Ha Yeong Kim

Scaffold-free parathyroid tissue engineering using tonsil-derived mesenchymal stem cells

UNLABELLED 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 7days and differentiation medium (containing activin A and sonic hedgehog) for next 7days. 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 (57.7-128.2pg/mL) and ionized calcium (0.70-1.15mmol/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 (3months), 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.

Expression of tenocyte lineage-related factors from tonsil-derived mesenchymal stem cells

Human palatine tonsil-derived mesenchymal stem cells (TMSCs) are known to be a new source of progenitor cells. Using waste tissue after tonsillectomy as a cell provider can be the biggest benefit of TMSCs, compared with other stem cells. The purpose of this study was to investigate tenogenic differentiation of TMSCs and to access the differential effects of transforming growth factor beta 3 (TGF-β3) on the tenogenesis of TMSCs. Human tonsil was obtained after tonsillectomy. Using a cytometric analysis, we were able to find that the TMSCs had typical mesenchymal stem cell markers: positive for CD73, CD90, and CD105, and negative for CD14, CD34, and CD45. Using TGF-β3, the expressions of tenocyte-specific genes and proteins, such as collagen type 1 (COL1), tenomodulin (TNMD), and scleraxis (SCX), were measured by a quantitative polymerase chain reaction (PCR), immunofluorescence staining, immunohistochemistry and Western blot analyses. Quantitative PCR assay showed that TGF-β3 significantly increased the expressions of tenocyte lineage marker genes, including COL1, TNMD, and SCX, at a 3-day treatment, compared with control. However, these increases were not found at long-term exposures (7 or 10 days), except that TNMD expression was maintained at 50 ng/mL at a 7-day exposure to TGF-β3. Like genes, the protein expression levels of COL1, TNMD, and SCX were also induced in TGF-β3-treated TMSCs in a 3-day treatment, which were maintained for 10 days, as evidenced by immunofluorescence staining, immunohistochemistry and Western blot analyses. This study demonstrated that TMSCs in tenogenic stimulation with TGF-β3 have a high tenogenic differentiation potential.

Standardization of A Physiologic Hypoparathyroidism Animal Model

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.

Feasibility of autologous plasma gel for tonsil-derived stem cell therapeutics in hypoparathyroidism

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.

Fabrication and characterization of 3D-printed elastic auricular scaffolds: A pilot study: 3D-Printed Elastic Auricular Scaffold

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.

Double intratibial injection of human tonsil-derived mesenchymal stromal cells recovers postmenopausal osteoporotic bone mass

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.

Therapeutic potential of tonsil-derived mesenchymal stem cells in dextran sulfate sodium-induced experimental murine colitis

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.