Akio Kishida

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.

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.

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.

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.

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.

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 M0 were assessed by IL-1β 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.

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.

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.