Study design and characterization of the candidate scaffolds

To analyze the cellular regulations of the candidate scaffolds and provide fundamental guidance for scaffold design targeting suture-bony composite defects, a series of investigations were conducted. The detailed flowchart of this study is displayed in Fig. 1a. For the characterization of candidate scaffolds, photographs depicted the general appearance in both dry and wet conditions (Fig. 1b). Scanning electron microscopy (SEM) observations showed the porous microstructures of GelMA and CTS under lyophilized conditions (Fig. 1c). In a hydrated state, GelMA formed a compact gel, whereas CTS formed a physically porous gel (Fig. 1b). Unlike these two, PLA existed in the form of electrospinning membranes (Fig. 1b, c). Energy-dispersive X-ray spectroscopy (EDS) mapping indicated that GelMA and CTS comprised C, O, and N elements, whereas PLA contained only C and O elements (Fig. 1d). The mechanical properties were assessed through frequency sweeps and cycle testing (Fig. 1e, f). Figure 1e demonstrated that the storage modulus remained consistent as the frequency increased, suggesting the formation of stable network structures in the three scaffolds,31 among which PLA exhibited the highest storage modulus (Fig. 1e, g). CTS displayed the most favorable performance under cyclically applied external forces, indicating its superior structural stability compared with the other two (Fig. 1f, g). For thermal stability, thermogravimetric analysis (TGA) results showed GelMA and CTS with weight reduction peaks below 100 °C (Fig. S1a). In vitro degradation tests indicated no degradation of CTS and PLA in phosphate-buffered saline (PBS) at 37 °C during the 8 weeks (Fig. S1b). Combining the findings of these two, the thermal stability order of the three scaffolds was PLA, CTS, and GelMA (Fig. 1g). As for cytotoxicity in vitro, MSC could adhere to and proliferate on the scaffolds, with the highest cell adhesion count on PLA, followed by GelMA and CTS (Fig. 1g and S2a, d). No adverse influence of scaffolds was observed on the survival of co-cultured MSC by live-dead assays (Fig. S2b, c). In terms of biosafety in vivo, measurements of rat body weight, head length, head width, cranial length, and cranial width 6 weeks post-surgery revealed no significant differences among the groups (Fig. S3a). Meanwhile, microscopic examination of histological images exhibited the absence of evident tissue harm or pathological alterations across primary organs (Fig. S3b). Moreover, blood routine and biochemical analysis were performed, revealing no substantial alterations attributable to the scaffold implantation (Fig. S3c). Collectively, these findings confirmed the excellent biocompatibility of the three scaffolds (Fig. 1g).

Fig. 1

Study strategy and characterization of the candidate scaffolds. a The flowchart of this study (created with Biorender.com). MSC, mesenchymal stem cells; OB, osteoblasts. b Photographs of GelMA, CTS, and PLA in dry and wet conditions. c SEM displaying the microstructures of GelMA, CTS, and PLA. d EDS elemental mapping of carbon (C), oxygen (O), and nitrogen (N) elements in the corresponding SEM images. The pie charts and the numbers in the images display the elemental proportions. Frequency sweeps (e, storage modulus G’ vs frequency) and mechanical cycle testing (f) of GelMA, CTS, and PLA scaffolds. g Comprehensive performance of each scaffold. In the radar chart evaluation, multiple criteria including (relative) cell adhesion, biocompatibility, (relative) modulus, thermal stability, and (relative) mechanical stability were considered to compare the performance of each scaffold

Phenotypic screening identified PLA as suture mesenchyme-regenerative scaffolds

To preliminarily compare the overall performance of the scaffolds in vivo, 2 × 4 mm rectangular defects were generated along the coronal sutures of rats, followed by the transplantation of scaffolds (Fig. 2a). In Sprague-Dawley (SD) rats, apart from the posterior frontal suture, all the other sutures remain open the whole life.32 However, concerning the current surgical model, a complete fusion of the coronal suture was observed 6 months postoperatively (Fig. S4a, b). This is consistent with the reported risk of cranial suture loss following calvarial defects,20 necessitating interventions to regenerate mesenchymal tissue and maintain suture patency. 6 weeks after scaffold implantation, micro-computed tomography (µCT) images and accompanying quantitative data displayed an inhibitory tendency on suture closure in PLA (Fig. 2b, c). Further histological analysis revealed persistent, undegraded GelMA and CTS components within the defects (Fig. 2f, g). Conversely, PLA displayed near-complete degradation by 6 weeks (Fig. 2f, g). Cellular infiltration was observed along the direction of PLA folds at the 2-week time point (Fig. 2d, e) accompanied by blue-stained nuclei penetrating the spinning’s interior (Fig. 2e). At 6 weeks, NC (suture-bony composite defects without scaffold implantation) and GelMA exhibited mineralized fibers and mature bone at the defect center, indicating possible suture closure risks in the future (Fig. 2g). In contrast, PLA featured abundant nascent mesenchymal tissues in the defects (Fig. 2g), suggesting capabilities to restore mesenchymal tissue and maintain suture patency. To quantify the regenerated hard tissue, 5 regions from the osteogenic front and 5 regions from the defect center of Masson’s images (Fig. 2g) were randomly selected (Fig. 6a). The statistical analysis of the total 10 regions revealed no significant differences in mineralized fibers and bone quantity among groups (Fig. 2h). However, when assessing separately, PLA exhibited a notable reduction in central ossification, while no significant distinctions were observed at the repairing forefront (Fig. 2i). Based on these animal and histological detections, PLA was preliminarily speculated to have potential in suture-mesenchyme regeneration.

Fig. 2

Phenotypic performance of the scaffolds at animal and histological levels. a Schematic illustration of a suture-bony composite defect across coronal sutures (a rectangular defect = 2 mm × 4 mm) in SD rats. b Representative µCT images and cross-sectional views of suture-bony composite defects 6 weeks postoperatively. The cross-sections depict the locations indicated by the white dashed lines in 3D images. c Quantitative analysis of defect closure 6 weeks post-surgery through µCT assessment. n = 6 replicates/group. H&E (d, f) and Masson’s trichrome staining (e, g) of suture-bony composite defects after scaffold implantation for 2 weeks (d, e) and 6 weeks (f, g). The high-magnification images are from the selected regions (black boxes) in the low-magnification images. h, i Statistical data for (g). Blue-stained mineralized fibers and/or mature bone were quantitively analyzed. Statistical analysis was conducted for the entire 10 randomly selected regions (h), followed by separate analyses for 5 osteogenic front views and 5 defect center views (i). NC, suture-bony composite defects without scaffold implantation. Data are expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001

Transcriptomic analysis verified the mesenchyme-regenerative capacity of PLA

To well know the efficacy of PLA in regenerating mesenchyme and inhibiting ossification at the transcriptome level, we subjected Cd45- mesenchymal lineage cells, sorted from digested nascent tissue (Fig. 3a), to RNA sequencing (RNA-seq). The findings revealed significant differences among sample groups, while within-group variations were minimal (Fig. 3a and S5a–c), indicating distinct biological functions of each scaffold. The heatmap illustrated the distinctive expression pattern of differentially expressed genes (DEGs) among the groups (Fig. 3b). Of note, PLA demonstrated a significantly higher number of upregulated DEGs, with counts of 1273, 1870, and 1866 compared to NC, GelMA, and CTS, respectively (Figs. 3c and S5d). Among these upregulated DEGs, we observed a spectrum of stemness-related markers such as Cd200, Itgav (Cd51), Cd44, Eng, and Thy1 (Fig. 3c),8,33 providing indications of PLA’s capacity for MSC recruitment. Furthermore, the FPKM (fragments per kilobase of transcript per Million mapped reads) values of genes associated with chemokines and cytokines exhibited an increase in PLA (Fig. 3c). Concurrently, chemokine/cytokine activity and cytokine-cytokine receptor interaction emerged as top up-enrichment GO (Gene Ontology) terms and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway for PLA (Fig. 3d, f). 19 upregulated DEGs in chemokine/cytokine activity (Fig. 3e) and 28 upregulated DEGs in cytokine-cytokine receptor interaction (Fig. 3g) were identified in all three comparisons (PLA-vs-NC, PLA-vs-GelMA, and PLA-vs-CTS), highlighting the pivotal role of PLA in establishing a microenvironment for cell communication mediated by chemokines and cytokines, thereby contributing to the active mesenchyme regeneration.

Fig. 3

Transcriptome analysis of scaffold-mediated MSC recruitment. a Workflow of the RNA-Seq experiment with three major steps: nascent tissue isolation, Cd45- cell sorting (Gating strategy of FCM for Cd45- cells), and RNA-seq analysis (PCA, Principal component analysis). b Heatmap analysis of DEGs. c Volcano plot illustrating the DEGs in PLA compared with NC, GelMA, and CTS, respectively. The upregulated DEGs and other genes of interest are labeled. d The top 20 up-enrichment GO terms of cellular component and molecular function in the PLA group compared with NC, GelMA, and CTS groups. e Venn diagram displaying common upregulated DEGs in chemokine/cytokine activity (GO:0008009 and GO:0005125) of the three comparisons (PLA-vs-NC, PLA-vs-GelMA, and PLA-vs-CTS). f The top 15 up-enrichment KEGG pathways in the PLA group compared with NC, GelMA, and CTS groups. g Venn diagram illustrating common upregulated DEGs in cytokine-cytokine receptor interaction (rno04060) of the three comparisons (PLA-vs-NC, PLA-vs-GelMA, and PLA-vs-CTS). NC, suture-bony composite defects without scaffold implantation

As for tissue ossification, GO enrichment was applied to assess the function of downregulated DEGs related to sclerotization (Fig. S5e). Comparative analysis with NC revealed a downregulation in biological processes such as ossification, bone growth, endochondral bone growth, and bone morphogenesis in PLA (Fig. 4a). The consistent downregulation of ossification by PLA, in comparison with other scaffolds, demonstrated a decrease in OB proliferation compared to GelMA and a reduction in OB proliferation and differentiation compared to CTS (Fig. 4a). Additionally, 6 downregulated DEGs associated with sclerotization were uncovered in all three comparisons (PLA-vs-NC, PLA-vs-GelMA, and PLA-vs-CTS). All these align with the observed phenotype of inhibited diffuse ossification in the center of suture-bony composite defects implanted with PLA.

Fig. 4

Transcriptome analysis of ossification and cellular decoding on fibrogenesis. a The down-enrichment GO terms related to ossification in the PLA group compared with NC, GelMA, and CTS groups. b Venn diagram of common downregulated DEGs associated with sclerotization in the three comparisons (PLA-vs-NC, PLA-vs-GelMA, and PLA-vs-CTS). IF images of fibroblast marker S100A4 (in green) in defect regions at 2 weeks (c) and 6 weeks (e) post-surgery. The high-magnification images are from the selected regions (white boxes) in the low-magnification images. Scar bar, 250 μm in low magnification and 50 μm in high magnification. d, f Statistical data for (c, e), respectively. NC, suture-bony composite defect without scaffold implantation. Data are expressed as mean ± SD. **P < 0.01; ***P < 0.001

Cellular decoding indicated no inductive fibrogenesis by PLA in suture-bony composite defects

Fibrosis, defined as the excessive accumulation of extracellular matrix components, signifies an adverse outcome of various organ injuries such as the heart, lung, skin, liver, kidney, and bone.34,35 Scaffold implantation may trigger fibrogenesis at the interface, impeding tissue integration and causing permanent scar restoration.36 To eliminate this problem, we employed immunofluorescence (IF) staining with S100A4, a distinctive fibroblast marker commonly employed to monitor tissue fibrosis,37 on tissue sections. The findings revealed that the restorative process of NC did not inherently generate substantial fibrous tissue (Fig. 4c–f). At 2 weeks post-surgery, only limited fibrous tissue was observed within the defects of each group (Fig. 4c, d). However, by 6 weeks, improper scaffolds like GelMA and CTS significantly triggered fibrogenesis (Fig. 4e, f). Unlike these two, PLA did not induce the formation of fibrotic mesenchymal tissue, with no significant difference in the proportion of S100A4high cells compared to NC (Fig. 4e, f).

Cellular decoding displayed enhanced MSC ingrowth and self-renewal by PLA

Subsequently, we examined whether cells preserved within the defects expressed mesenchymal stem and progenitor cell markers. As depicted in Fig. 5, NC exhibited restricted MSC ingrowth and maintenance at both 2-week and 6-week time points. Additionally, the efficacy of GelMA and CTS in attracting MSC is not satisfactory either (Fig. 5). Whereas, PLA recruited a significantly higher number of Cd51+Cd200+ skeletal stem/progenitor cells 2 weeks post-operation (Fig. 5a, b). These cells migrated towards the defect center following the coiling direction of PLA and were also observed inside the spinning (Fig. 5a). By 6 weeks, PLA sustained a notably enlarged population of Cd51+Cd200+ cells, far from that of the other groups (Fig. 5e, f). Conforming to protein-level findings, transcriptomic analysis identified significant upregulation of Cd51 and Cd200 in PLA (Fig. 5i). Distinct from Cd51+Cd200+ cells, only a minority of MSC in the defects were derived from the periosteum at 2 weeks (Fig. 5c). Compared with other groups, PLA attracted a relatively higher number of Ctsk+ MSC at 2 weeks, predominantly located in the thickened periosteum, with some also migrating along PLA towards the defect center (Fig. 5c, d). When progressing to 6 weeks, abundant Ctsk+ MSC were present within the newborn tissue of PLA (Fig. 5g, h). Nonetheless, the genetic level of Ctsk in neonatal tissue did not increase simultaneously (Fig. 5i), which might be attributed to the conclusion of the high-expression phase of Ctsk. Given that the proliferation marker Ki67 was broadly expressed within the defects of PLA at both 2 weeks (Fig. 6d, e) and 6 weeks (Fig. 7a, c, f, h), the substantial existence of MSC at 6 weeks may arise from the rapid self-renewal of early ingrowth cells. To enhance the confirmation, single-cell suspensions were prepared from the newborn tissue of suture-bony composite defects implanted with PLA for 6 weeks. Flow cytometry (FCM) results showed that the digested cells comprised 43.5% Cd51+ cells, 57.0% Ctsk+ cells, and 86.8% Pdgfrα+ cells (Fig. 7i). Thus, it can be concluded that PLA promoted MSC ingrowth and self-renewal, contributing to the reconstruction of suture mesenchyme.

Fig. 5

Cellular decoding on MSC identification in the newborn tissue. IF images showing the co-staining of skeletal stem and progenitor cells marker Cd51 (in green) and Cd200 (in red) in suture-bony composites at 2 weeks (a) and 6 weeks (e) post-surgery. b, f Statistical data for (a, e), respectively. IF images showing the periosteum-derived MSC marker Ctsk (in green) at 2 weeks (c) and 6 weeks (g) post-surgery. d, h Statistical data for (c, g). The high-magnification images are from the selected regions (white boxes) in the corresponding low-magnification images. Scar bar, 250 μm in low magnification and 50 μm in high magnification. i Transcriptome heatmap demonstrating the FPKM values of Cd200, Cd51, and Ctsk. NC, suture-bony composite defect without scaffold implantation. Data are expressed as mean ± SD. ***P < 0.001

Fig. 6

Cellular decoding on osteogenic lineage commitment in early stages. a Schematic representation of the random selection area at the osteogenic front or the defect center of suture-bony composite defect. b IF images displaying the co-staining of OB marker Sp7 (in green) and proliferation marker Ki67 (in red) at the osteogenic front 2 weeks post-surgery. The high-magnification images are from the selected regions (white boxes) in the low-magnification images. Scar bar, 250 μm in low magnification and 50 μm in high magnification. c Statistical data of Sp7+ cells at the osteogenic front (b). d IF images displaying the co-staining of Sp7 (in green) and Ki67 (in red) at the defect center 2 weeks post-surgery. The images are magnified from the selected regions (yellow boxes in b). Scar bar, 50 μm. e Statistical data of Ki67+ cells at the defect center (d). NC, suture-bony composite defect without scaffold implantation. Data are expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 7

Cellular decoding on osteogenic lineage commitment in the later period. a IF images displaying the co-staining of OB marker Sp7 (in green) and proliferation marker Ki67 (in red) at the osteogenic front 6 weeks post-surgery. The high-magnification images are from the selected regions (white boxes) in the low-magnification images. Scar bar, 250 μm in low magnification and 50 μm in high magnification. Statistical data of Sp7+ cells (b), Ki67+ cells (c), Sp7+Ki67+ cells (d), and Sp7+Ki67+ cell proportion (e) at the osteogenic front (a). f IF images displaying the co-staining of Sp7 (in green) and Ki67 (in red) at the defect center 6 weeks post-surgery. The images are magnified from the selected regions (yellow boxes in a). Scar bar, 50 μm. Statistical data of Sp7+ cells (g) and Ki67+ cells (h) at the defect center (f). i FCM analysis of single-cell suspensions prepared from the suture-bony composite defects implanted with PLA for 6 weeks. j Schematic diagram summarizing the cellular composition within the suture-bony composite defects of NC, GelMA, CTS, and PLA at 2 weeks and 6 weeks post-surgery. NC, suture-bony composite defect without scaffold implantation. Data are expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001

Spatial-temporal decoding of osteogenic lineage commitment dynamics in PLA

Next, the osteogenic lineage commitment occurred at the osteogenic front or the defect center was detected separately referring to the region selection method illustrated in Fig. 6a. At the early time point, OB were solely detected at the osteogenic front of each group (Fig. 6b). Among them, CTS significantly promoted the osteogenic orientation at the repairing forefronts (Fig. 6b, c). Surprisingly, at the later time point, there were more Sp7+ cells (Fig. 7a, b) as well as Sp7+Ki67+ cells (Fig. 7a, d) at the osteogenic front of PLA, ensuring robust osseointegration between PLA and the bony edge and possibly contributing to the subsequent bone healing. Despite this, PLA demonstrated the lowest proportion of Sp7+Ki67+ cells (Fig. 7e), suggesting that most cells at the osteogenic front of PLA remained undifferentiated by 6 weeks. Meanwhile, PLA exhibited the lowest count of Sp7+ cells at the defect center (Fig. 7f, g), re-verifying its potential to restore suture mesenchyme and maintain its patency. All the cellular decoding outcomes were summarized in a schematic diagram (Fig. 7j), offering a comprehensive understanding of cellular composition within newborn tissue of suture-bony composite defects implanted with different scaffolds.

Mechanistic validation of PLA’s cellular manipulation on suture-bony complex reconstruction in vitro

In vivo, PLA exhibited spatiotemporal regulation of osteogenic lineage commitment, with strong edge osteogenesis but minimal central ossification in suture-bony composite defects (Fig. 7). Correspondingly, we established two models in vitro (Fig. 8). First, we simulated PLA’s effects at the defect center by seeding undifferentiated MSC onto the scaffold surface. Genetic analysis revealed that PLA impeded the osteogenic differentiation of MSC with significantly lower levels of Alp, Runx2, Sp7, Col1, and Bsp (Fig. 8a). Besides, following 7 days of culture, a limited number of MSC on PLA developed into sizable cell colonies by SEM (Fig. 8c), suggesting that MSC on PLA could replicate and self-renew in the absence of osteogenic induction (Fig. 8c). Hence, PLA sustained MSC self-renewal and inhibited their osteogenic differentiation, with the presence of abundant proliferating MSC (Fig. 5) and limited OB (Fig. 7) observed at the defect center in vivo. To verify the osteogenic-inductive properties of PLA at the osteogenic front, a parallel model was adopted. Briefly, MSC were cultured in osteogenic medium (OM) for 5 days to generate OB (Fig. S7) and subsequently seeded onto PLA. As shown in Fig. 8b, PLA promoted the expression of middle/late osteogenic genes (Col1, Bsp, Mepe, and Phex) in OB. That is to say, for differentiated cells, PLA maintained and even augmented their osteogenic orientation (Fig. 8b). Collectively, in the potent and persistent osteoinductive environment in vitro, PLA demonstrated cell-specific actions on MSC and OB. As for the relevant in vivo setting, osteogenic signals are concentrated at the osteogenic front and attenuated at the defect center, accounting for the spatiotemporal effects of PLA.

Fig. 8

Mechanistic validation of PLA’s control of cell fate in vitro. RT-qPCR detecting osteogenic gene expressions in MSC (a) or OB (b). Each sample was examined in triplicate. Gapdh was used as the internal control. c SEM imaging capturing cell attachment at 48 h, followed by colony formation after 7 days. For colony formation detection, MSC were incubated in the complete medium. d EDS elemental mapping of calcium (Ca), phosphorus (P), carbon (C), oxygen (O), and nitrogen (N) in the corresponding SEM images of MSC treated with (+OM) or without (-OM) osteogenic medium. The pie charts and the numbers in the images display the elemental proportions. The gray region represented the sum of C, O, and N elements. NC, MSC seeded on glass slides or in blank wells. PLA, MSC seeded on PLA. Data are expressed as mean ± SD. **P < 0.01; ***P < 0.001

By prolonging osteogenic induction in vitro, MSC on PLA exhibited the capacity to form mineralized nodules (Fig. 8d). SEM images revealed comparable rough surfaces of mineralized nodules on glass slides (NC) and PLA, with EDS mapping indicating similar Ca and P deposition (Fig. 8d). Persistent osteogenic induction in vitro prompts partial MSC to adopt osteogenic commitment, thereby contributing to the formation of mineralized nodules. Similarly, osteogenic signals at the osteogenic front ensure the proper osteointegration. As the reconstruction of suture-bony composite defects proceeds, the signals gradually decline to a basal level, leading to slower hard tissue restoration and the formation of regenerated sutures by residual nascent mesenchyme.