MRL tendons heal with reduced adhesions and improved biomechanical properties
We first sought to compare the extent of adhesion formation and repair strength of healing flexor tendons in the C57 and MRL mice. The healing of partially lacerated deep digital flexor tendons (DDFT) was qualitatively assessed using hematoxylin-stained axial tendon sections. In general, the MRL tendons appeared to respond to the injury with a reduced cellular response compared to the C57 tendons. Specifically, there were no gross morphological differences in the uninjured (Fig. 1A,E), where the tendon bundles were sparsely populated by cells, and the tendons were encased with a very thin synovial sheath (black arrows in Fig. 1a,e). Fourteen days post injury (dpi), injured tendons in both mouse strains experienced substantial hyperplasia in the surrounding synovial sheath (black arrows, Fig. 1b,f), while no significant changes in cell number in the tendon proper were generally observed (yellow arrows Fig. 1B,F). At 28 dpi, substantial increases in cell density in both the tendon bundle and the peritendinous adhesions were observed in the C57 mice but not the MRL mice (yellow arrows, Fig. 1C,G). At this stage, the synovial hyperplasia began to resolve in the MRL mice but persisted in the C57 mice (black arrows, Fig. 1c,g). By 56 dpi, the injured MRL tendons appeared to have restored uninjured morphology where the tendon bundle was sparsely cellular and encased by a thin synovial membrane (Fig. 1H,h). In contrast, while the cellularity of the C57 tendon bundle decreased, the surrounding synovial sheath still demonstrated a hyperplasia morphology at 56 dpi (Fig. 1D,d). Quantification of the tendon perimeter adhered to the surrounding sheath and tissues (Fig. 1I), cellular density (Fig. 1J) in the tendon bundle demonstrated significant increases in adhesions and cellularity in the C57 tendons at 14 dpi and 28 dpi, respectively, compared to the MRL tendons. In addition, the synovial space was significantly increased in the MRL tendon at 14 and 28 dpi compared to the C57 tendons (Fig. 1K), while the cross sectional area of the MRL tendons was significantly larger than the C57 tendon at all time points (Fig. 1L).
MRL flexor tendons heal with reduced peritendinous adhesions. Representative (2X) micrographs (A–H) of hematoxylin-stained cross-sections of the digits of C57 and MRL mice before and after zone II injury up to 56 days post injury (dpi). High magnification (×20) regions of interest (a–h) are representative of five levels analyzed per tendon. Black arrows identify the thin synovial sheath in uninjured tendons, and the hyperplasia that ensues immediately after injury in both strains. The synovial sheath hyperplasia appears to persist in the C57 tendons up to 56 dpi, but resolves in MRL tendons by 28 dpi. Yellow arrows indicate regions of increased cellularity in the injured tendon and peritendinous tissues evident by the punctate, intense hematoxylin staining. Histomorphometric quantification of the healing tendon shows reduced adhesions (I) and tendon hyperplasia (J), as well as increased synovial space (K) and tendon area (L) in the MRL mouse. The length of the tendon perimeter adhered to the subcutaneous tissue and cell density are significantly reduced at 14- and 28- dpi in the MRL, respectively (N = 4 animals with n = 3–5 images (technical replicates) per animal).
To test whether the changes in cell density over time and between strains are attributable to cell proliferation, immunohistochemical staining for Proliferating Cell Nuclear Antigen (PCNA) was performed (Fig. 2A) and demonstrated activation of cellular proliferation in the tendon bundle and the peritendinous tissues following injury, which peaked at 14 dpi. Furthermore, quantifying the percentage of PCNA+ cells at all timepoints after injury revealed increased cellular proliferation in the healing C57 tendons relative to the MRL tendons (Fig. 2B).
MRL flexor tendons heal with reduced cellular proliferation. (A) Representative images of proliferating cell nuclear antigen (PCNA) immunohistochemical (IHC) staining conducted before injury and over time post injury on 5 µm transverse paraffin sections and counterstained with hematoxylin, and then quantified (B) for percent PCNA-positive cells within the tendon and the surrounding peritendinous tissues (N = 5 per group, with n = 3–5 images (technical replicates) per animal). Scale bar = 200 microns.
The functional properties of injured tendons were evaluated using biomechanical testing, which involved displacement-controlled tensile stretching of the tendon to failure at 0.05 mm/s. In terms of the structural properties, the injury resulted in significant decreases in peak tensile force and stiffness at 14 dpi in both the C57 and the MRL tendons. In contrast to the C57 tendons, which exhibited sustained reductions in peak tensile force and stiffness up to 56 dpi, the MRL tendons recovered uninjured levels of peak tensile force by 28 dpi (Fig. 3A) and were significantly stiffer (Fig. 3B) than the C57 tendons at all time points up to 56 dpi. Since the cross-section area of the uninjured MRL tendons are approximately 20% larger than the C57 tendons, we computed the corresponding stress–strain tensile curves and determined the effects of injury on the size-independent material properties. At 14 dpi, the tensile strength (peak stress) of the MRL was significantly reduced compared to uninjured tendon and the C57 injured tendon. The tensile strength of the C57 continued to decline at 28 dpi but recovered at 56 dpi. In contrast, the MRL tendon recovered the tensile strength to levels comparable to uninjured tendon as early as 28 dpi (Fig. 3C). Injury resulted in significant reductions in the elastic modulus of the C57 tendon, which did not improve over 56 dpi. While the elastic modulus of the uninjured MRL tendon was significantly reduced compared to the uninjured C57, injury resulted in reductions in the elastic modulus on 14, 28, and 56 dpi, which were not significantly different than uninjured tendon (Fig. 3D). Collectively, these data support the conclusion that MRL flexor tendons heal with reduced peritendinous adhesions and cellular proliferation and accelerated recovery of strength and stiffness.
MRL flexor tendons heal with accelerated recovery of strength and stiffness (A) Maximum force, (B) strength, (C) stiffness and (D) elastic modulus determined from displacement-controlled tensile testing of uninjured and injured flexor tendons of C57BL/6 J and MRL mice (N = 4–6). Asterisks indicate significant mouse strain differences (p < 0.05). a and b indicate significant differences (p < 0.05) from uninjured within C57 and MRL mouse strains respectively.
Altered inflammatory, fibrotic, and cell cycle regulation in C57 and MRL tendon healing
To gain insights into the differences in the biological drivers of the tendon injury response between the C57 and MRL mice, we performed next generation sequencing of bulk tissue RNA (RNA-seq) at 7 days post-injury, which we reasoned to be a time point that defines the transition from the acute inflammatory phase to the reparative phases. Based on a twofold change (± 2FC) and adjusted p value < 0.05 differential expression criteria relative to healthy uninjured tendon, 891 genes were differentially expressed (594 upregulated and 297 downregulated) in the C57 tendons, and 1358 genes were differentially expressed (747 upregulated and 611 downregulated) in the MRL tendons. Of those, 406 genes (DEG) were differentially expressed, relative to respective uninjured controls, in both mice. We identified 22 significantly enriched Reactome pathways in the injured C57 tendon and 51 significantly enriched pathways in the injured MRL tendon based on False Discovery Rate (FDR) < 0.05. Functional annotation of these enriched gene sets identified remarkable differences in the transcriptional activity, wherein the gene sets enriched in the C57 injured tendon were functionally linked primarily to fibrosis, immune system signaling and extracellular matrix (ECM) organization (Fig. 4A), while the gene sets enriched in the MRL injured tendon were dominated by cell cycle pathways (Fig. 4B). Closer inspection of the top 15 Reactome pathways enriched in the C57 injured tendons (Fig. 5A) identified cytokine signaling, including interleukins 4, 10, and 13, and neutrophil degranulation as significantly enriched immune system pathways, and collagen formation (biosynthesis and assembly), ECM proteoglycans, ECM degradation, and integrin and non-integrin cell-ECM interactions as significantly enriched ECM organization pathways. In contrast, the top 15 Reactome pathways enriched in the MRL injured tendons (Fig. 5B) identified numerous cell cycle pathways related to mitotic phase, cell cycle checkpoints, and G1-S phase transition, as well as cell signaling by Rho GTPases and immune system signaling by interleukins and neutrophil degranulation.
Functional annotation based on Reactome Pathways enrichment of 891 and 1358 differentially expressed genes (DEG) at 7 days post tendon injury in the C57 and MRL mice, respectively. Bubble plots of number of gene terms (x-axis) versus odds ratio (y-axis) represent 22 significantly enriched pathways in the injured C57 tendon (A) and 51 significantly enriched pathways in the injured MRL tendon (B) based on False Discovery Rate (FDR) < 0.05.
Bubble plots of the top 15 Reactome pathways enriched in the injured C57 tendon (A) and the MRL tendon (B), respectively. Bubble fill color represents the Reactome Pathway Root, while the size of the bubble represents -Log10(FDR) in A & B and the number of genes represented in the enriched pathway in C&D, respectively.
To investigate differences in activation of myofibroblasts and macrophages as drivers of fibrotic scarring that could explain these transcriptional differences, histology and immunofluorescent staining against α-SMA+ myofibroblasts, F4/80+ macrophages, and BCL-2 expressing cells were performed on C57 and MRL tendons at 7 dpi. Visual examination of Masson’s trichrome stained injured tendon sections at this time point did not reveal any significant morphological differences in the injured tendons (Fig. 6A,B), except for increases in the vascularization of the scar tissue immediately surrounding the injured C57 tendon, which was notable in α-SMA+ smooth muscle cells surrounding the blood vessels (Fig. 6C). In addition to the increased vascularization in the C57 scars, activated (α-SMA+) myofibroblasts and F4/80+ macrophages could be observed in both the injured C57 tendon and the surrounding scar tissue (Fig. 6C and insets). Of note was the observation of a subset of cells co-expressing both SMA and F4/80 in the injured C57 tendon (yellow staining in Fig. 6c′), which could implicate the activation of macrophage–myofibroblast transition (MMT) as a feature of fibrotic tendon healing. In contrast, fewer α-SMA+ myofibroblasts and F4/80+ macrophages could be observed in the injured MRL tendon, but were abundant in the surrounding scar tissue (Fig. 6D and insets). No localized expression of α-SMA and F4/80 could be observed in the injured MRL tendon. BCL-2 expression was notably abundant in the injured C57 tendon, but appeared to be reduced in the surrounding scar tissue (Fig. 6E and insets) as well as in the MRL injured tendon and surrounding scar tissues (Fig. 6F and insets).
MRL Tendons heal with reduced myofibroblast and macrophage activation and reduced BCL2 expression at 7 dpi. Histology using Masson’s trichrome (A,B) and immunofluorescent (IF) staining against α-SMA+ myofibroblasts and F4/80+ macrophages(C,D), and BCL-2 expressing cells (E,F) were performed on injured C57 and MRL tendons at 7 dpi. IF shows increased vascularization in the C57 scars and activated (α-SMA+) myofibroblasts and F4/80+ macrophages in both the injured C57 tendon and the surrounding scar tissue (Insets c′ and c′′). A subset of cells co-expressing both SMA and F4/80 in the injured C57 tendon (Inset c′). Fewer α-SMA+ myofibroblasts and F4/80+ macrophages are observed but no localized expression of α-SMA and F4/80 is evident in the injured MRL tendon (Inset d′), but SMA + myofibroblasts and F4/80+ macrophages were abundant in the surrounding scar tissue (Inset d′′). BCL-2 expression was notably abundant in the injured C57 tendon (Inset e′), but appeared to be reduced in the surrounding scar tissue (Inset e′′) as well as in the MRL injured tendon and surrounding scar tissues (Insets f′ and f′′). Scale bars = 200 micron in main figures and 50 microns in insets. In panels (A) and (B), T denotes tendon, P denotes pulley, S denotes scar.
Dichotomous roles for TGB1 in the C57 and MRL tendons
Ingenuity pathways analysis (IPA) of active or inhibited upstream transcriptional regulators were also examined to provide additional insights into the observed gene expression changes driving the altered mechanobiology of tendon healing. In the C57 injured tendons, the top 20 activated and inhibited upstream regulators (Fig. 7A) were associated with 184 and 62 unique genes, respectively, that enriched Gene Ontology (GO) terms related to chemotaxis and cell motility and migration, ECM organization, sprouting angiogenesis and blood vessel morphogenesis, positive regulation of cell growth and proliferation, inflammatory responses, collagen catabolism and regulation of phosphatidylinositol- and ERK1/2-signaling (Table S1). In the MRL injured tendon, the top 20 activated and inhibited upstream regulators (Fig. 7B) were associated with 325 and 304 unique genes, respectively, that enriched GO terms related to regulation of cell cycle, including mitotic cell cycle, sister chromatid cohesion, G1/S transition of mitotic cell cycle, mitotic cytokinesis, DNA replication, chromosome segregation, regulation of cell cycle, microtubule-based movement, metaphase plate congression, mitotic spindle organization, G2/M transition of mitotic cell cycle, negative regulation of cell cycle, and extracellular matrix organization (Table S2). Of particular note, was the inhibition of upstream regulators TP53, CDKN1A (p21), CDKN2A (p16), which are associated with DNA damage response and cell cycle arrest.
Ingenuity Pathway Analysis (IPA) Upstream Regulators during tendon healing in the C57 (A) and MRL (B) mice. Activation (Z-score ≥ 2, p value of overlap < 0.05) or inhibition (Z-score ≤ − 2, p value of overlap < 0.05) was determined relative to uninjured tendon gene expression in either strain.
TGFB1 was a significantly activated Upstream Regulator in both the C57 (Activation Z-score = 3.11 and p value = 6.4 × 10–13) and MRL (Activation Z-score = 2.97 and p value = 1.1 × 10–27) injured tendons. GO functional annotation revealed that activation of TGF-β1 drives different biological pathways in the C57 and MRL injured tendons. In the C57 injured tendons, the 76 target genes of TGFB1 (Table S3) enriched GO pathways related to cytokine and chemokine signaling, ECM organization, and cell motility and migration (Fig. 8A). In contrast, 160 target genes of TGFB1 in the MRL (Table S4) enriched GO pathways associated with not only ECM organization and cytokine signaling but mostly with regulation of cell cycle, mitosis, and DNA binding and transcription (Fig. 8B).
Biological Process Gene Ontology (GO) of 76 genes contributing to the activation of TGFB1 Upstream Regulator in the healing C57 tendon (A). Biological Process Gene Ontology (GO) of 160 genes contributing to the activation of TGFB1 Upstream Regulator in the healing MRL tendon (B). Bubble area corresponds to the number of genes in the enriched GO biological process.
Reduced circulating cytokines and chemokines in the MRL injured mice
We have previously shown that the putative senescence marker plasminogen activator inhibitor 1 (PAI-1), downstream of TGF-β, is elevated in fibrotic tendon tissue in humans and mice30. In subsequent studies, we observed increases in the pro-survival signal BCL-2 in injured tendon, which appeared to be linked to inflammatory nuclear factor κB (NF-κB) signaling31. Therefore, in order to evaluate the roles of the immune response and systemic inflammatory signaling on the tendon healing, sera were extracted from whole blood draws from mice at different time points up to 14 dpi and analyzed for senescence associated secretory phenotype (SASP) proteins including inflammatory cytokines/chemokines, TGF-βs, and MMPs. In general, C57 and MRL mice exhibit similar circulatory concentrations of serum proteins in their uninjured state (Fig. 9A). Upon injury, the temporal changes in the circulating proteins in the C57 mice were in general more pronounced than in the MRL mice. Analyzed proteins were clustered in 4 major classes based on Pearson’s correlations (Fig. 9B). In the first cluster, the chemokine CXCL9 (MIG) showed no significant difference over time in the C57 but increased significantly over time in the MRL injured tendons. Cluster 2 proteins showed no significant differences over time or between C57 and MRL mice, which included TGF-βs (1,2,3), MMP-3 and CCL 4 (MIP-1β). Cluster 3 proteins, which included IL-1α, -12 (P40), -13, MMP-2 and -8, CSF1 (M-CSF) and 3, CCL5 (RANTES) and 11 (Eotaxin), CXCL2 (MIP-2) and 5 (LIX), were significantly higher in the C57 mice compared to the MRL mice, but did not vary significantly over time. Cluster 4 proteins showed significant changes over time in the C57 mice only, peaking at either 1 dpi (IL-2 and -9, MMP-12, and CCL3 (MIP-1α)) or 5 dpi (IL-1β, -2, -4, -5, -6, -7, 10, -12 (P70), -17, TNF-α, LIF, MMP-9, CCL2 (MCP-1) and CXCL1(KC) and 10 (IP-10)). In contrast circulation levels of these proteins were significantly lower and remained mostly unchanged over time in the MRL mice. Protein association networks of cluster 3 (Fig. 9C) and cluster 4 (Fig. 9D) circulating proteins visualized in String DB identified strong functional associations, which involved cytokine signaling in immune system and more specifically signaling by interleukins, chemokine signaling and activation of matrix metalloproteinases.
Serum protein analysis indicates temporal systemic upregulation of several circulating enzymes, cytokines, and chemokines in the C57 mice following tendon injury, but not in the MRL mice. (A) Baseline concentrations of serum proteins pre-injury plotted in order of concentration shows no significant differences between C57 and MRL mice. (B) Heatmaps for circulating serum enzymes, cytokines, and chemokines concentration (normalized to day 0 C57 controls) changes over time post flexor tendon injury (dark red indicates > + threefold increase and dark blue indicated > − threefold decrease compared to preinjury serum concentration). Dendrogram represent clustering based on Pearson’s correlations. Each box represents the average of N = 3 mice at each time point. String DB representation of physical and functional association network of cluster 3 (C) and cluster 4 (D) proteins.
