A Three-Dimensional Printed Polycaprolactone-Biphasic-Calcium-Phosphate Scaffold Combined with Adipose-Derived Stem Cells Cultured in Xenogeneic Serum-Free Media for the Treatment of Bone Defects

Abstract

The efficacy of a three-dimensional printed polycaprolactone-biphasic-calcium-phosphate scaffold (PCL-BCP TDP scaffold) seeded with adipose-derived stem cells (ADSCs), which were cultured in xenogeneic serum-free media (XSFM) to enhance bone formation, was assessed in vitro and in animal models. The ADSCs were isolated from the buccal fat tissue of six patients using enzymatic digestion and the plastic adherence method. The proliferation and osteogenic differentiation of the cells cultured in XSFM when seeded on the scaffolds were assessed and compared with those of cells cultured in a medium containing fetal bovine serum (FBS). The cell-scaffold constructs were cultured in XSFM and were implanted into calvarial defects in thirty-six Wistar rats to assess new bone regeneration. The proliferation and osteogenic differentiation of the cells in the XSFM medium were notably better than that of the cells in the FBS medium. However, the efficacy of the constructs in enhancing new bone formation in the calvarial defects of rats was not statistically different to that achieved using the scaffolds alone. In conclusion, the PCL-BCP TDP scaffolds were biocompatible and suitable for use as an osteoconductive framework. The XSFM medium could support the proliferation and differentiation of ADSCs in vitro. However, the cell-scaffold constructs had no benefit in the enhancement of new bone formation in animal models.

Keywords: adipose; biphasic calcium phosphate; polycaprolactone; scaffold; stem cells.

Figure 1

The preview architectures of the scaffold prior to the printing process; (A) top view and (B) perspective view.

Figure 2

The compression forces (indicated by arrows) were applied to the superior aspect (A,B) and the lateral aspect (C,D) of the scaffolds.

Figure 3

A schematic diagram showing an overview of the in vitro experiments.

Figure 4

The pictures demonstrate the surgical sites of Group A (A) and Group C (B). The defects were covered with collagen membrane before suturing (C).

Figure 5

The architecture of the PCL–BCP TDP scaffold; (A) top view, (B) the scaffold was cut into round-shaped specimens for the experiments, (C) magnified picture of the scaffold specimen and (D) magnified picture of the lateral aspect of the scaffold. The scale bars represent 1 mm.

Figure 6

The SEM pictures demonstrate the architectures of the PCL–BCP TDP scaffolds. (A) top view, (B) lateral view and (C): image shows the BCP particles depositing on the surfaces of the scaffold (arrows). (D): The AR-stained BCP particles are seen as black spots throughout the surfaces of the scaffolds in the fluorescent microscope image.

Figure 7

FTIR spectra for BCP, PCL and PCL-30%BCP composite. (A) The bands of the PCL–BCP composite showed no change when compared with the peaks of BCP and PCL raw materials. (B) The bands of the PCL-BCP composite were not changed when compared to those of each material.

Figure 8

(A) The cross-sectional SEM image shows large voids inside the scaffold filament (indicated by arrows). (B) The magnified image of the box demonstrates immiscible blending of the BCP crystals and the PCL matrix in the box. (C) EDX analysis shows the high peaks of calcium (Ca) and phosphate (P) on the scaffold surfaces.

Figure 9

Morphologies of the adherent cells at day 21 taken via a phase-contrast microscope (DS-Fi2-U3, Nikon, Tokyo, Japan) with magnification 10×. (A) XSF group and (B) FBS group. More spindle-shaped morphologies and higher density of the cells in the XSF group were detected compared with those in the FBS group.

Figure 10

The images of flow cytometry analysis demonstrated the expression profiles of the MSC markers, hematopoietic markers, CD271 and CD146 in the XSF group.

Figure 11

The bar graph shows the numbers of viable cells in the constructs in the XSF and FBS groups over 21 days. The growth of the cells in the XSF group was higher than that of the cells in the FBS group at all time points. The significant difference was detected at day 14 (* p < 0.05).

Figure 12

The fold change of gene expression of the cells in the constructs over 21 days; (A) Col-1, (B) BSP, (C) ALP, (D) OPN, (E) RUNX-2, (F) BMP-2 and (G) OCN. On day 7, the levels of the osteogenic differentiation genes in the XSF–OS group, with the exception of Col-1, were notably higher than those in the FBS–OS and control groups. The genes significantly downregulated on day 14. The significant differences were at p < 0.05 (*) and p < 0.01 (**), compared with the control group.

Figure 13

The SEM images of the cell–scaffold constructs in the XSF–OS group (A,C,E) and the FBS–OS group (B,D,F) at culture days 3 (A,B), 14 (C,D) and 21 (E,F). The cells in both groups attached and grew well on the scaffold surfaces. Dense multilayer cell sheets were observed throughout the scaffolds from day 14 and the morphologies of the cells were difficult to identify.

Figure 14

The bar graph demonstrates the new bone VFs for all groups. The new bone VFs in Group C were greater than those in the other groups, whereas those in Group D were less than the other groups over the observation period (*, p < 0.05 against group C). From week 4, the new bone volumes in Group C were significantly greater than those in Group A (*, p < 0.05). The new bone formation in Group A was less than that in Group B at all time points (p > 0.05).

Figure 15

The µ-CT-constructed images demonstrate new bone formation within the defects; (A–C): Group A, (D–F): Group B, (G–I): Group C, and (J–L): Group D. The new bone formation in Groups A–C was clearly greater than in Group D. At week 8, the newly formed bone in Group A–C almost filled the entire roof of the defects, whereas in Group D, some new bone foci were detected in the middle part of the defects.

Figure 16

Histological features of the calvarial defects at week 2; (A): Group A, (B): Group B, (C): Group C, and (D): Group D. In Groups A and B, the scaffolds and the covering collagen membranes were surrounded by dense fibrous tissue and chronic inflammatory cells. New bone regeneration was detected extending from the periphery host bone (see boxes). In Group C, bone graft fragments were observed along the defect, which had less inflammatory cell infiltration. In Group D, newly formed bone was detected at the margins of the host bone. H = host bone, NB = new bone, SC = scaffold, MB = the collagen membranes, BG = bone graft.

Figure 17

Histological features of the calvarial defects at week 4; (A): Group A, (B): Group B, (C): Group C, and (D): Group D. The areas of new bone formation within the defects in Groups A–C were larger than in week 2. In Groups A and B, the newly formed bone came from the peripheries and seemed to regenerate along the roofs of the defects (see boxes), whereas that in Group C was generally found within the middle portions of the defect (*). Remnants of the collagen membranes (MB) were still detected in all Groups. In Group D, newly formed bone regenerated from the peripheries of the defects (see box). H = host bone, NB = new bone, SC = scaffold.

Figure 18

Histological sections of the calvarial defects at week 8; (A): Group A, (B): Group B, (C): Group C, and (D): Group D. In Groups A and B, the larger areas of newly formed bone were detected in some areas within the scaffold frameworks (see boxes). New bone bridging of the defects was found only in Group C. In Group D, newly formed bone was detected along the areas of the collagen membrane remnants (MB). H = host bone, NB = new bone, SC = scaffold.