Hydroxyapatite and β-tricalcium phosphate have been clinically applied as artificial bone materials due to their high biocompatibility. The development of artificial bones requires the verification of safety and efficacy through animal experiments; however, from the viewpoint of animal welfare, it is necessary to reduce the number of animal experiments. In this study, we utilized machine learning to construct a model that estimates the bone-forming ability of bioceramics from material fabrication conditions, material properties, and in vivo experimental conditions. We succeeded in constructing two models: ‘Model 1′, which predicts material properties from their fabrication conditions, and ‘Model 2′, which predicts the bone-formation rate from material properties and in vivo experimental conditions. The inclusion of full width at half maximum (FWHM) in the feature of Model 2 showed an improvement in accuracy. Furthermore, the results of the feature importance showed that the FWHMs were the most important. By an inverse analysis of the two models, we proposed candidates for material fabrication conditions to achieve target values of the bone-formation rate. Under the proposed conditions, the material properties of the fabricated material were consistent with the estimated material properties. Furthermore, a comparison between bone-formation rates after 12 weeks of implantation in the porcine tibia and the estimated bone-formation rate. This result showed that the actual bone-formation rates existed within the error range of the estimated bone-formation rates, indicating that machine learning consistently predicts the results of animal experiments using material fabrication conditions. We believe that these findings will lead to the establishment of alternative animal experiments to replace animal experiments in the development of artificial bones.

Pancreatic islet xenotransplantation is a potential treatment for diabetes mellitus, and porcine pancreas may provide a readily available source of islets. Islets in juvenile pigs are smaller than those in young adult pigs, but the insulin content is very similar. In addition, as juvenile pigs are more easily reared in uncontaminated conditions, many researchers have conducted studies using pancreatic islets from juvenile pigs. We aimed to analyze the distributions of endocrine cell clusters by comprehensively evaluating juvenile porcine pancreatic development and to propose an appropriate age at which islets could be isolated from the juvenile porcine pancreas. Splenic (SL) and duodenal lobe (DL) samples were collected from the pancreases of pigs aged 0–180 days (n = 3/day after birth). The chronological changes in endocrine cell clustering were analyzed in relation to morphological changes, cell characterization, numbers, islet areas, and gene expression. In juvenile pigs aged 0–21 days, the pancreas contained numerous endocrine cells, and compact islets appeared from 21 days of age. Well-defined small islets were seen at 28 days of age, and the clusters were denser in the SL than in the DL. At 35 days of age, the islets were morphologically similar to those observed at 180 days of age, and the greater number of islets was similar to that seen at 90 days of age. The differences in the islets’ cytoarchitecture between the lobes were negligible. The expression of β-cell-related genes was higher in the juvenile pancreas than in the adult pancreas, and the expression of neurogenin-3 decreased dramatically over time. These findings may have implications for attempts to refine the most appropriate age for islet isolation from porcine donors. Focusing on porcine pancreatic islets isolated at around 35 days after birth may offer benefits regarding their xenotransplantation potential.

The present study aimed at establishing a new cryopreservation method for mouse pancreatic islets by vitrification using hollow fibers as a container. A unique feature of the hollow fiber vitrification (HFV) method is that this method achieves stable vitrification using a minimum volume of cryoprotectant (CPA) solution, thereby ensuring high viability of the islets. The cytotoxicity, optimum composition, and concentration of the CPAs for vitrifying islets were examined. The viability, functional-integrity of vitrified islets were evaluated in comparison with those vitrified by conventional methods. Insulin secretion was measured in vitro by a static incubation assay and the metabolic functions was tested after transplantation into Streptozotocin-induced diabetic mice. The combination of 15% dimethyl sulfoxide+15% ethylene glycol resulted in the best CPA solution for the HFV of islets. HFV showed the highest viability in comparison to 2 vitrification methods, open pulled straws and vitrification with EDT324 solution. The vitrified islets stably expressed β-cells markers NeuroD, Pancreatic and duodenal homeobox-1, and MafA. Transplantation of the vitrified islets achieved euglycemia of the host diabetic mice and response to an intraperitoneal glucose tolerance test to a similar extent as non-vitrified transplanted islets. The HFV method allows for efficient long-term cryopreservation of islets. ^* MN and HM contributed equally to this work.

Feasibility of large experimental animal models in testing novel therapeutic strategies for diabetes

To overcome organ shortage during transplantation, interspecies organ generation via blastocyst complementation has been proposed, although not yet in evolutionarily distant species. To establish high levels of chimerism, low chimerism is required early in development, followed by high chimerism, to effectively complement the organ niche. Very few human cells are expected to contribute to chimerism in heterologous animals. Previous studies had demonstrated increased donor chimerism in both intra- and interspecies chimeras in rodents, using insulin-like growth factor 1 receptor (Igf1r) knockout (KO) mice; deletion of the Igf1r gene in the mouse host embryo created a cell-competitive niche. The current study aimed to generate IGF1R–KO pigs and evaluate whether they have the same phenotype as Igf1r-KO mice. To generate IGF1R–KO pigs, genome-editing molecules were injected into the cytoplasm of pig zygotes. The fetuses were evaluated at 104 days of gestation. IGF1R–KO pigs were generated successfully. Their phenotypes were almost identical to those of Igf1r-KO mice, including small lungs and enlarged endodermal organs in fetuses, and they were highly reproducible. Pigs may allow the generation of organs using blastocyst complementation with developmentally-compatible xenogeneic pluripotent stem cells over a large evolutionary distance.