AMPK activation enhances osteoblast differentiation on a titanium disc via autophagy.

PURPOSE: The acquisition of osseointegration during implant therapy is slower and poorer in patients with diabetes compared with healthy persons. The serum concentration of adiponectin in patients with type II diabetes is lower than that of healthy persons via the suppression of AMP-activated protein kinase (AMPK). Therefore, we hypothesized that the AMPK activation enhances bone formation around implants, resulting in the improved acquisition of osseointegration. The purpose of this study was to evaluate the impact of AMPK activation on osteoblast differentiation and its mechanism of downstream signaling on titanium disc (Ti). METHODS: Confluent mouse pre-osteoblasts (MC3T3-E1) cells (1 × 105 cells/well) were cultured with BMP-2 for osteoblast differentiation, in the presence or absence AICAR, an AMPK activator. We examined the effects of AMPK activation on osteoblast differentiation and the underlying mechanism on a Ti using a CCK8 assay, a luciferase assay, quantitative RT-PCR, and western blotting. RESULTS: Although the proliferation rate of osteoblasts was not different between a Ti and a tissue culture polystyrene dish, the addition of AICAR, AMPK activator slightly enhanced osteoblast proliferation on the Ti. AICAR enhanced the BMP-2-dependent transcriptional activity on the Ti, leading to upregulation in the expression of osteogenesis-associated molecules. AICAR simultaneously upregulated the expression of autophagy-associated molecules on the Ti, especially LC3-II. AdipoRon, an adiponectin receptor type1/type2 activator activated AMPK, and upregulated osteogenesis-associated molecules on Ti. CONCLUSIONS: AMPK activation enhances osteoblast differentiation on a Ti via autophagy, suggesting that it promotes the acquisition of osseointegration during implant therapy.

Design of palatal and lingual augmentation prostheses by using an intraoral scanner for a patient after a glossectomy: A clinical report.

Computer-aided design and computer-aided manufacturing was used to fabricate palatal and lingual augmentation prostheses for a patient with dysphagia after a glossectomy. The function of these prostheses was comparable with that of those fabricated by conventional methods. The patient outcome suggests that an intraoral scanner can be effectively used for the fabrication of augmentation prostheses for patients with dysphagia and a high risk of aspiration.

The effect of AMP kinase activation on differentiation and maturation of osteoblast cultured on titanium plate.

Background/purpose: 5' Adenosine monophosphate-activated protein kinase (AMPK) is known as an enzyme that maintains intracellular homeostasis and has various biological activity. The purpose of this study is evaluation effect of AMPK activation on implant prognosis. Materials & methods: MC3T3-E1 osteoblast-like cells were cultured on titanium using a 24-well plate. The experimental group was divided into the following 3 groups: (1) the normal culture group (control group), (2) the osteogenic induction group, and (3) the osteogenic induction + AMPK activation group. The cell counts were measured; real-time PCR was used to assess the expression of ALP and Osterix as osteogenic related genes at Day 0,7,14 and 21 after experiments. Additionally, ALP activity and calcification were assessed. Results: The results of the real-time PCR assessments revealed that the expression of ALP, which is a marker for the initial stages of calcification, was significantly increased by AMPK activation compared to the normal culture or osteogenic induction. A significant increase was also observed in the expression of Osterix, which is a marker for the later stages of calcification. Because significant increases were observed in ALP activity and calcification potential, this suggested that AMPK activation could elicit an increase in osteoblast calcification potential. Conclusion: AMPK activation promotes implant peripheral osteoblast differentiation and maturation and enhances calcification. Our results suggest that AMPK activation may help to maintain implant stability.

Space-making effect for new bone formation by suppressing scar contraction of mucosal epithelium of rat tooth extraction wound using diode laser and CO2 laser treatment.

Background/purpose: As an extraction wound closes, the mucosal epithelium of the tooth extraction wound impedes the space for new bone formation by invading into the extraction socket. Thus, the height of the alveolar crest decreases, causing significant depression of the alveolar mucosa. In this study, we created a rat tooth extraction model and examined the effects of laser irradiation by CO2 and diode on the dynamics of myofibroblast expression through α-SMA, and TGF-ß1. Materials and methods: After tooth extraction of five-week-old male Wistar rats, they were divided into two laser treatment groups (CO2 laser or diode laser was irradiated into tooth extraction socket) and non-laser treatment group (control group). Surrounding tissues, including the extraction socket, were removed at 3, 5, 7, and 21 days after tooth extraction and the expression of α-SMA and TGF-ß1 was verified using immunohistological techniques (6 animals in each group and each period, 72 animals in total). Results: α-SMA-positive cells and TGF-ß1-positive areas were significantly lower in the two laser treatment groups than in the control group. Particularly, the diode group almost had no TGF-ß1-positive areas on the 21st day when healing after tooth extraction was deemed to be completed. Conclusion: Both CO2 and diode laser irradiation of tooth extraction wounds decreases α-SMA-positive cells and TGF-ß1-positive areas. Further, it causes a decrease in myofibroblast expression and suppresses the invasion of mucosal epithelium into the extraction socket. Therefore, laser irradiation may exert a space-making effect for new bone formation and also contribute to socket preservation.

Application of a new scan body for face-driven fixed prosthetics.

OBJECTIVE: The current method of digitally designing dental prostheses mainly focuses on intra-oral soft and hard tissues, although the harmony of the facial soft tissue and the prosthesis is crucial, especially for esthetics. Here, we introduce a new method of digitally designing dental prostheses using a new device that generates a virtual patient and incorporates facial features into the prosthetic design. MATERIALS AND METHODS: A new extra-oral scan body for facial scanning was designed and developed. A definitive edentulous maxilla implant cast with four extra-oral scan bodies (regions: maxillary left and right lateral incisors, maxillary left and right premolars) was placed in the mouth of a dental mannequin. The dental mannequin was scanned with and without the extra-oral scan bodies. For reference data, an impression of the maxilla was taken and scanned with a laboratory scanner. By superimposing each acquired data, a virtual patient was generated, and the spatial location of the abutments relative to the face was clarified. Identifying the accurate location of the abutments enabled to design face-driven dental prosthesis. RESULTS: Based on the color-coded deviation map created by the data acquired from conventional and extra-oral scan bodies, the divergence of the two data was mostly within 0.1 mm, which proves that the extra-oral scan bodies were as accurate as conventional scan bodies. Therefore, the facial scan data and the scan data of the oral cavity were successfully superimposed, which allowed to generate a virtual patient to design face-driven prosthesis. CONCLUSION: The new method is effective for designing high-quality face-driven prostheses, especially when treating a patient with a full-arch implant-fixed prosthesis.

Effects of ytterbium laser surface treatment on the bonding of two resin cements to zirconia.

Monolithic zirconia crowns bonded to zirconia abutments have become more commonly used in the construction of cement-retained implant superstructures. The present study aimed to examine the effects of laser surface treatments on the bond strength of two resin cements to zirconia. Three types of surfaces were examined: untreated, alumina blasted, and ytterbium laser treated; and two types of resin cements: 4-META/MMA-TBB resin cement and composite resin cement. Half of the specimens were subjected to a thermocycling process. Subsequently, a shear bond test was carried out. In addition, surface roughness was measured for each surface type. The results showed that laser treatment increased zirconia surface roughness and that laser treatment significantly increased shear bond strength after the thermocycling of both cement types compared to no treatment. Our experimental results suggested that ytterbium laser surface treatment of zirconia increased the bond strength of resin cements.

Bond durability and surface states of titanium, Ti-6Al-4V alloy, and zirconia for implant materials.

PURPOSE: Screw-retained implant crowns used as dental implants comprise a zirconia coping and titanium base bonded using resin cement. These devices are prone to debonding failures. This study investigated the bond characteristics of implant materials based on shear bond strength (SBS) and surface characteristics. METHODS: Chemically pure (CP) titanium grade-4 (Ti), Ti-6Al-4V alloy (Ti-6Al-4V), and tetragonal polycrystalline zirconia (zirconia) were evaluated as adherent materials. Plates of each material were polished, primed for the respective resin cements, and cemented using either methyl methacrylate-based resin cement (Super-Bond) or composite-based resin cement (Panavia). The cemented samples were subjected to 10,000 thermocycles alternating between 5 and 55 °C, and the SBS were obtained before and after thermocycling. The sample surfaces were characterized based on surface observations, roughness, and free energy (SFE). RESULTS: The SBSs of all materials bonded using Panavia were significantly compromised during thermocycling and reached zero. Although the SBSs of Ti and Ti-6Al-4V bonded using Super-Bond were not significantly affected by thermocycling, those of zirconia decreased significantly. The bond durability between zirconia and Super-Bond was improved via alumina air-abrasion, which caused no significant loss of SBS after thermocycling. Surface analyses of the air-abraded zirconia validated these results and confirmed that its surface roughness and SFE were significantly increased. CONCLUSION: The bond durability between resin cement and zirconia was lower than that between Ti and Ti-6Al-4V. The alumina air-abrasion pretreatment of zirconia improved the SFE and surface roughness, thereby enhancing bond durability.

Three-dimensional spheroids of dedifferentiated fat cells enhance bone regeneration.

INTRODUCTION: Mesenchymal stromal/stem cells (MSCs) are multipotent, self-renewing cells that are extensively used in tissue engineering. Dedifferentiated fat (DFAT) cells are derived from adipose tissues and are similar to MSCs. Three-dimensional (3D) spheroid cultures comprising MSCs mimic the biological microenvironment more accurately than two-dimensional cultures; however, it remains unclear whether DFAT cells in 3D spheroids possess high osteogenerative ability. Furthermore, it is unclear whether DFAT cells from 3D spheroids transplanted into calvarial bone defects are as effective as those from two-dimensional (2D) monolayers in promoting bone regeneration. METHODS: We compared the in vitro osteogenic potential of rat DFAT cells cultured under osteogenic conditions in 3D spheroids with that in 2D monolayers. Furthermore, to elucidate the ability of 3D spheroid DFAT cells to promote bone healing, we examined the in vivo osteogenic potential of transplanting DFAT cells from 3D spheroids or 2D monolayers into a rat calvarial defect model. RESULTS: Osteoblast differentiation stimulated by bone morphogenetic protein-2 (BMP-2) or osteogenesis-inducing medium upregulated osteogenesis-related molecules in 3D spheroid DFAT cells compared with 2D monolayer DFAT cells. BMP-2 activated phosphorylation in the canonical Smad 1/5 pathways in 3D spheroid DFAT cells but phosphorylated ERK1/2 and Smad2 in 2D monolayer DFAT cells. Regardless of osteogenic stimulation, the transplantation of 3D DFAT spheroid cells into rat calvarial defects promoted new bone formation at a greater extent than that of 2D DFAT cells. CONCLUSIONS: Compared with 2D DFAT cells, 3D DFAT spheroid cells promote osteoblast differentiation and new bone formation via canonical Smad 1/5 signaling pathways. These results indicate that transplantation of DFAT cells from 3D spheroids, but not 2D monolayers, accelerates bone healing.

Enhancement of jaw bone regeneration via ERK1/2 activation using dedifferentiated fat cells.

BACKGROUND AIMS: Mesenchymal stem/stromal cells (MSCs) are multipotent and self-renewing cells that are extensively used in tissue engineering. Adipose tissues are known to be the source of two types of MSCs; namely, adipose tissue-derived MSCs (ASCs) and dedifferentiated fat (DFAT) cells. Although ASCs are sometimes transplanted for clinical cytotherapy, the effects of DFAT cell transplantation on mandibular bone healing remain unclear. METHODS: The authors assessed whether DFAT cells have osteogenerative potential compared with ASCs in rats in vitro. In addition, to elucidate the ability of DFAT cells to regenerate the jaw bone, the authors examined the effects of DFAT cells on new bone formation in a mandibular defect model in (i) 30-week-old rats and (ii) ovariectomy-induced osteoporotic rats in vivo. RESULTS: Osteoblast differentiation with bone morphogenetic protein 2 (BMP-2) or osteogenesis-induced medium upregulated the osteogenesis-related molecules in DFAT cells compared with those in ASCs. BMP-2 activated the phosphorylation signaling pathways of ERK1/2 and Smad2 in DFAT cells, but minor Smad1/5/9 activation was noted in ASCs. The transplantation of DFAT cells into normal or ovariectomy-induced osteoporotic rats with mandibular defects promoted new bone formation compared with that seen with ASCs. CONCLUSIONS: DFAT cells promoted osteoblast differentiation and new bone formation through ERK1/2 and Smad2 signaling pathways in vitro. The transplantation of DFAT cells promoted new mandibular bone formation in vivo compared with that seen with ASCs. These results suggest that transplantation of ERK1/2-activated DFAT cells shorten the mandibular bone healing process in cytotherapy.

Intramedullary injury combined with osteoporosis therapeutics regulates targeted local osteogenesis.

Bone marrow ablation prompts transient bone formation in nearly the entire medullary cavity before marrow regeneration occurs. Here, we establish a procedure to direct bone formation in a desired particular site within the medullary cavity for support of biomedical devices. Local intramedullary injury was performed in the tibiae of rats and parathyroid hormone (PTH), alendronate, or saline was administered. Newly generated bone in the medulla was assessed by micro-CT and histology. To evaluate the function of newly generated bone, animals received intramedullary injury in tibiae followed by daily PTH. At day-14, implants were placed in the endocortical bone and the bone response to the implants was assessed. The fate of newly generated bone was compared with and without implants. We found that neither intramedullary injury nor medication alone resulted in bone formation. However, when combined, substantial bone was generated locally inside the diaphyseal medulla. Newly formed bone disappeared without implant placement but was retained with implants. Bone was especially retained around and between the implants. This study found that local bone marrow disruption followed by PTH or alendronate generated substantial cancellous bone locally in the diaphyseal medulla. This approach offers promise as a tissue engineering tool in medicine and dentistry.

Effect of DNA/protamine complex paste on bone augmentation of the mandible: A pilot study on dogs.

OBJECTIVE: Our previous studies found that a salmon DNA-based scaffold containing protamine promoted bone regeneration of the calvarial defects of rats. The aim of the present pilot study was to examine the influence of the DNA/protamine (DP) complex on bone regeneration of a saddle type, alveolar ridge defects of the dog mandible. DESIGN: Alveolar ridge defects were performed in the mandibles of five adult female beagles. The following three treatment modalities were randomly allocated: (1) the DP complex paste, (2) a beta-tricalcium phosphate (ß-TCP), and (3) a blank (control). Healing of bone defects were evaluated by periapical radiography, micro-computed tomography (micro-CT), and histology. RESULTS: Periodical radiographic images revealed that a higher percentage of regenerated bone height was consistently achieved in the DP group, as compared with blank controls. All three-dimensional, sagittal, and coronal images of micro-CT showed increased amounts of newly formed bone and a greater bone volume/ tissue volume ratio, as compared with the blank and ß-TCP groups. In contrast, there was no significant difference in bone mineral density among the groups. Histological analysis confirmed that the alveolar bone defects were filled with newly formed bone with mature and compact properties in the DP group. CONCLUSIONS: These findings indicate that the DP complexes enhanced regeneration of vertical alveolar bone defects of the dog mandible.

Bacterial-induced maternal interleukin-17A pathway promotes autistic-like behaviors in mouse offspring.

Maternal immune activation (MIA) by an infection is considered to be an important environmental factor of fetal brain development. Recent animal model on MIA induced by polyinosinic:polycytidylic acid, a mimic of viral infection, demonstrates that maternal IL-17A signaling is required for the development of autism spectrum disorder (ASD)-like behaviors of offspring. However, there is little information on bacterial infection. In this study, we aim to elucidate the influence of MIA induced by lipopolysaccharide (LPS) to mimic a bacterial infection on fetal brain development. We demonstrated that LPS-induced MIA promoted ASD-like behaviors in mouse offspring. We further found that LPS exposure induced acute phase immune response: elevation of serum IL-17A levels in MIA mothers, upregulation of Il17a mRNA expression and increase of IL-17A-producing γδ T cells in the uterus, and upregulation of Il17ra mRNA expression in the fetal brain. Blocking of IL-17A in LPS-induced MIA ameliorated ASD-like behaviors in offspring. Our data suggest that bacterial-induced maternal IL-17A pathway promotes ASD-like behaviors in offspring.

Degradation rate of DNA scaffolds and bone regeneration.

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, 107B: 122-128, 2019.

Prospective multicenter non-randomized controlled study on intraosseous stability and healing period for dental implants in the posterior region.

BACKGROUND: A current implant body surface was treated with "rough processing" by sandblasting and acid etching for the purposes of obtaining more reliable osseointegration and shortening the treatment period. Various reports have examined the healing period with the use of these implant bodies, but a consensus opinion has not yet been obtained. The purpose of this study is to evaluate the relationship between insertion torque (IT) and implant stability quotient (ISQ) at implant treatment using the current rough-surfaced implant. We evaluated the implant treatment sites with ISQ values, IT values, and voxel values. METHODS: Participants in this study comprised 26 patients (10 males, 16 females; mean age, 55.5 years) who received posterior region dental implants at Tokyo Dental College Hospital or Fukuoka Dental College Hospital. For all participants, pretreatment computed tomography and determination of bone quality from voxel values were performed. Thirty-two implant bodies were inserted into the posterior region, and insertion torque was measured. ISQ was also measured at 0, 2, 4, 6, 8, and 12 weeks postoperatively. RESULTS: Eight implant bodies in the maxilla and 24 in the mandible were inserted. All ISQ values increased, exceeding 60 by 6 weeks postoperatively. For insertion torque < 30 N cm, ISQ increased significantly after 8 weeks. For ≥ 30 N cm, the ratio at which high ISQ values appeared increased significantly after 6 weeks. Compared with the treatment area with insertion torque < 40 N cm, the treatment area ≥ 40 N cm showed a significantly higher voxel value. CONCLUSIONS: No significant relationship was found between the insertion torque value and the ISQ value. Also, it was suggested that the ISQ value was considered to be an important indicator for observing the treatment state of the implant.

Third molar transplantation combined with an osteotome sinus lift - two case reports.

We report here on our application of the socket lift technique to create a transplant socket for the transplanted maxillary molar. These outcomes suggest that this technique is likely to be effective for tooth transplantation when the maxillary sinus bottom is close to the alveolar crest.

Salmon DNA Accelerates Bone Regeneration by Inducing Osteoblast Migration.

The initial step of bone regeneration requires the migration of osteogenic cells to defective sites. Our previous studies suggest that a salmon DNA-based scaffold can promote the bone regeneration of calvarial defects in rats. We speculate that the salmon DNA may possess osteoinductive properties, including the homing of migrating osteogenic cells. In the present study, we investigated the influence of the salmon DNA on osteoblastic differentiation and induction of osteoblast migration using MG63 cells (human preosteoblasts) in vitro. Moreover, we analyzed the bone regeneration of a critical-sized in vivo calvarial bone defect (CSD) model in rats. The salmon DNA enhanced both mRNA and protein expression of the osteogenesis-related factors, runt-related transcription factor 2 (Runx2), alkaline phosphatase, and osterix (OSX) in the MG63 cells, compared with the cultivation using osteogenic induction medium alone. From the histochemical and immunohistochemical assays using frozen sections of the bone defects from animals that were implanted with DNA disks, many cells were found to express aldehyde dehydrogenase 1, one of the markers for mesenchymal stem cells. In addition, OSX was observed in the replaced connective tissue of the bone defects. These findings indicate that the DNA induced the migration and accumulation of osteogenic cells to the regenerative tissue. Furthermore, an in vitro transwell migration assay showed that the addition of DNA enhanced an induction of osteoblast migration, compared with the medium alone. The implantation of the DNA disks promoted bone regeneration in the CSD of rats, compared with that of collagen disks. These results indicate that the salmon DNA enhanced osteoblastic differentiation and induction of migration, resulting in the facilitation of bone regeneration.

Accelerated Osteogenic Differentiation and Bone Formation on Zirconia with Surface Grooves Created with Fiber Laser Irradiation.

BACKGROUND: Modification of the surface topography of biomaterials is a critical factor for the proliferation and differentiation of osteoblasts. Elucidating the biological response to surface roughening is necessary for clinical application of zirconia implants. PURPOSE: To investigate the effects of fiber laser-induced zirconia surface roughening on cultured osteoblast-like cell morphology, proliferation, differentiation, and calcification, and on in vivo bone formation. MATERIALS AND METHODS: Sixty-six machine-surfaced yttria-tetragonal zirconia polycrystal plates (S-Zr) and 16 machine-surfaced implants were used as controls. We prepared 66 rough plates (R-Zr) and 16 rough implants by surface treatment using a fiber laser. RESULTS: MC3T3-E1 cells spread well in all directions on S-Zr, whereas elongated cells with poorly organized actin stress fibers were observed on R-Zr. Cell proliferation was significantly greater on R-Zr than on S-Zr. The Runx2 mRNA level increased time dependently in osteogenic culture condition. Alkaline phosphatase activity and osteocalcin mRNA levels were higher on R-Zr compared with S-Zr. Alizarin red S staining revealed greater calcification on R-Zr than on S-Zr. Laser treatment of zirconia implant bodies placed in rat tibiae increased the bone-implant contact ratio and removal torque considerably. CONCLUSIONS: Our results suggest that fiber laser irradiation produces adequate surface roughening of zirconia ceramics to support osseointegration.

Intermittent administration of parathyroid hormone ameliorates periapical lesions in mice.

INTRODUCTION: Intermittent administration of parathyroid hormone (PTH) promotes oral osseous wound healing and protects against ligature-induced alveolar bone loss. However, its therapeutic value on periapical periodontitis is unknown. The goal of this study was to determine the effect of intermittent PTH administration on the progression of periapical periodontitis. METHODS: Seven lymphotoxin alpha-deficient mice received pulp exposures of mandibular first and second molars. Exposed pulp in the right mandible was covered with plaque-contaminated fibrin, whereas exposed pulp in the left mandible was left open. After 4 weeks, the periapical tissues were examined to determine the effect of plaque-contaminated fibrin to induce periapical lesions. Fourteen mice received pulp exposure covered with plaque-contaminated fibrin. PTH (40 µg/kg/d) was administered intermittently to half of the mice for 3 weeks beginning 1 week after pulp exposure. The remaining half received saline injections as the vehicle control. At sacrifice, mandibles and tibiae were harvested and processed for histologic examination. Evaluation of neutrophils and blood vessels was performed after staining with immunofluorescence, and periradicular bone was histomorphometrically analyzed. RESULTS: The exposed pulp covered with plaque-contaminated fibrin resulted in significantly larger periapical lesions compared with the control. Intermittent PTH administration reduced the size of periapical lesions significantly. Significantly less neutrophil infiltration around the root apex was found in PTH-treated animals compared with the control. CONCLUSIONS: PTH treatment suppressed periapical inflammation by reducing neutrophil infiltration and protected against tissue destruction by periapical periodontitis.

Photothermal stress triggered by near infrared-irradiated carbon nanotubes promotes bone deposition in rat calvarial 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.

Mesenchymal stem cell spheroids exhibit enhanced in-vitro and in-vivo osteoregenerative potential.

BACKGROUND: Mesenchymal stem cells (MSCs) are a favored cell source for regenerative medicine because of their multilinage potential. However, the conventional monolayer technique used to culture MSCs, inadequately overcomes their low differentiation capacity. Culture of MSCs in multicellular spheroids, more accurately mimics the in-vivo microenvironment; thus, resolving this problem. In this study, we assessed whether the osteoregenerative potential of MSC spheroids is greater than that of monolayer MSCs. RESULTS: MSC spheroids were generated from rat MSCs (rMSCs) using low-binding plates. Real-time reverse transcription-polymerase chain reaction and immunocytochemical analysis indicated that osteogenic properties were accelerated in MSC spheroids compared with monolayer rMSCs when treated with an osteoblast-inducer reagent for 7 days. Moreover, increased calcium deposition was visualized in MSC spheroids using Alizarin red staining. In a rat calvarial defect model, micro-computed tomography and histological assays showed that MSC spheroid-engrafted defects experienced enhanced bone regeneration. CONCLUSIONS: Our in-vitro and in-vivo results reveal that MSCs in the spheroid culture exhibit enhanced osteoregenerative efficiency compared with monolayer MSCs.