Bone regeneration in minipigs by intrafibrillarly-mineralized collagen loaded with autologous periodontal ligament stem cells - PubMed (original) (raw)

Bone regeneration in minipigs by intrafibrillarly-mineralized collagen loaded with autologous periodontal ligament stem cells

Ci Zhang et al. Sci Rep. 2017.

Abstract

Biomimetic intrafibrillarly-mineralized collagen (IMC) is a promising scaffold for bone regeneration because of its structural and functional similarity to natural bone. The objective of this study was to evaluate the bone regeneration potential of IMC loaded with autologous periodontal ligament stem cells (PDLSCs) in large bone defects in minipigs. A macroporous IMC with a bone-like subfibrillar nanostructure was fabricated using a biomimetic bottom-up approach. Non-healing full thickness defects were established on the cranial bone in minipigs, and IMC and hydroxyapatite (HA) scaffolds seeded with autologous PDLSCs were implanted into these defects. Computed tomographic imaging, histology staining, and atomic force microscopy were applied to evaluate to the quantity, micro/nano structures, and mechanical performance of the neo-bone after 12 weeks of implantation. Compared with HA, IMC showed superior regeneration properties characterized by the profuse deposition of new bony structures with a normal architecture and vascularization. Immunohistochemistry showed that the runt-related transcription factor 2 and transcription factor Osterix were highly expressed in the neo-bone formed by IMC. Furthermore, the nanostructure and nanomechanics of the neo-bone formed by IMC were similar to that of natural bone. This study provides strong evidence for the future clinical applications of the IMC-based bone grafts.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

Figure 1

Morphological and elemental analysis of different scaffolds. (A) Representative (left) low- and (right) high-magnification SEM images of cross-sections of IMC and HA scaffolds. (B) EDS in different scaffolds, confirming the presence of apatite crystallites within the collagen fibrils in the IMC scaffold. Inset: AFM morphology of IMC showing obvious cross-banding patterns.

Figure 2

Figure 2

Representative SEM images of PDLSCs cultured on different scaffolds. (A) On day 3, PDLSCs attached to all the scaffolds, but only secreted ECM (black arrows) on the IMC scaffold. (B) On day 7, abundant fibrous ECM was secreted in the IMC group and calcified nodules deposited on the cell surface in the HA group.

Figure 3

Figure 3

Surgical procedure for producing a non-healing defect and representative CT images of bone regeneration by different scaffolds. (A) A non-healing defect of 2 cm width × 3 cm length × 0.5 cm depth in minipig cranium. (B) The defects were respectively filled with HA and IMC scaffolds seeded with PDLSCs. (C,D) Representative 3-D reconstructed images in the control (C) and experimental groups (D). (E–G) Representative cross-section images of defect area after implantation with IMC (E) and HA (F), and without implants (G) for 12 weeks. (H) Volume analysis of the defect area based on CT results. Groups labeled with star are significantly different (P < 0.05).

Figure 4

Figure 4

HE and Masson’s trichrome staining of defect area after implantation with different scaffolds for 12 weeks. Plenty neo-bone with osteon and vessles was formed by IMC, whereas limited newly-formed bone and lots of residual scaffolds were observed in the HA group. A small amount of newly-formed bone was seen around the defect margin in the control group. B: natural bone; S: scaffold; NB: new bone; DM: defect margin (black dotted line); O: osteon; V: vessel.

Figure 5

Figure 5

Immunohistochemical staining of Runx2, Osx, and TGF-β1 in the defect areas of the two in vivo groups. Runx2, Osx and TGF-β1 were highly expressed in the IMC group, whereas weak or negative staining was observed in the HA group.

Figure 6

Figure 6

Nanomechanics of bone tissue by AFM. (A–C) Representative 3-D AFM property maps of natural bone (A), and newly-formed bone in the IMC group (B) and HA group (C). (D) Quantitative analysis of Young’s modulus in different groups. Groups labeled with star are significantly different (*P < 0.05).

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