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. 2019 May 31;9(14):4241-4254.
doi: 10.7150/thno.35285. eCollection 2019.

A Combination of Oxo-M and 4-PPBP as a potential regenerative therapeutics for tendon injury

Solaiman Tarafder  1 , Christopher Ricupero  1 , Sumeet Minhas  1 , Rebecca J Yu  1 , Ashleigh D Alex  1 , Chang H Lee  1
Affiliations

A Combination of Oxo-M and 4-PPBP as a potential regenerative therapeutics for tendon injury

Solaiman Tarafder et al. Theranostics. .

Abstract

Tendons injuries frequently result in scar-like tissue with poor biochemical structure and mechanical properties. We have recently reported that CD146+ perivascular originated tendon stem/progenitor cells (TSCs), playing critical roles in tendon healing. Here, we identified highly efficient small molecules that selectively activate endogenous TSCs for tendon regeneration. Methods: From a pool of ERK1/2 and FAK agonists, Oxo-M and 4-PPBP were identified, and their roles in tenogenic differentiation of TSCs and in vivo tendon healing were investigated. Controlled delivery of Oxo-M and 4-PPBP was applied via PLGA µS. Signaling studies were conducted to determine the mechanism for specificity of Oxo-M and 4-PPBP to CD146+ TSCs. Results: A combination of Oxo-M and 4-PPBP synergistically increased the expressions of tendon-related gene markers in TSCs. In vivo, delivery of Oxo-M and 4-PPBP significantly enhanced healing of fully transected rat patellar tendons (PT), with functional restoration and reorganization of collagen fibrous structure. Our signaling study suggested that Oxo-M and 4-PPBP specifically targets CD146+ TSCs via non-neuronal muscarinic acetylcholine receptors (AChR) and σ1 receptor (σ1) signaling. Principal conclusions: Our findings demonstrate a significant potential of Oxo-M and 4-PPBP as a regenerative therapeutics for tendon injuries.

Keywords: Endogenous stem/progenitor cells; Regenerative Medicine; Small molecules; Tendon.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Fig 1
Fig 1
Oxo-M and 4-PPBP (A) inducing tenogenic differentiation of TSCs via FAK and ERK1/2 signaling. Tendon-related gene expressions were induced in TSCs by 1 wk treatment of Oxo-M, 4-PPBP, and Oxo-M + 4-PPBP (O+P) (B). The treatment with 4-PPBP significantly increased COL-I and COL-III whereas Oxo-M increased Tn-C, VIM, and Scx. A combination of Oxo-M and 4-PPBP increased all the tested gene markers up to 8 - 38 folds on par with CTGF (n = 5 per group, *: p<0.001 compared to control, #: p<0.001 compared to all the other groups).
Fig 2
Fig 2
Western blot show FAK and ERK phosphorylation induced by Oxo-M and 4-PPBP (A). siRNA transfection of FAK and ERK significantly reduced FAK and ERK1/2 mRNA expressions as compared to scrambled siRNA and no siRNA controls (B & C) (n = 5 per group; *: p<0.001 compared to all the other groups). FAK and ERK1/2 knockdown (KD) significantly attenuated the Oxo-M and 4-PPBP induced expressions of tendon-related gene (D) (n = 5 per group; *: p<0.001 compared to control - scrambled siRNA).
Fig 3
Fig 3
Tendon healing by Oxo-M and 4-PPBP. In contrast to the enlarged scarred healing in control (A), Oxo-M and 4-PPBP delivery resulted in native-like tendon by 2 wks (B). H&E (C) and Masson's Trichrome staining (D) further showed that Oxo-M and 4-PPBP delivery improved tendon healing with reorganized collagen fibers in comparison with disrupted scar-like tissues with control, Oxo-M, or 4-PPBP alone. Immunofluorescence show the increased number of endogenous CD146+ TSCs undergoing tenogenic differentiation expressing proCOL-I and Scx by 1 wk post-op in comparison with control, Oxo-M alone and 4-PPBP alone (E). Relative collagen quantity from imaging processing was significantly higher with Oxo-M and 4-PPBP delivery (F). Total cell numbers were significantly lower with Oxo-M and 4-PPBP delivery than control, Oxo-M alone or 4-PPBP alone by 1 wk (G) (n = 10 sections per tissue sample; *: p<0.01 compared to other groups in the same time point; #: p<0.01 compared to 1 wk data).
Fig 4
Fig 4
Circular-polarized image of Picrosirius Red (PR) stained tendon sections (A) showed densely aligned collagen fibers with Oxo-M and 4-PPBP delivery. Automated digital image processing demonstrated narrower fiber orientation with Oxo-M and 4-PPBP similar to native (B). In contrast, control, Oxo-M alone and 4-PPBP alone showed spread histogram of angular orientation of collagen fibers (B). Quantitatively, angular deviation (AD) was significantly lower with Oxo-M and 4-PPBP delivery in comparison with control, Oxo-M alone, and 4-PPBP alone (B) (n = 6 per group; *: p<0.001 compared to control, Oxo-M, and 4-PPBP, #:p<0.05 compared to Oxo-M + 4-PPBP). Consistently, tensile stiffness was significantly higher with Oxo-M and 4-PPBP as compared to control (C) (n = 5 per group, *: p<0.001 compared to control, #: p<0.001 compared to Oxo-M + 4-PPBP).
Fig 5
Fig 5
Controlled delivery of of Oxo-M and 4-PPBP for tendon regeneration. Oxo-M and 4-PPBP were encapsulated in PLGA µS (A) by double-emulsion technique. In order to measure concentration of released Oxo-M and 4-PPBP, adsorption maxima (λmax) was determined using UV-Vis spectroscopy as 230 nm and 207 nm for Oxo-M and 4-PPBP, respectively (B). PLGA µS provided sustained release of Oxo-M and 4-PPBP up to 7 days (C). A transwell co-culture experiment showed that Oxo-M and 4-PPBP released from PLGA µS increased collage deposition in CD146+ TSCs by 2 wks, suggesting preservation of their bioactivities after microencapsulation (D).
Fig 6
Fig 6
Effect of controlled delivery of Oxo-M and 4-PPBP on tendon healing. By 1 and 2 wks in vivo, PR staining showed the collagen density and alignment improved with Oxo-M µS and 4-PPBP µS in comparison with direct delivery of Oxo-M and 4-PPBP via fibrin gel (A). Control groups ended with scar-like, disrupt collagen structure (A). Imaging processing of collagen fibers showed that the angular distribution of collagen fibers was narrower with Oxo-M µS and 4-PPBP µS, closer to the native level (B). Angular deviation (AD) was also significantly lower with Oxo-M µS and 4-PPBP µS than direct delivery of Oxo-M and 4-PPBP (C). Consistently, tensile stiffness of tendons delivered with Oxo-M µS and 4-PPBP µS significantly increased as compared to direct application of Oxo-M and 4-PPBP (D) (n = 5 per group; #: p<0.001 compared to all the groups, *: p<0.01).
Fig 7
Fig 7
Specificity of Oxo-M and 4-PPBP to CD146+ TSCs. Immunofluorescence show abundant expression of σ1R, ChAT, and AChRs M1 - M4 in CD146+ TSC rather than CD146- tendon cells, except for AChRs M2 (A). Antibody-based functional assays showed that tenogenic gene expressions by Oxo-M and 4-PPBP in TSCs were significantly attenuated by blocking σ1R, ChAT, and AChRs M1 and M4 (B) (n = 5 each group; *: p<0.01 compared to control).
Fig 8
Fig 8
The siRNA KD of σ1R and AChRs M1 and M4. The transfection of siRNA significantly reduced σ1R and AChRs M1 and M4 expressions in comparison with controls (A) (n = 5 per group; *p<0.001). The siRNA KD significantly reduced the Oxo-M and 4-PPBP elevated expressions of COL-I, COL-III, VIM, and TnmD by 1 wk treatment (B). The Tn-C and SCX and expressions were significantly lowered by AChR M4 KD but not by σ1R KD. AChR M1 KD failed to result in significant difference in the expressions of Tn-C, VIM, TnmD and SCX (B) (n = 5 per group; *: p<0.01 compared to Control - scrambled siRNA).
Fig 9
Fig 9
Speculated mechanism for the specificity of Oxo-M and 4-PPBP to TS
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