Bone morphogenetic protein-12 inducing tenogenic differentiation of mesenchymal stem cells enhances healing of linea alba incision

Abstract

The objective of this study was to investigate the curative effects of mesenchymal stem cells' tenogenic differentiation on linea alba incision healing induced by bone morphogenetic protein-12. Mesenchymal stem cells were isolated and induced by 10 ng/ml of bone morphogenetic protein-12 for 48 h. Expression of scleraxis, collagen I and collagen III were examined at 48 h, 5 and 7 days to investigate the tenogenic differentiation. The expression of scleraxis increases continually even in the absence of bone morphogenetic protein-12 for 5 days (P<0.01). The expression of collagen I and III requires persistent inducing. Then fifty Sprague-Dawley rats were randomly divided into five groups: negative control, positive control, sham group, native mesenchymal stem cells and tenogenically differentiated mesenchymal stem cells. Tensiometric testing and modified semiquantitative histological analysis were performed to explore the curative effects. The tension levels in the positive control, sham, native mesenchymal stem cells and tenogenically differentiated mesenchymal stem cells were 44, 41.8, 51.6 and 69.7%, respectively, compared with the negative control. Tenogenically differentiated mesenchymal stem cells exhibited a greater increase in tension compared with positive control, sham and native mesenchymal stem cell groups (P<0.05). From the sections stained with Masson's Trichrome, collagen organization and amount of tenogenically differentiated mesenchymal stem cells was better than the other three groups (P<0.05). In conclusion, mesenchymal stem cells' tenogenic differentiation induced by bone morphogenetic protein-12 can enhance linea alba incision healing.

Keywords: bone morphogenetic protein-12; incisional hernia; linea alba incision; mesenchymal stem cells.

Figure 1.

In vivo implantation. (A) Preparation of MSC-sponge complex with MSCs growing on the sponge; (B) Creation of linea alba incision; (C) Closure of the incision with MSC-sponge complex embedded into the incision; (D) Sample of healed linea alba incision with tenogenically differentiated MSCs. MSCs, mesenchymal stem cells.

Figure 2.

Tensiometric testing. (A) Special pruning of linea alba sample with 2 mm of the midline scar preserved; (B) Tensiometric test using Instron tensiometric machine.

Figure 3.

BMP-12 induces MSCs tenocyte differentiation. Increasing expression of SCX persists even if BMP-12 is removed. However, increasing expression of collagen I and collagen III require persistent induction by BMP-12. *P<0.05. BMP, bone morphogenetic protein; MSCs, mesenchymal stem cells; SCX, scleraxis.

Figure 4.

Induced MSCs enhance ultimate tensile strength of linea alba incision. Group E of tenogenically differentiated MSCs exhibits more increased tension than positive control group B, sham group C and native MSCs group D, with increases of 58.6, 66.9 and 35.1%, respectively (P<0.05). *P<0.05. Group A did not undergo any operation and nothing was implanted. Group B only underwent operation, but did not have anything implanted. Group C underwent operation, but only sponge scaffold was implanted into the linea alba incision. Group D underwent operation and sponge scaffold with native MSCs were implanted. Group E underwent operation and sponge scaffold with tenogenically differentiated MSCs were implanted. MSCs, mesenchymal stem cells.

Figure 5.

Masson's Trichrome staining. (A) Negative control, (B) positive control, (C) sham group, (D) native MSCs and (E) tenogenically differentiated MSCs. Group E of tenogenically differentiated MSCs demonstrates rich and well aligned collagen fibrous matrix along the transversal (tensile) axis of the incision. By contrast, the fibrous matrix in positive control group B and sham group C are significantly disorderly in organization. Organization and amount of collagen fibrous of native MSCs group D are better than group B and C, but worse than group E, which is corresponding to the semiquantitative histological analysis (Table III). Aa, Ba, Ca, Da, Ea ×40; Ab, Bb, Cb, Db, Eb ×200. MSCs, mesenchymal stem cells.