Bringing a Gene-Activated Bone Substitute Into Clinical Practice: From Bench to Bedside

Abstract

Bone grafting and reconstruction are still challenging in clinical practice because of the limitations of bone autografts and the drawbacks of currently approved bone substitutes. We thus developed a gene-activated bone substitute based on octacalcium phosphate and naked plasmid DNA carrying the vascular endothelial growth factor gene. This advanced combined therapy medicinal product had no cytotoxic effects in vitro, slightly decreased bone marrow mesenchymal stromal cell (MSC) doubling time, and was characterized by a prolonged level of gene construct delivery in vivo in a luciferase bioimaging assay. In the model of critically sized cranial bone defects in rabbits, the gene-activated matrix increased bone tissue formation through angiogenesis induction. After preclinical studies, we conducted an open-label non-randomized clinical trial (NCT03076138). The primary study outcome was the proportion of patients with newly formed bone tissue within the surgical area as measured by computed tomography within 6 months after surgery. The main secondary outcomes included frequencies of adverse events (AEs) and serious adverse events (SAEs) as well as the surgical failure rate. After completing the clinical trial, the patients had dental implants placed in the bone grafting area, and trephine biopsy samples were collected. In total, 20 patients with alveolar ridge atrophy (n = 16) and jaw bone defects (n = 4) were enrolled in the study. There were no AEs or SAEs during the clinical trial or the follow-up period (30 months). In all patients, newly formed tissues with a bone density of 908.13 ± 114.40 HU were detected within the zone of bone grafting. There were no significant differences between the subgroups of patients with atrophy and bone defects: 915.28 ± 125.85 and 879.56 ± 48.36 HU, respectively (p = 0.60). Histological analysis showed that the bone grafting area comprised newly formed bone tissue with some fragments of the gene-activated bone substitute partially resorbed and integrated with bone, without fibrous tissue in between. The preclinical data and clinical trial results proved the feasibility, safety, and efficacy of the investigated material for jaw bone grafting, allowing us to bring the world's first gene-activated bone substitute from bench to bedside.

Keywords: bone substitute; clinical trial; gene-activated matrix; octacalcium phosphate; osteogenesis; plasmid DNA; vascular endothelial growth factor.

Figure 1

In vitro biocompatibility assay. (A) MSC-cultures, 3 days of co-incubation with the investigated materials; (B) number of dead cells; (C) cell culture doubling time. Scale bar: 50 μm. The asterisks above the lines connecting the groups indicate statistically significant differences (p < 0.05).

Figure 2

Plasmid DNA transfection efficacy in vivo. (A) Luminescence detection, day 7: 1 – OCP with Luc-carrying plasmid DNA, 2 –solution of plasmid DNA with Luc gene, 3 – OCP without plasmid DNA, 4 – alginate hydrogel with Luc-carrying plasmid DNA, 5 – hyaluronic acid hydrogel with Luc-carrying plasmid DNA; (B) dynamics of the luminescence level (p/s/cm²/sr). Vital luminescence bioimaging. The asterisks above the lines connecting the groups indicate statistically significant differences (p < 0.05). In the groups of OCP and pDNA-VEGF, only background luminescence was detected.

Figure 3

Critically sized bone defect repair in rabbits. CT data of parietal bones with circular defect segmentation. The asterisks above the lines connecting the groups indicate statistically significant differences (p < 0.05).

Figure 4

Critically sized bone defect repair in rabbits. (A) Full-defect histological images; (B) histological images from the central part of the defects at higher magnification: 1 – remaining fragments of implanted materials, 2 – newly formed bone tissue, 3 – fibrous tissue, H&E staining, scale bar: 300 μm; (C) quantitative evaluation of angiogenesis and bone formation; bone tissue rate is defined as the percentage of newly formed bone tissue in the total square of the defect (normal rate for the rabbit parietal bone measured with this method – 53 ± 3.5%). Bone defects were mostly filled by fibrous tissue in the empty defect group confirming the model to be critical-sized. The asterisks above the lines connecting the groups indicate statistically significant differences (p < 0.05).

Figure 5

Clinical trial with illustration of the typical indication for bone grafting: (A) scheme of the clinical trial design, (B) granules of the gene-activated bone substitute in the blood clot before implantation into the bone defect; (C) the space (indicated by the white arrow) opened to be filled in by the gene-activated bone substitute in the sinus-lifting procedure; (D) granules of the implanted gene-activated bone substitute (indicated by the asterisk) within the bone cavity.

Figure 6

Bone grafting results in patients with unilateral alveolar ridge atrophy (A) and bone defect caused by radicular cyst (B). On the left – CT scans, on the right – histological images of the trephine biopsies: 1 – gene-activated bone substitute fragments, 2 – newly formed bone tissue, 3 – fibrous tissue, Mallory trichrome staining.

Figure 7

Bone grafting results in patients with bilateral alveolar ridge atrophy. (A) The patient completed the clinical trial, on the left – CT scans: upper image – coronal view, bottom images – sagittal view, on the right – histological images of the trephine biopsy; (B) another patient completed the clinical trial, on the left – CT scans: upper image – coronal view, 6 months after surgery, bottom images – coronal view, 10 months after surgery and 4 months after dental implant placement; on the right – histological images of the trephine biopsy. 1 – gene-activated bone substitute fragments, 2 – newly formed bone tissue. H&E staining.