Tendons and ligaments are crucial connective tissues linking bones and muscles, yet achieving full functional recovery after injury remains challenging. We investigated the characteristics of tendon stem/progenitor cells (TSPCs) by focusing on the declining tendon repair capacity with growth. Using single-cell RNA sequencing on Achilles tendon cells from 2-and 6-week-old mice, we identified Cd55 and Cd248 as novel surface antigen markers for TSPCs. Combining single-cell RNA sequencing with single-nucleus RNA and ATAC sequencing analyses revealed that Cd55 and Cd248 positive fractions in tendon tissue represent TSPCs, as confirmed by their expression of established TSPC markers, with this population decreasing at 6 weeks. We also identified candidate upstream transcription factors regulating these fractions. Functional analyses of isolated CD55/CD248 positive cells demonstrated high clonogenic potential and tendon differentiation capacity, forming functional tendon-like tissue in vitro . This study establishes CD55 and CD248 as novel TSPC surface antigens, potentially advancing tendon regenerative medicine and contributing to the development of new treatment strategies for tendon and ligament injuries.

The development of decellularized vascular tissues for tissue engineering and vascular implants presents a promising approach to creating functional blood vessels. However, effective endothelialization with human endothelial cells remains challenging. This study examined the endothelialization of decellularized porcine aortas using human induced pluripotent stem (hiPS) cell-derived endothelial cells. Various decellularization methods were used to create vessels with different luminal surface properties. hiPS-derived endothelial cells seeded on these vessels showed adhesion and alignment, particularly on those decellularized with the high hydrostatic pressure (HHP) method. These cells expressed higher levels of EphrinB2 compared to those cultured on flat surfaces, suggesting they respond to the vessel’s topographical features. The results indicate that hiPS-derived endothelial cells can adhere to and orient on decellularized porcine aortas, mimicking arterial endothelial behavior. For translational applications, achieving complete endothelial coverage by hiPSC-derived endothelial cells requires the establishment of optimal culture and seeding conditions. Nevertheless, these results highlight the importance of luminal surface topography and suggest that decellularized vascular tissues and implants can be designed with patterned surfaces to support endothelial behavior.

Decellularized tissues are used as biomaterials for transplantation. Many decellularized tissues in clinical applications are prepared using surfactants; however, we have developed a new decellularization method that uses subcritical dimethyl ether (DME) instead of surfactants. Subcritical DME perfusion is usually used for lipid extraction; therefore, by perfusing tissues with subcritical DME, phospholipid cell membranes may be destroyed. DME vaporizes at room temperature and pressure, therefore, it is expected that it will not remain in the decellularized tissues and will not be toxic. In this study, subcritical DME was perfused into the porcine dermis, and the sample was subjected to DNA degradation to produce a subcritical DME-decellularized dermis. The subcritical DME-decellularized dermis showed good cell response in vitro and in vivo. In addition, we investigated the mechanism of the subcritical DME decellularization method and found that surfactants dissolve the entire cell and almost remove it; however, subcritical DME causes minor damage to the cell membrane and removes the cell nucleus through DNase treatment while leaving some of the cell membrane intact. These results suggest that subcritical DME-decellularized dermis is nontoxic and has the potential to develop highly functional decellularized tissues, such as extracellular vesicles, unlike decellularized dermis prepared with surfactants.

Decellularized tissue refers to extracellular matrix (ECM) derived from living tissue by removing the cellular components and is used for tissue regeneration. Various decellularized tissue sheets and powders, such as the dermis, urinary bladder matrix, and small intestinal submucosa, have been clinically used as covering and prosthetic materials. Moreover, there is growing interest in the use of decellularized tissue for soft-hard interregional tissue regeneration, including in the ligament-bone, tendon-bone, and periodontal ligament-bone interfaces. The focus in these applications lies in the mechanical properties of the decellularized tissue. Decellularized ligaments and tendons have been developed using various decellularization methods, with a focus on maintaining their shape and mechanical properties, and have been applied orthotopically or ectopically to ligaments and tendons. In the ligament-bone interface, it is suggested that decellularized ligament and tendon are regenerated through the migration and rearrangement of host cells, which is referred to as “in situ tissue regeneration.” It is also proposed that decellularized tissue can be used to prepare the complex structure of soft-hard interregional tissue, which consists of an ECM and cell populations with gradual change. In this case, the decellularized soft tissues of ligaments, tendons, pericardium, and others are fabricated and modified with hard tissue components to mimic the gradual structure of soft-hard interregional tissue. In this review, we present a detailed discussion of the regeneration of soft-hard interregional tissue using decellularized tissue.

Background: Analyzing the human bone marrow microenvironment requires an in vivo model that reflects the human bone marrow microenvironment. Introducing a human bone marrow mesenchymal stem cell (MSC) line into decellularized cancellous bone (DCB) is a first step in forming such a bone marrow model. Our goal with this research is identifying factors that promote the penetration of MSCs into DCBs in an ex vivo setting. Methods: We introduced the CRISPR Knock Out (GeCKO v2) library to identify candidate genes in UE7T-9 cell line (MSC line) for DCB penetration. We established a candidate gene-knockout UE7T-9 cell for validation and evaluated its penetration into DCB (measured distance of randomly selected 100 cells), proliferation (MTS assay), migration (scratch assay), and ancorage-independent growth (soft agar assay). RNA sequencing was performed to analyze changes in gene expression comprehensively. Results: We identified Serine/Arginine Repetitive Matrix 4 (SRRM4) knockout (KO) in the UE7T-9 cell as a candidate factor for bone penetration. SRRM4 KO promoted DCB penetration (3.1–7.1 times deeper, each p ≤ 1.91 × 10−24), cell migration (p = 0.039), and ancorage-independent growth (2.5 times in colony count, 7.1 times in colony size, each p = 0.001) but retained stem cell characteristics. Conclusions: SRRM4 KO is a newly defined factor of UE7T-9 cell penetrating into DCB. SRRM4 KO UE7T-9 cells may be used to analyze hematological diseases such as myelodysplastic neoplasms.

Tooth replantation is often compromised by the loss of periodontal ligament (PDL) integrity, leading to inflammatory root resorption and treatment failure. In this study, we investigated the regenerative potential of decellularized porcine pericardium (DPP), manufactured by either high hydrostatic pressure or surfactant decellularization. After evaluating the structural integrity and mechanical characteristics of the DPP, it was wrapped around the roots of extracted rat teeth to observe the biological response. PDL cells migrated onto the surface of DPP, establishing connection between DPP and PDL tissue. When rat molars were replanted with DPP wrapped around the root, recellularization of DPP and restoration of PDL were observed, along with the gradual augmentation of periostin-positive area within the PDL space. Severity of root resorption and inflammation was suppressed in DPP-wrapped roots. These findings suggest that DPP supports PDL regeneration and can serve as a clinically applicable material for improving tooth replantation outcomes.

The adhesion between an implant and a wound could result in over-bleeding when attempting to separate the two. To address this issue, a cell-repelling implant is preferred. In this study, a cell-repelling membrane was prepared by modifying decellularized porcine pericardium with multi-arm polyethylene glycol. With this modification technology, we switched the surface properties of the decellularized porcine pericardium from cell-adhering to cell-repelling. The result showed that this pericardium was successfully modified without any effect on the original properties of the pericardium and also maintained a low inflammatory response. The level of cell adhesion on the surface of the membrane was significantly reduced.

Understanding the interaction between macrophages and biomaterials is important for the creation of new biomaterials and the development of technologies to control macrophage function. Since macrophages are strongly adhesive, caution is required when performing in vitro evaluations. Similarly, when THP-1 cells, macrophage precursor cells, are differentiated into macrophages using phorbol-12-myristate-13-acetate (PMA), it becomes difficult to detach them from the adherent substrate, which has been a problem on investigation of immunological responses to biomaterials. In this study, the interaction of THP-1 cell-differentiated macrophages with biomaterials was analyzed based on a new method of seeding THP-1 cells. THP-1 cells were cultured in static and rotation culture without and with PMA. In undifferentiated THP-1 cells, there was no change in cellular function between static and rotation cultures. In rotation culture with PMA, THP-1 cells differentiated and formed macrophage aggregates. IL-1β and MRC1 expression in macrophage aggregates was examined after differentiation and M1/M2 polarization. Macrophage aggregates in rotation culture tended to be polarized toward M2 macrophages compared with those in static culture. In the evaluation of the responses of macrophage aggregates to several kinds of polymeric materials, macrophage aggregates showed different changes in MRC1 expression over time at 30, 50, and 70 rpm. Rotation speed of 30 rpm was considered most appropriate condition in that it gave stable results with the same trend as obtained with static culture. The use of macrophage aggregates obtained by rotational culture is expected to provide new insights into the evaluation of inflammatory properties of biomaterials.

Implanting physical barrier materials to separate wounds from their surroundings is a promising strategy for preventing postoperative adhesions. Herein, we develop a material that switches from an anti-adhesive surface to an adhesive surface, preventing adhesion in the early stage of transplantation and then promoting recellularization. In this study, 2-arm, 4-arm, and 8-arm poly(ethylene glycol) succinimidyl glutarate (2-, 4-, 8-arm PEG-NHS) were used to modify the surface of decellularized porcine and bovine pericardium. The number of free amines on the surface of each material significantly decreased following modification regardless of the reaction molar ratio of NH2 and NHS, the number of PEG molecule branches, and the animal species of the decellularized tissue. The structure and mechanical properties of the pericardium were maintained after modification with PEG molecules. The time taken for the PEG molecules to detach through hydrolysis of the ester bonds differed between the samples, which resulted in different cell repulsion periods. By adjusting the reaction molar ratio, the number of PEG molecule branches, and the animal species of the decellularized pericardium, the duration of cell repulsion can be controlled and is expected to provide an anti-adhesion material for a variety of surgical procedures.

Corneal opacity and deformation, which often require corneal transplantation for treatment, are among the leading causes of monocular blindness. To restore corneal clarity and integrity, there is a need for an artificial stroma that not only matches the transparency of donated human cornea but also effectively integrates to the corneal tissue. In this study, a transparent decellularized cornea was successfully developed using the high hydrostatic pressure method with processing conditions optimized for corneal decellularization. Biochemical analyses demonstrated the effective removal of cellular components from the transparent decellularized corneas, while preserving collagen and glycosaminoglycans. Proteome analysis also revealed that core matrisome and matrisome-associated proteins remained following decellularization, similar to the composition observed in untreated corneas. The light transmittance of the transparent decellularized corneas was 86.4 ± 1.5% in the visible region, comparable to that of donated human corneas. No complications, such as angiogenesis, were observed following interlamellar corneal transplantation in rabbits. The grafts were almost imperceptible immediately following surgery and achieved complete transparency within a few days, becoming indistinguishable even under a microscope. The transparent decellularized cornea presented here has promising potential as a material for application in lamellar keratoplasty.

In post-adhesion surgery, there is a clinical need for anti-adhesion membranes specifically designed for the liver, given the limited efficacy of current commercial products. To address this demand, we present a membrane suitable for liver surgery applications, fabricated through the modification of decellularized porcine pericardium with 20 KDa hexaglycerol octa (succinimidyloxyglutaryl) polyoxyethylene (8-arm PEGNHS). We also developed an optimized modification procedure to produce a high-performance anti-adhesion barrier. The modified membrane significantly inhibited fibroblast cell adherence while maintaining minimal levels of inflammation. By optimizing the modification ratio, we successfully controlled post-adhesion formation. Notably, the 8-arm PEG-modified pericardium with a molar ratio of 5 exhibited the ability to effectively prevent post-adhesion formation on the liver compared to both the control and Seprafilm®, with a low adhesion score of 0.5 out of 3.0. Histological analysis further confirmed its potential for easy separation. Furthermore, the membrane demonstrated regenerative capabilities, as evidenced by the proliferation of mesothelial cells on its surface, endowing anti-adhesion properties between the abdominal wall and liver. These findings highlight the membrane's potential as a reliable barrier for repeated liver resection procedures that require the removal of the membrane multiple times.

Hyaluronic acid (HA) has garnered much attention in the development of novel hydrogels. Hydrogels, as drug delivery systems, are very important in tissue engineering applications. In this study, we developed a novel HA nanogel containing a cholesterol and maleimide derivative (HAMICH) and its corresponding crosslinked hydrogel (HAMICH gel) to encapsulate drugs for their subsequent release. HAMICH gels self-assemble into nanoparticles via hydrophobic interactions. Dynamic light scattering analysis of HAMICH revealed that the particle size tended to decrease with increasing degree of cholesterol moiety substitution. The HAMICH gel was prepared through a Michael addition reaction between HAMICH and pentaerythritol tetra(mercaptoethyl)polyoxyethylene. The concentration of HAMICH needed for gelation depends on the degree of cholesterol moiety substitution; the higher the substitution degree is, the greater the concentration of HAMICH needed. The HAMICH gel exhibited less swelling and a smaller volume change than the gel with an unmodified cholesterol moiety in phosphate-buffered saline (pH 7.4). The HAMICH gel displayed enhanced peptide and protein trapping abilities without hydrogel swelling, suggesting its potential as a HA hydrogel for biomedical applications.

Decellularized tissues are used as transplant materials and scaffolding in regenerative medicine. Histological evaluation is used to assess decellularization and reveal residual cell nuclei and changes in the structure of the extracellular matrix. However, qualitative evaluation depends on the subjectivity of the evaluator. Therefore, in this study, an AI-based image classification method for objective evaluation of histological decellularization was developed and used to evaluate decellularization in stained images. Two image classifications were performed: untreated aorta and high hydrostatic pressure (HHP)-decellularized aorta, and untreated aorta and sodium dodecyl sulfate (SDS)-decellularized aorta. Both sets of images were classified with high accuracy. Accuracy, precision, recall [true positive rate (TPR)], false positive rate (FPR), F1-score, and area under the receiver operating characteristics curve (AUC-ROC) of the two classifications indicated that the AI-based classification method developed in this study accurately assessed decellularization. However, the TPR revealed that untreated aortas had a higher probability of being misidentified as HHP-decellularized aortas than as SDS-decellularized aortas. One factor that may have contributed to the misidentification of images of untreated aortas as those of decellularized aortas was that feature weighting was performed on other features other than the presence or absence of cell nuclei. Heatmaps were generated based on the results of image classification of stained images of decellularized aortas prepared by the two decellularization techniques. Therefore, the uniformity of decellularization could be visualized. The method developed in this study allows quantification of decellularization heterogeneity within decellularized tissues, which was previously unquantifiable. This method can be adapted to a wide variety of decellularized tissues and may contribute to rapid and efficient identification of decellularized tissues.

Evaluation of biomaterial properties using THP-1 cells require the establishment of standardized protocols. Two potential methods are available, which differ in the timing of cell contact with the material; i.e. cell differentiation occurring simultaneous with or prior to polarization. No reports have examined the activation state of macrophages during differentiation. The aim of this study was to clarify the effects of biomaterials on THP-1 cells during differentiation. THP-1 cells were seeded on polymeric materials in the absence and presence of phorbol-12-myristate-13-acetate, and M1/M2 polarization was induced. The differentiation from THP-1 cells into macrophages was evaluated by loss of proliferation and acquisition of adhesion. The activation levels and M1/M2 polarization of MO were assessed by IL-10 and MRC1 mRNA expression. Undifferentiated THP-1 cells were not markedly stimulated by interaction with biomaterials. However, THP-1 cells seeded on all the test materials differentiated into macrophages, and the macrophages polarized into different activated states depending on the material. These findings revealed the effects of material stimulation on macrophage activation state during differentiation. These results suggest that the step of cell differentiation and the step of contact with the material should be separated during systematic evaluation of biomaterials.

Investigation of biological response to materials is important in understanding their biocompatibility and cell -material interactions for biomaterial applications. Macrophages are important for early biological response. Responses of macrophages to materials have previously been investigated by quantitating inflammatory and anti-inflammatory cytokines using ELISA and RT-PCR assays, and by assessing phenotype changes using flow cytometry and immunohistochemistry. In this study, we developed a method to evaluate the proinflammatory response to polymeric materials using a macrophage cell line (THP-1) genetically tagged with a luminescent peptide (HiBiT). The gene for the luminescent peptide was inserted into IL -1,8 in THP-1 cells using the CRISPR/Cas9 system. Upon stimulation of HiBiT-tagged THP-1 cells with lipopolysaccharide, IL -10 secretion could be detected using highly sensitive measurement of luminescence as well as using ELISA and RT-PCR assays. We found that IL -10 production by HiBiT-tagged THP-1 cells differed in response to nylon, cellulose, and polytetrafluoroethylene. Moreover, the time course of IL -10 secretion also differed for these materials. These results indicate that IL -10 production over time in HiBiT-tagged THP-1 cells exposed to a material can be measured. We believe that this method for evaluation of proinflammatory response using genetically engineered macrophages would complement ELISA and RT-PCR in investigating cellular response to different materials.

With the current worldwide increasing use of plastics year by year, nanoplastics (NPs) have become a global threat to environmental and public health concerns. Among plastics, polypropylene (PP) is widely used in industrial and medical applications. Owing to the lack of validated detection methods and standard materials for PP NPs, understanding the impact of PP NPs on the environmental and biological systems is still limited. Here, isotactic polypropylene (iPP) was fabricated into oxidized polypropylene micro/nanoplastics (OPPs) via a thermal oxidation using hydrogen peroxide (H2O2) under various heating temperatures. The resulting OPPs were investigated in terms of the size distribution, surface chemistry, morphology, and thermal property as well as their concentration-dependent cytotoxicity to a human intestinal epithelial cell line (Caco-2), which could be a route to uptake NPs into the body through the food chain. The average diameters of the OPPs decrease with increasing reaction temperature. The OPPs obtained at 175 °C (OPP175) were spherical in shape and had a rough surface, with size distributions of approximately 0.14 ± 0.02 μm. A significant increase in the carbonyl content of the oxidized product was confirmed by Fourier transform infrared and X-ray photoelectron spectroscopy analyses. Caco-2 cells were exposed to OPP175 in a dose-dependent manner, and a significant loss of cell viability occurred at the concentration of 100 μg/mL. Thus, this study provides a fundamental approach for the fabrication of a model of NPs for the urgently demanded in vitro and in vivo studies to assess the potential impact of NPs on biological systems.

Microplastic (MP) pollution is a global environmental problem. To understand the biological effects of MPs on humans, it is essential to evaluate the response of human cells to model plastic particles that mimic environmental MPs in a sensitive and non-invasive manner. In this study, we investigated the preparation of poly(ethylene terephthalate) (PET) fragments with properties similar to those of environmental MPs by combining photo-oxidative degradation via ultraviolet (UV) irradiation with mechanical pulverization and hydrolysis via ultrasound (US) exposure. Combination of UV and US treatments decreased the particle size of PET fragments to 10.2µm and increased their crystallinity and Young's modulus to 35.7% and 0.73GPa, respectively, while untreated PET fragments showed the particle size of 18.9µm, the crystallinity of 33.7%, and Young’s modulus of 0.48GPa. In addition, an increase in negative surface potential and O/C ratio were observed for UV/US-treated PET fragments, suggesting surface oxidation via UV/US treatment. Cytokine secretion from human macrophages was evaluated by a highly sensitive inflammation evaluation system using the HiBiT-based chemiluminescence detection method developed by genome editing technology. UV/US-treated PET fragments induced a 1.4 times higher level of inflammatory cytokine secretion on inflammatory macrophages than untreated ones, suggesting that the biological responses of PET fragments could be influenced by changes in material properties via oxidation. In conclusion, UV/US treatment enables efficient preparation of model plastic particles and is expected to provide new insights into the evaluation of biological effects using human cells. (240 words)

Nanogels are candidate biomaterials for tissue engineering and drug delivery. In the present study, a cholesterol–hyaluronic acid hydrogel was developed, and the pro-inflammatory response of macrophages to the hydrogel was investigated to determine its use in biomedical applications. Hyaluronic acid modified with cholesterol (modification rate: 0–15%) and maleimide (Chol-HA) was synthesized. The Chol-HA nanogel was formed through self-assembly via hydrophobic cholesterol interactions in aqueous solution. The Chol-HA hydrogel was formed through chemical crosslinking of the Chol-HA nanogel via a Michael addition reaction between the maleimide and thiol groups of 4arm−PEGSH. We found that the Chol-HA hydrogels with 5, 10, and 15% cholesterol inhibited the pro-inflammatory response of HiBiT−THP−1 cells, suggesting that the cholesterol contributed to the macrophage response. Furthermore, Interleukin 4 (IL−4) encapsulated in the hydrogel of the Chol-HA nanogel enhanced the inhibition of the inflammatory response in HiBiT-THP-1 cells. These results provide useful insights into the biomedical applications of hydrogels.