The strong barrier function of the blood–brain barrier (BBB) protects the central nervous system (CNS) from xenobiotic substances, while the expression of selective transporters controls the transportation of nutrients between the blood and brain. As a result, the delivery of drugs to the CNS and prediction of the ability of specific drugs to penetrate the BBB can be difficult. Although in vivo pharmacokinetic analysis using rodents is a commonly used method for predicting human BBB permeability, novel in vitro BBB models, such as Transwell models, have been developed recently. Induced pluripotent stem cells (iPSCs) have the potential to differentiate into various types of cells, and protocols for the differentiation of iPSCs to generate brain microvascular endothelial cells (BMECs) have been reported. The use of iPSCs makes it easy to scale-up iPSC-derived BMECs (iBMECs) and enables production of BBB disease models by using iPSCs from multiple donors with disease, which are advantageous properties compared with models that utilize primary BMECs (pBMECs). There has been little research on the value of iBMECs for predicting BBB permeability. This study focused on the similarity of iBMECs to pBMECs and investigated the ability of iPSC-BBB models (monoculture and coculture) to predict in vivo human BBB permeability using iBMECs. iBMECs express BMEC markers (e.g., VE-cadherin and claudin-5) and influx/efflux transporters (e.g., Glut-1, SLC7A5, CD220, P-gp, ABCG2, and MRP-1) and exhibit high barrier function (transendothelial electrical resistance, >1000 Ω × cm2) as well as similar transporter expression profiles to pBMECs. We determined that the efflux activity using P-glycoprotein (P-gp) transporter is not sufficient in iBMECs, while in drug permeability tests, iPSC-derived BBB models showed a higher correlation with in vivo human BBB permeability compared with a rat BBB model and the Caco-2 model. In a comparison between monoculture and coculture models, the coculture BBB model showed higher efflux activity for compounds with low CNS permeability (e.g., verapamil and thioridazine). In conclusion, iPSC-BBB models make it possible to predict BBB permeability, and employing coculturing can improve iPSC-BBB function.

Autografts and allografts are currently considered the gold standard for grafting surgery; however, to meet the growing demand in fast-aging societies, synthetic biomaterials will play an increasingly important role. Here we report a biodegradable scaffold material composed of recombinant polypeptide based on the human type I collagen alpha 1 chain (RCPhC1) as a source of hydrogel-based graft materials. The flexibility to engineer ideal characteristics for bone grafts was demonstrated. The critical internal isotropic pore structure was generated through a designed thin-layer freeze casting process. The optimized biodegradation rate was controlled by dehydrothermal crosslinking by adjusting the amino acid composition of RCPhC1. As a result, RCPhC1 bone grafts manufactured by a highly scalable streamlined production protocol induced robust regeneration of mature bone tissue while being completely resorbed in pre-clinical animal models.

Bone graft materials provide a scaffold for migrating cells for bone regeneration. One of the major challenges is to support adequate neovascularization in the graft materials and bone tissue. Vascular endothelial cells have been shown to recognize the integrin-binding Arg-Gly-Asp (RGD) sequence in natural extracellular matrix (ECM) molecules. Here, we report a bone graft material composed of an RGD-enriched recombinant polypeptide based on human type I collagen alpha 1 chain (RCPhC1) and propose a category of bone graft materials called the recombinant bone matrix. RCPhC1 demonstrated significantly increased human umbilical vein endothelial cell attachment in vitro and was further processed through freeze casting and heat crosslinking processes to generate porous granular bone graft, in which RGD sequences remained canonical. When grafted in the rat model, RCPhC1 bone graft demonstrated a uniquely increased presence of CD34+ endothelial cells within the graft material. Bone tissue was found directly in contact with the pore structure of RCPhC1 bone graft, resulting in the regeneration of large bone tissue. By contrast, the combined demineralized and decellularized bone allograft containing bone collagen in the ECM did not show vascular formation within the graft material. When applied to canine tooth extraction socket, RCPhC1 bone graft rapidly induced highly vascularized regenerating tissues, which became a mature bone with the bone marrow tissue. These results indicate that RCPhC1 bone graft is a promising material and generated highly active bone tissues, which rapidly matured.

A Correction to this paper has been published: https://doi.org/10.1038/s43246-020-00111-0