Tomo Yonezawa

Inkjet‐bioprinted acrylated peptides and PEG hydrogel with human mesenchymal stem cells promote robust bone and cartilage formation with minimal printhead clogging

Inkjet bioprinting is one of the most promising additive manufacturing approaches for tissue fabrication with the advantages of high speed, high resolution, and low cost. The limitation of this technology is the potential damage to the printed cells and frequent clogging of the printhead. Here we developed acrylated peptides and co-printed with acrylated poly(ethylene glycol) (PEG) hydrogel with simultaneous photopolymerization. At the same time, the bone marrow-derived human mesenchymal stem cells (hMSCs) were precisely printed during the scaffold fabrication process so the cells were delivered simultaneously with minimal UV exposure. The multiple steps of scaffold synthesis and cell encapsulation were successfully combined into one single step using bioprinting. The resulted peptide-conjugated PEG scaffold demonstrated excellent biocompatibility, with a cell viability of 87.9 ± 5.3%. Nozzle clogging was minimized due to the low viscosity of the PEG polymer. With osteogenic and chondrogenic differentiation, the bioprinted bone and cartilage tissue demonstrated excellent mineral and cartilage matrix deposition, as well as significantly increased mechanical properties. Strikingly, the bioprinted PEG-peptide scaffold dramatically inhibited hMSC hypertrophy during chondrogenic differentiation. Collectively, bioprinted PEG-peptide scaffold and hMSCs significantly enhanced osteogenic and chondrogenic differentiation for robust bone and cartilage formation with minimal printhead clogging.

Bioactive nanoparticles stimulate bone tissue formation in bioprinted three-dimensional scaffold and human mesenchymal stem cells.

Bioprinting based on thermal inkjet printing is a promising but unexplored approach in bone tissue engineering. Appropriate cell types and suitable biomaterial scaffolds are two critical factors to generate successful bioprinted tissue. This study was undertaken in order to evaluate bioactive ceramic nanoparticles in stimulating osteogenesis of printed bone marrow-derived human mesenchymal stem cells (hMSCs) in poly(ethylene glycol)dimethacrylate (PEGDMA) scaffold. hMSCs suspended in PEGDMA were co-printed with nanoparticles of bioactive glass (BG) and hydroxyapatite (HA) under simultaneous polymerization so the printed substrates were delivered with highly accurate placement in three-dimensional (3D) locations. hMSCs interacted with HA showed the highest cell viability (86.62 ± 6.02%) and increased compressive modulus (358.91 ± 48.05 kPa) after 21 days in culture among all groups. Biochemical analysis showed the most collagen production and highest alkaline phosphatase activity in PEG-HA group, which is consistent with gene expression determined by quantitative PCR. Massons trichrome staining also showed the most collagen deposition in PEG-HA scaffold. Therefore, HA is more effective comparing to BG for hMSCs osteogenesis in bioprinted bone constructs. Combining with our previous experience in vasculature, cartilage, and muscle bioprinting, this technology demonstrates the capacity for both soft and hard tissue engineering with biomimetic structures.

Improved properties of bone and cartilage tissue from 3D inkjet-bioprinted human mesenchymal stem cells by simultaneous deposition and photocrosslinking in PEG-GelMA

ObjectivesBioprinting of bone and cartilage suffers from low mechanical properties. Here we have developed a unique inkjet bioprinting approach of creating mechanically strong bone and cartilage tissue constructs using poly(ethylene glycol) dimethacrylate, gelatin methacrylate, and human MSCs.ResultsThe printed hMSCs were evenly distributed in the polymerized PEG-GelMA scaffold during layer-by-layer assembly. The procedure showed a good biocompatibility with >80% of the cells surviving the printing process and the resulting constructs provided strong mechanical support to the embedded cells. The printed mesenchymal stem cells showed an excellent osteogenic and chondrogenic differentiation capacity. Both osteogenic and chondrogenic differentiation as determined by specific gene and protein expression analysis (RUNX2, SP7, DLX5, ALPL, Col1A1, IBSP, BGLAP, SPP1, Col10A1, MMP13, SOX9, Col2A1, ACAN) was improved by PEG-GelMA in comparison to PEG alone. These observations were consistent with the histological evaluation.ConclusionsInkjet bioprinted-hMSCs in simultaneously photocrosslinked PEG-GelMA hydrogel scaffolds demonstrated an improvement of mechanical properties and osteogenic and chondrogenic differentiation, suggesting its promising potential for usage in bone and cartilage tissue engineering.

3D bioprinting and the current applications in tissue engineering

Bioprinting as an enabling technology for tissue engineering possesses the promises to fabricate highly mimicked tissue or organs with digital control. As one of the biofabrication approaches, bioprinting has the advantages of high throughput and precise control of both scaffold and cells. Therefore, this technology is not only ideal for translational medicine but also for basic research applications. Bioprinting has already been widely applied to construct functional tissues such as vasculature, muscle, cartilage, and bone. In this review, the authors introduce the most popular techniques currently applied in bioprinting, as well as the various bioprinting processes. In addition, the composition of bioink including scaffolds and cells are described. Furthermore, the most current applications in organ and tissue bioprinting are introduced. The authors also discuss the challenges we are currently facing and the great potential of bioprinting. This technology has the capacity not only in complex tissue structure fabrication based on the converted medical images, but also as an efficient tool for drug discovery and preclinical testing. One of the most promising future advances of bioprinting is to develop a standard medical device with the capacity of treating patients directly on the repairing site, which requires the development of automation and robotic technology, as well as our further understanding of biomaterials and stem cell biology to integrate various printing mechanisms for multi‐phasic tissue engineering.

TNF‐α Induces Caspase‐1 Activation Independently of Simultaneously Induced NLRP3 in 3T3‐L1 Cells

The intracellular cysteine protease caspase‐1 is critically involved in obesity‐induced inflammation in adipose tissue. A substantial body of evidence from immune cells, such as macrophages, has shown that caspase‐1 activation depends largely on a protein complex, called the NLRP3 inflammasome, which consists of the NOD‐like receptor (NLR) family protein NLRP3, the adaptor protein ASC, and caspase‐1 itself. However, it is not fully understood how caspase‐1 activation is regulated within adipocytes upon inflammatory stimuli. In this study, we show that TNF‐α‐induced activation of caspase‐1 is accompanied by robust induction of NLRP3 in 3T3‐L1 adipocytes but that caspase‐1 activation may not depend on the NLRP3 inflammasome. Treatment of 3T3‐L1 cells with TNF‐α induced mRNA expression and activation of caspase‐1. Although the basal expression of NLRP3 and ASC was undetectable in unstimulated cells, TNF‐α strongly induced NLRP3 expression but did not induce ASC expression. Interestingly, inhibitors of the ERK MAP kinase pathway strongly suppressed NLRP3 expression but did not suppress the expression and activation of caspase‐1 induced by TNF‐α, suggesting that NLRP3 is dispensable for TNF‐α‐induced caspase‐1 activation. Moreover, we did not detect the basal and TNF‐α‐induced expression of other NLR proteins (NLRP1a, NLRP1b, and NLRC4), which do not necessarily require ASC for caspase‐1 activation. These results suggest that TNF‐α induces caspase‐1 activation in an inflammasome‐independent manner in 3T3‐L1 cells and that the ERK‐dependent expression of NLRP3 may play a role independently of its canonical role as a component of inflammasomes. J. Cell. Physiol. 231: 2761–2767, 2016.

TRIM39R, but not TRIM39B, regulates type I interferon response

Behcets disease (BD) is a chronic relapsing inflammatory autoimmune disease characterized by recurrent oral and genital ulcers, skin legions and uveitis and its pathogenesis is not fully elucidated. Previously we identified that two novel susceptible SNPs are associated with BD. One is located in putative RNF39 promoter region, another is located on TRIM39 coding exon. In this study, in order to identify the molecular function of TRIM39, we established gain-of-function of TRIM39 related genes and thus, performed microarray analysis. Our results indicate that TRIM39R, but not TRIM39B, regulates type I interferon response.

Role of Fibulin 3 in Aging-Related Joint Changes and Osteoarthritis Pathogenesis in Human and Mouse Knee Cartilage

The EFEMP1 gene encoding fibulin 3 is specifically expressed in the superficial zone (SZ) of articular cartilage. The aims of this study were to examine the expression patterns of fibulin 3 in the knee joints during aging and during osteoarthritis (OA) and to determine the role of fibulin 3 in the pathogenesis of OA.

Fibulin-3 in joint aging and osteoarthritis pathogenesis

The EFEMP1 gene encoding fibulin 3 is specifically expressed in the superficial zone (SZ) of articular cartilage. The aims of this study were to examine the expression patterns of fibulin 3 in the knee joints during aging and during osteoarthritis (OA) and to determine the role of fibulin 3 in the pathogenesis of OA.

Establishment of novel reporter cells stably maintaining transcription factor-driven human secreted alkaline phosphatase expression.

BACKGROUND Transcriptional regulation is a very important and pivotal function in myriad biological responses. Thus, methods to determine transcriptional activity are required in not only basic medical research but also in drug discovery. We established novel reporter constructs using human secreted embryonic alkaline phosphatase (SEAP) and Epstein-Barr virus nuclear antigen (EBNA) 1, which can maintain constructs synchronized to host cell replication. METHODS We established nuclear factor-kappa B (NFkB) or interferon regulatory factor (IRF) driven SEAP expression constructs and then, introduced them into culture cells. RESULTS The cells maintain reporter constructs for a long period in the culture and produce SEAP into culture supernatant in response to each specific ligand such as lipopolysaccharide (LPS) and interferon- beta. Measuring SEAP with chemiluminescence makes it possible to get high standard dynamic range applying to high-throughput screening in drug discovery in both 96 and 384 well format. We can also use it to determine transcriptional activity in the cells transfected with expression plasmid or treated with various toll-like receptor (TLR) ligands in a concentration-dependent manner and time-dependent manner. Finally, we demonstrated drug screening using a number of natural products library. CONCLUSION We for the first time established the two novel reporter cells and validated their quality and accuracy enough to carry out drug screening.