Leucine rich repeat containing 32 accelerates tenogenic differentiation of tendon-derived stem cells and promotes Achilles tendon repair in rats

Abstract

Although many surgical or non-operative therapies have been developed to treat Achilles tendon injuries, the prognosis of which is often unsatisfactory. Recently, biologic approaches using multipotent stem cells like tendon-derived stem cells (TDSCs) pose a possible treatment option. To evaluate whether the Leucine rich repeat containing 32 (Lrrc32) affects the tenogenic differentiation of TDSCs and thus promotes Achilles tendon healing. TDSCs were infected with the recombinant Lrrc32-overexpressing lentivirus (LV-Lrrc32) and then locally injected into the injured site of rat. Four weeks after surgery, the Achilles tendon tissue (~0.5 cm) around the injured area was harvested for analysis. Pathological results showed that Lrrc32-overexpressing TDSCs significantly improved the morphological changes of the injured tendons. Specifically, the increased collagen-I expression and hydroxyproline content in extracellular matrix, and more orderly arrangement of the regenerated collagen fibers were observed in the Lrrc32 overexpression group. Moreover, 4 weeks after injection of Lrrc32-overexpressing TDSCs, the expression of tenocyte-related genes such as tenomodulin (Tnmd), scleraxis (Scx) and decorin (Dcn) were upregulated in the area of the healing tendon. These findings indicated that Lrrc32 promoted the tenogenic differentiation of TDSCs in vivo. Additionally, Lrrc32 overexpression also increased the expression of TGF-β1 and p-SMAD2/3, suggesting that the beneficial effects of Lrrc32 on tendon repair might be associated with the expression of TGF-β1 and p-SMAD2/3. Our findings collectively revealed that Lrrc32-overexpressed TDSCs promoted tendon healing more effectively than TDSCs alone.

Keywords: Achilles tendon healing; Leucine rich repeat containing 32 (Lrrc32); tendon-derived stem cells (TDSCs); tenogenic differentiation.

Fig. 1.

Lrrc32 overexpression promoted tendon repair in vivo. (a) Achilles tendon injury model of rat. Triangle: calcaneus. (b) Quantification of hydroxyproline by ELISA (n=6 per group). (c) Histological evaluation of the area of the healing tendon (6 rats per group were used for histology analysis): hematoxylin and eosin (H&E) staining (the upper row); Masson’s trichrome staining (the middle row); immunohistochemical staining for collagen-I (the lower row). #: healing tendon cells. Magnification 100×, scale bar=200 µm. TDSCs: tendon-derived stem cells; Lrrc32: Leucine rich repeat containing 32; Control: Normal rat without Achilles tendon injury; NC: Achilles tendon injury and PBS-applied group; TDSCs+LV-NC: Achilles tendon injury and TDSCs+LV-NC-applied group; TDSCs+LV-Lrrc32: Achilles tendon injury and TDSCs+LV-Lrrc32-applied group. *P<0.05, ***P<0.001.

Fig. 2.

Characterization of parental TDSCs and lentivirus mediated Lrrc32 overexpression in TDSCs. (a) MSCs surface maker (CD29, CD44, CD90 and CD45) expression of TDSCs was detected by flow cytometer. (b) Light microscopy observation of the morphology of normal TDSCs (parental), and the differentiation assays were performed to analyze the osteogenic (Alizarin red staining), adipogenic (Oil Red O staining) and chondrogenic (Safranin O staining) differentiation of TDSCs. Magnification 200×, scale bar=100 µm. Lrrc32 mRNA and protein levels were analyzed by (c) qRT-PCR and (d) western blotting (n=6). TDSCs: tendon-derived stem cells; Lrrc32: Leucine rich repeat containing 32. ***P<0.001.

Fig. 3.

Lrrc32 overexpression promoted TDSC tenogenic differentiation. qRT-PCR analysis of transcript levels of (a)Tnmd, (b)Scx and (c)Dcn in Achilles tendon tissue (n=6 per group). (d) Immunofluorescent staining for Tnmd. Magnification 400×, scale bar=50 µm. TDSCs: tendon-derived stem cells; Lrrc32: Leucine rich repeat containing 32; Control: Normal rat without Achilles tendon injury; NC: Achilles tendon injury and PBS-applied group; TDSCs+LV-NC: Achilles tendon injury and TDSCs+LV-NC-applied group; TDSCs+LV-Lrrc32: Achilles tendon injury and TDSCs+LV-Lrrc32-applied group. *P<0.05, **P<0.01.

Fig. 4.

Lrrc32 overexpression increased the expressions of TGF-β1 and p-SMAD2/3. (a) TGF-β1, (b) SMAD2/3 and p-SMAD2/3 protein levels were analyzed by western blotting (n=6 per group). TDSCs: tendon-derived stem cells; Lrrc32: Leucine rich repeat containing 32; Control: Normal rat without Achilles tendon injury; NC: Achilles tendon injury and PBS-applied group; TDSCs+LV-NC: Achilles tendon injury and TDSCs+LV-NC-applied group; TDSCs+LV-Lrrc32: Achilles tendon injury and TDSCs+LV-Lrrc32-applied group. **P<0.01.