Ann Surg Treat Res.  2024 Jul;107(1):50-57. 10.4174/astr.2024.107.1.50.

Scaffold-based synergistic enhancement of stem cell effects for therapeutic angiogenesis in critical limb ischemia: an experimental animal study

• Park HS
• Choi GH
• Jung TW
• Lee T

• KMID: 2557580
• DOI: http://doi.org/10.4174/astr.2024.107.1.50

Abstract

Purpose
Stem cell-based therapies are considered an alternative approach for critical limb ischemia (CLI) patients with limited or exhausted options, yet their clinical use is limited by the lack of sustainability and unclear mechanism of action. In this study, a substance P-conjugated scaffold was injected with mesenchymal stem cells (MSCs) into an animal model of CLI to verify whether angiogenesis could be enhanced.
Methods
A self-assembling peptide (SAP) was conjugated with substance P, known to have the ability to recruit host stem cells into the site of action. This SAP was injected with MSCs into ischemic hindlimbs of rats, and the presence of MSCs was verified by immunohistochemical (IHC) staining of MSC-specific markers at days 7, 14, and 28. The degree of angiogenesis, cell apoptosis, and fibrosis was also quantified.
Results
Substance P-conjugated SAP was able to recruit intrinsic MSCs into the ischemic site of action. When injected in combination with MSCs, the presence of both injected and recruited MSCs was found in the ischemic tissues by double IHC staining. This in turn led to a higher degree of angiogenesis, less cell apoptosis, and less tissue fibrosis compared to the other groups at all time points.
Conclusion
The combination of substance P-conjugated SAP and MSCs was able to enhance angiogenesis and tissue repair, which was achieved by the additive effect from exogenously administered and intrinsically recruited MSCs. This scaffold-based intrinsic recruitment approach could be a viable option to enhance the therapeutic effects in patients with CLI.

Keyword

Angiogenesis; Chronic limb-threatening ischemia; Mesenchymal stem cells; Scaffold; Substance P

Figure

• 

  Fig. 1 Fluorescent immunohistochemical staining for stem cell markers (A) CD105, (B) CD90, and (C) CD29 from ischemic hindlimbs injected with rat MSC (tagged with green fluorescent protein), RSP, and a combination of MSC and RSP (10× magnification). MSC, mesenchymal stem cell; RSP, substance P conjugated to RADA 16-II; DAPI, 4′,6-diamidino-2-phenylindole.

• 

  Fig. 2 Immunohistochemical staining and quantification of angiogenesis markers for MSC, RSP, MSC + RSP, and control. (A) CD31. (B) von Willebrand factor (vWF). (C) α-smooth muscle actin (α-SMA). MSC, mesenchymal stem cell; RSP, substance P conjugated to RADA 16-II; DAPI, 4′,6-diamidino-2-phenylindole. *P < 0.05. †P < 0.05 for control vs. all other groups.

• 

  Fig. 3 (A) Fluorescent terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) staining and (B) quantification of TUNEL-positive stains for evaluation of apoptosis for MSC, RSP, MSC + RSP, and control. MSC, mesenchymal stem cell; RSP, substance P conjugated to RADA 16-II; DAPI, 4′,6-diamidino-2-phenylindole. *P < 0.05. †P < 0.05 for control vs. all other groups.

• 

  Fig. 4 (A) Representative Masson’s trichrome staining showing fibrosis between muscle fibers (10× magnification) and (B) quantification of degree of fibrosis from immunohistochemical stains for evaluation of fibrosis for MSC, RSP, MSC + RSP, and control. MSC, mesenchymal stem cell; RSP, substance P conjugated to RADA 16-II. *P < 0.05. †P < 0.05 for control vs. all other groups.

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