Two chiral matrices were synthesized according to our previous study (Jiang et al., 2022). The chiral material p-Ph-(D- Phe-NHCH₂CH₂OCH₂CH₂OH)₂ was named D and p-Ph-(L- Phe-NHCH₂CH₂OCH₂CH₂OH)₂ was named L. Chiral matrices used for in vivo implantation were prepared 1 day in advance. DMEM (Cyagen Biosciences, Inc.) were mixed into concentrated solution of the gelator in DMSO. After the chiral matrices were formed, DMEM were added to keep the matrices moist.

The cranial bone defect model was created in healthy 5-week old SD rats (Wang et al., 2022; Guo and He, 2023). All protocols and procedures are compliant with the guidelines prescribed by the Association for Assessment and Accreditation of Laboratory Animal Care, approved by the Peking University Health Centre Institutional Animal Care and Use Committee and Peking University Health Centre Ethics Committee (LA2019320). Each group was comprised of five rats. The rats were anesthetized with 1% (w/v) sodium pentobarbital solution (40 mg/kg). After disinfection and incision, a critically-sized defect of 5 mm was created on the rat crania by a trephine bar. The periosteum was completely removed. The freshly-formed cranial defects were then injected with pre-formed chiral matrices. The total injection volume for each rat was maintained at 100 μL. Then non-resorbable membranes were used to guide tissue regeneration. Penicillin was administered to prevent infection following surgery. Three days and 7 days after the surgery, the rats were euthanized using CO₂ asphyxiation, and tissue fluids from the defect area were collected. Protease inhibitor was added at 10% (v/v) of the lysate, and after centrifuging the mixture at 14,100 × g for 30 min, the supernatant was collected for LC-MS/MS analysis.

The analysis of tryptic peptides was conducted using nanoflow LC-MS/MS technology, which involved the use of a quadrupole Orbitrap mass spectrometer (Q Exactive HF-X, Thermo Fisher Scientific, Bremen, Germany) coupled with an EASY nLC 1,200 ultra-high pressure system (Thermo Fisher Scientific) via a nano-electrospray ion source. Five hundred ng of peptides were loaded onto a 25 cm column (150 μm inner diameter), which was packed with ReproSil-Pur C18-AQ 1.9-µm silica beads (Beijing Qinglian Biotech Co., Ltd., Beijing, China). Solvent A was composed of 0.1% (v/v) formic acid in water, and solvent B was composed of 80% (v/v) ACN and 0.1% (v/v) formic acid in water. Peptides were separated using a gradient from 8% to 12% B in 5 min, then 12%–30% B in 33 min, followed by a step up to 40% in 7 min, and a 15 min wash at 95% B at a flow rate of 600 nL per minute. The total duration of the run was 60 min, during which time the column was kept at a temperature of 60°C using an in-house-developed oven. The mass spectrometer was operated in “top-40” data-dependent mode, wherein the Orbitrap mass analyzer (120,000 resolutions, 350–1,500 m/z range) was used to collect MS spectra, with an automatic gain control (AGC) target of 3E6 and a maximum ion injection time of 80 m. The most intense ions from the full scan were isolated with an isolation width of 1.6 m/z, following higher-energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 27, MS/MS spectra were collected in the Orbitrap (15,000 resolution) with an AGC target of 5E4 and a maximum ion injection time of 45 m. Precursor dynamic exclusion was enabled for 16 s.

To create 3D matrices containing murine RAW264.7 macrophage cell line (Cyagen Biosciences, Inc.), a concentrated gelator solution was first prepared by mixing DMSO with gelator powder until complete dissolution. Then RAW264.7 cells (3 million ml⁻¹) suspended in DMEM (Cyagen Biosciences, Inc.) were mixed with the concentrated solution and added into a 48-well plate. The final gelator concentration was 3 mg/mL, with a final DMSO concentration of 3.3% (v/v). Within a few minutes, self-supporting matrices were rapidly formed. After the formation of matrices, additional DMEM was then added, and the matrices were transferred to a humidified incubator under standard culture conditions (37°C, 5% CO₂).

RAW264.7 cells were cultured in 48-well plates at a density of 1 × 10⁶ cells/mL for 1 day. Following culture, cells were rinsed with phosphate-buffered saline (PBS) and fixed with 4% (w/v) paraformaldehyde at room temperature for 30 min. The samples were then permeabilized with 0.1% (w/v) Triton X-100 (diluted with PBS) for 10 min and blocked with 3% (w/v) bovine serum albumin (BSA; diluted with PBS) for 20 min at room temperature. BSA of 3% (w/v) was then used as blocking solution to minimize nonspecific immunostaining. After removing the permeabilization solution, cells were rinsed with PBS for 5 min at room temperature. Then polyclonal rabbit anti-Akt1 (1:200; Abcam) were added to the samples in 5% (w/v) BSA in PBS and incubated overnight at 4°C. Cells were rinsed thoroughly and then incubated for 1 h in the dark with the secondary antibody goat anti-rabbit IgG H&L Alexa Fluor 488 pre-adsorbed (1:500; Abcam). Phalloidin (Sigma) was used for cytoskeletal staining, and 4’, 6-Diamidino-2-phenylindole (DAPI; Sigma) was used to stain the cell nuclei. A confocal laser scanning microscope (Leica) was utilized to capture images.

Cells were isolated by centrifugation at 1,000 rpm for 5 min. Then, 100% methanol was added to fix and permeate the cells. The samples were washed three times with PBS. Primary antibody specific to Akt1 (1:200, Santa Cruz), was added and incubated for 30 min, followed by rinsing with PBS for three times. Subsequently, goat anti-mouse IgG H&L Alexa Fluor 488 fluorescent secondary antibody (1:200, Abcam) was added, incubated for 30 min, and then rinsed with PBS for three times. PE fluorescently labeled primary antibody CD206 (1:200, eBioscience) or Rho (1:200, Abcam) was then added and incubated for 30 min, followed by rinsing with PBS three times. The cell suspension was then prepared using a filter and loaded on the machine. The expression levels of the various protein markers were detected using a flow cytometer, and FlowJo software was utilized for data analysis.

Statistical analyses were conducted using SPSS (version 26), and figures were generated using GraphPad Prism (version 9.0). Statistical significance was analyzed with a two-tailed unpaired Student’s t test or analysis of variance (ANOVA) for multiple comparisons.

Our previous study revealed that left-handed nanofibrils strongly stimulate M2 polarization of macrophages via mechanotransduction both in vivo and in vitro, in contrast to right-handed nanofibrils (Jiang et al., 2022). Additionally, left-handed nanofibrils also exerted a favorable impact on bone regeneration (Wei et al., 2019). To better understand the protein components of the bone defect areas biased by chiral matrices, we obtained tissue fluids from the defect regions (Figure 1E), and conducted protein sequencing on 20 samples (2 chiralities × 2 time points × 5 replicates), as shown in Supplementary Figure S1B. A comparison of the left-handed group with the right-handed group revealed a total of 78 differentially expressed proteins (DEPs) on day 3, and 49 DEPs on day 7, with no overlap between the groups (refer to Supplementarys Figures S1C, D). The heatmaps generated for the DEPs on day 3 and day 7 demonstrated clear expression differences between the left-handed and right-handed groups (refer to Figures 1A, B). The volcano plots for identified differentially-expressed proteins on day 3 and day 7 showed both the statistical significance and fold-change of the proteins (refer to Figures 1C, D). On day 3, 74 proteins displayed significantly increased expression, while 4 proteins were downregulated in the left-handed group (Supplementary Table S1). On day 7, 29 proteins were upregulated while 20 were downregulated, with significant changes in expression levels in the left-handed group (Supplementary Table S1). Hence, identified proteins affected by chirality within the bone defect areas can be clearly distinguished.

To analyze the specific biological functions of the DEPs on day 3, GO, KEGG and reactome pathway analyses were all performed. The results of the GO analysis revealed that the upregulated proteins in the left-handed group were mainly involved in cell adhesion, including cell-cell adhesion, cell-cell adherent junctions, cell projection organization, leading edge cell differentiation, focal adhesion, and cadherin binding. Additionally, regeneration-related processes were enhanced as well, such as extracellular matrix, positive regulation of transforming growth factor beta receptor signaling pathway, regulation of T-cell antigen processing and presentation, cell response to interleukin-4, regulation of growth plate cartilage chondrocyte proliferation, BMP binding, and type I transforming growth factor beta receptor binding (Figure 2A). The KEGG analysis showed that pathways directly related to mechanotransduction, such as AMPK, PI3K-Akt, Jak-STAT, Ras, and MAPK signaling pathways, were all enriched, confirming the upregulation of cell adhesion pathways in the left-handed group. Consistent with the GO analysis, various regeneration-related pathways such as osteoclast differentiation, VEGF, and T cell receptor signaling pathways were enriched in the KEGG analysis (Figure 2B). The reactome enrichment analysis was used to establish a functional network that linked the identified cell adhesion proteins. Pathways were classified based on their biological processes, cellular components, and molecular functions. The identified pathways were prominent in biological processes, and the signaling pathways with the highest number of identified proteins were related to membrane (21 DEPs) and protein binding (17 DEPs) functions. The enrichment of focal adhesions may be due to previously-established relationships between fibronectin recognition and integrin activation (Jiang et al., 2022), which implies the same initial cell sensing process to chirality. Furthermore, two pathways related to Rho GTPases (regulation of GTPase activity, and regulation of Rho-dependent protein serine/threonine kinase activity) were enriched as well (Figure 2C). These results suggest that Rho GTPases, acknowledged as significant mediators of intracellular mechanotransduction (Xie et al., 2023), could have a function in the transmission of chiral cues. The column and heatmap analyses revealed that the proteins involved in cell adhesion, such as Crip2, Col4a2, Was, Bzw1, Clic4, Lpl, Arf4, Sh3gl1, and Akt1, were significantly upregulated in the left-handed group. Similarly, regeneration-related proteins, such as Cdkn1c, Was, and Carm1, were also upregulated, which was consistent with the GO and KEGG analysis (Figures 2D–F). Our findings thus indicate that the left-hand matrix significantly upregulated cell adhesion and enhanced regeneration on the third day post-transplantation, which is consistent with previous studies (Dou and Feng, 2017).

The same methods were used to examine the functions of DEPs on day 7. A decrease in the number of DEPs was observed, as depicted in Supplementary Figure S1C. However, DEPs associated with GTPase remained upregulated in the left-handed group, including proteins involved in GTP binding, GTPase activity, GDP binding, and small GTPase mediated signal transduction, as demonstrated in Figures 3A, C. This observation thus suggests that GTPase may play a crucial role in the regeneration process from day 3 to day 7. Furthermore, the upregulation of matrix remodeling (extracellular matrix glycoproteins and cell adhesion) and mechanotransduction (positive regulation of ERK1 and ERK2 cascade, cytoskeletal protein binding, and focal adhesion) processes in the left-handed group were retained on day 7. Notably, KEGG analysis identified the enrichment of the TGF-beta signaling pathway, which may facilitate tissue repair (Perez et al., 2018). Among the pathways highlighted in Figure 3C, small GTPase mediated signal transduction exhibited the highest number of DEPs. Interestingly, newly-emerged enriched pathways such as bone mineralization, positive regulation of chondrocyte differentiation, epithelial to mesenchymal transition, and negative regulation of cell migration suggested better long-term bone healing processes in the left-handed group. Several GTP and GDP-related proteins, including Rab14, Sar1a, and Rab5c, were upregulated in the left-handed group, reinforcing the upregulation of Rho GTPase pathways (Schwartz et al., 2008; Barbera et al., 2019). The expression levels of cell adhesion-related proteins, such as Ccl6, Tpm1 and Pabpc1, were also higher in the left-handed group, as depicted in Figures 3D–F. Overall, these observations highlight the significant upregulation of GTPase-related pathways during the early phase of bone regeneration.

To investigate the interactions of DEPs between day 3 and day 7, we employed the STRING tool to analyze protein-protein interaction (PPI) relationships for the target proteins. From the 127 DEPs, we selected 23 DEPs that exhibited the greatest significance for further analysis. The results revealed that Akt1 played a central role in protein-protein analysis among all the DEPs from day 3 and day 7, highlighting its importance in the chirality-biased regeneration process (as illustrated in Figure 4A). The pairwise protein correlation heatmap demonstrated that most adhesion proteins were positively correlated with each other and with Akt1, further validating its central role (as shown in Figure 4B). The bubble plot illustrated that the screened 23 DEPs involved in cell-cell adhesion and GTP-binding signaling were mostly upregulated in the left-handed group (Figure 4C). Construction of the PPI networks separately for day 3 and day 7 showed that Akt1 and other adhesion proteins were situated within the central nodes of the network on day 3, whereas GTPase-related proteins were located at the center on day 7 (as seen in Figures 4D, E). As such, Akt1 and Rho GTPase were the crucial and central components of protein interactions on day 3 and day 7, respectively.

To validate protein sequencing results of the functions of Akt1 and Rho GTPase in the regeneration process, we cultured macrophages (RAW 264.7) in chiral matrices. Results from immunofluorescence staining indicated that Akt1 is significantly upregulated in the left-handed group, and inhibiting Akt1 using A-674563 downregulated Akt1 expression in both the left-handed and right-handed groups (Figure 5A). Additionally, data obtained from flow cytometry testing indicated that inhibiting Rho using Fasudil (HA-1077) HCl greatly suppressed downstream Akt1 expression (Figure 5B). Furthermore, inhibiting Akt1 led to a reduction in the expression of CD206, the macrophage M2 marker (Figure 5C). Besides, downstream Was, Clic4, Adipoq and Rab showed a positive effect on tissue regeneration (Jiang et al., 2010; Lovren et al., 2010; Dewidar et al., 2015; Warner et al., 2019). Hence, we could conclude that both Rho and Akt1 played a crucial role in chirality-mediated macrophage polarization, which in result impacted the regeneration process (Figure 5D).