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A DNA/protamine complex powder, prepared from the reaction between DNA and protamine sulfate solutions, was mixed with water to make a paste. The ALP activity of MC 3T3 E1 cultured on a thin film of the complex paste was higher than that cultured on a plastic well. It was found that DNA/protamine complex induced new bone formation from the results of micro-computed tomography ( -CT) images and conventional histological sections with hematoxylin and eosin staining in rat cranial defect tests. -CT image analyses showed that newly formed bone areas in defects for DNA/protamine complex were significantly higher than those for sham operation (controls) two and three months after surgery. Although the area of red-appearing osteoids reduced with increasing times after surgery, stimulated new bone formation areas were observed around newly formed bone in histological sections stained with Villanuva osteochrome bone stain. Therefore, DNA/protamine complex paste with a biodegradable property will be a useful injectable biomaterial for the repair of bone defects.

The bone regenerative healing process is often prolonged, with a high risk of infection particularly in elderly and diseased patients. A reduction in healing process time usually requires mechanical stress devices, chemical cues, or laser/thermal therapies. Although these approaches have been used extensively for the reduction of bone healing time, the exact mechanisms involved in thermal stress-induced bone regeneration remain unclear. In this study, we investigated the effect of optimal hyperthermia on rat calvarial defects in vivo and on osteogenesis in vitro. Photothermal stress stimulation was carried out using a new photothermal device, composed of an alginate gel including in carbon nanotubes and their irradiator with near-infrared light. Photothermal stress (15 min at 42℃, every day), trigged by near-infrared-induced carbon nanotube, promoted bone deposition in critical-sized calvarial defects compared with nonthermal stress controls. We recently reported that our novel DNA/protamine complex scaffold induces bone regeneration in calvarial defects. In this study, photothermal stress upregulated bone deposition in DNA/protamine-engrafted calvarial defects. Furthermore, photothermal stress significantly induced expression of osteogenic related genes in a time-dependent manner, including alkaline phosphatase, osterix, and osteocalcin. This was observed in DNA/protamine cells, which were expanded from regenerated tissue engrafted into the DNA/protamine scaffold, as well as in human MG63 preosteoblasts. In summary, this novel carbon nanotube-based photothermal stress approach upregulated expression of osteogenic-related genes in preosteoblasts, resulting in promotion of mineral deposition for enhanced bone repair.

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Scaffolds implanted into bone defect sites must achieve optimal biodegradation rates while appropriately filling the void as new bone formation progresses. We recently developed a unique biomaterial consisting of salmon deoxyribose nucleic acid (DNA) and protamine, which can be used as an osteoconductive scaffold for tissue engineering. The aim of the present study was to elucidate how the degradation rate of the scaffold affects bone regeneration. We examined the relationships between the degradation rate of salmon DNA scaffolds and new bone formation using a rat skin flank subcutaneous model and rat calvarial defect model. The degradation rates of the scaffolds were proportional to the durations of pretreatment with ultraviolet (UV) light irradiation. The biodegradation rates of the scaffolds were also dependent on the duration of UV irradiation, as tested a subcutaneous tissue implantation. Scaffolds irradiated with UV light for 0.5 h maintained gradual biodegradation of phosphate compared with scaffolds irradiated for 0 or 3 h. In the calvarial defect model, we found that new bone formation was higher in rats treated with scaffolds irradiated with UV light for 0.5 h compared with those irradiated with UV light for 0 or 3.0 h. The present results suggest that bioengineering of scaffolds for biodegradation is important to regenerate bone. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018.