Bone regeneration of minipig mandibular defect by adipose derived mesenchymal stem cells seeded tri-calcium phosphate- poly(D,L-lactide-co-glycolide) scaffolds

Abstract

Reconstruction of bone defects represents a serious issue for orthopaedic and maxillofacial surgeons, especially in extensive bone loss. Adipose-derived mesenchymal stem cells (ADSCs) with tri-calcium phosphates (TCP) are widely used for bone regeneration facilitating the formation of bone extracellular matrix to promote reparative osteogenesis. The present study assessed the potential of cell-scaffold constructs for the regeneration of extensive mandibular bone defects in a minipig model. Sixteen skeletally mature miniature pigs were divided into two groups: Control group and scaffolds seeded with osteogenic differentiated pADSCs (n = 8/group). TCP-PLGA scaffolds with or without cells were integrated in the mandibular critical size defects and fixed by titanium osteosynthesis plates. After 12 weeks, ADSCs seeded scaffolds (n = 7) demonstrated significantly higher bone volume (34.8% ± 4.80%) than scaffolds implanted without cells (n = 6, 22.4% ± 9.85%) in the micro-CT (p < 0.05). Moreover, an increased amount of osteocalcin deposition was found in the test group in comparison to the control group (27.98 ± 2.81% vs 17.10 ± 3.57%, p < 0.001). In conclusion, ADSCs seeding on ceramic/polymer scaffolds improves bone regeneration in large mandibular defects. However, further improvement with regard to the osteogenic capacity is necessary to transfer this concept into clinical use.

Figure 1

Scaffold characterization. The morphology of tri-calcium phophate poly(D,L-lactic-co-glycolic) acid (TCP-PLGA) scaffold observed by scanning electron microscopy (SEM) at low (a–c) and high magnifications (d–f) with interconnecting channels measuring about 450–500 μm. Scale bars represent 1 mm (a), 100 μm (b,c) and 50 μm (d), 10 µm (e) and 5 µm (f).

Figure 2

Alizarin Red and Live/Dead staining of pADSCs with quantification. pADSCs were cultured for 14 days in osteogenic differentiation medium in monolayer and on the scaffold. (a) Calcium deposition is shown as red colour by Alizarin Red staining of pADSCs cultured with or without osteogenic induction media (n = 3). Scale bar = 1 μm. (b) Quantification of the intensity of red colour in the monolayer (p < 0.0001). (c) Quantification of the intensity of red colour of Alizarin Red staining on the scaffold (surface vs. centre) at day 1 and day 14. Alizarin red staining reveals mineralized matrix both on the scaffold surface (green bars) and in the centre of the scaffolds (red bars, p = 0.53). (d) Cell viability by live/dead cell staining on TCP-PLGA scaffolds demonstrating the distribution of living (green) and dead (red) pADSCs on the surface vs. center. The green fluorescence represent living cells while red fluorescence indicate dead cells. (e) Quantification of ratio of living to dead cells on the scaffold surface where green represents the living cells and red represents the dead cells (p = 0.0225). (f) Quantification of ratio of living to dead cells in the scaffold center where green represents the living cells and red represents the dead cells (p < 0.0001). Scale bar = 2 μm. Data presented from 3 different pigs plated in triplicates.

Figure 3

MicroCT (µCT) reconstructions and quantification of bone volume at 12 weeks after implantation in mandibular defects of minipig. (a) 3D reconstruction of the defect areas filled with the empty scaffold. (b) Transverse µCT of the defect area with the empty scaffold. (c) µCT quantification of the relative residual soft tissue volume to the total volume (RV/TV, p = 0.1766).). (d) 3D reconstruction of the defect areas filled with the pADSC-seeded scaffold. (e) Transverse µCT of the defect area with the pADSC-seeded scaffold. (f) µCT quantification of bone volume to the total volume of the former defect area (BV/TV, p < 0.05). Green arrows represent the bone of the mandible. Red arrows represent the scaffold implanted in the critical size mandibular defect. Yellow arrow represents de novo bone formation in the critical size mandibular defect.

Figure 4

Bone regeneration capacity in the mandibular bone defect of minipigs evaluated by histological analysis and immunohistochemical staining at 12 weeks after implantation. (a) Haematoxylin and Eosin (H&E)-stained sections of the empty and seeded scaffolds at 5x and 10× magnifications at 12 weeks with scale bar = 500 µm and 100 µm respectively. H&E stain showed in-vivo new bone tissue formation with osteocytes. (b) Immunohistochemical staining for Osteocalcin in the empty and pADSC-seeded scaffolds at 5x and 10× magnifications (scale bar = 500 and 100 µm respectively). (c) Quantification of Osteocalcin staining. The area of bone labelling positive for OC was recorded in % of total bone area. Significantly higher amount of osteocalcin deposition was found in the test group (p < 0.001). Data presented as means ± SD (n = 3).

Figure 5

Timeline and graphical abstract of the experimental study. (a) Timeline and summary of the experiment. (b) Graphical abstract of the different working steps done in the experiment. Creating of the mandibular critical size defect, Fabrication of the scaffolds, isolation, cultivation and osteogenic differentiation of the cells, Implantation of cell-loaded scaffolds and healing, Radiographic, histological and immunohistochemical staining of the regenerated defects.

Figure 6

Surgical establishment of the mandibular critical size defect in the minipig model for implantation of empty and pADSCs-seeded TCP-PLGA scaffolds. An osteoperiosteal segmental mandibular defect of 1 cm length was made after adjustment of the titanium plate. (a) Submandibular skin incision with reflection of the mucoperiosteal flap exposing the body of the mandible with predetermining the dimension of the osseous defect using a ruler. (b) An initial cuts were done in the mandibular bone outlining the critical size defect. (c) Creating the mandibular critical size defect by cutting through the bone using reciprocating saw. (d) Fixation of the mandible with a load-bearing osteosynthesis plate to guarantee stability and avoid mandibular fractures. Scale bar = 1 mm.

Figure 7

CAD/CAM workflow for the fabrication of the scaffold. (a–f) Exemplary depiction of a mandibular critical size defect, measuring about 6 cm³. Computer-aided design (CAD) of the scaffold and computer-aided manufacturing (CAM) of a corresponding scaffold fitted into the defect area. (g–i) The printed scaffold which is then fitted into the mandibular defect with fixation by a load-bearing osteosynthesis plate. Scale bar = 1 mm.

Figure 8

Implantation of the scaffolds in the critical size mandibular defects. The mandibular defects were reconstructed by implantation of empty and pADSC-seeded scaffolds in the minipig. (a) Reflection of the flap with exposure of the critical size defect. (b) The cell-scaffold construct being transported to the operating room in 50 ml falcon tubes with cell culture media. (c) Implantation of the scaffolds either empty scaffolds or cell seeded scaffolds in the critical size defect. (d) Fixation of the scaffold using a load-bearing osteosynthesis plate. Scale bars = 1 mm.