Application of a laparoscopic device for cell-derived sheet transplantation on the liver in a porcine model.

BACKGROUND: Cell-derived sheets are of global interest for regenerative therapy. Transplanting a sheet for abdominal organs requires a device for laparoscopic delivery to minimize invasiveness. Here, using a porcine model, we aimed to confirm the feasibility of a device developed to deliver sheets to the thoracic cavity in a laparoscopic transplantation procedure. MATERIAL AND METHODS: We used the device to transplant human skeletal myoblast cell sheets onto the liver and measured extra-corporeal, intra-abdominal, and total procedure times for sheet transplantation. Tissues, including the liver and the sheet, were collected two days after transplantation and analyzed histologically. RESULTS: In all experiments (n = 27), all sheets were successfully placed at target locations. The mean (± standard deviation) extra-corporeal, intra-abdominal, and total procedure times were 44 ± 29, 33 ± 12, and 77 ± 36 s, respectively. We found no difference between the two surgeons in procedure times. Histological analyses showed no liver damage with the transplantation and that sheets were transplanted closely onto the liver tissue without gaps. CONCLUSION: We confirmed the feasibility of a simple universal device to transplant cell-derived sheets via laparoscopic surgery. This device could support a minimally invasive procedure for sheet transplantation.

A first-in-human clinical study of laparoscopic autologous myoblast sheet transplantation to prevent delayed perforation after duodenal endoscopic mucosal dissection.

BACKGROUND: The detection rate of superficial non-ampullary duodenal epithelial tumors (SNADETs) has recently been increasing. Large tumors may contain malignant lesions and early therapeutic intervention is recommended. Endoscopic mucosal dissection (ESD) is considered a feasible treatment modality, however, the anatomical and physiological characteristics of the duodenum create a risk of postoperative perforation after ESD. METHODS: To explore whether myoblast sheet transplantation could prevent delayed perforation after ESD, a first-in-human (FIH) clinical trial of laparoscopic autologous myoblast sheet transplantation after duodenal ESD was launched. Autologous myoblast sheets fabricated from muscle tissue obtained seven weeks before ESD were transplanted laparoscopically onto the serous side of the ESD. The primary endpoints were the onset of peritonitis due to delayed perforation within three days after surgery and all adverse events during the follow-up period. RESULTS: Three patients with SNADETs ≥ 20 mm in size underwent transplantation of a myoblast sheet onto the serous side of the duodenum after ESD. In case 1, The patient's postoperative course was uneventful. Endoscopy and abdominal computed tomography revealed no signs of delayed perforation. Despite incomplete mucosal closure in case 2, and multiple micro perforations during ESD in case 3, cell sheet transplantation could prevent the postoperative massive perforation after ESD, and endoscopy on day 49 after transplantation revealed no stenosis. CONCLUSIONS: This clinical trial showed the safety, efficacy, and procedural operability of this novel regenerative medicine approach involving transplanting an autologous myoblast sheet laparoscopically onto the serosa after ESD in cases with a high risk of delayed perforation. This result indicates the potential application of cell sheet medicine in treating various abdominal organs and conditions with minimal invasiveness in the future. TRIAL REGISTRATION: jRCT, jRCT2073210094. Registered November 8 2021, https://jrct.niph.go.jp/latest-detail/jRCT2073210094 .

Real-time observations of lithium battery reactions-operando neutron diffraction analysis during practical operation.

Among the energy storage devices for applications in electric vehicles and stationary uses, lithium batteries typically deliver high performance. However, there is still a missing link between the engineering developments for large-scale batteries and the fundamental science of each battery component. Elucidating reaction mechanisms under practical operation is crucial for future battery technology. Here, we report an operando diffraction technique that uses high-intensity neutrons to detect reactions in non-equilibrium states driven by high-current operation in commercial 18650 cells. The experimental system comprising a time-of-flight diffractometer with automated Rietveld analysis was developed to collect and analyse diffraction data produced by sequential charge and discharge processes. Furthermore, observations under high current drain revealed inhomogeneous reactions, a structural relaxation after discharge, and a shift in the lithium concentration ranges with cycling in the electrode matrix. The technique provides valuable information required for the development of advanced batteries.

Application of laser-induced bone therapy by carbon dioxide laser irradiation in implant therapy.

This study evaluated the application of laser-induced bone therapy (LIBT) to reduce implant healing time in rat tibia. Twenty 10-week-old female Sprague-Dawlay rats were used. The rats received laser irradiation (laser group) or sham operation (control group) on either side of the tibia. Five days after invasion, titanium implants were inserted in proximal tibia. Five, 10, and 20 days after implant placement, tibiae were collected. After taking micro-CT and performing a torque test, the tibiae were decalcified and 8-µm-thick sections were prepared. Specimens were stained with hematoxylin and eosin. Results. Micro-CT images, removal torque values, and histomorphometric analysis data demonstrated a significantly accelerated bone formation in the laser group earlier in the healing process. Conclusion. The use of laser irradiation was effective in promoting bone formation and acquiring osseointegration of titanium implants inserted in rat tibia. LIBT may be suitable for use in implant therapy.

Spatiotemporal expression of sclerostin in odontoblasts during embryonic mouse tooth morphogenesis.

INTRODUCTION: Sclerostin is the product of the SOST gene. Loss-of-function mutations in the SOST gene result in a high bone mass phenotype, thus confirming that sclerostin is a negative regulator of bone mass. SOST knockdown in humans also causes oral and dental malformations. However, the relationship between sclerostin and tooth development is unclear. METHODS: Using immunohistochemical techniques, we investigated sclerostin expression during fetal mouse tooth development and adult mouse tooth morphogenesis. RESULTS: Sclerostin was expressed in the secretory odontoblasts located along the ameloblasts of fetal mouse tooth germ and adult incisor. Sclerostin expression was also observed in the fetal and adult osteocytes in the jaw bone. CONCLUSION: These results suggest that sclerostin, one of the important regulatory factors of differentiated odontoblast function, may usable in vital pulp therapy.

Lymphocyte enhancer-binding factor 1: an essential factor in odontoblastic differentiation of dental pulp cells enzymatically isolated from rat incisors.

Lymphocyte enhancer-binding factor 1 (Lef1), a HMG-domain protein, is thought to play important roles in inductive tissue interaction during tooth development. Lef1 knockdown in mice causes arrest at the bud stage in tooth development. As this gene participates in the regulation of a large and diverse set of peptide growth factors in ectomesenchymal cell differentiation of dental papilla, Lef1 appears to be a key factor in odontoblast differentiation. However, the relationship between Lef1 and odontoblast differentiation is still unclear. To analyze the biological roles of Lef1 in regulating odontoblast differentiation, we transiently overexpressed or suppressed Lef1 in cultured dental pulp cells. Lef1-overexpressing cells expressed higher levels of dentin sialoprotein (DSPP), osteocalcin and alkaline phosphatase (ALP) mRNA and formed larger numbers of mineralized nodules compared to control cells. However, Msx-1 expression or cell proliferation was unaffected by overexpression of Lef1. To further examine the role of Lef1 in dental pulp cells, we knocked down Lef1 expression in dental pulp cells using short interfering RNA (siRNA). Transient expression of siRNA against Lef1 markedly reduced Lef1 mRNA levels, and Lef1-suppressed cells expressed lower levels of DSPP, osteocalcin and ALP mRNA compared to control cells. Furthermore, the formation of mineralized nodules was inhibited by siRNA against Lef1; however, neither Msx-1 expression or cell proliferation was inhibited by siRNA against Lef1. These results outline the role of Lef1 in accelerating odontoblast differentiation by regulating DSP and osteocalcin mRNA expression in dental pulp cells, confirming that Lef1 is a key factor for odontoblast differentiation.

Immunohistochemical localization of barx2 in the developing fetal mouse submandibular glands.

The development of mouse submandibular gland (SMG) begins at embryonic day 11.5-12 (E11.5-12), during which successive rounds of epithelial clefting and branching create complex epithelial tree-like structures. Homeobox genes regulate place-dependent morphogenesis, including epithelial-mesenchymal interactions, and control the expression patterns of signaling molecules. The Barx2 containing Homeobox exerts several key roles in development. Some studies have shown that the Barx2 plays important roles in the epithelial-mesenchymal interactions of organogenesis. However, the mechanisms of Barx2 associated with the development of SMG are obscure. In this study, we demonstrated for the first time the exact spatial and temporal Barx2 expression pattern in SMG epithelial tissue during development using immunohistochemical staining and Real-Time quantitative PCR. Barx2 was expressed in the nucleus of the epithelial cells located in the proliferative and differentiative regions of the developing SMG during the early development stages (E11.5-E13.5). After the E14.5-time period, the expression gradually decreased, and at E16.5, expression mostly disappeared despite the fact that evidence of cytodifferentiation, such as the appearance of proacinar cells, distinct lumen formation, and secretory products, was beginning to be observed. Results of Real-Time PCR demonstrated that the amount of Barx2 mRNA expression in SMG was maximal on E14.5, and gradually decreased by E18.5. These results indicate that Barx2 is associated with early stage epithelial tissue development, and can be a useful epithelial marker of the SMG during early developmental stages.

Modulation of branching morphogenesis of fetal mouse submandibular gland by sodium ascorbate and epigallocatechin gallate.

As an initial step to study the effect of antioxidants on the oral environment, we here investigated how sodium ascorbate and (-)-epigallocatechin 3-O-gallate (EGCG) affect the branching morphogenesis of the fetal mouse submandibular gland (SMG). When mouse SMG was prepared from the embryo at 13-day post prenatal stage and cultured, gradual development of branching morphogenesis was observed. Addition of sodium ascorbate affected this morphological change in a bimodal fashion. At lower concentrations of sodium ascorbate (0.25 approximately 2.27 mM), the branching morphogenesis was slightly but significantly (about 60%) enhanced, whereas at higher concentrations of sodium ascorbate (6.82 approximately 10.1 mM), the branching morphogenesis was inhibited. The addition of EGCG failed to stimulate, but inhibited the branching morphogenesis in a dose-dependent manner. These data support that the addition of a lower concentration of sodium ascorbate is essential to stimulate the growth of SMG, and that sodium ascorbate, but not all antioxidants, induces hormesis (beneficial action at lower concentration) in the present SMG system.

Inhibition of branching morphogenesis of mouse fetal submandibular gland by sodium fluoride--protection by epidermal growth factor.

As an initial step to study the effect of sodium fluoride on the oral environment, we investigated how sodium fluoride (NaF) affects the branching morphogenesis of fetal mouse submandibular gland (SMG). When mouse SMG was prepared from the embryo at 13 days post prenatal stage and cultured, gradual development of branching morphogenesis was observed. Addition of NaF affected this morphological change in bimodal fashions. At a lower concentration of NaF (< 2 microM), the branching morphogenesis was slightly enhanced, whereas at a higher concentration of NaF (4 - 8 microM), it was almost completely inhibited. The inhibitory effect of NaF at the higher concentration was abrogated by stimultaneous addition of epidermal growth factor (EGF), but not by 5alpha-dihydrotestosterone (DHT) or insulin-like growth factor (IGF). These data demonstrate that EGF can effectively reduce the cytotoxic activity of NaF at micromolar concentration.