Injectable bone tissue engineering using expanded mesenchymal stem cells

Abstract

Patients suffering from bone defects are often treated with autologous bone transplants, but this therapy can cause many complications. New approaches are therefore needed to improve treatment for bone defects, and stem cell therapy presents an exciting alternative approach. Although extensive evidence from basic studies using stem cells has been reported, few clinical applications using stem cells for bone tissue engineering have been developed. We investigated whether injectable tissue-engineered bone (TEB) composed of mesenchymal stem cells (MSCs) and platelet-rich plasma was able to regenerate functional bone in alveolar deficiencies. We performed these studies in animals and subsequently carried out large-scale clinical studies in patients with long-term follow-up; these showed good bone formation using minimally invasive MSC transplantation. All patients exhibited significantly improved bone volume with no side effects. Newly formed bone areas at 3 months were significantly increased over the preoperation baseline (p < .001) and reached levels equivalent to that of native bone. No significant bone resorption occurred during long-term follow-up. Injectable TEB restored masticatory function in patients. This novel clinical approach represents an effective therapeutic utilization of bone tissue engineering.

Figure 1. Bone regeneration using TEB in canine bone defect models

(A) The mean newly formed bone areas of TEB, ABG, and PRP implanted groups and no implant controls at 2, 4 and 8 weeks post-transplantation. Data shown in the bar graph are the means ± s.d. (B) GFP-expressing BMMSCs were created using a retroviral construct in order to trace the distribution of transplanted TEB. (C-E) GFP-expressing BMMSCs (green) were identified in the grafted area at 2, 4 and 8 weeks after transplantation and osteoblasts (ob) lined up beside the regenerated bone, osteocytes (oc) within it and marrow (m) were positive for GFP (magnification x 200). (F-M) SEM evaluations of control, PRP, ABG and TEB implants were recorded at 2 and 4 weeks after transplantation. Increased bone formation (b) was observed in the ABG and TEB groups compared to control and PRP groups. (N-R) The effect of TEB transplantation in periodontitis models and representative histological images of the native tissue (N), GTR group (O), TEB transplantation group (P). (Q, R) GFP-expressing BMMSCs were detected within the regenerated tissue.

Figure 2. Transplantation of BMMSCs viability and clinical histological observation in human patients.

(A) Treatment protocol schema using injectable TEB. (B) Time course of hBMMSCs viability in TEB. Data are shown as mean ± s.d. (C-F) Representative image of live and dead staining of TEB. (G-J) The assessment of the microstructure of TEB using SEM. (G’-J’) Higher magnification images of G-J. hBMMSCs are indicated by red arrowhead. Scale bars, 20 μm (G-J); 7.5 μm (G’-J’). (K-M) Representative histological images of human biopsy samples in ABG, NB, and TEB. Scale bar, 500 μm. Biopsy samples were analyzed by HE staining (N-P) and osteocalcin (OCN) immunostaining (Q-S). Scale bar, 100 μm. (T) The newly formed bone areas of a biopsy sample that was obtained at implant placement surgery. Data are shown as mean ± s.d.

Figure 3. Clinical outcomes of TEB transplantation in human patients

(A-H) Representative images of GBR. Most of the implant threads were exposed (A). TEB (*) was transplanted into the bone cavity (B). In second-stage surgery, all space was completely filled with hard, bone-like tissue (*) (C). (D) The final prostheses. (E-G) X-ray images taken at post-operation immediately, 6 months, and 5 years. (H) HE staining of a biopsy sample at second-stage surgery. (i-p) Representative images of SFE. The maxillary bone was insufficient to place dental implants (I). After maxillary sinus floor augmentation and implants placement (J), TEB was transplanted into the sinus cavity where the implant fixture was exposed. At second-stage surgery, adequate bone regeneration was observed, and it was filled with newly formed bone (*) (K). (L) The final prostheses. (M-P) CT image taken before surgery, at post-operation 6 months, 2 years. HE staining of a biopsy sample at second-stage surgery. (Q-X) Representative images of socket preservation. After tooth extraction (Q), TEB (*) was transplanted into the socket (R). At the time of the re-entry and dental implant placement procedures, the bone defect was fully filled with hard bone tissue (*) (S, T). (U) The final prostheses. (V-X) CT image taken at post-operation immediately, 3 months and the first-stage surgery for dental implantation. (Y-HH) Representative images of periodontal regeneration. Before surgery, deep periodontal probing depth (14 mm) and intraosseous defect (8 mm) with severe tooth mobility (Degree 3) was observed (Y, Z). TEB (*) was transplanted into the defect (AA). At 6 months after TEB surgery, the bone defect was filled with bone (*) and no tooth mobility was found (BB). cc shows magnified view at 5 years. (DD-HH) CT image taken at post-operation immediately, 3, 6 months, 1 year, and 5 years.