Hydrogel characterization
The gelatin microgels that are used in this research are polydisperse with the average diameter of ~ 200 µm28. Microgels are physically crosslinked and are stable in an aqueous solution at 25 °C for at least 24 h (Fig. S1). At 28 °C and 30 °C, microgels start to lose their structure after 30 and 5 min, respectively. Covalent crosslinking of the microgels by mTG results in a stable microporous hydrogel which remains stable throughout culture with MSCs at 37 °C for at least one month.
Void fraction, rheological properties, and equilibrium swelling ratio of swelled hydrogels were examined (Fig. S2). On average, swelled microporous hydrogels (PGELs) had a void fraction of 0.32 and had comparable stiffness as nonporous hydrogel (NGEL).The storage modulus remained stable over increasing angular frequency indicating stable chemical crosslinking34. On average, NGEL had a higher swelling ratio than PGEL, but this difference was not significant. In addition to these characterizations, detailed rheology of gelation, SEM images, injectability of PGELs and enzymatic degradation have been previously described33.
Cell viability, proliferation and morphological changes
The potential for the PGELs to support cell encapsulation was explored through live/dead and alamarBlue proliferation assays, (Fig. 2) and Lactate Dehydrogenase (LDH) cytotoxicity assays (Fig. S3). Cells encapsulated in PGEL demonstrated high viability with robust cell spreading as early as 1 day post encapsulation (Fig. 2a), while cells in the NGEL remained highly spherical due to the entrapment by the surrounding polymers (Fig. 2b). The cells in the NGEL can fully spread only by matrix degradation or stress relaxation of the surrounding polymers. Live/dead assay on day 14 showed a continuation of these trends (Fig. 2c,d), and cells encapsulated in both conditions began to spread more compared with day 1. Cell proliferation in the PGEL was markedly higher than the NGEL (Fig. 2g). Cytotoxicity during the encapsulation process was low for both PGEL and NGEL (Fig. S3), demonstrating the biocompatibility of mTG crosslinking of gelatin. When the cells were cultured in osteogenic differentiation media, MSCs in the PGEL adopted a more complex random morphology (Fig. 2e) compared to the cells cultured in the growth media (Fig. 2c). In contrast, morphological changes of the cells encapsulated in the NGEL were less noticeable due to cell entrapment (Fig. 2d, Fig. 2f). Quantification of cell circularity confirms these observations. Notably, cell circularity in the PGEL was much lower than NGEL for all conditions due to cell spreading. Additionally, cells encapsulated in PGEL had increased circularity in response to differentiation, which is in contrast to the NGEL condition, where cell circularity decreased. MSC differentiation is affected by cell morphology35,36. Our findings highlight the importance of cell morphological changes during differentiation, and that when unobstructed, cells adopt morphology associated with these changes.
MSC growth in differing 3D culture conditions. Representative Z projections of living (green) and dead (red) cell staining of MSCs encapsulated in microporous (a, c, e) and nonporous (b, d, f) hydrogel in growth medium for day 1 (a, b), and day 14 (c, d), or incubated in osteogenic differentiation medium for 14 days (e, f). MSC proliferation was quantified by alamarBlue assay (g). Circularity of living cells in the microporous (P) and nonporous (NP) hydrogels incubated in growth medium (GM) for either 1 or 14 days, or differentiation medium (DM) for 14 days. *** p < 0.001 (Tukey’s HSD). Scale bar = 50 µm.
Morphology of encapsulated cells was further examined by staining for actin cytoskeleton and nuclei (Fig. 3). In accordance with Live/Dead imaging, encapsulated cells rapidly spread in the PGEL condition as early as 1 day after encapsulation (Fig. 3a). By comparison, cells encapsulated in NGEL displayed minimal spreading on day 1 (Fig. 3b). After 7 days of culture, minimal changes were observed in the spreading behavior in the PGEL condition (Fig. 3c), and cells had begun to spread in the NGEL condition (Fig. 3d). These results confirm the rapid adhesion and spreading behavior of cells encapsulated in PGEL, and rapid formation of actin stress fibers in this condition. The formation of actin stress fibers is known to enable mechanotransduction-mediated osteogenesis, suggesting PGEL may enhance MSC osteogenic differentiation37. In a prior study, in contrast with 2D systems, spreading of MSCs in nonporous hydrogels decreased with increasing stiffness26. The utility of PGEL circumvents this restriction by providing macroscopic, interconnected pore space for encapsulated cells.
Cytoskeletal organization of encapsulated cells. Z-projection images of cell nuclei (blue) and actin cytoskeleton (red) after 1 (a, b) and 7 (c, d) days of culture, after encapsulation in the microporous (a, c) or nonporous (b, d) environment. Scale = 200 µm.
Osteogenic differentiation examined by EDS
Cell morphology, and calcium mineral deposition due to osteogenic differentiation, were observed under SEM and EDS (Fig. S4). Cells appear morphologically distinct, after incubation in osteogenic differentiation media. The increase in calcium and phosphorous in hydrogels incubated in osteogenic differentiation media is attributed to bone mineral deposition, which indicated encapsulated cells had successfully differentiated into osteoblasts.
Biochemical characterization of osteogenic differentiation
Osteogenic differentiation of MSCs encapsulated in PGEL and NGEL was examined by alkaline phosphatase (ALP) and calcium assays after 14 days of incubation in osteogenic differentiation medium (Fig. 4).
Microporous hydrogel enhances mesenchymal stem cell osteogenic differentiation. After 14 days of incubation in osteogenic differentiation medium: Alkaline phosphatase staining of cells encapsulated in (a) microporous (PGEL) and (b) nonporous (NGEL) hydrogels. (c) Alkaline phosphatase activity normalized to DNA content, and (d) calcium mineral deposition in equal volumes of cell-encapsulated hydrogels, normalized to DNA content. Scale bar = 200 µm, inset scale bar = 1 mm.
ALP staining shows a contrast between cells encapsulated in PGEL (Fig. 4a) and NGEL (Fig. 4b), where cells encapsulated in PGEL had significantly higher ALP activity. ALP is an enzyme involved with the mineralization of bone tissue, and is a marker of early MSC osteogenic differentiation38. This microscopic observation is consistent with quantitative results, which show ALP activity and calcium deposition increased by about a factor of 4 on a per cell basis (Fig. 4c, d) for cells encapsulated in PGEL in comparison to NGEL. Calcium deposition is indicative of mature osteoblasts, demonstrating that encapsulation in PGEL improved mineral deposition over the culture period.
Considering the identical material and comparable stiffness of PGEL and NGEL33, these results highlight the importance of differing 3D micro-environments for the control of MSC osteogenesis. More specifically, PGELs allow rapid morphological changes of the encapsulated cells and direct cell–cell physical contacts through the interconnected micropore network, which may have promoted osteogenesis and calcium mineral deposition. Whether the differing pore structure affects nutrient transport to encapsulated cells is unclear, as cells encapsulated in PGEL are clustered at a high local cell density in the pore space, compared with homogeneous cell distribution in NGEL.
Transcriptomic analysis by RNA-seq
RNA-Seq was used to examine the changes in gene expression of MSCs encapsulated in the different 3D environments. RNA from MSCs encapsulated in PGEL and NGEL was extracted at 3 days (P3, NP3), and 14 days (P14, NP14) after encapsulation to assess early and late differentiation (Figs. 5,6). PCA analysis (Fig. 5a) shows a clear trend based on sample condition and time, indicating that gene expression changed substantially depending on the 3D environment, and on the duration of differentiation. The number of differentially expressed genes between groups (Fig. 5b) aligns with PCA analysis, confirming the central role the 3D environment played in differential gene expression. Genes commonly related to osteogenic differentiation (Fig. 5c-f) show a trend that osteogenic differentiation was increased in the PGEL, and increased over incubation time. Integrin binding sialoprotein (IBSP) expression (Fig. 5e) is notable as a late stage marker of osteogenesis. These results confirm the increase in osteogenesis for cells encapsulated in the PGEL condition. Expression data for selected genes related to osteogenesis, cell adhesions, cytoskeletal organization, cell–cell connections, ECM remodeling and deposition are shown in Fig. 6.
Differential gene expression identified by RNA-Seq. (a) PCA of sample set. PC1 and PC2 account for 43% and 22% of variance, respectively. (b) Number of differentially expressed genes between groups, comparing culture condition and time. (c-e) Expression of genes directly related to osteogenesis, collagen type I alpha chain 1 (COL1A1), integrin binding sialoprotein (IBSP), alkaline phosphatase, biomineralization related (ALPL), and osteomodulin (OMD). Abbreviations: NP3 (Nonporous, day 3), P3 (Microporous, day 3), NP14 (Nonporous, day 14), P14 (Porous, day 14). Data shown are median, bounded by the interquartile range.
Gene expression for genes of interest in different 3D environments and time points, for genes related to osteogenesis (a), mechanotransduction (b, c, d, e, f), cystoskeleton production and organization (g, h, I, j), cell–cell connections (k, l, m, n, o, p, q), extracellular matrix production and matrix remodeling (r, s, t). Data is separated between culture condition and time point (NP3 = nonporous day 3, P3 = porous day 3, NP14 = nonporous day 14, P14 = porous day 14). (a) Secreted Protein Acidic and Cysteine Rich (SPARC), (b) Integrin Subunit Alpha 5 (ITGA5), (c) Vinculin (VCL), (d) Paxillin (PXN), (e) Runt-related Transcription Factor 2 (RUNX2), (f) Ras Homolog Family Member A (RHOA), (g) Microfibril Associated Protein 5 (MFAP5), (h) ENAH Actin Regulator (ENAH), (i) Actin Beta (ACTB), (j) Nexilin F-Actin Binding Protein (NEXN), (k) Cadherin 2 (CDH2), (l) Cadherin 11 (CDH11), (m) Gap Junction Protein Delta 3 (GJD3), (n) Activated Leukocyte Cell Adhesion Molecule (ALCAM), (o) Notch Receptor 1 (NOTCH1), (p) Frizzled Class Receptor 4 (FZD4), (q) catenin beta 1 (CTNNB1), (r) Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase 1 (PLOD1), (s) Lysyl Oxidase (LOX), (t) Matrix Metalloproteinase 2 (MMP2).
Osteogenesis genes
Osteonectin (SPARC) is a protein involved in calcium mineral deposition, which had increased expression in PGEL, providing further evidence of the increase in osteogenesis for these cells.
Cell adhesion, focal adhesion genes
Increase in Integrin Subunit Alpha 5 (ITGA5) expression, related to integrin α5β1 (one of the primary integrins involved in binding to gelatin), in PGEL is consistent with an increase in cell spreading as visualized in confocal images39. However, expression of proteins related to focal adhesions and focal adhesion-mediated signaling overall did not show a clear trend (PXN, RUNX2, YAP1), though vinculin (VCL) expression was upregulated in the PGELs, and with increasing culture length. In similar 3D matrices, it was previously reported that differences in gene expression of mechanotransduction-related genes was diminished as length of culture increased40, which could explain this trend. Additionally, while cell spreading is higher for cells in PGEL, substrate stiffness is similar between PGEL and NGEL, which may have resulted in insignificant differences in the expression of these genes, due to the well-known relationship between substrate stiffness and focal adhesion formation41.
Cytoskeletal organization genes
Gene expression related to cytoskeletal organization (MFAP5, ENAH, ACTB, NEXN) shows a general trend of increased expression for cells encapsulated in PGEL, and an increase in expression over the culture period, likely as a product of increased cell spreading.
Cell–cell connection genes
Among the genes related to cell connections, an increase in CDH11 expression in PGEL was noted. On 2D surfaces, higher CDH11 expression correlated with higher osteogenesis of MSCs19. CDH11 expression was constant for cells encapsulated in the NGEL, though expression at day 3 was higher than in PGEL. Among gap-junction proteins, which have been previously implicated to regulate MSC differentiation42, GJD3 was highly expressed, and had increased expression for cells in PGEL.
Wnt/Notch signaling genes
CTNNB1, NOTCH1, FZD4 are involved in cell signaling pathways (Wnt/Notch). We hypothesize that over the culture period, cells encapsulated in PGEL increased the number of cell–cell connections as cell density in the hydrogel increased, leading to increased expression of cell–cell connection-related genes, and potentially associated pathways, such as the Notch pathway. However, gene expression for cells encapsulated in the NGEL were generally prevented from making these connections, and expression of cell–cell connection related genes remained constant as a result. Additionally, CTNNB1 and FZD4 participate in Wnt signaling, which mediates mechanical stretching-induced osteogenesis43, which may have been influenced by the differing microenvironments, and has been previously implicated to mediate osteogenesis for MSC aggregates on differing biomaterial substrates44.
ECM remodeling genes
Lysyl hydroxylase 1 (PLOD1) and lysyl oxidase (LOX) are involved with collagen production, indicating ECM deposition was increased in PGEL in comparison to NGEL. We hypothesize that the open pore space may enable more rapid production of ECM, as cells in this condition do not need to degrade the surrounding matrix. Matrix metalloproteinase 2 and 9 (MMP2 and MMP9) are gelatinases, some of the primary means for cells to degrade gelatin. Cells encapsulated in NGEL may need to degrade the surrounding polymer mesh for division, spreading, and new ECM production, likely leading to the observed increased production of MMP2. MMP9 expression was not detected in any sample groups by RNA-seq. In addition to the selected genes, other genes of interest are shown in Fig S5. Statistical significance of all graphed gene expression comparisons are displayed in Fig S6.
GSEA was used to investigate differences in pathway activity between sample groups. The most recently updated KEGG, REACTOME, and GO databases were used. For each pairwise comparison, top pathways sorted by normalized enrichment score (NES) were plotted (Fig S7), and selected pathways relevant to our investigation are shown in Fig. 7. In addition, the full data table is supplied as additional supplementary information. While several highlighted pathways appear to be unrelated to MSC differentiation, many pathways relevant to cell behavior scored highly, in agreement with our observations at the single gene level. On day 3, pathways related to integrin-cell surface interactions, actin and laminin binding, focal adhesions, and adherens junctions have higher gene expression in the PGEL condition than in the NGEL condition (Fig. 7a). On day 14, many identified pathways continue to be upregulated in comparison to the NGEL condition, including adherens junctions, focal adhesions, actin assembly, and integrin-cell surface interactions, indicating the effect of the 3D environment on encapsulated cells continued to affect cell behavior throughout the culture period (Fig. 7b). In the NGEL condition on day 3, pathways related to ECM degradation and binding (Fig. 7a), and on day 14, mechanosensing (Fig. 7b), were highlighted.
Normalized Enrichment Score (NES) of selected upregulated pathways in pairwise comparisons between (a) nonporous day 3 (NP3) and porous day 3 (P3), and (b) nonporous day 14 (NP14) and porous day 14 (P14), given by GSEA.
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- Source: https://www.nature.com/articles/s41598-024-65731-9