated. None of the six rabbits showed any sign of respiratory distress. Endoscopic examination revealed that respiratory epithelium completely covered the regenerated trachea and there were no signs of collapse or blockage. A patent luminal contour of the trachea was observed on the computed tomography scan in all six rabbits and the reconstructed areas were not narrow compared to normal adjacent trachea. Histologic examination showed that neo-cartilage was successfully produced with minimal inflammation or granulation tissue. Ciliary beating frequency of the regenerated epithelium was not significantly different from the normal adjacent mucosa. MSCs cultured with a PCP scaffold successfully restored not only the shape but also the function of the trachea without any graft rejection.

Nonthermal plasma (NTP) is generated by ionization of neutral gas molecules, which results in a mixture of energy particles including electrons and ions. Recent progress in the understanding of NTP has led to its application in the treatment of various diseases, including cancer. However, the molecular mechanisms of NTP-induced cell death are unclear. The purpose of this study was to evaluate the molecular mechanism of NTP in the induction of apoptosis of head and neck cancer (HNC) cells. The effects of NTP on apoptosis were investigated using MTT, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling, Annexin V assays, and western blot analysis. The cells were examined for production of reactive oxygen species (ROS) using DCFCA or MitoSOX staining, intracellular signaling, and an animal model. NTP reduced HNC cell viability in a dose-dependent manner and induced apoptosis. NTP resulted in alteration of mitochondrial membrane potential and accumulation of intracellular ROS generated from the mitochondria in HNC cells. Blockade of ROS production by N-acetyl-L-cysteine inhibited NTP-induced apoptosis. NTP led to the phosphorylation of c-JUN N-terminal kinase (JNK) and p38, but not extracellular-regulated kinase. Treatment with JNK and p38 inhibitors alleviated NTP-induced apoptosis via ROS generation. Taken together, these results show that NTP induced apoptosis of HNC cells by a mechanism involving MAPK-dependent mitochondrial ROS. NTP inhibited the growth of pre-established FaDu tumors in a nude mouse xenograft model and resulted in accumulation of intracellular ROS. In conclusion, NTP induced apoptosis in HNC cells through a novel mechanism involving MAPK-mediated mitochondrial ROS. These findings show the therapeutic potential of NTP in HNC.