Background
Tendinopathies like those of the superficial digital flexor tendon (SDFT) result in major leg lameness and are debilitating for horses of all disciplines [
1,
2]. For both acute and chronic tendinopathies, like those of the SDFT, a closer look at the pathology associated with tendon injury oftentimes implicates alterations in extracellular matrix (ECM) regulators of collagen fibrillogenesis and organization [
3]. Alterations in the expression of ECM regulators lead to changes in biomechanical properties that impact the strength and stability of these energy storing tendons [
4]. Due to this, novel therapeutics are necessary since complete repair is unlikely and further injury is a major concern [
2,
3].
Small leucine-rich repeat proteoglycans (SLRPs) are a class of regulatory molecules that are essential for collagen organization in tendon development, maturation, and repair [
5]. The contributions of SLRPs have been particularly well-characterized in tendons [
6‐
12]. Besides directly affecting collagen fibrillogenesis, SLRPs like biglycan (BGN) and decorin (DCN) play roles in determining how tissue niche impacts cell biology, including: 1) the differentiation status of tendon progenitors in health and pathology [
13]; 2) inflammatory regulation as Damage Associated Molecular Pattern proteins interacting with Toll-like receptors [
14]; 3) recruitment of cells to sites of tissue repair or regeneration [
15]; and 4) sequestration of growth factors essential for generation and maintenance of the tendon phenotype [
13]. Previous work has demonstrated that the absence of these SLRPs dramatically affects tendon repair outcomes with BGN essential early in repair and DCN crucial later in tendon repair [
7,
9]. Interestingly, expression of
BGN and
DCN in mature animals decreases after an injury and never recovers to the level seen during development and maturation [
11], suggesting that low BGN and/or DCN may contribute to the impaired injury response.
After a mature tendon is injured, repair occurs as a result of extrinsic and intrinsic influences. Leukocytes and fibroblasts migrate into the lesion early in repair [
15]. Post-injury, these fibroblasts originate from the extrinsic paratenon and have demonstrated distinct differences in marker expression and tenogenic potential as compared to the tendon proper fibroblast cell population [
16‐
20]. Thus, when considering therapeutic interventions for tendon repair, both cell populations should be included since the role of each cell type remains unresolved.
Recognizing the value of BGN and DCN in tendon development and maturation and their subsequent decline at the time of repair, we hypothesize that addition of BGN or DCN to the tendon matrix would improve tendon formation. To test this hypothesis, equine tendon proper (TP) and peritenon (PERI) cells were seeded in an in vitro fibrin-based three-dimensional tendon construct model in which the gel contained two differing amounts of either exogenous bovine purified BGN or exogenous bovine purified DCN. The effects of the exogenous BGN or DCN on biomechanics, electron microscopic ultrastructure, collagen content, and gene expression were determined.
Discussion
Embedding small leucine-rich proteoglycans within a fibrin gel affected features of the engineered tendons. When considering gene expression, the supplementation of either exogenous biglycan or decorin had a greater effect on the tenogenic capacity of the equine peritenon cells than they did on tendon proper cells. Yet, biomechanical properties were bolstered by supplementation of SLRPs for both cell types to varying degrees. PERI cells supplemented with bBGN or bDCN showed significant or approaching significant increased Young’s modulus, and ultimate tensile strength in PERI cells increased with the addition of 25 nM bBGN. Moreover, tendon proper cell-seeded constructs had increased Young’s modulus for and 25 nM bDCN, and nearly significant increases for 5 nM bBGN and 5 nM bDCN. These results suggest that SLRP supplementation can have positive tenogenic effects on extrinsic PERI cells and intrinsic TP cells.
Cells within a connective tissue can be affected by changes in their tissue niche. For example, if biglycan and decorin expression are absent during development the resulting changes affect the fibril structure with a shift toward larger diameters. In addition to alterations in mechanical properties, such as a failure at lower loads, decreased stiffness, and increase in percent relaxation, knocking out expression of biglycan and decorin affects collagen fiber realignment with a slower response to load [
7,
9,
12]. Other knockout SLRP models, including biglycan, decorin, and double biglycan and fibromodulin, have varying degrees of apparent phenotypes including accelerated degeneration of articular cartilage, subchondral sclerosis, reduced growth rate of bone with decreased bone mass, and disruption of proper collagen fibrillogenesis [
47‐
50]. Conversely, supplementation of SLRPs provides evidence for crucial roles in: signaling pathways, such as the
TGF-β (transforming growth factor beta),
WNT,
TLR (toll-like receptor),
EGFR (epithelial growth factor receptor) internalization, and Akt -dependant/−independent; collagenase shielding; collagen fibrillogenesis in the form of wound healing and scar mitigation; and proteoglycan regulation [
26,
50‐
59].
When evaluating gene expression of the PERI supplemented cells, 5 nM bDCN showed significant increases in
BGN, FMOD, and
Scleraxis (
SCX) and a significant decrease in
CSPG4. The increase in tendon specific markers may be the result of regulation in the TGFβ pathway with decreased activation of ERK1/2 resulting in increased expression of
SCX and subsequently SLRPs like
BGN by TGFβ [
60,
61]. Although the cross-linking marker
LOX tends to decrease, biomechanics (UTS and Young’s Modulus) increase and the fibril distribution is shifted to smaller fibrils indicating that more collagen fibrils are being produced (supported by a trend towards increased
COL1A1 expression) but the fibrils are not maturing and cross-linking remains low (Fig.
5, Fig.
S-3). Previous studies in
DCN knockout mouse models identified an increase in fibril diameter with subsequent decrease in elastic and viscoelastic properties while alterations of the dermatan sulfate side chains had no effect on mechanical properties indicating that the decorin core protein itself is essential for the organization of collagen and its resulting tissue mechanics [
12,
26,
47]. In contrast to DCN, the 5 nM bBGN supplementation produced increases in
CSPG4 expression, suggesting that although
BGN and
DCN have similar signaling pathways in collagen fibrillogenesis they are antagonistic in perivascularization. In breast carcinoma cells,
DCN had an anti-angiogenic effect while
BGN in bone fractures increased pro-angiogenic signals such as
VEGFA (vascular epithelial growth factor A) showing that
BGN and
DCN have antagonistic effects, thus explaining the difference in
CSPG4 expression between bBGN and bDCN in PERI cells [
62,
63]. This would indicate that during fibrillogenesis following an injury,
DCN may play a vital role in mitigating scar formation by preventing vessel growth allowing improved tissue function [
64,
65].
Tendon proper and peritenon cells responded differently to supplementation. Such variations could be due to the differences in niche composition within which these cells exist in vivo as well as differences in cell origins, both of which could affect the tenogenic capacity of these cell types [
18‐
20,
66]. The tendon proper niche consists of a stiff, relatively acellular and hypoxic environment with cells undergoing mechanical load and recoil activating characteristic signaling molecules such as
TGFβ and
Egr1/2 through induction of
SCX [
67]. Other major regulators of tendon maturation and differentiation include
GDF5 (growth and differentiation factor 5) and mohawk (
MKX) in addition to the SLRPs such as fibromodulin, and biglycan [
13,
67,
68]. The peritenon niche is not as clearly defined but is comprised of cells which: (1) express perivascular markers such as endomucin (
EMCN), CD34, and
CD45; (2) secrete stimulatory factors during repair; (3) express matrix remodeling-related genes (matrix metalloprotease,
MMP1 and
MMP3, and
COL3A1); and (4) possess high cellular phenotypic heterogeneity [
18‐
20,
66].
Peritenon cells had a more pronounced response to SLRP supplementation, particularly for DCN. This suggests that DCN may contribute to the peritenon cells transitioning into a tendon-like phenotype after the initial inflammatory response or that DCN aids in the collagen fibril assembly in the extracellular matrix. These functions could be instrumental for tendon repair since peritenon cells are a highly mobile cell type, reacting immediately in response to an injury. Additionally, from the expression and biomechanics data, DCN supplementation could improve peritenon cell utility in engineered tendon grafts. Though tenocytes (TP cells) might seem to be good tissue engineering candidates, it is interesting to compare the response of PERI cells to SLRP supplementation relative to the non-supplemented TP control. When PERI cell-seeded constructs supplemented with SLRPs were compared to TP control constructs – tenocytes that might be used in grafts. Increases in UTS, Young’s modulus, and MTL were significant or approaching significance with all four doses of SLRPs used (Fig. S-
1). Relative to TP control, the constructs with PERI cells had greater collagen content or increased levels approaching significance (Fig. S-
1). Moreover, PERI cell-derived constructs had similar matrix assembly marker expression levels (Fig.
S-6). Although there were no differences in fibril density or mean diameter, bBGN and bDCN supplemented peritenon cells displayed a shift towards larger fibrils which partially explains the increased UTS, Young’s modulus, and MTL (Fig. S-
1,
S-4,
S-5). The expression of
CSPG4 in bDCN supplemented peritenon constructs was similar control TP cell-derived construct levels, which supports a shift away from a perivascular-like phenotype. This suggests the utility of DCN, in particular for peritenon cells, as a phenotype influencing signaling molecule capable of affecting cells of both regions in the tendon. This finding could have implications during injury repair and cell selection for engineered grafts. Many findings in this study support the supplementation of SLRPs like BGN and DCN in therapeutic strategies. Further studies are required to discern the exact mechanisms by which supplemental SLRPs are affecting PERI cells.
This study has a number of important limitations. First, purified bovine proteins were used with the equine cells instead of equine-derived SLRPs. Second, cellular responses to SLRP supplementation are being described in an in vitro model where the active agent is continuously present in the matrix which would not be the case in vivo in a potentially pathological or inflammatory environment. Third, construct numbers were limited, and thus extensive histological analysis, analyses of other SLRPs, and the combinatorial effects of BGN ad DCN were omitted. Fourth, the tendon cells were isolated from horses with a range of ages and breeds. Fifth, the findings are based upon cells of the equine superficial digital flexor tendon. Therefore, the results might not translate to other tendons and ligaments. Finally, a limited n = 5 horses were used in to compare controls and treatments individually to determine if defined and hypothesized improvements in tendon formation were seen with SLRP supplementation. While this might limit the statistical power of the study, it allows for preliminary answers into the efficacy of SLRP supplementation for improving tendon formation. Moving forward, the evaluation of SLRPs in an in vivo injury model could provide further insight into this novel therapeutic intervention for injury repair for equine athletes of all disciplines. Moreover, our findings lend support to further studies that include the incorporation of SLRPs in tendon engineering strategies.
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