Chirality Regulates Macrophage Polarization for Tissue Regeneration Through Bidirectionally Modulating WTAP‐Mediated m6A Modification

Chirality, a fundamental design feature at all levels of life organization, has shown great capability in guiding cell‐fate determination and tissue regeneration. Chirality‐directed differentiation programs involve remarkable changes in transcriptional networks, yet whether epigenetic regulatory events are also required is less well understood. Herein, by combining high‐throughput m6A MeRIP sequencing, RIP‐qPCR, and gene modulation techniques, it is demonstrated that biomimetic chiral nanofibrils can bidirectionally regulate wilms tumor 1 associated protein (WTAP)‐mediated m6A RNA methylation to guide macrophages polarization for tissue regeneration. The biomimic chiral nanofibril hydrogels are fabricated using a self‐assembly approach based on C2‐symmetric phenylalanine derivatives. In vitro and in vivo studies indicated that the L‐nanofibrils exhibit a greater propensity to promote M2‐macrophage polarization than D‐nanofibrils, thereby favoring osteogenesis. Then, m6A‐MeRIP sequencing revealed a unique chirality‐dependent m6A methylation level in macrophages, which was gated by a competitive pair of CCM3‐FAK through enantioselectively integrin recognition. L‐nanofibrils considerably suppressed WTAP‐mediated m6A methylation by promoting Itgα3‐FAK expression and paxillin‐driven mechanotransduction, whereas D‐nanofibrils induced WTAP‐mediated m6A methylation by favoring ItgαV‐CCM3 expression and suppressing mechanotransduction. Furthermore, the gain‐of‐function and loss‐of‐function experiments and RIP‐qPCR demonstrate that the WTAP expression could direct macrophage polarization via manipulating the functional molecule B7‐H3 (CD276). Thus, a mechano‐epigenetic mechanism of chirality‐mediated cell‐fate determination is unveiled, which holds remarkable promise in the realm of assisted regenerative material design.


Introduction
[7] The transfer of chiral information from the molecular to cellular and multicellular levels was postulated to be the initial step of symmetry breaking. [3,8,9][12] Illuminated by the significant influences of endogeneous chirality on life activities, the effectiveness and safety of exogenous chirality with in vivo application evolved as a key concern. [13]ince the 1960s, the chiral drug sorting has become strictly in demand because of the high incidence of drug-induced malformations in pregnancies. [14]More recently, the critical role of food chirality has also come to light as it has been observed that the chiral conversion of l-amino acids to d-configurations by Chirality, a fundamental design feature at all levels of life organization, has shown great capability in guiding cell-fate determination and tissue regeneration.Chirality-directed differentiation programs involve remarkable changes in transcriptional networks, yet whether epigenetic regulatory events are also required is less well understood.Herein, by combining high-throughput m 6 A MeRIP sequencing, RIP-qPCR, and gene modulation techniques, it is demonstrated that biomimetic chiral nanofibrils can bidirectionally regulate wilms tumor 1 associated protein (WTAP)-mediated m 6 A RNA methylation to guide macrophages polarization for tissue regeneration.The biomimic chiral nanofibril hydrogels are fabricated using a self-assembly approach based on C2-symmetric phenylalanine derivatives.In vitro and in vivo studies indicated that the L-nanofibrils exhibit a greater propensity to promote M2-macrophage polarization than D-nanofibrils, thereby favoring osteogenesis.Then, m 6 A-MeRIP sequencing revealed a unique chirality-dependent m 6 A methylation level in macrophages, which was gated by a competitive pair of CCM3-FAK through enantioselectively integrin recognition.L-nanofibrils considerably suppressed WTAP-mediated m 6 A methylation by promoting Itgα3-FAK expression and paxillin-driven mechanotransduction, whereas D-nanofibrils induced WTAP-mediated m 6 A methylation by favoring ItgαV-CCM3 expression and suppressing mechanotransduction.Furthermore, the gain-of-function and loss-of-function experiments and RIP-qPCR demonstrate that the WTAP expression could direct macrophage polarization via manipulating the functional molecule B7-H3 (CD276).Thus, a mechano-epigenetic mechanism of chirality-mediated cell-fate determination is unveiled, which holds remarkable promise in the realm of assisted regenerative material design.
intestinal bacteria can modulate immune responses in mammals. [15,16]Therefore, the significance of chirality in regenerative materials, as a critical part of successful regenerative medicine aimed at restoring tissue defects to physiological functions, should also be taken into account.
As a framework for tissue regeneration, materials have to provide microenvironmental niches that can act in concert with the host cells to trigger a series of biochemical cascades. [17][20][21] It is well established that chiral materials can effectively modulate macrophage behavior. [22,23]Chiral 1D nanoparticles were reported to regulate immune cell maturation, with left-handed nanoparticles showing enhanced efficiency. [24]D poly-lysine-modified PPFU films could govern macrophage polarization on the material surface, with right-handed polylysine exerting a more pronounced influence on M2 polarization than the left-handed variant. [25]In contrast, 3D hydrogels exhibited distinctive trends in which left-handed chiral environments deftly guided M2 differentiation to facilitate bone restoration. [22]hese striking results further requested the urgency to figure out the molecular mechanism underlying chirality-mediated cell fate commitment. [26,27]Existing researches have shown that chirality could significantly modulate signaling at the RNA transcriptional level to manipulate cellular behaviors. [28][31][32] Thus far, the upstream epigenetic regulatory mechanisms of chirality-mediated cell fate determination remain elusive, presenting great challenges in designing chiral regenerative materials with precise biological functions.
][35] Suppressing the expression of the m 6 A methyltransferase METTL3 not only curtails M1 macrophage proliferation but also fosters their transition to the M2 phenotype. [32,36,37]Silencing of the demethylase fat mass and obesity-associated (FTO) disrupts the polarization of M2 macrophages. [38]In addition, the m 6 A modification was also intricately manipulated by the microenvironment niches. [39]aterial mechanics can regulate the expression of m 6 A modification enzymes in epithelial cells, thereby leaving an indelible mark on their differentiation lineage. [40]The cationic microenvironment induced by metal-organic framework nanoparticles promoted tumor treatment efficiency by upregulating the m 6 A modification levels. [41]Similarly, the local hypoxic microenvironment regulated by the OsSx-PEG nanocatalyst extensively augmented RNA m 6 A methylation. [42]Therefore, we postulated a hypothesis that chirality, a critical microenvironmental niche, governs macrophage polarization by modulating m 6 A epigenetic modifications. [32,33]n this study, by combining high-throughput m 6 A MeRIP sequencing, RIP-qPCR, and gene modulation techniques, we revealed the chirality biased transcriptome-wide profiling of m 6 A modifications and elucidated the mechano-epigenetic mechanism of chirality-mediated cell-fate determination.We found that the biomimetic chiral nanofibrils bidirectionally regulated Wilms tumor 1 associated protein (WTAP)-mediated m 6 A RNA methylation to guide macrophages polarization for tissue regeneration.In addition, the chirality-dependent m 6 A methylation level was gated by the competitive pair CCM3-FAK through enantioselectively integrin recognition.Furthermore, we mapped the chiral-sensitive m 6 A-modified sites on the functional molecule contributions to M2 polarization (Figure 1A).Our in-depth exploration of the intricacies behind chirality-gated m 6 A dual modifications during macrophage polarization lays the groundwork for the advancement of biomaterials capable of finely tuning cellular behaviors.

The L-Nanofibrils Microenvironment Promotes Osteogenesis by Inducing M2 Macrophages Differentiation
Leveraging the environmental cues provided by chiral niches offers a potential solution to address the challenges associated with establishing a blueprint for efficient regeneration. [26]In this study, we elucidated the comprehensive landscape of gene expression cascades associated with chirality-guided macrophage lineage diversification, providing crucial insights into leveraging biomimetic extracellular matrices to precisely modulate the immune microenvironment and facilitate the development of advanced regenerative materials.
Using the self-assembly of C2 symmetric phenylalanine derivative gelators, we synthesized chiral nanostructured hydrogels and evidenced their chirality through circular dichroism (CD) spectra and scanning electron microscopy (SEM). [22]The L-nanofibrils (L-nano, left-handed nanofibrils) and D-nanofibrils (D-nano, right-handed nanofibrils) exhibited a mirror symmetry with a fiber diameter of approximately 100 nm.The CD spectra also indicated that both L-nano and D-nano showed symmetry, with an isodichroic point and a zero crossing at a wavelength of 239 nm (Figure 1B).The SEM images revealed that the hydrogel matrix had a chiral nanofiber ultrastructure (Figure 1C).Additionally, rheological tests revealed that both L-nano and D-nano had similar viscoelasticity (storage modulus G 0 ≈9700 Pa and loss modulus G 00 ≈2200 Pa) (Figure 1D).Consequently, the L-nano and D-nano, with their similar physical properties, present an optimal microenvironment for examining the influence of the 3D chiral structure on macrophages.
To examine the effect of chirality on bone regeneration in vivo, we created a femur defect model in mice and compared the bone repair ability following the implantation of the L-nano and D-nano groups (Figure 1E).Four weeks after implantation, 3D micro-CT scans indicated that the L-nano group had more new bone formation and a more prominent external callus surrounding the defect area compared with D-nano group.Quantitative analysis showed that the bone mineral density (BMD) value in the L-nano group was considerably higher than that in the D-nano group (Figure 1F).Consistent with this, Masson staining revealed that the L-nano group showed a higher density and greater number of dark blue collagen fibers in the bone defect, with a thicker and more extensive morphology.Conversely, the D-nano group showed less dense and more sparse staining of collagen fibers (Figure 1G).These findings suggest that L-nanofibrils effectively promote bone repair compared to D-nanofibrils.Meanwhile, cellular analysis revealed that a large number of CD68þ macrophages infiltrated in the area around the bone defects, indicating that macrophages were predominantly engaged in the process of bone repair (Figure 1H).Immunofluorescence and western blot results further showed that the expression level of the M2 surface marker CD206 was higher in the L-nano group than in the D-nano group (Figure 1I and S1, Supporting Information).In addition, q-PCR analysis showed that the M2 polarization associated anti-inflammatory factors, such as IL-10 and Arg-1, were markedly increased in the L-nanofibril-cultured condition as compared to the D-nano group.On the contrary, there were no significant differences in the expression of the M1 macrophages polarization-related proinflammatory factors, IL-6 and TNF-a (Figure S1, Supporting Information).Thus, our results indicated that L-nanofibrils exhibited a greater propensity to promote M2-macrophage polarization than D-nanofibrils, thereby favoring osteogenesis.

Chiral Microenvironment Regulates Intracellular FAK/CCM3 Signaling Axis Involving Integrins, Which in Turn Regulates Macrophage Polarization and Promotes Osteogenesis
We further investigated how macrophages initially interpret chiral nanostructures and employed molecular biological techniques to explore the downstream cascade in the macrophage response to chiral fibers.The staining results showed that the expression of integrin α3 on macrophages was enhanced in L-nanofibrils after 12 h culturing in chiral hydrogels, while the expression of integrin αV was increased in D-nanofibrils (Figure 2A,B and S2, Supporting Information).[50] Our previous studies have reported that L-nanofibers can expose more arg-gly-asp sequences to the FN, which is beneficial for FN activation. [22,27]Hence, the above results further explained that L and D nanofibers in this study activated integrin α3 and αV, respectively.Integrins are heterodimeric cell surface receptors that facilitate the mechanical connection between cells and the extracellular matrix, which can regulate intracellular mechanotransduction. [51]ur results demonstrated that FAK was highly expressed in L-nanofibrils (Figure 2C), whereas CCM3 was highly expressed in D-nanofibrils (Figure S3, Supporting Information).In addition, the expression of CCM3 on the cell membrane was reversed by treatment with the FAK inhibitor PF562271 (Figure 2D).These results suggested that the mutual competition between FAK and CCM3 might be the gatekeeper for chirality recognition.Meanwhile, we observed a more prominent nuclear translocation of the YAP protein in L-nanofibers than in D-nanofibers (Figure S4, Supporting Information).Previous studies have shown that CCM3 is located in focal adhesions and competes with FAK to bind paxillin, thereby weakening FAK activation and YAP/TAZ signaling. [52]In addition, integrin α3 has been shown to activate FAK through the c-Src/ERK signaling pathway, and integrin αV is synergistically and exclusively upregulated in cerebrovascular malformations characterized by highly expressed CCM3 family molecules. [53,54]These results indicate that the L and D nanofibers selectively activate the corresponding integrins and regulate downstream cascade reactions of macrophages through competition between CCM3 and FAK in focal adhesions.
Considering the aforementioned results, we inferred that silencing CCM3 could rescue the phenotype and function of macrophages cultured in D-nanofibrils to levels comparable to those in L-nanofibrils.The typical M2 surface marker CD206 and anti-inflammatory factor Arg-1 were extensively upregulated after suppressing CCM3 expression, while proinflammatory factor TNF-α was downregulated (Figure 2E and S5, Supporting Information).Reconstructed immunofluorescences showed an increase in protrusions in the L-nanofibrils, particularly after silencing of CCM3, which was consistent with the morphological characteristics of M2 macrophages (Figure S6, Supporting Information).Co-culture of CCM3-silenced bone marrowderived macrophages (BMDM) and bone marrow stromal cells (BMSCs) in a transwell system showed that inhibiting CCM3 in BMDM increased mineralized nodule formation in BMSC (Figure 2F-H).These results demonstrated that CCM3 knockdown rescues chirality-derived macrophage M2 polarization and osteogenesis.The above findings indicated that the diverse macrophage polarization is initiated by chirality-dependent activation of the competitive pair CCM3-FAK through heterogeneous integrin recognition.

Chiral Microenvironment Regulates Macrophage Polarization and Promotes Osteogenesis through WTAP Modification
Cell fate determinantion is the result of coordinated regulation at different levels, and post-transcriptional regulation plays a crucial role in this process. [55]Therefore, exploring the posttranscriptional mechanisms of gene expression, such as mRNA modification, is crucial for understanding the regulatory processes underlying cell fate differentiation.M 6 A modification is highly susceptible to biomechanical signals, thereby affecting cellular fate and function. [56,57]To gain a better understanding of the molecular mechanism by which m 6 A contributes to the chirality regulation of macrophage polarization and the promotion of ossification, we conducted genetic-and cellular-level studies.The qRT-PCR, western blotting, and immunofluorescence staining analyses all consistently demonstrated that the gene and protein expression of WTAP was lower in the L-nanofibril hydrogel than in the D-nanofibrils (Figure 3A-C and S7, Supporting Information).WTAP is a writer enzyme that forms a complex with METTL3 and METTL14 to catalyze RNA methylation modification of RNA. [58]The expression of Mettl3 and Mettl14, which are related to m 6 A modification, was also downregulated in the Lnanofibrils (Figure S8, Supporting Information).To gain deeper insight into the effect of m 6 A modification mediated by WTAP on the macrophage phenotype and function in a chiral environment, we performed in vitro and in vivo experiments to validate its regulatory role.When WTAP was knocked out using CRISPR-Cas9 in BMDM (Figure S9, Supporting Information), the immunofluorescence and real-time fluorescence quantitative PCR (qPCR) results showed that the expression of the typical M2 marker CD206 and the anti-inflammatory factor IL10 was increased, while the expression of the proinflammatory factors iNOS and CCR7 decreased (Figure 3D,E).Conversely, overexpression of WTAP inhibited M2 polarization of macrophages in both L-and D-nanofibrils (Figure S10, Supporting Information).These protein secretion and gene expression results indicated that L-nanofibril-mediated regulation of m 6 A levels could impact the stability and translation of mRNAs encoding for proinflammatory cytokines, resulting in reduced inflammation.Previous studies have shown that the level of m 6 A modification is markedly increased in proinflammatory macrophages induced by lipopolysaccharides. [59] Knockdown of key m 6 A modification enzyme METTL14 inhibits macrophage inflammation. [60]Immune regulatory drugs, on the other hand, exert anti-inflammatory effects by inhibiting the expression of WTAP. [61]The m 6 A methylation is also involved in the programming of macrophages in tumors and inflammation progression. [62]Together, these results demonstrated that the diverse macrophage polarizations can be attributed to the chirality-dependent m 6 A modification.
After determining that L-nano and D-nano regulate the expression of WTAP results in distinct BMDM differentiation in vitro, we envisioned that combinations of WTAP-depleted macrophages (MF) and chiral matrices could enhance tissue regeneration in vivo.We implanted macrophage-laden chiral hydrogels into mouse femur bone defects, which are considered a model of bone formation (Figure 4A).As demonstrated by the microcomputed tomography (micro CT) scans demonstrated, the WTAP KO-MF/L-nano matrix group showed the highest amount of new bone formation at 2 and 4 weeks.After 2 weeks, the presence of newly formed bone was detected in both the WT-MF/ L-nano and WTAP KO-MF/D-nano groups, with several regions of bone isolated from host bone tissues, representing the abundance of multiple centers of new bone formation within the femur defect.Notably, the complete repair of the bone defect was already evident in the WTAP KO-MF/L-nano group.However, fewer isolated bone regions were formed after delivery in the wild-type MF/D-nano groups.After 4 weeks, full bone defect repair was observed in the L-nano group and WTAP KO-MF/D-nano groups.Quantitative analysis revealed that the WTAP KO-MF/L-nano group exhibited the highest bone volume and BMD values.The WT-MF/L-nano and WTAP KO-MF/ D-nano groups exhibited comparable bone volumes and BMD values, whereas the WT-MF/D-nano group exhibited the least bone regeneration.Overall, chiral hydrogels containing WTAP-knockout macrophages exhibited superior repair efficacy in both L-nano and D-nano, compared to the wild-type cell group (Figure 4B,C).Histological examination with HE and Masson staining also showed that the WTAP KO-MF/L-nano group had the highest density and greatest number of dark blue collagen fibers in the bone defect.In addition to lesser new bone formation, bone maturation was slower and characterized by the lowest number of thick collagen bundles in the WT-MF/D-nano group.These findings suggest that the hydrogel complex loaded with WTAP knockout macrophages was beneficial for bone regeneration (Figure 4D).In vitro transwell co-culture experiments consistently showed that BMSCs co-cultured with WTAP knockout BMDM had more mineralized nodules by Alizarin red staining, demonstrating that the WTAP-knockout macrophages had the most obvious effect on promoting BMSC osteogenesis (Figure 4E,F).These results suggest that the m 6 A writer enzyme, WTAP, is a key epigenetic modifier of mRNA in macrophages via chiral-dependent mechanical signals, which affect the macrophage signaling pathways that regulate bone regeneration.Given the observed impact of the FAK-CCM3 balance on macrophage polarization in a chiral environment, we further explored the downstream m 6 A gene expression following the disinhibition of mechanical transduction.Protein analysis demonstrated that silencing of CCM3 led to a marked decrease in WTAP expression in D-nanofibrils, resembling the L-nanofibrils levels (Figure S11, Supporting Information).This result revealed that chirality-dependent FAK-CCM3 equilibrium displacement can modulate the WTAP levels, providing a mechanistic understanding of how chirality controls m 6 A modifications in macrophages.These findings are in agreement with previous research that has identified m 6 A as a responsive hub to hemodynamic forces stimuli, which highlights the importance of biomechanical cue-dependent m 6 A RNA modifications in regulating cellular function. [56]L-nanofibrils promote Itgα3-FAK expression and enhance mechanotransduction, leading to reduced m 6 A methylation, while D-nanofibrils favor ItgαV-CCM3 expression and suppress mechanotransduction, resulting in increased m 6 A methylation.These findings suggest that chiralitydependent mechano-epigenetic regulation critically influences cellular transcript features.To determine the targeted mRNA of m 6 A modification during macrophage polarization in chirality environment, we performed m 6 A-MeRIP sequencing on mRNA from macrophages grown in L-nano and D-nanofibrils.Overall, the majority declines in peaks in the two groups were mainly located near the 3 0 UTR region.Most m 6 A peaks were located in the 3 0 -UTR (52.61% in the D-nano group, 49.93% in the L-nano group), followed by the CDS (13.57% in the D-nano group, 16.09% in the L-nano group) Figure 5.The methylation modification levels of CD276 were reduced and the gene expression was increased in the L-nanofibrils, which maintained the mRNA stability.A) The distribution of m 6 A-containing peaks across mRNA was displayed in metageneplot.B) Representative pie chart of peak distribution exhibited the proportion of total m 6 A peaks in the indicated regions including 5 0 -untranslated region (5 0 -UTR), 3 0 -untranslated region (3 0 -UTR), and exon.C) Heat map indicated that there were differences in the expression of m 6 A-related enzymes between L-nano and D-nano groups.D) CD276 was anchored by analyzing genes with reduced m 6 A modification but increased gene expression.E) KEGG enrichment scatter plot showed that genes in L-nano group involved in pathways, which related to inflammation inhibition and tissue repair including TGF-β, PI3K-Akt, and FoxO.F) SRAMP prediction showed the prediction score distribution along the query sequence.G) RIP-qPCR showed the relative enrichment of CD276 mRNA.H) IGV (Integrated Genomics Viewer) showed a higher m6A modification peak for TGF-β in the D-nano group.and the 5 0 UTR (33.82% in D-nano group and 33.99% in L-nano group) (Figure 5A,B).Our results revealed a significant difference in the modification peak of L-nano and D-nano 3 0 -UTR, with L-nanofibrils showing a significantly lower peak.This observation is noteworthy because m 6 A modification, which occurs on adenosine residues at the N 6 position, is known to be highly enriched in the 3 0 untranslated region (UTR) of mRNA. [63]s such, the lower peak of m 6 A modification in L-nanofibrils 3 0 -UTR compared to D-nanofibrils suggests that L-nanofibrils inhibit the post-transcriptional m 6 A modification of in macrophages.The heat map results showed that the demethylationrelated enzymes ALKBH5 and FTO were highly expressed in the L-nano group, whereas the methylation-related enzymes WTAP, YTHDF2, and METTL3 were highly expressed in the D-nano group, which reinforce the phenotype results (Figure 5C).Epigenetic modifications play an important role in regulating gene expression and are often associated with heritable changes without altering DNA sequence. [64,65]Methylation decreases mRNA stability, whereas demethylation increases mRNA stability. [65]Notably, there was an enrichment of genes that exhibited reduced m 6 A peaks but increased expression in the L-nanofibrils (Figure S12, Supporting Information).Among these, immune checkpoints such as B7-H3 (CD276), which belong to the B7 family and have been reported to be essential for maintaining immune homeostasis and regeneration, [66] were identified (Figure 5D).Functional enrichment analysis of multi-omic profiling genes, which exhibited reduced m 6 A peaks but increased expression in the presence of L-nanofibrils, revealed their involvement in pathways associated with inflammation inhibition and tissue repair promotion.These pathways include TGF-β, PI3K-Akt, and FoxO (Figure 5E).
In a previous study, silencing FTO expression with siRNA considerably downregulated the expression of B7 family molecules. [67,68]However, the regulatory mechanisms of m 6 A modification on the expression levels of B7 family molecules in macrophages are still under investigation.CD276 (B7-H3), a component of the B7 family, is implicated in crucial biological processes such as homeostasis and regeneration. [69]Mechanistically, we investigated the m 6 A modification mechanism underlying CD276 and conducted verification experiments.The potential m 6 A modification site of CD276 was predicted by using SRAMP [70] and verified using RIP-qPCR in wild-type and WTAP-knockout macrophages cultured in a chiral environment (Figure 5F,G).To verify the WTAP-mediated m 6 A modification of CD276 mRNA, we performed the m 6 A-RNA immunoprecipitation (RIP) assay and detected the m 6 A level in CD276 mRNA using real-time polymerase chain eaction (PCR).As result, WTAP-KO macrophages in L-nanofibrils showed the lowest levels of m 6 A in CD276 mRNA (Figure 5G).Additionally, Me-RIP results showed a higher m 6 A modification peak for CD276 in the D-nanofibrils than in L-nanofibrils (Figure 5H).Our results suggest that the methylation modification levels of CD276 are reduced and their expression increases in L-nanofibrils, maintaining mRNA stability by inhibiting m 6 A modification, thus promoting the expression of B7 family molecules.Further research is required to fully understand the role of CD276 expression, influenced by the chiral environment, in promoting bone tissue regeneration through epigenetic modifications.Concordantly, a protein interaction network revealed that molecules with decreased m 6 A modification and increased expression were linked to tissue regeneration pathways and factors, such as MMP14 and Col6a1 (Figure S13, Supporting Information), which are related to osteoblast induction.To verify the regulatory effect of m 6 A methylation on CD276 expression, qPCR and immunofluorescencer results showed that in WTAP-knockdown BMDM cells, CD276 gene expression was significantly upregulated and fluorescence intensity was considerably enhanced (Figure 6A,B).This indicates that decrease in m 6 A methylation levels could facilitate the upregulation of CD276 gene expression.To prove that CD276 is a functional molecule of macrophages in the tissue repair process, we studied the effect of CD276 expression on the BMDM-mediated osteogenesis.Alizarin red staining showed that after co-culturing with CD276-silenced BMDM and bone marrow stromal cells (BMSCs), calcium salt deposition on the cell surface was markedly decreased, and the osteogenic level of BMSCs was reduced (Figure 6C-E).These results suggested that CD276 is a crucial functional factor of BMDM in promoting BMSC osteogenesis.Based on the premise that chiral regulation of the m 6 A modification of CD276 promotes osteogenesis, we aimed to elucidate whether CD276 governs the phenotype of M2 differentiation.By using an siRNA to inhibit CD276 expression, we demonstrated that CD276 has a direct regulatory effect on the M2 phenotype.Specifically, immunofluorescence results showed that the fluorescence intensity of CD206 was markedly reduced after CD276 silencing (Figure 6F).Collectively, these data demonstrate that the chiral microenvironment modulates the phenotypical and functional characteristics of macrophages by regulating the expression of CD276.Our study demonstrated that L-nanofibrils suppress WTAP and reduce m 6 A methylation levels, further promoting CD276 production and inducing M2 macrophages.These findings were supported by m 6 A methylation analysis and MeRIP sequencing data, highlighting the importance of chirality-dependent mechanoepigenetic regulation in shaping cellular transcript features.

Conclusion
Taken together, this study uncovered the mechanisms underlying the chirality-niched bidirectional regulation of global m 6 A RNA methylation and the site-specific modified functional proteins guiding macrophages polarization.These findings revealed that chiral-dependent WTAP expression orchestrates reciprocal control over m 6 A methylation, specifically mediated by the competitive pair CCM3-FAK, which is regulated through chiral-selective integrin activation.Significantly, L-nanofibrils demonstrating a greater contribution to tissue regeneration.This study offers a comprehensive understanding of chiralitydirected macrophage polarization, which is useful in the design of valuable functional materials and enables precise modulation of cellular behavior.

Figure 1 .
Figure 1.L-nanofibrils promoted M2-macrophage polarization than D-nanofibrils to favor osteogenesis.A) Diagram represented the chiralitydependent mechano-epigenetic mechanism in immune regulation and osteogenesis promotion.B) Circular dichroism spectrum results exhibited good symmetry between L and D nanofibrils.C) Scanning electron microscope (SEM) showed the symmetrical chiral ultrastructure of L and D nanofibrils.Scale bars: 100 nm.D) Rheological tests revealed that both L and D nanofibrils had similar storage modulus (G 0 ≈ 9700 Pa) and loss modulus (G 00 ≈ 2200 Pa).E) Diagram showed the construction of bone defect and implantation of L and D nanofibrils.F) Micro-CT results indicated that L-nano group had more new bone formation, and quantitative analysis also showed that the BMD value in the L-nano group was significantly higher than in the D-nano group.Scale bars: 1 mm.G) Masson staining of tissue sections of mouse femoral defect showed a higher density of dark blue collagen fibers in L-nano group than in D-nano group.Scale bar: 200 μm.H) Immunofluorescence staining results indicated a high number of CD68þ macrophages were infiltrated in the area around the bone defects.Scale bar: 100 μm; 20 μm.BD: bone defect.I) Immunofluorescence results indicated an increased expression of the M2-related protein CD206 in the L-nano group.Scale bars: 5 μm.Data are the means AE SEM.*P < 0.05; two-tailed unpaired Student's t-test.

Figure 2 .
Figure 2. Macrophage and function in D-nano and L-nano group were enantioselectively gated by a competitive pair of CCM3-FAK through heterogenerous integrin recognition.A) Immunofluorescence results showed that the expression of Integrin αV was higher in D-nano group than that in L-nano group.B) Immunofluorescence results showed that the expression of Integrin α3 was higher in L-nano group than that in D-nano group.C) Immunofluorescence results manifested that the expression of CCM3 in L-nano group was lower than that in D-nano group.D) After using FAK inhibitor PF562271, immunofluorescence results demonstrated that the expression of CCM3 increased in both L-nano and D-nano groups.E) QPCR results indicated a sharply increased expression of M2-related genes in both L-nano and D-nano groups after CCM3 silencing.F) Diagram showed the coculturing of BMSC and CCM3-silenced BMDM by transwell assay.G) Inhibiting CCM3 in BMDM increased mineralized nodule formation of BMSC.H) Quantitative analysis showed that the osteogenic ability of BMSC was the highest in L-nano/CCM3 silencing group.Scale bars: 5 μm (A)-(D); 100 μm (G).Data are the means AE SEM.*P < 0.05; one-way ANOVA.

Figure 3 .
Figure 3. Chirality regulated the expression of WTAP results in distinct BMDM polarization.A) The qPCR results showed that the expression of WTAP was significantly lower in the L-nano group than that in D-nano group.B) Western blot results indicated that the protein expression of WTAP in the L-nano group was lower.C) Immunofluorescence results indicated that WTAP gene expression in the L-nano group was significantly lower.D,E) Immunofluorescence and qPCR results indicated a sharply increased expression of M2-related genes in both L-nano and D-nano groups after WTAP knockdown.Scale bars: 5 μm.Data are the means AE SEM.*P < 0.05; one-way ANOVA.

Figure 4 .
Figure 4. WTAP-depleted macrophage-laden chiral hydrogels enhanced tissue regeneration in vivo.A) Diagram showed the construction of bone defect and the implantation of BMDM-laden hydrogel.B,C) The Micro-CT and quantitative analysis indicated the highest bone volume and bone mineral density (BMD) values in the WTAP KO-MF/L-nano group.Scale bar: 1 mm.D) The HE and Masson staining results of the mouse femoral tissue sections showed that the WTAP KO-MF/L-nano group had a highest density and greatest number of dark blue collagen fibers at the bone defect.E,F) Alizarin red staining showed that WTAP KO-MF/L-nano group had the most mineralized nodules.Scale bar: 400 μm (D); 100 μm (E).Data are the means AE SEM.*P < 0.05; one-way ANOVA.

2. 4 .
Downstream Genes, CD276, Regulated by m 6 A Are Associated with the Polarization of BMDM Toward the M2 Phenotype and the Promotion of Osteogenesis

Figure 6 .
Figure 6.By regulating the expression of CD276, chiral microenvironment modulated the phenotypical and functional characteristics of macrophages.A,B) QPCR and immunofluorescence results showed that the expression of CD276 in L-nano group increased after the knockdown of WTAP.C) Diagram showed the coculturing of BMSC and CD276-silenced BMDM by transwell assay.D,E) Alizarin red staining showed that after co-culturing with CD276silenced BMDMs and BMSCs, calcium salt deposition on the cell surface was significantly decreased.F) Immunofluorescence results showed that the fluorescence intensity of CD206 was significantly reduced after CD276 was silenced.G) Diagram showed the molecular process of chirality affecting the phenotype and function of macrophages.Scale bars: 5 μm; Data are the means AE SEM.*P < 0.05; one-way ANOVA.