Reconstruction of mandibular defects with autogenous bone and decellularized bovine bone grafts with freeze-dried bone marrow stem cell paracrine factors

Abstract

The gold standard following segmental mandibulectomy is vascularized autologous bone graft in the form of the fibula flap. However, in bone reconstruction the use of autogenous bone does not always guarantee a successful outcome. The aim of the present investigation was to develop a novel biologically active bone (BAB) graft, and to use it for the reconstruction of large size defects of the mandible bone following tumor resection. In the first part of the present study, biologically active bone graft was developed by using human freeze-dried bone marrow stem cells (BMSCs) paracrine factors and three-dimensional bone scaffold derived from cancellous bovine bone following decellularization. In the second part of the research, one male and three female patients with primary tumors of the mandible underwent hemimandibulectomy. The mandibular bone defects following tumor resection were reconstructed with autogenous rib grafts in three patients and BAB graft was used in one patient. The graft-host interfaces were covered with decellularized human amnion/chorion membrane graft. All patients were followed-up every five months following the reconstruction of the mandible, with no complications observed. Preliminary clinical investigations demonstrated that a BAB graft containing freeze-dried BMSC paracrine factors may be used for the reconstruction of large mandibular bone defects following tumor resection.

Keywords: decellularized human amniotic membrane; freeze-dried bone marrow stem cell paracrine factors; hemimandibulectomy; reconstruction of mandibular bone defects; three-dimensional bone scaffold.

Figure 1.

Bovine bone prior to and following decellularization, lyophilization and seeding with human freeze-dried bone marrow stem cells. (A) Stereoscopic microscope image of native bovine trabecular bone (х16 magnification), (B) decellularized bovine bone graft (х16 magnification) and (C) porous frontal surface of the bone graft (х32 magnification). (D) Micro computed tomography image of decellularized bone tissue (scale bar, 1 mm). (E) Stereoscopic microscope image of rear surface of decellularized and lyophilized bovine bone graft placed in RPMI-1640 medium (х32 magnification) and (F) bone marrow stem cells seeded on the surface of the decellularized bone graft (х56 magnification). (G) Native and (H) decellularized bovine bone graft stained with hematoxylin and eosin (magnification, ×400). (I) Cluster of differentiation 105/endoglin (×100 magnification), (J) bone morphogenetic protein-2, (×100 magnification) (K) collagen type Iα1 (×100 magnification) and (L) fibronectin (×100 magnification) expression in biologically active bone grafts with freeze-dried bone marrow stem cells.

Figure 2.

Scanning electron microscopy of bone graft and freeze-dried human bone marrow stem cells. (A and B) Human bone marrow stem cell prior to (magnification, ×500 and ×2,000, respectively) and (C and D) following freeze-drying (magnification, ×500 and ×2,000, respectively). (E and F) Scanning electron micrograph of the bone graft (magnification ×1,000 and ×2,000, respectively). (G and H) Scanning electron micrograph of bone marrow stem cells seeded on the decellularized bone graft (magnification, ×500 and ×1,000, respectively).

Figure 3.

Relative gene expression levels in isolated freeze-dried BMSCs and decellularized bovine bone grafts seeded with freeze-dried BMSCs. Gene expression analysis demonstrated that the BAB grafts containing freeze-dried BMSCs expressed a large number of varying growth factors, in particular osteocalcin and osteopontin. BMSCs, bone marrow stem cells; BAB, biologically active bone; EGF, epidermal growth factor; FGF, fibroblast growth factor; VEGF, vascular endothelial growth factor; BMP, bone morphogenetic protein.

Figure 4.

Reconstruction of mandibular bone defects with autogenous rib grafts and decellularized bovine bone grafts seeded with freeze-dried bone marrow stem cell paracrine factors. (A) X-ray of the patient diagnosed with osteoblastoma in the right portion of the mandibular body. (B) Reconstruction of the mandible with an autogenous VI rib graft. (C) Autogenous rib graft, titanium plate and host mandibular bone covered with a decellularized human amnion/chorion membrane graft. (D) Postoperative X-ray after five months of follow-up. (E) Postoperative X-ray following left hemimandibulectomy in a patient with osteoblastic osteosarcoma. (F-H) Reconstruction of the mandibular defect with a titanium plate and biologically active bone graft seeded with freeze-dried bone marrow stem cell paracrine factors. (I) Decellularized human amnion/chorion membrane graft used to cover the defect. (J-M) Thoracodorsal flap on the vascular pedicle was used for complete closure of the soft tissue without tension. (N and O) Pre- and postoperative appearance following mandibular reconstruction with the preserved contour of the mandible and face. (P) X-ray image five months subsequent to mandible bone defect reconstruction demonstrating bone volume maintenance.