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BACKGROUND: Respiratory mucosa defects result in airway obstruction and infection, requiring subsequent functional recovery of the respiratory epithelium. Because site-specific extracellular matrix (ECM) facilitates restoration of organ function by promoting cellular migration and engraftment, previous studies considered decellularized trachea an ideal ECM; however, incomplete cell removal from cartilage and mucosal-architecture destruction are frequently reported. Here, we developed a decellularization protocol and applied it to the respiratory mucosa of separated porcine tracheas. METHODS: The trachea was divided into groups according to decellularization protocol: native mucosa, freezing–thawing (FT), FT followed by the use of Perasafe-based chemical agents before mucosal separation (wFTP), after mucosal separation (mFTP), and followed by DNase decellularization (mFTD). Decellularization efficacy was evaluated by DNA quantification and hematoxylin and eosin staining, and ECM content of the scaffold was evaluated by histologic analysis and glycosaminoglycan and collagen assays. Biocompatibility was assessed by cell-viability assay and in vivo transplantation. RESULTS: The mFTP mucosa showed low antigenicity and maintained the ECM to form a proper microstructure. Additionally, tonsil-derived stem cells remained viable when cultured with or seeded onto mFTP mucosa, and the in vivo host response showed a constructive pattern following implantation of the mFTP scaffolds. CONCLUSION: These results demonstrated that xenogenic acellular respiratory mucosa matrix displayed suitable biocompatibility as a scaffold material for respiratory mucosa engineering.

Background and aims Osteoporosis, which is a disease characterized by weakening of the bone, affects a large portion of the senior population. The current therapeutic options for osteoporosis have side effects, and there is no effective treatment for severe osteoporosis. Thus, we urgently need new treatment strategies, such as topical therapies and/or safe and effective stem cell therapies. Methods We investigated the therapeutic potential of directly injecting human tonsil-derived mesenchymal stem cells (TMSC) into the right proximal tibias of ovariectomized postmenopausal osteoporosis model mice. Injections were given once (1×) or twice (2×) during the 3-month experimental period. At the end of the experiment, micro-computed tomographic images revealed some improvement in the proximal tibias and more significant improvement in the femoral heads of treated mice. Results Osteogenic effect was qualitatively and quantitatively more pronounced in TMSC/2×-treated mice. Furthermore, TMSC/2× mice exhibited significant recovery of the serum osteocalcin level, which is pathologically elevated in osteoporosis, and increased serum alkaline phosphatase, which indicates bone formation. TMSC therapy was generally well tolerated and caused no apparent toxicity in the experimental mice. Moreover, TMSC therapy reduced visceral fat. Conclusion Our results demonstrate that double injection of TMSC directly into the proximal tibia triggers recovery of osteoporosis, and thus could be a potential therapeutic approach for severe bone loss.

Cereblon (CRBN) has a pleiotropic role in important cellular processes and is a potential therapeutic target in several diseases, including mental retardation, cancer, and metabolic disorders. The role of CRBN in polymicrobial sepsis induced by cecal ligation and puncture (CLP) was investigated using CRBN-deficient (KO) mice. Survival following CLP was significantly higher in KO mice compared to wild-type (WT) controls (50% vs 0% at day 6 after CLP). The improved survival of KO mice was accompanied by reduced peripheral blood bacterial load and lung injury. Serum tumor necrosis factor (TNF)-α and high mobility group box 1 (HMGB1) concentrations were significantly lower in KO mice than in WT mice. Peritoneal macrophages from KO mice with CLP-induced septic mouse had higher levels of activation of AMPK and heme oxygenase-1 (HO-1). Forced expression of CRBN in macrophage of KO mice suppressed activation of 5′ adenosine monophosphate-activated protein kinase (AMPK) and HO-1 and augmented expression of TNF-α and HMGB1 as inhibition of AMPK by compound C. These studies demonstrate the contribution of CRBN expression to the pathogenesis of CLP-induced sepsis and peritoneal macrophage responses and suggest a novel therapeutic modality for polymicrobial sepsis.

To restore damaged parathyroid function, parathyroid tissue engineering is the best option. Previously, we reported that differentiated tonsil-derived mesenchymal stem cells (dTMSC) restore in vivo parathyroid function, but only if they are embedded in a scaffold. Because of the limited biocompatibility of Matrigel, however, here we developed a more clinically applicable, scaffold-free parathyroid regeneration system. Scaffold-free dTMSC spheroids were engineered in concave microwell plates made of polydimethylsiloxane in control culture medium for the first 7 days and differentiation medium (containing activin A and sonic hedgehog) for next 7 days. The size of dTMSC spheroids showed a gradual and significant decrease up to day 5, whereafter it decreased much less. Cells in dTMSC spheroids were highly viable (>80%). They expressed high levels of intact parathyroid hormone (iPTH), the parathyroid secretory protein 1, and cell adhesion molecule, N-cadherin. Furthermore, dTMSC spheroids-implanted parathyroidectomized (PTX) rats revealed higher survival rates (50%) over a 3-month period with physiological levels of both serum iPTH (57.7–128.2 pg/mL) and ionized calcium (0.70–1.15 mmol/L), compared with PTX rats treated with either vehicle or undifferentiated TMSC spheroids. This is the first report of a scaffold-free, human stem cell-based parathyroid tissue engineering and represents a more clinically feasible strategy for hypoparathyroidism treatment than those requiring scaffolds. Statement of Significance Herein, we have for the first time developed a scaffold-free parathyroid tissue spheroids using differentiated tonsil-derived mesenchymal stem cells (dTMSC) to restore in vivo parathyroid cell functions. This new strategy is effective, even for long periods (3 months), and is thus likely to be more feasible in clinic for hypoparathyroidism treatment. Development of TMSC spheroids may also provide a convenient and efficient scaffold-free platform for researchers investigating conditions involving abnormal calcium homeostasis, such as osteoporosis.

Objective In this study, we assessed the effectiveness of a tonsil-derived mesenchymal stem cell (TMSC)-transplanted polycaprolactone/beta-tricalcium phosphate prosthesis (specifically designed for easier fixing and grafting with a single scaffold) on rabbit mandible osteogenesis. Methods The mandibles of 18 rabbits were exposed, and 10 × 8-mm bone defects were made. Two rabbits did not receive implants; four were reconstructed with the scaffold control (SC) (SC group); four were reconstructed with scaffolds soaked in peripheral blood (PB) (PB group); four were reconstructed with TMSC-transplanted scaffolds (TMSC group); and four were reconstructed with differentiated osteocyte-transplanted scaffolds (DOC) (DOC group). Each rabbit was sacrificed 12 weeks after surgery, and the area of new bone formation was investigated by mechanical testing, histology, and micro-computed tomography. Results More extended and denser new bone masses were observed in the TMSC and DOC groups, although fibrosis and vascular formation levels were similar in all groups, suggesting that the dual-structured scaffold alone provides a good environment for bone attachment and regeneration. The bone volumes of representative scaffolds from the SC, PB, TMSC, and DOC groups were 43.12, 48.35, 53.10, and 57.44% of the total volumes, respectively. Conclusion The design of the scaffold resulted in effective osteogenesis, and TMSCs showed osteogenic potency, indicating that their combination could enable effective bone regeneration. Level of evidence NA Laryngoscope, 130:358-366, 2020.

Hypoparathyroidism is a deficiency of the parathyroid hormone (PTH) in the body. We previously reported the possibility of treating it using tonsil-derived mesenchymal stem cells (TMSCs) differentiated into PTH-releasing cells. The purpose of this study was to evaluate the feasibility of using autologous plasma gel as scaffold material in treatment of hypoparathyroidism with TMSC. We obtained plasma by venous sampling of autologous blood and centrifuged and fabricated the plasma gel using a sinusoidal pattern heating machine. After we created the hypoparathyroidism animal model, we administered undifferentiated TMSCs and TMSCs differentiated into parathyroid cells at each rat dorsum by intramuscular injection with and without the plasma gel. In the plasma gel groups, intact PTH was detected from on day 21 after TMSC injection; we did not detect intact PTH in the groups that were only transplanted with TMSCs during the entire experimental period. Serum calcium was higher and phosphorous was lower in the TMSC with plasma gel groups than in the groups with TMSCs alone. We detected PTH and chromogranin A in the TMSC-plasma gel-transplanted areas on immunohistochemistry and immunofluorescence stain. Plasma gel can be considered as a cell-delivery scaffold for treating hypoparathyroidism with tonsil-derived mesenchymal stem cells.

Aesthetic reconstruction of the external ear is challenging due to the complex anatomical shape of the auricle. Recently, artificial scaffolds such as Medpor (Stryker, Kalamasoo, MI, USA) have become widely used in ear reconstruction. However, the Medpor scaffold is stiffer than the natural ear, which may lead to discomfort, and moreover has uniform design for every patient. In this study, we investigated whether three-dimensional (3D)-printed artificial polyurethane (PU) scaffolds are suitable for auricular reconstruction.PU scaffolds were fabricated using 3D printing according to a design derived from a digital imaging and communications in medicine (DICOM) image of the human auricle. The microstructure of the scaffolds was observed using scanning electron microscopy, and the porosity was examined. Cell proliferation on the scaffolds was assessed in vitro using tonsil-derived mesenchymal stem cells to evaluate the biocompatibility of the scaffolds. The scaffolds were implanted in C57BL/6 mice, and histological analysis was performed.The structural study revealed that the 3D-printed porous PU scaffolds have rectangular microstructure with regular pitch and line, as well as high porosity (56.46% ± 10.22%) with a pore diameter of 200 µm. The mechanical properties of the 3D-printed PU scaffolds were similar to those of the human auricle cartilage. Cell proliferation on the PU scaffolds was greater than that on Medpor scaffolds. Histological evaluation demonstrated that the porous parts of the PU scaffolds became filled with collagen and vascular tissue.Elastic, porous PU scaffolds can be obtained using 3D printing, have biomechanical properties similar to those of the natural ear, and are suitable for use in auricular reconstruction.NA Laryngoscope, 129:351-357, 2019.

Parathyroid cells release parathyroid hormone (PTH), which controls calcium homeostasis. Loss of parathyroid cells results in hypoparathyroidism and consequent low-turnover bone disease. Here, we investigated whether our recently-established human tonsil-derived mesenchymal stem cells (TMSC) restore in vivo parathyroid cell function in rats with parathyroidectomy (PTX). Compared with undifferentiated control TMSC, TMSC differentiated with activin A and soluble sonic hedgehog induced a significant release of PTH as early as day 7, with increased PTH release occurring in response to lower calcium levels and vice versa. Released PTH increased osteocalcin expression and alizarin red S staining in preosteoblastic cells, indicating its functional activity. PTX rats fed calcium-free diet only survived for ∼10 days. Subcutaneous injection with TMSC alone did not increase their survival rates, regardless of differentiation. However, survival rates increased for up to 28 days in response to TMSC embedded in Matrigel (TMSC-MA), showing 40% and 80% in control and differentiated TMSC-MA, respectively. When compared with continuous increases by control TMSC-MA, stable levels of secreted PTH and serum ionized calcium were found in PTX rats with differentiated TMSC-MA. This is the first report that differentiated TMSC resemble parathyroid cells and, if embedded in Matrigel, restore in vivo parathyroid function.

Three new canthinone type alkaloids, canthin-6-one-1-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside (1), canthin-6-one-1-O-[6-O-(3-hydroxy-3-methylglutaryl)]-β-D-glucopyranoside (2) and canthin-6-one-1-O-[2-β-D-apiofuranosyl-6-O-(3-hydroxy-3-methylglutaryl)]-β-D-glucopyranoside (3) were isolated from the stem barks of Ailanthus altissima together with four quassinoids (4-7), seven phenylpropanoids (8-14) and a lignan of previously known structure (15). The inflammatory activities of the 15 isolates were screened on LPS-induced nitric oxide (NO), a proinflammatory mediator, in RAW 264.7 cells.

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A ready-made, acellular patch-type prosthesis is desirable in repairing partial tracheal defects in the clinical setting. However, many of these prostheses may not show proper biological integration and biomechanical function when they are transplanted. In this study, we developed a novel 3D printed polyurethane (PU) tracheal scaffold with micro-scale architecture to allow host tissue infiltration and adequate biomechanical properties to withstand physiological tracheal condition. A half-pipe shaped PU scaffold (1.8 cm of height, 0.18 cm thickness, and 2 cm of diameter) was fabricated by 3D printing of PU 200 μm PU beam. The 3D printed tracheal scaffolds consisted of a porous inner microstructure with 200 × 200 × 200 μm3 sized pores and a non-porous outer layer. The mechanical properties of the scaffolds were 3.21 ± 1.02 MPa of ultimate tensile strength, 2.81 ± 0.58 MPa of Young's modulus, and 725% ± 41% of elongation at break. To examine the function of the 3D printed tracheal scaffolds in vivo, the scaffolds were implanted into 1.0 × 0.7 cm2 sized anterior tracheal defect of rabbits. After implantation, bronchoscopic examinations revealed that the implanted tracheal scaffolds were patent for a 16 week-period. Histologic findings showed that re-epithelialization after 4 weeks of implantation and ciliated respiratory epithelium with ciliary beating after 8 weeks of implantation were observed at the lumen of the implanted tracheal scaffolds. The ingrowth of the connective tissue into the scaffolds was observed at 4 weeks after implantation. The biomechanical properties of the implanted tracheal scaffolds were continually maintained for 16 week-period. The results demonstrated that 3D printed tracheal scaffold could provide an alternative solution as a therapeutic treatment for partial tracheal defects.

Ideal hypoparathyroidism animal models are a prerequisite to developing new treatment modalities for this disorder. The purpose of this study was to evaluate the feasibility of a model whereby rats were parathyroidectomized (PTX) using a fluorescent-identification method and the ideal calcium content of the diet was determined. Thirty male rats were divided into surgical sham (SHAM, n = 5) and PTX plus 0, 0.5, and 2% calcium diet groups (PTX-FC (n = 5), PTX-NC (n = 10), and PTX-HC (n = 10), respectively). Serum parathyroid hormone levels decreased to non-detectable levels in all PTX groups. All animals in the PTX-FC group died within 4 days after the operation. All animals survived when supplied calcium in the diet. However, serum calcium levels were higher in the PTX-HC than the SHAM group. The PTX-NC group demonstrated the most representative modeling of primary hypothyroidism. Serum calcium levels decreased and phosphorus levels increased, and bone volume was increased. All animals survived without further treatment and did not show nephrotoxicity including calcium deposits. These findings demonstrate that PTX animal models produced by using the fluorescent-identification method, and fed a 0.5% calcium diet, are appropriate for hypoparathyroidism treatment studies.

Surgical transplantation of parathyroid tissue into the forearm muscle is one of the most commonly used surgical techniques. While simple, the procedure suffers from drawbacks. This study evaluated the feasibility of thermoreversible gel as an injectable carrier for parathyroid autotransplantation. Polyethyleneglycol-polyalanine-co-phenylalanine (PEG-PAF) thermoreversible gel (sol form at 4 °C, gel form at 37 °C) were manufactured. Thirty-eight Sprague-Dawley rats were divided into two groups (19 control, C group; 19 experimental, P group). The parathyroid glands of rats were excised. Parathyroid tissues were transplanted into the muscle pocket in sternocleidomastoid muscle in the C group. In the P group, the tissues were injected into the same muscle mixed with 0.3 ml thermoreversible gel. The serum levels of parathyroid hormone (PTH), ionized calcium, and phosphorous were measured before surgical procedure, on 7, 21, 56, and 70 days after surgery. Histology and immunohistochemistry were performed. Preoperative median PTH level of the C and the P group were 60.80 and 43.85 pg/ml, respectively (p = 0.641). Seventy days after surgery, median PTH level was 32.8 and 25.61 pg/ml, respectively. On day 70, the PTH level was restored by 54 % in the C group and 56 % in the P group compared to the preoperative value (p = 0.620). There were no significant intergroup differences in the ionized calcium/phosphorous level. Histology and immunohistochemistry revealed the successful transplantation of parathyroid tissues into the muscles in both groups. In conclusion, the PEG-PAF-based thermoreversible gel is a good candidate carrier material for intramuscular parathyroid autotransplantation.

The therapeutic potential of tonsil-derived mesenchymal stem cells (TMSC) prepared from human tonsillar tissue has been studied in animal models for several diseases such as hepatic injury, hypoparathyroidism, diabetes and muscle dystrophy. In this study, we examined the therapeutic effects of TMSC in a dextran sulfate sodium (DSS)-induced colitis model. TMSC were injected in DSS-induced colitis mice via intraperitoneal injection twice (TMSC[x2]) or four times (TMSC[x4]). Control mice were injected with either phosphate-buffered saline or human embryonic kidney 293 cells. Body weight, stool condition and disease activity index (DAI) were examined daily. Colon length, histologic grading, and mRNA expression of pro-inflammatory cytokines, interleukin 1β (IL-1β), IL-6, IL-17 and tumor necrosis factor α, and anti-inflammatory cytokines, IL-10, IL-11 and IL-13, were also measured. Our results showed a significant improvement in survival rates and body weight gain in colitis mice injected with TMSC[x2] or TMSC[x4]. Injection with TMSC also significantly decreased DAI scores throughout the experimental period; at the end of experiment, almost complete reversal of DAI scores to normal was found in colitis mice treated with TMSC[x4]. Colon length was also significantly recovered in colitis mice treated with TMSC[x4]. However, histopathological alterations induced by DSS treatment were not apparently improved by injection with TMSC. Finally, treatment with TMSC[x4] significantly reversed the mRNA levels of IL-1β and IL-6, although expression of all pro-inflammatory cytokines tested was induced in colitis mice. Under our experimental conditions, however, no apparent alterations in the mRNA levels of all the anti-inflammatory cytokines tested were found. In conclusion, our findings demonstrate that multiple injections with TMSC produced a therapeutic effect in a mouse model of DSS-induced colitis.