From Synthesis to Clinical Trial: Novel Bioinductive Calcium Deficient HA/β-TCP Bone Grafting Nanomaterial

Abstract

Maxillary sinus augmentation is a commonly used procedure for the placement of dental implants. However, the use of natural and synthetic materials in this procedure has resulted in postoperative complications ranging from 12% to 38%. To address this issue, we developed a novel calcium deficient HA/β-TCP bone grafting nanomaterial using a two-step synthesis method with appropriate structural and chemical parameters for sinus lifting applications. We demonstrated that our nanomaterial exhibits high biocompatibility, enhances cell proliferation, and stimulates collagen expression. Furthermore, the degradation of β-TCP in our nanomaterial promotes blood clot formation, which supports cell aggregation and new bone growth. In a clinical trial involving eight cases, we observed the formation of compact bone tissue 8 months after the operation, allowing for the successful installation of dental implants without any early postoperative complications. Our results suggest that our novel bone grafting nanomaterial has the potential to improve the success rate of maxillary sinus augmentation procedures.

Keywords: HA/β-TCP; bionanomaterials; bionanotechnology; bone grafting nanomaterials; clinical cases; dental implants; maxillary sinus lifting; nanostructures; scaffolds; synthetic bone materials.

Figure 1

Operative procedure for bone defect plastic. 1—prepared area before the operation; 2—general view of the operative area (a)—bone before defect formation; 3—general view of bone defect formation stage (b)—bone defect; and 4—general view of the last operation stage (c)—defect filled with bone grafting nanomaterial.

Figure 2

Sinus lift procedure (description is in the text). 1—Right maxilla, a defect in the dental arch; 2—The trapezoidal mucosal–periosteal flap has been formed and detached, and the alveolar process and anterior wall of the right maxillary sinus have been skeletonized; 3—A window has been formed in the anterior wall of the right maxillary sinus; 4—Mobilization of the Schneiderian membrane to the height of the planned augmentation; 5—Loading, distribution, and condensation of the graft into the area of the lower wall of the right maxillary sinus; 6—Control of the degree of vascularization of the recipient site and control of the integrity of the Schneiderian membrane; 7—Introduction of the APRF membrane under the mucosal–periosteal flap in the area of the window on the anterior wall of the maxillary sinus; 8—The mucosal–periosteal flap is placed back in its original position and the wound is tightly sutured.

Figure 3

Scanning electron microscopy of HA/β-TCP bioactive nanomaterial (left, Magnification ×40) with cross-section image (right, Magnification ×100).

Figure 4

FTIR analysis of bioactive bone graft.

Figure 5

XRD of synthesized calcium deficient hydroxyapatite as prepared dried powder (upper image) and sintered powder at 900 °C (lower image).

Figure 6

Human osteoblast cell viability assay during 7 days of cultivation with HA/β-TCP bioactive nanomaterial (A) with the collagen production assay in weeks 1 and 2 after cell seeding (B). *—statistical significance (p ≤ 0.05 between control end experimental groups). Image of blood clot formation within one minute after the material interacted with human blood (C) with SEM image of HA/β-TCP bioactive nanomaterial (D) after the blood interaction experiment.

Figure 7

Histological evaluation of bone defect zone after bone trauma on days 7, 14, and 28 in the control group (A–C) and after application of HA/β-TCP bioactive material (D–F). Arrows demonstrate remnants of HA/β-TCP nanomaterial. Hematoxylin and eosin staining. Magnification ×100.

Figure 8

CT scans of a patient with complete edentulism of the upper jaw and a deficiency in bone tissue volume in the lateral sections of the upper jaw before sinus lifting (A); 6 months after the procedure (B,C); and after the dental implant operation (D) with bone sample harvesting (E). Yellow arrow—bone deficiency site; red arrow—HA/β-TCP bioactive nanomaterial; green arrow—place of bone sampling.

Figure 9

Structural components of the sinus augmentation zones. Osteogenesis features at the place of sinus augmentation are visible (A). Numerous bone trabeculae (BT) of variable thicknesses and structures are found around the remnants of HA/β-TCP (NG). The spaces between bone trabeculae are filled with connective tissue (CT). The structural assessment of the tissue from the biopsy (B) revealed equal volumes of bone trabeculae (BT) and connective tissues (CT) and small remnants of HA/β-TCP (NG). (A)—histological specimen of the biopsy material from the zones of augmentation. Staining with hematoxylin and eosin. Magnification ×40.

Figure 10

Heterogeneity of bone trabeculae structure and maturity within the zones of augmentation. Most of the bone trabeculae corresponded to a 2–3 score of osteogenesis. Remnants of HA/β-TCP were mostly resorbed and surrounded by primary bone (A), with irregular matrix formation and osteocyte distribution that was later replaced by secondary bone (B), with well-seen plates and regular orientation of osteocytes in the lacunae (C), with further osteocyte formation around channels with blood vessels (D). (A,B)—Toluidine blue staining, demonstrating newly formed bone trabeculae formed by primary bone, with further replacement by secondary bone. Magnification ×100. (C,D)—Hematoxylin and eosin staining, representing maturation of bone trabecules made by secondary bone with osteons. Magnification ×200.

Figure 11

Cell types inside the zones of sinus augmentation. Numerous macrophages (A) and osteoclast macrophages (B) were found on the surface and between the bone trabeculae. Augmentation of sinuses by HA/β-TCP was associated with the differentiation and recruitment of osteogenic cells (C,D) found within bone tissue and in the connective tissue between trabeculae. Bone remodeling was also accompanied by angiogenesis (E). (A)—numerous CD163+ macrophages around and between trabeculae, IHC, magnification ×40; (B)—CD68+ osteoclasts on the surface of the resorbed trabeculae, IHC, magnification ×400; (C,D)—osteogenic cells (SATB2+) around and between newly formed trabeculae, IHC, magnification ×100 and ×400, respectively. (E)—CD34+ endothelial cells reflecting angiogenesis, IHC, magnification ×400. (F)—bar chart, demonstrating the semi-quantitative scores of different cell counts.

Figure 12

Mild inflammatory infiltration of the periodontal connective tissue at the place of augmentation (A) with few T cytotoxic cells (B) and a lack of Treg lymphocytes. (A)—hematoxylin and eosin staining. Magnification ×100. (B)—bar chart representing the scores of osteogenesis and immune reaction to HA/β-TCP at the zones of augmentation. (C,D)—immunohistochemistry using monoclonal antibodies to CD8 and FOXP3. Magnification ×400.