Emilio Satoshi Hara

Phospholipids play key roles in bone formation, with phosphatidylserine (PS) reportedly inducing more rapid mineralisation than phosphatidylcholine (PC); however, the underlying mechanisms remains unclear. This study investigated PS and PC mineralisation using experimental methods and computational chemistry. The stationary points in the potential energy surfaces of the reactions were preliminarily found using a neural network potential (PreFerred Potential in Matlantis) capable of predicting the interaction energies for arbitrary combinations of atoms, and then refined through density functional theory calculations (Gaussian16, at the B3LYP/6-31G(d,p) level of theory). When hydrolysis reactions were assumed to be the initial step in the mineralisation of phospholipids, the results were consistent with empirical analysis. PS was found to be more easily hydrolised than PC, primarily owing to the presence of a labile proton in the NH₃ ⁺ group of serine that facilitates proton transfer, enhancing hydrolysis of PS at lower energy thresholds. Specifically, when a single phospholipid was considered, three distinct hydrolysis routes were identified: between serine (or choline) and phosphate, between glycerol and phosphate, and between an aliphatic carbon chain and the glycerol backbone. In particular, the initial steps of hydrolysis involved the formation of a pentavalent phosphate intermediate. When calculations were performed with two adjacent phospholipid molecules, the loosely bound proton (H⁺) in the NH₃ ⁺ group could be readily transferred either to the P–O bond linking serine to the phosphate group; or to the P–O bond connecting the phosphate to glycerol in a neighboring PS6 molecule. These findings reveal the important roles of serine NH₃ ⁺ in facilitating hydrolysis of PS, and provide insights for designing novel molecules to accelerate bone regeneration.

Ectopic bone marrow (eBM) holds tremendous potential as an artificial organ, serving not only in stem cell transplantation therapies but also as a controlled experimental system for analyzing cellular dynamics and interactions between cells and the matrix during the formation, maintenance, and aging of BM. Although bone morphogenetic protein-2 (BMP-2) has been reported to induce eBM formation, it remains unproven whether BMP-2–induced eBM (BMP-eBM) can provide a functional BM niche that is comparable with native BM in long bones (LB-BM). In this study, through the use of single-cell RNA sequencing and transplantation models, we demonstrate that BMP-eBM displays a microstructure, cellular composition, and functional hematopoiesis similar to LB-BM. BMP-eBM establishes an optimized microenvironment capable of supporting hematopoietic stem cells and CXC chemokine ligand 12 (CXCL12)–abundant reticular (CAR) cells, which are critical components of the BM niche. BMP-eBM was able to significantly restore survival in irradiated mice. Through parabiosis and cell transplantation experiments, we identified that in situ adipose tissue–derived CD51highCxcl12-GFP– cells are the principal source of CAR cells within BMP-eBM. Furthermore, BMP-eBM can be isolated and after preconditioning, retransplanted as an independent, functional hematopoietic organ. In conclusion, our study confirms that BMP-eBM functions effectively as a hematopoietic organ, capable of supporting and maintaining a functional BM niche. These findings underscore BMP-2 as a crucial molecule for eBM generation and suggest its potential for addressing BM-related diseases and for use as a platform for in vitro and ex vivo biomedical applications.

The pathology of medication-related osteonecrosis of the jaw (MRONJ), often associated with antiresorptive therapy, is still not fully understood. Osteocyte networks are known to play a critical role in maintaining bone homeostasis and repair, but the exact condition of these networks in MRONJ is unknown. On the other hand, the local application of E-coli-derived Recombinant Human Bone Morphogenetic Protein 2/β-Tricalcium phosphate (E-rhBMP-2/β-TCP) has been shown to promote bone regeneration and mitigate osteonecrosis in MRONJ-like mouse models, indicating its potential therapeutic application for the treatment of MRONJ. However, the detailed effect of BMP-2 treatment on restoring bone integrity, including its osteocyte network, in an MRONJ condition remains unclear. Therefore, in the present study, by applying a scanning electron microscope (SEM) analysis and a 3D osteocyte network reconstruction workflow on the alveolar bone surrounding the tooth extraction socket of an MRONJ-like mouse model, we examined the effectiveness of BMP-2/β-TCP therapy on the alleviation of MRONJ-related bone necrosis with a particular focus on the osteocyte network and alveolar bone microstructure (microcrack accumulation). The 3D osteocyte dendritic analysis showed a significant decrease in osteocyte dendritic parameters along with a delay in bone remodeling in the MRONJ group compared to the healthy counterpart. The SEM analysis also revealed a notable increase in the number of microcracks in the alveolar bone surface in the MRONJ group compared to the healthy group. In contrast, all of those parameters were restored in the E-rhBMP-2/β-TCP-treated group to levels that were almost similar to those in the healthy group. In summary, our study reveals that MRONJ induces osteocyte network degradation and microcrack accumulation, while application of E-rhBMP-2/β-TCP can restore a compromised osteocyte network and abrogate microcrack accumulation in MRONJ.

Controlling stem cell behavior at the material interface is crucial for the development of novel technologies in stem cell biology and regenerative medicine. The composition and presentation of bio-factors on a surface strongly influence the activity of stem cells. Herein, we designed an electroactive surface that mimics the initial process of trabecular bone formation, by immobilizing chondrocyte-derived plasma membrane nanofragments (PMNFs) on its surface for rapid mineralization within 2 days. Moreover, the electroactive surface was based on the conducting polymer polypyrrole (PPy), which enabled dynamic control of the presentation of PMNFs on the surface via electrochemical redox switching, further resulting in the formation of bone minerals with different morphologies. Furthermore, bone minerals with contrasting surface morphologies had differential effects on the differentiation of human bone marrow-derived stem cells (hBMSCs) cultured on the surface. Together, this electroactive surface showed multifunctional characteristics, not only allowing dynamic control of PMNF presentation but also promoting the formation of bone minerals with different morphologies within 2 days. This electroactive substrate could be valuable for more precise control of stem cell growth and differentiation, and further development of more suitable microenvironments containing bone apatite for housing a bone marrow stem cell niche, such as biochips/bone-on-chips. Graphical abstract

Soft robotics has attracted great attention owing to their immense potential especially in human–robot interfaces. However, the compliant property of soft robotics alone, without stiff elements, restricts their applications under load‐bearing conditions. Herein, biohybrid soft actuators, that create their own bone‐like rigid layer and thus alter their stiffness from soft to hard, are designed. Fabrication of the actuators is based on polydimethylsiloxane (PDMS) with an Au film to make a soft substrate onto which polypyrrole (PPy) doped with poly(4‐styrenesulfonic‐co‐maleic acid) sodium salt (PSA) is electropolymerized. The PDMS/Au/PPy(PSA) actuator is then functionalized, chemically and physically, with plasma membrane nanofragments (PMNFs) that induce bone formation within 3 days, without using cells. The resulting stiffness change decreases the actuator displacement; yet a thin stiff layer cannot completely stop the actuator's movement, while a relatively thick segment can, but results in partial delamination the actuator. To overcome the delamination, an additional rough Au layer is electroplated to improve the adhesion of the PPy onto the substrate. Finally, an alginate gel functionalized with PMNFs is used to create a thicker mineral layer mimicking the collagen‐apatite bone structure, which completely suppresses the actuator movement without causing any structural damage.

Phospholipids co-localized with collagen were found at the early stages of dentin mineralization in mouse first molar. Plasma membrane phospholipids induced rapid mineralization of collagen.

Meckel's cartilage, a cartilage rod present in the mandible during developmental stages, shows a unique developmental fate: while the anterior and posterior portions undergo ossification, the middle part degenerates. Previously, it was shown that a stiff environment promoted cartilage degeneration in the middle region, while a soft environment enhanced the mineralization in the anterior region of Meckel's cartilage. This study aims to elucidate the spatio‐temporal changes in the mechanosensing properties of Meckel's cartilage during its early developmental stages and clarify the mechanotransduction‐related mechanisms involved in its degeneration. The results show that the expression of Hippo pathway effector yes‐associated protein (YAP) is only detectable in the Meckel's cartilage onward embryonic day (E)14.5, indicating that mechanosensing is dependent on the tissue developmental stage. Consistently, microenvironmental stiffness‐induced cartilage degeneration can only be induced in cartilages onward E14.5, but not in those at earlier developmental stages. Expressions of integrin‐β1 and cartilage matrix‐degrading enzymes, matrix metalloproteinase 1 (MMP‐1) and MMP‐13, are significantly enhanced in the degeneration area. Moreover, verteporfin (YAP inhibitor) and integrin‐β1 antibody block the substrate stiffness‐induced degeneration by suppressing the expressions of MMP‐1 and MMP‐13. These data provide new insights into the interplay between biochemical and mechanical cues determining the fate of Meckel's cartilage.

Inspired by the dynamic process of initial bone development, in which a soft tissue turns into a solid load‐bearing structure, the fabrication, optimization, and characterization of bioinduced variable‐stiffness actuators that can morph in various shapes and change their properties from soft to rigid are hereby presented. Bilayer devices are prepared by combining the electromechanically active properties of polypyrrole with the compliant behavior of alginate gels that are uniquely functionalized with cell‐derived plasma membrane nanofragments (PMNFs), previously shown to mineralize within 2 days, which promotes the mineralization in the gel layer to achieve the soft to stiff change by growing their own bone. The mineralized actuator shows an evident frozen state compared to the movement before mineralization. Next, patterned devices show programmed directional and fixated morphing. These variable‐stiffness devices can wrap around and, after the PMNF‐induced mineralization in and on the gel layer, adhere and integrate onto bone tissue. The developed biohybrid variable‐stiffness actuators can be used in soft (micro‐)robotics and as potential tools for bone repair or bone tissue engineering.

Medication-related osteonecrosis of the jaw (MRONJ) is related to impaired bone healing conditions in the maxillomandibular bone region as a complication of bisphosphonate intake. Although there are several hypotheses for the onset of MRONJ symptoms, one of the possible causes is the inhibition of bone turnover and blood supply leading to bone necrosis. The optimal treatment strategy for MRONJ has not been established either. BMP-2, a member of the TGF-β superfamily, is well known for regulating bone remodeling and homeostasis prenatally and postnatally. Therefore, the objectives of this study were to evaluate whether cyclophosphamide/zoledronate (CY/ZA) induces necrosis of the bone surrounding the tooth extraction socket, and to examine the therapeutic potential of BMP-2 in combination with the hard osteoinductive biomaterial, β-tricalcium phosphate (β-TCP), in the prevention and treatment of alveolar bone loss around the tooth extraction socket in MRONJ-like mice models. First, CY/ZA was intraperitoneally administered for three weeks, and alveolar bone necrosis was evaluated before and after tooth extraction. Next, the effect of BMP-2/β-TCP was investigated in both MRONJ-like prevention and treatment models. In the prevention model, CY/ZA was continuously administered for four weeks after BMP-2/β-TCP transplantation. In the treatment model, CY/ZA administration was suspended after transplantation of BMP-2/β-TCP. The results showed that CY/ZA induced a significant decrease in the number of empty lacunae, a sign of bone necrosis, in the alveolar bone around the tooth extraction socket after tooth extraction. Histological analysis showed a significant decrease in the necrotic alveolar bone around tooth extraction sockets in the BMP-2/β-TCP transplantation group compared to the non-transplanted control group in both MRONJ-like prevention and treatment models. However, bone mineral density, determined by micro-CT analysis, was significantly higher in the BMP-2/β-TCP transplanted group than in the control group in the prevention model only. These results clarified that alveolar bone necrosis around tooth extraction sockets can be induced after surgical intervention under CY/ZA administration. In addition, transplantation of BMP-2/β-TCP reduced the necrotic alveolar bone around the tooth extraction socket. Therefore, a combination of BMP-2/β-TCP could be an alternative approach for both prevention and treatment of MRONJ-like symptoms.