Self-assembling peptide nanofibers coupled with neuropeptide substance P for bone tissue engineering

Abstract

The number of patients requiring flat bone transplantation continues to increase worldwide. Cell transplantation has been successfully applied clinically; however, it causes another defect site and the time requirements to harvest cells and expand them are considerable. In this study, KLD12/KLD12-SP (KLD12+KLD12-substance P [SP]) was designed to mimic endogenous tissue-healing processes. The structures of KLD12, KLD12-SP, and KLD12/KLD12-SP were observed by transmission electron microscopy and circular dichroism spectra. KLD12/KLD12-SP nanofibers (5-10 nm) were created under physiological conditions by formation of a β-sheet structure. The ability of mesenchymal stem cells (MSCs) to recruit KLD12/KLD12-SP was observed by using an in vivo fluorescence imaging system. Labeled human bone marrow stromal cells supplied via an intravenous injection were recruited to the scaffold containing KLD12/KLD12-SP. Polylactic acid/beta-tricalcium phosphate (PLA/β-TCP) scaffolds filled with KLD12/KLD12-SP were applied to repair calvarial defects. The composite constructs (groups: defect, PLA/β-TCP, PLA/β-TCP/KLD12, and PLA/β-TCP/KLD12/KLD12-SP) were implanted into rat defect sites. Bone tissue regeneration was evaluated by observing gross morphology by hematoxylin and eosin and Masson's trichrome staining at 12 and 24 weeks after surgery. Gross morphology showed that the defect site was filled with new tissue that was integrated with host tissue in the KLD12/KLD12-SP group. In addition, from the staining data, cells were recruited to the defect site and lacunae structures formed in the KLD12/KLD12-SP group. From these results, the PLA/β-TCP+KLD12/KLD12-SP composite construct was considered for enhancement of bone tissue regeneration without cell transplantation.

FIG. 1.

Structural analysis. Transmission electron microscopy image of (A) KLD12, (B) KLD12-SP, and (C) bioactive KLD12. (D) Circular dichroism spectra of various peptides. Scale bar: 100 nm. SP, substance P. Color images available online at www.liebertpub.com/tea

FIG. 2.

In vitro MSC migration assay. (A) Sucrose (negative control). (B) KLD12. (C) Soluble SP. (D) Bioactive KLD12. (E) Quantitative analysis (*p<0.05). Scale bar: 100 μm. MSCs, mesenchymal stem cells. Color images available online at www.liebertpub.com/tea

FIG. 3.

(A) In vivo real-time hBMSC recruiting assay. Movement of labeled hBMSCs to the scaffolds containing bioactive KLD12, KLD12, or PBS at 6 weeks. (B) Optical images of the scaffolds explanted after 6 weeks. (C) Quantitative analysis of optical intensity (*p<0.05). (D) MSC recruitment of bioactive KLD12-SP peptides. Representative images of the scaffolds containing bioactive KLD12, KLD12, or PBS after 2 weeks (nonlabeled hBMSCs were used). (E) Neo-live labeled hBMSCs at 6 weeks after subcutaneous implantation of the scaffolds. Scale bar: 100 μm. hBMSCs, human bone marrow stromal cells; PBS, phosphate-buffered saline. Color images available online at www.liebertpub.com/tea

FIG. 4.

Bone reconstruction of each group in the calvarial model at 12 and 24 weeks. Scale bar: 3 mm. Color images available online at www.liebertpub.com/tea

FIG. 5.

(A) Hematoxylin and eosin staining for each group (defect, PBS, KLD12, and bioactive KLD12) of the calvarial model at 12 and 24 weeks. The images of all groups were taken on the edge and center (arrow: interface between host tissue and new tissue, asterisk: new bone tissue [lacunae structure]). (B) Masson's trichrome staining for each group (defect, PBS, KLD12, and bioactive KLD12) of the calvarial model at 12 and 24 weeks. Photographs of the edge and center were taken for all groups. (C) Quantitative analysis of the stained area (collagenous and noncollagenous tissue) (*p<0.05). Scale bar: 50 μm. Color images available online at www.liebertpub.com/tea

FIG. 6.

MSC recruitment of in vivo samples. (A) Center at 12 weeks. (B) Edge at 12 weeks. (C) Center at 24 weeks. (D) Edge at 24 weeks (arrow: interface between host and new tissue). Scale bar: 100 μm. Color images available online at www.liebertpub.com/tea

FIG. 7.

Immunofluorescence staining of type 1 collagen and osteocalcin. (A) Center at 12 weeks. (B) Edge at 12 weeks. (C) Center at 24 weeks. (D) Edge at 24 weeks (arrow: interface between host and new tissue). Scale bar: 100 μm. Color images available online at www.liebertpub.com/tea