Abstract Nuclear morphology plays a critical role in regulating gene expression and cell functions. While most research has focused on the direct effects of nuclear morphology on cell fate, its impact on the cell secretome and surrounding cells remains largely unexplored. In this study, we fabricate implants with a micropillar topography using methacrylated poly(octamethylene citrate)/hydroxyapatite (mPOC/HA) composites to investigate how micropillar-induced nuclear deformation influences cell secretome for osteogenesis and cranial bone regeneration. In vitro, cells with deformed nuclei show enhanced secretion of proteins that support extracellular matrix (ECM) organization, which promotes osteogenic differentiation in neighboring mesenchymal stromal cells (MSCs). In a female mouse model with critical-size cranial defects, nuclear-deformed MSCs on micropillar mPOC/HA implants elevate Col1a2 expression, contributing to bone matrix formation, and drive cell differentiation toward osteogenic progenitor cells. These findings indicate that micropillars modulate the secretome of hMSCs, thereby influencing the fate of surrounding cells through matricrine effects. Subject terms: Mesenchymal stem cells, Regenerative medicine, Biomedical materials __________________________________________________________________ Nuclear morphology plays a critical role in regulating gene expression and cell function. Here, Wang et al. report that topography-induced nuclear deformation enhances the secretome of hMSCs, promoting extracellular matrix (ECM) organization and facilitating bone regeneration through matricrine effects. Introduction The nucleus is a dynamic organelle that changes its morphology in response to the cell’s status^[65]1. Its morphology has a critical influence on nuclear mechanics, chromatin organization, gene expression, cell functionality and disease development^[66]2–[67]5. Abnormal nuclear morphologies, such as invagination and blebbing, have functional implications in several human disorders, including cancer, accelerated aging, thyroid disorders, and different types of neuro-muscular diseases^[68]6,[69]7. In addition, severe nuclear deformation is also observed during tissue development, cell migration, proliferation, and differentiation^[70]2. To manipulate nuclear morphology, various biophysical tools have been developed, including atomic force microscopy (AFM) nanoindentation, optical, magnetic, and acoustic tweezers, microfluidic devices, micropipette aspiration, plate compression, substrate deformation, and surface topography modulation, referred to as microtopography engineering^[71]8–[72]15. Among these methods, microtopography engineering of materials is can be readily applicable to implantable medical devices and has broad implications for regenerative engineering. One commonly used approach is the fabrication of pillar structures, which are employed to deform cell nuclei and study nuclear properties such as mechanics and deformability^[73]16. These micropillar designs have been utilized to manipulate various cell functions, including migration, adhesion, proliferation, and differentiation^[74]17–[75]20. A design featuring 5 × 5 μm² micropillars with 5 μm spacing has been shown to significantly enhance the osteogenic differentiation of MSCs, highlighting the considerable potential of surface engineering for advancing bone regeneration^[76]20,[77]21. A wide range of materials can be used to create micropillar structures, such as poly-L-lactic acid (PLLA), poly(lactide-co-glycolide) (PLGA), OrmoComp (an organic-inorganic hybrid polymer), and methacrylated poly(octamethylene citrate) (mPOC)^[78]20–[79]23. Among these options, mPOC is particularly suitable for bone regeneration due to its major component, citrate, which acts as a metabolic factor to enhance the osteogenesis of mesenchymal stromal cells (MSCs)^[80]24. Additionally, a series of products made from citrate-based biomaterials (CBBs), including Citrelock, Citrefix, and Citregraft, have been cleared by the FDA for musculoskeletal regeneration in patients, further demonstrating the clinical efficacy of CBBs. Implantation of mPOC micropillars in a mouse cranial defect model demonstrated its bone regenerative potential in vivo^[81]21. However, the volume of regenerated bone remains limited, highlighting the need for further development of implant to enhance the efficacy of bone regeneration. More importantly, the majority of the new bone does not directly contact the implants; instead, it forms with a noticeable gap between the implant and the regenerated tissue. This observation inspired us to consider that nuclear deformation on micropillar implants may influence surrounding cells through the modulation of their secretomes. Bioactive molecules secreted by cells are crucial for intercellular communication, affecting various biological processes such as inflammation, cell survival, differentiation, and tissue regeneration^[82]25,[83]26. The success of many cell and exosome-based therapies depends on the cellular secretome^[84]27, which can be modulated by surface topography. For example, surfaces featuring grooves, roughness, or spiral patterns have been shown to influence the secretory profile of MSCs, primarily affecting immune regulation^[85]28. Additionally, the cytokine secretion profile of stromal cells, including MSCs and kidney-derived perivascular stromal cells (kPSCs), is closely linked to cell morphology, which is regulated by the unique surface structures^[86]29. Despite reports highlighting the influence of surface topography on secretion, the impact of nuclear morphogenesis, regulated by topography, on cellular secretion remains unclear. Additionally, in vivo testing of regeneration is necessary to advance the clinical application of surface engineering. Hydroxyapatite (HA) is a naturally occurring mineral form of calcium apatite, widely utilized in bone regeneration due to its exceptional biocompatibility, osteoconductivity, and structural similarity to the mineral component of bone^[87]30. The incorporation of HA with mPOC potentially combines the advantages of both materials in bone repair, thereby enhancing bone formation and offering a promising clinical option for future orthopedic implants. In this study, we fabricate micropillars to manipulate nuclear morphology and investigate their effects on the secretome of human mesenchymal stromal cells (hMSCs), as well as test their regenerative efficacy for bone tissue in vivo. Our results show that mPOC/HA micropillars facilitate osteogenic differentiation of hMSCs compared to flat mPOC/HA samples in vitro. Secretome analysis reveals that hMSCs with deformed nuclei exhibite higher expression levels of bioactive factors associated with extracellular matrix (ECM) components and organization, as well as ossification. In vivo, both mPOC/HA flat and micropillar scaffolds seeded with hMSCs result in new bone formation; however, the micropillar group demonstrates significantly greater new bone volume and regenerated tissue thickness. Spatial transcriptomic analysis further confirms elevated expression of genes related to the regulation of ECM structures, consistent with the secretome analysis results. These findings suggest that the influence of nuclear deformation on the osteogenesis of hMSCs operates through similar mechanisms in both in vitro and in vivo environments. Therefore, using microtopography engineering of scaffolds to control nuclear morphology and materials science approaches to mimic native bone composition is a promising approach to enhance bone regeneration. Results Influence of micropillar structures on physical and chemical properties of mPOC/HA implants mPOC prepolymer was synthesized according to our previous report^[88]31, and its successful synthesis was confirmed via the nuclear magnetic resonance (1H NMR) spectrum (Supplementary Fig. [89]1a–c). The size of HA nanoparticles is around 100 nm, as characterized by dynamic light scattering (DLS) (Supplementary Fig. [90]1d). To mimic the nature of bone composition^[91]32, 60% (w/w) HA was mixed with mPOC, and the slurry was used to fabricate flat and micropillar implants using a combination of UV lithography and the contact printing method (Fig. [92]1a). The square micropillars, with dimensions of 5 by 5 in side length and spacing, were fabricated (Fig. [93]1b). The height of the micropillars is around 8 μm, which can cause significant nuclear deformation (Fig. [94]1c, d)^[95]22. Fourier transform infrared (FTIR) spectrum shows a similar typical peak of functional groups in mPOC and mPOC/HA implants (Supplementary Fig. [96]1e). The surface roughness of the implants was scanned using an atomic force microscope (AFM) (Fig. [97]1e). The analysis result indicates that the topography didn’t affect the surface roughness of the implants (Fig. [98]1f). Additionally, we tested the hydrophilicity of flat and micropillar implants via water contact angle measurement (Supplementary Fig. [99]2). Although, at the initial state, the flat surface was more hydrophilic, there was no significant difference in the water contact angle after a 5-minute stabilization process. Fig. 1. Fabrication of surface-engineered mPOC/HA implants. [100]Fig. 1 [101]Open in a new tab a Illustration shows the combination of UV lithography and contact printing to fabricate free-standing mPOC/HA micropillars. b SEM image shows the micropillar structures made of mPOC/HA. c Optical microscope image and d cross-section analysis of mPOC/HA micropillars. e Surface scanning of flat and micropillar implants by AFM. f Surface roughness of flat and micropillar implants. N.S., no significant difference, n = 3 biological replicates. g Degradation test and h calcium release of flat and micropillar mPOC/HA implants. N.S., no significant difference, n = 4 biological replicates, insert plot shows the initial release of calcium within 24 h. i. Representative images of flat and micropillar implants at different time points after accelerated degradation. Data are presented as mean ± SD. Values from two groups were compared using a non-paired Student’s t-test (two-sided). Source data is provided as a Source Data file. The mechanical properties of the implants were tested using the nano-indentation method. The force-indentation curve of the flat sample has a sharper slope, indicating it is stiffer than the micropillar sample (Supplementary Fig. [102]3a). The Young’s Modulus of the flat sample (0.95 ± 0.12 GPa) is significantly higher than that of the micropillars (0.48 ± 0.02 GPa) and the lateral modulus of the micropillars (46.88 ± 1.49 MPa) (Supplementary Fig. [103]3b, c). However, based on a previous report, the high modulus of the substrates is beyond the threshold that cells can distinguish and does not have an influence on nuclear morphology manipulation^[104]33,[105]34. Accelerated degradation and calcium release tests of the implants were performed in DPBS at 75 °C with agitation^[106]35. There is a burst weight loss and calcium release of both flat and micropillar samples at day 1, followed by a gradual change until day 10, and another increase in the degradation and calcium release rate from day 10 to 14 (Fig. [107]1g, h). The micropillar structure enhanced the degradation and calcium release, but not significantly. According to the images of the samples captured at different time points, the initial burst degradation and calcium release can be attributed to the fast surface erosion of both scaffolds, as many small pores can be observed on their surfaces (Supplementary Fig. [108]4). From day 10 to 14, scaffolds started break into pieces that may lead to another burst degradation and calcium release (Fig. [109]1i). The micropillars exhibited slight deformation in both the xy and z directions after degradation, though the changes were not significant (Supplementary Fig. [110]5). Additionally, the structures transformed from outward convex to inward concave shapes. Nuclear deformation facilitates osteogenic differentiation of hMSCs hMSCs were cultured on the flat and micropillar mPOC/HA surfaces in osteogenic medium and stained for F-actin and nuclei after 3 days (Fig. [111]2a). Noticeable deformation in both the nucleus and cytoskeleton was observed, consistent with mPOC micropillars^[112]21. The Nuclear shape index (NSI) was calculated to assess the degree of nuclear deformation^[113]22. A significantly lower NSI value, indicating more severe deformation, was found in the micropillar group (Fig. [114]2b). Confocal images were then employed to evaluate the 3D geometry of cell nuclei (Fig. [115]2c). 3D reconstruction analysis revealed that several geometric parameters, including nuclear volume, surface area, and project area, were significantly decreased on micropillars, while nuclear height was significantly increased (Fig. [116]2d and Supplementary Fig. [117]4). Fig. 2. Nuclear deformation promotes osteogenic differentiation of hMSCs. [118]Fig. 2 [119]Open in a new tab a Staining of nucleus (green) and F-actin (red) of hMSCs on flat and micropillar mPOC/HA surfaces. Insert: high magnification of cell nucleus. Dashed lines indicate micropillars. b Analysis of nuclear shape index of hMSCs. n = 117 (flat) and 132 (pillar) collected from 3 biological replicates, ****p < 0.0001. c Orthogonal view of cell nucleus on flat and micropillar surfaces. d Nuclear volume analysis based on 3D construction of the confocal images of cell nuclei. n = 35 cells collected from 3 biological replicates, ****p < 0.0001. e Initial cell adhesions on flat and micropillar surfaces. n = 5 biological replicates, N.S., no significant difference. f SEM images show the cell adhesions on flat and micropillar mPOC/HA surfaces. g Live/dead staining of hMSCs on flat and micropillar surfaces at 72 h in osteogenic medium. h Cell metabolic activity of cells on flat and micropillar surfaces tested by a MTT assay. n = 5 biological replicates, ****p < 0.0001. i Cell proliferation tested via DNA content after 72 h induction. n = 5 biological replicates, N.S., no significant difference. j ALP staining of hMSCs on flat and micropillar surfaces after 7 d induction. k ALP activity test of cells after 7 d osteogenic induction. n = 3 biological replicates. l Blot images of osteogenic marker OCN and RUNX2 in cells cultured on flat and micropillar implants. GAPDH is shown as a control. Quantification (m) OCN and (n). RUNX2 according to Western blot tests. n = 3 biological replicates, ****p < 0.0001. Data are presented as mean ± SD. Values from two groups were compared using a non-paired Student’s t-test (two-sided). Source data are provided as a Source Data file. We then investigated the impact of micropillars on cell adhesion, a crucial aspect for manipulating cell function^[120]36. Initial cell attachment tests revealed that the micropillar structure did not influence cell attachment on the implants (Fig. [121]2e). SEM imaging of cell adhesion demonstrated that cells formed lamellipodia on flat surfaces but exhibited more retraction fibers on micropillars (Fig. [122]2f). The retraction fibers were observed on the top, side, and bottom of micropillars, indicating that cells were sensing the 2.5D environment using these antennae-like structures^[123]17. The majority of cells were found to be viable on both flat and micropillar substrates, as evidenced by live/dead staining (Fig. [124]2g and Supplementary Fig. [125]5). While the micropillars reduced cell metabolic activity (Fig. [126]2h), there was no significant impact on cell proliferation after 3 days of culture (Fig. [127]2i). To assess the impact of mPOC/HA micropillars on the osteogenesis of hMSCs, we stained ALP (alkaline phosphate) on substrates with both flat and micropillar structures (Fig. [128]2j). Quantification results demonstrated a significant increase in ALP activity on the micropillars (Fig. [129]2k). Furthermore, additional osteogenic differentiation markers of hMSCs, including RUNX2 and osteocalcin (OCN), were quantified through western blot analysis (Fig. [130]2l). The quantification of these proteins revealed a significant increase in both RUNX2 and OCN in cells on micropillars, confirming that the structures can effectively promote the osteogenic differentiation of hMSCs (Fig. [131]2m, n)^[132]20–[133]22. Micropillars modulate the secretome of hMSCs that regulate extracellular matrix formation Previously, we demonstrated the ability of micropillar implants to enhance in vivo bone formation^[134]21. However, the newly formed bone was not in close contact with the implant. Consequently, we hypothesized that nuclear deformation on micropillars might impact cellular secretion, thereby influencing osteogenesis through the secretome. To test this hypothesis, secretome analysis was conducted using medium collected from flat and micropillar samples. Differences in protein secretion levels between the two groups were depicted through a volcano plot, revealing a significant influence of nuclear deformation on the secretome (Fig. [135]3a, b). Gene ontology (GO) analysis was performed to annotate the significantly altered proteins in relevant processes^[136]37. Top changes in cellular component, molecular functions, biological processes, and biological pathways indicated that micropillars predominantly affected extracellular matrix (ECM)-related processes (Fig. [137]3c and Supplementary Figs. [138]8–[139]10). Moreover, ossification and collagen fibril organization were identified as biological processes significantly overrepresented by differentially expressed proteins (Fig. [140]3d). The heatmap plot of proteins associated with collagen-containing extracellular matrix and ossification showed predominant upregulation on micropillars (Fig. [141]3e). The linkages of proteins and GO terms in biological process highlighted that ECM organization forms the largest cluster and is closely associated with the ossification process (Fig. [142]3f). Fig. 3. Secretome of hMSCs on flat and micropillar mPOC/HA surfaces. [143]Fig. 3 [144]Open in a new tab a PCA plot of differentially expressed proteins secreted by hMSCs on flat and micropillars. Cyan: flat; Red: micropillar. b Volcano plot of proteins secreted by hMSCs seeded on micropillars compared to the flat surface. Blue and orange dots indicate significantly downregulated and upregulated proteins secreted by cells on micropillars compared to those on flat surface. Grey dots indicate non-significantly changed proteins. A threshold of expression greater than 2 times fold-change with p < 0.05 was considered to be significant (non-paired Student’s t-test (two-sided)). Proteins that are related with collagen-ECM pathways are labeled. c Top 4 significantly enriched GO terms and Pathways identified through over-representation analysis using the one-sided Fisher’s exact test. Significance was determined based on adjusted p-values < 0.05 (FDR < 5%, Benjamini-Hochberg). ***p < 0.001. d The most significantly enriched Biological Processes (one-sided Fisher’s exact test, adjusted p-values < 0.05 (FDR < 5%, Benjamini-Hochberg)). e Heatmap of proteins that are related to collagen-containing extracellular matrix and ossification. F indicates flat samples and P indicates pillar samples, n = 3 biological replicates for each group. f The linkages of proteins and GO terms in biological processes related to collagen fibers, ECM, and ossification as a network. g Heatmap of the top 15 enriched terms plotted based on Reactome pathway analysis. Source data are provided as a Source Data file. Reactome pathway analysis was further conducted to assess potential downstream effects of secretome changes on micropillars^[145]38. Results indicated that pathways related to ECM organization, ECM proteoglycans, and collagen fibril crosslinking were among the top 15 pathways significantly overrepresented by differential expressed pathways (DEP), predominantly showing upregulation (Fig. [146]3g and Supplementary Fig. [147]11). We also noticed an upregulation in the degradation of the ECM on micropillars, indicating enhanced ECM remodeling which a crucial factor for tissue regeneration^[148]39. These findings suggest that micropillars can influence the ECM formation of hMSCs through matricrine effects. Additionally, we performed proteomic analysis using cells cultured on flat and micropillar mPOC/HA scaffolds (Supplementary Fig. [149]12). PCA and volcano plots indicated significant influences of nuclear deformation on protein expression. Pathway analysis revealed significant changes in many cell proliferation-related processes, consistent with previous transcriptomic tests on micropillars^[150]21. Nuclear deformed cells facilitate osteogenic differentiation of undeformed cells by affecting ECM Since the micropillar surfaces can modulate the secretome of hMSCs, we investigated whether the deformed cells could influence the osteogenic differentiation of undeformed cells using a transwell assay (Fig. [151]4a). The flat and micropillar mPOC/HA surfaces were fabricated at the bottom of cell culture plates to manipulate the nuclear morphology of hMSCs, while undeformed hMSCs were seeded on a transwell membrane with 400 nm nanopores, allowing the exchange of growth factors. After cell attachment, all samples were cultured in osteogenic induction medium. ALP staining of the cells on the transwell membrane showed a higher number of ALP-positive cells when co-cultured with nuclear-deformed cells, indicating enhanced osteogenic differentiation (Fig. [152]4b, c). Additionally, Alizarin Red S (ARS) staining confirmed increased calcium deposition—a key step in osteogenesis—when the cells were cultured above the micropillar-treated cells (Fig. [153]4d, e). Based on the secretome analysis, hMSCs on micropillars appear to promote osteogenesis in the transwell culture by secreting proteins that enhance ECM structure and organization. Collagen staining revealed higher coverage, stronger staining intensity, and more interconnected collagen network structures in the transwell co-cultured with micropillar-treated cells (Fig. [154]4f, g). In addition, energy dispersive X-ray spectroscopy (EDS) images showed more Ca and P deposition in the transwell co-cultured with micropillar-treated cells (Fig. [155]4h). Together with the secretome analysis, these findings suggest that the proteins secreted by cells with deformed nuclei improve ECM organization in undeformed cells, thereby promoting osteogenesis. Fig. 4. The paracrine effect of cells with/without nuclear deformation tested through transwell assay. [156]Fig. 4 [157]Open in a new tab a Schematic illustration of the experiment setup. b ALP staining and (c). quantification of ALP-positive cells on transwell membrane incubated with undeformed and deformed MSCs (n = 3 biological replicates). d ARS staining and e. quantification of cells on transwell membrane incubated with undeformed and deformed MSCs (n = 6 biological replicates). (f) Immunofluorescence staining images of collagen in the ECM of cells on the transwell membrane incubated with undeformed and deformed MSCs. g The coverage of collagen was analyzed according to the staining images (n = 4 biological replicates). h EDS images showing Ca, P, and SEM images of cells on the transwell membrane incubated with undeformed and deformed MSCs. Data are presented as mean ± SD. Values from two groups were compared using a non-paired Student’s t-test (two-sided). Source data is provided as a Source Data file. mPOC/HA micropillar implant promotes bone formation in vivo To test the in vivo regeneration efficacy of mPOC/HA scaffolds, we created a critical size cranial defect model in nude mice. Two 4 mm diameter critical defects were made on the left and right sides of the skull tissue for the implantation of flat and micropillar scaffolds, respectively (Fig. [158]5a). The scaffolds were seeded with hMSCs for 24 h to allow for cell attachment and nuclear deformation (Fig. [159]5b). After 12 weeks, micro CT was performed to evaluate the bone formation in the living animals. Based on the images, newly formed bone can be observed in the defect area with both flat and micropillar mPOC/HA implants (Fig. [160]5c and Supplementary Fig. [161]11). Furthermore, larger bone segments were observed with the micropillar implant treatment. Quantification results confirmed a significantly increased bone volume with micropillar implant treatment (Fig. [162]5d). Fig. 5. mPOC/HA micropillar implant promotes bone regeneration in vivo. [163]Fig. 5 [164]Open in a new tab a Image shows implantation of hMSC seeded flat and micropillar mPOC/HA scaffolds. b Staining images of nuclei (green) and F-actin (red) of cells on the implants. c Representative μCT images of a typical animal implanted with hMSC-seeded flat (left) and micropillar (right) scaffolds at 12-weeks post-surgery. d Regenerated bone volume in the defect region (n  =  5 animals). e Trichrome staining of the defect tissue treated with flat and micropillar implants. f Average thickness of regenerated tissues with implantation of flat and micropillar scaffolds (n  =  5 animals). IHC staining of osteogenic marker, g OPN and h. OCN, in regenerated tissues with flat and micropillar implants. Data are presented as mean ± SD. Values from two groups were compared using non-paired Student’s t-test (two-sided). Source data is provided as a Source Data file. Histology analysis was further performed to evaluate the influences of flat and micropillar mPOC/HA implants on bone regeneration. Trichrome staining images revealed that defects treated with micropillar implants exhibited more osteoid tissue (Fig. [165]5e and Supplementary Fig. [166]12). Moreover, both flat and micropillar mPOC/HA implants showed evidence of newly formed bone tissue, indicating enhanced bone regeneration compared to the mPOC alone scaffold. As no bone segment was observed with flat mPOC implant treatment^[167]21. The thickness of the regenerated tissue was quantified, and the results demonstrated a significant enhancement with micropillar implant treatment (Fig. [168]5f). Positive staining of osteogenesis markers, including osteopontin (OPN) and osteocalcin (OCN), was observed throughout the regenerated tissues with both flat and micropillar implants, indicating osteoid tissue formation (Fig. [169]5g, h). The tissue appeared more compact in the micropillar group compared to the flat group. Furthermore, regenerated bone segments were more frequently observed with micropillar implant treatment. It has been reported that athymic nude mice retain an innate immune system, including macrophages, which contribute to bone regeneration^[170]40. Therefore, we further assessed macrophage activation in the regenerated tissue by staining for three markers: F4/80 (a pan-macrophage marker), CD86 (an M1 macrophage marker), and CD163 (an M2 macrophage marker), to evaluate macrophage polarization (Supplementary Fig. [171]15)^[172]35. The results indicate a slight increase in overall macrophage expression and a decrease in the M1/M2 ratio; however, these changes were not statistically significant. Micropillar implants facilitated bone regeneration in vivo via regulation of ECM organization and stem cell differentiation Histological analyses showed more new bone formation with micropillar implants, although the new bone tissue did not directly interact with the micropillar surfaces. To further investigate the transcription profile of the regenerated tissue, we performed spatial transcriptomics (ST) analyses with both flat and pillar samples (Supplementary Fig. [173]16). ST represents a powerful tool to investigate the cellular environment and tissue organization by providing a detailed map of gene expression within the native tissue context^[174]41. Differential gene expression (DGE) analysis revealed changes in expression levels between the two groups. Although only a few genes showed significant differences, all of them were related to ECM structure or organization (Supplementary Fig. [175]16). Notably, the expression of Col1a2, critical for type I collagen formation (comprising 90% of the bone matrix), was enhanced in the micropillar group (Fig. [176]6a). This expression showed a gradient, increasing toward the dura layer, possibly due to the osteogenic contribution of dura cells^[177]42. We then plotted a heatmap showing the top 10 up-regulated and down-regulated differentially expressed genes (pillar vs. flat) in comparison with those in native skull bone (Fig. [178]6b). The heatmap indicated that the tissue regenerated with micropillar implants had expression patterns more similar to native skull bone than the flat group. Gene Ontology (GO) analysis of DGEs was further performed to annotate their relevant biological processes (Fig. [179]6c). Protein localization to extracellular matrix and crosslinking of collagen fibrils were among the top 5 up-regulated processes in the micropillar group. These results are consistent with the secretome test, all indicating that micropillar structures can influence ECM organization via matricrine effects. Fig. 6. Spatial transcriptomic analysis of tissues regenerated with flat and micropillar implants. [180]Fig. 6 [181]Open in a new tab a Spatial plot of Col1a2 expression profile in tissues regenerated with flat mPOC/HA implant and micropillar mPOC/HA implant. Arrow indicates enhanced expression around dura layer. b The heatmap showing the top ten up- and down-regulated DEGs (pillar vs flat) in tissues regenerated with flat mPOC/HA implant, micropillar mPOC/HA implant, and native skull tissue. c Gene Ontology analysis results based on the top 100 up-regulated genes (pillar vs flat). The results are colored by q, false-discovery-rate-adjusted p-value. d Deconvoluted cell types in each spatial capture location in flat and micropillar groups. Each pie chart shows the deconvoluted cell type proportions of the capture location. e Bar plots of the cell type proportions in tissues regenerated with flat mPOC/HA implant and micropillar mPOC/HA implant. LMPs, MSCs, and fibroblasts are the predominant cell types. f Violin plot of the proportion of LMPs in flat (100 capture locations) and micropillar (69 capture locations) groups. The boxplots display medians and quartiles, with whiskers extending to 1.5 times the interquartile range, and the violin plot outlines represent the kernel probability density. The p-value from a two-sided Wilcoxon rank-sum test is shown. g Top enriched processes associated with LMP compared with other cell lineages. LMP: late mesenchymal progenitor cells; MSC: mesenchymal stromal cells; OLC: MSC-descendant osteolineage cells. The results are colored by q, false-discovery-rate-adjusted p-value. Source data are provided as a Source Data file. To further investigate the relationship between cell type composition and the regenerated tissues, we performed cellular deconvolution on the ST data using single-cell RNA sequencing (scRNA-seq) references from