A novel injectable fibromodulin-releasing granular hydrogel for tendon healing and functional recovery

Abstract

A crucial component of the musculoskeletal system, the tendon is one of the most commonly injured tissues in the body. In severe cases, the ruptured tendon leads to permanent dysfunction. Although many efforts have been devoted to seeking a safe and efficient treatment for enhancing tendon healing, currently existing treatments have not yet achieved a major clinical improvement. Here, an injectable granular hyaluronic acid (gHA)-hydrogel is engineered to deliver fibromodulin (FMOD)-a bioactive extracellular matrix (ECM) that enhances tenocyte mobility and optimizes the surrounding ECM assembly for tendon healing. The FMOD-releasing granular HA (FMOD/gHA)-hydrogel exhibits unique characteristics that are desired for both patients and health providers, such as permitting a microinvasive application and displaying a burst-to-sustained two-phase release of FMOD, which leads to a prompt FMOD delivery followed by a constant dose-maintaining period. Importantly, the generated FMOD-releasing granular HA hydrogel significantly augmented tendon-healing in a fully-ruptured rat's Achilles tendon model histologically, mechanically, and functionally. Particularly, the breaking strength of the wounded tendon and the gait performance of treated rats returns to the same normal level as the healthy controls. In summary, a novel effective FMOD/gHA-hydrogel is developed in response to the urgent demand for promoting tendon healing.

Keywords: fibromodulin; functional reconstruction; granular hydrogel; hydrogel microparticles; tendon wound healing.

FIGURE 1

Fibromodulin (FMOD) improved tenocyte mobility. FMOD enhanced both tenocyte migration (a) and invasion (b). Scale bar = 200 μm (a) or 100 μm (b). Data are shown as mean ± SD, n = 6 (a) and 4 (b), respectively. Two‐sample t‐tests were used for statistical analyses. **p < 0.005.

FIGURE 2

Fibromodulin (FMOD) modulated tenocyte gene expression. Relative expressions of MMP2 (a), MMP9 (b), MMP14 (c), MMP3 (d), Col1α1 (e), Col3α1 (f), and LOX (g) against GAPDH were normalized to the initial pre‐treatment values. Data are shown as mean ± SD, n = 3. Two‐sample t‐tests were used for statistical analysis. *p < 0.05; **p < 0.005.

FIGURE 3

Fibromodulin (FMOD)‐releasing granular HA hydrogel was engineered based on a delicately designed blueprint. Four percent high molecular weight (HMW) HA solution (a) was cross‐linked with DVS (b). The formed bulk HA hydrogel was mechanically fragmented to fabricate HA hydrogel microparticles (HMPs) with a fine and fiber‐like porous structure (c), and then lyophilized to pack into granular HA hydrogel (d). The granular HA hydrogel was reconstituted with FMOD protein prior to use (e).

FIGURE 4

(FMOD)/gHA‐hydrogel was intensively characterized including storage and loss modulus measurements of cross‐linked HA‐hydrogel (a), the swelling ability of the gHA‐hydrogel (b), the viscosity of the reconstituted granular HA hydrogel at different gel concentrations (c), and the release profile of FMOD from the reconstituted gHA‐hydrogel (d), which fitted the Korsmeyer–Peppas kinetic model (e) and the distribution of FMOD in the reconstituted gHA‐hydrogel (f). Data are shown as mean ± SD, n = 8 (a), 3 (b–d, f). Mann–Whitney U tests (a) and two‐sample t‐tests (c) were used for statistical analyses. *p < 0.05; **p < 0.005.

FIGURE 5

Fibromodulin (FMOD)/gHA‐hydrogel reduced scar formation in adult rat tendon wounds on 21 days post‐injury. Representative H&E staining (a) and picrosirius red (PSR) staining coped polarized light microscopy (PLM) (b) photographs showed FMOD/gHA‐hydrogel‐treated wound have more organized collagen fibrils compared to PBS‐reconstituted gHA‐hydrogel control. Moreover, immunofluorescence staining presented more tenocytes in FMOD/gHA‐hydrogel‐treated tendons (c). Scale bar = 200 μm (a, b) or 25 μm (c).

FIGURE 6

Fibromodulin (FMOD)/gHA‐hydrogel significantly improved the outcome of rat Achilles tendon healing mechanically. A significantly higher strength recovery ratio (a) and stiffness recovery ratio (b) was found in the FMOD/gHA‐hydrogel‐treated tendons in comparison with the control group on 21 days post‐injury. Data are shown as mean ± SD, n = 6. Mann–Whitney U tests were used for statistical analyses. *p < 0.05.

FIGURE 7

Gait test illustrated the functional recovery of the tendon wounded rats. A gait test apparatus was made according to Cesar S Mendes et al. (a), and the rat's gaits were recorded by videos (b) according to scheduled time points (c). Three parameters, PL (pawprint length), TS (the distance between the first and fifth toes or toe spreading), and IT (the distance between the second and fourth toes or intermediary toes), were measured for AFI calculation (d). Pawprints of unwounded and wounded rats were significantly different (e). AFI were normalized to untreated rats at day 0 (f). Data were shown as mean ± SD, n = 4. Mann–Whitney U tests were used for statistical analyses. *p < 0.05.